ASBMR 29th Annual Meeting

1182

p63 Plays a Central Role in Cartilage Development by Directly Regulating Key Genes for Chondrogenesis. T. Ikeda¹, H. Kawaguchi², T. Saito², A. Kan², F. Yano¹, M. Ushita², Y. Kawasaki², K. Nakamura*², U. Chung¹, ¹Div of Tissue Engineering, Graduate School of Medicine, The Univ of Tokyo, Tokyo, Japan, ²Orthop Surg, Graduate School of Medicine, The Univ of Tokyo, Tokyo, Japan.

To understand the molecular network underlying chondrogenic differentiation, we identified the embryonic SOX6 promoter and its core enhancer CES6. Using the 4 × CES6 as bait for bio-panning of cartilage-derived phage display libraries, we identified a p53 family gene p63 as a CES6-binding transcription factor. Luciferase-reporter assay revealed that p63 transactivated not only SOX6 promoter, but also promoters of type II collagen (COL2A1) and SOX9. Among the three alternative splicing variants α, β and γ, p63β exhibited the strongest activity. When multi-tissue expression was evaluated by real-time RT-PCR, p63 was mainly expressed in the tracheal cartilage, testis and prostate. Overexpression of p63 in non-chondrogenic cell line HeLa caused rapid induction of SOX6 and COL2A1 expressions, which were 2-3 fold greater than that by overexpression of SCOW, the representative transcription factor for chondrogenesis. Furthermore, retroviral overexpression of p63 into de-differentiated mouse costal cells after a long period of culture induced their re-differentiation to chondrocytes with enhanced Alcian blue staining and increased COL2A1, SOX6 and SOX9 mRNA levels. By combining luciferase-reporter assay, electrophoretic mobility shift assay, and chromatin immunoprecipitation, we identified the direct binding sites of p63β in CES6 of SOX6 and in the proximal promoters of the SOX9 and COL2A1. The identified sequences were 1-2 bp different from the known p53 binding motif “RRRCWWGYYY”. To further learn the endogenous function of p63, we investigated the skeleton of p63 knockout (p63-/-) mouse embryos that exhibited severe short limb deformities. The expressions of SOX6, SOX9 and COL2A1 were rarely detectable in limb buds of p63-/- embryos around El2-13 by whole mount in situ hybridization. Although a small cartilage was barely formed after E17, the SOX6, SOX9 and COL2A1 expressions were decreased compared to those in the wild-type littermates. Since BrdU uptake was comparable between p63-/- and wild-type limb buds, the impaired cartilage development by the p63 deficiency may be due to a defect in chondrogenic differentiation, but not in proliferation, of mesenchymal cells. In conclusion, we identified a novel chondrogenic transcription factor p63 which may play a central role in cartilage development by directly regulating key genes for chondrogenesis like SOX6, SOX9 and COL2A1. Further understanding of molecular network related to p63 will herald a new era of cartilage regenerative medicine.

Disclosures: T. Ikeda. None.

1183

Atf4 Regulates Chondrocyte Proliferation and Differentiation by Direct Targeting Indian Hedgehog Transcription. W. Wang*, X. Yang, Center for Bone Biology, Departments of Medicine and Pharmacology, Vanderbilt University, Nashville, TN, USA.

Indian Hedgehog plays a critical role in growth plate biology, and understanding the regulation of its expression is important for our understanding of endochondral bone formation. In studies on Atf4, a CREB-related transcription factor required for osteoblast differentiation and function (Yang & Karsenty, 2004), we noted evidence that Atf°4 also affects cartilage biology. For example, Atf4-deficient (Atf4^−/−) mice are small in size with shorter limbs suggesting a defect in endochondral bone formation. We found that Atf4 whose cell-specificity is regulated at protein level (Yang et al., 2004) is expressed in chondrocytes by Western blot analysis using an antibody against Atf4. To determine whether Atf4 mutant mice display abnormalities of growth plate chondrocytes, we examined Atf4^−/− newborns in details by histological analysis, BrdU-labeling of dividing cells, and in situ hybridization. Compared to their wildtype counterparts, Atf4^−/− newborns exhibit 1) a delay in hypertrophic mineralization, 2) an expansion of the hypertrophic zone, 3) a decrease in BrdU positive proliferating chondrocytes, and 4) a dramatic decrease in the expression of the Indian Hedgehog (Ihh), a key regulator coordinating the chondrocyte proliferation and differentiation in endochondral bone formation in the prehypertrophic chondrocytes. Consistent with these in situ hybridization results, we also detected a decrease in Ihh mRNA expression in the Atf4^−/− chondrocytes. Based on these observations we hypothesize that Atf4 may play a central role in controlling chondrocyte proliferation and differentiation by direct targeting Ihh transcription. To test this hypothesis, we cloned a 5 kb promoter fragment of the Ihh gene from a BAC genomic DNA clone and made 5 Ihh promoter driven-luciferase reporter constructs that contain different lengths of the Ihh promoter sequences. Co-transfection assays using these reporter constructs and Atf4 expression vector showed that ATF4 indeed was able to transactivate the Ihh promoter with maximal observed activation in a reporter construct containing 742 base-pairs of the Ihh proximal promoter sequence, pIhh742-Luc. By visual scanning we found 9 putative Atf4 binding sites (Al to A9) within this fragment. Gel retardation assays showed that Atf4 bound strongly to A9 and weakly to A1 to A8. Deletion of A9 led to a decrease in ATF4 transactivation activity on the pIhh742-Luc by 60%, suggesting that Atf4 activates the Ihh gene through A9. Taken together, our findings identify Atf4 as a physiological activator of Ihh gene and a regulator of chondrocyte proliferation and differentiation by directly targeting Ihh gene transcription.

Disclosures: W. Wang, None.

1184

Trpsl Transcription Factor Regulates Chondrocyte Hypertrophy and Perichondria! Mineralization through Repression of Runx2. D. Napierala¹, K. Sam*¹, R. Morello¹, T. Bertin*¹, O. Zheng¹, T. Sibai*¹, R. Shivdasani*², B. Lee¹, ¹Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA, ²Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA, USA.

Mutations involving the TRPS1 gene cause tricho-rhino-phalangeal syndrome (TRPS) characterized by short stature, cone-shaped epiphyses, premature closure of growth plates and distinctive craniofacial appearance. During mouse skeletal development Trpsl is highly expressed in chondrogenic mesenchymal condensations, prehypertrophic chondrocytes, joint mesenchyme and perichondrium. Trpsl is a transcription repressor that contains nine zinc-fingers including GATA-type and Ikaros-type domains. To elucidate the role of Trpsl in endochondral bone formation, we analyzed Trpsl mutant mice deleted for the GATA DNA-binding domain (Trps±GT mice).

Histological analysis of long bones of E18.5 embryos showed reduced size of bone marrow cavity due to expansion of cartilaginous part in homozygous Trps±GT mice. Morphometric analyses and RNA in situ hybridization demonstrated that zones of proliferating, prehypertrophic and hypertrophic chondrocytes are elongated in TrpsAGT mice in comparison with WT littermates. Moreover, the Trpsl mutant mice demonstrated advanced mineralization of perichondrium. Both mineralization and chondrocytes maturation are regulated by the Runx2 transcription factor, therefore we tested the potential interaction of Trpsl and Runx2 in vitro. Cotransfection experiments demonstrated that Trpsl can repress the Runx2-mediated transactivation of the target reporter. Moreover, the C-terminal part of the Trpsl protein physically interacts with the runt domain of Runx2 in the GST pull-down assay. In conclusion, our data indicate that Trpsl regulates the growth plate mineralization and chondrocyte hypertrophy, and that could be partially through the repression of the Runx2 protein function.

Disclosures: D. Napierala, None.

This study received funding from: N1DCR.

1185

Identification of a Novel Chondrogenic Factor Sorting Nexin 19 Using a Real-Time Fluorescence Monitoring Cell Line ATDC5-S2RD5. A. Kan, T. Ikeda, T. Saito, U. Chung, H. Kawaguchi, Sensory & Motor System Medicine, Tissue Engineering, University of Tokyo, Tokyo, Japan.

To identify chondrogenic factors, the present study initially attempted to establish a monitoring system that could detect chondrogenic differentiation in a precise, noninvasive and real-time manner. For this purpose, we identified a 49 bp region including a SOX9 binding enhancer in intron 1 of type II collagen (COL2A1) gene, which was highly conserved among human, mouse and rat. We then established several lines of mouse chondrogenic ATDC5 cells stably transfected with four tandem copies of the 49 bp region cloned upstream of the COL2A1 promoter and DsRed2 fluorescence reporter gene. Among them, we selected a clone ATDC5-S2RD5 that fluoresced not only upon chondrogenic stimulation by insulin, but also by BMP-2, TGFβ, and SOX9 / SOX6 co-transfection. This cell line was confirmed to show increases in COL2 mRNA level and Alcian blue staining by the stimulations above. We then created retroviral expression libraries from human tracheal cartilage and mouse embryos, and applied the ATDC5-S2RD5 cell line to the expression cloning for their screening. Among several genes that caused fluorescence in the system, sorting nexin (SNX) 19, a member of the SNX family, intracellular transportation proteins whose insufficiency causes skeletal dysplasia in human, exhibited strong fluorescence. Multi-tissue expression analysis by real-time RT-PCR confirmed the SNX 19 expression in cartilage, and the subcellular localization was shown to be limited in the cytoplasm by immunostaining with an anti-myc-FITC antibody in HeLa cells overexpressing myc-SNX19. To learn the functional relevance of SNX 19 to chondrogenic differentiation, we established ATDC5 cells and mouse primary costal cells overexpressing SNX 19 or the siRNA through retroviral transfection. COL2 mRNA level by real-time RT-PCR and the promoter activity by luciferase-reporter assay were increased by the SNX 19 overexpression, whereas they were decreased by the knockdown. Among signaling molecules ERK, p38 MAPK, JNK, Akt, IkB, Smads and GSK3β, the SNX19 overexpression enhanced phosphorylation of p38. SB203580, a specific inhibitor of p38, suppressed COL2 expression induced by the SNX 19 overexpression. In conclusion, we established a real-time fluorescence monitoring cell line for chondrogenesis ATDC5-S2RD5. Using it, we identified a novel chondrogenic factor SNX 19 which was a cytoplasmic protein related to the p38 signaling. Further screening of molecules using this monitoring cell line will elucidate the molecular network underlying chondrogenic differentiation.

Disclosures: A. Kan, None.

1186

Osteal Macrophages: Novel Regulators of Bone Formation. L, J. Raggatt¹, M. H Chang*¹, K. A. Alexander*¹, J. S. Kuliwaba², N. L. Fazzalari², D. A. Hume*¹, A. R. Pettit¹, ¹Institute for Molecular Bioscience, Cooperative Research Centre for Chronic Inflammatory Diseases, University of Queensland, Brisbane, QLD, Australia, ²Institute of Medical and Veterinary Sciences and Hanson Institute, University of Adelaide, Adelaide, SA, Australia.

Resident tissue macrophages are an integral component of many tissues and are important in immunity, homeostasis and repair. Macrophages have been implicated in both bone destruction and osteophyte formation in bone diseases, suggesting these cells may also be associated with osteal tissues. We report here that resident tissue macrophages (referred to as OsteoMacs) are present on the endosteal surface of human trabecular bone. We have extended these observations using the murine tissue macrophage marker F4/80 to investigate fully the anatomical distribution of OsteoMacs in mouse bones. Immunohistochemistry demonstrated F4/80⁺ OsteoMacs are intercalated in bone lining tissues. OsteoMacs were also closely associated with bone remodelling units in the secondary spongiosa but are not TRAP⁺ mononuclear osteoclast precursors. Importantly, OsteoMacs formed a cell layer over bone forming osteoblasts (OB, collagen type 1⁺ and osteocalcin⁺) on growing cortical surfaces. Histomorphometric analysis showed that on cortical endosteal surfaces 75.9±5.3% of the OB bone surface was covered by a F4/80⁺ cell [canopy], indicating that OsteoMacs and OB are intimately associated in vivo and that OsteoMacs may regulate OB function. We examined the effect of OsteoMacs on OB functional responses to elevated extracellular calcium (eCa²⁺), as eCa²⁺ is unique to the bone microenvironment and induces macrophage expression of the osteo-inductive molecule BMP-2. Using a co-culture system of macrophages with OB, in either direct contact or separated using transwells, we demonstrated that OB mineralization occured in response to eCa²⁺ only in the presence of macrophages. At no stage were OB exposed to the classical differentiation agents ascorbate and beta-glycerophosphate, yet macrophage enhanced OB mineralization occurred rapidly (7 days). Conditioned medium from eCa²⁺ stimulated macrophages also induced mineralization of OB cultures, indicating that macrophages produce a soluble factor that enhances OB mineralization. However addition of recombinant noggin or soluble BMPR1 to co-cultures did not inhibit macrophage-induced OB mineralization, indicating that BMP-2 is not the osteo-inductive molecule. These observations suggest macrophages detect changes in eCa²⁺ and consequently produce soluble factors) that drive OB mineralization. We propose that OsteoMacs are an integral component of osteal tissues and play a novel role in bone homeostasis through regulating OB function and orchestrating bone formation.

Disclosures: L.J. Raggatt, None.

1187

Upregulation of Calcium-sensing Receptor Gene Expression by the Proinflammatory Cytokine, Interleukin-6: In Vivo and In Vitro Studies. L. Canaff*, K. Schorr*, G. N. Hendy, Medicine, McGill University, Montreal, PQ, Canada.

Increased calcium-sensing receptor (CASR) expression in parathyroid gland, thyroid C-cells, and kidney tubule leads to a decrease in the extracellular calcium set-point, thereby reducing parathyroid hormone (PTH) secretion and renal calcium reabsorption and increasing calcitonin secretion resulting in reduced circulating calcium levels. Hypocalcemia is common in critically ill patients with burn injury and/or sepsis. In such patients, circulating levels of cytokines are increased, with those of interleukin-6 (IL-6) being most marked. In the present study, we have examined the ability of IL-6 to affect the expression of the CASR. In vivo, in the rat, parathyroid, thyroid and kidney CASR mRNA levels increased after intraperitoneal injection of IL-6. Peak values occurred at 16h and remained elevated at 24h. This was associated with decreased circulating calcium and PTH levels. Further in vitro studies were conducted. The human CASR gene has two promoters (P1 and P2) yielding transcripts having alternative 5′ untranslated regions (1A and 1B), but encoding the same receptor protein. Activities of human CASR P1 and P2 promoter/ luciferase reporter constructs transfected into proximal tubule HKC cells and TT thyroid C-cells were stimulated ∼2-3 fold by IL-6. The PI construct contains 1439 bp upstream of the transcription start (+1), the 638 bp exon 1A and 242 bp of exon 2 before the ATG translation start site. Transfection of PI deleted and mutated constructs into TT cells revealed that a Stat element within exon 1A accounted for ∼70% of the induction by IL-6. Cotransfection of Stat 1 (Y701F) or Stat3 (Y705F) dominant negative constructs markedly reduced (∼70%) IL-6-stimulated upregulation of the PI promoter. Pretreatment of TT cells with the MAPK pathway inhibitor U0126 (10 microM) led to significant loss (∼30%) of IL-6 upregulation of all the PI promoter constructs including an exon 1A Stat mutant. The P2 construct has 460 bp upstream of the transcription start (+1), the 259 bp exon 1B and 242 bp of exon 2 before the ATG translation start site. Transfection of P2 deletion constructs into TT cells showed that sequence within −341 to +220 is required for IL-6 responsiveness. There are no Stat elements in the P2 promoter or exon IB but Spl elements are present at the transcription start site (+1). Cotransfection of Spl and/or Statl or Stat3 dominant negative constructs abolished IL-6 responsiveness of the P2 promoter. In conclusion, a variety of elements in the CASR gene promoters mediate transcriptional upregulation by IL-6, with Stat and MAPK independently and additively controlling promoter P1, and Stat regulating promoter P2 via Spl.

Disclosures: L. Canaff, None.

1188

The Role of Caspase-3 in the Anabolic Actions of PTH in Bone. J. Yamashita¹, D. Yang¹, K. Yamashita*¹, E. T. Keller², L. K. McCauley¹, ¹University of Michigan School of Dentistry, Ann Arbor, MI, USA, ²University of Michigan Medical School, Ann Arbor, Ml, USA.

Caspase-3 is a crucial component in the signaling of programmed cell death, cell survival, and cell cycle in a variety of cells. It has been reported that caspase-3 functions in osteogenic differentiation of bone marrow stromal cells and that the activation of caspase-3 is essential for osteoclast differentiation. Intermittent PTH is a powerful bone anabolic agent, but the exact mechanisms of PTH action are unclear. It has been hypothesized that PTH may extend the life span of osteoblasts by suppressing apoptosis and the long-living osteoblasts would contribute to enhanced mineral matrix deposition. Because caspase-3 is a pro-apoptotic factor, suppression of caspase-3 could enhance the anabolic effect of PTH in bone. However, the role of caspase-3 in PTH actions is not known. In this study we tested the hypothesis that inhibition of caspase-3 enhances anabolic actions of PTH in bone. Caspase-3 deficient mice were genotyped and the expression of caspase-3 protein verified by Western blotting of spleen lysate. Histomorphometric analysis of the tibiae of day-21 mice showed no significant difference in bone area (%) between caspase-3 wild type (+/+) and knockout (-/-) mice. Caivarial osteoblasts were isolated from caspase-3 +/+ and -/- mice. Cell enumeration showed no difference between caspase-3 +/+ and -/-osteoblasts over the 9day culture period. To characterize apoptosis of osteoblasts, cells were treated with staurosporine and relative cell numbers versus control were assessed. Caspase-3 -/- osteoblasts ex vivo showed more resistance to staurosporine-induced cell death than +/+, suggesting an important role of casapase-3 in osteoblast survival. To determine the role of caspase-3 in bone formation in response to PTH, daily administration of PTH (50μg/kg/day) was performed on +/+ and -/- mice from day-4 to day-21 of age. Peripheral quantitative computed tomography and histomorphometric analyses showed that both caspase-3 +/+ and -/- mice responded to PTH with substantially increased bone area when compared to vehicle-control groups. Such increased bone mass was more significantly pronounced in caspase-3 -/- mice than +/+ (p<0.05). To further determine whether the inhibition of caspase-3 augments anabolic effect of PTH in osteoporotic bone, caspase-3 +/+ and -/- mice (9 weeks old) were ovariectomized and PTH (80μg/kg/day) administered for 21 days. Daily PTH administration significantly increased bone mass in both genotypes with greater anabolic effect in -/- than +/+ bone. These results suggest that the inhibition of caspase-3 may enhance the anabolic actions of PTH in bone. Caspase-3 could be a target for the prevention and treatment of osteoporosis in conjunction with PTH therapy.

Disclosures: J. Yamashita, None.

1189

Dissection of the Mechanisms of PTH-mediated Inhibition of Sodium-dependent Phosphate Transport Using a Long-acting PTH(l-28) Analog. S. Nagai, M. Okazaki*, J. T. Potts, H. Jüppner, T. J. Gardella, Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA.

PTH reduces the reabsorption of inorganic phosphate (Pi) largely through its effects on sodium-dependent phosphate transporters (NaPi-IIa and NaPi-IIc) in renal proximal tubule (PT) cells. The mechanisms by which PTH regulates transporter function in these cells are not clear, particularly in terms of the signaling and protein-trafficking pathways utilized. We now investigated these processes by utilizing a new PTH analog, [Ala¹,12,Aib³,Gln¹⁰,Har¹¹,Trp¹⁴,Arg¹⁹]hPTH(l-28)NH₂ {M-PTH(l-28)} that binds to the PTH receptor more stably than does PTH(l-34). The capacity of the analog to inhibit phosphate uptake in opossum kidney (OK) cells, a model PT cell line, was first assessed by a routinely used 4-hour incubation protocol. M-PTH(l-28) inhibited Pi uptake to the same extent (∼60%) as did PTH(l-34) (ligand concentrations = 1×10⁻⁷ M). Phosphate transport was then assessed at various time points after incubating the cells with ligand for only 10 minutes followed by extensive rinsing before measuring Pi uptake. Using this protocol, Pi uptake was maximally inhibited by PTH(l-34) two hours after wash-out and returned to basal by 24 hours. In cells treated with M-PTH(l-28), Pi uptake was again maximally inhibited at 2 hours, but remained suppressed for at least 24 hours. Analysis of cAMP production showed that the response to PTH(l-34) diminished to basal by 1 hour after wash-out, whereas that of M-PTH(l-28) continued for at least 6 hours. PTH(l-34) is known to induce die removal of NaPi-IIa from the surface of OK cells; confocal microscopy showed that whereas NaPi-IIa surface expression was restored by 24 hours in cells treated with PTH(l-34), it was completely absent in cells treated with M-PTH(l-28). To explore these differences in ligand activity in vivo, PTH(l-34) and M-PTH(l-28) were injected subcutaneously into mice, and serum Pi was measured. Both ligands suppressed Pi levels maximally at 2 hours, but where Pi levels returned to normal by 4 hours in PTH(1-34)-treated mice, they remained suppressed for 6 hours in M-PTH(l-28)-treated mice. The data show that persistent binding of a PTH agonist to the PTH receptor in renal PT cells can lead to sustained cAMP signaling and sustained down-regulation of NaPi-IIa. The mechanisms by which PTH regulates NaPi-IIa expression and function in PT cells are incompletely understood, but the formation of cAMP appears to be a key signaling event. Moreover, the recovery process occurs more slowly than expected, particularly when assessed with the new analog, M-PTH(l-28). This new PTH analog should be useful in exploring the underlying mechanisms further.

Disclosures: S. Nagai, None.

1190

Analysis of PTH-PTH Receptor Interaction Mechanisms Using a New, Long-Acting PTH(l-28) Analog Reveals Selective Binding to Distinct PTH Receptor Conformations and Biological Consequences In Vivo. M. Okazaki*, S. Nagai, T. Dean*, J. T. Potts, T. J. Gardella, Endocrine Unit, Massachusetts General Hospital, Boston, MA, USA.

The PTH/PTHrP receptor (PTHR) is likely to exist in multiple protein conformations. This raises the possibility that different PTH or PTHrP ligands could bind selectively to distinct PTHR conformations and thereby produce different biological effects. Based on recent radioligand binding data, we hypothesize that the PTHR can adopt a novel PTHR conformation, termed R0, that is uncoupled from heterotrimeric G protein but yet can form highly stable complexes with certain PTH ligands, including PTH(l-34). In contrast, PTHrP(l-36) and M-PTH(I-15) (M = Aibl,3,Gln10,Har11,Ala12,Trp14), bind only poorly to R0, and instead bind predominantly to PTHRs in a G protein-coupled conformation (RG). We further hypothesize that R0 can isomerize to RG, and thus contribute to ligand activity. For example, stable binding of a ligand to R0 could result in signaling responses in cells long after the initial ligand-binding event. To test these hypotheses, we investigated the structural features in the ligand and receptor that underlie stable binding to R0 in vitro, and we explored the biological relevance of such binding in vivo. One analog to emerge from the studies was [Ala1,12,Aib3,Gln10,Har11,Trp14,Arg19]hPTH(l-28)NH2 {M-PTH(l-28)}. In R0 binding assays, performed using membranes expressing the rat PTHR, 1251-PTH(l-34) tracer radioligand, and in the presence of GTPI³S, M-PTH(l-28) bound with 80-fold higher affinity than did PTH(l-34), and more than 200-fold higher than did M-PTH(1-15) or unmodified PTH(l-28). In ROS17/2.8 cells, M-PTH(l-28) produced a more prolonged cAMP response than did the latter three peptides. When injected subcutaneously into mice (50 nmole/Kg), M-PTH(l-28) produced a more prolonged hypercalcaemic response than did PTH(l-34): blood Ca++ induced by PTH(l-34) peaked at 2 hours and returned to vehicle control at 4 hours; whereas, that induced by M-PTH(1-28) peaked at 3 hr, and did not return to control until at least 6 hours. Overall, the studies support the hypothesis that the PTHR can form a stable R0 conformation, and that the extent to which a ligand (e.g. PTH or PTHrP) binds this conformation can determine the biological actions of that ligand in vivo. The capacity of a ligand to bind to R0 requires interaction to the both the juxtamembrane and N-terminal regions of the PTHR, because both the “M” modifications and the 16-28 segment of M-PTH(l-28) are required. Further exploration of the conformational states possible for the PTHR should improve our understanding of how this receptor functions in normal and pathological physiology.

Disclosures: M. Okazaki, Chugai Pharmaceutical Co., Ltd 2.

This study received funding from: NIH DDK 11794.

1191

Parathyroid Hormone Expression Is Negatively Regulated by FGF23 in Primary Bovine Parathyroid Cells. T. Krajisnik¹, P. Bjorklund*², R. Marsell*², O. Ljunggren*¹, G Akerstrom*², K. B. Jonsson*², G Westin*², T. E. Larsson¹, ¹Uppsala University Hospital, Dept. of Medical Sciences, Uppsala, Sweden, ²Uppsala University Hospital, Dept. of Surgical Sciences, Uppsala, Sweden.

Fibroblast Growth Factor-23 (FGF23) is a circulating protein that regulates serum levels of inorganic phosphate as well as l,25(OH)2D3. FGF23 is elevated in chronic kidney disease (CKD), as a response to the prevailing hyperphosphatemia. In addition, high serum FGF23 in CKD is a predictor of future secondary hyperparathyroidism, although the reason for this association is unknown. The objective of the current study was to investigate the effect of FGF23 on parathyroid glands in vitro. For this purpose, we used primary isolates of bovine parathyroid cells. Recombinant FGF23 was produced by transient transfection of the hFGF23(R176Q)-pcDNA3.1-V5-His-TOPO plasmid into COS-7 cells. Conditioned serum-free medium was collected and analyzed for intact FGF23 concentration using an intact FGF23 ELISA. Control medium was obtained by transfection of the pcDNA3.1(+) expression vector. Importantly, we found that Klotho, a FGF23 receptor co-factor, was abundantly expressed in bovine parathyroid cells, as detected by RT-PCR. FGF23-stimulation of parathyroid cells at concentrations of 400-2000 pg/mL was followed by a 2-7-fold increase in Egr-1 mRNA level, a marker of FGF23 signaling (p<0.05). FGF23 potently and dose-dependently decreased the PTH mRNA level within 12 hours in the concentration range 400-2000 pg/mL (p<0.01). In agreement, FGF23 dose-dependently decreased PTH protein secretion into conditioned media at concentrations 700-2000 pg/mL (p<0.05). Finally, FGF23 did not alter cell viability or apoptosis, as determined by flow cytometry analysis. A small but significant increase in cell proliferation was observed (p0.001), as measured by tritium-labelled thymidine incorporation. We conclude that FGF23 is a potent negative regulator of PTH mRNA expression and protein secretion. Our data indicate that increased FGF23 levels in CKD may, in part, inhibit secondary hyperparathyroidism. This potentially defines a novel function of FGF23 in CKD, besides the previously established role in ameliorating hyperphosphatemia by promoting renal Pi excretion.

Disclosures: T. Krajisnik, None.

1192

Geranylgeranylation Promotes Osteoblast Survival and Is Involved in Anti-apoptotic Effects of PTH. T. Yoshida*, P. H. Stern Department of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.

We have previously described a Ga12/Gα13-RhoA-phospholipase D pathway in osteoblastic cells, and found that the pathway is stimulated by parathyroid hormone (PTH). It was therefore of interest to determine the role of the pathway in osteoblast cell function and in parathyroid hormone action on osteoblasts. Studies in other tissues indicate that the pathway is involved in cell survival and influences the apoptotic cascade. Covalent attachment of geranylgeranyl moieties from the donor geranylgeranylpyrophosphate (GGPP) leads to the activation of RhoA, and several studies show that reducing the cellular pool of GGPP by inhibition of HMG-CoA reductase leads to apoptosis. These reports led us to investigate whether the pathway, and specifically geranylgeranylation, could be involved in osteoblast survival and in the anti-apoptotic effects of PTH. We used the geranylgeranyl group donor geranylgeraniol (GGOH) and the geranylgeranyltransferase inhibitor GGTI-2166 as tools to study this. Preosteoblastic MC3T3-E1 cells were seeded at a density of 250,000 cells/well in 6-well plates, and serum starved for 24 hr, a stimulus leading to apoptosis and elevated caspase-3 activity, a central step in the apoptosis cascade. Subsequent treatment with PTH for 6 hr markedly decreased caspase-3 activity. Effects were seen at PTH concentrations as low as 10 pM. In contrast to the effects of short-term exposure, overnight treatment with PTH was not effective. The response to PTH was density-dependent and was attenuated in higher density cultures. GGOH (10 uM) mimicked the effect of PTH, decreasing caspase-3 activity in serum-starved cells. The effects of PTH or fetal bovine serum to decrease caspase-3 activity were antagonized by GGTI-2166, 10 and 30 uM. In cultures of primary mouse osteoblasts, PTH promoted cell survival after serum starvation, as measured by trypan blue exclusion, and the geranylgeranyltransferase inhibitor GGTI-2166 antagonized the protective effect of PTH. These results suggest that GGPP is critical for osteoblast survival. In addition to direct effects through Gal2/Gal3-coupled receptors, we speculate that the PTH effect on survival and on PLD could be mediated through increases in a cellular pool of GGPP.

Disclosures: T. Yoshida, None.

1193

Decreases in Cortical Thickness, and not Changes in Trabecular Microstructure, Are Associated with the Pubertal Increase in Forearm Fractures in Girls. S. Kirmani, L. McCready*, M. Holets*, P. R. Fischer*, B, L. Riggs, L. J. Melton, S. Khosla, Mayo Clinic, Rochester, MN, USA.

We previously demonstrated that forearm fractures in girls peak between age 8 and 11 years, during the time of maximal pubertal growth (JAMA 2003; 290:1479). The structural basis for this increase is unclear but may be related to the transient cortical thinning and increased porosity related to increased calcium demands during maximal growth. It is also possible that there may be alterations in trabecular microstructure during puberty that reduce bone strength. To address these possibilities, we used high-resolution 3D pQCT (XtremeCT, Scanco, voxel size ∼90 microns) to assess trabecular and cortical bone parameters in healthy girls aged 7-16 yrs (n=46) without a prior history of fracture. Subjects were classified into 4 groups based on bone age (BA) assessed using hand and wrist x-rays: Group I (BA, 6-8 yrs, n = 8), Group II (BA, 9-11 yrs, n = 15), Group III (BA, 12-14 yrs, n = 13), and Group IV (BA, 15-16 yrs, n = 10). There were no significant differences (P > 0.2) in trabecular parameters (bone volume/total volume, BV7TV; trabecular number, Tb.N; or trabecular thickness, Tb.Th) through the course of puberty, expressed as a percentage of the mean value in Group I (Panel A of figure). By contrast, there were marked but transient decreases in cortical thickness (C.Th) which decreased by 50% during puberty (P < 0.005 for group I vs. group III, P = 0.07 for group I vs. group II) before rising sharply by 89% at the end of puberty (P < 0.005 for group I vs. group IV). This pattern was a mirror image of the previously described rise in distal forearm fractures in this population (Panel B), and there were similar changes in cortical vBMD (data not shown).

Our findings, which represent the first direct assessment of trabecular and cortical structure during growth in children, thus demonstrate that: (1) perhaps surprisingly, puberty is associated with minimal changes in trabecular microstructure, suggesting that these parameters may be established very early in life; and (2) the transient increase in distal forearm fractures during growth in girls is largely explained by temporary cortical thinning (and perhaps increased cortical porosity). Further studies are needed to define the mechanisms underlying these alterations in cortical parameters as well as preventive measures that may attenuate these changes and, hence, distal forearm fractures during growth.

Disclosures: S. Kirmani, None.

This study received funding from: Thrasher Research Fund.

1194

Vitamin D Receptor Gene Polymorphisms Modulate the Musculoskeletal Response to Vitamin D Supplementation in Healthy Girls. A, Arabi¹, Z. Mahfoud*², L. Zahed*³, J, Maalouf¹, M. Nabulsi*⁴, M. Choucair*¹, G. El-Hajj Fuleihan¹, ¹Calcium Metabolism and Osteoporosis Program, American University of Beirut, Beirut, Lebanon, ²Epidemiology and Population Health, American University of Beirut, Beirut, Lebanon, ³Pathology and Laboratory Medicine, American University of Beirut, Beirut, Lebanon, ⁴Pediatrics, American University of Beirut, Beirut, Lebanon.

Associations between Vitamin D receptor (VDR) genotypes and bone mass have been reported. Our group showed a beneficial effect of vitamin D (Vit D) supplementation on bone mass in girls¹. This effect was mediated by changes in height, bone area and lean mass'. Whether the musculoskeletal response to Vit D is modulated by polymorphisms in VDR gene has not been previously assessed.

The relationship between VDR gene polymorphisms and the musculo-skeletal response to Vit D supplementation was evaluated in 168 girls (10-17 years), randomly assigned to placebo (n=55), Vit D3 1400 IU/week (n=58) or Vit D3 14000 IU/week (n=55), for one year¹. Calcium intake was similar between groups¹.

VDR genotypes were determined using BsmI, TaqI and Apal restriction enzymes. Bone mass at the spine (LS), hip, foreami (FA) and total body (TB), and lean mass were measured by DXA at baseline and at one year.

Because the response to Vit D3 was not dose dependent¹, Vit D3 groups (low and high dose) were combined in the analyses. VDR gene polymorphisms using Bsml restriction enzyme were associated with percent changes in height, LS BMC, femoral neck BMC (FN BMC) and TB BMC, FN area, TB area, TB BMD and FA BMD in the Vit D3 group but not in the placebo group. The least increments were observed in the BB genotype (Table). There was no difference in changes in lean mass between genotypes. The relationship between VDR genotypes and changes in BMD and BMC remained significant after adjustment for menarcheal status, changes in lean mass, in height or in bone area. Similar relationships between skeletal response and VDR were obtained with Taq1 restriction enzyme. The least increments were observed in the tt genotype. No such relationship was observed when VDR genotypes were determined using Apal restriction enzyme.

In conclusion, plymorphisms in the VDR gene may modulate the skeletal response to vitamin D supplementation in healthy adolescent girls.

¹ El-Hajj Fuleihan et al, J Clin Endocrinol Metab, 2006.

Table showing the percent changes BMC in placebo or Vitamin D3 group, according VDR genotype

Table  .  

*; # Significant differences between the two genotypes by ANOVA and post-hoc analyses.

Disclosures: A. Arabi, None.

This study received funding from: Nestle Foundation; Merck KGaA.

1195

Growth and Response to Active Vitamin D and Phosphorus Treatment Is Highly Associated with Haplotype of the Vitamin D Receptor Promoter in X-linked Hypophosphatemic Rickets. F. Jehan, M. Nguven*, O. Walrant-Debray*, C. Sinding*, M. Garabedian*, Hopital Saint Vincent de Paul, Inserm U561, Paris, France.

X-linked hypophosphatemic rickets is a monogenic disease characterized by bone deformities, growth retardation, dental anomalies, urinary phosphate wasting, low serum 1,25-dihydroxyvitamin D3 and hypocalciuria. Treatment with phosphorus and 1-hydroxylated forms of vitamin D cures, or prevents, bone deformities, and improves calcium/phosphorus status. However, its beneficial effect on growth retardation remains controversial, mainly due to wide variations of individual expressivity of the disease. We have hypothesized that natural genetic variations (polymorphisms and/or haplotypes) could explain some of the variability in growth impairment and treatment responses. We considered variants in the promoter region of the vitamin D receptor (VDR) gene as a good target, since we found association between VDR promoter variants and growth in healthy adolescents girls (d'Alesio et al., Hum Mol Genet, 2005 14: 3539–48), and since variants influencing VDR expression are likely to influence responses to vitamin D treatment in hypophosphatemic patients.

Methods. We analyzed the VDR promoter haplotype structure in a large cohort of hypophosphatemic rickets patients treated since early childhood with phosphate and 1-hydroxylated vitamin D derivatives (n=52 including 16 patients now adults), that we have clinically and biologically followed from diagnosis on.

Results: 1) A clear association was observed between adult height (expressed as SD scores) and VDR Hapl haplotype (Hapl+: −0.82±0.40, Hapl-; −2.59±0.26; mean ± SE; p<0.004). The Hapl effect on growth was highly significant as early as 4 years of age (p<0.004). It was independent of the gender, vitamin D and phosphate daily dosages, and age at onset of treatment. No such effect was observed by breaking the cohort with the two other major haplotypes of the VDR promoter.

2) Urinary calcium excretion increased with the l-(OH)D dosage in all patients during childhood. But the sensitivity to treatment was clearly Hapl dependent. With I microgram per day of 1-OH vitamin D, the mean urinary calcium/creatinine ratio for each patient was normal in Hapl+ children (0.36±0.20 mmol/mmol, n=26) and already elevated in Hapl-children (0.76±0.35, n=10, mean * SD, p<0.0001 vs. Hapl+). Other biological values were similar in the two Hapl groups (serum calcium, phosphorus, 25-(OH)D, and 1,25-(OH)2D).

Conclusion: variations in the clinical and biological expressivity of die hypophosphatemic rickets may be explained, at least in part, by natural genetic variants on the VDR promoter that may regulate the VDR protein expression, and hence the patient sensitivity to vitamin D treatment.

Disclosures: F. Jehan, None.

1196

Sequencing of Collagen I in Three Patients with Multiple Fractures of Unknown Aetiology Reveals Novel Mutations Causing Osteogenesis Imperfecta with Normal to High BMD, K. Lindahl*, H. Brandstrdm, A, Kindmark*, Ö. Liunggren*, Medical Sciences, Endocrinology, Uppsala, Sweden.

Osteogenesis imperfecta (OI) is a heterogeneous genetic disorder with an incidence of approximately 1/15 000 individuals. In general, patients with OI have low Bone Mineral Density (BMD) and an increased susceptibility to fracture. Clinical features of OI also include blue sclerae, dentinogenesis imperfecta, deafness and hyper mobile joints. Phenotypes range from mild to lethal. In more than 90% of cases the disorder is due to a dominant mutation in one of the two genes that encode collagen type I, Coll AI and CollA2. Mild OI is usually the result of a mutation that leads to a premature stop codon, which subsequently leads to too little, but structurally normal collagen. In contrast, the more severe OI phenotypes are often de-novo mutations leading to an exchange of a glycine residue for an amino acid with a bulkier side chain. Patients with multiple fractures, but no other clinical signs of OI, pose a diagnostic problem, especially when BMD is normal. It is debated whether collagen sequencing should be performed as a routine in these cases.

In this study, we have sequenced Coll a 1 and Colla2 in three patients who had suffered more than 15 fractures, but had a BMD in the normal to high range. None of the patients had any clinical signs of OI and despite extensive investigation neither of the patients had a clinical diagnosis explaining their fragile bones.

DNA was extracted from whole blood using a commercially available kit. Exons and flanking intron sequences of COL1A1 and COL1A2 were sequenced using standardised sequencing with ABI3130XL using 110 primer pairs.

Three novel mutations were found: Aspl219Asn, was detected at the splice site of the carboxy-terminal propeptide of the COL1A1 gene, Glnl206Termination in COL1A1 and Gly757Ser in COL1A2. Control sequence ruled out the possibility of a naturally occurring polymorphism and the mutations were not previously reported in the collagen database. These findings indicate that sequencing of collagen type I is valuable in patients with multiple fractures of unknown aetiology. Furthermore, BMD is not necessarily low or strictly correlated to bone strength in OI.

Disclosures: K. Lindahl, None.

1197

Bone Traits Are Determined Early in Life but Adapt During Growth to Accommodate Local Loading Circumstances. Q. Wang¹, S. Cheng², E. Seeman¹, ¹Medicine/Austin Health, University of Melbourne, Melbourne, VIC, Australia, ²Department of Health Sciences, University of Jyvaskyla, Jyvaskyla, Finland.

Bone structure determines the loads tolerated but the loads imposed also determine its structure. We hypothesized that: l)the position of an individual's bone trait is established prior to puberty and remained in the position. 2) bone shape changes to accommodate prevailing loads, 3)bone strength and the loads imposed increase in proportion with age so their ratio is independent of age. In 258 10–13yrs healthy girls, peripheral quantitative computed tomography (Stratec XCT-2000) was used to determine the cross sectional structure of the left tibial shaft and muscle size during 2 years. Mothers of 108 of these girls were also studied. Height, lower leg length, bone traits such as total tibial cross sectional area (CSA) and cortical thickness increased with age but trait variances - the magnitude of the dispersion around the age-specific mean did not. The relative position of bone trait, whether at the 5th, 50th or 95th percentile, was established prior to puberty. In 60-70% individuals their bone traits remained in their percentile after 2 years; the other 30-40% individuals their bone trait's percentile increased or decreased. The change in the percentile of muscle CSA or in lean tissue mass (from DXA) correlated with the change in the percentile of bone traits (r=0.5-0.6). Periosteal apposition added more than twice the amount of bone anteriorly (18 mg) and posteriorly (10 mg) than medially (5 mg) and laterally (4 mg) increasing tibial ellipticity. Bending strength of tibial shaft thus increased more in the antero-posterior (Imax) than medio-lateral direction (Imin); the ratio imax/Imin increased by 7% over the 2 years (p<0.001) and was 13% higher in the mothers than in girls at baseline (p O.001) (Fig). Imax increased with age and correlated with the estimated load imposed (tibial length × weight) (r2=0.73). The ratio Imax/estimated load was independent of age reflecting proportional age-related increases in the structural determinants of bone strength and the load imposed.

In conclusion, we infer that skeletal structure in one individual as a result of genetic variability is already expressed before puberty. In this setting, skeletal development is adaptive throughout growth so that load applied modifies bone shape accommodating that load efficiently.

Disclosures: Q. Wang, None.

This study received funding from: Academy of Finland.

1198

Extraction Socket Healing in Pediatric Patients Treated with Pamidronate: A Retrospective Chart Review, C. J. Chahine*¹, M. Cheung*², T. W. Head*¹, S. Schwartz*³, F. Rauch², F. H. Glorieux², ¹Oral and Maxillofacial Surgery, McGill University, Montreal, PQ, Canada, 2Genetics Unit, Shriners Hospital for Children, Montreal, PQ, Canada, Pediatric Dentistry, Montreal Children's Hospital, Montreal, PQ, Canada. Osteonecrosis of the jaw (ONJ) has been described in adults who have received bisphosphonates, but its occurrence in children has not been reported. In the present study we evaluated whether ONJ has occurred in any of the pediatric patients who received pamidronate therapy at the Shriners Hospital for Children in Montreal. 338 patients had received pamidronate treatment. Dental follow up data were obtained on 49 patients (16 females) who had undergone tooth extraction (age at last follow up: 2 to 24 years, mean 12.8 years). The indication for pamidronate use included osteogenesis imperfecta (N = 46), fibrous dysplasia (N = I), hypercalcemia (N = 1), and muscular dystrophy (N = 1). A total of 181 teeth had been extracted (145 primary teeth, 36 permanent teeth). 19 of the teeth required surgical extraction with flap elevation, bone removal, and tooth sectioning. Prophylactic antibiotic use was implemented in 10 patients. The indication for dental extraction was impacted teeth (N = 16), fractured teeth (N = 11), decay or odontogenic abscess (N = 38), retained primary teeth (N = 67), and ectopic eruption or pre-orthodontic therapy (N = 49). The mean cumulative dose of pamidronate prior to dental extraction was 40.6 mg/kg. The mean duration of pamidronate treatment prior to dental extraction was 4.5 years. 100 teeth were extracted during active treatment. The mean interval of time between the last pamidronate dose prior to dental extraction and first pamidronate dose following dental extraction was 55 and 61 days, respectively. 81 teeth were extracted an average of 1.6 years after the last dose of pamidronate. All of the patients were followed postoperatively with a mean of 2.6 years (range: 1 month to 10 years). Adequate healing as judged by the treating dentist's clinical evaluation was noted on post-operative visits in all patients. There was no evidence of delayed healing, exposed bone, or ONJ in any patient. In conclusion, we did not detect any cases of ONJ in this large pediatric patient population with long-term pamidronate exposure.

Disclosures: C.J. Chahine, None.

1199

Large-scale Analysis of Association Between Polymorphisms in the LRP-5 and −6 Genes and Bone Mineral Density and Fracture, J. B. J. van Meurs*¹, T. A. Trikalinos*², S. H. Ralston*³, F. Rivadeneira*¹, J. P. A. loannidis*², A. G Uitterlinden*¹, The Genomos Study Group*⁴, ¹Internal Medicine, Erasmus Medical Center, Rotterdam, The Netherlands, ²Hygiene and Epidemiology, University of Ioannina, Ioannina, Greece, ³Rheumatic Diseases, University of Edinburgh, Rotterdam, United Kingdom, ⁴For more information:, http://www.genomos.eu/, The Netherlands.

Objective: Mutations in the low-density lipoprotein receptor-related protein 5 (LRP5) gene cause rare syndromes characterized by altered bone mass. Common LRP5 variants may affect bone mass and fracture risk, but the magnitude of the association has been debated. The objective of this study was to generate large-scale evidence on whether common variants of LRP5 and its close homologue LRP6 are associated with bone mineral density (BMD) and fracture-risk.

Methods: We performed a prospective meta-analysis of participant-level data from 18 centers on 37,760 men and women. We assessed the association of common LRP5 (Val667Met, Alal330Val) and LRP6 (Ilel062Val) variants with lumbar spine (LS) BMD, femoral neck (FN) BMD, all prevalent fractures, and prevalent vertebral fractures. Genotyping was standardized across centers. Unadjusted analyses with generalized linear mixed models allowed for random effects across center-gender strata. Adjustments for sex, age, weight, height, menopausal status and hormone replacement therapy were also performed. Fracture risks were also adjusted for BMD.

Results: LS-BMD decreased by 0.02 g/cm² per 667Met allele copy (p=3.3 × 10⁻⁸), and by 0.014 g/cm² per 1330Val copy (p=2.6 × 10⁹). The corresponding decrease for FN-BMD was 0.011 and 0.008 g/cm² (p-5.0′10⁶ and p=3.8*10⁻³). For both LS and FN this corresponds to approximately 0.1 SD difference in BMD. For fractures, the strongest association was seen for vertebral fracture risk (OR=1.26 [95%CI: 1.05–1.24] and 1.12 [95%CI: 1.01-1.24] per allele for Val667Met and Alal330Val, respectively), but an effect was present also for all fractures (OR 1.14 [95%CI:1.05-1.24] and 1.06 [95%C1:1.01–1.12] per allele, respectively). The fracture risks were partly attenuated by adjustment for differences in BMD. Haplotype analysis of the two LRP5 variants indicated independent effects of both variants on BMD. The LRP6 Ilel062Val polymorphism was not associated with any of the phenotypes.

Conclusions: In this very large genetic association study of more than 37,000 subjects we show that common variants of the LRP5-gene are consistently associated with both BMD and fracture risk across different Caucasian populations. The magnitude of the effect is modest, but highly significant. LRP5 is the first gene to reach a genome-wide significance level for a phenotype related to osteoporosis.

Disclosures: J.B.J, van Meurs, None.

This study received funding from: European Commission, grant QLK6-CT-2002-02629.

1200

Serum DHEA Is Independently of Estradiol and Testosterone Related to Incident Fractures in Elderly Men - The MrOS Sweden Study. C. Ohlsson¹, L, Vandenput¹, A. Holmberg², H. Mallmin³, E. Orwoll⁴, A. Oden*⁵, A. Eriksson¹, M, Lorentzon¹, M. Stenstrom*¹, M. K. Karlsson², Ö. Liunggren⁶, F. Labrie*⁷, D. MellstrÖm¹, ¹Center for Bone Research at die Sahlgrenska Academy, Dpts of Internal Medicine and Geriatrics, Gothenburg University, Gothenburg, Sweden, ²Dpt of Orthopaedics, Malmo University Hospital, Lund University, MalmO, Sweden, ³Dpt of Surgical Sciences, Uppsala University, Uppsala, Sweden, ⁴Bone and Mineral Unit, Oregon Health and Sciences University, Portland, OR, USA, ⁵Consulting Statistician, Gothenburg, Sweden, ⁶Dpt of Medical Sciences, Uppsala University, Uppsala, Sweden, ⁷Laboratory of Molecular Endocrinology and Oncology, Laval University, Quebec, PQ, Canada.

The enzymes required to transform adrenal-derived dehydroepiandrosterone (DHEA) into androgens and/or estrogens are expressed in several peripheral target tissues such as bone and muscle. The impact of serum DHEA for bone health in elderly men is unclear. The aim of the present study was to determine the relationship between serum DHEA and incident fractures in elderly men of MrOS Sweden (n=30I4, 75.4 ± 3.2 years). Incident X-ray verified fractures at major non-vertebral osteoporosis-related sites (hip 39, distal radius 29, proximal humerus 16 and pelvis 8 fractures) were identified in 82 subjects with an average follow-up of 3.3 years. Serum levels of DHEA, estradiol and testosterone were analyzed by validated gas chromatography/mass spectrometry assays (n=2640). Serum SHBG was measured using an immunoradiometric assay.

Multivariate proportional hazards regression analysis was used to assess the independent relationship of DHEA to fracture risk. In age-adjusted analyses, baseline DHEA was inversely related to incident fracture risk (hazard ratio (HR) per SD decrease in DHEA = 1.43; CI 1.17-1.75). As serum estradiol and serum testosterone were inversely and SHBG was directly related to incident fracture risk, these parameters were included in the analyses of the independent relationship between DHEA and incident fracture risk. Interestingly, the relationship between DHEA and incident fracture risk was not affected by adjustment for estradiol, testosterone and SHBG (HR per SD decrease in DHEA = 1.40; CI 1.13–1.74). Neither inclusion of total hip BMD (HR per SD decrease in DHEA = 1.36; CI 1.11–1.67) nor inclusion of BMI(HR per SD decrease in DHEA = 1.45;CI 1.18-1.77) in the model affected the association between DHEA and incident fractures. In conclusion DHEA is an independent predictor of incident non-vertebral fractures at major osteoporosis-related sites in elderly men. Because this association was independent from changes in BMD, one may speculate that adrenal-derived DHEA could influence neuromuscular function and thereby fracture risk in elderly men.

Disclosures: C. Ohlsson. None.

1201

Enhancement of Prognosis of Osteoporotic Fractures by Genetic Marker: Contribution of the Collagen I alpha 1 Gene. B. H. Tran*, N. D. Nguyen. S. A. Frost*, J. R. Center, J. A. Eisman, T. V. Nguven, Bone and Mineral Research Program, Garvan Institute of Medical Research, Sydney, Australia.

Given the time-invariant nature of the genetic information and BMD and age-related nature of fracture risk, a short-term absolute risk approach is more meaningful and appropriate than a relative risk-based approach. The present study was aimed at developing a prognostic model for predicting 5-y and 10-y of fracture risks in elderly women by incorporating genetic marker information.

COLIA1 genotypes (GG, GT and TT) were determined in 915 women of Caucasian background aged 60+ years as at 1989, who have been followed for up to 17 years (1989-2006). Low trauma, non-pathological fractures were recorded. Femoral neck bone mineral density (FNBMD) was measured by dual energy X-ray absorptiometry (GE-LUNAR DXA) at baseline and expressed as T-scores. A series of nomograms for predicting 5-year and 10-year risk of fractures including any fracture, hip and vertebral fractures for an individual woman were developed by using Cox's proportional hazards model.

The proportion of individuals with GG, GT and TT genotypes in the population of 63%, 32% and 5%, respectively, which was consistent with Hardy-Weinberg equilibrium law. In the multivariable Cox's proportional hazards model, the risk ratio for COLIA1 TT (vs. G allele) and any, hip and vertebral fractures were 3.07 (1.47–6.43), 7.07 (2.37–21.14) and 4.45 (1.89–10.47), respectively, after adjusting for age and FNBMD. The area under the curves for prediction models for any fracture, hip and vertebral fractures were 0.68, 0.85 and 0.69, respectively. Using the models' parameters, various nomograms were constructed for individualizing the risk of any, hip and vertebral fractures. A woman with the TT genotype had an absolute fracture risk equivalent to a 20-year older counterpart without the TT genotype independence of BMD. Similarly, a woman homozygous for the T allele had an absolute risk approximately equivalent to a woman with the same age without the genotype but with 1 SD lower in FNBMD.

These data suggest that in women the effect of COLIA1 TT genotype (5% in the population) is equivalent to an effect of 20-year increase in age or one SD lower in BMD. The incorporation of COLIA1 genotypes could enhance the short-term prognosis of osteoporotic fracture in women.

Figure Figure:. 5-y risk of any fracture in women, stratified by age, BMD and COLIA1 genotype

Disclosures: B.H. Tran, None.

This study received funding from: National Health and Medical Research Council, Australia.

1202

Serum 25 Hydroxy Vitamin D 25(OH) and the Risk of Hip Fractures: The Women's Health Initiative (WHI). J. A. Cauley*¹, A. LaCroix*², L. Wu*², J Lee*³, M. Horowitz*¹, D. Bauer*⁴, R. Jackson*⁵, M, Danielson¹, M, LeBoff*⁶, F. Stranczyk*¹, S. Cummings*⁸, ¹University of Pittsburgh, Pittsburgh, PA, USA, ²University of Washington, Seattle, WA, USA, ³University of California, Davis, Davis, CA, USA, ⁴University of California San Francisco, San Francisco, CA, USA, ⁵Ohio State University, Ohio, OH, USA, ⁶Harvard University, Cambridge, MA, USA, ⁷University of Southern California, Los Angelos, CA, USA, ⁸University of San Francisco, San Francisco, CA, USA.

Vitamin D deficiency is common in older persons, especially in home based nonulations. in general medical innatients and in women with acute hin fracture. To test the community dwelling women, we preformed a nested case control study in the WHI Observational Study Cohort. We excluded women who had a history (hx) of hip fracture or used hormones or other osteoporosis medications at baseline. A total of 39,793 women met these criteria. We randomly chose 400 women who had an incident hip fracture, confirmed by medical record over a median of 7.1 y. A control was selected for each case, matched on baseline age (+/- one year), race/ethnicity and date of blood draw (within 75 days). 25(OH)D was measured in fasting baseline serum using DiaSorin kits. The sensitivity of the assay was l.Sng/ml; interassay coefficients of variation ranged 8.6% to 12.5%. Conditional logistic regression models were conducted to evaluate the association between hip fractures and Vitamin D. Odds ratio (OR) with 95% confidence intervals (CI) were estimated per SD and across quartiles of 25(OH)D (defined by the distribution in controls) with p values for tests of linear trend. We adjusted for potential confounders. The mean age of cases and controls was 70.8 years; 95% were White. 25(OH)D levels ranged from 3.7 to 48.6 ng/ml. The mean (SD) Vitamin D level (ng/ml) in the cases was 22.4 (8.1) and 23.8 (7.2) in the controls, p=0.007. The risk of hip fractures was 77% higher among women whose 25(OH)D was <19ng/ml (Table). Additional adjustment for other risk factors for fracture had no effect. In conclusion, low serum 25(OH)D levels are an independent prognostic risk factor for hip facture in community dwelling postmenopausal women. Odds ratio (95% CI) for hip fracture

Table  .  

*Multivariate adjustment includes age, BMI, parental hx of hip fracture, personal hx of fractures, smoking, alcohol, calcium and Vitamin D intake.

Disclosures: J.A. Cauley, Merck & Company 2, 8: Novartis Pharmaceuticals 2, S: Eli Lilly & Company 2, 5, 8; Pfizer Pharmaceuticals 2.

This study received funding from: NHLBI.

1203

Variation in the FLNB Gene Regulates Bone Density in Two Populations of Caucasian Women. S. G Wilson¹, B. H. Mullin*¹, M. R. Jones*¹, I. M. Dick¹, F. Dudbridge*², T. P. Spector³, R. L. Prince⁴. ¹Endocrinology, Sir Charles Gairdner Hospital, Nedlands, Australia, ²MRC Biostatistics Unit, Cambridge, United Kingdom, ³Twin Research and Genetic Epidemiology, King's College London, London, United Kingdom, ⁴Medicine and Pharmacology, The University of Western Australia, Nedlands, Australia.

Based on our previous linkage data showing that genes in the chromosome 3p14–21 region are associated with osteoporosis¹ and data that mutations of the Filamin B (FLNB) gene are the cause of osteochondrodysplasias including Larsen's Syndrome, Boomerang Dysplasia and atelosteogenesis type I & III, recently confirmed by KO studies in mice, we have undertaken a detailed study of the effects of variation in the FLNB gene sequence on bone structure. Filamin B is a cytoskeleton protein involved in actin polymerisation and participates in its interaction with signal transduction pathways. Thus the aim of this study was to evaluate the role of variation in the FLNB gene in regulating bone mass and the propensity to osteoporotic fracture. This hypothesis was tested in two large cohorts, firstly 771 women from the GENOS sib-pair study, identified on the basis of an osteoporotic proband and previously used in linkage analysis, and secondly a population-based cohort of 1192 unrelated Caucasian women from the CAIFOS/CARES study. Hip bone density was measured by DXA (Hologic QDR 4500). Genotyping for the family study was by GoldenGate Assay (Illumina Inc.) and in the population study used MALDI-ToF (Sequenom Inc.). Association between BMD and genotype in the family study was assessed using the FBAT program and in the population-based cohort used ANOVA. Genotype effect on fracture rate was examined using a Chi-square test. Haplotype analysis was undertaken using UNPHASED software. Nine SNPs covering the LD in the FLNB gene were studied in the family-based cohort, of which four (rs7637505, rs9822918, rs2177153 and rs2001972) showed an association with age corrected BMD at both femoral neck (P = 0.02-0.0002) and total hip (P = 0.02–0.0006). Six SNPs were taken forward to the population-based replication study of which, rs9822918 (P = 0.02–0.003) and rs2177153 (P = 0.02) showed association with BMD. The difference in mean BMD for alleles of rs2177153 was 3.9–44.9%. Fewer prevalent fractures were observed in individuals homozygous for the G allele of rs2177153 (P = 0.015). Our data shows that genetic variation in the FLNB gene is associated with differences in bone mass and this has been replicated in two independent cohorts of Caucasian women. These results suggest that understanding the mechanism may lead to greater insight into bone cell biology and new therapies.

1. Wilson SG et al., 2003 Am. J. Hum. Genet. 72:144–155.

Disclosures: S.G Wilson, None.

This study received Junding from: NHMRC Project No. 294402.

1204

Genetic and Environmental Effects on Muscle Cross-Sectional Area and Structural Strength of Tibia in Older Female Twins. T. Mikkola*¹, SL Sipila*², T. Rantanen*¹, H. Sievänen³, H. Suominen¹, J. Kaprio*⁴, M. Koskenvuo*⁴, M. Kauppinen*², A. Heinonen¹, ¹Department of Health Sciences, University of Jyväskyla, Jyväskyla, Finland, ²Finnish Centre for Interdisciplinary Gerontology, University of Jyväskyla, Jyväskyla, Finland, ³Bone Research Group, UKK Institute for Health Promotion Research, Tampere, Finland, ⁴Department of Public Health, University of Helsinki, Helsinki, Finland.

The aims of this study were to estimate to what extent individual differences in cross-sectional area of the lower leg muscles (mCSA), bending strength of tibial shaft (BSIb) and compressive strength of distal tibia (BSIc) are influenced by genetic and environmental factors in older women, and to investigate whether the associations between these traits are due to common genetic or environmental factors.

The mCSA, BSIb and BSIc values were measured with peripheral quantitative computed tomography from 102 monozygotic (MZ) and 113 dizygotic (DZ) 63–76-year-old female twin pairs. Quantitative genetic models were used to decompose the phenotypic variances into common and trait-specific additive genetic (A), shared environmental (shared by the sisters, C) and individual environmental (unique to each sister, E) factors.

The pairwise correlations of the MZ and DZ twins were 0.73 and 0.42 for mCSA, 0.73 and 0.50 for BSIb and 0.73 and 0.56 for BSIc, respectively. The age-adjusted multivariate independent pathway model showed that the total relative contributions of A, C and E were 75%, 0% and 25% for mCSA, 55%, 20% and 25% for BSIb and 40%, 37% and 23% for BSIc, respectively. In addition, the model showed that a genetic factor, common to all three traits accounted for 75% (95% CI: 66–82%) of the variance in mCSA, 17% (8–27%) of that in BSIb and 8% (2–17%) of that in BSIc. BSIb and BSIc had a shared environmental factor in common which accounted for 20% (9–35%) and 37% (20–53%) of the variances in BSIb and BSIc, respectively. A common individual environmental factor accounted for 5% (2-11%) of the variance in mCSA, 22% (13–33%) of that in BSIb and 13% (7–22%) of that in BSIc. The remaining variances were attributable to trait-specific genetic factors and trait-specific individual environmental factors.

In older women, cross-sectional area of the lower leg muscles, tibial bending strength and tibial compressive strength share common genetic and environmental effects. The total influence of genes is strong on muscle area and weaker on tibial strength indices. These results suggest that in older women the same genetic and environmental factors may predispose to or protect from both sarcopenia and bone fragility.

Disclosures: T. Mikkola, None.

This study received funding from: Finnish Ministry of Education.

1205

Effect of Denosumab on Bone Mineral Density and Bone Turnover Markers: 48-Month Results. P. Miller¹, M. Bolognese*², E. M. Lewiecki*³, M. McClung⁴, B. Ding*⁵, Y. Liu⁵, J. San Martin⁵, ¹Colorado Center for Bone Research, Lakewood, CO, USA, ²Bethesda Health Research Center, Bethesda, MD, USA, ³New Mexico Clinical Research & Osteoporosis Center, Albuquerque, NM, USA, ⁴Oregon Osteoporosis Center, Portland, OR, USA, ⁵Amgen Inc., Thousand Oaks, CA, USA.

Denosumab is an investigational fully human monoclonal antibody that reduces bone turnover by inhibiting RANKL, an essential mediator of osteoclast formation, function, and survival. An ongoing phase 2 study in postmenopausal women with low bone mineral density (BMD) showed that denosumab increased BMD and reduced bone turnover markers (BTM) during the first 24 months of therapy. We report 48-month results of denosumab continuous treatment, discontinuation, and rechallenge.

After 24 months, subjects were reassigned. Those who continued on denosumab were switched from other denosumab dosing regimens to 60 mg Q6M (continuous treatment). Those who received denosumab 210 mg Q6M were switched to placebo (PBO) for the rest of the study (discontinuation), and those on 30 mg Q3M were switched to 12 months of PBO followed by 12 months of 60 mg Q6M (rechallenge). Subjects in the PBO arm remained on PBO, and those in the alendronate (ALN) arm discontinued ALN and were followed. Changes in BMD, BTM, and safety were assessed at 48 months.

BMD data from 229 subjects were available for this interim analysis. After 48 months, continuous denosumab treatment resulted in sustained BMD gains from baseline. Lumbar spine BMD % change from baseline at month 48 was 10.6% for denosumab vs −2.7% for PBO (between-group difference: 13.4%, P < .001). BMD also increased at the total hip with denosumab (5.8% vs −2.9%; between-group difference: 8.7%, P < .001; Figure). Denosumab discontinuation was associated with a BMD decrease to values near baseline. Rechallenge with denosumab increased BMD to an extent similar to initial therapy. In subjects who discontinued ALN, a slight decline of BMD was observed. Continuous denosumab therapy led to sustained reduction of BTM from baseline. BTM increased upon denosumab discontinuation and decreased with rechallenge. Adverse event rates were similar among treatment groups over 48 months.

These data indicate that RANKL inhibition with denosumab led to continued gains in BMD and suppression of BTM relative to PBO over 48 months, which appeared to be reversible with discontinuation and responsive with rechallenge.

Disclosures: P. Miller, Amgen Inc. 2, 5, 8; P&GP 2, 5, 8; sanofi-aventis 2, 5, 8; Roche 2, 5,8.

This study received funding from: Amgen Inc.

1206

Efficacy of Bazedoxifene in Reducing New Vertebral Fracture Risk in Postmenopausal Women with Osteoporosis: Results From a 3-Year, Randomized, Placebo- and Active-Controlled Clinical Trial. S. L. Silverman¹, C. Christiansen², H. K. Genant³, J. R. Zanchetta⁴, I. Valter*⁵, T.J. de Villiers*⁶, G. Constantine*⁷, A. A. Chines⁷, ¹Osteoporosis Medical Center, Clinical Research Center, Beverly Hills, CA, USA, ²Center for Clinical and Basic Research (CCBR), Ballerup, Denmark, ³University of California, San Francisco, and Synarc, San Francisco, CA, USA, ⁴University of El Salvador, Metabolic Research Institute, Buenos Aires, Argentina, ⁵CCBR, Tallinn, Estonia, ⁶Panorama MediClinic, Capetown, South Africa, ⁷Wyeth Pharmaceuticals, Collegeville, PA, USA.

Bazedoxifene (BZA), a novel selective estrogen receptor modulator (SERM), is currently in clinical development for the prevention and treatment of postmenopausal osteoporosis. We report the results of a 3-year, Phase III study that evaluated the effect of BZA therapy on the incidence of new vertebral fracture compared with placebo (PBO) and raloxifene (RLX) in postmenopausal women with osteoporosis.

This study enrolled generally healthy postmenopausal women aged 55-85 years with lumbar spine (LS) or femoral neck (FN) T-scores <-2.5 and no prevalent vertebral fractures or LS or FN T-scores

−4.0 with prevalent vertebral fractures. Participants were randomized to receive 20 mg/d or 40 mg/d BZA, 60 mg/d RLX, or PBO and received supplemental calcium (1200 mg) and vitamin D (400 1U). The primary efficacy outcome was the incidence of new vertebral fractures after 36 months; secondary outcomes included non-vertebral fractures (NVFs). A total of 7492 women (mean age ± SD, 66.4 ± 6.7 years) were randomized and received >1 dose of study medication. At baseline, mean LS T-score was −2.4, mean total hip T-score was

-1.4, and 56% of women had >1 prevalent vertebral fracture (mostly mild). The 3-year incidences of new vertebral fracture, based on Kaplan-Meier estimates, were 2.3%, 2.5%, 2.3%, and 4.1% in the BZA 20 mg, BZA 40 mg, RLX 60 mg, and PBO groups, respectively, with a relative risk reduction for new vertebral fracture of 42% (P=0.015), 37% (P=0.031), and 42% (P=0.012), respectively, versus PBO. There was overall no treatment effect on NVFs. In a post-hoc analysis of women with FN T-score <-3.0 or >1 moderate or multiple vertebral fracture (n=1782), NVF incidence was 3.0%, 3.8%, 5.9% and 6.3% in BZA 20 mg, 40 mg, RLX 60 mg and PBO groups, respectively. Relative to PBO, BZA 20 mg reduced NVF incidence by 52% (P=0.045). Similar reduction was observed when both BZA doses were combined (46% reduction, P=0.037).

We conclude that BZA treatment significantly reduces the risk of new vertebral fractures, and in subjects at higher risk for fractures, BZA is associated with a significant reduction in NVFs.

Disclosures: S.L. Silverman, Novartis, Eli Lilly, Wyeth, Roche, Procter & Gamble, Merck

2; Roche, Novartis, Merck, Procter & Gamble, Wyeth 5: Eli Lilly, Merck, Procter & Gamble, Roche 8; Compumed (member, Board of Directors) 9. This study received funding from: Wyeth Pharmaceuticals.

1207

The Effect of Calcium plus Vitamin D Supplement on Hip Geometric Structures: Results from the Women's Health Initiative CaD Trial. Z. Chen¹, T. J. Beck², N. C. Wright¹, A. Z. LaCroix³, J. A. Caulev⁴, C. E. Lewis⁵, T. Bassford*¹, M. S. LeBoff⁶, R. D. Jackson*⁷, ¹U of Arizona, Tucson, AZ, USA, ²Johns Hopkins Univ., Baltimore, MD, USA, ³Fred Hutchinson Cancer Research Center, Seattle, WA, USA, ⁴U of Pittsburgh, Pittsburgh, PA, USA, ⁵U of Alabama, Birmingham, AL, USA, ⁶Brigham and Women's Hospital, Boston, MA, USA, ⁷Ohio State Univ., Columbus, OH, USA.

A previous report has shown that calcium and vitamin D (CaD) supplementation caused a small but significantly improvement in bone mineral density (BMD) among healthy postmenopausal women who participated in the CaD trial of the U.S. nationwide Women's Health Initiative Study (WHI). The purpose of this study was to investigate the effect of CaD intervention on femoral bone strength as measured by hip structure analysis (HSA) in the same sample. The WHI CaD trial was designed for women to take either the CaD supplement (containing calcium carbonate (500 mg as elemental calcium) with vitamin D₃ (200 IU)) or matching placebo twice daily (GlaxoSmithKline Consumer Healthcare, Parsippany, NJ) depending on their randomization assignment. The analysis included participants (n = 1,731) at three WHI bone density centers. HSA in the femoral narrow neck (FNN), intertrochanter (IT), and femoral shaft (FS) regions were done using the validated Beck's method on hip scans from dual-energy x-ray absorptiometry. Women in the CaD arm were similar to women in the placebo arm in baseline characteristics, such as age (62.2 vs. 62.3 year), height (161.81 vs. 161.75 cm) and body weight (76.2 vs. 75.2 kg). Percent changes in HSA measurements at FNN from baseline to year3 are presented in the Figure  ,  . Results from multiple linear regression analysis after adjusting for baseline HSA measure suggest significant CaD intervention effects on HSA measurements in the FNN region during the first three years of the intervention (p<0.05). Overall CaD reduced the expansion of outer diameter (OD) and increased buckling ratio (BR). Meanwhile, section modulus (SM) and cortical thickness (CT) were significantly improved by CaD intervention. Higher cross-sectional area (CSA) were also observed in the CaD arm, but the intervention effect was not statistical significant. Adjusting for hormone interventions did not change the findings. Interestingly, the CaD effect on bone strength was limited to the FNN region. In conclusion, CaD intervention has significant and skeletal-site specific benefits on bone strength.

Figure Figure 1.. Percent change in HSA measurments by intervention from baseline do year3

Disclosures: Z. Chen, None.

This study received funding from: N1AMS R01-AR0494U.

1208

Denosumab Increases Bone Mineral Density in Patients with Rheumatoid Arthritis: 12-month Results. R. K. Dore*¹, E. Hurd*^2., W. Palmer*³, W Shergy*⁴, N. Lane*⁵, S. Cohen*⁶, L. Zhou*⁷, R. Newmark^7., W. Tsuhji*⁷, ¹Robin K. Dore, Inc., Anaheim, CA, USA, ²Arthritis Centers of Texas, Dallas, TX, USA, ³Westroads Medical Group, Omaha, NE, USA, ⁴Rheumatology Associates of North Alabama, Huntsville, AL, USA, ⁵UC Davis Medical Center, Sacramento, CA, USA, ⁶Metroplex Center for Clinical Research, Dallas, TX, USA, ⁷Amgen, Inc., Thousand Oaks, CA, USA.

Rheumatoid arthritis (RA) is characterized by articular and periarticular bone erosions and systemic bone loss. RANKL is a key factor implicated in the bone destruction associated with many diseases, including RA. In clinical studies, denosumab, a fully human monoclonal antibody, inhibits RANKL and increases bone mineral density (BMD) in postmenopausal women with low bone mass. As part of a study to determine the effects of denosumab on joint destruction in patients with RA, the effect of denosumab on systemic bone loss was examined.

Patients with mild to moderately active RA for at least 6 months with manifest erosions and maintained on methotrexate (MTX) were randomly assigned to treatment with subcutaneous injections of placebo or denosumab (60 or 180 mg) every 6 months. BMD was measured by dual-energy x-ray absorptiometry (DXA) at baseline, 1 month, 6 months, and 12 months. Adverse events were monitored throughout the study.

A total of 227 patients were enrolled in the study (78 placebo, 73 denosumab 60 mg, 76 denosumab 180 mg). Average lumbar spine BMD T-score at baseline was (mean ± SD) −0.50 ± 1.58. Approximately one-third of patients used steroids at screening and one-fifth of all patients used bisphosphonates during the study; uses of both were balanced among groups. An additional 5% of patients in each treatment group had previously used bisphosphonates. At 12 months, denosumab significantly increased BMD at the lumbar spine, total hip, femoral neck, and trochanter compared with placebo (Table). Adverse events occurred with similar frequency among the treatment groups. Flare of RA was the most common adverse event.

All statistical comparisons were based on a repeated-measures model adjusting for treatment, visit, baseline use of steroid, previous use of biologic, baseline value, and visit by treatment interaction.

In summary, denosumab treatment increased BMD in patients with RA at 12 months and showed similar frequency and severity of adverse events across treatment groups. These results are consistent with those observed at 6 months.

Table  .  

Disclosures: R.K. Dore, Merck 2, 5, 8: Eli Lilly 2, 5, 8; Roche 2, 5, 8, Amgen 2.

This study received funding from: Amgen, Inc.

1209

Efficacy of Bazedoxifene for Prevention of Postmenopausal Osteoporosis: Results of a 2-Year, Phase III, Placebo- and Active-Controlled Study. P. D. Miller¹, C. Christiansen², H. C. Hoeck*², D. L. Kendler³, E. M. Lewiecki⁴, G. Woodson⁵, M. Ciesielska*⁶, A. A. Chines⁶, G Constantine*⁶, P. D. Delmas⁷, ¹University of Colorado Medical Center, Denver, CO, USA, ²Center for Clinical and Basic Research, Ballerup, Denmark, ³Osteoporosis Research Centre, Vancouver, BC, Canada, ⁴New Mexico Clinical Research & Osteoporosis Center, Inc, Albuquerque, NM, USA, ⁵Atlanta Research Center, Decatur, GA, USA, ⁶Wyeth Pharmaceuticals, Collegeville, PA, USA, ⁷University of Lyon and INSERM Research Unit 831, Lyon, France.

Bazedoxifene (BZA) is a novel selective estrogen receptor modulator (SERM) currently in clinical development as monotherapy for prevention and treatment of postmenopausal osteoporosis and in combination with conjugated estrogens for the treatment of menopausal symptoms and prevention of postmenopausal osteoporosis. In preclinical studies, BZA maintained skeletal mass without stimulation of the mammary gland or endometrial tissue. This 2-year, Phase III study was designed to assess the efficacy and safety of 3 BZA doses compared with placebo (PBO) and raloxifene (RLX) in the prevention of postmenopausal osteoporosis.

Healthy postmenopausal women (N=1583; mean age: 57 y) with lumbar spine or femoral neck BMD T-scores no less than-2.5 (mean: −1.2) were randomized to 1 of 5 groups: BZA 10, 20, or 40 mg, PBO, or RLX 60 mg. All women received 600 mg elemental calcium. The primary outcome was percent change in lumbar spine BMD at 24 months; secondary outcomes included BMD at other skeletal sites and serum bone turnover markers.

All BZA doses and RLX prevented bone loss, whereas PBO was associated with significant reductions in BMD. The difference in percent change of lumbar spine BMD from baseline to 24 months relative to PBO was 1.08%, 1.41%, 1.49%, and 1.50% for 10 mg, 20 mg, 40 mg BZA and 60 mg RLX, respectively (P<0.001 for all comparisons). Comparable BMD responses were observed with BZA at other skeletal sites. Significant decreases in serum osteocalcin and C-telopeptide levels from baseline and relative to PBO were observed as early as 3 months and remained sustained through study end (P<0.001). By month 24, median serum osteocalcin levels decreased from baseline by 21%, 22%, 22%, and 27% with BZA 10, 20, and 40 mg, and RLX, respectively, and 6% with PBO (P<0.001 vs baseline for each); median serum C-telopeptide levels decreased by 25%, 24%, 22%, and 32% with respective BZA doses and RLX and 13% with PBO (P<0.001 vs baseline for each). Overall, BZA was well tolerated and exhibited a favorable safety profile.

In conclusion, this study demonstrated that treatment with BZA, a new SERM, prevented bone loss, reduced bone turnover, and was generally well tolerated in postmenopausal women with normal or low BMD.

Disclosures: P.D. Miller, Procter & Gamble Pharmaceuticals, Sanofi-Aventis Pharmaceuticals, Roche Pharmaceuticals, Eli Lilly, Merck & Co., Novartis Pharmaceuticals, Amgen 2; Procter & Gamble Pharmaceuticals, Sanofi-Aventis Pharmaceuticals, Roche Pharmaceuticals, Eli Lilly, Merck & Co., Novartis Pharmaceuticals, Amgen, NPS, GlaxoSmithKline 5, 8. This study received funding from: Wyeth Pharmaceuticals.

1210

The Effect of a Single Large Oral Dose of Cholecalciferol on Serum 25-hydroxyvitamin D Levels in Humans. M. llahi, L. A, G Armas, R. P. Heaney, Endocrinology, Diabetes & Metabolism, Creighton University Medical Center, Omaha, NE, USA.

We report results of work to quantify the response of 25-hydroxyvitamin D to a large oral dose of cholecalciferol. Large intermittent doses of vitamin D have been shown to reduce fracture risk and to elicit good patient compliance. However, the time course of the serum 25(OH)D response has never been measured and hence both the safety and the optimum frequency of dosing are uncertain.

This study included two groups, an older group of 20 subjects (age 61–84 yrs, 15 females, 5 males) and a younger group of 10 subjects (age 27–47yrs, 6 females, 4 males). The subjects were healthy with limited sun exposure. Data were collected from October to March. Subjects were dosed at baseline with 100,000 IU of cholecalciferol as a single oral dose. Blood for 25-hydroxyvitamin D was drawn throughout the ensuing 16 weeks. Blood for calcium was drawn through the first 5 days.

Baseline levels of 25{OH)D were 59.4 nmol/L (median; interquartile range 55.6-75.1) in the older group and 63.7 nmol/L (median; interquartile range 57.1-78.4) in the younger group. 25(OH)D increased to 92.1 nmol/L (median; interquartile range 81.1-104.6) in the older age group, and to 102.8 nmol/L (median; interquartile range 91.6-126.2) in the younger age group. Both groups reached peak 25(OH)D levels at day 7. In most of the subjects the mean 25(OH)D remained above 80 nmol/L for 70 days. Only two subjects in the older age group did not reach 80 nmol/L throughout the study. The incremental rise of 25(OH)D in the older age group was 28.7 nmol/L (median; interquartile range 24.0-37.9). In the younger age group the incremental rise of 25(OH)D was 36.5 nmol/L (median; interquartile range 28.0-52.1). Calcium at baseline for both the older and younger group was 9.5 mg/dl (median; interquartile range 9.4-9.8). Calcium levels during the first week of supplementation remained below baseline.

In summary, one large oral dose of 100,000 IU of cholecalciferol raises and maintains 25(OH)D above 80 nmol/L in most subjects for 70 days without raising serum calcium levels. No subject achieved a 25(OH)D level above what would commonly be found in outdoor workers at the end of summer. Even a dose this large will not raise 25(OH)D above 80 nmol/L in more severely vitamin D deficient subjects. We conclude that doses of 100,000 IU cholecalciferol are safe and that the optimum frequency of dosing is at least every 60–70 days.

Disclosures: M. Ilahi, None.

This study received'funding from: Research Fund of Creighton University.

1211

Aktl Contributes to Maintenance of Bone Mass and Turnover by Inhibiting Apoptosis of Osteoblasts Through Fox03a/Bim Axis. N. Kawamura, F. Kugimiva*, T. Kadowaki*, K. Nakamura, U. Chung, H. Kawaguchi, Sensory & Motor System Medicine, Tissue Engineering, and Metabolic Diseases, University of Tokyo, Tokyo, Japan.

We found that mice lacking a phosphoinositide-dependent serine-threonine protein kinase Aktl (Aktl-/-) exhibited low-turnover osteopenia through dysfunctions of osteoblasts and osteoclasts: the former through increased susceptibility to apoptosis and decreased differentiation with impaired transactivity of Runxi, and the latter through suppressed RANKL expression in osteoblasts and cell-autonomous defects in osteoclasts. This study sought to learn the molecular mechanism underlying the enhanced apoptosis in Aktl-/- osteoblasts. Among apoptosis-related molecules that are known to be possible substrates of Akt, transcription factors Fox03a and FoxOl were expressed in primary osteoblasts and MC3T3-E1 cells. Although these factors showed nuclear entry after serum deprivation, only that of Fox03a was enhanced in Aktl-/- osteoblasts as compared to wild-type (WT) osteoblasts. Meanwhile, overexpression of the constitutively active form of Aktl (AktlCA) in osteoblasts stimulated the cytoplasmic Fox03a phosphorylation and inhibited the nuclear entry. The increased apoptosis and caspase-3 activity of Aktl-/-osteoblasts after serum deprivation were restored by introduction of a dominant-negative form of Fox03a (Fox03aDN). Among the candidate molecules of the transcriptional target of Fox03a: Fas ligand, Bim, and Bcl-xL, only Bim mRNA and protein levels were increased after serum deprivation, followed by the induction of cleaved caspase-3, in osteoblasts. The cleaved caspase-3 induction was suppressed by silencing of Bim through RNA interference. Actinomycin D canceled the Bim increase after serum deprivation, indicating the transcriptional regulation. The Bim promoter activity determined by luciferase-reporter assay was enhanced by Fox03a, and endogenous Bim expression was stimulated by overexpression of Fox03a while inhibited by Fox03aDN, indicating that Bim is a transcriptional target of Fox03a in osteoblasts. AktlCA overexpression suppressed the Bim transactivation by Fox03a and endogenous Bim induction after serum deprivation. The Bim induction which was higher in Aktl-/- osteoblasts than WT was restored by the Fox03aDN transfection. We therefore conclude that Aktl inhibits osteoblast apoptosis through phosphorylation of Fox03a and prevention of the nuclear entry, leading to impaired transactivation of pro-apoptotic Bim. Taken all together, Aktl was established as a crucial regulator of osteoblasts and osteoclasts to maintain bone mass and turnover via several pathways.

Disclosures: N. Kawamura, None.

1212

Dimorphic Effects of Notch Signaling in Bone Homeostasis. F. Engin¹, L Yang¹, G Zhou¹, T. Benin*¹, M. Jiang*¹, Y. Chen*¹, L. Wang², H. Zheng*¹, Z. Yao³, B. Boyce³, B. Lee⁴, ¹Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA, ²Pediatrics, Baylor College of Medicine, Houston, TX, USA, ³Pathology and Laboratory Medicine, University of Rochester, Rochester, NY, USA, ⁴Molecular and Human Genetics, Baylor College of Medicine, Howard Hughes Medical Institute, Houston, TX, USA.

Evolutionarily conserved Notch signaling plays a critical role in diverse developmental and physiological processes including cell fate determination, differentiation, proliferation, and apoptosis. Dysregulation of Notch pathway has been associated with many different diseases including spondylocostal dysostosis and cancer. However, its in vivo function during mesenchymal cell differentiation, and specifically in bone homeostasis remains largely unknown. Here, we show that osteoblast-specific gain of Notch 1 function results in severe osteosclerosis. Transgenic mice over-expressing Notchl intra cellular domain (N1ICD) from the Collal promoter have increased proliferation of immature osteoblasts that produce immature woven bone. Under these pathological conditions, Notch stimulates early osteoblastic proliferation by up-regulating Cyclin D, Cyclin E and Osterix. Notch also regulates osteoblastic terminal differentiation by directly binding Runx2, an essential transcription factor for osteoblastogenesis, and repressing its transactivation function. Consistent with this proliferative effect, human osteosarcomas show evidence of increased Notch signaling and its inhibition by a gamma-secretase inhibitor in vitro decreases the proliferation of human osteosarcoma cells. In contrast, loss of all physiologic Notch signaling in osteoblasts, generated by deletion of Presenilin 1 and 2 in osteoblasts, is associated with late onset, age-related osteoporosis. Double knock-out mice show decreased expression of Osteoprotegrin (Opg) expression indicating an increased osteoblast-dependent osteoclastic activity. Moreover, co-culture and flow cytometric analyses reveal increased differentiation of osteoclast precursors explaining the low bone mass phenotype in these mice. Together, these findings highlight the potential dimorphic effects of Notch signaling in bone homeostasis, and importantly, they may provide direction for novel therapeutic applications.

Disclosures: F. Engin, None.

This study received funding from: HIN grants ESI 1253 (B. Lee), HD226S7 (B. Lee), DEO

1213

Osteoblasts in Adult Bone use Slug for Regeneration and Repair but not Remodelling, J. G Mount*, S. Allen*, M. Muzylak*, L. E. Lanyon, A. Goodship*, I. M. McGonnell*, J. S. Price, Veterinary Basic Sciences, Royal Veterinary College, London, United Kingdom.

The zinc finger transcription factor Slug was first described as a marker of pre-migratory and migratory neural crest in the embryo. It is a member of the highly conserved Snail family of transcription factors, originally thought to be involved exclusively in developmental functions. Snail family members have not been identified in adult bone and have not been ascribed any function in osteoblasts. We recently identified a range of neural crest markers, including Slug, in the periosteum from which deer antlers regenerate (these are the only bones that repeatedly regenerate in adult mammals). This led us to further explore the role of Slug in bone. We also studied the relationship between Slug and Wnt signaling since there is a LEF/TCF binding site in Slug's promoter and during development it is regulated by Wnts.

Immunocytochemistry was used to co-localize Slug and activated P-catenin (“Peat”) in mesenchymal progenitors in the foetal primoridiae of antlers and in osteoblasts on the frontal bone on which antlers develop. Slug and “peat also co-localized” in osteoblasts in foetal long bones. Slug could not be detected in any adult deer or rodent bones, however, in healing adult rat fractures it was identified in periosteal progenitors. Similarly, in regenerating adult antlers Slug and apcat localized in mesenchymal progenitors and in osteoblasts. This suggests that Slug may have distinct roles at early and late stages of skeletal cell differentiation. Interestingly, Slug was not detected in cartilage in regenerating antlers, nor in cartilage in fracture callus. This indicates that its down-regulation is required for chondrogenesis.

In order to investigate the relationship between Slug expression, osteogenesis and chondrogenesis, periosteum-derived progenitor cells (PDPs) were cultured under osteogenenic or chondrogenic conditions. Slug expression increased as osteoblast differentiation increased (determined by changes in Osterix and Runx-2 expression), whereas its expression decreased with chondrocyte differentiation (determined by changes in Sox9 and type II collagen). Wnt/p eaten in signaling is known to control cell fate determination in mesenchymal cells and in PDP cells activation of Wnt signaling by LiCI decreased Slug expression.

In conclusion, we have shown, to our knowledge for the first time, that in adults Slug has a role in osteoblasts during bone regeneration and repair, but not remodeling. We also present evidence that Slug is involved in the regulation of mesenchymal cell fate by Wnt/p catenin signaling. The study also affirms that in adults developmental mechanisms are recapitulated during bone regeneration and fracture repair.

Disclosures: J.G Mount, None.

This study received funding from: BBSRC.

1214

MicroRNA Expression Profiling and Function During In Vitro Osteogenesis. G J. Spencer*, P. O. Genever*, Department of Biology, University of York, York, United Kingdom.

Understanding molecular mechanisms that control osteogenic differentiation is central to the development of novel therapies aimed at bone remodelling such as osteoporosis. Many studies have successfully identified critical factors involved in initiating and maintaining lineage-specific transcriptional changes although the role of post-transcriptional gene silencing during osteoblast differentiation remains unclear. MicroRNAs (miRNAs) are a superfamily of evolutionary conserved, small non-coding RNAs that bind to the 3′ UTR of mRNAs inhibiting protein translation and influencing a broad range of biological roles, instrumental in conferring tissue identity and function. In this study we aimed to determine expression and investigate the function of miRNAs during osteogenesis in vitro. Expression was determined by microarray analysis of 984 known and predicted miRNAs using RNA isolated from undifferentiated rat primary calvarial osteoblast progenitors or cells induced to differentiate in the presence of osteogenic supplements (dexamethasone, ascorbic acid and p-gycerolphosphate) over 21 days. Differentiation was confirmed by induction of alkaline phosphatase, von-Kossa positive mineralization and qRT-PCR, revealing temporally relevant changes in expression of osteogenic markers (cbfa-1, alkaline phosphatase, type-1 collagen, osteopontin and osteocalcin). Analyses revealed constitutive expression of 20 miRNAs, 5 miRNAs specifically expressed in osteoblast progenitors but down regulated during osteogenesis and 35 miRNAs absent from progenitors and expressed in mature osteoblasts. The functional role of miRNAs was determined using anti-mir microRNA inhibitors transfected into rat calvarial osteoblast progenitors before osteogenic stimulation. Inhibition of miRNA, Iet7i, constitutively expressed throughout osteogenesis, significantly reduced alkaline phosphatase activity and impaired osteoblast differentiation as determined by changes in gene expression by qRT-PCR. In contrast, inhibition of progenitor specific mir298 promoted osteoblast differentiation and reduced osteoblast numbers as determined by viability assays. Bioinformatic analyses of Iet7i and mir298 targets using predictive algorithms revealed components of the Wnt, Notch and BMP signaling pathways, suggesting the endogenous function of Iet7i in promoting osteoblast differentiation, and mir298 in inhibiting differentiation and promoting proliferation may be mediated by specific regulation of these signaling pathways. Together these data provide evidence for an important functional role of miRNAs during osteogenesis which may have pervasive implications for our understanding of bone biology.

Disclosures: GJ. Spencer, None.

This study received[funding from: The Wellcome Trust.

1215

Pericytes Derived from Adipose Tissue; Characterization and Evaluation of their Osteogenic Potential. I. Kalaizic, L. Wans¹, X. Jiang*¹, K, Lamothe*², L. H. Aguila*², P. W. Rowe¹. ¹Dept. of Reconstructive Sciences, Uni. of Conn. Health Center, Farmington, CT, USA, ²Dept. of Immunology, Uni. of Conn. Health Center, Farmington, CT, USA.

We have shown that pericytes/myofibroblast expand following osteoblast ablation. This was assessed by a dramatic increase in a population of cells within the bone marrow that was positive for smooth muscle actin (SMAA), SM22 and exhibited a myofibroblastic phenotype. To further substantiate these observations and test the hypothesis that pericytes from non-calcified tissues have osteoprogenitor potential we have obtained SMAA-GFP transgenic mice that direct GFP towards smooth muscle cells and pericytes. We have characterized the GFP expression in bone and other tissues. A few SMAA-GFP positive cells detected in bone were localized to microvasculature in the periosteum and in a few locations within the trabecular area within bone marrow space. We have confirmed the close relationship of SMAA+ cells in periosteum with the underlying endothelial cells by CD31 immunohistochemistry. Utilizing SMAA-GFP, we observed rich vascular network in muscle and adipose tissue. In adipose tissue, two levels of SMAA+ cells were closely associated with CD31 + staining; a smooth muscle layer of blood vessels, and extensive capillary network.

Phenotypic analysis against cell surface markers expressed in mesenchymal progenitor cells showed mat 50% of freshly isolated SMAA-GFP+ cells from adipose tissue were CD45-/Scal+. Culturing for 6 days enriched this CD45-/SMAA+/Scal+ mesenchymal progenitor population to 80%. In addition, SMAA+ cells were negative for c-kit and CDllb.

Bone marrow and adipose derived stromal cells isolated from SMAA-GFP mice were purified by FACS to generate SMAA-GFP+ and GFP- populations. Following induction to osteogenesis and adipogenesis, expression of bone and fat markers was restricted to SMAA-GFP+ population. This in vitro data confirmed that SMAA-GFP+ cells differentiated into osteoblasts and adipocytes. To confirm the ability of SMAA-GFP positive cells derived from adipose tissue to generate mature osteoblasts in an in vivo model, we have utilized dual transgenic mice obtained by breeding a SMAA(green) with osteoblast specific Col2.3(blue). Following FACS isolation, pure SMAA-GFP+/2.3GFP-cells were transplanted by direct intra marrow injection into GFP negative recipient mice. One month after the injection histological analysis of transplanted bones reveals the presence of numerous osteoblasts (Col2.3GFP+) localized to bone surface and closely associated with new bone formation as detected by underlying deposition of xylenol orange. Our result confirms that a pericyte population from adipose tissue has osteoprogenitor potential with the ability to generate mature bone lineage cells.

Disclosures: I. Katajzic, None.

1216

Oxidative Stress-induced Vascular Calcification Is Associated with Increased Expression of Receptor Activator of Nuclear Factor kB Ligand (RANKL). C. Pyiai*¹, X. Feng¹, T. L. Clemens¹, J. M. McDonald², Y. Chen¹, ¹Pathology, MCP, University of Alabama at Birmingham, Birmingham, AL, USA, ²Pathology and VA medical Center, University of Alabama at Birmingham, Birmingham, AL, USA.

Oxidative stress plays a critical role in the pathogenesis of atherosclerosis, including the development of atherovascular calcification, a prominent feature of atherosclerosis. Enhanced osteogenic differentiation of vascular smooth muscle cells (VSMC) is associated with oxidative stress in vitro. Further, increased expression of RANKL is present in calcified vessels. Thus, we determined the effects of oxidative stress on the expression of RANKL and its role in VSMC calcification.

VSMC were explanted from the aorta of C57BL6 mice and the smooth muscle (SM) cell identity was confirmed by immunofluorescent staining with SM a-actin antibody. VSMC were exposed to osteogenic media for 3 weeks and mineralization was determined by von Kossa staining. H202, at concentrations of 0.05 mM to 0.4 mM, were used to induce oxidative stress. The expression of RANKL, alkaline phosphatase (ALP), type I collagen (Col I), and osteocalcin (OC) was determined by real-time PCR and RT-PCR. The effect of oxidative stress on RANKL promoter activity was determined with a serial of deletion mutants of RANKL promoter- luciferase reporters. All experiments were performed with VSMC at passage 5 to 8. H,0, increased intracellular peroxide production in VSMC as determined by dichlorofluorescein fluorescence. VSMC mineralization was induced by H202 in a dose-dependent manner (fold increase at 0.05 mM=1.5±0.7, 0.3 mM=6.0±1.4, and 0.4 mM=16.0±1.4 compared with control, n = 4, p<0.05). Increased expression of bone-associated proteins including ALP, Col I and OC and decreased expression of SM-a actin were demonstrated when VSMC were exposed to 0.4 mM H₂0₂ (fold increase in ALP=8.7± 1.2, Col I=6.6±0.9, and OC=5.3±1.4 compared with control, n = 4, p<0.05). In addition, a 33-fold increase in the expression of RANKL and no change in osteoprotegerin (OPG) was demonstrated in VSMC exposed to 0.4 mM H202 (n = 4). Luciferase-reporter assay revealed an oxidative stress-responsive region between −400 and −200 in RANKL promoter. Addition of RANKL (100 ng/ml) or OPG (50 ng/ml) protein into the osteogenic media did not affect VSMC calcification; and oxidative stress-enhanced calcification was not blocked in VSMC from RANKL knock out mice.

In summary, oxidative stress decreased the expression of SM a-actin and increased the expression of bone-associated proteins and calcification in VSMC. Oxidative stress induced the expression of RANKL via its promoter region located between −400 and −200. Elevation of RANKL during VSMC calcification did not mediate the effect of oxidative stress on VSMC calcification.

Disclosures: Y. Chen, None.

1217

Androgens Act Directly via the Androgen Receptor in Mineralizing Osteoblasts to Regulate Bone Turnover and Maintain Trabecular Bone in Male Mice. R. A. Davev¹, M. Chiu*¹, C. Chiang*¹, A. J. Moore*², E. Doust*¹, C. Ma*¹, T. L. Clemens³, H. A. Morris², J. D. Zajac¹, ¹Medicine, AH, University of Melbourne, Heidelberg, Victoria, Australia, ²Hanson Institute, Adelaide, Australia, ³Pathology, University of Alabama at Birmingham, Birmingham, AL, USA.

Androgens play a key role in skeletal growth and bone maintenance, however the relative contribution of their actions via the androgen receptor (AR) versus the estrogen receptor (ER) remains unclear. Our recent data using Collagen lal 2.3kb-AR knockout mice (KO) provides strong evidence for a role of androgens acting directly via the AR in mature osteoblasts to maintain trabecular bone (1). The aim of this study was to define the target cells of androgen action via the AR in bone using mice in which the DNA-binding actions of the AR are deleted specifically in terminally differentiated, mineralizing osteoblasts (mOBL-ARKOs).

mOBL-ARKO mice were generated by breeding mice with a floxed exon 3 of the AR gene (2) with osteocalcin-Cre mice. The phenotype of male mOBL-ARKOs and littermate controls (wild-type, AR floxed, osteocalcin-Cre) was assessed by histomorphometry in die distal femoral metaphysis at 6,12 and 24 wks of age (n=10/group).

mOBL-ARKOs had normal femur length, body weight, testis and seminal vesicle mass compared to controls. Deletion of the AR specifically in mineralizing osteoblasts resulted in 22-38% decreases in trabecular number (TbN) at 6, 12 and 24 wks of age (PO.05) in the secondary spongiosa. At 6 wks of age, this loss of TbN resulted in a 36% decrease in trabecular bone volume (BV/TV) (PO.01). In contrast, BV/TV in mOBL-ARKOs did not differ from controls at 12 and 24 wks of age, as they were able to maintain or increase trabecular thickness (PO.05), suggesting a compensatory mechanism to prevent further bone loss in the absence of AR action in mineralizing osteoblasts. The loss of TbN observed at all ages is likely to be due to increased bone resorption, evident from 12 wks of age as measured by serum X-laps (P<0.05). Although the mean X-laps was higher in mOBL-ARKOs at 6 wks of age (mean±SE; wild-type: 92±6; mOBL-ARKO: 122±15, n=10), this did not reach significance (P=0.07). Trabecular bone parameters in the primary spongiosa were unaffected in mOBL-ARKOs indicating that the accrual of new bone is not dependent on the DNA-binding actions of the AR in mineralizing osteoblasts. Our findings conclusively demonstrate that in addition to the well-established actions of androgens via aromatisation to estrogen, androgens also act directly via the AR in osteoblasts. We have now shown that androgens also act directly via the AR in mineralising osteoblasts to maintain trabecular bone mass by regulating bone turnover.

References

1) Notini AJ, et al. JBMR 2007; 22:347-56;

2) Notini AJ et al. J Mol Endo 2005; 35:547-55.

Disclosures: R.A. Davey, None.

1218

Local Over Expression of Aromatase in Osteoblasts Leads to Clearly Increased Bone Mass. K. Sjogren*, N. Andersson, S. Moverare-Skrtic*, C. Swanson, C. Ohlsson, Division of Internal Medicine, Medicine, Goteborg, Sweden.

Testosterone (T) is of importance for the regulation of peak bone mass and age-dependent bone loss in males. The effects of T on bone can either be direct or mediated via local aromatization of T to estrogen (E2), followed by activation of estrogen receptors (ERs) in osteoblasts.

To investigate the importance of high local formation of E2 in osteoblasts, we developed a transgenic mouse model that over-expresses the human aromatase gene under the control of the rat type I alpha I procollagen promoter (Coll-Aro mice). This promoter will direct the aromatase expression to late osteoblasts in bone.

Measurements of aromatase expression in bone by quantitative real-time PCR showed that the system works and that Coll-Aro mice have a high and specific expression of human aromatase in bone. The bone specific expression of aromatase was supported by that serum E2 levels were unaffected in both male and female Coll-Aro mice and that the uterine weight was unaffected in female mice. At 11 weeks of age, male Coll-Aro mice exhibited a dramatic increase in cortical bone mineral content as analysed in the diaphyseal region in femur (+28%, p<0.001) while there was no effect in female mice as measured by peripheral quantitative computed tomography (pQCT). The increased cortical bone mineral content in the male Coll-Aro mice was mainly due to increased cortical thickness (+21%, pO.OOl) but also to a slightly increased cortical volumetric BMD (+6%, p< 0.001). Detailed analysis of the tibia by uCT revealed an increase in trabecular thickness (+9%, p<0.05) in male Coll-Aro mice.

In conclusion, we show that bone-specific anabolic estrogenic effects can be achieved without systemic adverse effects by increasing the expression of aromatase specifically in bone. Based on the fact that aromatase expression in osteoblasts and breast/uterus is dependent on different promoters one may speculate that development of tissue specific inducers of aromatase expression in bone might result in bone-anabolic effects without systemic adverse effects.

Disclosures: K. Sjogren, None.

1219

Modulation of Mouse RANKL Gene Expression by Runx2 and Vitamin D3. R. Kitazawa, K. Mori, T. Kondo, S. Kitazawa, Division of Pathology, Kobe University, Graduate School of Medicine, Kobe, Japan.

The expression of receptor activator of nuclear factor-kB ligand (RANKL) in osteoblastic cells is regulated by bone-seeking hormones such as PTH and vitamin D3. Runx2 is a master gene for osteobalstic differentiation and, at the same time, modulates osteoclastogenesis by regulating RANKL gene through the proximal (-378/-354, −214/-194 and −200/-180) and the distal (conserved noncoding sequence located 76 kb upstream of exon 1) runx2 sites. To elucidate the precise mechanism whereby runx2 and vitamin D3 regulate RANKL expression, we studied the function of runx2 on chromatin structure and on the proximal binding sites using mouse osteoblastic cell lines derived from normal (ST2) and runx2-deficient mice (C6).

Quantitative RT-PCR revealed that although the expression of RANKL mRNA in the steady-state was higher in runx2-deficient C6 cells than in ST2, it increased 20-fold in ST2, but only 1.8-fold in C6 by an 8-hour D3 treatment. Additionally, runx2 knock down in ST2 by siRNA resulted in an increase in the steady-state expression of RANKL mRNA and a decrease in the inducible effect of D3. Conversely, restoration of Runx2 by the Tet-On system in C6 decreased steady-state RANKL expression and increased the inducible effect of vitamin D3.

The exogenous transfected 2 kb promoter-reporter construct (2k-luc) showed 60% of the steady-state activity in C6 compared with that in ST2. Vitamin D3 increased the promoter activity of 2k-luc in ST2, but not in C6. Forced expression of runx2 by Tet-On system in C6 increased the steady-state activity of 2k-luc and its response to D3. Transfection of the mutated constructs with any one, two or all of the three proximal runx2 sites, depending on the number of mutated sites, decreased both the steady-state activity and the inducible effect of vitamin D3 in ST2, but not in C6. These data suggest that runx2 may exert a positive effect on the activity of the proximal 2kb promoter and its response to vitamin D3. We then assessed the acetylation status of the area spanning 40 kb upstream of the basic promoter in ST2 and C6 by Chip assay with anti-acetylated H3 and H4 histone antibodies. Whereas H3 and H4 histone acetylation was detected even in the steady-state in C6, it was detected only in the presence of vitamin D3 in ST2, suggesting that constitutive histone acetylation of the 5′flanking region of the RANKL gene due to runx2-deficiency in C6 may cause its higher expression at the steady-state than in ST2.

Although runx2 may suppress RANKL gene by condensing chromatin structure, it exerts a positive effect in D3-induced RANKL transcription when the proximal runx2 sites are accessible to runx2 by D3 treatment. Thus, RANKL expression in stromal/ osteoblastic cells is keenly regulated in response to vitamin D3 by transactivating the gene at two different levels.

Disclosures: R. Kitazawa, None.

This study receivedfunding from: Japanese Ministry of Education. Sports and Culture.

1220

Sex Steroids Hormone Receptors in Osteoclasts Mediate Osteoprotecitve Effects by Regulating Its Life Cycle. Y. Imai¹, T. Nakamura², H Matsumoto*³, S. Takeda⁴, K. Igarashi*⁵, T. Fukuda*², Y. Yamamoto*², J Kanno*⁵, K. Takaoka⁶, T. J. Martin⁷, P. Chambon*⁸, S. Kato³, ¹Laboratory of Nuclear Signaling, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Department of Orthopaedic Surgery, Osaka City University Graduate School of Medicine, Tokyo, Japan, ²Laboratory of Nuclear Signaling, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan, ³Laboratory of Nuclear Signaling, Institute of Molecular and Cellular Biosciences, The University of Tokyo, ERATO, JST, Tokyo, Japan, ⁴Tokyo Medical and Dental University, Tokyo, Japan, ⁵Division of Cellular and Molecular Toxicology, National Institute of Health Sciences, Tokyo, Japan, ⁶Department of Orthopaedic Surgery, Osaka City University Graduate School of Medicine, Osaka, Japan, ⁷St. Vincent's Institute of Medical Research, Fitzroy, Australia, ⁸Institut de Genetique et de Biologie Moleculaire et Cellulaire and Insitut Clinique dela Souris, Illkirch, France.

Sex steroids, estrogen and androgen, have the osteoprotective effect and prevents bone loss such as post-menopausal osteoporosis. However the molecular mechanism of how these are accomplished remains to be elucidated. Here we report critical roles of osteoclastic androgen receptor (AR) in males and osteoclastic estrogen receptor alpha (ERα) in females in mediating the actions of sex steroids on bone.

We selectively ablated AR and ERa in differentiated osteoclasts (AR^δc/y and ERα^δOc/δOc) using Cathepsin K-Cre knock-in mice. AR^δOc/Y males exhibited clear bone loss in plain X-ray and 3D-CT, similar to the osteoporotic bone phenotype. Also in DEXA, femurs of AR^δ/Y males showed low bone mineral density. Bone histomorphometric analysis revealed significant increase of osteoclast surface, osteoclast number and eroded surface in AR^δOc/Y males when compared to the control. In addition, MAR and BFR were increased in AR^δOc/Y males, these results suggested that AR^δOc/Y males had a high turnover osteoporotic phenotype. In In vitro study, quantitative RT-PCR showed that mRNA expression of Fas ligand (FasL) was facilitated in AR-overexpressed primary cultured osteoclasts treated with DHT, although the expression of Fas was not altered. Likewise, osteoclastic ERa appeared to mediate the osteoprotective estrogen action in females, but not males. From these findings, we presume that the osteoprotective actions of sex steroids are mediated at least in part through osteoclastic AR in male and ERa in female, and that sex steroids regulate the life span of mature osteoclasts by controlling Fas/FasL related cell death signaling.

Disclosures: Y. Imai. None.

This study received funding from: JST, ERATO.

1221

Control of Estradiol-Directed Gene Transactivation by an Intracellular Estrogen Binding Protein and an Estrogen Response Element Binding Protein. H. Chen. M. Hewison. J. S. Adams., Endocrinology, Diabetes and Metabolism, Cedars-Sinai Medical Center, Los Angeles, CA, USA.

Experiments of nature have helped to illuminate the key pathways associated with metabolism and signal transduction of steroid hormones. In recent studies we have used the steroid hormone resistance exhibited by New World primates (NWP) to identify entirely novel mechanisms associated with intracellular trafficking and DNA targeting of steroid hormones. NWP exhibit resistance to estrogens that is associated with overexpression of an estrogen response element binding protein (ERE-BP) and an intracellular estradiol binding protein (IEBP). The NWP ERE-BP shows homology with human heterogeneous nuclear ribonucleoprotein C like protein, whilst IEBP shows homology with human hsp27. Although ERE-BP acts as a competitor for ERE occupancy by liganded estrogen receptor-alpha (ER), the function of IEBP/hsp27 is less clear. In new studies using human breast cells we show that IEBP/hsp27 can regulate estrogen signaling: 1) as a protein chaperone for ER; 2) as a cytosolic decoy for estradiol (E₂) binding; 3) by binding to ERE-BP and modulating the temporal organization of ERE occupancy by the ER and ERE-BP. IEBP/ hsp27 was able to bind E2 with relatively high affinity (Kd=0.25 nM). This was similar to that observed with estren (0.1 nM), whereas the selective estrogen receptor modulator tamoxifen showed no binding to IEBP/hsp27. Studies using ER^+VI: MCF-7 cells showed that, in the absence of E₂, IEBP/hsp27 and ER were localized predominantly in the cytoplasm, with both translocating to the nucleus in the presence of E₂. GST-pulldown analyses confirmed binding of ER to IEBP/hsp27 but also revealed indirect association between ER and ERE-BP. Protein-protein interaction of IEBP/hsp27 and ERE-BP was confirmed by immunolocalization, as well as yeast 2-hybrid and GST-pulldown analyses. These studies also showed that normal E₂ responses in MCF-7 cells involve cycling of the IEBP/hsp27-ERE-BP complex between the cytosol and nucleus. Over-expression of either IEBP/hsp27 or ERE-BP suppressed E₂-mediated transcription in ER^+VI: cells. This was associated with abnormal subcellular distribution of the IEBP/hsp27-ERE-BP complex, which resulted in concomitant dysregulation of ERE occupancy by ER and ERE-BP as determined by chromatin immunoprecipitation. We hypothesize that IEBP/hsp27 and ERE-BP not only cause hormone resistance in NWP but are also crucial to normal estrogen signaling in human cells. In particular, protein-protein interaction between IEBP/hsp27 and ERE-BP in both cytosolic and nuclear compartments appears to be essential to the regulation of their individual actions on ER signaling.

Disclosures: H. Chen, None.

1222

Estradiol Mediates Rescue of the Dominant-negative Effects of Estrogen Response Element-binding Protein In Vivo. H. Chen, M. Hewison, L. Nguven*, J. S. Adams, Endocrinology, Diabetes and Metabolism, Cedars-Sinai Medical Center, Los Angeles, CA, USA.

Biological responses to estrogens are dependent on the integrated actions of various proteins, including the estrogen receptor (ER), that regulate the transcription of estrogen response element (ERE)-containing target genes. We have identified a naturally occurring antagonist of this mechanism that functions by competing with ER for ERE occupancy. The hnRNPC-like protein, termed an ERE-binding protein (ERE-BP), is constitutively over-expressed in cells from estrogen-resistant New World primates. In order to verify that ERE-BP legislates estrogen resistance in vivo, we generated transgenic mice that specifically over-express varying levels of ERE-BP in breast tissue under the control of whey acidic protein gene promoter. Female transgenic mice with high levels of ERE-BP were unable to nurse their pups, none of which survived to weaning unless were fostered. Mice expressing intermediate and low levels of ERE-BP in breast had progressively more estrogen-driven breast development and lactational capacity. We hypothesized that escape from the dominant-negative action of ERE-BP on the breast was dictated by relative balance in ERE-BP expression levels and the concentration of estrogen available to the dominant-positive-acting ER. To test this hypothesis, four-week-old ERE-BP-high female mice were implanted subcutaneously with a capsule containing 7.2mg/21 day-release estradiol (E2) or placebo. Analysis of 8-week-old mice breast whole-mounts and histology showed that E2-treated ERE-BP mice exhibited a 4.5-fold increase in mammary gland branch number (p<0.00l) and 3.7-fold increase in duct number (p<0.05) compared with the placebo-infused controls. Analysis of postpartum breast whole-mounts and histology showed that E2-treated ERE-BP mice demonstrated more than a doubling in milk-containing lumina and a 3.0-fold increase in lobule-alveolae and duct formation (p<0.02) compared to placebo-infused ERE-BP mice, consistent with improved mammary gland differentiation. These data emphasize that 1 ] ERE-BP is a potent attenuator of estrogen-directed bioactivity and 2] increased circulating levels of E2 can rescue the host from the suppressive effects of ERE-BP in vivo, confirming in vitro data from our group. In summary, we have shown for the first time that there exists a reciprocal, dominant-positive:dominant-negative relationship between E2-liganded ER and ERE-BP in vivo. Although our experiments have focused on breast tissue, we hypothesize that ERE-BP is a crucial determinant of ERE-directed estrogen responses throughout the body.

Disclosures: H. Chen, None.

1223

Bax-deficient Mice Exhibit Marked Increase in Callus Size and Cartilage During Endochondral Repair of Femur Fractures. C. H. Rundle, X. Wang*, J. E. Wergedal, M. H. C. Sheng, R. M. Porte*, K. H. W. Lau, S. Mohan, Research (151), J.L. Pettis VAMC, Loma Linda, CA, USA.

While it is clear that endochondral bone formation during fracture repair involves the spatial and temporal regulation of the proliferation and differentiation of multiple cell types, there is suggestive evidence that the spatial and temporal regulation of apoptosis is also critical. Accordingly, this study evaluated the effects of Bax (a pro-apoptotic gene) deficiency on fracture repair. Healing of femoral fractures (created by three-pointed bending) in adult Bax KO [in a C57BL/6 (B6) genetic background] and B6 control mice was monitored by pQCT and histomorphometry. Bax KO mice developed larger calluses with increased cartilage area [3-fold at 7 days (p<0.003); 2-fold at 14 days (p<0.05)]. The bone mineral content (BMC) of Bax KO calluses was also greater than B6 calluses [∼2-fold each at 14 days (p<0.004), 21 days (p<0.04), and 28 days (p<0.0008)]. Cell counts within the cartilage area of the healing calluses showed that the enlarged cartilage in the Bax KO callus was due to an increase in chondroblast number and not cell size. To evaluate whether the BAX deficiency-related impairment of apoptosis accounted for the difference, the number of apoptotic cells in the cartilage within the healing callus was determined by the TUNEL assay. There was no significant difference in the percentage of apoptotic cells, indicating that the large increase in chondroblast number could not be due to differences in chondroblast apoptosis alone and suggesting that Bax deficiency also increased chondroblast proliferation during early fracture healing. There were also more osteoclasts on the woven bone surfaces of Bax KO mice during callus remodeling at 21 days (25%, p<0.004) and 28 days (33%, p<0.05). The callus size and BMC of Bax KO mice returned to B6 levels at 35 days. To identify a mechanism for the increase in chondroblasts, whole genome gene expression analysis was performed at 14 days post-fracture. There were no significant expression changes in genes associated with mitochondrial apoptosis, with the exception of the Bik-like gene (2.3-fold increase, p<0.05), suggesting a compensatory function for this gene. Consistent with the greater prehypertrophic chondrocyte content of early fracture cartilage, the expression of collagen-2 and collagen-9 (3-fold, p<0.05 for each), but not collagen-10, was elevated. Conclusions: 1) Bax deficiency enhanced cartilage formation, bone formation and remodeling during fracture repair, suggesting that Bax is a negative regulator of these processes, and 2) Bax deficiency increased chondroblast proliferation, suggesting that Bax may regulate fracture repair through a mechanism independent of its pro-apoptotic action.

Disclosures: C.H. Rundle, None.

1224

Type III Sodium-Dependent Phosphate Transporter Controls Endochondral Ossification Through Regulating Chondrocyte Apoptosis. A. Sugita¹, S. Kawai¹, T. Havashibara*¹, H. Yoshikawa², T. Yoneda¹, ¹Biochemistry, Osaka University Graduate School of Dentistry, Suita, Japan, 2Orthopedics, Osaka University Graduate School of Medicine, Suita, Japan.

Previous studies reported that phosphate levels in growth plate increased as chondrocyte differentiation advances. Cartilage differentiation is markedly disturbed in X-linked hypophosphatemia (XLH), a dominant disorder of phosphate homeostasis. These results suggest a critical role of phosphate in chondrocyte differentiation. Extracellular phosphate is incorporated into cells through the sodium-dependent phosphate transporter (NPT). Growth plate chondrocytes express type Ha NPT (Npt2a) and type III NPT (Pit-1). It is therefore likely that these NPTs are involved in the regulation of chondrocyte differentiation. However, which NPT is a major player is currently unknown. Phosphonoformic acid (PFA), a non-selective competitive inhibitor of NPT, reduced 3:P uptake and suppressed apoptosis in mouse primary rib chondrocytes. PFA also inhibited chondrocyte apoptosis in vivo. Of interest, PFA decreased intracellular levels of ATP, which is essential for the activation of caspase-9 and caspase-3, leading to the progression of apoptosis. To investigate the specific role of Npt2a and Pit-1 in chondrocyte apoptosis, we knocked-down Npt2a or Pit-1 using small interfering double-stranded RNA (siRNA). siPit-1 significantly decreased 32P uptake, intracellular ATP levels, caspase-9 and caspase-3 activity, followed by reduced apoptosis. As a consequence, calcification of chondrocytes was diminished. In contrast, siNPT2a showed no effects. Both siPit-1 and siNPT2a did not affect early stages of chondrocyte differentiation. Finally, we examined the effects of Pit-1 overexpression on apoptosis in chondrocytes of Hyp mice that are a mouse homologue of human XLH and exhibit reduced Pit-1 expression and apoptosis in cartilage compared with wild-type mice. Overexpression of Pit-1 significantly increased J2P uptake in Hyp chondrocytes. In conjunction, it also increased intracellular ATP levels, caspase-9 and caspase-3 activation and apoptosis in Hyp chondrocytes. In conclusion, our results suggest phosphate uptake through Pit-1 specifically modulates the activation of ATP-dependent caspase signaling pathway, which in turn controls apoptosis and calcification of chondrocytes, thereby regulates endochondral ossification.

Disclosures: A. Sugita, None.

1225

The 7B2 Protein Regulates FGF-23 Degradation and Production in X-linked Hypophosphatemia (XLH). B. Yuan*, M. Takaiwa*, Y._Xing*, J.J. Meudt*, M. K. Drezner, Department of Medicine, Endocrinology Section, University of Wisconsin, Madison, WI, USA.

The mechanism by which inactivating PHEX mutations increase serum FGF-23 remains unknown. However, FGF-23 may be processed at a subtilisin-like proprotein convertase (SPC) cleavage stie (RXXR). Indeed, we reported mice treated 5 days with an SPC inhibitor, Decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone (Dec), develop inhibition of FGF-23 degradation and elevation of serum FGF-23, as well as abnormal Pi homeostasis and vitamin D metabolism, characteristic of XLH. Yet, these studies did not reveal increased FGF-23 mRNA, failing to recapitulate the mechanism underlying abnormal serum FGF-23 in XLH. Thus, we further investigated if abnormal SPC activity can create both aberrant FGF-23 cleavage and production. Initially, we explored if SPC2 and helper protein7B2 truly alter FGF-23 degradation, using HEK293 cells transfected with recombinant FGF-23 cDNA and co-transfected with SPC2 and 7B2 alone or together. After 48 hours Western blotting indicated neither SPC2 nor 7B2 alone altered FGF-23 degradation. However, transfection with SPC2 and 7B2 increased C-terminal fragments of FGF-23 in cell culture medium (2.14±0.18 vs 1.0±0.08; p<0.0l) and decreased intact FGF-23 in cell lysates (0.48±0.4 vs 1.02±0.2; p<0.05), confirming that diminished SPC2/7B2 activity may increase serum FGF-23 levels. Subsequently, we explored if SPC2/7B2 inhibition altered FGF-23 mRNA production. Incubation of immortalized osteoblasts with 25 p.M Dec for 12h inhibited FGF-23 degradation, but more importantly increased FGF-23 mRNA (3.9±0.9 vs 1.2±0.2; p<0.001). Surprisingly, altered FGF-23 degradation and production corresponded with decreased 7B2 active protein (O.53±0.01 vs 1.77±0.1; p<0.001) and mRNA (0.6U0.09 vs l.04±0.14; p<0.001). These observations suggest that decreased 7B2 not only reduces FGF-23 degradation but up-regulates production. To confirm this possibility we assessed 7B2 in hyp-mice, the murine homolog of XLH. The hyp-mice have decreased osseous production of 7B2 mRNA (0.45±0.02 vs 1.06±0.05; p<0.01) and reduced 7B2 active protein (0.68±0.04 vs 1.0±0.08; p<0.01). Further, we found that administration of 25 uM Dec to normal mice for 12-days not only resulted in the HYP biochemical phenotype, but in a significant decreased in ossous 7B2 mRNA (0.84±0.05 vs 1.0±0.12; P<0.05), an expected decrease of FGF-23 degradation and an increase in FGF-23 mRNA (2.23±0.12 vs I.16±0.02; p<0.01). Our observations indicate that decreased 7B2 production and accordingly 7B2 active protein in osteoblasts result in the sustained elevation of circulating FGF-23 in XLH due both to the direct effects of SPC2/7B2 on FGF-23 cleavage and to likely downstream effects of SPC2/7B2, which increase FGF-23 mRNA production.

Disclosures: B. Yuan, None.

This study received[funding from: NIH.

1226

Abnormal Bone Mineralization, Osteoclastogenesis & Bone Turnover in Transgenic Mice Overexpressing MEPE. V. David¹, A. Martin¹, L. W, Fisher², A. Hedge¹, P. S. N. Rowe¹, ¹Internal Medicine, Kansas University Medical Center, Kansas City, KS, USA, 2NIDCR-NIH, Bethesda, MD, USA.

Matrix extracellular phosphoglycoprotein (MEPE) is a SIBLING-protein involved in bone-mineralization. Although increased MEPE expression occurs in several bone-mineral and renal-phosphate handling diseases, its precise physiological role is unknown. In this study, we constructed transgenic-mice overexpressing murine-MEPE (Collal 2.3kb promoter) and used them as a model-system to test whether MEPE influences bone-mineralization in vivo. We analyzed bone-phenotypes of male-transgenic (TGC) and wild type (WT) C57BI6 newborn, 5 week-old and 19 week-old adult mice (n=10).

Protein (westem-blot) and mRNA (RT-PCR) measurements confirmed a 50X over-expression of MEPE in bone. Newborn mice overexpressing MEPE displayed a low bone-mass phenotype (see figure, 53% decrease in BMD, p<0.001). Abnormal bone-growth also occurred at 5 weeks as assessed by femur length (5% decrease, p<0.01) and growth plate width (+26%; p<0.05). In older animals. X-ray densitometry and 3D-microtomography analyzes (uCT) showed MEPE over-expression results in a lower tibial bone-mineral density (6% decrease, p<0.01) and cortical thickness (10% decrease, p<0.01). In contrast, TGC mice had a significantly reduced vertebral trabecular bone-volume (8%, p<0.05). Despite an increase in PHEX (+300%, p<0.05) and other osteocyte and osteoblast specific markers, MEPE over-expression resulted in reduced bone-remodeling. Specifically, TGC mice displayed lowered bone-formation rate (34% decrease in BFR/BS, p<0.05), reduced osteoclast number (33% decrease N.Oc./B.Pm, p<0.01), as well as serum and urinary bone-resorption markers (CTX and PYD, −50%, p<0.05). Also, BMSCs from MEPE transgenic-mice failed to mineralize in vitro as measured by alizarin-red staining. Interestingly, a 5X increased mRNA expression of DMP1 (a related SIBLING-protein) occurred in transgenic bone.

In summary, our findings show bone over-expression of MEPE negatively impacts bone-mineralization, hinders bone-growth and reduces bone-resorption and modeling age-dependently. Also, an indirect or direct negative effect on osteoclastogenesis plays a key role in the transgenic phenotype.

Disclosures: V. David, None.

This study received funding from: ROl-ARS 1598-01 & R03-DE015900-01.

1227

FGF Receptors 3 and 4 Are Not Physiologically Relevant FGF23 Targets in the Kidney. S. Liu, L. Vierthaler*, W. Tang*, J. Zhou*, L. D. Quarles, Internal Medicine/The Kidney Institute, University of Kansas Medical Center, Kansas City, KS, USA.

Fibroblast growth factor 23 (FGF23) is a phosphaturic factor that exerts its biological effects on the kidney proximal tubule, leading to suppression of the sodium-dependent phosphate transport and production of l,25(OH)₂D and to rickets/osteomalcia. FGF23 activation of target tissues appears to require the co-expression of FGF receptors and klotho, a transmembrane beta-glucuronidase. Klotho binds to the c splice isoforms of FGF receptors 1, 3 and 4, and the resulting FGF receptorklotho complex creates the functional FGF23 receptor. The proximal tubule restricted location of the FGF receptor 3c implicates this receptor as the physiologically relevant target for FGF23 in the kidney. To confirm the role of FGF receptor 3 in mediating FGF23-dependent kidney effects, we crossed Hyp mice, which have elevated circulating FGF23 levels due to an inactivating Phex mutation, onto FGFR3-null mice. Consistent with their high circulating FGF23 levels, Hyp mice compared to wild-type (WT) littermates exhibited low serum phosphate (5.5 ± 0.2 mg/dL in Hyp vs. 8.9 ± 0.2 mg/dL in WT, P < 0.01) and serum 1,25 (OH)₂D levels (132 ± 25 pM in Hyp vs. 197 ▪ 23 pM in WT, P = 0.08), reduced Npt2a expression in the proximal tubules, and low bone mineral density (BMD) due to the presence of osteomalacia. In contrast, neither the serum phosphate nor 1,25(OH)₂D levels were altered in FGF receptor 3 null mice. More importantly, ablation of the FGF receptor 3 in Hyp mice failed to correct the hypophosphatemia, aberrant l,25(OH)₂D levels, reduced Npt2a expression or decreased BMD. In similar studies, ablation of FGF receptor 4 in Hyp mice also failed to correct the effects of excess FGF23. To explore the role of FGFR1 and klotho in mediating the proximal tubule response to FGF23, we performed immunohistology of mouse kidneys using FGFR1 and klotho specific antibodies. Consistent with previous reports, we found that both FGFR1 and klotho are predominately expressed in the distal rather than the proximal tubule. Our findings suggest that neither FGFR3 nor FGFR4 are responsible for the renal actions of FGF23. Moreover, the co-localization FGFR1 with klotho in the distal tubule would require the presence of a distal to proximal tubular autocrine pathway to explain FGF23 inhibition of renal phosphate absorption and l,25(OH) ₂D production by the proximal tubule. Further studies are needed to determine whether FGFRlc in the distal tubule or a novel receptor in the proximal tubule mediate the renal effect of FGF23.

Disclosures: S. Liu, None.

This study received funding from: R01-AR45955 and P20 RR-17708.

1228

Alox5 Null Mice Have Reduced Cortical Bone Density, Altered Bone Turnover and Low Serum IGF-I. V. E. DeMambro¹, L. G Horton¹, C. L. Ackert-Bicknel¹1, W. G Beamer¹, M. C. Horowitz², C. J. Rosen¹, ¹The Jackson Laboratory, Bar Harbor, ME, USA, 2Yale University School of Medicine, New Haven, CT, USA.

We previously reported mat a congenic mouse strain, B6.C3-6T, which carries a 30 cM QTL region from C3H on Chr 6. B6.C3-6T, has reduced serum IGF-I, decreased areal BMD and low trabecular BV/TV, with enhanced marrow adiposity. A candidate gene in this QTL is AloxS (Lipoxygenase 5), which by sequencing in 6T, has a truncated 3′UTR. AloxS was also reported to be a prime candidate gene for BMD and fat mass in a B6 × DBA analysis, although phenotyping was limited in the Alox5 nulls (-/-) by small numbers of mice(l). To test the hypothesis that Alox5 polymorphisms could contribute to the 6T phenotype, we raised Alox5-/ - (n=20) mice on a pure B6 background and compared them with littermate +/+ (n=20) controls. At 16 weeks, male and female Alox5-/- had decreased aBMD (p<0.05) by DXA; % body fat was lower in the male -/- vs +/+ (p<0.05) but did not differ in the females. Femoral pQCT revealed that both male and female Alox5-/- mice had decreased total vBMD, reduced periosteal and endosteal circumferences, and thinner cortices (p<0.05 vs gender matched +/+). Femur length was not affected. MicroCT40 of the femur revealed a decrease in both Bone Area and Bone Surface at the midshaft of female and male -/- mice, while analysis of the distal femur showed that male Alox5-/- mice had slightly reduced BWTV (p=0.08 vs +/+) due to a decrease in trabecular thickness (p<0.05 vs +/+), without changes in trabecular number. Female Alox5-/-mice had an increase in distal femoral BV/TV (p<0.05 vs +/+) and connectivity density (p<0.05 vs +/+) with a trend towards increased trabecular number and decreased trabecular spacing. Histomorphometry of female -/- tibias at 8wks of age showed decreased osteoblasts/osteoid perimeter (p=0.02 vs +/+) and a slightly reduced Bone Surface/Bone Volume (p=0.10 vs +/+ controls). Both male and female Alox5-/- had a 15-20% reduction in serum IGF-1 (p<0.05 vs +/ +) while osteocalcin levels tended to be lower in the females -/- (p=O.IO vs +/+) but not in the males -/-. In vitro studies of marrow stromal and bone marrow cells from lOwk AloxS-/-females demonstrated reduced CFU-AP+ colonies (p<0.05 vs +/+), as well as less TRAP+ multinucleated osteoclasts compared to controls. In summary, Alox5-/- mice have in vivo and in vitro evidence of altered bone turn over, as well as compartment specific changes in the skeleton. Polymorphisms in the Alox5 gene may contribute to the unique skeletal phenotype of 6T, and may be important in peak bone acquisition.

Disclosures: V.E. DeMambro, None.

This study received funding from: NIAMS 45433.

1229

Weekly Administration of a Novel Parathyroid Hormone - Collagen Binding Domain Fusion Protein Increases Bone Mineral Density by More Than 15 Percent in Normal Mice. T. Ponnapakkam¹, O. Matsushita*², L Sakon*³, R. C. Gensure¹, ¹Pediatrics, Ochsner Clinic Foundation, New Orleans, LA, USA, ²Microbiology and Parasitology, Kitasato University School of Medicine, Tokyo, Japan, ³Chemistry and Biochemistry, University of Arkansas, Fayetteville, AR, USA.

Pararthyroid hormone (PTH) is an anabolic bone agent which has been shown to be superior to bisphosphonate compounds in the treatment of osteoporosis; however, bisphosphonates are currently the first line treatment for this disorder primarily because of dosing convenience and more favorable side-effect profile. Bisphosphonates accumulate in the bone, allowing them to be efficacious with weekly/monthly oral dosing or yearly intravenous dosing. PTH, on the other hand, is quickly metabolized in the bloodstream and must therefore be given by daily subcutaneous injection. To prolong the retention of PTH in the bone, and thus prolong is duration of action and minimizing systemic side-effects, we have synthesized a fusion protein (PTH-CBD) of human PTH(l–33) and the collagen binding domain of ColH collagenase, derived from Clostridium histolyticum. In-vitro collagen binding assays indicate that the PTH-CBD fusion protein retains its ability to bind collagen. PTH-CBD also stimulates cAMP accumulation with similar potency and efficacy to human PTH(l–34) in LL-CPK cells stably transfected with the parathyroid hormone receptor (PTH-CBD: Kd = 2×10–8 M, Emax=390 +/-54 pmol/well; PTH(l–34): Kd=1.5xlO-8M, Emax 357 +/- 60 pmol/well). Weekly injections of PTH-CBD in normal young female C57BL/6J mice (Jackson Laboratories) for 8 weeks resulted in a 16.7 +/- 4.5 percent (p<0.01, N=6) increase in spinal bone mineral density (BMD), vs. 7.3 +/- 7.1 percent for PTH( 1-34) (NS, N=6) and 4.9 +/- 6.7 percent for vehicle control (NS, N=8). BMD was also measured in excised spines after sacrifice, and there was again a significant increase seen after PTH-CBD treatment vs. vehicle control (15.4% increase, 74.8+/- 3.0 vs. 64.9 +/-1.5 mg/cm2, p<0.05). The PTH(l-34) group showed no significant change in BMD vs. vehicle control (67J+/-2.5 vs. 64.9 +/-1.5 mg/cm2, NS). There were no side effects observed in either treatment group; the animals did not develop hypercalcemia and there was no evidence of bone tumors in whole-body DEXA scans or in excised bone tissues at the end of the study. In-vivo efficacy of longer (i.e. monthly) dosing intervals of PTH-CBD is currently being assessed. This novel fusion protein represents an application of a new concept in drug design, combining individual protein domains to create an agent with unique properties. PTH-CBD allows the superior anabolic effect of parathyroid hormone in the treatment of osteoporosis to be obtained with a more convenient dosing interval, at least in mice.

Disclosures: R.C. Gensure, None.

1230

ACE-011, A Soluble Activin Receptor Type IIA Fusion Protein, Increases BMD and Improves Microarchitecture in Cynomolgus Monkeys. R. J. Fajardo¹, M. L. Bouxsein¹, A. E. Pearsall*², A. Grinberg*², M. Davies*², L Monnell*², J. Ucran*², M. Barazza*², C. Kelly*², P. Khanzode*², K, Underwood*², R. Kumar*², R. S. Pearsall², ¹Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA, ²Acceleron Pharma, Cambridge, MA, USA.

Until recently, the role of activin, a member of the TGF-β superfamily, in bone metabolism was unclear. We have reported that treatment with an activin antagonist, a soluble form of the extra cellular domain of activin type IIA receptor (ActRIIA) fused to a murine IgG-Fc fragment, increases bone formation in normal mice and reverses bone loss in OVX mice. However, the pharmokinetics of human ActRIIA-IgG-Fc (ACE-011) and its effects on bone mass in a larger animal model with a physiology more similar to humans has been lacking. To address this, female young adult Cynomolgus monkeys were used 1) to assess the pharmacokinetic (PK) properties and tolerability of ACE-011, and 2) to assess skeletal effects of ACE-011 treatment. In the PK study, a single dose of ACE-011 (1, 10 and 30 mg/kg) was administered by subcutaneous (SC) injection. Tolerability and PK parameters were assessed over a 28-day period. ACE-011 showed linear pharmacokinetic parameters across the 3 dose levels tested in the 28-day study. The half-life of ACE-011 was found to be 7 to 10 days. A single subcutaneous dose of ACE-011 up to 30 mg/kg was well tolerated, as were multiple doses (7) at 10 mg/kg over a 3-month period. In the efficacy study, monkeys were treated with ACE-011(10 mg/kg, SC, bi-weekly) or VEH for 3 months (n=5/gr). At the conclusion of the study bones were harvested for ex vivo assessment of bone density and structure using DXA, pQCT, and uCT. Compared to VEH, 3-months treatment with ACE-011 administration significantly increased BMD, trabecular bone density and structure in the axial and appendicular skeleton. BMD of the 5th lumbar vertebral body by DXA was 13.2% higher in ACE-011 vs. VEH (p<0.01). MicroCT analysis of the same bone revealed that ACE-011 treatment led to an increase in trabecular BV/TV (+16.2%, p<0.01), trabecular number (+13%, p=0.07), and a decrease in structure model index (7 fold decrease, p<0.01) relative to VEH. At the distal femur, compared to VEH, BMD by DXA was increased 15.3% (p=0.052), whereas trabecular bone density by pQCT was increased 79.5% (p<0.01). In summary, these results indicate that soluble ActRIIA (ACE-011) has a PK profile consistent with intermittent dosing, and that biweekly treatment with ACE-011 significantly improves bone density and structure at multiple skeletal sites in monkeys. These data provide strong rationale for further investigations into the use of ACE-011 as a new anabolic agent for the treatment of skeletal fragility.

Disclosures: R.S. Pearsall, Acceleron Pharma 3.

This study received funding from: Acceleron Pharma.

1231

Treatment with an Anti-Sclerostin Antibody Directly Stimulates Bone Formation in a Dose-Dependent Manner in Ovariectomized Rats with Established Osteopenia. X. Li, K. S. Warmington*, O. Niu*, M. Grisanti, H. Tan. W. S. Simonet, P. J. Kostenuik, C. Paszty, H. Z. Ke, Amgen Inc., Thousand Oaks, CA, USA.

We previously reported that sclerostin inhibition via an anti-sclerostin antibody (Scl-Ab) increased bone formation, restored bone mass and bone strength in aged ovariectomized (OVX) rats with established osteopenia. In the present study, using osteopenic OVX rats, we examined the effects of various Scl-Ab doses on bone mass and bone formation. Six-month-old female SD rats were sham-operated or OVX and left untreated for 5 months. OVX rats were then treated with vehicle or Scl-Ab at 1, 2.5, 5,10, 25 mg/kg (twice a week, s.c.) for 5 weeks (10/group). Xylenol orange (XO, 90 mg/kg) was injected s.c. the day before and the day of treatment initiation in order to detect mineralization and bone formation for the initial effects of treatment. Calcein and tetracycline were injected s.c. 12 and 2 days before necropsy, respectively. Histomorphometric analysis at the 2nd lumbar vertebral body showed that Scl-Ab treated groups had greater trabecular bone volume (BV/TV, +14 to +69%) in a dose-dependent manner compared with OVX control. There was a significantly higher osteoblast surface in OVX rats treated with 5 or 25 mg/kg of Scl-Ab compared with OVX control. However, osteoclast surface did not differ significantly between all doses of Scl-Ab treated OVX rats and OVX control. Qualitatively, OVX rats treated with Scl-Ab had more bone surface labeled with XO than Sham or OVX rats treated with vehicle. Almost all of the XO labeled bone surface in Scl-Ab treated animals was followed by new bone formation and calcein-tetracycline labels, indicating that Scl-Ab directly stimulates the trabecular bone formation without prior activation of bone resorption. Scl-Ab treated OVX rats had greater mineralizing surface (MS/BS, +76 to +288%), mineral apposition rate (MAR, +8 to +60%) and bone formation rate (BFR/BS, +101 to +546%) according to the measurement of calcein-tetracycline labels in a dose-dependent manner compared with OVX control. Qualitatively, an “over-filling” in trabecular bone remodeling was observed, illustrating that Scl-Ab increases bone formation on trabecular remodeling surfaces. These results reveal that treatment with a Scl-Ab in OVX rats increases the formation mode of trabecular bone modeling and also increases bone formation on trabecular remodeling surfaces without increasing bone resorption.

Disclosures: X. Li, Amgen Inc. 1, 3.

This study received funding from: Amgen Inc.

1232

Sclerostin Inhibition Leads to Increased Periosteal and Endocortical Bone Formation as well as Decreased Cortical Porosity in Aged Ovariectomized Rats. O. Niu*¹, K. S. Warmington*¹, M. Grisanti¹, H. Tan¹, M. S. Ominsky¹, W. S. Simonet¹, M. Robinson*², P. J. Kostenuik¹, H. Z. Ke¹, C. Paszty¹, X. Li¹, ¹Amgen Inc., Thousand Oaks, CA, USA, 2UCB-CellTech, Slough, United Kingdom.

We previously reported that treatment with a sclerostin antibody (Scl-Ab) stimulated trabecular bone formation, restored bone mass and bone strength in aged ovariectomized (OVX) rats. Cortical bone is known to be a major contributor to bone strength, so we used dynamic histomorphometry to examine the effects of sclerostin inhibition on cortical bone formation. Six-month-old female SD rats were sham-operated or OVX and left untreated for 13 months. OVX rats were then treated s.c. with vehicle or a Scl-Ab at 25 mg/kg, twice a week, for 5 weeks (9-12/group). Calcein and tetracycline were given by s.c. injection 10 and 3 days before necropsy, respectively. Left femurs were collected and femoral midshaft cross-sections were prepared for dynamic histomorphometry. Scl-Ab treated OVX rats had decreased marrow cavity area (-17%) and increased cortical bone area (+14%) compared with OVX controls. Mineralizing surface (MS/BS), mineral apposition rate (MAR) and bone formation rate (BFR/ BS) were determined. Scl-Ab-treated OVX rats had significantly greater bone formation on periosteal (MS/BS +155%, MAR +84%, BFR/BS +339%) and endocortical surfaces (MS/BS +372%, MAR +145%, BFR/BS +913%) compared with OVX controls. In addition to examining periosteal and endosteal surfaces, we looked at the effect of Scl-Ab treatment on intracortical surfaces. Cortical porosity was quantitated by determining the total porosity area for all pores with a diameter greater than 40 um. Expressed as percent of cortical area (Ct.Ar), the OVX control group had increased porosity area (Po.Ar/Ct.Ar, +1124%) as compared to sham controls. As compared to OVX control rats, Scl-Ab treatment decreased porosity area (to sham levels) and significantly increased MS/BS (+205%), MAR (+49%) and BFR/BS (+504%) on the intracortical surfaces. These results suggest that the decreased porosity in Scl-Ab treated OVX rats is due to increased bone formation on the intracortical surfaces. In summary, sclerostin inhibition stimulated periosteal and endocortical bone formation, and also reduced cortical porosity by increasing bone formation on intracortical surfaces of aged OVX rats with established osteopenia. These results suggest that treatment with anti-sclerostin antibody has the potential to improve cortical geometry and bone strength by promoting bone formation on periosteal, endocortical and intracortical surfaces in patients with low bone mass.

Disclosures: X. Li, Amgen Inc. 1, 3.

This study received funding from: Amgen, Inc.

1233

Comparison of a Soluble Activin Receptor Type IIA (ACTRIIA), PTH and Zoledronate, for Treatment of Established Bone Loss in OVX Mice. E. Rosen*¹, E. Canalis², L. Stadmever*², E. Corv*¹, V. Glatt¹, R. S. Pearsall³, M, L. Bouxsein*¹, ¹Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, MA, USA, ²St. Francis Hospital and Medical Center, Hartford, CT, USA, 'Acceleron Pharma, Cambridge, MA, USA.

We previously reported that treatment with an activin antagonist, a soluble form of the extra cellular domain of activin receptor type Ha fused to a murine IgG-Fc fragment (RAP-011), induced new bone formation and reversed bone loss in OVX mice. Here, we compare the skeletal effects of RAP-011 to known anabolic (PTH(l-34)) and antiresorptive (zoledronate, ZOL) therapies. 8-wk old female C57BL/6 mice were OVX or SHAM-OVX, and allowed to lose bone for 8 wks. OVX mice were then randomized to receive RAP-011(10 mg/kg, I.P. 2×/ wk), PTH (80 ug/kg, sc, 5x/wk), ZOL (single I.P. injection, 20 ug/kg) or vehicle (VEH, I.P., 2×/ wk) for 6 weeks (n=8-10 / gr). In two other groups (VEH and RAP-011), treatment was stopped after 6 wks, and mice were sacrificed 6 wks later. Skeletal response was assessed by PIXImus, microCT and histomorphometry. Total body BMD increased significantly in PTH, RAP-011, and ZOL versus baseline but was unchanged in VEH and SHAM. By microCT, trabecular (Tb) BV/TV at the distal femur was higher than VEH in RAP-011 and ZOL, but not PTH. Histomorphometry of the distal femur revealed that bone formation rate was increased only in RAP-011 (1.4-fold, p=0.03 vs VEH). Osteoblast number increased in PTH (1.4-fold, p=0.014) but not in RAP-011 or ZOL, while osteoclast number decreased in RAP-011 (p=0.03) and ZOL (p<0.01), but not PTH. Eroded surface was increased in PTH, but not other treatments. At the 5th lumbar vertebral body, Tb BV/TV was significantly greater in RAP-011 (+30%), PTH (+35%) and ZOL (+21%) than VEH (p<0.001 for all). Tb number was increased in PTH and RAP-011, but not ZOL. Vertebral compressive strength was higher in all treatment groups compared to VEH (p<0.001 for all), whereas work-to-failure was significantly increased by PTH and RAP-011, but not ZOL. Six weeks after cessation of treatment, vertebral and femoral Tb BV/TV remained increased in RAP-011 vs VEH. Altogether these data indicate that in adult OVX mice with established bone loss, treatment with an activin antagonist (RAP-011) increases bone volume, trabecular number and bone formation rate without enhancing bone resorption. The pattern of skeletal response to RAP-011 differed from PTH and from ZOL, suggesting that activin inhibition represents a novel mechanism for restoration of bone mass following estrogen deficiency.

Disclosures: M.L. Bouxsein, Acceleron Pharma 2, 5.

This study received funding from: Acceleron Pharma.

1234

Intermittent PTH Has Increased Anabolic Effects in Cyclooxygenase-2 Knockout Mice. M. Xu. O, Gao*, O. Voznesenskv*, S. Choudharv, V. Diaz-Doran*, L. Raisz, C. Pilbeam, Endocrine Division and Musculoskeletal Institute, University of Connecticut Health Center, Farmington, CT, USA.

Because PTH induces cyclooxygenase (COX)-2 and prostaglandin (PG) E₂ production and because PGE₂ and PTH can both increase bone formation, we hypothesized that COX-2 knockout (KO) mice would have decreased anabolic response to PTH compared to wild type (WT) mice. Five-mo old male WT (27) and KO (24) mice in a CD-I background were injected with vehicle (control) or PTH (80 ùg/kg,l-34 hPTH) daily for 22 d and euthanized 3 h after the last injection. Bone mineral density (BMD) was measured by DXA in vivo at beginning and end. KO mice weighed 10% less than WT mice (p<0.01) and had 20% less body fat than WT mice (p<0.0l). There was no difference in femur BMD between WT and KO before PTH. Comparing each mouse to itself, PTH increased femur BMD 6.0% (p<0.0l) in WT mice and 11.4% (p<0.01) in KO mice, and the increase was significantly greater (p<0.05) in KO mice. PTH increased the BMD of ex vivo vertebrae (L1-L5) 11% (p<0.05) in KO mice but only 6% (NS) in WT mice. On uCT of ex vivo bones no change in B V/TV was seen in PTH-treated WT or KO mice. However, PTH increased trabecular thickness in vertebrae and distal femurs 15-20% (p<0.01) in WT mice and 21-25% (pO.Ol) in KO mice. Femur cortical thickness (pO.01) and area (p<0.05) were less in KO (0.25 mm, 1.15 mm2) compared to WT mice (0.29 mm, 1.28 mm2). PTH increased cortical thickness and area 25-30% (p<0.01) in KO mice but the increase (3-7%) in WT was not significant. Dynamic histomorphometry of the distal femur showed that, compared to controls, PTH significantly (p<0.01) increased (1) MAR from 0.85 tol .85 u/d in WT mice and from 0.78 to 2.46 u/d in KO mice and (2) BFR/BS from 0.23 to 0.77 u3/u2/d in WT mice and from 0.24 to 1.32 u3/u2/d in KO mice. PTH increased resorption similarly in WT and KO mice. Compared to controls, PTH increased (1) osteoclast surface in WT(7.4%vs 17.9%, p<0.01) and KO mice (6.7% vs 13.0%, p<0.01); (2) serum calcium 10% (p<0.05) in both WT and KO mice; and (3) serum TRAP levels 1.8- to 2-fold (p<0.01) in both WT and KO mice. Analysis of mRNA in tibiae by real time PCR at end of experiment showed no differences in COX-1 in any group. Alkaline phosphatase, Runx2, and RANKL mRNA were increased and OPG mRNA decreased by PTH in both WT and KO mice. Compared to controls, PTH decreased SOST mRNA in KO mice (RQ values, 8.0 vs 2.6, p<0.01) but not significantly in WT mice (RQ values, 6.7 vs 4.1). In summary, PTH increased BMD of femur and spine to a greater degree in COX-2 KO mice than in COX-2 WT mice. Effects of PTH on trabecular bone volume were similar in WT and KO mice. We speculate that absence of COX-2 expression may increase the anabolic response to PTH primarily in cortical bone.

Disclosures: M. Xu, None.

This study received funding from: NIH DK-48361 and AR-47673.

1235

Deletion of Steroid Receptor Coactivator-2 (TIF-2) Results in Skeletal Resistance to Estrogen but a High Bone Mass Phenotype Due to Concomitant Resistance to PPARγ. U. l. Moedder¹, D. G Fraser*¹, A. Sanva¹, M. Gehin*², P. Chambon*², B. W. O'Mallev*³, C. J. Rosen⁴, T. C. Spelsberg¹, S. Khosla¹. ¹Mayo Clinic, Rochester, MN, USA, ²Institut de Genetique et de Biologie Moleculaire et Cellulaire, Illkirch, France, ³Baylor College of Medicine, Houston, TX, USA, ⁴Jackson Labs, Bar Harbor, ME, USA.

Both the estrogen receptor (ER) and PPARγ regulate bone metabolism and there is increasing evidence for cross-talk between these receptors. Clinically, PPARγ agonists are associated with increased bone loss and fractures in postmenopausal women, but not in men. Since steroid receptor coactivator (SRC)-2 enhances both ER and PPARγ activity, we examined the consequences of deletion of SRC-2 on bone using SRC-2 knock out (KO) mice. To assess the skeletal response to estrogen (E), wild type (WT) or SRC-2 KO mice (n = 7-10 per group) were studied following sham surgery, ovariectomy (ovx) or ovx following treatment with a physiological concentration of E2 (2.5 μg/kg/d) for 2 months. While both WT and SRC-2 KO mice lost similar amounts of bone following ovx, the same dose of E2 was much less effective in preventing ovx-induced bone loss in the SRC-2 KO compared to the WT mice (Figure  ).

Despite this defect in E action, SRC-2 KO mice clearly had a high bone mass phenotype under basal conditions (BV/TV [mean + SEM] at the femur metaphysis of 22.2 ± 3.4 %, vs. 12.4 ± 1.3 % for the WT, P = 0.02) with a marked decrease in bone marrow adipocytes (AV/TV of 0.6 ± 0.1 % vs. 1.3 ± 0.3 % for the WT, P = 0.03). These data suggest that, in the SRC-2 KO mice, endogenous PPARγ ligands may be less effective at inhibiting bone formation. Consistent with this, in the presence of the PPARy agonist, rosiglitazone (1 uM), bone marrow cultures from SRC-2 KO mice formed significantly more mineralized nodules (by 93 ± 34 %, P = 0.02) compared to cultures from WT mice. Collectively, these findings demonstrate that SRC-2 may be a key coregulator linking ER and PPARγ signaling pathways in bone. While loss of SRC-2 leads to skeletal E resistance, the concomitant resistance to PPARy appears to be dominant, resulting in a net increase in bone mass and reduction in bone marrow adipocytes. To the extent that the ER and PPARy may compete for limiting cellular concentrations of SRC-2, loss of E following the menopause may lead to the observed susceptibility to thiazolidinesdiones (TZD)-induced bone loss in postmenopausal women by increasing the availability of SRC-2 to enhance PPARy signaling in bone.

Disclosures: U.I. Moedder, None.

1236

Increased Bone Mineral Density in Transgenic Mice Over-expressing Tissue-nonspecific Alkaline Phosphatase. M. C. Yadav*¹, S. Narisawa*¹, K. Johnson*², R. Terkeltaub*², N. Pleshko Camacho³, J. L. Millan¹, ¹Oncodevelopmental Biology, Bumham Institute for Medical Research, La Jolla, CA, USA, 2VAMC/UCSD, La Jolla, CA, USA, 'The Hospital for Special Surgery, New York, NY, USA.

We have recently reported that the rickets and osteomalacia characteristic in tissue-nonspecific alkaline phosphatase (TNAP)-deficient mice (Akp2-/- mice) results from highly increased levels of the calcification inhibitor PPi, a natural substrate of TN AP, and from the concomitant increase in the expression of skeletal osteopontin (OPN), another calcification inhibitorl. These studies suggested the possibility of manipulating the PPi/ OPN axis as a means of affecting calcification. In this paper, we have tested this axis by surmising that transgenic mice over-expressing TNAP might be able to achieve tissular expression of TNAP sufficiently high to be able to lower circulating PPi and OPN concentrations to enhance bone mineral density (BMD) in these animals. Transgenic mice were generated by expressing human TNAP cDNA under control of the Apolipoprotein E promoter, which drives expression of TNAP primarily in the post-natal liver2. We examined the expression levels of TNAP in tissues from mice carrying one copy or two copies of the ApoE-Tnap transgene and also from [Akp2-/-; ApoE-Tnap] mice, and examined the ability of their primary osteoblasts to calcify in culture. MicroCT analysis was used to measure BMD in long bones, vertebrae and calvaria. TNAP expression in ApoE-Tnap mice was major in the liver and kidney as expected, with lower but yet detectable levels in bone, brain and lung. Serum AP concentrations were 10 to 50-fold higher than age-matched sibling control wild-type (WT) mice. As predicted, serum levels of PPi and OPN were reduced in the transgenic animals. Furthermore, microCT analysis of femur, vertebrae and calvaria revealed higher BMD in cancellous bone of ApoE-Tnap+ and ApoE-Tnap+/+ mice compared to WT mice. Thus, we show that increases in tissular and circulating levels of TNAP lead to higher BMD by reducing the effective levels of the calcification inhibitors PPi and OPN. These data provide a mechanistic interpretation for the correlation between AP and BMD that has been observed in humans and mice. Furthermore, these studies suggest that administering recombinant TNAP may serve as a novel therapeutic approach for osteoporosis.

lHarmey et al., Elevated osteopontin levels contribute to the hypophosphatasia phenotype in Akp2-/- mice. J. Bone Min. Res. 21: 1377-1386 (2006).

2Murshed et al., Broadly expressed genes accounts for the special restriction of ECM mineralization to bone. Genes Dev. 19: 1093-1104 (2005).

Disclosures: M.C. Yadav, None.

1237

The Retinoblastoma-Related Pocket Protein Rbl2 Is a Candidate Gene for a Quantitative Trait Locus Affecting Peak Bone Mass and Strength in Mice. R. F. Klein, E. A. Larson*, W. J. Wagoner*, D. A. Olson*, A. S. Carlos*, Bone and Mineral Unit, Oregon Health & Science University, Portland, OR, USA.

We previously identified a QTL on chromosome 8 (Chr 8) associated with variation in peak bone mineral density (BMD) in C57BL/6 (B6) and DBA/2 (D2) mice. A chromosomal fragment containing the Chr 8 QTL interval (61.6 Mb - 111.7 Mb) was transferred from D2 onto a B6 genetic background by selective breeding. Comparison of the resulting congenic strain (B6.D2 D″M″231″3) with the background B6 strain, established that the D2 interval on Chr 8 conferred increased bone mass and strength. Peak whole body, femoral and vertebral BMD (determined by DXA in 20-wk-old male mice) were, respectively, 11%, 23% and 16% greater in the congenic (D2 allele-bearing; n=10) mice compared to the background B6 (n=9) mice (all t-tests p < 0.0001). MicroCT analysis of mid-shaft femoral geometry revealed 18% greater cross-sectional area, 10% greater cortical thickness and 47% increased moment of inertia in congenic mice compared to background mice (all p < 0.0001). Consistent with the increased bone density and geometry, femoral failure load (determined by 3-point bending) of the congenic mice was increased 40% compared to the background mice (p < 0.0001). The Chr 8 introgressed region is homologous with a human genomic region (16qll.l-q23) linked with variation in both bone density and circulating osteocalcin levels suggesting that the effects on bone mass at this chromosomal region may be mediated through genes affecting bone remodeling. Serum osteocalcin levels (determined by R1A) were increased 53% (92 ± 5 ng/ml vs. 60 ± 4 ng/ml, p < 0.0001) in the B6.D2^D8Mit231,113 congenic strain compared to the background strain. Based on synteny between human genome sequence and the murine Chr 8 introgressed region, we identified Rbl2 (residing at 16q 12.1 in the human genome and at 94 Mb on Chr 8 in the mouse genome), which encodes the retinoblastoma pi30 pocket protein, as a positional candidate gene for the observed variation in bone traits. Sequencing of the Rbl2 gene identified 5 nonsynonymous amino acid variants, one of which (exon 20; position 1023) involved a substitution of alanine (B6) for serine (D2). The transcriptional activity of pi 30 is influenced by post-translational phosphorylation at serine and threonine residues raising the possibility that this polymorphism may alter pl30 function. Given the important role the family of retinoblastoma proteins plays in coordinating skeletal development through their effects on osteogenic differentiation, we hypothesize that Rbl2 is an important determinant of bone mass in mice and that inherited variation in the activity of retinoblastoma proteins may explain some proportion of the natural variation in peak bone density and strength in humans.

Disclosures: R.F. Klein, Merck & Co. 8; Procter & Gamble, Inc. 8; sanofi-aventis 8; Eli Lilly & Co., Inc. 8.

1238

Activation of Renin-Angiotensin System Induces Osteoporosis Independently of Hypertension. Y. Asaba*¹, M. Ito², K. Watanabe¹, S. Takeshita¹, J. Ishida*³, Y. Nimura*⁴, A. Fukamizu*³, K. Ikeda¹, ¹Department of Bone and Joint Disease, National Center for Geriatrics and Gerontology (NCGG), Obu, Japan, ²Department of Radiology, Nagasaki University School of Medicine, Nagasaki, Japan, ³Center for Tsukuba Advanced Research Alliance (TARA), University of Tsukuba, Tsukuba, Japan, ⁴Department of Surgery, Nagoya University Graduate School of Medicine, Nagoya, Japan.

Osteoporosis and cardiovascular disease are major health problems worldwide. Although low BMD has been associated with deaths from stroke, the relationship between blood pressure and BMD is controversial and the underlying mechanism remains elusive. We have studied the role of the renin-angiotensin system (RAS) in bone metabolism using Tsukuba Hypertensive Mice (THM) overexpressing the human renin and angiotensinogen genes. THM exhibited elevated circulating angiotensin II (Ang II), high blood pressure (140 +/- 10 mmHg vs. 118 +/- 10 mmHg in WT) and low trabecular bone volume, as determined by microCT-40, at 3 and 6 months old. The trabeculae of THM were characterized by microarchitectural alterations such as decreases in trabecular thickness and connectivity and increases in trabeculr separation and structure model index. Bone histomorphometry at the proximal tibia revealed the number of osteoclasts and eroded surface were significantly elevated compared with wild-type mice, and the bone formation rate was also increased, pointing to a state of high bone turnover. Interestingly, single transgenic mice overexpressing the human renin gene were normotensive but exhibited lower bone mass. Together with the expression of angiotensin converting enzyme (ACE) in bone by RT-PCR, it was suggested that local RAS is sufficient to induce osteoporosis independently of hypertension. Ang II had no direct effect on osteoclastogenesis from isolated bone marrow macrophages (with RANKL/M-CSF), but stimulated the formation of TRAP-positive osteoclasts in co-cultures of bone marrow cells and osteoblasts, pointing to an indirect mechanism through osteoblasts. Both the ATI and AT2 receptors were expressed in osteoblasts, in which Ang II had increased RANKL and VEGF expression. Knockdown of AT2 by siRNA inhibited osteoclastogenesis in co-culture, while that of ATI caused a paradoxical increase, suggesting a functional interaction between the 2 receptors. Accordingly, treatment of THM mice in vivo with an ACE inhibitor (enalapri!) improved but an ATI blocker (losartan) exacerbated osteoporosis. In conclusion, activated RAS is a contributing factor to the pathogenesis of osteoporosis, and certain anti-RAS drugs may be an effective therapy for the co-morbidity of hypertension and osteoporosis with aging.

Disclosures: Y. Asaba, None.

1239

Effects of Testosterone and Estrogen on Bone Resorption Markers and RANKL mRNA Levels in Bone Marrow Osteoblastic Cells in Men. A. Sanval, K. Hoey*, J. Peterson*, E. Atkinson*, L. McCready*, M. Oursler, S. Khosla, Mayo Clinic, Rochester, MN, USA.

Previous studies have shown that in men, estrogen (E) may be a more potent inhibitor of bone resorption than testosterone (T), although this remains controversial. In addition, while it is clear that RANKL is a key regulator of bone resorption, possible effects of T and E on RANKL production in men have not been defined. Thus, to characterize further possible differential effects of T and E on bone resorption and on RANKL production in vivo, we studied 59 elderly men (mean age, 67 yrs) using a previously validated experimental paradigm in which the men were made hypogondal with GnRH, endogenous E production was suppressed with the aromatase inhibitor letrozole, and sex steroid levels maintained with exogenous T or E supplementation. Following serum collections for the bone resorption markers, CTx and TRAP 5b, the men had withdrawal of both T and E (-T, -E), were replaced with T alone (+T, -E) but with continuation of letrozole to block aromatization of T to E, E alone (-T, +E) or both (+T, +E) for 3 weeks and the serum collections were repeated. In addition, bone marrow aspirates were performed and osteoblast lineage cells isolated by magnetic activated cell sorting (MACS) using an antibody against alkaline phosphatase (AP). RNA was extracted from the cells and RANKL mRNA levels quantified using quantitative PCR. Panel A shows the percent changes in serum CTx, panel B shows the percent changes in serum TRAP 5b, and panel C shows the relative RANKL mRNA levels in the AP positive cells isolated from the subjects in the 4 groups.

As is evident, while there were clear effects of E on both resorption markers, T did not regulate either marker. The pattern of changes in RANKL mRNA levels in the AP positive cells was similar to the changes in the bone esorption markers, although due to the variability of these measurements, there was only a trend for an E effect. Collectively, these findings are consistent with the hypothesis that, at physiological concentrations, E has greater suppressive effects on bone resorption than T and that these differences are likely mediated, at least in part, by regulation of RANKL production. The use of more highly purified cell populations (e.g., additional purification using fluorescent activated cell sorting following MACS) should reduce the variability of the mRNA assessments and allow for clearer definition of the mediators of sex steroid action in vivo.

Disclosures: A. Sanyat, None.

This study received finding from: NIH AG04875.

1240

Increased Plasma Osteoprotegerin (OPG) Concentrations Are Associated with Indices of Bone Strength of the Hip: The Framingham Study. E. J. Samelson*¹, K. E. Broe¹, S. Demissie*², E. J. Benjamin*³, R. S. Vasan*³, L Massaro*³, S. Kathiresan*³, C. J. O'Donnell*³, T. J. Beck*⁴, D Karasik*¹, D. P. Kiel*¹, ¹Inst for Aging Research, Hebrew SeniorLife, Boston, MA, USA, ²Biostatistics, BUSPH, Boston, MA, USA, ³Framingham Heart Study, Framingham, MA, USA, ⁴Johns Hopkins, Baltimore, MA, USA.

OPG is an important regulator of bone turnover though its effects on osteoclastogenesis, yet little is known about the significance of circulating OPG concentrations with respect to bone strength in humans. Therefore, we evaluated the cross-sectional association between plasma OPG and femoral neck bone density (FN BMD) and geometry in a large cohort of women and men.

Participants included 1379 post-menopausal women and 1165 men, age 50+ years, in the Framingham Offspring Study. Information was obtained from exams performed 1996-2001. DXA (Lunar DPX-L) scans were used to evaluate FN BMD and geometry. Hip structure analysis was used to determine subperiosteal width, section modulus (bending strength), cross-sectional area (compression strength), and BMD at the narrow neck (NN) region. Plasma OPG concentrations were measured by ELISA (Biomedica, Austria). Sex-specific analysis of covariance was used to calculate means and assess linear trend in BMD and geometry values across OPG quartiles, adjusted for age, height, body mass index, current smoking, diabetes, coronary heart disease, osteoporosis medications, estrogen use (women), and NN BMD (for geometry outcomes).

Mean age of participants was 64 years (range, 50-89 years). OPG concentrations were greater in women (median 5.75 pmol/L; interquartile range (IQR) 4.85-6.84) than men (median 5.32; IQR 4.38-6.47) and increased with age. Multivariable-adjusted mean FN BMD in women increased from the lowest to the highest OPG quartile (Table; trend, p<0.01). However, no linear trend between FN BMD and OPG was observed in men (trend, p=0.34). Section modulus, width, and cross-sectional area increased with OPG in men (trend, pO.OI), whereas no association between hip geometry indices and OPG was observed in women.

Increased OPG concentrations were associated greater bone density in women and with favorable geometry features in men. These results suggest that higher OPG concentration may indicate greater skeletal strength in women and men, possibly through reducing bone loss in women and through increasing periosteal apposition in men.

Table  .  

Adjusted for age, height, body mass index, currenl smoking, diabetes, coronary heart disease, osteoporosis medications, estrogen use (women).

“Additionally adjusted for NN BMD.

Disclosures: EJ. Samelson, None.

1241

Coadaptation Among Trabecular and Cortical Traits Contribute to Genetic Variation Affecting Bone Size in Recombinant Inbred Mouse Vertebrae. S. M. Tommasini¹, B. Hu*², J. H. Nadeau*³, K. J. Jepsen². ¹CUNY Graduate Center, New York, NY, USA, ²Mount Sinai School of Medicine, New York, NY, USA, ³Case Western Reserve University School of Medicine, Cleveland, OH, USA.

A previous study showed that randomization of A/J (A) and C57BL/6J (B) genomic regions resulted in adult Recombinant Inbred (RI) mice having mechanically functional femora with different sets of morphological and tissue-quality traits. This suggested that multiple traits were coordinated to ensure mechanical functionality. Analysis of RI strains has the potential for determining if similar biological controls exist for the construction of vertebrae. To test the hypothesis that trabecular, cortical, and compositional traits of the vertebral body are functionally related, we examined vertebrae from 20 genetically randomized female AXB/BXA RI mouse strains (n=5/genotype; age=16wks). Trabecular traits (bone volume fraction, Tb BV/TV; number; thickness; spacing), cortical traits (cortical thickness, CtTh), tissue mineral density (TMDn), and total vertebral volume (TtV = bone vol + marrow vol) of the L4 vertebral body were measured using micro-CT (GEMS). A correlation analysis using the mean values for each RI strain revealed that 30% of the correlations examined were significant. Thus, many bone traits covaried after genetic randomization. A Path Analysis was conducted using the mean Z-scores of each RI strain. Directed paths (Fig 1) identified important functional interactions among total vertebral size, cortical thickness, and mineral density similar to that observed in femora, such that larger vertebrae (greater TtV) tended to have thinner cortices with lower mineral content, whereas smaller vertebrae had thicker cortices with higher mineral content. Further, the Path Analysis highlighted important functional relationships between cortical and trabecular components such that vertebrae with thicker cortices had higher Tb BV/TV and Tb TMDn, and vertebrae with thinner cortices had lower Tb BV/TV and Tb TMDn. These results suggest that there are critical biological controls that co-adapt the set of cortical, trabecular, and compositional bone traits so that vertebrae are sufficiently stiff for daily loading. Therefore, this approach provides important new insight showing how genetic variation in trabecular architecture is functionally related to genetic variation in cortical traits.

Figure Figure 1.. Path analysis showing causal (straight arrows) and non-causal (curved arrows) relationships among body weight, total vertebral volume (TtV), cortical thickness (CtTh), cortical tissue mineral density (Ct TMDn), trabecular bone volume fraction (Tb BV/TV), and trabecular tissue mineral density (Tb TMDn). Path coefficients based on Z-transformed data.

Disclosures: S.M. Tommasini, None.

1242

Correlation Between Growth Patterns and Material Composition Leads to Preferred Sets of Adult Bone Traits. C. Price, H. W. Courtland, K. J. Jepsen, Mount Sinai School of Medicine, New York, NY, USA.

The specific combination of bone size, shape, and material composition observed in adulthood is predictive of whole bone mechanical properties and fracture risk. However, it is unknown how certain combinations of adult bone traits arise. Knowledge of how specific trait combinations arise is important because while most sets of bone traits satisfy the mechanical demands of daily loading certain sets are preferred under extreme loading conditions. Therefore, the goal of this investigation was to determine how different combinations of adult bone traits develop in inbred mouse femora.

Variability in periosteal and endosteal surface growth results in different combinations of adult bone traits. To determine how surface movement influences bone shape and size we plotted femoral polar moment of inertia (J_o) against cortical area (CtAr) for A, B6, and C3H inbred mice from 1 to 112-days of age. Power law regression analysis (J_o = A*CtAr^B) demonstrated that B6 mice constructed femora in a more “structurally efficient” manner resulting in a greater femoral J_o for a given CtAr. A and C3H femora exhibited similar structural efficiencies of growth that were reduced in comparison to B6. Analysis of bone surface movement demonstrated that strain-specific differences in the structural efficiency of growth resulted from variability in the relative rates of periosteal and endosteal expansion.

To test if the structural efficiency of growth could be extrapolated from adult data sets we validated a generalized power law regression with a fixed exponent (J_o = A*CtAr^1.44) that utilized only adult bone CtAr and J_o values. We observed significant variability in structural efficiency of growth (A) among a cohort of 8 inbred mouse strains and 18 A, B6, and C3H reciprocal cross offspring/founders. Using this panel we also identified a significant correlation between the structural efficiency (SE) of growth and measures of ash content (ash = −0.09*SE + 0.70, r² = 0.30, p<0.01). Thus, femora with more efficient growth patterns exhibited lower ash content values than those with less efficient growth patterns. This correlation between measures of material composition and structural efficiency was further confirmed by an analysis of previously published data including 29 different inbred mouse strains [1]. Together, these results suggest that surface growth patterns and material composition are co-adapted during bone growth. This investigation also provides new insight into bone adaptation and bone fragility and suggests mechanisms by which growth patterns can be used to establish preferred sets of (or robust) adult bone traits (high CtAr, high J_o, and low ash).

[1] Wergedal, et al, 2005 Bone

Disclosures: C. Price, None.

1243

Heritability of Bone Microstructure in Women. S. L. Ferrari, T. Chevallev, L. P. Boniour, R. Rizzoli, Div. of Bone Diseases, WHO Collaborating Center for Osteoporosis Prevention, Geneva University Hospital, Geneva, Switzerland.

Areal bone mineral density (BMD) has a 60-80% heritability (h2) in humans. Bone microarchitecture, another major constituent of bone strength, is also highly heritable in inbred strains of mice. Because bone microstructure is difficult to assess in humans, the proportion of the population-based variance for trabecular and cortical bone traits explained by additive genetic factors is currently unknown. We measured cortical (Ct) and trabecular (Tb) bone structure at distal radius and tibia in 103 mother-daughter pairs using high-resolution (82 microns) peripheral computed tomography (HR-pQCT, Scanco, Switzerland). BMD was evaluated by DXA at radius, hip and spine. H2 (%) was calculated as twice the beta coefficient of the linear regression between maternal and offspring values. The sample consisted of 103 young European-Caucasian females (mean age +/-SD, 20.4 +/ −0.6 yrs) and their mothers, of whom 53 were pre- (48.1 +/-3.2 yrs) and 50 postmenopausal (53.3 +/-3.2 yrs; 5.0 +/-3.4 yrs since menopause, YSM). H2 estimates for body weight, height and BMD were in the expected range, from a lowest 42% and 50% for total and ultradistal (UD) radius BMD, to a highest 70% and 84% for total hip and trochanter BMD, respectively. Heritability of cross-sectional area (CSA), as evaluated by HR-pQCT, was 62% at radius and as high as 96% at tibia, a weight-bearing site. Concerning tibia bone microstructure, h2 values equal or greater than 50% were found for Tb bone density (BV/ TV), Tb number (TbN), as well as Tb and Ct thickness (TbTh, CtTh), whereas h2 was much lower for cortical volumetric density (D.Ct). However, TbTh and D.Ct had h2 values of 79% and 48%, respectively, in daughters with pre-menopausal mothers, but of less than 20% in those with post-menopausal mothers, indicating that increased bone turnover may promptly overcome genetic effects on these traits. At radius, h2 of 50% or greater was found for Tb BV/TV and TbN in all pairs, and for CtTh and D.Ct in pre-menopausal mother-daughter pairs only. Since DXA and HR-pQCT evaluate the same ROI at distal radius, bone microstructural traits were then adjusted by multiple linear regressions for UD radius BMD in addition to height, weight, age and YSM, leading to the following adjusted h2 values at radius: BV/TV, 52%, TbN, 49%, CtTh, 58%, CSA, 44%, D.Ct 32% and TbTh < 10%.

In summary, we report for the first time the heritability of bone microstructure in humans, including the evidence that additive genetic effects may contribute specifically to these traits, i.e. independently of body size and BMD. Heritability for BMD, CSA and TbTh being more robust at weight-bearing sites, it suggests a genetic influence on the skeletal response to loading.

Disclosures: S.L. Ferrari, None.

This study received funding from: Swiss National Science Foundation.

1244

Spatio-Temporal Dynamics of a Single Bone Remodeling Unit. M. D. Ryser*¹, N. Nigam*¹, S. V. Komarova², ¹Mathematics and Statistics, McGill University, Montreal, PQ, Canada, ²Dentistry, McGill University, Montreal, PQ, Canada.

Bone remodeling occurs asynchronously at multiple sites in the mature skeleton and involves bone resorption by osteoclasts, followed by formation of new bone by osteoblasts. At each location, osteoclasts and osteoblasts are organized in Bone Remodeling Units (BRUs), which contain 10-20 osteoclasts in the leading front followed by 1000 – 2000 osteoblasts. BRUs exist much longer than individual osteoclasts and osteoblasts, traveling for 2-6 mm at a rate of 20-40 urn/day. To analyze BRU progression, we developed a mathematical model that describes changes in osteoclast and osteoblast numbers in time and space while taking into account autocrine and paracrine interactions among these cells. We assumed that basal level of a pro-resorptive cytokine RANKL may change locally due to microfracture or external stimulus. The changes in osteoclast numbers were modeled to result from osteoclast formation, death and movement via diffusion and convection in response to RANKL field. The cutting cone was modeled as a free-boundary value problem with appropriate Stefan conditions. The changes in osteoblast numbers were modeled to result from osteoblast formation and death, whereas the necessity for osteoblast active movement was investigated. RANKL antagonist OPG was modeled to be produced by mature osteoblasts and to diffuse with different rates both through bone tissue and through the liquid interface between osteoblasts and osteoclasts. The evolution of the cutting cone arising from this model was studied using numerical simulations. We have found that our model successfully recapitulates spatial and temporal dynamics observed in vivo in a cross-section of the bone. The formation and movement of osteoclasts were most strongly affected by RANKL produced by cells resident to quiescent bone, such as lining cells, osteocytes and stromal cells. Coupling of osteoclasts to osteoblasts allowed for sufficient recruitment of osteoblasts to remodeled surfaces, in such way that a diffusion term describing the directional movement of osteoblasts was not necessary. OPG diffusion through bone was critical in determining the direction of BRU movement. RANKL produced by osteoblasts was found to have little effect on activation of BRU, but was most important in preventing OPG from diffusing through the liquid int between osteoblasts and osteoclasts, and inhibiting osteoclast activation. Thus, our model demonstrates that taking into account spatial organization of BRUs provides new insights into the roles of RANKL and OPG in regulating bone turnover. In the future, this model will allow in silico analysis of the impact of cytokines, growth factors and potential therapies on the process of one remodeling.

Disclosures: M.D. Ryser, None.

This study received funding from: C1HR/IMHA/TAS and NSERC.

1245

Rapamycin Differentially Alters the Skeletal Response to PTH and Mechanical Loading., P. J. Niziolek*¹, S. M. Murthy*², S. N. Ellis*², K. B. Sukhija*³, T. A. Hornberger*³, C. H. Turner¹, A. G. Robling²¹Department of Biomedical Engineering, Purdue University, Indianapolis, IN, USA, ²Department of Anatomy, Indiana University, Indianapolis, IN, USA, ³Department of Bioengineering, University of California - San Diego, San Diego, CA, USA.

Exercise-induced skeletal muscle hypertrophy requires signaling through the mTOR pathway. Likewise, bone cells may respond to exercise through the mTOR pathway, but this hypothesis has not yet been tested. PTH-induced bone hypertrophy requires IGF signaling (an upstream effector of mTOR), but the downstream components of this PTH-stimulated cascade are less certain. We investigated whether the bone-building effects of intermittent PTH require functional mTOR signaling, by treating mice with rapamycin, a selective inhibitor of mTOR activity, during a 6-wk course of daily PTH. We also investigated whether the anabolic effects of mechanical stimulation in bone require functional mTOR signaling, as they do for skeletal muscle, by subjecting mice to ulnar loading in the presence of rapamycin.

Female C57BL/6J mice were implanted with sustained-release rapamycin (or placebo) pellets, then treated with [A] either 0 (vehicle), 30, 60, or 90 μg/kg PTH 1–34 7dy/wk for 6 wk, or [B] mechanical loading of the right ulna for 3 consecutive days, (2 Hz; 2400 με). PTH treated mice were measured for skeletal effects using DEXA, pQCT, and μCT; serum Trap 5b was measured via ELISA. Loaded mice were measured for MS/BS, MAR, and BFR/BS in the right and left ulnar midshaft via fluorochrome histomorphometry.

Rapamycin reduced BMC modestly but significantly. PTH induced significant dose-responsive gains in bone mass in the presence of rapamycin, albeit to a lesser degree than in placebo pellet animals. Trabecular bone mass and morphology in the distal femur (BV/ TV, Tb.N, vBMD) followed similar trends as was observed for whole-body DEXA. Serum Trap 5b measurements indicated that resorption was enhanced significantly in the rapamycin-treated mice, particularly in those given PTH. Mechanical loading increased mineralizing surface similarly in rapamycin and non-rapamycin treated mice, but rapamycin treatment significantly suppressed load-induced mineral apposition rates and bone formation rates by 34% and 38% (p<0.05), respectively.

The anabolic effects of PTH appear to occur in the presence of rapamycin. Most of the disparity in PTH-induced bone gain between rapamycin and non-rapamycin treated mice can be attributed to the enhanced resorptive response induced by rapamycin. The reduced osteogenic response to mechanical loading in the rapamycin-treated mice suggests that mTOR participates in bone cell mechanical signal transduction cascades. Our data indicate that bone and muscle share a common exercise-induced pathway.

Disclosures: P.J. Niziolek, None.

1246

Temporal Pattern of Gene Expression and Histology of Stress Fracture Healing in the Rat Ulna-Loading Model.. L. J. Kidd*¹, A. Stephens*¹, J.S. Kuliwaba*², N. L. Fazzalari², M. R. Forwood¹¹School of Biomedical Science, The University of Queensland, Brisbane, Australia, ²Division of Tissue Pathology, Institute of Medical and Veterinary Science and Hanson Institute, Adelaide, Australia.

Rat ulnar loading has become a central tool in the investigation of bone fatigue injury and remodelling, however this model has not yet been fully characterised. The aim of this study was to undertake a detailed examination of the histology, histomorphometry and gene expression of the healing and remodelling process initiated by rat ulnar fatigue-loading. In a group of 30 female Wistar rats cyclic ulnar loading was stopped at a fatigue level of 10% increase in displacement. Ulnae were harvested 2, 4 or 6 weeks following loading. All animals developed a fatigue fracture of the distal diaphysis of the loaded ulna. These were of a consistent configuration and position with a “U” shaped, incomplete, “stress” facture on the medial cortex of the bone. Fracture healing involved direct remodelling that progressed along the fracture line as well as woven bone proliferation at the site of the fracture. Remodelling was greatest at 2 weeks and it originated at the region of the fracture line closest to the periosteal exit.

Real-time PCR was performed on the ulnae from 40 rats in which a stress fracture was created. Rats were euthanized following 4 hours, 24 hours, 4 days, 7 days and 14 days (n=8/group). A control group did not undergo loading. At 4 hours post fracture there was peak increased mRNA expression (p0.05), compared to non-loaded controls, for IL-6 (400 fold increase), OPG (11 fold), COX-2 (6 fold) and VEGF (2 fold). At 24 hours there was peak mRNA expression of IL-11 (48 fold). At 4 days there was a significant increase in mRNA expression of SDF-1 (4 fold), SOST (3 fold), and BMP-2 (6 fold). At 7 days there was peak mRNA expression of RANK-L (12 fold). Other genes that showed a marginal increase in mRNA expression were BAX, Bcl-2, Collagen 10, COX-1, IGF-1, Runx-2, TNFa and IL-1, all with 2–3 fold increases, peaking at 4 days post fracture. mRNA expression of RANK-L, OPG and COX-2 was also increased in the opposite, unloaded, left limb suggesting a systemic response in bones distant to the site of injury.

These results show that rat ulna-loading is an excellent model for creating stress fractures and initiating remodelling. The gene expression results demonstrated a clear temporal cascade of important signalling events that occurred during fracture healing and remodelling. Dramatic, early up-regulation of IL-6 and IL-11 suggests their central role in initiating later signalling events. Prominent, up-regulation of COX-2, VEGF, OPG, SDF-1, BMP-2 and SOST prior to peak expression of RANK-L indicates the likely importance of these factors in mediating and possibly co-ordinating directed remodelling.

Disclosures: L.J. Kidd, None.

This study received funding from: NHMRC.

1247

GSK-3β Inhibits Osteoblastic Bone Formation Through Suppression of Runx2 Transcriptional Activity by the Phosphorylation at a Specific Site., M. Hirata, F. Kugimiva, N. Kawamura, S. Ohba, K. Nakamura*, H. Kawaguchi, U. Chung. Sensory & Motor System Medicine & Tissue Engineering, University of Tokyo, Tokyo, Japan.

GSK-3β is known to be a kinase involved in bone formation signalings like Wnt and Akt. To investigate the role of GSK-3β in bone, we initially investigated the skeletal phenotype of GSK-3β-deficient mice. Although the homozygous deficient mice were embryonically lethal, the heterozygous deficient (Gsk-3β+/-) mice developed and grew normally; however, bone densitometry, 3D-μCT, and histomorphometric analyses revealed that Gsk-3β+/- mice showed increased bone mass with enhanced bone formation as compared to the wild-type littermates. In Gsk-3β+/- calvarial osteoblast cultures, the differentiation and function determined by ALP, Alizarin red and von Kossa stainings, and mRNA levels of osteoblastic markers were enhanced, while the proliferation was unaffected. Similarly, addition of lithium chloride or SB216763, selective inhibitors of GSK-3β, stimulated the bone formation parameters above. Meanwhile, overexpression of wild-type GSK-3β or a constitutively active form of GSK-3β (CA-GSK-3β) suppressed the parameters, although the kinase-inactive form of GSK-3β (KI-GSK-3β) overexpression did not affect it. Neither CA-GSK-3β overexpression, genetic GSK-3β insufficiency, nor GSK-3β inhibitors altered the expression or subcellular localization of Runx2, a master gene for osteoblast differentiation. However, the luciferase reporter assay revealed that the Runx2-dependent transcription was attenuated by wild-type GSK-3β or CA-GSK-3β overexpression, but not by KI-GSK-3β, whereas it was enhanced by GSK-3β inhibitors. Binding with the oligonucleotide probe of OSE2 / osteocalcin promoter by electrophoretic mobility shift assay was enhanced in nuclear extracts from Runx2-overexpressing osteoblasts from Gsk-3β+/-, as compared to those from wild-type. To learn the contribution of phosphorylation of Runx2 by GSK-3β, we generated phosphorylation-deficient mutants of Runx2 at the five consensus sites, and found that a specific mutant at S369–S373–S377 cancelled the inhibition of Runx2 transactivity by CA-GSK-3β. Finally, when Runx2+/ mice were crossed with Gsk-3β+/- mice to generate the compound deficient mice (Gsk-3β+/-;Runx2+/) or were administered lithium chloride from E7.5, the cleidocranial dysplasia was significantly rescued, confirming the interaction between Runx2 and GSK-3β in vivo as well. In conclusion, GSK-3β was shown to inhibit the I Runx2 transcriptional activity through the phosphorylation at S369–S373–S377, causing the suppression of osteoblast differentiation and bone formation.

Disclosures: M. Hirata, None.

1248

Overexpression of DeltΔFosB Decreases Adipose Mass in a Non-Cell Autonomous Manner by Increasing Energy Expenditure Independently of Bone Cells., G C. Rowe*¹, L. Neff*¹, T. Green*², H. Saito*¹, C. Choi*³, G. Shulman*³, E. Nestler*², W. Home¹, R. Baron¹Orthopaedics, Yale University, New Haven, CT, USA, 2Psychiatry, University of Texas Southwestern Medical Center, Dallas, TX, USA, 'Internal Medicine, Yale University, New Haven, CT, USA.

Overexpression of ΔFosB in mice under the control of the enolase 2 promoter (EN02), which drives expression in bone, fat and the brain, results in increased bone formation and decreased adipose mass, due to a reduction of the size of the individual adipocytes and not their number. To determine whether this effect on fat was linked to the bone phenotype, we generated mice which overexpress ΔFosB under the control of the osteocalcin promoter (OG2). These mice failed to recapitulate the adipose phenotype, demonstrating that the effect on adipocytes is independent of the increased bone formation. Since differentiation of the adipocytes was unimpaired we then explored whether the function of the adipocyte or the metabolic status of the EN02-ΔFosB mice may be altered. To determine if the decrease in adipose mass was due to a cell autonomous effect within the adipocyte we generated a mouse overexpressing ΔFosB under the control of the adipocyte protein 2 promoter (aP2). Again, these mice did not recapitulate the decrease in adipose mass observed in the EN02-ΔFosB mice, eliminating the possibility that the decrease in size of the adipocytes was due to a cell-autonomous defect. These results lead us to explore the metabolic status of the EN02-ΔFosB mice: in contrast to the OG2-ΔFosB and aP2-ΔFosB mice, the EN02-ΔFosB mice exhibited an improved glucose tolerance and an increased sensitivity to insulin. Furthermore metabolic analysis of the EN02-ΔFosB mice revealed an increase in energy expenditure that was not present in the aP2-ΔFosB mice. Since the effect of ΔFosB on the regulation of adipose mass and energy expenditure was not due to its expression in either the osteoblast or adipocyte lineages, these results opened the possibility of a central effect of ΔFosB. We found that ΔFosB is overexpressed in the arcuate nucleus (ARC) of the hypothalamus in the EN02-ΔFosB mice. Since the hypothalamus has been identified as a key regulator of both adipose and bone mass, we then examined the possibility of ΔFosB regulating adipose mass centrally Stereotaxic injections were performed using adeno-associated viruses to target ΔFosB to the ARC of the hypothalamus. Six weeks post injection the mice injected with ΔFosB exhibited a significant reduction in body weight compared to control injected mice. This suggests that ΔFosB is able to alter adipose mass in a non-cell autonomous manner by altering the hypothalamic regulation of energy expenditure independent of the osteoblast.

Disclosures: GC. Rowe, None.

1249

Inhibition of Wnt Signaling by Osterix., C. Zhang¹, J. Lyons*², K. Sinha*¹, P. McCrea*², X. Zhou*¹, B. de Crombrugghe¹¹Molecular Genetics, University of Texas MD Anderson Cancer Center, Houston, TX, USA, ²Biochemistry and Molecular Biology, University of Texas MD Anderson Cancer Center, Houston, TX, USA.

During mouse embryonic development both the osteoblast-specific transcription factor Osterix (Osx) and Wnt/p-catenin signaling are required for osteoblast differentiation. To explore whether Osx also plays a role in control of osteoblast proliferation, we compared BrdU incorporation in wild type and Osx-null E18.S calvaria and examined the proliferation rates of primary calvarial cells isolated from wild type and Osx-null E18.5 embryos. BrdU incorporation in calvaria and proliferation of calvarial cells were markedly increased in Ox-null mice. In addition, overexpression of Osx in C2C12 mesenchymal cells resulted in slower growth. These experiments suggested the hypothesis that Osx could control osteoblast proliferation.

A microarray comparison of RNA expression profiles of wild type and Osx-null calvarial cells from E18.5 embryos revealed that expression of the Wnt inhibitor, Dkkl, which is high in wild type osteoblasts, was abolished in calvarial cells of Osx-null embryos. Moreover, expression of several Wnt target genes such as c-Myc and cyclin Dl increased in Osx-null calvarial cells. These findings suggested the hypothesis that Osx might inhibit Wnt pathway activity. Several lines of evidence, indeed, support this hypothesis. In vitro transfection studies in HEK293 cells demonstrated that Osx activated the Dkkl promoter. In other transfection experiments Osx inhibited expression of the β-catenin-induced TOPFLASH reporter. In addition, Osx also inhibited β-catenin-induced secondary axis formation in Xenopus embryos. Moreover, in calvaria of Osx^−/− mice, which harbor the TOPGAL reporter transgene, β-galactosidase activity of the reporter was increased, suggesting that Osx inhibits the Wnt pathway in osteoblasts in vivo. Coimmunoprecipitation experiments showed that Osx interacted with Tcf, a DNA-binding partner of β-catenin. Gel shift assay indicated that Osx was able to disrupt Tcf binding to DNA. Thus our observations suggest that Osx has the ability to inhibit Wnt-β-catenin signaling through several mechanisms. We speculate that the proposed inhibition by Osx of osteoblast proliferation might be a consequence of the Osx-mediated control of Wnt-β-catenin activity.

Disclosures: C. Zhang, None.

1250

Zfp521, a D2D FosB-interacting Protein, Is a Novel Inhibitor of Runx2 Activity with Opposite Effects on Osteoblasts and Bone Formation In Vitro and In Vivo.E. Hesse, M. Wu*, G C. Rowe*, L. Neff*, W. C. Horne, R, Baron. Orthopaedics and Cell Biology, Yale University School of Medicine, New Haven, CT, USA.

The identification of novel molecules or pathways that contribute to the regulation of bone formation in vivo is essential for the development of future anabolic drugs. Over-expression of ΔFosB leads to a striking osteosclerotic phenotype in vivo and to the increased expression of osteoblast (OB) markers and bone nodule formation in vitro (Sabatakos, Nat Med, 6:985, 2000). We recently reported that the further N-terminally truncated and API transcription-inactive isoform A2ΔFosB induces the same phenotype as ΔFosB in vivo. We therefore proceeded to a yeast two-hybrid screen and identified the zinc finger protein Zfp521, a 180 kDa protein consisting of 30 kruppel-like zinc fingers, as a A2ΔFosB-interacting protein. In situ hybridization and immunocytochemistry showed that Zfp521 is expressed at the periphery of mesenchymal condensations as early as day EI2.5. At later stages of bone development, Zfp521 is strongly expressed in chondroblast and OB precursors in the perichondrium and periosteum, as well as in prehypertrophic chondrocytes, OB and osteocytes. In vitro, Zfp521 expression repressed, whereas RNAi-mediated depletion of Zfp521 enhanced, in vitro OB differentiation, nodule formation and mineralization. Co-immunoprecipitation studies revealed that Runx2 interacts with Zfp521, and we identified zinc fingers 6–10 and 26–30 as interacting sites. Moreover, in vitro overexpression of Runx2 dose-dependently rescued the Zfp521 induced repression of the OB phenotype, suggesting that Zfp521 acts as a Runx2 antagonist. Consistent with this hypothesis, Zfp521 strongly repressed the transcriptional activity of Runx2 in a reporter assay as well as the expression of Runx2 target genes (Runx2 itself, alkaline phosphatase, and other OB markers). Surprisingly, and despite the fact that Zfp521 inhibits OB differentiation in vitro, over-expressing Zfp52I in transgenic mice under the control of the OB-specific osteocalcin promoter resulted in a marked increase in bone volume and bone formation as measured by histomorphometry. Therefore, based on the well known functional duality of Runx2, which is required for OB differentiation at early stages of bone development while inhibiting OB maturation at later stages (Liu, J Cell Biol, 155:157, 2001; Geoffroy, Mol Cell Biol, 22:6222, 2002), we propose that Zfp52l both inhibits early OB differentiation and promotes late stages of OB maturation and bone formation by antagonizing Runx2 activity. Thus, the balance between Zfp521 and Runx2 could determine the rate of osteoblast differentiation and bone formation during development and in the adult skeleton.

Disclosures: E. Hesse, None.

1251

Cox-2 Deficiency Impairs Periosteal Progenitor Cell Activation and Differentiation: Rescued by Ep4 Agonist., X. Zhang, C. Xie*, M. Xue*, A, Naik*, E. Schwarz*, R. J. O'Keefe. Orthopaedics, University of Rochester, Rochester, NY, USA.

We previously showed that genetic ablation of cydooxygenase-2 (COX-2) resulted in delayed and defective fracture healing in mice. In this study, we evaluated COX-2 expression in the bone repair microenvironment and its critical role for activation and expansion of local progenitor cells following skeletal injury. In a stabilized femoral fracture healing model, COX-2 mRNA was increased at day 3 prior to chondrogenesis and peaked at day 7 in early chondroprogenitors and proliferating chondrocytes. Coincident with COX-2 expression, initiation and completion of periosteum mediated endochondral bone repair was markedly delayed in COX-2^−/− mice, as evidenced by delayed appearance of PCNA positive periosteal progenitor cells at day 3, marked reduction of Col2al gene expression at day 5, and deficient angiogenesis and osteogenesis at day 14. The deficient activation of periosteum bone repair in COX-2^−/− mice resulted in the development of fibrotic non-unions in live 4mm segmental cortical bone graft transplantations. Minimal periosteal response was found on the wild-type (WT) or COX-2-/- (KO) graft when transplanted into a KO host. In contrast, the transplantation of a wild type live graft into a wild type host defect resulted in abundant bone formation and neovascularization on grafted surface. An intermediate amount of periosteal bone formation with increased amount of cartilage formation was found on the graft when transplanting a KO graft into a WT host, suggesting the requirement of COX-2 in the local microenvironment for the normal initiation and progression of periosteal bone repair. To rescue the deficient periosteal response in COX-2-/- mice, an EP4 agonist CP-734432 was administered via periosteal injection. The delivery of EP4 agonists during the early phase of fracture healing reversed the impaired fracture healing in COX-2-/- mice. PGE2 and agonists of EP2 or EP4 receptor potently increased phosphorylation of ERK1/2 in mouse bone marrow stromal cells and primary sternal chondrocyte cultures. The activation of the M APK/ERK pathway led to rapid induction of early growth response gene (EGR-1). Treatment with PGE2 and EP agonists also induced the phosphorylation of GSK3P at serine 9 in the same cultures with further induction of the active form of p-catenin. Taken together, our data strongly suggest that transient induction of COX-2 functions as an essential early signal for initiation and completion of endochondral bone healing through targeting of the periosteal progenitor cell population. These signals are necessary for early events in repair and are a potential target for the treatment of impaired skeletal healing.

Disclosures: X. Zhang, None.

This study received funding from: AR051469, AR46545, MTF.

1252

TRIP-1: An Important Co-Regulator of Bone Formation in Skeletal Remodeling., D. Metz-Estrella*, T. Sheu*, R. Mroczek*, J. Puzas. Orthopaedics, University of Rochester, Rochester, NY, USA.

TGFbeta receptor interacting protein (TRIP-1) is an intracellular signaling cofactor with high affinity for fragments of tartrate resistant acid phosphatase (TRAP). TRAP, when bound to TRIP-1 activates the TGFbeta signaling pathway and leads to an increase in markers of bone formation in osteoblasts. This may be one way that osteoblasts recognize resorption lacunae during remodeling. Pull down assays also show that TRIP-1 interacts with intermediates of the BMP pathway (i.e. Smadl) and that this interaction is inversely related to the level of activation in the pathway. Based on these findings we hypothesize that TRIP-1 is a pleiotropic regulator of Smad-dependent pathways during bone remodeling.

To further investigate the mechanism by which TRIP-1 influenced signaling we identified regions in the Smads that bind to TRIP-1. Our results show that the MH1 domain of Smad3 is mainly involved in the physical interaction with TRIP-1 and that this interaction leads to a large increase in its phosphorylation.

In regard to its pleiotropic nature TRIP-1 also activates a BMP-specific reporter in osteoblasts. When this same reporter is transfected into Smad4-deficient cells, TRIP-1 expression leads to a greater than 50 fold induction. Moreover, co-localization experiments have shown that TRIP-1 associates with phosphorylated Smads 1, 5, 8 in the nuclei of osteoblasts.

To understand the in vivo role of TRIP-1 we used a mouse model in which TRIP-1 can be over expressed in osteoblasts using the TetOn system. In these mice, induction of TRIP-1 expression in the calvarium led to an approximate three fold increase in bone volume (p<0.001) and a three fold increase in mineral apposition rate (p<0.001). These results provide evidence to conclude that TRIP-1 is an important co-regulator of the Smad pathways and may control, in part, bone formation at sites of remodeling.

Disclosures: J. Puzas, None.

1253

Serum 1GFBP-2 (IGF binding protein-2) Is a Marker of Bone Turnover; In Vivo Evidence from the IGFBP-2 Null Male Mouse.. V. E. DeMambro¹, D. Clemmons², W. G Beamer¹, M. L. Bouxsein³, E. Canalist⁴, C. J. Rosen¹¹The Jackson Laboratory, Bar Harbor, ME, USA, ²University of North Carolina, Chapel Hill, NC, USA, ³Beth Israel Deaconess Medical Center, Boston, MA, USA, ⁴St. Francis Hospital & Medical Center, Hartford, CT, USA.

IGFBP-2 is a 36kD IGF binding protein with strong affinity for hydroxyapatite and extracellular Matrix proteins. It circulates in relatively high concentrations, and increases with age, and malnutrition. IGFBP-2 is expressed in most tissues including bone and combined with IGF-I can stimulate bone formation in experimental animals. A recent study highlighted a direct correlation between serum IGFBP-2 and bone turnover markers in both men and women. However, the mechanism of this effect is unknown. To understand the role of IGFBP-2 in the mammalian skeleton we generated IGFBP-2 null (-/-) mice that were backcrossed 10 generations onto C57BL/6J. We then performed skeletal and metabolic phenotyping at 8 and 16 weeks and compared the nulls to +/+ littermates. Male BP2-/- mice had shorter femurs at 16 wks and had greater % body fat than +/+ (p<0.05). However, they were not insulin resistant and had normal glucose tolerance. Serum IGF-1 in the BP2-A mice was 10% higher than +/+ (p<0.05) at 8 but not 16 wks. Hepatic expression of IGFBP-1,3,4,5,6 was higher in the -/- at 6wks than +/+ mice (p<0.05). BP2-/- mice at 8 weeks had reduced total bone area and cortical thickness by microCT with a 17% reduction in trabecular BV/TV vs +/+ (p<0.01). This was almost entirely due to reduced trabecular thickness (p<0.01) rather than change in trabecular #. Serum osteocalcin was suppressed by 40% in BP2-/- vs +/+ (p<0.01) and both CFU-AP+ pre-OBs and TRAP+ osteoclasts were significantly less abundant in vitro than +/+ cells from littermates. Moreover, null mice have 20% smaller spleens and less B220+ mononuclear cells in marrow and spleen than +/+. Histomophometry at 16 wks revealed there were significantly less osteoblasts/BPm (pO.Ol) and less osteoclasts/BPm (p<0.05) in BP2-/- than +/+ mice. Mineralizing surface/bone surface in BP2-/- was markedly reduced (p<0.00l), as was the overall bone formation rate/BSd (-45%, p<0.001) but MAR was not different. In summary, IGFBP-2 null mice have a skeletal phenotype that is directly related to bone turnover and mostly likely begins with impaired OB recruitment. Recent work suggests that IGFBP-2 can bind to the avb5 integrin receptor, independent of IGF-I, and may suppress PTEN expression. Conversely, IGFBP-2 may mediate its action by transporting IGF-1 to its receptor on stromal cells leading to enhanced OB differentiation. Studies are ongoing to assess whether recombinant IGF-I or IGFBP-2 can rescue the in vitro or in vivo phenotypes. Nonetheless, circulating IGFBP-2 may become a useful marker of bone turnover.

Disclosures: V.E. DeMambro, None.

This study received finding from: NIAMS 45433.

1254

Chemokine Receptor CCR-1 Deficient Mice Have a High Turnover Osteoporosis that Is Greater in Females than in Males., J. Lorenzo., L Kalinowski*, S. Jastrzebski*, S. K. Lee*. Medicine, University of Connecticut Health Center, Farmington, CT, USA.

Chemokine receptor CCR1 is expressed on osteoclasts and binds the ligands CCL3 (macrophage inflammatory protein 1α, MlP-1α) and CCL9 (MlP-1γ). Previous in vitro results demonstrated that MIP-1α and MIP-1γ stimulate osteoclast formation and mobility. To better understand the in vivo effects of CCR1 and its ligands on bone, we examined mice that were globally deficient in CCR1 (CCR1 KO). Animals were examined for changes in bone mass, bone turnover and the ability of bone marrow cells to form osteoclast-like cells (OCL) in vitro. CCR1 KO and wild type (WT) mice were in a C57BL/ 6 background. Bone mass was measured by micro-CT. Changes in bone cells were determined by histomorphometry. Global bone turnover was assessed by measuring serum c-terminal telopeptide (CTX), a measure of bone resorption, and osteocalcin (OCN), a measure of bone formation. In vitro osteoclastogenesis was assayed in bone marrow cultures, treated with M-CSF and RANKL (30 ng/ml for each) for 4 days. Mice were examined at 9–10 weeks of age and females (F) and males (M) were analyzed separately. Based on the previous in vitro data, we anticipated that CCR1 KO mice would have increased bone mass and low turnover. Instead we found that both F and M CCR! KO mice had decreased vertebral trabecular bone mass, with the greatest effect in females (BV/TV was decreased 15.8% in F and 8.4% in M, trabecular thickness was decreased 12.2% in F and 3% in M, p<0.05 for all). Femoral cortical bone thickness was also significantly decreased in F and M CCR1 KO mice by 10% for both (p<0.05). There was no significant difference in femoral trabecular bone mass between either F or M CCR1 KO and WT. In the femurs of F mice there were increases in the osteoclast surfaces (29.2%), the number of osteoclasts per unit bone surface (22.8%) and osteoblast surfaces (43.3%) (p<0.05 for all). In the femurs of M mice there were no significant differences in histomorphometric indices between CCR1 KO and WT. CCR1 KO mice also had increased serum turnover markers. In F CCR1 KO mice serum CTX was 36% greater while OCN was increased by 49% over WT (p<0.05 for both). Increases in serum CTX and OCN in M CCR] KO mice were also seen but were less (20% and 39%, respectively, p<0.05 for both). OCL formation in vitro in bone marrow cell cultures was similar in F CCR1 KO and WT cultures. In contrast, in M cells, there was an 18% decrease in OCL formation relative to WT cells (p<0.05). These results demonstrate that CCR1 KO mice have a high turnover osteoporotic phenotype, which is markedly greater in female than in male mice. These findings imply that complex mechanisms, which are not dependent on the ability of CCR1 ligands to stimulate osteoclasts, are responsible for the observed bone phenotype of CCRI KO mice.

Disclosures: J. Lorenzo, None.

1255

Selective Deletion of the Membrane-bound Colony Stimulating Factor 1 Isoform In Vivo Does Not Affect Estrogen-deficiency Bone Loss in Mice., G. Yao, J. Wu*, K. Insogna. Yale University, New Haven, CT, USA.

It has been reported that a neutralizing antibody to CSF1 completely prevents ovariectomy (OVX)-induced bone loss in mice. There are two isoforms of CSF1, soluble (sCSFl) and membrane-bound (mCSFl), but their individual biological functions are unclear. It has been reported that estrogen-withdrawal increases levels of IL-1 and TNF in bone marrow, which induce the formation of a stromal cell population producing high levels of secreted CSF1. However, others have found that estrogen-withdrawal selectively up-regulates expression of mCSFl and down-regulates sCSFl expression in rat bone marrow cultures. To explore the role of CSF1 isoforms in estrogen-deficiency bone loss, we have generated mCSFl knock-out mice, lsoform-specific RT-PCR confirmed the absence of transcripts for mCSFl in bone tissue isolated from these animals and western blot analysis demonstrated similar amounts of sCSF 1 secreted by osteoblasts isolated from mCSFl k/o and wild-type mice. The mCSFl k/o mice had higher femur BMD than wild-type controls. Five month-old mCSFl k/o and wild-type female mice underwent ovariectomy (OVX) or sham-OVX. One month after surgery, femoral and spinal BMD (determined by DEXA) were reduced by 7% and 5% respectively in OVX-wild-type animals as compared to sham-OVX wild-type mice. OVX mCSF-1 k/o mice showed similar 5% and 8% reductions in femoral and spinal BMD respectively compared to sham-OVX mCSF-1 k/o animals. We next used real time PCR to quantitate expression of transcripts for mCSFl and sCSFl in RNA isolated from bones of OVX or sham-OVX wild-type mice. When data from three separate experiments were analyzed, OVX induced a significant 4-fold increase in the expression of sCSFl while mCSFl expression was either unchanged or changed minimally. In summary, our findings indicate important non-redundant functions for the two isoforms of CSF1. In particular, it appears that the mCSFl isoform is not involved in estrogen-deficiency bone loss although this isoform is essential for normal bone remodeling since, in its absence, bone density is increased. In contrast our data suggest that sCSFl could play a key role in estrogen-deficiency bone loss.

Disclosures: G Yao, None.

This study received finding from: NIH.

1256

Point Mutation of Endofin at PPlc-Binding Domain (F872A) Stimulates Bone Formation in Transgenic Mice: A Negative Feedback Regulation of BMP Signaling.. F. Zhang*¹, W. Shi*¹, T. Oiu¹, X. Wu¹, J. Chen*², J. Si*³, N. Wan¹, X. Cao¹¹Pathology, University of Alabama at Birmingham, Birmingham, AL, USA, ²Pharmacology, Tongji Medical School, Wuhan, China, ³Pathology, Shihezi University School of Medicine, Shihezi, China.

Smad anchor for receptor activation (SARA) facilitates TGFβ signaling by recruiting and presenting Smad2/3 to the receptor complex. SARA does not bind Smadl and hence does not enhance bone morphogenetic protein (BMP) signaling. We identified Endosome-associated FYVE-domain protein (endofin) acts as a Smad anchor for receptor activation in BMP signaling. We have shown that endofin binds Smadl preferentially and enhances Smadl phosphorylation and nuclear localization upon BMP stimulation. Silencing of endofin by RNAi resulted in a reduction in BMP-dependent Smadl phosphorylation. We also demonstrate that endofin contains a protein-phosphatase-binding motif, which recruits protein phosphotasel (PPlc) to negatively modulate BMP signals through receptor dephosphorylation of BMP type I receptor. Point mutation of endofin at PPlc-binding domain (F872A) almost abolished the interaction between PPlc and endofin. As a result, BMP signaling was sensitized and osteoblast differentiation was enhanced.

To further determine whether this endofin mutant affects osteoblast activity and bone formation in vivo, transgenic mice were generated in which this mutant (F872A) was overexpressed in osteoblasts driven by 2.3 kb Col I promoter. Significant bone formation in transgenic mice was observed. The expression of the mutant endofin (F872A) was detected in 4 different transgenic lines with Western blot. X-ray analysis revealed that bone density of the entire skeleton is elevated at 4-month-old age of all 4 transgenic lines. Analysis of femur by μCT shows that trabecular volume (BV/TV) is increased by 11 percent more in comparison to their wild type littermates. Consistently, the trabecular bone thickness and number are also significantly increased, whereas the trabecular bone separation is decreased. To examine whether the effect of this endofin mutant on bone formation is due to BMP signaling change, Smadl phosphorylation was detected in osteoblasts. Immunostaining of tibia sections demonstrate that the level of phosphorylated Smadl was enhanced in osteoblasts from the transgenic mice. Taken together, the recruitment of PP1 c to endofin functions as a negative feedback regulation of the BMP signaling pathway. Disruption of PPlc binding motif of endofin by point mutation leads to an elevated level of phosphorylated Smadl and augment of bone formation in transgenic mice.

Disclosures: F. Zhang, None.

1257

Molecular Mechanisms for BMP-2 Transcription by the Hedgehog Pathway.M. Zhao¹, R. L. Chandler*², J. Liu*¹, G R. Mundy¹, D. P. Mortlock*²¹Medicine/Clinical Pharmacology, Vanderbilt University, Nashville, TN, USA, ²Molecular Physiology & Biophysics, Vanderbilt University, Nashville, TN, USA.

The Hedgehog pathway is essential for normal developmental patterning from drosophila to man. This pathway mediates effects via the Gli family of transcriptional activators, and one of its major downstream target genes is BMP-2. We have previously examined the molecular mechanisms by which Gli2 transactivates BMP-2 through the 5′ flanking promoter region from −2712 to +165 (Zhao et al., 2006, MCB), but did not find good evidence for discrete functional binding sites. We have thus now examined much larger genomic regions flanking the murine BMP-2 gene. Two BAC clones, namely 5′BAC-LacZ and 3′BAC-LacZ, spanning from −185 to +53kb and −2.7 to +207kb of upstream and downstream respectively of the gene, were modified by inserting a LacZ reporter cassette in the place of exon 3 of BMP-2 coding region. Using transgenic mice which contain the BAC-LacZ genes, we have found that each BAC clone directs distinct BMP-2 expression patterns. Strikingly, we found that the regulatory sequence located more than 53kb 3′ to the promoter is required for BMP-2 expression in osteoblast progenitors during skeletal development. To refine the location of the osteoblastic element, we have generated transgenics that carry 3′BAC-LacZ with different deletions. We found that deletion of+132 to +168kb region completely abolished LacZ-positive osteoblasts in long bones. Finally, a conserved 656bp region, namely ECR1, located within +155 to +I60kb from the BMP-2 promoter, was identified as an osteoblast-specific enhancer of BMP-2 expression, shown by LacZ staining in both endochondral and intramembranous bones of ECR1 transgenic mice. Sequence analysis of the ECR1 fragment shows a putative Gli-response element. Thus, we examined the effect of GH2 on ECR1 regulation of BMP-2 transcription. We made two chimeric reporter constructs by linking ECR1 to the BMP-2 promoter (2.7kb) or SV40 promoter (0.2kb). Luciferase assays showed that ECR1 addition increased Gli2 stimulation of BMP-2 promoter activity. We also found that GH2 significantly enhanced the activities of 3′BAC-LacZ and ECRl-LacZ genes that were stably transfected into the osteoblastic MC3T3-E1 cells. These results strongly suggest that the 3′ genomic sequence from +155 to +160kb of BMP-2 gene, covering a cis-enhancer ECR1, is required for osteoblast progenitor-specific BMP-2 expression, and the Hedgehog signaling mediator Gli2 enhances BMP-2 transcription in osteoblasts through this region. The identification of this enhancer should clarify regulatory mechanisms that control BMP-2 transcription in osteoblasts during skeletal development and postnatal bone formation.

Disclosures: M. Zhao, None.

1258

Absence of sFRPl, an Antagonist of Wnt Signaling Accelerates Fracture Healing., T. Gaur¹, J. J. Wixted*², S.Hussain*¹, D. Avers*², P. V. N. Bodine³, G. S. Stein¹, J. B. Lian¹¹Department of Cell Biology and Cancer Center, University of Massachusetts Medical School, Worcester, MA, USA, ²Department of Orthopedics, University of Massachusetts Medical School, Worcester, MA, USA, ³Women's Health and Musculoskeletal Biology, Wyeth Research, Collegeville, PA, USA.

Bone fractures are the frequent consequence of trauma and pathological bone conditions (osteoporosis, diabetes and cancer). Fracture healing is a complex regenerative process and delayed process in aging and diseased bone results in clinical complications. Several clinical studies as well as transgenic mouse models have shown that the Wnt pathway supports bone formation. We examined the role of Wnt signaling in improving fracture repair using a knockout mouse model for the Wnt antagonist secreted frizzled related protein 1 (sFRP1-KO). We also used the chemical inducer LiCI to activate canonical Wnt signaling in wild type mice. A transverse fracture in the right tibia was generated in WT and sFRPl-KO mice. Healing was monitored at regular intervals by radiographic analyses. Bone samples were collected at 7, 11, 14, 21 and 28 days post fracture for histological and gene expression analyses for assessing the progression of bone formation and remodeling. Fracture healing was achieved completely in WT mice by 28 days post fracture but significantly accelerated in the sFRPl-KO mouse with appearance of new bone formation beginning at day 11. Both radiographic and histological analyses showed that on day 7, the initial callus appeared similar, but by day 11, reduced callus formation with increased cartilage tissue and trabecular bone bridging the fracture occurred in sFRP 1 -KO mice. After day 11, bone remodeling (by TRAP staining) was much more robust in the sFRPl-KO. Radiography identified union of the fracture by day 21 in the sFRPl-KO mice, in contrast to the WT fracture which still exhibited presence of cartilaginous callus along with formation of new bone. Enhanced fracture healing was observed in WT mice treated with LiCI. Gene expression analyses using RNA from callus tissues showed reduced TNFa levels suggesting less inflammation in KO mice fractures. Lower levels of chondrocyte proliferation markers (Col2al and Sox9) and mild induction of bone markers (Runx2, osteocalcin, osteopontin and Collal) were observed in sFRPl-KO mice, consistent with less chondroid tissue and more bone formation in sFRPl-KO mice by histology. Bone remodeling markers, MMP9, TRAP and VEGF were slightly elevated in sFRP1-KO mice, suggesting mechanisms for accelerated healing. Our studies establish a direct improvement of fracture repair by increasingWnt signaling and also provide a possibility for modulation of the Wnt pathway to achieve better fracture healing in compromised clinical cases.

Disclosures: T. Gaur, Wyeth Research, Inc. 2.

This study received funding from: Wyeth Research.

1259

Enzyme Replacement Therapy for Murine Hypophosphatasia., J. L. Millan¹, S. Narisawa*¹, I. Lemire*², T. P. Loisel*², G Boileau*³, P. Leonard*², M. D. McKee⁴, P. Crine², K. Johnson*⁵, R. Terkeltaub*⁵, N. Pleshko Camacho⁶, M. P. Whyte⁷¹Oncodevelopmental Biology, Burnham Institute for Medical Research, La Jolla, CA, USA, ²Enobia Pharma, Inc., Montreal, PQ, Canada, ³University of Montreal, Montreal, PQ, Canada, ⁴McGill University, Montreal, PQ, Canada, ⁵VAMC/UCSD, La Jolla, CA, USA, ⁶The Hospital for Special Surgery, New York, NY, USA, ⁷Shriners Hospitals for Children and Washington University, St. Louis, MO, USA.

Hypophosphatasia (HPP) is the inbom-error-of-metabolism that features rickets or osteomalacia due to loss-of-function mutation within the tissue-nonspecific alkaline phosphatase (TNALP) gene. Consequently, there is extracellular accumulation of the mineralization inhibitor inorganic pyrophosphate (PPi), one of TNALP's natural substrates. HPP features a remarkable range of severity spanning (most severe to mildest) perinatal, infantile, childhood, adult, and odontohypophosphatasia forms, classified (historically) according to age at diagnosis. TNALP null mice (Akp2-/-) phenocopy infantile HPP remarkably well, as they are bom with a normally mineralized skeleton, but develop radiographically apparent rickets at about 6 days of age, and die between day 12-16 suffering severe skeletal hypomineralization and episodes of apnea and epileptic seizures attributable to disturbances in pyridoxal 5/-phosphate (vitamin B6) metabolism. There is no established medical therapy for HPP. Case reports of enzyme replacement therapy (ERT) using intravenous (i.v.) infusions of ALP-rich plasma from Paget's bone disease patients and purified placental ALP have described failure to rescue affected infants. It seems that ALP activity must be increased not in the circulation, but in the skeleton itself. This hypothesis is supported by beneficial outcomes for two girls with infantile HPP following marrow cell transplantation where perhaps some TNALP-containing cells were introduced throughout the skeleton. Here, we report survival without significant skeletal or dental disease or seizures in TNALP knockout mice undergoing enzyme replacement therapy (ERT) from birth with a bone-targeted form of recombinant TNALP (sALP-FcDIO). Short-term and long-term efficacy studies with daily s.c. injections of 1,2, or 8.2 mg/kg sALP-FcDIO for 15,19 and 52 days showed homing of sALP-FcDlO to bone tissue and normal circulating PPi concentrations with preservation of skeletal and dental architecture (assessed by radiographs, microCT, and histology) as well as increased life span and well-being. This bone-homing, recombinant form of human TNALP prevents lethal HPP in TNALP knockout mice. Our findings represent the first successful use of ERT for a heritable primary disease of the skeleton, and are a foundation for therapeutic trials for human HPP.

Disclosures: J.L. Millan, Enobia Pharma, Inc. 5.

This study received funding from: Enobia Pharma, Inc.

1260

Treatment of Osteogenesis Imperfecta in Adults with Teriparatide: Results of an 18-month Prospective Observational Trial.J. D. Ringe¹, H. Faber*¹, E. Farahmand*¹, T. N. Nickelsen²¹Medizinische Klinik IV, Klinikum Leverkusen, Leverkusen, Germany, ²Medical Department, Lilly Deutschland GmbH, Bad Homburg, Germany.

Osteogenesis imperfecta (01) is a genetic disorder of connective tissue characterized by brittle bones and a susceptibility to fracture from normal impacts of daily living or inadequate trauma. It has been shown that an antiresorptive therapy with bisphosphonates has beneficial effects on the clinical outcome of Ol, especially in children. So far there is a lack of data on the use of osteoanabolic drugs in this disorder.

The aim of the ongoing “Teriparatide in Osteogenesis Imperfecta” trial (TO1-trial) is to study the efficacy of an anabolic treatment with teriparatide (rhPTH-1–34) in adults with clinically symptomatic disease. In this 18-month prospective, observational, single center trial we included 10 patients (6 men, 4 women) above the age of 30 years (mean age 44.9) with a T-score <-3.0 at the lumbar spine and < −2.0 at the total hip, and at least one prevalent vertebral and one non-vertebral fracture. Fluoride, bisphosphonates or alfacalcidol had been used previously over different intervals, but during the last 6 months only calcium and vitamin D were given. During the study, all patients received daily subcutaneous injections of 20μg teriparatide plus oral supplements of 1200 mg calcium and 800 IU vitamin D per day.

Baseline characteristics of patients included a mean height of 154.1 cm, mean weight 66.3 kg, average T-scores of-4.03 at the lumbar spine and −3.24 at the hip, and a mean number of 1.5 new vertebral and 0.8 non-vertebral fractures per patient during the last year before intervention.

BMD measured at 6-month intervals showed highly significant increases at both sites and amounted to an average gain of 10.8% at the lumbar spine and 8.4% at the total hip after 18 months. During the 18 months we observed only one new vertebral and two non-vertebral fractures. This is remarkable in relation to the respective average fracture rates during the last year before starting teriparatide injections. There was a significant decrease in back pain. Moderate, transient adverse events occurred in 5 of 10 patients. All patients will be followed during a subsequent antiresorptive therapy up to month 36. These very positive preliminary results should encourage further, controlled trials with an anabolic therapy in osteogenesis imperfecta.

Disclosures: J.D. Ringe, Eli Lilly and Company 8.

1261

Mutations in CRTAP or P3H1 Cause Dysregulation of Prolyl-3-hydroxylation and Recessive Osteogenesis Imperfecta.D. Baldridge*¹, U. Schwarze*², R. Morello¹, J. Lennington*¹, T. K. Bertin*¹, P. R. Eyre², M. Weis*², A. Green*³, J. Walsh*³, D. Lambert*³, P. Krakow*⁴, P. L. Rimoin*⁴, P. H. Cohn*⁴, P. H. Byers*², B. Lee¹¹Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA, ²University of Washington, Seattle, WA, USA, ³Our Lady's Hospital, Dublin, Ireland, ⁴Cedars Sinai Medical Center, Los Angeles, CA, USA.

Osteogenesis imperfecta (OI) or brittle bone disease is a genetic condition caused by mutations in the type I collagen genes (COl1A1 or COL1A2). Quantitative or structural type I collagen alterations lead to recurrent fractures. ProlyL 3-hydroxylases, such as P3H1, are recently identified enzymes that hydroxylate a unique proline residue in the cc-helical domain of fibrillar collagens like type 1 collagen. CRTAP, or cartilage-associated protein, interacts with P3H1, and Crtap null mice display an Ol-like phenotype and lack fibrillar collagen prolyl 3-hydroxylation. The purpose of this study is to identify additional genetic causes of osteogenesis imperfecta.

In our study of 70 OI subjects we report a spectrum of recessively-inherited phenotypes, including Ol types II and III, caused by mutations in either CRTAP (two patients) or mutations in P3H1 (six patients). The latter group includes a mutation in the Irish Traveller population, a community with a high degree of consanguinity and which has a prevalence of OI of approximately 2%. The mutations lead to loss-of-function, including premature termination, frameshift, and splice site alterations that result in a severe decrease of mRNA, as well as decreased collagen protyl-3-hydroxylation by mass spectrometry, as demonstrated in patient fibroblasts. MS analysis of collagen from bone shows a 50% decrease in prolyl-3-hydroxylation in a patient with a P3H1 mutation, while a patient with a CRTAP mutation shows a 95% reduction. This suggests that there may be partial functional redundancy among the family of prolyl-3-hydroxylase enzymes and therefore as yet undiscovered less severe phenotypes from loss of function of each of these genes. However, expression of the cofactor CRTAP may be essential for complete enzymatic activity of the complex in vivo. Clinically, one patient with a P3H1 mutation was treated with IV bisphosphonates and showed prolonged survival. Hence, osteoclastic inhibition may be a viable strategy for treatment of OI due to abnormal 3-prolyl-hydroxylation.

These results demonstrate a genetic basis for recessive OI and help to explain the apparent recurrence of OI to unaffected parents previously attributed primarily to germline mosaicism. In summary, loss of function of CRTAP or P3H1 leads to the severe OI phenotype including types II and III.

Disclosures: D. Baldridge, None.

1262

Expression of Osteocalcin by Circulating Endothelial Progenitor Cells Predicts Endothelial Dysfunction or Structural Coronary Artery Disease.U. I Moedder, M Gössl*, A. Lerman*, S. Khosla. Mayo Clinic, Rochester, MN. USA

Increasing evidence indicates a link between bone and the vasculature, and bone marrow as well as circulating cells with osteogenic potential have been identified by staining for the osteoblastic marker, osteocalcin (OCN) Endothelial progenitor cells (EPCs) contribute to vascular repair, but repair of vascular injury may result in calcification, which is also a marker for future coronary events A previous preliminary study (Bull Exp Biol Med 139 266,2005) found that circulating cells staining for another bone-related protein, osteonectin, were present in the circulation of 7 subjects with coronary artery disease (CAD) but absent from the circulation of 4 control subjects Thus in the present study, we tested whether patients with coronary atherosclerosis had increases in circulating EPCs expressing an osteogenic phenotype, as assessed by OCN staining We studied 59 patients undergoing invasive coronary assessment Subjects were defined as controls (no significant structural coronary lesions, normal endothelial function, n = 11) versus two groups with coronary atherosclerosis early coronary atherosclerosis (defined as no significant structural coronary lesions but abnormal endothelial function, n = 19) and late coronary atherosclerosis (defined as severe, multi-vessel coronary artery disease [CAD], n = 29) Peripheral blood mononuclear cells were analyzed using flow cytometry following staining for EPC markers (CD 133, CD34, vascular endothelial growth factor receptor 2/kinase insert domain receptor [KDR]) and OCN Compared to controls, patients with early or late coronary atherosclerosis had significantly higher percentages of EPCs which co-stained for OCN, particularly in the population containing relatively late EPCs (CD133-/CD34+/KDR+, by 8- and 5-fold, P = 0 0006 and 0 012, respectively) In unadjusted logistic regression models, a doubling of these OCN positive EPCs was associated with an odds ratio of 1 8 (95% confidence interval, 1 2–2 7) for having CAD, following adjustment for age, sex, and statin use, the corresponding odds ratio was 4 2 (95% confidence interval, 1 5–12 1), by comparison, the analogous odds ratios for the currently used marker for coronary atherosclerosis, high sensitivity C-reacUve protein, were 09 (0 7–1 3) and 1 2 (0 7 1 9), unadjusted and age, sex, and statin use adjusted, respectively Thus, a higher percentage of EPCs co-stains for OCN in patients with coronary atherosclerosis as compared to subjects with normal endothelial function and no structural CAD These findings have potential implications for the mechanisms of vascular calcification and for the development of novel markers for coronary disease

Disclosures U.I. Moedder None

1263

A Novel Central Mechanism in Uremic Bone Disease.W. Cheung*¹, C. Vanek², U T Iwamec*³, R. T. Turner*³, R. Klein*², R. Mak*¹¹Pediatrics, University of California at San Piego, La Jolla, CA, USA, ²Bone and Mineral Unit, Oregon Health and Science University, Portland, OR, USA, ³Nutrition and Exercise Sciences, Oregon State University, Corvallis, OR, USA.

Bone disease is common in patients with uremia In this study, we tested whether leptin signaling, via the hypothalamic melanocortin system, is an important cause of uremic bone disease We performed sub-total nephrectomy (N) or sham operation (S) in 8-week-old wild-type (WT, C57BL/6J), leptin deficient (ob/ob), leptin receptor deficient (db/db) and melanocortin receptor 4 knockout (MC4-RKO) mice Additional WT-N mice were treated with agouti-related protein (AgRP, a MC4-R antagonist) or vehicle (V) Secondary hyperparathyroidism in uremic mice was minimized by administering a low phosphorus and high calcium diet WT-N mice were fed ad libitum and all other mice were pair-fed to WT-N mice Changes in whole body composition were assessed by DXA at the beginning and at the end of the 6-week study Excised left femoral composition was determined by DXA and x-ray microtomorgraphic scanning Femoral strength was assessed by 3-point bending Architecture of right femora was analyzed by mCT WT-N, ob/ob-N, db/db-N, MC4-RKO-N, WT-N/V and WT-N/AgRP mice were uremic based on increased blood urea nitrogen and creatinine levels WT-N mice displayed a uremic bone phenotype characterized by decreased femoral length, bone mineral content/density and failure load mCT evaluation demonstrated reduced total femoral volume, cortical area and thickness in WT-N mice The uremic bone phenotype was attenuated in ob/ob and db/db mice, nephrectomy in these animals did not result in significant differences in femoral bone composition and architecture compared to controls Uremic bone disease was also attenuated by central MC4-R deficiency MC4-RKO N mice had no change in bone parameters compared with MC4-RKO-S mice We also tested the effect of central MC4-R antagonism in the WT-N mice Uremic bone disease in WT-N was ameliorated by central administration of AgRP In summary, leptin signaling through the central melanocortin system may play an important role in the pathogenesis of uremic bone disease These findings may have significant clinical and pharmacotherapeutic implications 

Table  .  

Disclosures W Cheung None

1264

Threonine 137 Is an Important Determinant of Sodium-Phosphate Co-transport in Human NaPi-IIc.G Jaureguiberry*¹, T, O, Carpenter², £. Forman*³, H. Juppner¹, C. Bergwitz¹¹Endocrine Unit, Massachusetts General Hospital, Boston, MA, USA, ²Dept. of Pediatrics and Endocrine Unit, Yale School of Medicine, New Haven, CT, USA, ³Dept. Anesthesiology, Massachusetts General Hospital, Boston, MA, USA.

We recently reported homozygous and compound heterozygous loss-of-function mutations in SLC34A3/NaPi-IIc, as the cause for hereditary hypophosphatemic rickets with hypercalciuria (HHRH). A previously reported male affected by HHRH (Pediatrics 1989; 84:276-280) was found to carry a novel SLC34A3 mutation, T137M c.410C>T(p.T137M)), on the maternal allele and the deletion g.4225_50del on his paternal allele. In addition to rickets and typical biochemical abnormalities, he also suffered from recurrent kidney stones prior to therapy with phosphate or vitamin D analogs. His mother, who is a heterozygous carrier of Tl 37M, also had one renal stone during pregnancy, but both parents were otherwise healthy. To permit functional analysis of T137M and g.4225_50del in vitro, we generated expression plasmids encoding enhanced green-fluorescence protein (EGFP) followed, in-frame, by cDNAs encoding wild-type or mutant human NaPi-IIc, i.e. EGFP-hNaPi-IIc, EGFP-[M137]hNaPi-IIc, or EGFP-[V446Stop]hNaPi-IIc. The V446Stop mutant showed a complete loss-of-function when assayed for surface expression and sodium-dependent [33P]-uptake in Xenopus oocytes. Conversely, EGFP-[M137]hNaPi-IIc was well expressed in apical patches, the renal brush border membrane equivalent of Opossum kidney cells, and it was properly inserted into the cell membrane when the mutant cRNA was injected into oocytes. However, when quantified by confocal microscopy, oocyte membrane fluorescence was reduced to 50% compared to EGFP-hNaPi-IIc. After correction for surface expression, the rate of [33P]-uptake by oocytes mediated by EGFP-[M137]hNaPi-IIc was decreased by an additional 60%. In comparison to wild-type, function of this NaPi-IIc mutant was thus reduced to 30%, which, taken together with complete loss-of-function by g.4225_50del on his second allele appears to be sufficient to explain the phenotype in our patient. Two-electrode studies indicated that EGFP-[M137]hNaPi-IIc is non-electrogenic and the stoichiomeby of simultaneous [33P]- and [22Na]-uptake was unaltered when compared to EGFP-hNaPi-IIc. However, EGFP-[M137]hNaPi-Ilc displayed a significant phosphate-independent inward-directed sodium-leak, which appears to be insensitive to phosphonoformic acid (PFA) and changes in extracellular pH, factors known to inhibit sodium-dependent phosphate co-transport. Ml37 may thus reduce the rate of phosphate uptake by uncoupling sodium-phosphate co-transport, suggesting that this amino acid residue has an important functional role in human NaPi-IIc.

Disclosures: G Jaureguiberry, None.

This study received funding from: National Kidney Foundation.

1265

Alendronate Impairs Angiogenesis and Bone Formation During the Early Stages of Bone Healing in an Animal Model for Osteonecrosis of the Jaw.J. I. Aguirre, S. M. Vanegas*, M. K. Altaian*, S. E. Franz*, M. E. Leal, T. J. Wronski. Physiological Sciences, University of Florida, Gainesville, FL, USA.

Osteonecrosis of the jaw (ONJ) is a bone disorder characterized by persistent areas of exposed, necrotic bone in the oral cavity after jaw trauma, dental surgery, or tooth extraction. ONJ is most frequently observed in cancer patients that receive high doses of nitrogen-containing bisphosphonates (BPs) IV to inhibit bone metastases. It has also been reported to occur, although with lower incidence, in postmenopausal women undergoing treatment for osteoporosis with lower doses of orally-administered BPs. Despite the association of BPs with ONJ, the pathophysiology of this condition is not well understood. Since tooth extraction has been identified as a risk factor for ONJ, we propose to use BP-treated rats as a novel animal model to test the hypothesis that BPs inhibit angiogenesis and delay filling of the root socket with new bone after tooth extraction. Intact female rats at 2.5 months of age were injected SC twice weekly with either vehicle, a low dose of alendronate (15 μg/kg bw), or a high dose of alendronate (150 μg/kg bw). The 10-fold difference in these doses is roughly equivalent to the difference between osteoporosis and oncology doses for other BPs. Treatments were administered for 3 weeks before the first mandibular molar was extracted in each rat, and continued during a post-extraction period of 10 days. The left mandibles were then collected to assess the effects of alendronate on bone healing by determining osteo- and angiogenesis within the distal root socket by quantitative histomorphometry. We found that both the low and high doses of alendronate decreased woven bone volume within the root socket by approximately 50% compared to vehicle-treated rats. In addition, alendronate induced significant decreases in osteoid and osteoblast surfaces by approximately 45% and 30% at the low and high doses, respectively. In contrast, no significant difference was observed in osteoclast surface with either dose. Remarkably, both the low and high doses of alendronate decreased blood vessel area by 30% and 45%, and blood vessel number by 40% and 35%, respectively. Along the mesial and distal alveolar bone surfaces at the margins of the distal root socket, both doses of alendronate significantly decreased mineralizing surface, but no difference was observed in osteoclast surface. These findings indicate that alendronate impairs angiogenesis and bone formation during the early stages of bone healing after tooth extraction in female rats. Furthermore, these findings provide evidence for use of tooth extraction in rats as an animal model for BP-associated ONJ.

Disclosures: J.I. Aguirre, None.

1266

Nitrates and BMD Among Canadian Men and Women.S. A. Jamal¹, D. Goltzman², D. A. Hanley³, A. Papaioannou⁴, J. C. Prior⁵, R. G. Josse¹¹Medicine, University of Toronto, Toronto, ON, Canada, ²Medicine, McGill University, Montreal, PQ, Canada, ³Medicine, University of Calgary, Calgary, AB, Canada, ⁴Medicine, McMaster University, Hamilton, ON, Canada, ⁵Medicine, University of British Columbia, Vancouver, BC, Canada.

Cross sectional studies suggest that compared to nonusers, postmenopausal women using nitrates have higher BMD and a decreased fracture risk. We used data from a population-based cohort study, The Canadian Multicentre Osteoporosis Study (CaMos), to determine the relationship between nitrate use and bone loss among men and women in a prospective manner. Subjects were 50 years and older, not using bone active agents at the baseline visit. Medication use was assessed by questionnaire at baseline and subjects classified as nitrate users if they used any of: isosorbide dinitrate, mononitrate or nitroglycverin at least once a day every day. We excluded subjects who reported use of nitroglycerin (tablet or spray) exclusively on as “as needed” (prn) basis. BMD at the lumbar spine, total hip and femoral neck was measured at baseline and five years later.

We used linear regression models, stratified by gender and adjusted for age and weight to determine the change in BMD at the total hip, femoral neck and lumbar spine (LI to L4) from baseline. Results are expressed as the annualized percent change in nitrate users compared with nonusers.

BMD measurements were obtained in 1449 men; 14 men reported nitrate use. BMD measurements were obtained in 2682; 35 women reported nitrate use. The majority of the cohort was Caucasian (95%). The mean age of the men in our cohort was 66.3 ± 9.5 years; the mean age of the women was 68.2 ± 9.3 years. The men weighed 81.2 ± 13.8 kg and the women weighed: 68.9 ±13.9 kg. Among both men and women, nitrate users were significantly older and weighed significantly less than nonusers.

At all BMD sites and among both men and women, nitrate users lost less bone mass than nonusers. For example, among men, nitrate users had a 1.3% (95% Confidence Interval [CI]: 0.6 to 3.7) annual increase in total hip BMD while nonusers lost 0.9% (95% CI: 0.5 to 1.1) at the total hip. At the lumbar spine nitrate users had a 1.2 % (95% CI: 0.5 to 4.1) increase in BMD while nonusers gained 0.6% (95% CI: 0.3 to 1.1) at the lumbar spine. Women who used nitrates had a 2.8% (95% CI: 0.4 to 5.2) increase in total hip BMD compared to nonusers who lost 1.9% (95% CI: 1.7 to 2.1). Nitrate users also had a 1.4 % (95% CI: 1.1 to 4.0) increase in lumbar spine BMD compared to nonusers who gained 0.5% (95% CI: 0.4 to 0.7).

Our analysis demonstrates that nitrate use is associated with increases in BMD among both men and women 50 years and older. These findings are consistent with earlier studies and strengthen the need for a randomized trial to determine the effects of nitrates on fracture.

Disclosures: S.A. Jamal, None.

1267

Bone Apposition in Patients on Teriparatide Treatment Is Preferably Directed to Skeletal Regions of Local Structural Weakness: Assessment by High Resolution CT Based Finite Element Analysis In Vivo.C. Graeff¹, P. Zysset*², F. Marin*³, C. C. Glüer¹¹Medical Physics, Diagnostic Radiology, Universitätsklinikum Schleswig Holstein, Campus Kiel, Kiel, Germany, ²Institute for Lightweight Design and Structural Biomechanics, Vienna University of Technology, Vienna, Austria, ³Medical Research, Eli Lilly & Co, Europe, Madrid, Spain.

The concepts of skeletal adaptation to loads, such as Wolffs law and Frost's mechanostat, are well established. High resolution computed tomography (HRCT) combined with finite element (FE) analysis technique provide new tools to study these concepts non-invasively in vivo. We investigated at which locations within vertebral bodies new bone is preferentially added under teriparatide treatment, hypothesizing that bone apposition induced by teriparatide is not solely governed by existing bone material acting as a scaffold but is controlled by mechanical needs, e.g. happens preferentially in areas of high strain.

In 27 patients participating in the EUROFORS study we studied the relationship of local baseline strain and subsequent increases in bone mass during 6 months of teriparatide (TPTD, 20 &mUg per day) treatment. HRCT scans of T12 were performed at 0 and 6 months. Voxel by voxel, we correlated baseline FE-calculated strains (and, for comparison, local apparent bone volume as assessed by HRCT, appBV/TV) with subsequent locally site-matched increases in bone mineral density (BMD) by employing image registration methods. For the FE analysis the entire segmented vertebrae were converted into finite elements of 1.3 mm isotropic resolution and relative von Mises strains were calculated by dividing local strain by global strain applied to the whole vertebra.

Results shown in Figure 1 confirm our hypothesis: a sharp increase in BMD with increasing local strain, then converging (left) and a less pronounced upturned U-shaped relation to baseline app.BVATV

Figure Figure 1.. BMD changes as function of baseline strain (left) and appBV/TV (right). Each point represents 2.5% of the total number of voxels.

We conclude that bone apposition under TPTD treatment is not uniform but directed to skeletal regions of local structural weakness. This represents a microstructural, non-invasive, visual and quantitative demonstration of the validity of the biomechanical concepts of bone tissue response to local strains, in accordance with Wolffs law and Frost's mechanostat.

Disclosures: C. Graeff, None.

This study received funding from: Eli Lilly & Company.

1268

Changes in Circulating Osteoblast Lineage Cells Following PTH (1–34) Therapy and Correlation with Changes in Trabecular Bone Mass., S. Kirmani, M. Drake, U. Moedder, J. Peterson*, S. Khosla. Mayo Clinic, Rochester, MN, USA.

Using flow-cytometry, we previously demonstrated a five-fold increase in circulating osteoblast progenitor cells in pubertal boys compared to adult males (NEJM 352:1959–1966). We recently further classified these osteocalcin (OCN) positive cells into two distinct populations: small, relatively agranular cells that co-stained for the stem cell marker, CD34 (OCN^small, which may represent a less differentiated cell population), and larger, more granular cells that were CD34 negative (OCN^gran, which may represent a more differentiated cell population) (Bone 40:1370–1377). To evaluate the effects of PTH therapy on these cells, we studied 8 women at baseline, 3 months and 12 months after starting 20 meg/day rh 1–34 PTH (Forteo). All 8 subjects completed the baseline and 3 month visit, and 7 of the 8 completed the 12 month visit. Panel A in the figure shows the changes in the OCN^smalland OCN^gran cells. At 3 months, there was no significant change in the OCN^small cells, but a trend for an increase (of 26%, P = 0.14) in the OCN^gran cells. However, at 12 months, this pattern was reversed, with the OCN^small cells showing a marked increase (of 64%, P = 0.016), and the OCN^gran showing a decrease back to baseline levels. Interestingly, the percent change in OCN^gran cells between 0 and 12 months was highly positively correlated (R = 0.82, P = 0.02) with changes in bone volume/tissue volume (BV/TV) as assessed by high resolution 3D-pQCT (Xtreme CT, Scanco) in these patients (Panel B). The mean (± SEM) increase in BV/TV in the subjects who had an increase in the OCN^gran cells between baseline and 12 months was 10.8 ± 3.25% as compared to 1.7 ± 0.6% in the subjects who had a decrease in these cells (P = 0.066). To the extent that the OCN^small cells and OCN^small cells represent less as compared to more differentiated osteoprogenitor populations, respectively, our findings suggest that PTH initially expands the population of more mature osteoprogenitor cells, whereas over a longer period of time this effect wanes, and more immature cells are recruited along the osteoblast pathway. Sustained increases in the more differentiated cells appear to be predictive of larger gains in trabecular bone volume, although further studies characterizing the relative osteoblastic differentiation of the OCN^small vs. OCN^gran are needed to test this hypothesis.

Disclosures: S. Kirmani, None.

1269

Effects of 2 Year Teriparatide Treatment on 3-D Femoral Neck Bone Distribution, Geometry and Bone Strength: Results from the Eurofors Studystudy.J. Borggrefe*¹, C. Graeff¹, T. N. Nickelsen², F. Marin*², J. del Pino*³, J. Kekow*⁴, R. Möricke*⁵, C. C. Glüer¹¹Med. Phys., Diagn. Radiology, UKSH, Kiel, Germany, ²Eli Lilly & Co, Bad Homburg, Germany, ³Internal Medicine, Hospital ClÃnico, Salamanca, Spain, ⁴Fachkrankenhaus Rheumatologie, Vogelsang, Germany, ⁵Endokrinolog. Praxis, Magdeburg, Germany.

A DXA-based analysis of treatment-induced changes in bone strength of the proximal femur has limitations due to the complex 3-D bone structure, the projectional imaging and limited spatial resolution. We studied teriparatide (TPTD) induced changes in bone distribution, geometry (periosteal apposition occurring?), and bone strength based on 3-D QCT of the femoral neck.

We examined 53 patients treated for 24 months with TPTD (20 μg/d) in the EUROFORS study with 3 subgroups of women, based on prior antiresorptive treatment: treatment-naive; pre-treated; and pre-treated with inadequate response. In addition to DXA femoral neck BMD (aBMD [g/cm²]), CT scans of the proximal femur were obtained at 0,6,12, and 24 months and were analyzed with Mindways' QCTPRO BIT software. The QCT neck region was defined semiautomatically by six reformatted 1mm slices positioned perpendicular to the neck axis and with a consistent position along that axis based on neck excentricity criteria. Cortical (vBMDcor [mg/cm³]) and trabecular (vBMDtra [mg/cm³]) bone density were evaluated in regions segmented by a fixed threshold. Minimum section modulus (Zl [cm³]) was calculated as measure of bending strength, buckling ratio (BR) as measure of risk of buckling (high risk if BR>10), and cross sectional area (CSA [cm²]) to evaluate geometric changes.

Strength in bending and buckling did not change in the first 12 months and improved significantly at 24 months, independent of type of pre-treatment. No significant change in periosteal bone geometry was observed. Treatment effects were more favourable in individuals at higher baseline risk: the improvement in Zl was larger at lower baseline Zl; absolute BR reduction over 2 years was −0.8 in high risk subjects (baseline BR>10) compared to −0.2 in other subjects (p<.05), resulting in a 29% reduction of subjects (16 vs 21) in the high risk category.

We conclude that 24 months of TPTD treatment improves bone strength of the proximal femur both with regard to bending and buckling, with larger protective effects in subjects at higher risk. 

Table  . Baseline data and TPTD-induced changes; *:p<0.2,*:p<.05,**:p<.01,^β:p<.001

Disclosures: J. Borggrefe. None.

This study received funding from: Eli Lilly.

1270

Effect of Vitamin-D3 and Calcium on Fracture Risk in 65–71 Year Old Women in a 3-Year Randomized Clinical Trial - Preliminary Results of the OSTPRE-Fracture Prevention Study.K. T. J. Salovaara¹, M. Tuppurainen¹, T. Rikkonen¹, M. Kärkkäinen*¹, J. Sirola*¹, R. Honkanen*¹, E. Alhava*², H. P. Kröger³¹Bone and Cartilage Research Unit, University of Kuopio, Kuopio, Finland, ²Department of Surgery, University Hospital of Kuopio, Kuopio, Finland, ³Department of Orthopedic, Trauma and Handsurgery, University Hospital of Kuopio, Kuopio, Finland.

Although Ca+Vitamin D supplementation has been shown to prevent fractures in the elderly, the effect of such a supplementation on fracture prevention in younger ambulatory postmenopausal women is still debatable.

The aim of this population level intervention was to determine whether daily Vitamin-D3 800 IU and Ca 1000 mg administered in two daily doses (Calcichew-D3 forte 500 mg/ 400IU, Leiras Nycomed Ltd. Finland) could decrease the incidence of fractures in postmenopausal women.

The women within the OSTPRE-cohort who were 65 years or older on 30.11.2002 (n = 5553) were asked regarding their willingness to participate in a randomized controlled trial with questions about medications, health disorders and health behaviour. Of the questionnaires that were adequately completed with written consent, 3432 subjects were randomized to have either Ca+Vit D supplementation (n=1718) or no supplementation (n=1714) for 3 years. Primary end points were number of falls and fractures. Fracture data was collected from the entire cohort by telephone interviews once a year and also from a subsample every 4 months. All self reported fractures were included in the analysis. Risk of fracture (HR) was calculated using Cox proportional hazards model. The study was approved by the local ethics committee.

Mean age of women was 67.4 (SD 1.8, range 65–71). At baseline (n = 3432) there were no statistically significant differences in age, weight, bone mineral density, HRT-use, hormonal status, age at menopause, smoking habits, alcohol use or functional capacity. A total of 290 fractures were reported including 144 osteoporotic fractures (vertebral n=26, hip n=10, distal forearm n=82 and humerus n=26). The incidence of distal forearm fractures was lower (1.86 %) in the intervention group compared to the control group (2.68%) (p = 0.107, chi square test). In the Cox regression model (n = 2946) the hazard ratios (HR with 95% CI) in the intervention group compared to the control group were 0.84 (0.65–1.08) for all fractures, 0.74 (0.51–1.06) for osteoporotic fractures and 0.60 (0.37–0.98) for distal forearm fractures, respectively.

In conclusion, this population level intervention study suggests that daily Vitamin-D3 and Calcium supplementation has a trend towards fracture reduction in all fractures and particularly osteoporotic fractures in postmenopausal women.

Disclosures: K.T.J. Salovaara, None.

This study received funding from: Leiras Nycomed, Finnish Academy, Kuopio University Hospital EVO-grant.

1271

Long Term Risk Factors for Incident Vertebral Fractures In Healthy Older Women After 15 Years of Follow-up: The Study of Osteoporotic Fractures.T. A. Hillier¹, J. A. Cauley², M. C. Nevitt³, J. H. Rizzo*¹, K. L. Pedula*¹, K. K. Vesco*¹, M. C. Hochberg⁴, S. R. Cummings³¹Center for Health Research, Kaiser Permanente, Portland, OR, USA, ²U Pittsburgh, Pittsburgh, PA, USA, ³CPMC Research Institute and UCSF, San Francisco, CA, USA, ⁴U Maryland, Baltimore, MD, USA.

Vertebral fractures (Vfx) are associated with morbidity and mortality in older white women, yet little is known about their long-term risk factors.

We therefore performed a comprehensive assessment of potential risk factors in 2,286 women age 65 or older without baseline Vfx enrolled in the Study of Osteoporotic Fractures. Women who returned after 15 years of follow-up represented 76% (3572/4702) of survivors, 64% (2286/3572) of whom had repeat lateral thoracic and lumbar spine films. Incident VFx were defined as ≥20% and ≥4mm decrease in vertebral height at any level. More than 50 risk factors were evaluated, including known short-term predictors of vertebral fractures and long-term predictors of non-spine fracture. Risk factors associated univariately with incident VFx (p<0.10) were further tested using backward stepwise regression to determine the final multivariable logistic regression model.

During a mean of 15 (SD 0.70) years of follow-up, 324 (14%) of women developed an incident vertebral fracture. Age, BMD, history of fracture since age 50, and falls remained signficant risk factors (p<0.05) for incident VFx (Table). DXA BMD at total hip and spine were both significantly associated with incident VFx; spine BMD had a higher point estimate and was used in the final model. Other risk factors for non-spine fracture, such as alcohol, smoking, physical activity, and estrogen or glucocorticoid use, were not independent predictors.

Table  .  

*per standard deviation decrease

Among women who survived to age 80 and above, only four factors were independent predictors of new VFx during 15 years of follow-up. Our results suggest a single BMD is highly predictive of long-term vertebral fracture risk, and that efforts to prevent vertebral fracture in healthy older women should focus on BMD and on falls.

Disclosures: T.A. Hillier, None.

This study received funding from: the National Institute of Aging (NIA) and National Institute of Arthritis, Muscoloskelelal, and Skin Diseases (N1AMS): 2R01AG027574–22AI, R01 AG005407, R0IAG027576–22, 2R01AG005394–22A1, AG05407. AG05394, AR35583, AR35582, AR35584.

1272

Is Antidepressant Use Associated with Bone Loss or Fractures in Postmenopausal Women?L. Spangler*¹, D. Scholes¹, R. Brunner*², L Robbins*³, S. Reed⁴, K. Newton*¹, J. Melville*⁴, A. LaCroix⁵¹Group Health, Seattle, WA, USA, ²University of Nevada, Reno, NV, USA, ³University of California, Sacramento, CA, USA, ⁴University of Washington, Seattle, WA, USA, ⁵Fred Hutchinson Cancer Research Center, Seattle, WA, USA.

A link between depression and bone outcomes has been postulated. The association may be the result of common physiologic systems, similar risk factors, or medication use. Literature on the association between antidepressant medication (ADM) use and BMD is limited and most research on the association with fracture relies on automated data.

We assessed these associations in 93,676 postmenopausal women (50 to 79 years old) enrolled in the WHI Observational Study, followed an average of 7.4 years. Participants brought current medications to the interviews and ADM was recorded. We further categorized ADM as selective serotonin reuptake inhibitors (SSRIs) and all other types for some analyses. To assess self-reported fractures of the hip (adjudicated), spine, wrist, and “other” skeletal sites we used Cox survival models controlling for exercise, cardiovascular disease, analgesic use, and fracture history. To assess 3-year changes in hip, spine, and whole body BMD in 6441 women enrolled at the 3 densitometry clinics we used linear regression models that included baseline BMD and hormone therapy. In addition, all models adjusted for depressive symptoms (Burnam's screen), age, menopause duration, race, physical (unction, smoking, height and weight.

At baseline, 8% of women were using ADM (4% SSRI). Overall, no significant associations were seen between ADM and 3-year changes in BMD at any skeletal site after adjustment. There was a positive association between ADM and increase risk of self-reported spine fractures (HR for SSRI =1.25 CI .96 – 2.05; HR for other ADM = 1.45 CI 1.17–1.81) and fractures at other skeletal sites (HR for SSRI = 1.32 CI 1.21 – 1.45; HR for other ADM =1.16 CI 1.06 −1.27) (Table 1). Controlling for falls had little effect on these associations. No associations were observed between ADM and hip or wrist fracture. The results from this sizeable study of generally healthy postmenopausal women provide additional evidence for an association between ADM and increased risk of fracture at some skeletal sites.

Table Table 1.. Antidepressants use (ADM) and adjusted hazard ratios for fracture

Disclosures: L. Spangler, None.

1273

Functional Decline after Incident Wrist Fracture: The Study of Osteoporotic Fractures.B. J. Edwards¹, D. Dunlop¹, J. Song*¹, H. A. Fink², J. Cauley³¹Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA, ²Medicine, Geriatric Research Education and Clinical Center, Veterans Affairs Medical Center, Minneapolis, MN, USA, ³Medicine, University of Pittsburgh, Pittsburgh, PA, USA.

Wrist fractures are common fractures that occur in post-menopausal women. Little is known about the effect of wrist fractures on functional decline. Analyses included 7,694 participants of the Study of Osteoporotic Fractures (SOF), a multicenter observational study of women aged > 65 years. Functional activity was by self-report and measured ability to perform five activities: walking, climbing stairs, preparing meals, shopping, and doing heavy housework. The items are rated on a four point scale from no difficulty (0), mild (1) and moderate difficulty (2), to unable to perform (3). We calculated global function (0–15), and targeted upper extremity function (0–9 from cooking, heavy housekeeping and shopping).

Participants who did not sustain a vertebral or hip fracture or wrist fracture prior to baseline and participated in as at least one (range 1–5) follow-up visits through 1998. (N=7,694) We track wrist fracture status at each visit to utilize longitudinal information and to include records from women who contribute function data both prior to and following an incident wrist fracture. Longitudinal multiple regression analyses with generalized estimating equations (GEE) are used to estimate annual decline in function over time.

Age-adjusted average annual rates of global functional decline were almost 3 times greater following a wrist fracture and remained significantly higher controlling for BMI, and health status (Table 1), Similarly, wrist fractures resulted in significantly greater decline in upper extremity function (Table 2)

Table Table 1.. Average Annual Decline in Global Function by Incident Wrist Fracture Status

Table  . Average Annual Decline in Upper Extremity Function by Incident Wrist Fracture Status

Women with incident wrist fractures had statistically significant functional decline independent of age, BMI and baseline health.

Disclosures BJ Edward, Merck 2; Procter and Gamble 2 8; Roche 8; GSK 8 This study received funding from; NIH

1274

Fracture Risk Increases after Cancer Diagnosis in Postmenopausal Women: Results from the Women's Health Initiative.Z. Chen¹, M. Marictc¹, A. K. Aragaki*², C. P. Mouton*³, A. M. Lopez*⁴, L. A Arendell¹, T. Bassford*¹, R. T. Chlebowski⁵¹Univ of Arizona, Tucson, AZ, USA, ²Fred Hutchinson Cancer Research Center, Seattle, WA, USA, ³Howard University, Washington, DC, USA, ⁴Arizona Cancer Center, Tucson, AZ, USA, ⁵UCLA, Los Angeles, CA, USA.

The primary aim of this study was to investigate changes in fracture and fall risk after breast cancer (BC) or other cancer (OC) diagnosis This was a prospective study in the Women's Health Initiative (WHI) cohort Participants included 145,886 postmenopausal women who had no cancer history at baseline and were enrolled in either the Observational Study (OS) or the Clinical Trials (CT) They were followed up to 9 years and classified into no-cancer, incident BC and incident OC group Hip fractures were adjudicated for both OS and CT, while the remaining fractures were only adjudicated in the CT group Falls were self-reported Hazards ratio (HR) and confidence interval (CI) were computed from Cox Proportional Hazards Models The results suggest that fracture and fall risk increases after cancer diagnosis (Table I) In comparison to women without a cancer history, hip fracture risk was significantly higher after BC diagnosis (HR =1 51 CI(1 13–2 54)) or OC diagnosis (HR = 2 03 (I 62–2 54)), but the annual incidence rates of hip fractures before a cancer diagnosis were comparable between groups after adjusted for age, hormone use, health and other lifestyle factors Risk of fall also increased with either a BC (HR= 1 16(1 08–1 25)) or OC diagnosis (HR = 1 27(1 19–1 36)) Although fall was a risk factor for fracture, the increased number of falls did not explain the elevated hip fracture risk The relative risk for vertebral, forearm or total fractures were all elevated after either BC or OC diagnosis, however, the increased risk reached statistical significant (p<0 05) only for vertebral and total fractures in the other cancer group In conclusion, postmenopausal women have a significantly elevated risk for fracture and fall after a cancer diagnosis However, the increased risk of fall is not the major contributor to this higher fracture risk observed in the incident cancer cases Future study should investigate the impact of cancer prognosis and cancer treatment on the observed increased risk of fracture and fall associated with cancer.

Table  . Age-adjusted Annualized Percentages of Fractures and Falls Before and After Cancer Diagnosis (n= 146,959)

Disclosures M Maricic None

This study received funding from NIH

1275

Impact of Adherence to Osteoporosis Medications on Fracture Rates: A Population-based Study.S. Jaglal¹, D. Thiruchelvam*², G Hawker³¹Rehabilitation Science, University of Toronto, Toronto, ON, Canada, ²Institute for Clinical Evaluative Sciences, Toronto, ON, Canada, ³Medicine, University of Toronto, Toronto, ON, Canada.

The purpose of this study was to examine if current efforts at managing osteoporosis led to a reduction in fracture in elderly men and women in Ontario, Canada. Specifically we determined the impact of differing levels of adherence to osteoporosis medications on subsequent fracture in a population-based study. Ontario residents over the age of 65 have universal access to Medicare and to certain osteoporosis medications. We identified all persons aged 67 and older who initiated an osteoporosis medication between April 1,2002 - March 31, 2004 and who did not have a drug claim for an osteoporosis medication (bisphosphonate or SERM) during the 2 years prior to initiation date (N=90,723). We excluded patients with erroneous records (prescribed 1 pill for 1 day, n=866 or adherence > 120%, n = 4,605), prescription for salmon calcitonin (n=687), history of Paget's, epilepsy, cancer, pathological fracture (n=163), discharged to nursing home (n=2,458) or died before the end of the 2 year followup period from medication initiation (n=7,859) for a final cohort of 74,085 patients. The main predictor was refill compliance calculated using the Medication Possession Ratio (MPR) defined as the no. days supplied from first to last prescription / length of followup (730 days). This was defined at 4 cut-offs, 30%, 50%, 67% and 80%. The outcome was fracture in the period after the initiation of medication. There were 1751 (2.4%) fractures. Logistic regression analyses were conducted for each refill compliance cut-off adjusted for age, sex, BMD, no. of non-osteoporosis medications, and no. of physician visits in 2 year period prior to initiation of treatment, BMD after initiation of treatment, and Charlson co-morbidity index,. Only 80% and 67% refill compliance were statistically significantly associated with fracture reduction; Odds ratio (80%) = 0.85, p = 0.046; Odds ratio (67%) = 0.85, p = 0.013. Age <75 (OR = 0.63), being female (OR = 0.54), having had a BMD prior to treatment (OR = 0.85) and one after (OR = 0.89) were also statistically significantly associated with fracture reduction. In conclusion, patients who are at least 67% adherent with their osteoporosis medications over a two-year period are less likely to fracture. Other factors associated with fracture reduction are younger age, female sex, and BMD testing. These are important findings, which confirm at a population level previous reports based on selected clinical trials patients. We now have evidence to show decision-makers that if we continue our efforts to monitor treatment and increase adherence even bigger gains in fracture reduction might be possible.

Disclosures: S. Jaglal. None.

This study received funding from: Institute for Clinical Evaluation Sciences.

1276

Clinical Low-Trauma Fractures Cluster in Time in Postmenopausal Women.P. Geusens¹, T. van Geel*², G. Dinant*²¹Internal Medicine/ Rheumatology, University Hospital Maastricht and UHasselt, Maastricht, The Netherlands, ²Department of General Practice, University Maastricht, Maastricht, The Netherlands.

Objective. Postmenopausal women with a fracture history have an increased risk for new fractures. Some studies indicate that this risk is highest immediately after a fracture. We assessed during a 10-year follow-up the time relation between a first and second clinical fracture.

Study design. Population-based study in 10 general practice centers, including 4203 postmenopausal women, aged between 50 and 80 years at baseline, who completed a questionnaire about the incidence of radiographically confirmed clinical low-trauma fractures, analysed by multiple Cox regression.

Main outcome measures. The time frame between start of menopause, a first and second low-trauma clinical fracture.

Results. 924 women sustained at least one fracture after menopause, of whom 243 had a second fracture. The fracture probability for a first fracture within 1, 5, 10 and 20 years from the menopause on was 1.0 (0.7–1.3), 3.5 (3.0–4.1), 7.9 (7.1–8.7) and 19.0% (17.6–20.3). The fracture probability for a second fracture within 1, 5,10 and 20 years following a first fracture was 6.1 (4.5–7.6), 15.9 (13.4–18.4), 27.1 (23.8–30.5) and 40.2% (35,4–45.0), respectively.

Of all first fractures, 4.3% (3.0–5.6) occurred within one year after menopause, 15.5% (13.2–17.8) within 5 years and 33.9% (30.9–36.9) within 10 years. Of all second fractures, 22.6% (17.7–27.5) occurred within one year after a first fracture, 53.9% (48.1–59.8) within 5 years and 79.8% (75.2–84.5) within 10 years.

Conclusion. In postmenopausal women, low-trauma clinical fractures cluster in time. Twenty percent of second fractures occur within 1 year after a first fracture, half within 5 years and 80% within 10 years. Prevention of a second fracture is therefore indicated soon after a first fracture has occurred.

Disclosures: P. Geusens, None.

1277

Tri-partite Relationships Between Bone, Cartilage and Adipogenic Changes Contribute to Bone Loss.N. H, Kulkarni*¹, T. Wei*¹, B. Han*¹, A. Schreiweis*¹, K. A. Brune*¹, T. A. Christopher*¹, J. A. Wolos*¹, A, Harvey*¹, D. L. Sterchi*¹, B. Gitter*¹, S. Chandrasekhar*¹, T. J. Martin², H. U. Bryant¹, Y. L. Ma¹, P. G Mitchell*¹, J. E. Hale*¹, J. E. Onyia¹¹Eli Lilly and Company, Indianapolis, IN, USA, ²SV1MR, Fitzroy, Victoria, Australia.

Ovariectomy (Ovx) or estrogen withdrawal leads to an increase in bone turnover resulting in osteopenia. A hallmark of this process includes changes in bone, cartilage and adipogenic markers. While the bone markers are well studied in osteopenia, the regulation of cartilage and adipogenic markers remain poorly understood. Here, we present a comprehensive longitudinal evaluation of the anatomical (BMD), biochemical and molecular changes in bone, cartilage and adipogenic markers in rat Ovx model. Female, 6-month old rats were Ovx or sham-operated and sacrificed at 2,4, 6, 8 and 12 weeks post-Ovx. The left femur was analyzed for BMD and the right metaphyseal femora were used for mRNA analysis of bone, cartilage and adipogenic/lipid metabolism markers. Biochemical markers of bone and cartilage turnover (PINP, CTX-I, CTX-II) and leptin, an adipogenic marker, were measured in the serum. Ovx-induced decrease in distal metaphyseal BMD was evident at 2 weeks with a steady decline over 12 weeks and was consistent with observed histological decrease in bone volume and increase in marrow adiposity. Serum PINP, CTX-1 and CTX-II robustly increased 3.5-, 1.8-, and 4.7- fold respectively in 2 weeks and returned to near sham levels by 12 weeks. The levels of serum leptin increased, most evident in 6 weeks and was sustained over time consistent with increased adiposity. The mRNA levels of 15 osteogenic markers (alkaline phosphatase, collagens α, (I), α₁,(V), osteocalcin, etc) initially increased with maxima at 6 weeks and thereafter decreased below sham levels; whereas the expression levels of osteoblast-derived resorption indicator (OPG/RANKL ratio) steadily declined reminiscent of BMD. Interestingly, a steady decrease in the expression levels of chondrocytic markers (collagen α₁, (II), (X), aggrecan, COMP, cdrap) were observed. In contrast, markers of 7 adipogenic/ lipid metabolism (LPL, FABP4, SCD1, PEPCK, leptin, etc) increased significantly and persisted with time. Overall, gene expression changes in these markers were consistent with biochemical markers and pathological changes in Ovx-induced osteopenia. Gene expression markers (bone, cartilage and adipogenic) independently showed a correlation of r²=0.55; 0.58; 0.52 respectively to BMD, whereas a combination of these markers increased the correlation to H=0.91. These results suggest that the relationship between bone, cartilage, and adipogenic changes underscore osteopenia and should together be leveraged as markers to better model the disease process.

Disclosures: N.H. Kulkarni, Eli Lilly and Company 3.

1278

Bone Mineral Density in Hip and Spine by Quantitative CT and Previous History as Predictors of Incidental Low Trauma Fractures in Elderly Men and Women.K. Siggeirsdottir*¹, T. Aspelund*¹, G Sigurdsson¹, B. Jonsson*², B. Mogensen*³, G Eiriksdottir*¹, S. Sigurdsson*¹, L. Launer*⁴, T. Harris*⁴, T. F. Lang⁵, V. Gudnason*¹¹Icelandic Heart Association, IS-201 Kopavogur, Iceland, ²University Hospital, Malmö, Sweden, ³University of Iceland, 1S-101 Reykjavik, Iceland, ⁴National Institute on Aging, Bethesda, MD, USA, ⁵University of California, San Francisco, CA, USA.

The experience with QCT in prospective population studies is limited. We have studied the predictive power of QCT of hip and lumbar spine in the AGES-REYKJAVIK Study, a cohort of 67–85 year old men (n=829) and women (n=1088) drawn from an established population based cohort. 4 detector Siemens CT system with 1 mm slices was used and for this analysis integral BMD in femoral neck and vertebral centrum in L,+L2 was used. All low trauma fractures during 3.7 years follow-up were validated by medical and radiological records, altogether 44 fractures in 36 men and 121 fractures in 104 women. Baseline information on previous fractures, falls during the last twelve months, medications, body mass index and measured grip strength were among several variables included in a Cox-proportional hazards model (see table). 

Table  .  

* p<0.05, ** p<0.01

The results show that the risk of all osteoporotic fractures almost doubled in both sexes per one standard deviation below the mean in femoral neck BMD whereas lumbar spine BMD did not add further to the prediction. Increase in age after 67 seems to be mainly a risk factor amongst men. Previous osteoporotic fractures and falling during the last twelve months were strong risk factors in both sexes whereas grip strength reached only significance in women. The area under the ROC curve (aROC) was 0.83 (0.78–0.88) for men. using the above variables, and 0.72 (0.76–0.77) for women. Using hip BMD increased the aROC curve significantly both for men (p=0.03) and women (p=0.0230). Grip strength did not improve diagnostic capacity. We conclude that volumetric BMD at the femoral neck measured by QCT is an important risk factor for all osteoporotic fractures.

Disclosures: G Sigurdsson, None.

This study received funding from: NIH NO 1-AG-1–2100.

1279

Fracture Risk Index After Falling: A Selection Strategy for Fracture Reduction Through Falls Prevention in the Frail Elderly.P. N. Sambrook¹, J.S. Chen*¹, L. M. March¹, J. M. Simpson*², M. J. Seibel³¹Institute of Bone & Joint Research, University of Sydney, Sydney, Australia, ²School of Public Health, University of Sydney, Sydney, Australia, ³ANZAC Research Institute, University of Sydney, Sydney, Australia.

This study aims to develop and evaluate a fracture risk index after falling for use in the frail elderly. We assessed risk factors at baseline and prospectively recoded falls and falls-related fractures in 2005 institutionalized older people. Each fall was treated as a study unit and a fracture as an outcome. To account for within-subject correlation between falls, the generalized estimating equations approach (specified a binomial distribution and a log link) was used to identify fracture predicators after falling. Relative risk (RR) for a risk factor was assigned as a weight. A baseline weighting of one was given if a resident did not have the risk factor. Fracture score was calculated by multiplying the weights of all independent risk factors in a person. The score is a RR compared to residents without the risk factors. The subjects (1532 females & 473 males) were older and frail, with a mean age of 87 (SD = 7.1) years and 45% living in nursing homes. Over a mean of 1.55 years, 6646 falls were reported by 1342 subjects and 308 fractures by 270 subjects, giving an incidence rate of 214 and 9.9 per 100 person years for falls and fractures respectively. The independent risk factors were lower broadband ultrasound attenuation, lower weight, longer lower leg length, better balance, intermediate-care residence, a history of fracture and lack of report of a fall in the past year. The risk score corresponded well with fracture risk. Fracture rate was 1.1 per 100 falls for 13.8% of falls with a mean score of 3.1 compared to a rate of 9.9 for 14.4% of falls with a mean score of 20.0, a multiplicative increase of 8.8. In order to identify residents who would be at high risk of both falls and fractures from baseline, the risk score was multiplied by a falls risk score calculated using the falls risk index proposed by the Fracture Risk Epidemiology in the frail Elderly (FREE) study (Chen et. al. A multivariate regression model predicted falls in residents living in intermediate hostel care. J Clin. Epidemiol. 58:503–508, 2005). The results showed that 34% of highest risk residents (i.e. highest combined score) had incidence rates of 249 and 16.2 per 100 person years for falls and fractures respectively and accounted 56% of the total fractures. The rates were 30% and 125% higher in falls and fractures respectively compared to the rest of the sample. Prevention strategies on fracture reduction through fall prevention should be directed to those at highest risk in both falls and fractures. The index could help to rationalize the provision of falls prevention programs in the frail elderly.

Disclosures: P.N. Sambrook, None.

This study received Junding from: NHMCRC of Australia.

1280

Finite Element Analyses Based on In Vivo HR-pQCT Images of the Radius Tend to Improve Wrist Fracture Prediction.S. Boutroy*¹, B. Van Rietbergen*², E. Sornav-Rendu*¹, F. Munoz*¹, M. L. Bouxsein³, P. D. Delmas¹¹INSERM Unit 831 and Université de Lyon, Lyon, France, ²Department of Biomedical Engineering, Eindhoven University of Technology, Eindhoven, The Netherlands, ³Orthopedic Biomechanics Laboratory, Beth Israel Deaconess Medical Center, Boston, MA, USA.

We recently reported that attributes of cortical (Ct) and trabecular (Tb) architecture contribute to skeletal fragility, partially independently of areal BMD (Somay-Rendu, JBMR 2007; Boutroy, JCEM 2005). Integrated assessment of bone strength by finite element analysis (FEA) may further contribute to the prediction of fracture risk. To test this, we performed FEA on in vivo HR-pQCT images of the distal radius (XtremeCT, Scanco Medical AG) in 33 women (73 ± 6 yrs) who previously sustained a fragility fracture of the wrist (fx) and 33 age-matched controls, from the OFELY cohort. We found that areal BMD; total, Tb and Ct volumetric BMD; Tb number, separation, thickness and distribution; as well as Ct thickness were significantly worse in fx cases than controls, ranging from −6% to 73%. FEA-derived stiffness, apparent modulus and estimated failure load were 15-16% lower in fx than in controls (p < 0.05). In both groups, the load was mostly carried by the cortex, from 60% (distal region) to 91 % (proximal region). Tb bone carried 9 to 22% more load in controls than in fx, implying that the load distribution was more uniform in controls. To reduce the number of variables, we conducted a principal components (PC) analysis with varimax rotation in the 66 women. The first PC included stiffness, apparent modulus, estimated failure load, areal and volumetric BMD and Ct thickness, explaining 51% of the variance. PC2 combined Tb architecture: number, separation and distribution, and explained 13% of the variance. An additional 10% of the variance was explained by the distribution of the load between Ct and Tb bone (PC3). We used univariate logistic regression to compute odds ratio (OR) for the PC: PC1=2.27 [1.16–4.44], PC2=2.22 [0.97–5.09] and PC3=2.21 [0.99–4.98]. The OR for combination of PCI and PC2 was 4.72 [1.54–14.28], but the addition of PC3 did not reach significance (p=0.27). Altogether these results indicate that FEA may provide information about skeletal fragility and fracture risk not assessed by BMD or architecture measurements alone. In particular, the load distribution between Ct and Tb bone, assessed by FEA, seems very promising for improving wrist fracture prediction independently of BMD and microarchitecture. These data provide strong rationale for additional studies testing the ability of FEA based on HR-pQCT to predict fracture risk.

Disclosures: S. Boutroy. None.

1281

Height Loss Predicts Fractures in Middle Aged and Older Men and Women: European Prospective Investigation Into Cancer-Norfolk Population Cohort Study.A. Moayyeri*¹, A. Welch*¹, R. N. Luben*¹, N. J. Wareham*², S. Bingham*³, K. T. Khaw*¹¹Department of Public Health and Primary Care, Institute of Public Health, School of Clinical Medicine, The University of Cambridge, Cambridge, United Kingdom, ²MRC Epidemiology Unit, The University of Cambridge, Cambridge, United Kingdom, ³MRC Dunn Human Nutrition Unit, The University of Cambridge, Cambridge, United Kingdom.

With effective interventions available for fracture prevention, a major clinical issue is identification of individuals at greatest fracture risk for preventive interventions. Height loss among middle aged men and women can be easily measured in outpatient clinics and may contribute to fracture risk prediction. We aimed to assess measured height loss and fracture incidence in a prospective observational population study. Height was measured in men and women in the Norfolk cohort of the European Prospective Investigation into Cancer (EPIC-Norfolk) between 1993 and 1997 and was repeated between 1997 and 2000. Incident fractures up to 2006 were ascertained by hospital record linkage. Height loss and known risk factors of fracture were entered into Cox proportional hazards models to determine their independent contribution to the risk of fracture. In 14,921 men and women aged 42–82 years, during follow-up period of 7.1 (SD 0.7) years, there were 390 fractures, including 122 hip fractures. Annual height loss in the group of fracture sufferers (1.8 mm, SD 0.3) was significantly greater than other participants (0.9 mm, SD 0.2; p<0.001). Participants with annual height loss >0.5 cm had an age and sex-adjusted hazard ratio of any fracture of 1.76 (95%CI 1.16–2.67) and of hip fracture of 2.08 (95%CI 1.07–4.05) compared to those with no height loss. In multivariate models, 1 cm of height loss per year was associated with a hazard ratio of 1.86 (95% CI 1.28–2.72) for all fractures and 2.24 (95%CI 1.23–4.09) for hip fracture independent of age, sex, past history of fracture, smoking, body mass index, alcohol intake, and heel ultrasound measures. Annual height loss of 1 cm was comparable to having a past history of fracture and equivalent to being about 14 years older in chronological age in terms of magnitude of relationship with fracture risk. Middle aged and older men and women with annual height loss >0.5 cm are at increased risk of hip and total fracture. Serial height measurements can easily be incorporated in routine clinical practice and may contribute to the development of practical fracture risk assessment tools.

Disclosures: A. Moayyeri, None.

This study received funding from: Medical Research Council and Cancer Research UK.

1282

Hip Muscle Cross-Sectional Area and Attenuation: Association with Hip Fracture.T. F. Lang¹, X. Cheng*², C. Li*¹, A. Koyama*¹, T. Harris*³, Y. Lu*¹. ¹Radiology, University of California, San Francisco, San Francisco, CA, USA, ²Radiology, Beijing Ji Shui Tan Hospital, Beijing, China, ³Epidemiology and Biometry, National Institute on Aging, Bethesda, MD, USA.

Purpose: Falling is an important risk factor for hip fracture, and the strength of the hip musculature is crucial for recovery of stability after loss of balance. We hypothesized that the size (cross-sectional area (CSA) of hip muscle from hip QCT) and adipose infiltration of muscle (defined by CT x-ray attenuation of the hip muscles), acting as surrogate measures for hip muscle function, would correlate with hip fracture independently of age and body size.

Methods: To test the hypothesis, we compared CT scans of the contralateral hip in 45 women (mean age 74.71±5.94) imaged within 48 hours of their hip fractures to 66 age-matched control subjects (mean age 70.70±4.66). A volumetric CT scan encompassing the proximal part of the femur was analyzed for total percentage of fat tissue in the in the CT field of view and for the CSA and lean tissue Hounsfield Unit (HU) values of the hip extensor, abductor, adductor and flexor muscles. The CT scan was also analyzed to compute areal bone mineral density (aBMD) of the hip bone. Logistic regression analyses were employed to determine differences between fracture subjects and controls in body composition measures after adjustment for age, height, and BMI. Receiver-operator curve (ROC) analyses were employed to determine if combinations of aBMD and body composition were better discriminators of hip fracture than aBMD alone.

Results: Subjects with hip fractures were on average 5 years older (p<0.001) had 8% lower BMI (p<0.05), 8% lower percentage fat (p<0.0001) and roughly 20% lower extensor and adductor CSA values than controls (p<0.0001). Despite having lower total adiposity, they had higher muscle adiposity as indicated by lower lean tissue attenuation in all four muscle groups, with statistically significant differences in the adductor, abductor and flexor groups (0.0000Kp<0.02). After adjustment for age and BMI, the fracture subjects had lower total % fat, smaller extensor and adductor CSA values, and greater adipose infiltration (lower attenuation) in the adductor (0.00l<p<0.05). ROC analyses showed that models combining percent fat, muscle CSA and muscle attenuation values with aBMD had higher AUC values than models containing only aBMD (0.97 vs 0.88, pO.OOl).

Conclusions: Reduced total adiposity and increased hip muscle adiposity appear to be independent correlates of hip fracture, and combination of skeletal and body composition variables appears to enhance diagnostic efficacy compared to skeletal variables alone.

Disclosures: T.F. Lang, None.

This study received funding from: Eli Lilly.

1283

Deletion of Subunit bl in Osteoblast Reveals Novel Role for Calcineurin in Bone Formation.A. R. Venn_*, H. Yeo*, J. M. McDonald, T. L. Clemens, M. Zavzafoon. Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, USA.

We recently reported that the pharmacological inhibition of calcineurin (Cn) by low concentrations of Cyclosporin A increases osteoblast differentiation in vitro and bone mass in vivo. To determine whether Cn exerts direct actions in osteoblasts, we generated mice lacking Cnb1 (Cn regulatory subunit) in osteoblast (ΔCnb1^OB) using Cre-mediated recombination methods. Transgenic mice expressing Cre-recombinase, driven by human osteocalcin promoter, were crossed with homozygous mice that express loxP-flanked-Cnb1 (Cnb1^f7f). Micro-CT analysis of tibiae at three months showed that ΔCnb1^OB mice had dramatic increases in bone mass compared to controls. Histomorphometric analysis showed significant increases in mineral apposition rate (67%), bone volume (32%), trabecular thickness (29%) and osteoblast numbers (68%), as well as 40% decrease in osteoclast numbers as compared to the values from control mice. To delete Cnb1 in vitro, primary calvarial osteoblasts, harvested from Cnb1^f7f mice, were infected with adenovirus expressing the Cre-recombinase. Cre-expressing osteoblast had a complete inhibition of Cnb1 protein levels, but differentiated and mineralized more rapidly than control, GFP-expressing cells. Deletion of Cnb1 increased expression of osteoprotegrerin and decreased expression of RANKL. Co-culturing Cnb1-deficient osteoblast with wild type osteoclast demonstrated that osteoblast lacking Cnb1 failed to support osteoclast differentiation in vitro. Taken together, our findings demonstrate that the inhibition of Cnb1 in osteoblasts increases bone mass by directly increasing osteoblast differentiation and indirectly decreasing osteoclastogenesis.

Disclosures: A.R. Venn, None.

This study received funding from: NIH Grant P30-AR46031 and NIH grant P0I-CA098912.

1284

Conditional Knockouts in Early and Mature Osteoblasts Reveal a Critical Role for Ca²⁺ Receptors in Bone Development.W. Chang¹, C. Tu*¹, T. Chen*¹, B. Liu*¹, H. Elalieh*¹, M. Dvorak¹, T. Clemens², B. Kream³, B, Halloran¹, D. Bikle¹, D. Shoback¹¹Endocrine Research Unit, VAMC, University of California San Francisco, San Francisco, CA, USA, ²University of Alabama, Birmingham, AL, USA, ³University of Connecticut Health Center, Farmington, CT, USA.

Ca¹⁺ receptors (CaRs), which detect changes in extracellular [Ca²⁺] ([Ca²⁺]_e) in parathyroid and kidney cells, are also expressed in osteoblasts (OBs) during differentiation. In vitro studies support a role for CaRs in mediating high [Ca²⁺]_e-induced changes in proliferation, chemotaxis, and gene expression in OBs, but their impact on skeletal development in vivo is unclear. We generated mice with conditional knockout of CaRs in early committed or mature OBs by breeding a novel mouse line carrying floxed-CaR (CaR^flox/flox) gene alleles with transgenic mice expressing Cre recombinase (Cre) under the control of 2.3 kb type 1 collagen (2.3Coll-Cre) or 3.5 kb human osteocalcin (OC) promoters (OC-Cre), respectively. Resulting 2.3Coll-Cre/CaR^flox/flox and OC-Cre/CaR^flox/flox mice were born in normal Mendelian ratios with the 2.3Coll-Cre/CaR^flox/flox mice demonstrating severe growth retardation, clearly visible by 3 days of age. At 21 days of age, weights of 2.3Coll-Cre/CaR^flox/flox mice were =30% of their wild-type (wt) littermates. They had smaller, undermineralized skeletons by whole-mount alcian blue and alizarin red staining with significant reductions in bone volume (BV) and bone mineral density (BMD) in femur and vertebrae by micro-computed tomography (μCT). Goldner and von Kossa staining revealed excessive amounts of unmineralized osteoid in both metaphyses and diaphyses of the long bones from 2.3Coll-Cre/CaR^flox/flox vs wt mice. Defective mineralization was evident in newborns and persisted postnatally in 2.3Coll-Cre/CaR^flox/flox mice suggesting that CaRs are essential for both prenatal and postnatal bone development. Quantitative real-time PCR demonstrated significant reductions in OC RNA level (by =50–65%) and increases in RTJNX2 and RANKL expression (by = 50%) in calvariae and humeri from 2.3Coll-Cre/CaR^flox/flox vs wt mice without changes in RNA expression of Coll and osteopontin, supporting a role for CaRs in OB maturation. Width of the hypertrophic zone in growth plate was reduced in 2.3Coll-Cre/CaR^flox/flox mice, indicating delayed growth plate development which could contribute to their growth retardation. In contrast, growth curves of OC-Cre/CaR^flox/flox mice were comparable to wt littermates. Analyses of bone micro-architecture by uCT revealed no significant changes in BV, BMD, and structure parameters in the OC-Cre/CaR^flox/flox mice. These studies confirm a critical role for CaRs at early stages of bone development potentially by promoting OB maturation.

Disclosures: W. Chang, None.

1285

Inadequate Bone Adaptation in Bone Cell Specific Connexin43 Deficient Mice.Y. Zhang*, A. F. Taylor*, E. M. Paul*, A. Davison*, S. K. Bronson*, H, J. Donahue.. Penn State University, College of Medicine, Hershey, PA, USA.

Previous in vitro studies suggest that gap junction (GJ) deficiency affects bone metabolism possibly resulting in osteopenia. We examined the hypothesis that connexin 43 (Cx43), the predominate Cx in bone, deficiency in murine osteoblasts and osteocytes results in osteopenia. Osteoblast/osteocyte specific Cx43 knockout (KO) mice were generated by breeding Cx43 null mice (Cx43^+/−) with mice expressing Cre recombinase under the control of the human osteocalcin promoter (OC-Cre) and with mice in which the Cx43 gene is flanked by two loxP sites (Cx43^fl/fl). Our breeding strategy resulted in littermates with Cx43 deleted from osteoblasts (conditional knockout, KO, Cx43^fl/−;OC-Cre), a single functional allele of Cx43 in osteoblasts(heterozygous equivalent, HET, Cx43^fl/+;OC-Cre), or with two functional alleles in all cell types (wild type equivalent, WT, CX431^fl/+). All mice examined were 3 weeks old. GJ intercellular communication (GJIC) was dramatically reduced in osteoblastic cells isolated from KO relative to HET or WT, suggesting that we had indeed deleted Cx43 expression in osteoblastic cells. MicroCT of humeri and femora revealed that bones from KO mice were osteopenic, relative to WT or HET mice. KO displayed periosteal expansion, cortical thinning, increased porosity and an approximately 8% and 26% reduction in bone mineral density and bone volume over total volume, respectively. More importantly, three point bending revealed that the failure stress of femora from KO mice was reduced approximately 41% and 27% relative to WT and HT, respectively. Our results demonstrate successful conditional deletion of the Cx43 gene in osteoblasts and osteocytes and confirm previous in vitro data suggesting that Cx43 is largely responsible for GJIC between osteoblastic cells. More importantly, microCT and mechanical testing revealed osteopenia in osteoblast/osteocyte specific Cx43 deficient mice. The exact mechanism by which Cx43 deficiency results in osteopenia was not investigated in this study. However, the remarkable similarity between bones from KO mice and those from mice experiencing disuse osteopenia, together with our previous in vitro data demonstrating that GJIC contributes to bone cell response to load induced biophysical signals, suggests that bones from KO mice were unable to properly adapt to loads engendered during normal ambulation, which, as confirmed by behavioral studies, all genotypes examined were experiencing. In any case, these in vivo data confirm in vitro data suggesting Cx43 mediated GJIC is critical for normal bone adaptation and emphasize the physiological relevance of GJs in bone.

Disclosures: Y. Zhang, None.

This study received funding from: NIH.

1286

Low Bone Mass in Mice with Null Mutation in TG-interacting Factor (TGIF): Impaired Osteoblast Function.M. K. Crook¹, K. S. Mohammad¹, G. A. Clines¹, L. Bartholin*², T. Melhuish*², C. R. McKenna*¹, M. Niewolna¹, L. J. Suva³, A. Carver³, D. Wotton*², T. A. Guise¹¹Department of Internal Medicine, University of Virginia, Charlottesville, VA, USA, ²Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA, USA, ³University of Arkansas for Medical Sciences, Little Rock, AR, USA.

TGIF (TG interacting factor) is a transcriptional corepressor of both TGFβ and retinoic acid signaling. Mutations in the TGIF gene can lead to holoprosencephaly, a severe defect in craniofacial development. TGIF-null mice had developmental and skeletal abnormalities including growth retardation, domed skulls, twisted noses, and defects in vertebrae, ribs and sternum. Loss of TGIF may enhance responses to TGFβ and retinoic acid, both of which can adversely affect bone. Therefore, we studied bone remodeling in TGIF-/- and wild-type (WT) littermates in the C57BL/6J mice. Skeletal assessment revealed that the TGIF-/- mice had significantly less trabecular bone by DXA (PIXImus), microCT and histomorphometry. Low bone mass was associated with increased osteoclast activity, reduced osteoblast number and bone formation rates. Primary osteoblasts from TGIF-/-compared to WT mice had reduced expression of two late markers, osteocalcin and bone sialoprotein, and increased expression of the early marker Coll A, assessed by quantitative real-time PCR, suggesting that complete osteoblast differentiation was impaired in TGIF-/-mice. To evaluate if the low bone mass seen in TGIF -/- mice was due to enhanced TGFβ signaling, we treated TGIF -/- and WT mice for 10 weeks with a small molecule inhibitor of the TGFβ receptor I kinase, which significantly reduces TGFβ1-dependent phosphorylation of Smad proteins and increase bone mass in normal mice. As expected, inhibitor treatment versus vehicle significantly increased bone mineral density (BMD) in WT mice at total body (p=0.0005), tibia (p<0.0001), femur (p=0.0046) and spine (p=0.0005). In contrast, BMD was not increased at any site in the TGIF -/- mice treated with inhibitor versus vehicle. There was no difference in phosphoSmad2 staining in the bones of TGIF-/- compared to WT mice.

The data suggest that reduced bone mass in TGIF-/- mice is due to impaired osteoblast differentiation and increased osteoclast activity. We hypothesized that the effects were due to enhanced activity of TGFβ and/or retinoic acid due to loss of TGIF repression. However, we found no evidence of increased TGFβ signaling, nor were the bone abnormalities reversed by a TGFβ receptor kinase inhibitor. The mechanism responsible for the abnormal bone remodeling requires further elucidation and may involve increased retinoic acid signaling.

Disclosures: M.K. Crook, None.

1287

Skeletal Phenotype in Transgenic Mice Over-Expressing CTGF in Cells of the Osteoblast Lineage.J. A. Arnott, E. Nuglozeh*, K. B. Buck*, F. F. Safadi, S. N. Popoff. Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, PA, USA.

CTGF has recently emerged as an important growth factor in osteogenesis, demonstrated by its ability to promote proliferation, matrix production and differentiation in cultures of osteoblasts. Since most of the data concerning the role of CTGF in osteogenesis has come from in vitro studies, in this study we generated transgenic mice in which CTGF is over-expressed under control of the truncated 3.6kb collagen type I (pOBCol3.6) promoter (CTGF p^OBCol3.6 mice). This promoter was chosen because it is expressed early during osteoblast differentiation. The targeting vector used to generate transgenic mice also contained LacZ (to identify cells expressing the transgene) and an enhancer element to boost CTGF expression. The presence of the transgene was determined by PCR of tail DNA using transgene specific primers. Six lines were established by mating founder mice with C57/Blk6 wild type (WT) mice. Multiple tissues were used to examine specificity of transgene expression using PCR with transgene specific primers, followed by confirmation of CTGF mRNA expression levels by Northern blot analysis. Transgene expression was highest in long bone and calvaria, with lower levels of expression in other type I collagen producing tissues (lung and skin). Two of the transgenic lines with different CTGF expression levels were used for analysis of the skeletal phenotype. Mice from one line survive, however, mice from the other line die within a few days after birth. Line one showed a 3–4 fold (moderate expression) increase and line two showed a >7–8 fold (high expression) increase in CTGF protein levels in bone when compared to age matched WT mice. Histological and morphometric examination of the distal femoral metaphysis from TG mice with moderate over-expression of CTGF exhibited significant increases in trabecular bone volume associated with increased osteoid thickness and osteoblast activity/numbers compared to WT mice. Increased thickness of the periosteum with increased numbers of osteoprogenitor cells was also observed in TG compared to WT bone. Primary cultures of osteoblasts derived from these TG mice also exhibited enhanced differentiation (ALP staining and mineralization) compared to WT cultures. Surprisingly, examination of bones from transgenic mice over-expressing CTGF at very high levels demonstrated an increase in osteoclast number and size. These data suggest that the precise effects of CTGF on bone cell differentiation and function depend on the magnitude of CTGF over-expression. Moderate levels of CTGF have a direct effect on osteoblasts to promote bone formation, while high levels favor the formation of osteoclasts, perhaps indirectly through a RANK-L dependent mechanism.

Disclosures: S.N. Popoff, None.

1288

The Human Type la2 Procollagen Nuclear Targeting Sequence Together with Human Runx2 Enhancer Elements Drives Robust Expression in Transgenic Mouse Osteoblasts.D. D. Strong*, C. Strivers*, K. Howard*, Si Kleinman*, T. A. Linkhart. MDC, J.L. Pettis Memorial VA Med Ctr., Loma Linda, CA, USA.

Translocation of plasmid DNA into the nucleus is an important rate-limiting step affecting transgene expression levels in non-dividing cells. Inclusion of a SV40 viral DNA nuclear targeting sequence (DTS) in plasmid vectors enhances nuclear import efficiency and increases transgene delivery and expression in vitro and in vivo. Plasmid nuclear import involves cellular transcription factors that bind in the cytoplasm to viral DTSs and translocate the DNA by a nuclear localization signal/importin dependent process. We identified a sequence in the human type 1 alpha 2 procollagen promoter (hColl a2) (-312 to +45) that stimulated osteoblast (Ob) specific nuclear entry of plasmid DNA but not into the nuclei of fibroblasts or chondrocytes. We used this hColla2-based nuclear import sequence together with an enhancer sequence derived from the human Runx2 promoter to increase Ob-specific promoter activity and retain nuclear entry activity. We found that the hRunx2-hColla2 promoter linked to the luciferase reporter (pGL3Basic vector) demonstrated three-fold more activity than the hColla2 promoter alone in transient transfection assays of Obs. To test the specificity and strength of expression of the hColla2 and hRunx2-hColla2 promoters in vivo we took advantage of Cre-loxP technology and created transgenic (Tg) mouse lines to characterize expression patterns of the promoters in the postnatal skeleton. Specifically the Cre recombinase (Cre) transgene was linked to either the hColla2 or the hRunx-2-hColla2 promoter in the pTurbo-Cre vector (T. Ley) that expresses Cre fused to a nuclear localization signal sequence to facilitate recombination. Multiple lines of Tg mice were prepared and Cre expression was assessed by crossing Cre Tg mice with Rosa26LacZ (R26R) reporter mice to allow expression of beta-galactosidase following Cre activation. Using these mice, we found Cre activity in distinct cell populations in skeletal tissues, including Obs lining the bone surface and in osteocytes. The Runx2-hColla2 promoter expressed higher levels of Cre than the hColla2 promoter and Cre expression levels and patterns were comparable to mCollal-Cre Tg animals (from Dr. Karsenty) crossed to R26R animals and analyzed in parallel. The Tg lines showed that the Ob-specific nuclear entry sequence (± hRunx2 enhancer) was also a strong promoter in Ob lineage cells. The hColla2 promoter not only contains a novel Ob-specific DTS, useful for development of nonviral expression vectors to increase transgene expression in Obs but also acts as a strong promoter in vivo to drive Ob-specific transgene expression in skeletal tissues.

Disclosures: D.D. Strong, None.

1289

Tec Tyrosine Kinases Are Essential For Osteoclast Differentiation.M. Shinohara*, T. Koga*, K. Okamoto*, H, Takayanagi. Cell Signaling, Tokyo Medical and Dental University, Tokyo, Japan.

Osteoclasts are multinucleated cells that originate from bone marrow-derived monocyte/macrophage precursor cells (BMMs). The differentiation of BMMs to osteoclasts is mainly regulated by three signaling pathways activated by receptor activator of nuclear factor χB ligand (RANKL), macrophage colony-stimulating factor (M-CSF) and immunoreceptor tyrosine-based activation motif (ITAM). RANKL and its costimulatory receptors of the immunoglobulin superfamily cooperatively stimulate the tyrosine phosphorylation of ITAM in DNAX-activating protein 12 (DAP12) and Fc receptor common γ subunit (FcRγ). However it has been unclear how RANKL contributes to the activation of the tyrosine kinase cascade downstream of ITAM. In this study, a genome-wide screening for non-receptor tyrosine kinases revealed selective expression of Tec and Btk, which are essential for B cell development. Notably, Tec^−/−Btk^−/− mice exhibited an osteopetrotic phenotype due to severe impairment of osteoclast differentiation. In vitro analysis demonstrated that Tec and Btk are essentially required for the osteoclast differentiation in a cell autonomous manner. Tec kinases are activated by RANKL stimulation and recruited to a lipid raft to form an osteoclastogenic complex with RANK, adaptor proteins such as BLNK and ITAM-harboring adaptors, which mediates the activation of PLCγy-calcium signaling-NFATc 1 pathway. Furthermore, TeC^−/−Btk^−/− mice are resistant to ovariectomy-induced bone loss. Thus, this study identifies the importance of Tec kinases in the osteoclast differentiation and may provide a molecular basis for a novel therapeutic strategy for bone diseases.

Disclosures: M. Shinohara, None.

1290

Regulation of Skeletal Homeostasis by Anti-apoptotic Molecule Bcl-2.Y. Nagase¹, M. Iwasawa¹, T. Akiyama¹, Y. Kadono¹, T. Miyamoto², Y. Oshima¹, M. Nakamura¹T. Yasui¹, K. Nakamura¹, S. Tanaka¹¹Orthopaedic Surgery, University of Tokyo, Tokyo, Japan, ²Orthopaedic Surgery, Cell differentiation, Keio University School of Medicine, Tokyo, Japan.

Anti-apoptotic molecule Bcl-2 resides on mitochondrial outer membrane, and inhibits apoptosis by suppressing cytochrome c release from mitochondria. Although several studies have uncovered the importance of Bcl-2 in maintaining skeletal integrity, detailed cellular and molecular mechanisms still remain elusive. We investigated the role of Bcl-2 on bone metabolism by analyzing bcl-2-deficient mice. Bcl-2 -/- (KO) mice were dwarf at 3 weeks of age compared to wild type (WT) littermates, and radiographic analysis of long bones showed a sclerotic lesion at the metaphysis and an atrophic change at the diaphysis. The histomorphometric study revealed that KO mice showed an increase in primary spongiosa due to a decrease in the number of osteoclasts and eroded surface. Many KO osteoclasts were found unattached to primary spongiosa, whereas WT osteoclasts were largely juxtaposed to bone in vivo. The proportion of osteoclast precursors in bone marrow cells which were positive for c-Fms, Macl and c-Kit was not different between KO mice and WT littermates as determined by flow cytometry. However, NFATcl induction in response to RANKL stimulation was impaired in M-CSF-dependent osteoclast precursors obtained from KO mice, which led to a delayed differentiation into mature osteoclasts. KO osteoclasts exhibited an increased cell death and higher Caspase-3 activity after cytokine withdrawal, and their bone-resorbing activity was impaired as determined by pit formation assay as compared to WT osteoclasts in spite of their normal actin ring formation. These abnormalities were restored by retroviral introduction of Bcl-2. In addition to such cell-autonomous defect in bone resorption, bone formation of KO mice was also impaired, which caused an atrophic bone lesion at the diaphysis and reduced calcein double labeling. KO mice exhibited a decrease in osteoblast surface/bone surface, mineral apposition rate and bone formation rate by histomorphometric analysis. KO osteoblasts exhibited an increased cell death, and expression of osteoblastic markers such as type I collagen, alkaline phosphatase and osteocalcin was reduced in primary osteoblasts obtained from KO mouse calvaria, and their mineralization was attenuated. Serum deprivation induced Caspase-3 activation in KO osteoblasts at higher levels, which indicated an increased susceptibility to apoptosis. Collectively, Bcl-2 promoted the differentiation, activity and survival of both osteoclasts and osteoblasts, and plays an essential role in maintaining skeletal homeostasis.

Disclosures: Y Nagase, None.

1291

Distinct Roles of ITAM Signaling in Bone Remodeling in Various Bony Microenvironments.Y. Wu¹, J. Torchia*², W. Yao³, N. E. Lane³, L. L. Lanier¹, M. C. Nakamura², M. B. Humphrey⁴¹Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA, ²Medicine, VA Medical Center, San Francisco, CA, USA, ³Medicine, University of California, Davis, Sacramento, CA, USA, ⁴Medicine, University of Oklahoma Health Science Center, Oklahoma City, OK, USA.

Immunoreceptor tyrosine-based activation motif (ITAM) signaling mediated by DAP12 or Fcε receptor lγ chain (FcRγ) has been shown to be critical for osteoclast differentiation and maturation under normal physiological conditions. Their function in pathological conditions is unknown.

We studied the role of ITAM signaling during rapid bone remodeling induced by ovariectomy (OVX) in wild-type (WT), DAP 12-/-, FcRγ-/- and DAP12-/-FcRγ-/- mice. Six weeks after OVX, DAP12-/-FcRγ-/- mice showed resistance to lumbar vertebral body (LVB) trabecular bone loss, while WT, DAP12-/- and FcRγ-/- had significant LVB bone loss. In contrast, all ITAM-deficient mice responded to OVX with bone loss in both femur and tibia of approximately 40%, relative to basal bone volumes (Table 1). DAP12-/-FcRγ-/- mice showed the greatest absolute loss of trabecular bone after OVX, losing more than twice as much bone volume as WT, DAP 12-/- and FcRγ-/- (Table 1). Only WT mice developed significant cortical bone loss after OVX. In vitro studies showed microenvironmental changes induced by OVX are indispensable for enhanced osteoclast formation and function. Cytokine changes, including TGFp″ and TNFa, were able to induce osteoclastogenesis independent of RANKL in bone marrow-derived macrophages (BMMs) from WT but not DAP 12-/- and DAP12-/-FcRγ-/- mice. Follicle-stimulating hormone (FSH) stimulated RANKL induced osteoclast differentiation from BMMs in WT, but not DAP 12-/- and DAP12-/-FcRγ-/- mice.

Our study demonstrates that whereas ITAM signaling is critical for normal bone remodeling, estrogen deficiency induces an ITAM-independent bypass mechanism allowing for enhanced osteoclastogenesis and activation in specific bony microenvironments.

Table Table 1.. Bone loss in distal femur, six weeks after OVX, n=5 in each group.

Disclosures: Y. Wu. None.

This study received funding from: NIH. VAMC.

1292

Osteoclast Specific Ablation of Dicer Suppresses Bone Resorption and increases Bone Mass.F. Mizoguchi¹, T. Nakamura*², T. Havata¹, H. Hemmi*³, Y. Ezura¹, K. Nakashima*¹N. Miyasaka*⁴, B. Harfe*⁵, S. Kato*², M. Noda¹¹Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, 21st Century COE Program, Tokyo, Japan, ²Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo, Japan, ³MTT Program, Tokyo Medical and Dental University, Tokyo, Japan, ⁴department of Medicine and Rheumatology, Tokyo Medical and Dental University, Tokyo, Japan, ⁵Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, FL, USA.

Micro RNAs (miRNA) are endogenous non-coding RNAs of ∼22 nucleotides in length. MiRNAs regulate the expression of genes by binding 3*-UTR regions of the targets, and have been reported to be involved in fine tuning of biological events, such as cell proliferation, differentiation and cell death in both physiological and pathological conditions. Osteoclastogenesis is a complex process where multiple transcription factors and cytokines are involved. However, the role of miRNAs in osteoclastogenesis is totally unknown. It was reported that simple loss-of-function approaches will be less fruitful for studying miRNA function due to their high level of possible redundancy (B.Harfe. PNAS 2005). RNaselll enzyme Dicer is one of the key molecules that process pre-miRNAs to mature miRNAs while dicer knock out mice are embryonic lethal. To examine the role of miRNAs in osteoclastogenesis, we specifically knocked out dicer in these cells by crossing conditional allele (Dicer flox) with Cathepsin K-Cre knock-in allele. Mutant mice survived normally during perinatal period. However, during the growth period, body weight gain was less and body size was smaller by about 20 % than control Cre-negative littermates at the age of 7 weeks. Bone mineral density (BMD) levels at the distal 1/3 of the femora in mutant mice were increased by about 20 % than control littermates. In contrast, BMD levels of whole femora were similar between mutant mice and control mice. Thus, the effects of dicer KO appears to be more in cancellous bone. In fact, 3-dimensional micro CT analyses revealed that dicer deficiency in osteoclasts increased BV/TV levels by about 20% compared to control littermates. Further analyses revealed that 3D-thickness of the trabecular bone was specifically increased in mutant mice, while the levels in the number of trabeculi in mutant mice were similar to those in control mice. Histological analysis revealed that TRAP positive cells in trabecular bone (OcS/BS, N.Oc/BS) were decreased in mutant mice by about 30% compared to control littermates. These results indicated that dicer is required for the maintenance of osteoclastic activities in bone. Thus, miRNAs play a regulatory role in these cells to determine the levels of bone resorption.

Disclosures: F. Mizoguchi, None.

1293

Rab3D Recruits the Dynein Complex to Secretory Vesicles in Osteoclasts Through Direct Interaction with Tetex-1.N. J. Pavlos¹, J. Xu¹, A. Carrello*¹, R. Jahn*², M. H, Zheng¹¹Department of Orthopaedic Surgery, The University of Western Australia, Perth, Australia, ²department of Neurobiology, The Max Planck Institute for Biophysical Chemistry, Goettingen, Germany.

Targeted intracellular trafficking of osteolytic enzymes and degraded bone matrix substrates to and from the ruffled border of bone-resorbing osteoclasts requires coordinated interplay between carrier vesicles, motor proteins and the cytoskeletal network. We have previously shown that Rab3D, an exocytic-related small GTPase, regulates a post-TGN vesicle transport step that is required for the maintenance of the ruffled border membrane and bone resorption (1). Here, to investigate the complement of effectors through which Rab3D elicits its biological function, we have employed a yeast two-hybrid system to screen for candidate Rab3D-interacting proteins. Using the N-terminal of Rab3D as “bait” we identified Tetex-1, a 14 kDa light chain of the cytoplasmic dynein motor complex, as a putative effector molecule. Specific interaction between Rab3D and Tetex-1 was confirmed by GST-pull-down, immunoprecipitation and colocalization studies. Furthermore, yeast two-hybrid analysis demonstrated that Tetex-1 specifically associates with Rab3 family members via an interaction with the Switch II/GTP-binding motif. Consistently, bioluminescence resonance energy transfer (BRET) analyses demonstrated that Tetex-1 preferentially binds to the active (GTP-bound) forms of Rab3 GTPases in vivo. Moreover, overexpression of wild-type and constitutively active Rab3D led to the recruitment of Tetex-1 and other subunits of the dynein/dynactin complex to secretory vesicle membranes. Disruption of cytoplasmic dynein by dynamitin overexpression redistributed Golgi-associated Rab3D to peripheral vesicles. Similarly, depolymerization of microtubules with nocodazole altered the spatial distribution Rab3D-bearing vesicles and impaired Rab3D-Tectex-1 interaction in vivo. These data demonstrate that Tetex-1 is a bona fide Rab3 binding protein and that Tetex-1 may cooperate with Rab3D to recruit the dynein motor complex to membrane micro-domains, thereby regulating the sorting and microtubule-dependent targeting/recycling of post-Golgi secretory vesicles to/from the ruffled border during osteoclast-mediated bone resorption.

1. Pavlos, N.J., Xu, J, Riedel, D., Yeoh, J.S.G, Teitelbaum, S.L., Papadimitriou, J.M., Jahn, R. Ross, F.P., Zheng, M.H. (2005) Mol. Cell. Biol 25, 5253–5269

Disclosures: N.J. Pavlos, None.

1294

Mice with a Myeloid Lineage-Specific Deletion of IKKβ Demonstrate Both In Vivo and In Vitro Defects in Osteoclastogenesis.J. E. Otero*¹, M. Al-hawagri*¹, S. Dai*¹, J. Vacher*², M. Pasparakis*³, Y. Abu-Amer¹. ¹Orthopedic Surgery and Cell Biology & Physiology, Washington University School of Medicine, Saint Louis, MO, USA, ²Institut de Recherches Cliniques de Montreal, Montreal, PQ, Canada, ³Molecular Biology, EMBL, Monterotondo, Italy.

Nuclear Factor kappa B (NF-kB) is a family of transcription factors that regulates various cellular and physiological processes such as inflammatory signaling, immune system development, and cell survival. NF-kB activity is controlled by two upstream kinases, IKKα and IKKβ. NF-kB is also essential for osteoclast development and survival, evidenced by the osteopetrotic phenotype displayed by mice doubly deficient in subunits NF-kB1 and NF-kB2. The significance of upstream regulation of NF-kB in the context of osteoclastogenesis in vivo has been unclear given the embryonic and early postnatal lethal phenotypes of mice deficient in IKKβ and IKKα, respectively. Furthermore, the only published report of a conditional deletion of IKKβ relevant to osteoclastogenesis utilizes an inducible system, in which specificity of deletion is uncertain. We report for the first time that a myeloid lineage-specific deletion of IKKβ using the Cre-loxP system renders mice defective in both in vivo and in vitro osteoclastogenesis. Cre recombinase driven by the promoter for myeloid-specific marker, CD11b, deletes loxP-flanked IKKβ in bone marrow macrophages to a degree sufficient to drastically reduce the IKKβ mRNA level compared to that observed in CD11b Cre-negative, loxP-flanked IKKβ controls. Furthermore, IKKβ protein levels are undetectable in both bone marrow and spleen osteoclast precursors from CD11β Cre-positive, loxP-flanked IKKβ mice. The defect in osteoclastogenesis appears to be due to developmental and survival defects, since knockout bone marrow macrophages form less osteoclasts in response to RANKL and are also more sensitive to RANKL and pro-inflammatory cytokine, TNF-α, induced apoptosis. Furthermore, retroviral transduction of a dominant-negative form of IKKβ into wild-type bone marrow macrophages blocks basal and TNF-α stimulated osteoclastogenesis. Interestingly, although IKKβ protein is undetectable in spleen macrophages, no defect in in vitro osteoclastogenesis has been observed, which suggests that the microenvironment of macrophages in the spleen pre-programs osteoclast precursors to differentiate into osteoclasts independently from IKKβ activity. These findings support the conclusion that IKKβ is required for osteoclast development and survival in a cell-autonomous manner. Furthermore, our results substantiate IKKβ as a target in the treatment of inflammatory bone loss and validate the CD11 b promoter as a tool for transgenic expression of genes in cells of the myeloid lineage.

Disclosures: J.E. Otero, None.

1295

Conditional Ablation of Parathyroid Hormone Related Peptide (PTHrP) in Mammary Epithelial Cells Inhibits Breast Cancer Progression.J. Li*¹, A. Karaplis², P. Siegel*¹, V. Papavasiliou*¹, W. Muller*¹, R. Kremer¹. ¹Dept. of Medicine, McGill University Health Centre, McGill University, Montreal, PQ, Canada, ²Dept of Medicine, Jewish General Hospital, McGill University, Montreal, PQ, Canada.

Breast cancer, the second leading cause of cancer death in women, is frequently associated with increased circulating PTHrP levels in late stage of the disease. However, a causal relationship between breast tumor progression and PTHrP has not yet been established. Here, we used the Cre/LoxP recombination system to disrupt PTHrP function in the mammary epithelium of a transgenic mouse model of human breast cancer (PyVMT). In this model hyperplasia occurs at 4–5 weeks, adenocarcinoma at 7–8 weeks and pulmonary metastasis at 12–13 weeks in 100% of animals. Mice carrying a conditional PTHrP allele in which the fourth coding exon was flanked by LoxP recombination sites were backcrossed on an FVB background. These mice were first crossed with the PyVMT mammary tumor model and then with a separate transgenic strain expressing Cre in the mammary epithelium (MMTV/Cre) both on an FVB background. Targeted excision of the PTHrP allele was confirmed using molecular and histological approaches. Ablation of PTHrP in normal FVB animals did not interfere with the initial stages of mammary ductal outgrowth. Ablation of PTHrP in PyVMT animals significantly delayed tumor onset as demonstrated by Kaplan Meier analysis. At age 50 days, 50% of control animals (PyVMT-PTHrP^flox/flox-Cre and PyVMT-PTHrP^+/+-Cre⁺) had a palpable tumor as compared to age 67 days (p < 0.005) in heterozygous (PyVMT-PTHrPwn^+/flox-Cre⁺) and 78 days (p <0.001) in homozygous (PyVMT-PTHrP^flox/flox-Cre⁺) animals. In addition tumor growth slowed significantly over time with a significant reduction observed in both PyVMT^flox/+-Cre⁺ and Py VMT-PTHrP^flox/flox-Cre⁺ animals at all time points. Tumor weight at sacrifice (13 weeks) was significantly reduced in homozygous (67 ± 5% reduction, p< 0.001) and heterozygous animals (48 ± 8% reduction, p <0.005) as compared to controls. Finally, metastatic spread to lungs at sacrifice was seen in 14/14 control animals, 0/13 homozygous animals and 6/14 heterozygous animals. Molecular and immunohistochemical analysis of tumor tissues revealed an 80% inhibition of markers of tumor progression including cyclin D1, Neu/Erb2 and Ki67 in homozygous PyVMT-PTHrP^flox/flox-Cre⁺ animals and a 40% reduction in heterozygous PyVMT-PTHrP^flox/+-Cre⁺ animals as compared to controls.

In summary these studies provide a direct demonstration that PTHrP is critical for the initiation and progression of mammary tumorigenesis in vivo.

Disclosures: J. Li, None.

1296

The 57 kDa C-terminal Fragment of Dentin Matrix Protein 1 (DMP1) Retains All Biological Activity: Osteocytic Regulation of Pi Homeostasis Through FGF23.Y. Lu¹, S. Liu², S. Yu³, Y. Xie*³, J. Zhou*², L. D. Ouarles², L, F. Bonewald¹, J. Q. Feng³¹Oral Biology, School of Dentistry, UMKC, Kansas City, MO, USA, ²The Kidney Institute, KU Medical Center, Kansas City, KS, USA, ³Baylor College of Dentistry, Dallas, TX, USA.

Patients with Autosomal Recessive Hypophosphatemic Rickets (ARHR) were found to have mutations in DMP1. Like Dmpl -nullI mice, these patients exhibit osteomalacia, renal Pi wasting and elevated FGF23 expression in osteocytes. The intact DMP1 protein has never been purified from bone, only 37 kDa N-terminal and 57 kDa C-terminal fragments. To determine if one of these fragments is biologically active, we re-expressed the 57 kDa C-terminal fragment of DMP1 in Dmpl-null animals using the 3.6 kb Col 1 promoter. Re-expression of the full-length DMP1 was performed as a control. To validate successful re-expression in osteoblasts/osteocytes, in situ hybridization and immunohistochemistry were performed showing highly elevated expression in osteoblasts/osteocytes as compared to endogenous DMP1 which is more highly expressed in osteocytes. The Dmpl-null phenotype was fully corrected with introduction of either the full-length molecule (two independent lines) or the 57 kDa fragment (two independent lines). Defects in the Dmpl-null mice such as shortened long bones, disorganized growth plate, diffuse fluorochrome labeling of matrix, disorganized osteocyte lacuno-canalicular network, etc. where found to be corrected in either the full-length or the 57 kDa fragment rescued mice using approaches such as X-ray, uCT, procion red injection, TEM, backscatter SEM, von Kossa, and resin-casted SEM approaches. Biochemical analysis further confirmed that high FGF23 (13-fold elevation in Dmpl-null mice as compared to controls), low serum Pi (40% decrease), and low serum calcium (8.9% decrease) levels were restored to control levels in both full-length and 57 kDa animals by 7 weeks of age. Preliminary studies suggest DMP1 regulation of FGF23. In summary, our findings show that the 57 kDa fragment is the biologically active form of DMPI. We are in the process of determining if the biological activity of DMPI resides in the carboxy terminus as mutations were identified in the last 20 amino acids of DMPI in the ARHR patients.

Disclosures: Y. Lu, None.

This study received[funding from: NIH.

1297

Parathyroid Hormone Activates β-catenin Signaling Through LRP5/6.M. Wan, C. Yang*, H. Yuan*, X. Wu*, C. Lu*, C, Chang*, X. Cao. Pathology, University of Alabama at Birmingham, Birmingham, AL, USA.

Parathyroid hormone (PTH) plays a primary role in regulating calcium homeostasis and is the only anabolic treatment for osteoporosis in clinical. PTH (1–84) or its analog PTH (1–34) binds to the PTH/PTHrP receptor (PTH 1R) and activates Gαs and Gαq, leading to the activation of PKA and PKC respectively. However, the mechanism accounting for the anabolic actions of PTH on bone is still not clear. Here we show that PTH induces the recruitment of LRP5/6, coreceptors of Wnt canonical signaling, to PTH1R to stabilize (β-catenin, which is known to positively regulate osteoblast activity and bone formation. PTH stimulated β-catenin-responsive luciferase reporter and elevated the levels of β-catenin protein in both cytoplasm and nucleus in osteoblastic UMR106 cells and embryonic kidney epithelial HEK 293 cells, indicating that treatment of PTH stabilizes β-catenin protein and promotes its nucleus translocation. To further clarify the mechanism, immunoprecipitation assays demonstrated that PTH induced the intereaction of PTH1R with LRP5/6 as well as the formation of ternary complex of PTH ligand, PTH1R and LRP5/6. The extracellular domains of LRP5 or LRP6 act as dominant negative mutants to inhibit the formation of the triple complex. PTH-induced recruitment of LRP5/6 resulted in phosphorylation LRP5/6 at their intercellular PPPSP motifs, which is required for the subsequent binding of Axin to LRP5/6. Interestingly, addition of the inhibitors of either PKA or PKC blocked PTH-induced LRP5/6 phosphorylation, indicating that both PKA and PKC signals are required for optimal activation of LRP5/6 signaling. Moreover, PTH-induced activation of LRP5/6 was confirmed in xenopus system. Coinjection of PTH and PTH1R mRNAs significantly enhanced the efficiency of secondary axis induction by LRP5 and LRP6 to 18% and 25%, respectively.

Activation of LRP5/6-β-catenin signaling by PTH was also examined in vivo. Injection of PTH in rats stimulated phosphorylation of LRP5/6 in 30 minutes and peaked at 8 hours. Levels of both P-catenin and phosphorylated LRP5/6 were significantly increased in the osteoblasts of trabecular bone sections when PTH was intermittently administrated for 28 days in mice, correlating with increased bone formation. Neither β-catenin nor phosphorylated LRP5/6 level was stimulated with continuous PTH treatment for the same period of time. These findings reveal that coreceptors LRP5/6 mediate PTH-activated β-catenin signaling in a similar way as in canonical Wnt pathway in osteoblasts, and imply that this pathway is one of the key elements in mediating the anabolic bone effect of PTH.

Disclosures: M. Wan, None.

1298

T-lymphocytes Amplify the Anabolic Action of Intermittent PTH Treatment by Regulating Osteoclast Formation.M. Terauchi, Y. Gao, S. Galley, X. Yang*, K. Page*, E. Shah* M. N. Weitzmann, R. Pacifici.. Division of Endocrinology, Metabolism and Lipids, Emory University School of Medicine, Atlanta, GA, USA.

Intermittent PTH treatment (iPTH) stimulates bone formation by targeting stromal cells (SCs) and osteoblasts (OBs), but osteoclasts (OCs) are now known to be the source of a key permissive signal, as iPTH fails to exert its anabolic activity in the absence of OCs. T cells have been shown to be required for continuous PTH treatment to induce OC formation and bone resorption, suggesting the possibility that T cells may regulate the anabolic activity of iPTH by modulating OC formation. To investigate this hypothesis 5 week-old C57BL/6 T cell receptor β knock-out mice (TCRβ -/-), a strain which lacks αβ T cells, were treated with iPTH by injecting sq human PTH 1–34, 80 μ/kg/day for 4 weeks. Prospective in vivo BMD measurements by DXA revealed that iPTH caused a larger increase in femur BMD in congenic WT controls than in TCRβ -/- mice (45.5% vs. 33.8%, p<0.05). μCT analysis of the femoral metaphysis showed that iPTH induces a greater increase in trabecular BV/TV in WT than in TCRβ -/- mice (25.0% vs. 16.8%; p<0.05). Trabecular number, separation and thickness were also differentially affected by iPTH in WT and TCRβ -/- mice. The blunted response of TCRβ -/- mice to iPTH was confirmed by 4-point bending tests which showed that iPTH increases femoral stiffness in WT, but not in TCRβ -/- mice. Measurements of serum levels of osteocalcin, a marker of bone formation, and of CTX, a marker of resorption revealed that iPTH stimulates both bone formation and resorption in WT mice while it had no effect in TCRβ -/- mice. Furthermore, in vivo iPTH treatment failed to stimulate the formation of CFU-ALP, an index of the number of SCs with osteogenic potentials, in whole bone marrow (BM) from TCRp -/-. PTH also caused a 3-fold lower increase in ex vivo formation of OCs in TCRβ -/- mice as compared to WT mice. These findings demonstrate that T cells potentiate the capacity of iPTH to stimulate OB and OC formation. Additional FACS studies revealed that in vivo iPTH treatment increases by 2-fold intracellular RANKL levels in BM CD4+ and CD8+ T cells, while in vitro PTH treatment upregulates T cell RANKL expression in cocultures of T cells and SCs, but not in purified T cells alone. Thus, PTH stimulates T cell RANKL expression by targeting SCs. In summary, the data demonstrate that PTH stimulated SCs induce the production of RANKL by T cells, suggesting that T cell produced RANKL contributes to PTH induced osteoclastogenesis, and indicating that T cells potentiate the bone anabolic activity of iPTH by promoting the formation of OCs, a lineage required for iPTH to stimulate bone formation.

Disclosures: M. Terauchi, None.

1299

Movements Within the Parathyroid Hormone Receptor (PTH1R) Upon PTH Binding.B. E. Thomas¹, A. Wittelsberger¹, P. Monaghan*¹, P. F. Mierke*², M. Rosenblatt¹¹Tufts University School of Medicine, Boston, MA, USA, department of Molecular Pharmacology, Brown University, Providence, RI, USA.

Defining the nature of the parathyroid hormone (PTH)–PTH receptor (PTH1R) interaction is key to understanding the role of PTH in physiology, disease, and facilitating the discovery of therapeutic agents which work through the PTH1R. The PTH–PTHR1 interaction leads to receptor (Re) activation, signal transduction, and expression of PTH bioactivity. Since the hormone docks distant from intracellular G proteins, changes resulting from PTH binding to the extracellular face of PTH1R must converge into a conformational shift at the cytoplasmic PTH1R–G protein interface. We set out to determine the motions of transmembrane (TM) domains of PTH1R induced by PTH and the sequence of events that trigger Re activation. We engineered Cys-substitutions into TMs postulated to play a critical role in Re activation. Promotion of disulfide cross linking by PTH indicates that two TMs are brought sufficiently close to form a linkage not possible in the “inactive” state. We chose to study the dynamics of TM2 and TM7 motion by creating a series of PTH IR mutants, each of which contains a Cys pair with potential to span TM2 to TM7 superimposed on a PTH1R template which contains a c-myc “tag” for purposes of detection and a Factor Xa (FXa) cleavage site. The PTH1R template displays binding and PTH-stimulated adenylyl cyclase activity comparable to native PTH1R. PTH1R mutants were exposed to PTH-(1–34), followed by Factor Xa cleavage. The appearance/disappearance of a low MW band (∼23 kDa) was monitored using anti-c-myc antibody. A decreased quantity of the band indicates new disulfide formation. For one TM2/TM7 pair, F238C/F447C, mild oxidation promoted disulfide formation. But addition of PTH decreased disulfide linkage, indicating that the Cys pair in TM2 and TM7 are near each other in the inactive state of Re, but move apart upon binding PTH. These data are supported by molecular modeling insights: bound PTH causes disruption of interaction between residues at position 238 in TM2 and 447 in TM7, suggesting that F238 and F447 move apart to accommodate the bound hormone. For the K240C/F447 pair, disulfide linkage is promoted by PTH binding, indicating that the Cys pair moves even closer. One experiment provides demonstration of the sensitivity of this approach: placement of Cys in TM2 at position 241 instead of 240 precludes disulfide formation with Cys 447. Since the TMs have a helical structure, insertion of Cys one position removed places the sulfhydryl function one-third way around the helix, away from the favorable spatial arrangement of the 240/447 pair. These studies begin to elucidate the molecular mechanisms by which PTH triggers activation of PTHR1.

Disclosures: B.E. Thomas, None.

1300

Bone Mineralization and Vitamin D Inadequacy. Histomorphometrlc Analysis of Iliac Crest Bone Biopsies and Circulating 25-hydroiyvltamin D in 648 Patients.M. Priemel¹T. Kladde*¹, S. Kessler*¹, S. Seitz*¹, 1L Puschel*², T. Schinke¹, M. Amling¹¹Trauma Surgery and Center of Biomechanics, University Medical Center Hamburg, Hamburg, Germany, ²Forensic Medicine, University Medical Center Hamburg, Hamburg, Germany.

The estimated required circulating 25-hydroxyvitamin D (25[OH]D) to maintain skeletal health range between 12 and 40 ng/ml. This wide range is based on many studies that have used the relationship between low 25[OH]D and increased secretion of parathyroid hormone (PTH) to determine a plausible threshold. PTH is however only one measurable index of skeletal health and we reasoned, that histomorphometic analysis of iliac crest bone biopsies would be another and even more direct approach to assess bone health. Thus to get an answer as to the circulating levels of 25[OH]D that should be ensured, and to assess the prevalence of vitamin D insufficiency we measured 25[OH]D serum levels and performed transiliac crest bone biopsies (8mm diameter) in 648 patients (388 males, mean age: 58.8 years, and 260 females, mean age: 68.6 years) who had no signs of secondary osteopathies at autopsy. All biopsies were undecalcified embedded and histologically processed. Static structural analysis was performed using the Osteomeasure system according to ASBMR standard. Analysis included bone volume (BV/TV), osteoid volume (OV/BV), osteoid surface (OS/BS), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp). Statistical analysis consisted of testing for the lack of correlation and the similarity of the correlation coefficient in males and females. The histologic results demonstrate an unexpected high prevalence of mineralization defects. Indeed 37.35% of the analyzed patients presented with an OS/BS of more than 20%. Based on the definition described by Delling in 1975 that an OV/BV of >1.2% is pathologic 46.76% of the biopsies presented with manifest skeletal mineralization defects. Even a more conservative threshold with a pathologic increase in OV/BV of > 2% was detected in 27.62% of the patients. Most interestingly these mineralization defects were found independent of BV/TV, throughout all ages and affected both sexes. While we could not establish a minimum 25[OH]D level that was inevitably associated with manifest mineralization defects, we did not find pathologic accumulation of osteoid in any patient with circulating 25[OHJD levels above 30ng/m!. Therefore our data demonstrate that the prevalence of vitamin D deficiency and manifest mineralization defects is extremely high in the European population. In case of vitamin D supplementation, the dose given should ensure that circulating levels of 25[OH]D reach a threshold of 30 ng/ml.

Disclosures: M. Priemel, None.

O021

Marrow Adipogenesis and Osteohistogenesis Reflect Global Energy Utilization Through Activation of PPARG and Modulation by [Clock] Genes in a Genotype Specific Manner.C, L. Ackert-Bicknell¹, V. E. DeMambro¹, M. L. Bouxsein², M. C. Horowitz³, E. Canalis⁴C. J. Rosen¹¹The Jackson Laboratory, Bar Harbor, ME, USA, ²Beth Israel Deaconess Medical Center, Boston, ME, USA, ³Yale University School of Medicine, New Haven, CT, USA, ⁴St. Francis Hospital and Medical Center, Hartford, CT, USA.

The relationship between marrow adipogenesis and bone formation is critical for understanding osteoporosis. We generated a congenic mouse (6T) with C3H alleles from a QTL on mouse Chr 6 introgressed on a 99.5%C57BL/6J(B6) background. 6T have reduced BMD, impaired osteoblast(OB) differentiation, more marrow adiposity, but low body temp, and unresponsiveness (i.e. thermal output) to stimulation by a β3 agonist. An inversion in C3H, and polymorphisms in genes at the break point [115.38–116.38 Mb between Pparg and Alox5], prompted us to hypothesize that genes in this region influence OB and adipocyte(Ad) function. To test this, we fed 3 wk B6 and 6T mice an 11%(LF) or 60% fat(HF) diet for 13 wks and studied metabolic and skeletal parameters. On a HF diet, B6 gained wt and became insulin resistant with no change in BV/TV by μCT, while 6T showed no change in wt or insulin secretion, but lost trabecular bone (p<0.001 vs B6). Histomorphometry comparing HF to LF diets revealed that B6 had suppressed bone formation, reduced osteoclast #, increased marrow Ads (2.1 fold higher), but no change in BV/TV. In contrast 6T on HF had a 1.5 fold increase in OB#, but no change in Ad #, despite a 30% reduction in BV/TV. Expression profiling (using a 2×2 statistical design:genotype × diet)revealed that 21 genes were differentially regulated in liver; and 64 in bone (marrow+bone). Most were histones, but in 6T liver on HF, Ccrn41 (a [clock] gene) was 2 fold up-regulated;p= 1.0×10⁻⁶, while in 6T bone, uncoupling protein-1(Ucp-1) was increased nearly 3 fold; p= 1.0×10⁻⁶. 2 candidate genes within the 1Mb region on Chr 6 were suppressed in 6T bone on the HF diet: March 8, a membrane protein (p<0.0005,vs B6) and Rafl (p<0.007vs B6). In the liver, two downstream targets of Pparg, Fasn, and Srebp, as well as ephrinA1 and SOCS-2,were increased by a HF diet in 6T(p<0.05 vs B6). In summary, we found genotype-specific skeletal and marrow responses to HF. In 6T, HF caused bone loss despite recruitment of more OBs, but in B6, even with marrow adipogenesis, and a reduced bone formation rate, BV/TV was preserved. Expression profiles demonstrated that a HF diet activated Pparg in a strain-specific manner, likely through recruitment of co-activators that differentially influenced Ad and OB function. In 6T, greater UCP-1 expression (a marker of brown fat), and enhanced insulin sensitivity imply distinct physiologic roles for marrow fat (i.e. energy use:6T vs storage: B6) influenced by Chr 6 genes and modulated by [clock] proteins.

Disclosures: C.L. Ackert-Bicknell. None.

This study receivedfunding from: niams 45433

O064

Estrogen Receptor a Is Required for Strain-Related β-Catenin Signaling in Osteoblasts. V. J. Armstrong*, M. Muzvlak*, A. Sunters*, G Zaman*, L. K. Saxon, J. S. Price, L. E. Lanvon.. Veterinary Basic Sciences, The Royal Veterinary College, London, United Kingdom.

Wnt/β-catenin signaling has been implicated in the regulation of bone mass through its involvement in bone cells' response to their mechanical environment [1]. Since Estrogen Receptor a (ERα) is also involved in bones' response to loading [2], we investigated whether strain induced signaling through ERα uses the same pathway as β-catenin.

Western blot and immuno-cytochemical analysis showed that in ROS 17/2.8 cells a short period of dynamic strain in vitro increased the levels of activated β-catenin (^aβcat) in the cytoplasm and within I hour stimulated its translocation to the nucleus. These changes in βcat were paralleled by inhibitory phosphorylation of GSK-3β. Strain, and the GSK-3P inhibitor LiC1, also induced a significant increase in TCF/LEF transcriptional activity. In contrast, estrogen had no influence on the level or distribution of βcat, nor any effect on TCF/LEF activation. Nuclear translocation of βcat and TCF/LEF activation stimulated by both strain and LiCl were inhibited by the ER modulator IC1 182,780, which also reduced strain-induced nuclear accumulation of ERα. The ER modulator Tamoxifen also inhibited LiCl stimulated nuclear translocation of ^aβcat.

In primary cultures of osteoblast-like cells derived from the long bones of Wild Type mice and those lacking ERβ, β-catenin was similarly activated and translocated to the nucleus in response to strain and LiCl. In these cells this response was blocked by ICI 182,780. In contrast, in cultures of osteoblast-like cells from mice lacking ERα neither strain nor LiCl stimulated nuclear accumulation of ^aβcat. ICI 182,780 had no effect in these cells.

These data show that in osteoblastic cells exposure to strain causes similar activation of β-catenin, its translocation to the nucleus, and regulation of transcription as GSK-3β inhibition by LiCl. These changes require ERα but not ERβ. To our knowledge these are the first data to demonstrate that in osteoblasts ERα is required for these cells' responses to strain involving Wnt/β-catenin. Reduced effectiveness of bone cells' responses to bone loading, associated with decline in bio-available estrogen and ERα, may contribute to the failure to maintain structurally appropriate bone mass in osteoporosis in both men and women. This failure may in part be due to reduced effectiveness of Wnt/β-catenin signaling.

1. Sawakami K, et al (2006) J Biol Chem 281(33):23698

2. Lee K, et al (2003) Nature 424:389

Disclosures: V.J. Armstrong. None.

This study received funding from: BBSRC.

O099

Expression of OCZF Directed by the Cathepsin K Promoter Affects Bone Mass and Osteoclast Formation in Transgenic Mice.T. Shobuike*¹, T. Kukita², K. Nagata*², J. Teramachi*², M. Asagiri³, H. Takayanagi³, A. Kukita¹¹ Saga University, Saga, Japan, ²Oral Biological Science, Kyushu University, Fukuoka, Japan, ³Cell Signaling and COE Program, Tokyo Medical and Dental University, Tokyo, Japan.

We previously isolated the OCZF cDNA encoding Kat-6 antigen specifically expressed in rat osteoclast. The OCZF gene product is a member of POK protein family that contains two domains of POZ/BTB and zinc finger in their N- and C-terminal region respectively, and that is shown to be involved in various cellular processes. Suppression of OCZF by antisense RNA inhibits osteoclast differentiation in vitro, indicating that OCZF is involved in osteoclastogenesis, but in vivo role in the process is not clear. To this end we generated transgenic mice, which expressed OCZF under the control of mouse cathepsin K promoter. Nine transgenic mice were identified among the offspring by PCR and Southern blot analysis of mouse tail genomic DNA. Transgene expression was verified by RT-PCR using osteoclasts differentiated in vitro from bone marrow at 10 weeks of age. Two lines of the transgenic animals exhibited relatively high transgene expression, and were further investigated. The c-fos and NFATcl protein exhibit higher levels during differentiation into osteoclasts in vitro from bone marrow of the transgenic mice than that of normal littermates. Micro-CT and peripheral quantitative CT (pQCT) analyses and histomorphometry of femur were performed in mice at 6 weeks of age. Micro-CT imaging demonstrates a reduction in trabecular bone volume in the transgenic mice relative to normal littermates. pQCT analyses also demonstrate a reduction in bone mineral density and bone mineral content especially in trabecular bone with a decrease in strength strain index, whereas those in cortical bone were slightly affected. Consistently, von Kossa staining shows a smaller mineralized area in the transgenic mice than in normal littermates. Histological analyses with tartrate-resistant acid phosphatase staining and calcein labeling indicate an increase in the number of osteoclast, whereas bone formation rate did not significantly change. These results suggest that OCZF has an important role in osteoclastogenesis in vivo.

Disclosures: T. Shobuike. None.

0112

Transgenic Overexpression of Osteoclastic Protein-tyrosine Phosphatase, PTP-oc, in Cells of Osteoclastic Lineage Led to Increased Bone Resorption and Marked Reduction in Trabecular Bone Mass and Density in Adult Mice.M. H. C. Sheng, M. Amoui*, A. K. Srivastava, J. E. Wergedal, K. H. W. Lau. Loma Linda VAMC, Loma Linda, CA, USA.

Past in vitro studies have suggested that the structurally unique osteoclastic transmembrane PTP-oc acts as a positive regulator of osteoclast activity. This study sought to determine whether transgenic (TG) overexpression of PTP-oc in cells of osteoclastic lineage would increase (↑) bone resorption and decrease (↓) bone mass or density (BMD) in adult mice. To generate TG mice, a TG construct containing the rabbit PTP-oc cDNA driven by a tartrate-resistant acid phosphatase (TRACP)-IC promoter was injected into the pronucleus of fertilized ova, which were implanted into pseudo-pregnant B6D2F1 mice. TG founders, identified by a PCR-based genotyping assay using primers that span unique regions of TRACP-1C promoter and rabbit PTP-oc sequence, were bred with C57BL/6J mice to produce F2 mice. Ten-week-old male F2 progenies were extensively followed for pQCT, histological, and serum resorption marker analyses. The body weight of TG mice was 10% less (p<0.05, n=8 each) than that of wild-type (WT) littermates. pQCT analyses of the femur revealed that TG mice had 30% ↓ in trabecular BMD (p<0.002). Histologic analyses at the secondary spongiosa in the femur of 9 TG and 7 WT littermates confirmed that TG mice showed 35% ↓ in trabecular surface (Tb.Pm, pO.Ol), 27% ↓ in % trabecular area (Tb.Ar, p<0.05), 11% ↓ in trabecular number (Tb.N, p<0.05), and 36% ↑ in trabecular separation (Tb.Sp, p<0.005). The lumbar vertebra of 12 TG mice, when compared to 8 WT littermates, also showed 20% ↓ in Tb.Pm (p<0.005), 17% ↓ in Tb.N (p<0.003) and 35% ↑ in Tb.Sp (p<0.004). Consistent with an ↑ in bone resorption, the serum c-telopeptide level of TG mice (n=14) was 25% (p<0.05) higher than that of littermates (n=11). That the number of osteoclasts and the length of TRACP-labeled bone surface per total bone surface were not different suggested that TG overexpression of PTP-oc increased osteoclast activity rather than osteoclast differentiation. The same phenotype was confirmed in a second TG line. In congruent with an ↑ in osteoclastic activity, the average pit area created by osteoclasts derived from bone marrow cells of 10-week-old male TG mice in response to RANKL and mCSF in the pit formation assay was ∼50% greater (p=0.008) than those by osteoclasts of WT littermates (n=8 each). However, the TRACP activity per cellular protein in osteoclasts derived from the two groups of mice was not different. In summary, TG overexpression of PTP-oc in cells of osteoclastic lineage led to a marked reduction in trabecular bone mass in adult mice due to an ↑ in osteoclastic activity. These findings provide compelling in vivo evidence that PTP-oc is a positive regulator of osteoclastic activity.

Disclosures: M.H.C. Sheng, None.

This study received funding from: Veterans Administration.

0142

CITED1 Ablation Impairs Endochondral Bone Formation During Embryonic Development. D. Yang, J. Guo*, R, Bringhurst*. Endocrine Unit, Massachusetts General Hospital, Boston, MA, USA.

CITED1 (CBP/P300 interacting Transactivator with Glutamic Acid [E]/ Aspartic Acid [D]-Rich C-terminal domain) is a transcriptional cofactor located on the × chromosome. We previously observed that CITED1 expression is rapidly and transiently induced by FTHIR activation in osteoblasts and that cultured osteoblasts from mice lacking CITED1 display abnormal differentiation in vitro. To determine the possible role of CITED1 in early bone development, we have analyzed endochondral bone formation in tibiae from CITED1-knockout (KO) embryonic mice in which the CITED1 gene is replaced with a functional LacZ/Neo cassette and bred into the 129Sv background for five generations. The tibiae of CITED1 KO mice were shorter than those of wild type (WT) littermates, and bone volume and the extent of trabeculae in CITED1 KO tibiae at E18.5 and post-natal day 1 (P1) were significantly reduced, as shown by von Kossa staining and histology. At El 5.5, the regions of overall chondrogenesis and of chondrocyte hypertrophy, demonstrated by Col II and Col × expression respectively, were not different in WTs and KOs; but bone formation, shown by Col I in situ analysis, appeared slightly earlier inside the tibia and expanded wider in perichondral region in the WT. Osteopontin (OPN) is expressed by late hypertrophic chondrocytes and osteoblasts within the primary and secondary spongiosa. Relative to WT, the length of the central region of OPN expression was reduced in all KO bones from E15.5 through P1, accounting for the foreshortening of the overall bone length. In situ hybridyzation and LacZ staining analysis showed strong expression of CITED1 in this same central region of the developing bones but not in adjacent chondrocytes. We conclude that CITED1 expression, probably by cells of the osteoblast lineage, is important for normal endochondral bone formation and that loss of functional CITED1 expression leads to a reduction in the rate of bone formation. Whether this defect reflects impaired actvity of osteoblasts that normally express CITED1 and/or loss of an indirect effect on chondrocyte proliferation and/or differentiation, which could involve PTHrP, remains to be investigated.

Disclosures: D. Yang. None.

0157

Biglycan and Fibromodulin Control Bone Mass by Regulating Osteoclast Differentiation Through Bone Marrow Stromal Cells.Y. Bi*, T. M. Kilts*, A. C. Griffin*, M. F. Young. National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA.

Members of the Small Leucine-Rich Proteoglycan (SLRP) family populate numerous sites in the musculoskeletal system including tendon, cartilage and bone, but, their precise role in these tissues is still unclear. Previous work showed that mice with combined deficiencies in two SLRPs, biglycan (bgn) and fibromodulin (fmod), acquire osteoarthritis and ectopic tendon ossification. Here we show that the bgn-/0/fmod-/- mice have delayed bone formation during development judged by alizerin red/alcian blue staining and severe age-dependent osteopenia determined by X-ray and microCT analyses. In order to understand the molecular basis for this osteopenia, osteogenic bone marrow stromal cells (BMSCs) were isolated and examined. Colony-forming unit fibroblastic (CFU-F) analysis, which estimates the number of osteogenic stem cells, showed no significant differences between WT and bgn-/0/fmod-/- mice. Further, in vitro osteogenesis assays showed that Ca2+ accumulation was increased in the bgn-/0/fmod-/- BMSC cultures compared to WT BMSCs. Western analysis of the BMSC protein extracts showed that the bgn-/0/fmod-/-cells had increased levels of Runx2 compared to WT controls. The bgn-/0/fmod-/- BMSCs also had increased BMP-2 and TGF-betal signaling indicated by increased levels of p-smadl and p-smad2/3 in the presence of BMP-2 and TGF-betal compared to WT BMSCs, respectively. Furthermore, in vivo bone formation was not significantly different between WT and bgn-/0/fmod-/- BMSCs tranplanted subcutaneously into immune-compromised mice. From these data, we concluded that the decreased bone mass in bgn-/0/fmod-/- mice was not primarily due to a defect in osteogenesis. We next determined whether the osteopenia in bgn-/0/fmod-/- mice could be due to increased differentiation and/or activity of osteoclasts. To test this, sections of long bone and BMSC transplants were stained for Tartrate-Resistant Acid Phosphatase (TRAP), an osteoclast marker. The number of TRAP positive cells was higher in bone and in vivo BMSC transplants from bgn-/0/fmod-/-mice. Osteoclastogenesis was assessed by adding bone marrow suspensions to plates in which BMSC had formed individual colonies and showed that greater numbers of colonies in the bgn-/0/fmod-/- cultures supported formation of TRAP-positive multinucleated cells compared to WT. In summary, we have shown that bgn and fmod when depleted in combination have a negative influence on bone mass by increasing osteoclast formation and function and thereby pointing to the possibility that these SRLPs could be novel targets to modulate bone turnover.

Disclosures: Y. Bi. None.

This study received funding from: IRP-NIDCR. NIH.

0165

Rescue of MT1-MMP Expression in Cartilage Increases Survival, Chondrocyte Proliferation and Bone Formation in MT1-MMP Deficient Mice.L. Szabova*, S. Yamada* K. Holmbeck*. CSDB/MMPU, NIDCR, Bethesda, MD, USA.

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a potent collagenase essential for proper remodeling of collagen rich tissues. Mice deficient for MT1-MMP (KO) are dwarfs with severe fibrosis of connective tissues, bone loss and retention of embryonic cartilages in the skull due to impaired degradation of unmineralized type 1 and II collagens. Here we define the contribution of disrupted cartilage remodeling in the development of the MT1-MMP-deficient phenotype. Specifically, we created transgenic mice where MT1-MMP expression was selectively re-introduced in cartilage tissues using a transgene driven by the type II collagen promoter/enhancer. We have bred these mice into the MT1-MMP-deficient background and evaluated the developmental effects of MT1-MMP expression in the cartilage tissue of otherwise MT1-MMP deficient mice. Cartilage specific MT1-MMP expression in KO mice resulted in complete rescue of the pre-weaning death observed in KO mice, increased body weight and prolonged survival. Transgenic KO mice further displayed increased bone formation in the skull compared to KO mice. Strong expression of the MT1-MMP transgene increased chondrocyte proliferation in the epiphyseal growth plate, which resulted in increased growth of the long bones. Increased bone formation quite unexpectedly coincided with expression of the transgene in a subset of bone cells. Expression of type II collagen was documented in bone cells of normal mice using in situ hybridization and immunostaining thus documenting that transgene expression was not ectopic, but mirrored the normal expression pattern of type II collagen. This finding explains the increased bone formation observed in transgenic KO mice since MT1-MMP is necessary for maintaining the bone formation. In conclusion, reintroduction of MT1-MMP in the cartilage of MT1-MMP KO mice results not only in cartilage specific expression, but also directs transgene expression in a subset of bone cells thereby facilitating increased bone formation. These bone cells are most likely the descendants of a common progenitor of bone cells and chondrocytes expressing type II collagen and support our previous observation that some chondrocytes can differentiate into bone cells. Our data suggest that MT1-MMP provides type II expressing chondrocytes and bone cells with the necessary collagenolytic activity required for chondrocyte proliferation and bone formation in vivo.

Disclosures: L. Szabova. None.

0179

Sclerostin Overexpression Impairs Limb Patterning.N. M. Collette*¹, R, M. Harland*^2.G. G. Loots¹¹Biosciences and Biotechnology Division, Lawrence Livermoe National Laboratory, Livermore, CA, USA, ²Department of Molecular and Cellular Biology, University of California, Berkeley, CA, USA.

Selerostin (SOST) is a negative regulator of bone formation that has been described as both a BMP- and WNT- antagonist. Loss of SOST function causes sclerosteosis (MIM 269500), a condition of severe progressive bone overgrowth. Using transgenic mice expressing human SOST from a bacterial artificial chromosome (BAC) we have demonstrated that SOST over-expression causes decreased bone formation and results in osteopenia, similar to the over-expression of other BMP antagonists such as Noggin and Gremlin. These mice also exhibit severe limb patterning defects that are dose-dependent and range from the loss of a single posterior digit to the loss and/or fusion of many distal limb skeletal elements. The apical ectodermal ridge (AER) relays cell-cell signals to the underlying differentiating limb mesenchyme, a place of SOST expression initiated as early as E9.5. To determine how elevated levels of SOST impair proper limb patterning, we have examined AER and cartilage markers by measuring mRNA levels by in situ hybridization. We find FGF8, a major AER signaling molecule and underlying mesenchymal markers BMP2, Glil, Paxl, HoxD12 and gremlin expression to be perturbed in SOST transgenic mice. In addition, skeletal analysis of double mutant animals show that over-expression of SOST fails to complement loss of BMP antagonists (Gremlin and Noggin) essential for limb patterning and chondrogenesis, resulting in more dramatic skeletal defects than single mutants alone, suggesting that SOST action in the limb parallels the BMP-pathway. This work is supported by NIH ROl HD047853 and the work has been performed under the auspices of the U.S. Department of Energy by the University of California, Lawrence Livermore National Laboratory Contract No. W-7405-Eng-48. UCRL-ABS-229990

Disclosures: N.M. Collette, None.

0185

TNF-alpha Upregulates Aortic BMP2-Msx2-Wnt Signaling in Diabetic LDLR-/- Mice.J. S. Shao, C. F. Lai, Z. Al-Aly*, J. Cai*, E. Huang*, S. L. Cheng, D. A. Towler. Dept. of internal Medicine, Washington University School of Medicine, St. Louis, MO, USA.

Aortofemoral calcification is prevalent in type II diabetes (T2DM), tracking metabolic syndrome parameters and increasing the risk for lower extremity amputation. LDLR-/-mice fed high fat diets (HFD) develop obesity, T2DM, and accumulate aortic calcium - the latter mediated via osteogenic mechanisms that resemble craniofacial mineralization. HFD upregulate aortic BMP2 and Msx2-Wnt signaling cascades that promote mineralization in neural crest-derived skeletal tissues. Administration of recombinant purified BMP2 augments aortic Msx2-Wnt signaling in TOPGAL reporter mice, and promotes aortic calcium deposition in LDLR-/- mice. Low-grade inflammation, including elevated circulating TNF-alpha, is characteristic of obesity with T2DM, therefore, we examined the relationships between TNF-alpha and aortic BMP2-Msx2-Wnt signaling in the LDLR-/-model. HFD feeding promotes obesity, hyperglycemia, and hyperlipidemia – and upregulated serum TNF-alpha and haptoglobin in male LDLR-/- mice. Oxidative stress, reflected in serum 8-F-alpha-isoprostane (8-IsoP) levels, was increased, with concomitant upregulation of aortic BMP2 (2.5-fold), Msx2 (2.5-fold), Wnt3a (10-fold), and Wnt7a (14-fold) gene expression (all p < 0.01, 2-tailed t-test). Treatment of diabetic LDLR-/- mice with the TNF-alpha antagonist infliximab (10 ug / gm twice weekly, 5 animals per group) did not reduce obesity, hyperleptinemia, or hyperglycemia; however, serum 8-IsoP and haptoglobin levels were significantly decreased. Moreover, aortic BMP2, Msx2, Wnt3a, and Wnt7a expression and aortic calcium accumulation were concomitantly and significantly reduced by infliximab treatment (all p < 0.05). Administration of sodium salicylate, an alternative anti-inflammatory strategy, also reduced serum haptoglobin and aortic Msx2 gene expression. Finally, C57BL/6 mice with arterial smooth muscle TNF-alpha expression augmented by a SM22- TNF-alpha transgene accumulate significantly higher levels of aortic BMP2, Msx2, Wnt3a, and Wnt7a mRNAs vs. their non-transgenic sibling cohorts Furthermore, 80% of SM22-TNFα TOPGAL mice exhibit aortic beta-galactosidase reporter staining vs. 0% of their non-transgenic TOPGAL siblings (p=0.05 with Yates' chi-square correction), indicating net enhanced mural Wnt signaling by the SM22-TNFalpha transgene. Thus, inflammatory TNF-alpha signals promote pro-calcific BMP2-Msx2-Wnt programs in the aortic tissues of diabetic LDLR-/- mice. Strategies that inhibit inflammation-induced arterial BMP2-Msx2-Wnt signals may improve aortofemoral physiology and reduce lower extremity amputation risk in T2DM.

Disclosures: D.A. Towler, National Institutes of Health 2; Barnes-Jewish Hospital Foundation 2; Wyeth 5; GlaxoSmithKline 5.

This study received funding from: National Institutes of Health.

0223

Conditional Ablation of the Osteoblast Calcium-Sensing Receptor Causes Abnormalities in Skeletal Development and Mineralization.M. M. Dvorak¹, C. Tu*¹, H. Elalieh*¹, T. Chen*¹, B. Liu*¹, B. E. Kream*², D. D. Bikle¹, W. Chang¹, D. M. Shoback¹¹Department of Medicine, University of California, San Francisco, CA, USA, ²Department of Medicine, University of Connecticut Health Center, Farmington, CT, USA.

The calcium-sensing receptor (CaR) is a G protein-coupled receptor essential for maintenance of calcium homeostasis. Defining the role for the osteoblast CaR in vivo, using global full-length CaR knockout models, is complicated by metabolic disturbances and the potential for compensation by a CaR splice variant identified in these mice. To circumvent these issues, we generated Flox-CaR+/+ mice, in which loxP sites flank exon 7 that encodes the transmembrane and signaling domains of the receptor. Osteoblast-specific inactivation of the CaR was achieved by mating Flox-CaR+/+ mice to transgenic mice expressing Cre-recombinase, under control of the 3.6 kb fragment of the rat al(I) collagen promoter (Coll-Cre). ColI-Cre+/-/Flox-CaR+/+ mice exhibited growth delay from birth and died within four weeks. At 21 days, the skeletal phenotype was hallmarked by hypomineralization, evident by von Kossa staining and micro-computed tomography (μCT) analysis, which revealed significant reductions in bone volume/tissue volume (BV/TV; ↓∼75%), bone mineral density (BMD; ↓∼90%), segmented BMD (↓∼20%), trabecular number (↓∼40%), trabecular thickness (↓∼30%) and connectivity density (↓∼80%) in the secondary spongiosa of the distal femur (n=6, p<0.05, ANOVA), compared to controls. The changes were comparable, although less extensive in the L4 vertebra. The cortical compartment of the femur was also affected, with significant decreases in BV (↓∼60%), cortical thickness (↓∼70%), BMD (↓∼45%), and segmented BMD (↓∼15%) and markedly increased cortical porosity (∼70%), compared to controls (n=6, p<0.05, ANOVA). Histology of femora from ColI-Cre+/-/Flox-CaR+/+ mice revealed severe hyperosteoidosis (Goldner staining) as well as trabeculation of the cortex (Von Kossa staining). Reduced mineral content could be secondary to increased turnover, with inadequate mineralization of newly made osteoid. This is consistent with quantitative realtime PCR (qPCR) analysis of humeri and calvaria that indicate significant increases in markers of proliferation (ccndl), osteoblast differentiation (collagen I, alkaline phosphatase, osteopontin), mineralization (ankylosis protein, ectonucleotide pyrophosphatase/phosphodiesterase 1) and regulators of osteoclastogenesis (RANK-L, osteoprotegerin); (n=3-5, p<0.05, ANOVA). Our findings indicate a critical role for the skeletal CaR in the control of bone mineralization in early postnatal skeletal development.

Disclosures: M. M. Dvorak, None.

0262

Novel Vitamin D3 Analogs (DLAMs) Antagonize Bone Resorption Via Suppressing RANKL Expression in Osteoblasts.M. Inada, K. Tsukamoto*, M. Takita, M. Hirata, A. Hoshino*, T. Tominari* K. Nagasawa* C. Mivaura.. Department of Biotechnology and Life Scence, Tokyo University of Agriculture and Technology, Koganei, Tokyo, Japan.

Active vitamin D3, lα, 25(OH)2D3 (D3), has been proposed to regulate bone remodeling through bone formation and resorption that is maintaining calcium levels in blood serum. Recently new aspects of D3 activities have been reported in bone resorption, inhibition of osteoclast differentiation via interference of c-fos and NFATcl expression. The other aspect was found in D3 analogs, synthetic modification at C2 position of D3 that is enhancing bone formation and bone mass. Here we show newly synthesized analogs, lα,25(OH)2D3-26,23-lactam, (23S,25S)-DLAM-1P and DLAM-2P, that has a lactam moiety in the side chain, perfectly antagonized D3-induced osteoclast (Oc) differentiation. In computer docking simulation estimated DLAM-1P bind to VDR, lactam moiety in DLAM-1P may interfere VDR helix-12 folding at the site of Phe-422 (ligand binding domain). Oc formation was assessed by mouse co-culture system. We first examined antagonistic effects of DLAMs on Oc formation induced by D3. When simultaneous treatment of DLAM-1P (10-7M to 10-5M) and D3 (10-8M), DLAM-1P clearly suppressed the number of TRAP+ Ocs in a dose-dependent manner. To understand the mechanism of action of DLAM-1P, we analyzed mRNA expression of RANKL, a sole molecule for Oc differentiation. DLAM-1P clearly suppressed the D3-induced expression of RANKL mRNA in osteoblasts. In organ culture using mouse calvaria, bone resorbing activity (calcium release) induced by D3 was clearly suppressed by adding DRAM-1P that is associated with less induction of RANKL mRNA. The other analog DRAM-2P has shown similar activities to DRAM-1P. Therefore, DLAM analogs act on osteoblasts as an antagonist of D3 to suppress RANKL dependent Oc differentiation in the cell and the organ, suggesting the DLAMs are novel candidate for the treatment of pathological bone loss such like osteoprosis. Further modification will require for the therapeutic compound, enhancing bone formation and inhibition of RANKL expression beside minimum modification in DLAMs structure, e.g. installing substituents on its C2 position.

Disclosures: M. Inada, None.

0272

Characterization of the Bone Phenotype in C1C-7 Deficient Mice.A. V. Neutzskv-Wulff*¹, K. Henriksen¹, A. Snel*¹, T. J. Jentsch*², J. Fuhrmann*², P. Lange*², C. Christiansen³, M. A. Karsdal¹¹Nordic Bioscience, Herlev, Denmark, ²MDC/FMP, Berlin, Germany, ³CCBR, Ballerup, Denmark.

Loss of the chloride channel C1C-7 leads to severe osteopetrosis. C1C-7 is believed to play a role in the ability of the osteoclasts to acidify the resorption lacuna, and thereby their ability to resorb bone. We therefore examined the bone phenotype of C1C-7 knockout (KO) mice in vitro and in vivo in detail, and compared it to the phenotype of oc/oc mice. C1C-7 KO, oc/oc mice, and their corresponding wildtype littermates (WT) were sacrificed at 4-5 weeks of age. Bones and spleens were dissected and used for isolation of osteoclasts. The isolated cells were differentiated into mature osteoclasts on bone using M-CSF and RANKL. Cell culture supematants were collected for measurements of CTX-I, TRACP and gelatinase activity by zymography. Cells were fixed and TRACP stained and the resorption pits were counted. Biochemical markers of resorption (CTX-I), osteoclast number (TRACP 5b), and osteoblast activity (ALP) were measured in serum of CIC-7 KO, oc/oc mice and the corresponding WT. Osteoblastogenesis in vitro was investigated using calvaria! osteoblasts. In addition, bones were used for histological examination of TRACP positive osteoclasts with respect to number and morphology. Furthermore, osteoblast numbers and morphology were examined.

The osteoclasts from the CIC-7 KO mice were unable to resorb bone, as measured by CTX-I and by counting of resorption pits. Measurements of TRACP activity, as well as TRACP staining, showed the presence of equal numbers of osteoclasts in WT and KO cultures. Gelatinase activity was similar in both genotypes. Furthermore, the morphology of the KO cells was normal. Histological investigation of TRACP stained bone sections of both CIC-7 KO and oc/oc mice, indicated an elevated number of large osteoclasts present compared to WTs. The serum TRACP levels were increased by 250% in CIC-7 KO and oc/oc mice, whereas the resorption per osteoclast was reduced to 50% of the WT level. Finally, the serum ALP level in KO and oc/oc mice was increased by 30%, whereas no differences in osteoblast function were observed in vitro.

In summary, the osteoclasts from C1C-7 KO mice differentiate normally and form actin rings, but fail to resorb bone in vitro. In vivo, the osteoclasts are larger and more numerous, however, show no signs of resorption. Interestingly, an elevation of TRACP, a marked reduction in resorption per osteoclast, and elevated ALP level were observed in serum from both CIC-7 KO and oc/oc mice, showing increased signs of bone formation despite the low resorption. These findings indicate that the osteoclasts, and not their activity, control osteoblastic activity.

Disclosures: A.V. Neutzsky-Wulff, None.

0293

Parathyroid Hormone-Related Protein Induces Bone Pain Through Stimulation of Proton-Secretion in Osteoclasts.L. Wang*, H. Wakabavashi*, T. Hiraga*, T. Yoneda.. Department of Biochemistry, Osaka University Graduate School of Dentistry, Osaka, Japan.

Bone pain is the most common complication associated with bone metastases. Of note, clinical studies reported that inhibitors of osteoclasts such as bisphosphonates (BPs) efficiently reduced bone pain, suggesting a causative role of osteoclasts. Parathyroid hormone-related protein (PTH-rP), a potent stimulator of osteoclasts, has been implicated in bone metastasis. Activated osteoclasts are known to release protons via the a3 type vacuolar-H⁺-ATPase (a3 V-H⁺-ATPase) to dissolve bone minerals, thereby inducing acidosis in the neighboring environment. Acidosis is a well-known cause of pain. These results collectively suggest that PTH-rP is associated with bone pain through stimulating osteoclastic bone resorption. Repeated subcutaneous injections of PTH-rP caused increased osteoclastic bone resorption in the metatarsal bones and hyperalgesia in the hind-paw. Immunohistochemicat examination revealed that the hyperalgesia was associated with increased protein expression of the transient receptor potential channel-vanilloid subfamily member 1 (TRPV1) and phosphorylated-ERK (p-ERK) in the ipsi-lateral dorsal root ganglions (DRGs) and c-Fos in the spinal dorsal horn. The hyperalgesia and elevated protein expression were significantly reduced by a most potent BP zoledronic acid, recombinant human OPG, a specific inhibitor of the V-H⁺-ATPase FR167356 and an antagonist of TRPV1 activation I-RTX, respectively. Finally, TRPV1 knockout mice exhibited reduced PTH-rP-induced hyperalgesia. Relevant to bone pain in bone metastases of breast cancer, we developed an animal model in which the MDA-MB-231 human breast cancer cells that produces large amounts of PTH-rP were directly inoculated into the tibial marrow cavity in nude mice. These mice showed hyperalgesia in the ipsi-lateral hind-paw and a monoclonal antibody to PTH-rP reduced the hyperalgesia. These results suggest that PTH-rP produced by metastatic cancer cells plays a part in causing bone pain through stimulation of proton-secretion in osteoclasts, thereby activating the acid-sensing receptors such as TRPV1 that are expressed in the nociceptive neurons innervating bone.

Disclosures: L. Wang, None.

O309

Myeloma Cells Decrease EphB4 Expression in Osteoblasts; A Novel Mechanism for Regulation of Bone Formation in Multiple Myeloma.A. L. Bates*, G. R. Mundy, C. M. Edwards. Vanderbilt Center for Bone Biology, Vanderbilt University, Nashville, TN, USA.

Multiple myeloma is associated with a destructive osteolytic bone disease, characterized by an increase in osteoclastic bone resorption and a reduction in osteoblastic bone formation. The increase in bone resorption in myeloma is well characterized; however the precise cellular and molecular mechanisms which mediate the reduction in bone formation and the uncoupling of bone resorption from bone formation are poorly understood. Recently, a novel mechanism has been suggested for coupling between osteoblasts and osteoclasts during normal bone homeostasis due to bidirectional signaling between the ligand ephrin B2, expressed by osteoclasts, and its receptor EphB4, expressed by osteoblasts. The interaction between EphB4 and ephrin B2 resulted in inhibition of osteoclast activity by reverse signaling through ephrin B2 on osteoclasts and stimulation of osteoblast differentiation and bone formation by forward signaling through EphB4 on osteoblasts (Zhao et al. 2006). Since the normal coupling of bone resorption to bone formation is dysregulated in multiple myeloma, we hypothesized that this may be mediated by modifications in the EphB4/ephrin B2 receptor/ligand interaction. To study bone formation in multiple myeloma in vivo, we used the 5TGM1 murine model of myeloma. 5TGM1 myeloma cells were inoculated by i.v. injection into C57BlKaLwRij mice, resulting in homing of myeloma cells to the bone marrow, and development of an osteolytic bone disease. In addition to the well characterized increase in osteoclastic bone resorption, the bone disease was associated with a significant reduction in osteoblast number and rates of bone formation (p<0.01). Real time PCR demonstrated expression of EphB4 mRNA in 5TGM1 myeloma cells, C2C12 and 2T3 osteoblasts, with a 2-fold increase in expression in 5TGMI myeloma cells when compared with C2C12 or 2T3 ceils. Western blotting demonstrated expression of EphB4 protein in C2C12 and 2T3 cells. Treatment of C2C12 or 2T3 cells with conditioned media from 5TGM1 myeloma cells resulted in a significant reduction in expression of EphB4 in both C2C12 and 2T3 cells, suggesting that myeloma cells release a soluble factor which down-regulates EphB4 in osteoblasts. In conclusion, we have shown that the development of myeloma bone disease in the 5TGM1 murine model of myeloma is associated with a reduction in bone formation, and that 5TGM1 myeloma cells can down-regulate EphB4 expression in osteoblasts. This raises the possibility that the reduction in bone formation associated with myeloma bone disease is mediated by a reduction in EphB4 expression and thus disruption of the normal coupling of bone resorption and bone formation.

Disclosures: CM. Edwards, None.

0358

Endogenous Sex Hormones and Incident Fracture Risk in Older Men: The Dubbo Osteoporosis Epidemiology Study.C. Meier¹, T. V. Nguyen², D. J. Handelsman*³, C. Schindler*⁴, M. M. Kushnir*⁵, A. L. Rockwood*⁵, W. A. Meikle*⁶, J. R. Center², J. A. Eisman², M. J. Seibel⁷¹Division of Endocrinology, University Hospital Basel, Switzerland, ²Bone and Mineral Research Program, Garvan Institute of Medical Research, Sydney, Australia, ³Department of Andrology, ANZAC Research Institute, Sydney, Australia, ⁴Institute of Social and Preventive Medicine, University of Basel, Switzerland, ⁵ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA, ⁶Department of Medicine and Pathology, University of Utah, Salt Lake City, UT, USA, ⁷Bone Research Program, ANZAC Research Institute, Sydney, Australia.

One third of osteoporotic fractures occur in men. Data on the influence of gonadal hormones on incident fracture risk in elderly men are limited. The present study examined the prospective relationship between serum levels of testosterone (T) and estradiol (E2) and future fracture risk in elderly men as part of the long-term observational Dubbo Osteoporosis Epidemiology Study of community-dwelling men over 60 years followed prospectively since 1989. This analysis included men who had serum samples for baseline measurements (n=609, 70.6%) with follow-up of 5.8 years (up to 13 yrs) closing in 2005. Clinical risk factors, including bone mineral density (BMD) and lifestyle factors were assessed at baseline. Serum T and E2 were measured by tandem mass spectrometry (LC-MS/MS). The incidence of low-trauma symptomatic fracture was ascertained by X-ray record.

During follow-up, 113 men suffered at least one low-trauma fracture. Fracture risk was increased in men with reduced T levels (hazard ratio [HR] 1.33; 95%CI: 1.09, 1.62). After adjustment for SHBG, both low serum T (HR 1.48; 95%CI: 1.22, 1.78) and serum E2 (HR 1.21: 95%CI: 1.00, 1.47) were associated with increased overall fracture risk. After further adjustment for major fracture risk factors (age, weight or BMD, fracture history, smoking status, calcium intake and SHBG), lower T was still associated with increased risk of hip (HR 1.88; 95%CI: 1.24, 2.82) and non-vertebral (HR 1.32; 95%CI: 1.03, 1.68) fracture. By contrast, lower E2 was only associated with increased fracture risk in the presence of body weight (HR 1.25; 95% CI: 1.02, 1.54), but not at any site after adjustment for BMD (HR 1.19; 95% CI: 0.69, 1.03).

In community-dwelling men over 60 years of age, serum T, but not E2, is an independent predictor of osteoporotic fracture and its measurement may provide additional clinical information for the assessment of fracture risk in elderly men.

Disclosures: C. Meier, None.

O360

Respiratory Function Is Associated with Bone Ultrasound Measures and Hip Fracture: European Prospective Investigation Into Cancer-Norfolk Population Cohort Study.A. Moayyeri*¹, R. N. Luben*¹, S. Bingham*², A. Welch*¹, N. J. Wareham*³, S. Kaptoge¹, J. Reeve¹, K. T. Khaw*¹¹Department of Public Health and Primary Care, Institute of Public Health. School of Clinical Medicine, The University of Cambridge, Cambridge, United Kingdom, ²MRC Dunn Human Nutrition Unit, The University of Cambridge, Cambridge, United Kingdom, ³MRC Epidemiology Unit, The University of Cambridge, Cambridge, United Kingdom.

Forced expiratory volume in 1 second (FEV1), an easily obtainable measure of respiratory function in clinics, has been shown to be associated with physical activity. We hypothesized that FEV1 is linked with bone health. In the context of the European Prospective Investigation into Cancer-Norfolk study, 14,800 participants aged 42-81 in 1997-2000 were evaluated by spirometry and heel ultrasound and were followed for fracture outcomes up to July 2007. After excluding participants with history of pulmonary diseases, among 5,555 men and 6,935 women (mean age 62.1+/- 9.0), FEV1 significantly correlated with heel broadband ultrasound attenuation (BUA; Pearson r=0.403; p<0.001) and velocity of sound (VOS; r= 0.269; p<0.001). The association remained significant in sex-stratified linear regression models after adjustment for age, history of fracture, height, body mass index, smoking status and alcohol consumption (standardized Beta coefticient=0.057 and p<0.001 in men; Beta=0.075 and pO.OOl in women). Mean adjusted FEV1 among 109 hip fracture patients (2.00+/-0.60 liter) was significantly lower than that of other participants (2.49+/-0.71 liter; t-test p<0.001). In a Cox proportional hazards regression model, FEV1 was a significant predictor of hip fracture after adjustment for age, sex, history of fracture, height, body mass index, smoking and alcohol consumption (hazard ratio for 1 standard deviation [700 ml in 1 second] decrease in FEVI=1.39, 95%CI 1.03-1.88, p=0.029). Among 6197 current and former smoker participants, hazard ratio for 1 standard deviation decrease in FEV1 was 1.67 (95%CI 1.11 −2.57, p=0.014). Middle aged and older men and women with lower respiratory function appear to be at increased risk of osteoporosis and hip fracture. The observed association might be related to the level of physical activity or deformities in thoracic spine related to osteoporosis. Given the feasibility and affordability of spirometry in general practices, it can be used to improve the identification of high risk groups at the first point of care.

Disclosures: A. Moayyeri, None.

This study received funding from: Medical Research Council and Cancer Research UK.

O373

Changes in Hip Geometric Structures with Aging—Longitudinal Data Analysis from the Women's Health Initiative Observational Study.T. Bassford*¹, T. J. Beck², G. Wu*¹, J. A. Cauley³, A. Z. LaCroix*⁴, C. E. Lewis⁵, Z. Chen¹¹University of Arizona, Tucson, AZ, USA, ²Johns Hopkins University, Baltimore, MD, USA, ³University of Pittsburgh, Pittsburgh, PA, USA, ⁴Fred Hutchinson Cancer Research Center, Seattle, WA, USA, ⁵University of Alabama-Birmingham, Birmingham, AL, USA.

Fracture risk increases with age. In addition to bone loss, change in the distribution of bone mass is another significant factor associated with bone fragility. The objective of this study was to investigate the relationship between aging and changes in hip geometry derived from hip structure analysis (HSA). A prospective study in an ethnically diverse subcohort of 5,856 postmenopausal women enrolled in the Observational Study of the Women's Health Initiative was conducted. Eligible subjects were between 50 and 79 years of age and had completed bone scans using Dual-energy X-ray Absorptiometry (DXA). The Beck HSA software was used to estimate bone mineral density (BMD), cross-sectional area (CSA), outer diameter (OD), section modulus index (SM), average cortical thickness (CT) and buckling ratio (BR) for the femoral narrow neck (FNN), intertrochanter (IT) and proximal femoral shaft regions (FS). Analyses showed that BMD, CSA, SM and CT were all significantly lower and the OD and BR measures were significantly higher in the older age group in comparison to the younger age group at baseline (p<0.01). Results fan paired t-test indicated significant changes in all HSA measures from baseline to year 6 (Table). Interestingly, both the bone bending strength (SM) and the likelihood of cortical failure under compression (BR) increased significantly over this same time frame, suggesting a complex process with changes in the hip geometry associated with aging. The rates of changes in HSA measures varied by age group and by femoral site. For example, OD expansion in all regions was significantly larger in the 60–69 or 70–79 years age group in comparison to the 50–59 years age group. While significant age differences in the change of other HSA measures were only observed in the FNN region. In conclusion, hip geometric structures change significantly with aging and the rate of changes varies by age and femoral site. The impacts of these changes on hip bone strength are complicated and remained to be investigated.

Table  .  

*p-value from paired t-test

Disclosures: T. Bassford. None.

This study received funding from: NIAMS R01-AR049411.

0386

Oral Treatment with the Calcium Receptor Antagonist SB-423557 Causes PTH Release in Multiple Species and Positive Bone Forming Effects in the Rat.S. Kumar¹, X. Liang¹, J. A. Vasko*¹, G. B. Stroup², S. J. Hoffman¹, V. R, Vaden*², H. Haley*², J. Fox³, E. F. Nemeth³, A. M. Lago*², J. F. Callahan*², P. Bhatnayar*¹, W. F. Huffman*¹, M. Gowen²¹GlaxoSmithKline, Collegeville, PA, USA, ²GlaxoSmithKline, King of Prussia, PA, USA, ³NPS, Salt Lake City, UT, USA.

Antagonists of the parathyroid calcium receptor (calcilytics) stimulate the secretion of PTH. Previously, we demonstrated the ability of an orally active calcilytic compound to cause sustained increases in circulating levels of endogenous PTH and to stimulate bone formation and resorption (without a net increase in bone formation) in the ovariectomized (OVX) rat. In the present study, a prodrug approach has been used to preserve oral bioavailability and yield a calcilytic with a shorter half-life in vivo. SB-423557 is the ethyl ester prodrug of SB-423562 that, when administered orally to rats, dogs or monkeys, caused a dose-dependent, transient increase in circulating levels of endogenous PTH. In order to examine the bone forming effect of SB-423557, six-month-old female rats received OVX or sham surgery and were untreated for 6 weeks to allow osteopenia to develop and then treated orally daily with either vehicle, SB-423557 (50 mg/kg), or with rat PTH(l-34) (5 μg/kg SC) for 12 weeks. Plasma levels of PTH peaked at 10–60 min following oral administration of 50 mg/kg SB-423557 (3-fold, C_max of 40–60 pM) and returned to baseline by 2–3 hours. SC administration of rat PTH(l–34) resulted in a systemic C_max of 127-240 pM at 10 minutes post-injection.

SB-423557 significantly and completely prevented additional OVX-induced loss of BMD in the lumbar spine and partially prevented trabecular BMD loss in the proximal tibia by 39% (ns) compared to OVX controls. Histomorphometric analysis indicated greater trabecular bone area (36%, ns) in the spine and increased cortical area (72%) and endocortical bone formation rate (220%) with no effect on the eroded perimeter of the distal tibia in the SB-423557 treated rats compared to vehicle-treated OVX animals. Serum osteocalcin increased (ns) with SB-423557 treatment with no effect on urinary deoxypyridinoline levels. In addition, treatment with SB-423557 resulted in greater ultimate strength (ns), toughness, and elastic modulus of a lumbar vertebral body and at the femoral diaphysis compared to OVX controls. Treatment with PTH( 1–34) also completely prevented the OVX-induced loss in bone mass, BMD, and strength. These data provide a proof of principle for stimulation of bone formation following daily brief antagonism of the calcium receptor in the OVX rat and support the potential use of these agents to treat disorders of bone metabolism such as osteoporosis.

Disclosures: S. Kumar, GlaxoSmithKline 3.

O390

β2 Adrenergic Receptor Deficiency Enhances Bone Mass in by Antagonizing Against Aging-induced Bone Loss and Blunts Anabolic Effects of PTH on Osteoblasts.R. Hanyu*¹, Y. Saita*¹, J. Nagata*¹, Y. Izu*¹, T. Hayata*¹, H. Hemmi*², S. Takeda*³, Y. Ezura*¹, K. Nakashima*¹, H. Kurosawa*⁴, M. Noda*¹¹Department of Molecular Pharmacology, 21st Century COE Program, MRI, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo, Japan, ²MTT Program, MRI, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo, Japan, ³Department of Orthopedics, 21st Century COE Program, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo, Japan, ⁴Department of Orthopedics, Juntendo University school of Medicine, Bunkyo-ku Tokyo, Japan.

Soaring of the aged population in modern world increases the fraction of aged population with diseases and thus in bone field, leads to increase in osteoporosis incidence. Loss of bone mass is accelerated with age regardless of gender. Osteoporotic patients with their bone mass levels at their nadir require treatment to increase bone mass through the activation of bone formation. PTH has been proven to be efficacious in increasing bone mass. However, its efficacy in the highly aged population such as those over 80 years of age in human have not yet been fully elucidated. In mice, aged models of over one year old, indicate lower bone mass than younger adult animals regardless of the gender. Recently, sympathetic nervous system was reported to regulate bone mass in mice. However, these reports always indicate the effects of adrenergic system on young adult mice. Therefore, the purpose of this paper is examine the effects of β2 adrenergic system and PTH on the bone mass in aged mice. Aged (54 weeks old) female mice null for β2 adrenergic receptor (Adrβ2-/-) and wild type (WT) were used. Mice were treated with vehicle or PTH (80μg/kg/day sc) for 4 weeks (5 days/week). The base line bone mass levels in these aged mice were significantly reduced compared to young adult mice. Aged Adrβ2-/- mice exhibited higher total body BMD, trabecular bone volume fraction(BV/ TV), cortical bone volume compared with aged WT mice. In WT, PTH treatment significantly increased the levels of total body BMD, 3D- BV/TV, mineral apposition rate (MAR) and bone formation rate (BFR). In contrast, in aged Adrβ2-/- mice, PTH failed to increase the levels of BMD and BV/TV. Analyses on aged Adrβ2-/- mice also revealed that PTH failed to enhance BFR and MAR. Importantly, Adrβ2 deficiency increased cortical bone volume compared to WT while this parameter was no longer enhanced by PTH in the mutant mice. Regarding bone resorption side, Oc.S/BS, N.Oc/BS were simitar regardless of the genotype or PTH treatment. In conclusion, we found that in one year old aged female mice, β2 adrenergic receptor signal operates to reduce bone mass, while β2 adrenergic signaling is required for the anabolic actions of PTH on bone formation.

Disclosures: R. Hanyu, None.

O409

Decreased Bone Turnover and Porosity Are Associated with Improved Bone Strength in Ovariectomized (OVX) Cynomolgus Monkeys Treated with Denosumab, a Fully Human RANKL Antibody.M. S. Ominskv¹, L Schroeder*¹, J. Jolette*², S. Y. Smith², D. J. Farrell*², J. E. Atkinson¹, P. J. Kostenuik¹¹Amgen Inc., Thousand Oaks, CA, USA, ²Charles River Laboratories Preclinical Services Montreal, Inc., Montreal, PQ, Canada.

Denosumab (DMab), a fully human monoclonal antibody against RANKL, was previously shown to decrease biochemical markers of bone turnover and increase bone mineral density in adult OVX cynomolgus monkeys. We now report from that study the effects of DMab on bone turnover at the histologic level, and their relationships with bone strength.

One month after surgery, OVX cynos (9–16 years old) were treated with either vehicle (OVX-Veh) or DMab (25 or 50 mg/kg, SC, once/month) for 16 months (n=14-20/group). Sham controls were treated with vehicle (n=17). Double fluorochrome labels were injected prior to iliac and rib biopsies (at month 6 and 12), and prior to sacrifice. Histomorphometry was performed on these biopsies, the tibial diaphysis, and cancellous bone in L2 vertebra and the proximal femur.

Cancellous bone turnover was increased in OVX-Veh animals, based on significantly greater mineralizing surface (MS/BS), bone formation rate (BFR), and activation frequency at the lumbar spine, iliac crest, and femur neck (p<0.05 vs Sham). Both doses of DMab fully prevented these OVX-related changes while reducing values significantly below those of sham controls (p<0.05). Osteoclast and osteoblast surfaces were 78–100% lower in the lumbar spine, iliac crest, and femur neck of DMab groups, while eroded surface was decreased by 45–93% (p<0.05 vs OVX-Veh).

Cortical bone turnover was also increased in OVX-Veh cynos, as shown by significantly greater cortical porosity, labeled perimeter, and BFR at the endocortical and haversian surfaces of the tibial diaphysis and rib (p<0.05 vs Sham). Both doses of DMab fully prevented these OVX-related changes, while reducing labeled perimeter and BFR significantly below sham control levels (p<0.05).

Prolonged DMab-related turnover suppression was associated with significant increases in strength parameters at the lumbar vertebrae, femur neck, and femur diaphysis (p<0.05 vs OVX-Veh). Regression analysis of all groups combined demonstrated significant inverse relationships between the strength of L5-L6 cores (yield load) and mineralizing surface (r²=0.44) or eroded surface in L2 (r²=0.37; p<0.001). Cortical porosity in the tibia was negatively correlated with peak load at the femur diaphysis (r²=0.21; p<0.001).

In summary, denosumab treatment of OVX cynos was associated with significant decreases in histomorphometric indices of bone turnover. These changes were accompanied by significant improvements in bone strength at cortical and cancellous sites.

Disclosures: P.J. Kostenuik, Amgen, Inc. 1, 3.

This study received funding from: Amgen Inc.

O423

Efficacy of Adding Teriparatide versus Switching to Teriparatide in Postmenopausal Women with Osteoporosis Previously Treated with Raloxifene or Alendronate.F. Cosman¹, R. A. Wermers², C. Recknor³, K. F. Mauck*², L. Xie*⁴, E. V. Glass*⁴, J. H. Krege⁴¹Helen Hayes Hospital, West Haverstraw, NY, USA, ²Mayo Clinic, Rochester, MN, USA, ³United Osteoporosis Centers, Gainesville, GA, USA, ⁴Eli Lilly and Company, Indianapolis, IN, USA.

In patients previously treated long term with antiresorptive drugs, information regarding the relative efficacy of adding teriparatide (TPTD, 20 mcg/d) versus switching to TPTD is not available. Postmenopausal women with osteoporosis previously treated for at least 18 months with alendronate (ALN, 70 total mg/week, median treatment duration 37.3 months) or raloxifene (RLX, 60 mg/d, median treatment duration 36.9 months) were randomized to either add TPTD or switch to TPTD for 6 months. A preplanned 12-month extension of this study is currently ongoing. Efficacy results included markers of bone turnover and DXA BMD. Baseline BMD and other characteristics were well matched with the exception that previous ALN patients had lower baseline bone turnover than previous RLX patients (Table). Adding TPTD conferred smaller increases in bone turnover versus switching to TPTD; these differences were more marked between the groups previously treated with ALN. However, adding -PTD conferred greater increases in BMD versus switching to TPTD; these differences were again more marked between the groups previously treated with ALN. All regimens were well tolerated. In conclusion, an anabolic response to TPTD was observed in patients previously treated with ALN or RLX regardless of whether the previous antiresorptive drug was continued or discontinued. In general, greater bone turnover was achieved by switching from antiresorptive to I FID, while greater BMD increase was achieved by continuing antiresorptive during TPTD treatment.

Table Table:. Baseline and change from baseline after 6 Months of TPTD Treatment.

For bone markers, values are median baseline, median absolute change from baseline (% change). For BMD, values are mean baseline, mean absolute change from baseline (% change). BL, baseline; PINP, amino-terminal propeptide of type I collagen; BSAP, bone specific alkaline phosphatase; CTx, C-terminal telopeptide of type I collagen; LS, lumbar spine; FN, femoral neck; TH, total hip. P-values are based on percent changes. *p<0.001 and **p<0.05 within group from baseline; †P<0.001 and ‡P<0.05 between groups within a stratum.

Disclosures: F Cosman, Eli Lilly and Company 2, 5, 8; Merck 5, 8.

This study received funding from: Eli Lilly and Company.

O435

Mechanistic Bases of Bone Mineral Density Increase During Alendronate Therapy.D. Vashishth¹, P. Chavassieux², G Boivin², P. D. Delmas²¹INSERM Unite 831 Universite de Lyon France & Rensselaer Polytechnic Institute, Troy, NY, USA, ²INSERM Unite 831 Universite de Lyon, Lyon, France.

An increase in the mean degree of tissue mineralization (DMB) occurring through secondary mineralization has been proposed to increase bone mineral density (BMD) during bisphosphonate (BP) therapy [1]. In this study we conducted additional analyses on human iliac crest biopsies obtained as part of alendronate (ALN) clinical trials [2] to identify the mechanistic bases of BMD increase.

Out of a group of 16 patients on a three-year ALN-therapy [1], we identified two groups of 5 patients each showing lower (8.5 %) and higher (13.3%) bounds of BMD increase but no difference in baseline BMD. For all 10 patients, previously prepared microradiographs were reanalyzed to measure the mean degree of tissue mineralization (DMB) at the osteonal and interstitial compartments in both cortical and cancellous bone tissues. Based on a moving average analysis, six fields each of cortical and cancellous bone tissues were randomly selected for measurement from each biopsy. The average values for patients in each group within osteonal and interstitial compartments were compared between and across cortical and cancellous bone tissues. All DMB measurements were also tested for correlation with standard histomorphometric measures of bone structure (BV/TV, Tb.Th, Tb. Separation, Tb.N), osteoclast activity (EV/BV, E-Depth, Oc#/BS), osteoid (OS/BS, OV/BV, OTh), and bone formation (MAR, BFR/BS, FP) reported previously [2].

The low-BMD-gain group demonstrated no difference between the osteonal and interstitial bone compartments within cortical or cancellous bone tissues but demonstrated a higher DMB in cancellous than in cortical tissue (p<0.05). In contrast, the high-BMD-gain group showed higher DMB in interstitial than in osteonal compartment for both cortical and cancellous tissues as well as a higher DMB in cancellous than in cortical tissue (p<0.05). Out of all the DMB measures in cortical and cancellous tissues, only cortical bone interstitial level DMB correlated to bone formation rate (BFR/ BS) (r = −0.86; p = 0.006) and formation period (FP) (r = −0.75; p = 0.04). In conclusion, this study demonstrates that the effects of ALN-therapy are more evident in cancellous than in cortical bone. Moreover since the interstitial level DMB is at least partially dependent on the duration of secondary mineralization, and negatively correlated to formation parameters, slow bone formation rate and longer bone formation period produce conditions conducive to complete secondary mineralization and consequently larger BMD gain.

References: [1] Boivin et al. Bone. 2000 5:687–94. [2] Chavassieux et al. JCI1997 100:1475–80.

Disclosures: D. Vashishth, Merck 5.

This study received funding from: INSERM, France & NIH Grants AR49635, AG 20618.

O498

Thioredoxin-1 Overexpression Attenuates Streptozotocin-induced Diabetic Osteopenia in Mice: A Novel Role of Oxidative Stress and Therapeutic Implications.Y. Hamada*¹, H. Fujii*¹, R. Kitazawa², S. Kitazawa², M. Fukapawa¹¹Division of Nephrology and Kidney Center, Kobe University School of Medicine, Kobe, Japan, ²Division of Molecular Pathology, Department of Biomedical Informatics, Kobe University School of Medicine, Kobe, Japan.

Diabetes mellitus is associated with increased risk of osteopenia and bone fracture. However, the mechanisms accounting for diabetic bone disorder still remain to be clarified. Moreover, there are few effective treatments for this disease. We have previously reported that streptozotocin-induced diabetic mice develop low turnover osteopenia associated with increased oxidative stress in diabetic condition (BONE 2007). Therefore, in order to determine the role of oxidative stress in the development of diabetic osteopenia, we investigated the effect of thioredoxin-1 (TRX) overexpression, a major intracellular antioxidant, on the development of diabetic osteopenia using TRX transgenic mice (TRX-Tg). TRX-Tg was C57BL/6 mice that carry the human TRX transgene under the control of [β-actin promoter.

Eight-week-old male TRX-Tg and wild type littermates (WT) were intraperitoneally injected with either streptozotocin or vehicle alone. Mice were classified into four groups: 1) non-diabetic WT, 2) Non-diabetic TRX-Tg, 3) diabetic WT, and 4) diabetic TRX-Tg. After 12 weeks of streptozotocin treatment, the physical properties of femora, and the parameters of bone histomorphometry of tibiae were assessed. Oxidative stress in the whole body as well as in the bone was evaluated.

TRX overexpression did not affect either body weights or hemoglobin Alc levels both in the diabetic mice and in the non-diabetic mice. There were no significant differences in renal function, and serum levels of calcium, phosphate, and intact parathyroid hormone among four groups. Urinary excretion of 8-hydroxydeoxyguanosine (8-OHdG), a marker of oxidative DNA damage, was significantly elevated in diabetic WT, which was attenuated in diabetic TRX-Tg. Immunohistochemical staining for 8-OHdG was clearly intensified in the bone tissue of diabetic WT compared with non-diabetic WT. In contrast, staining was attenuated in diabetic TRX-Tg. TRX overexpression partially restored reduced bone mineral density and prevented the suppression of bone formation (OV/BV, OS/BS, Ob.S/BS, O.Th, MAR, and BFR/BS) observed in diabetic WT. These results suggest that increased oxidative stress in diabetic condition contributes to the development of diabetic osteopenia. Furthermore, our findings indicate that suppression of increased oxidative stress by TRX induction can be a therapeutic approach for treatment of diabetic osteopenia.

Disclosures: Y. Hamada, None.

O504

Time Sequence of Secondary Mineralization and Microhardness of Bone in an Ewe Model.Y. Bala*, D. Farlay*, C. Simi*, P. J. Meunier, P. D. Delmas, G Boivin. INSERM Unité 831, Universite de Lyon, Lyon, France.

The degree of mineralization is a major determinant of the mechanical resistance of bone (Follet et al. 2004, Bone 34:783–9). Mineralization begins by a rapid primary mineralization followed, after the full completion of the Basic Structural Units (BSUs), by a secondary mineralization phase, i.e., a slow and gradual maturation of the mineral component leading to complete mineralization of newly formed BSUs (Meunier & Boivin 1997, Bone 21:373–7). The duration of primary mineralization has been evaluated by several autors but the time sequence of secondary mineralization is still poorly investigated [Fuchs et al. 2005, J Bone Miner Res 20 (Suppl.l):325]. Our aim was to determine the time course of secondary bone mineralization in ewe, an animal model having an Haversian bone tissue with a remodeling activity close to the Human one (Chavassieux et al. 1997, Bone 20:451–5). 18 ewes (4.5±0.4 years, INRA, Theix, France) received every six months for 30 months fluorescent labelings following the schedule: TO double tetracycline, T6 single fluorescein, T12 triple tetracycline, T18 double fluorescein, T24 single tetracycline, T30 double alizarin labeling in order to date the age of the BSUs. Transiliac bone samples have been taken on each ewe after T18 and T30 labelings, then embedded in methyl methacrylate. Microradiography (Boivin & Meunier 2002, Calcif Tissue Int 70:503–11) performed on 100±1 μm-thick sections allowed the measurement of the focal Degree of Mineralization of Bone (DMB g/cm³) on 505 different BSUs in which the duration of the mineralization was precisely determined by labeling. Microhardness using a Vickers indenter under a load of 25g for 10s (Hv kg/mm²) was measured on 367 among the 505 BSUs [Bala et al. 2006, J Bone Miner Res 21(suppl.l):S332]. DMB measured at primary mineralization (0.76±0.10 g/cm³) significantly increased during the first 6 months (+26%, p<0.0001). Then, DMB increased more slowly until 30 months to reach a mean of 1.20±0.12 g/cm³. DMB measured at the end of the primary mineralization corresponded to 64% of the final mineralization. DMB after 6 and 12 months were 85% and 91% of the final DMB, respectively. Secondary mineralization is thus the fastest during the first year. Hv followed a similar trend, with a rapid increase during the first six months (+32%, p<0.0008), then a slow increase until 30 months. Hv and DMB are strongly and positively correlated (r²=0.52, p<0.000l). The time course of secondary mineralization can be divided into two parts having separated trends, a rapid increase during the first year then a slowdown of mineralization until 30 months. Mineralization explains a great part of the hardness of bone at BSU level.

Disclosures: G. Boivin, None.

S002

Oxidized Metabolites of Polyunsaturated Fatty Acids Stimulate Osteoblast Apoptosis via both PPARγ-dependent and -independent Mechanisms.R. L. Jilka, M. Almeida, R. Wynne*, L. Han, S. C. Manolagas. Center for Osteoporosis and Metabolic Bone Diseases, Central Arkansas Veterans Healthcare System, University of Arkansas for Medical Sciences, Little Rock, AR, USA.

Age-related loss of bone mass and strength in mice is accompanied by increased reactive oxygen species (ROS) in the bone marrow, increased osteoblast apoptosis, and decreased osteoblast number; but the underlying mechanisms are unknown. The level of expression of the lipoxygenase Aloxl5, and the nuclear hormone receptor PPARγ, is inversely related to bone mass in mice; and the expression of these genes increases with aging. Aloxl5, as well as ROS, oxidize polyunsaturated fatty acids (PUFAs) to generate PPARγ ligands such as 9-hydroxyoctadecadienoic acid (9-HODE) and 12-hydroxyeicosatetrenoic acid (12-HETE). Oxidized PUFAs are farther metabolized to form 4-hydroxynonenal (4-HNE), which can form pro-apoptotic protein and DNA adducts, and can further increase oxidative stress. Here, we examined the impact of these PUFA metabolites on the apoptosis of C2C12 uncommitted osteoblast progenitor cells and OB-6 osteoblastic cells. We found that 9-HODE or 12-HETE stimulated apoptosis of C2C12 cells after 6 hours and of OB-6 cells after 16 hours, as measured by caspase-3 activity. This effect required 0.5 to 5 μM of 9-HODE or 12-HETE, which is near their binding constant for PPARγ2. Using osteoblastic OB-6g2 cells that express PPARγ2 under the control of the tet-OFF promoter, we found that the pro-apoptotic effect of both metabolites required the presence of this transcription factor. Six hours of treatment with 4-HNE also stimulated apoptosis of C2C12 and OB-6 cells at physiologic concentrations (10–40 μM). Unlike 9-HODE and 12-HETE, however, the pro-apoptotic effect of 4-HNE did not require PPARγ2. Using pharmacologic inhibitors, we further determined that 4HNE-stimulated apoptosis required p38 MAP kinase, and that neither MEK nor JNK MAP kinases were involved. 4-HNE-induced apoptosis was prevented by the antioxidant N-acetyl cysteine. Moreover, treatment of C2C12 cells with 40 μM 4-HNE for 1 hour increased phosphorylation of p66^sbc, a ROS-sensitive adaptor protein known to directly activate apoptosis. Based on these findings, we hypothesize that an age-related increase in the production of oxidized lipids such as 9-HODE and 13-HETE stimulate apoptosis of osteoblasts and osteoblast progenitors via activation of PPARγ2; and downstream PUFA metabolites like 4-HNE further increase oxidative stress and stimulate apoptosis of these cells. In view of the role of lipid oxidation in atherosclerosis and increasing epidemiologic evidence linking atherosclerosis and osteoporosis, it is tempting to speculate that these diseases share a common pathologic basis.

Disclosures: R.L. Jilka, Radius Health, Inc. 1.

S004

The Role of Fas/Fas Ligand System in Estrogen Deficiency-induced Osteoporosis.N. Kovacic¹, V. Grubisic*¹, K. Mihovilovic*¹, I. K. Lukic¹, LX Grcevic*², V. Katavic*¹, P. Croucher³, A. Marusic*¹¹Department of Anatomy, Zagreb University School of Medicine, Zagreb, Croatia, ²Department of Physiology and Immunology, Zagreb University School of Medicine, Zagreb, Croatia, ³Academic unit of Bone Biology, University of Sheffield Medical School, Sheffield, United Kingdom.

The aim of this study was to estimate the role of Fas/Fas ligand system in vivo in the pathogenesis of estrogen deficiency induced bone loss.

We first analyzed the expression of Fas gene by quantitative PCR in bones and bone cell cultures from wild-type mice, four weeks after the ovariectomy (ovx). Then we performed ovx in mice deficient for Fas gene (Fas -/-) and their wild-type controls. After four weeks we analyzed: 1) standard histomorphometric parameters of their femora, 2) differentiation of osteoblast (Obl) and osteoclasts (Ocl) in vitro from their bone marrow progenitors. Obl and Ocl differentiation was estimated histochemically (number of alkaline phosphatase positive Obl colonies, and number of TRAP-positive Ocl-like cells), and according to the expression of Obl (Runx2, alkaline phosphatase, osteocalcin and osteprotegerin) and Ocl (RANK, calcitonine receptor) differentiation genes.

Our results showed that after four weeks, gene expression of Fas was increased in bone and mature Obi cultures from ovx (0.36±0.06 and 2.6±0.05 respectively) compared to sham operated animals (0.31±0.01 and 1.8±0.76, p<0.05). A mild decrease in Fas expression was also observed in the Ocl cultures from ovx animals. Trabecular bone volume was generally significantly higher in Fas -/- mice (p=0.01, t-test) than in wild-type controls. Furthermore, trabecular volume significantly decreased in wild-type mice four weeks after ovx (p=0.01, t-test), and remained unaltered in Fas -/- mice (p=0.67, t-test). Mean number of TRAP-positive Ocl on bone surface of wild-type mice increased four weeks after ovx (p=0.03), whereas this number was unchanged in Fas -/- mice (p=0.27). Ovx also significantly increased osteoclastogenesis in vitro in wild-type animals but this effect was absent in Fas -/- mice. Osteoblastogenesis in vitro was stimulated by ovx in both animal strains, but this effect was more pronounced in Fas -/- mice. Obl differentiation genes were had similar expression patterns in sham operated and ovx mice, although Fas -/- mice Obis generally had higher expression levels of Obl differentiation genes compared to wild-type controls. Our findings point to the conclusion that Fas/Fas ligand system may have an important role in the pathogenesis of postmenopausal osteoporosis and modulation of its effects on bone cells may contribute to the development of new strategies for osteoporosis treatment.

Disclosures: N. Kovacic, None.

S010

Osterix as a Regulator of Bone Formation and Maintenance.J. Kim¹, W. Baek*¹, M. Lee*¹, H. Akiyama*², Z. Zhang*³, B. de Crombrugghe³¹Department of Molecular Medicine, Kyungpook National University School of Medicine, Daegu, Republic of Korea, ²Department of Orthopaedics, Kyoto University, Kyoto, Japan, ³Department of Molecular Genetics, University of Texas M. D. Anderson Cancer Center, Houston, TX, USA.

Osterix (Osx) is a zinc-finger containing transcription factor which is essential for osteoblast differentiation and bone formation. Osx null mutant by gene targeting shows a complete absence of intramembranous and endochondral bone formation. Disruption of Osx has led to perinatal lethality, hence preventing the study for the importance of Osx in bones that are developing or formed. Here, we examined the role of Osx in developing bones after bone collar formation using the time-specific and site-specific Cre/loxP system. Osx was inactivated in osteoblasts by Collal-Cre with the activity of Cre recombinase under the control of 2.3-kb collagen promoter. Low bone mass was observed in mouse bones at the age of 2 months. Bone forming rate by Calcein double labeling was shown a remarkable reduction, while single labeling surface was increased in endosteum and trabecular of mouse lumbar. The analysis of Deoxypyridinoline (DPD) crosslinks in the urine, which reflects osteoclast activity in vivo, was not significantly changed in Osx-inactivated mice. These results demonstrated that Osx inactivation decreased the function of osteoblasts to synthesize and regulate the deposition and mineralization of bone extracellular matrix, whereas it was not affected to osteoclast differentiation and activity in vivo. We also studied the role of Osx in intact bones after birth by generating Osx inactivation in osteoblasts using inducible Cre system. Bone volume was decreased and bone forming rate was reduced in mice which Osx was inactivated in osteoblasts of already formed bones after birth by 4-OHT administration. Thus, this study suggests that Osx plays a significant role in regulating bone formation and maintenance. To better understand the role of Osx in cellular mechanisms, we are investigating the interrelation between osteoblasts and osteoclasts derived from Osx-inactivated mice.

Disclosures: J. Kim, None.

This study received funding from: Korea Research Foundation Grant funded by Korea Government (MOEHRD, Basic Research Promotion Fund) (KRF-2005–204–E00006) and Grain Korea 21 Project in 2007.

S016

Overexpression of Osteoblast Specific Angiopoietinl Increases Bone Mass.T. Suzuki*¹, T. Miyamoto², N. Fujita*³, M. Yagi*³, K. Ninomiya*¹, R, Iwasaki*⁴, Y. Toyama*³, T. Suda*³¹Cell Differentiation and Orthopaedics Surgery, Keio University, Tokyo, Japan, ²Orthopaedics Surgery and Musculoskeletal Reconstruction and Regeneration Surgery, Keio University, Tokyo, Japan, ³Orthopaedics Surgery, Keio University, Tokyo, Japan, ⁴Department of Dentistry and Oral Surgery, Keio University, Tokyo, Japan, ⁵Cell Differentiation, Keio University, Tokyo, Japan.

Angiogenesis has been considered to play an important role in the bone formation. However, relationship between angiogenesis and bone formation is poorly understood. Angiopoietin1(Angpt1) is an essential molecule of angiogenesis, is expressed in osteoblasts. To understand the effect of Angpt1 on bone formation in vivo, we generated a transgenic mouse overexpressing Angptl in osteoblasts under the control of mouse 2.3kb alpha 1 collagen promotor. 51 lines of offspring were generated by the embryo microinjection. The presence of the transgene in the offsprings was tested by PCR of tail DNA using transgene specific primers. 16 transgenic founder lines were established by mating founders with C57/BL6 wild type mice. Angpt1 expression was confirmed by RT-PCR with mRNA extracted from long bones, and Western blotting with protein extracted from calvaria. CD31-positive endothelial cells were increased in tibial trabecular area of 6-weeks-old Angptl transgenic mice compared with wild-type controls. Microcomputed tomography analysis showed that bone mineral density and bone volume/total volume were significantly increased, while trabecular spacing was significantly decreased in 8-weeks-old Angpt1 transgenic mice compared with wild-type controls. Immunohistochemistry using alkaline phosphatase (ALP) antibody showed increase of ALP activity in femoral trabecular area of 8-weeks-old Angptl transgenic mice compared with wild-type controls. Furthermore, tartrate resistant acid phosphatase (TRAP) staining showed TRAP activity of 8-weeks-old transgenic mice femoral trabecular area is higher than wild-type controls. Overall, these data showed that Angptl overexpression in osteoblasts induced angiogenesis of the trabecular area, which results in increased bone mass in vivo.

Disclosures: T. Suzuki, None.

S021

Marrow Adipogenesis and Osteoblastogenesis Reflect Global Energy Utilization Through Activation of PPARG and Modulation by [Clock] Genes in a Genotype Specific Manner.C. L. Ackert-Bicknell¹, V. E. DsMambiQ¹, M. L. Bouxsein², M. C. Horowitz³, E. Canalis⁴, C. J. Rosen¹¹The Jackson Laboratory, Bar Harbor, ME, USA, ²Beth Israel Deaconess Medical Center, Boston, ME, USA, ³Yale University School of Medicine, New Haven, CT, USA, ⁴St. Francis Hospital and Medical Center, Hartford, CT, USA.

The relationship between marrow adipogenesis and bone formation is critical for understanding osteoporosis. We generated a congenic mouse (6T) with C3H alleles from a QTL on mouse Chr 6 introgressed on a 99.5%C57BL/6J(B6) background. 6T have reduced BMD, impaired osteoblast(OB) differentiation, more marrow adiposity, but low body temp, and unresponsiveness (i.e. thermal output) to stimulation by a β3 agonist. An inversion in C3H, and polymorphisms in genes at the break point [115.38–116.38 Mb between Pparg and Alox5], prompted us to hypothesize that genes in this region influence OB and adipocyte(Ad) function. To test this, we fed 3 wk B6 and 6T mice an 11%(LF) or 60% fat(HF) diet for 13 wks and studied metabolic and skeletal parameters. On a HF diet, B6 gained wt and became insulin resistant with no change in BW/TV by μCT, while 6T showed no change in wt or insulin secretion, but lost trabecular bone (p<0.001 vs B6). Histomorphometry comparing HF to LF diets revealed that B6 had suppressed bone formation, reduced osteoclast #, increased marrow Ads (2.1 fold higher), but no change in BV/TV. In contrast 6T on HF had a 1.5 fold increase in OB#, but no change in Ad #, despite a 30% reduction in BV/TV. Expression profiling (using a 2×2 statistical design:genotype × diet)revealed that 21 genes were differentially regulated in liver; and 64 in bone (marrow+bone). Most were histones, but in 6T liver on HF, Ccrn41 (a [clock] gene) was 2 fold up-regulated;p=1.0xl0⁻⁶, while in 6T bone, uncoupling protein- l(Ucp-l) was increased nearly 3 fold; p=1.0xl0⁻⁶. 2 candidate genes within the 1Mb region on Chr 6 were suppressed in 6T bone on the HF diet: March 8, a membrane protein (p<0.0005,vs B6) and Rafl (p<0.007vs B6). In the liver, two downstream targets of Pparg, Fasn, and Srebp, as well as ephrinA1 and SOCS-2,were increased by a HF diet in 6T(p<0.05 vs B6). In summary, we found genotype-specific skeletal and marrow responses to HF. In 6T, HF caused bone loss despite recruitment of more OBs, but in B6, even with marrow adipogenesis, and a reduced bone formation rate, BV/TV was preserved. Expression profiles demonstrated that a HF diet activated Pparg in a strain-specific manner, likely through recruitment of co-activators that differentially influenced Ad and OB function. In 6T, greater UCP-1 expression (a marker of brown fat), and enhanced insulin sensitivity imply distinct physiologic roles for marrow fat (i.e. energy use:6T vs storage: B6) influenced by Chr 6 genes and modulated by [clock] proteins.

Disclosures: C.L. Ackert-Bicknell. None.

This study received funding from: NIAMS 45433

S023

Osterix Mediates Mesenchymal Progenitor Cell Differentiation into Osteoblasts Downstream of PTH During Bone Repair.L. A. Kaback*, Y. Jiang¹, A. Naik*, C. Hock*, E. M. Schwarz, S. Bukata, R. J. Q'Keefe, H. Drissi Orthopaedics, University of Rochester, Rochester, NY, USA.

We investigated the mechanisms underlying the effects of PTH on bone repair. We hypothesize that PTH accelerates fracture healing through induction of the zinc finger transcription factor Osterix (Osx), thereby inducing mesenchymal stem cell (MSC) commitment towards osteogenesis. Our real time RT-PCR data show that Osx transcripts are up-regulated upon intermittent PTH treatment of osteoblast progenitors 24, 36 and 72 hours in culture compared to untreated controls, concomitant with that of osteoblast phenotypic genes. Continuous PTH treatment strongly inhibits Osx mRNA levels and osteoblast markers at all time points. Daily injections of mice with the anabolic PTH drug, Forteo, or saline for up to 14 days was performed. Bone marrow derived MSCs cultured from Forteo treated and control mice revealed accelerated osteoblast maturation with increased alkaline phosphatase and Von Kossa staining. Analyses of RNA extracted from these MSCs shows that Osx expression in marrow cultures from Forteo treated mice was dramatically up-regulated compared to control samples. Furthermore, Runx2 levels were also up-regulated in the Forteo treated samples along with markers of osteoblast differentiation. Together, these results indicate that in vivo treatment with Forteo mimics the anabolic effects of PTH observed in vitro. The cellular distribution of Osx during fracture repair was determined immunohistochemically in response to Forteo. Our results show that after 7 days of Forteo exposure, Osx protein is maintained in the periosteal cells and confined in the immature chondrocytes while absent from the hypertrophic chondrocytes in the fracture callus. By 10 to 14 days post-fracture, Osx persists in mesenchymal progenitor cells as well as in the immature chondrocytes, and in osteoblasts embedded in the newly formed bone in the control calluses. However, this representation of Osx is intensified in the Forteo treated animals. We harvested RNA from fracture callus of mice treated or not with Forteo. Our real time RT-PCR data show a progressive induction of Osx transcripts which becomes maximal after 10 days of treatment, but is down-regulated 14 and 21 days post-fracture. Additionally, while Runx2 regulation parallels that of Osx in these calluses, type I collagen and osteocalcin continue to be up-regulated 14 days post-fracture. Together, our results suggest that PTH may accelerate fracture repair through MSC differentiation into mature osteoblasts in response to Osx enhanced expression. Signaling and transcriptional mechanisms behind this PTH mediated regulation of Osx is currently under investigation.

Disclosures: H. Drissi, None.

S025

Analysis of Krox20 Gene Regulation Reveals Srf Function in Bone and Cooperation with NFAT1.M. Frain*¹, A. Nordheim*², P. Chamay*¹¹Ecole Normale Superieure, Inserm U 784, Paris, France, ²Department of Molecular Biology, Tuebingen University, Institute of Cell Biology, Tuebingen, Germany.

The Krox20 gene encodes a zinc finger transcription factor that plays a key role in regulating bone formation. We have shown that Krox20 is expressed in a subpopulation of growth plate hypertrophic chondrocytes and in differentiating osteoblasts and that Krox20 conditional knockout mice develop severe osteopenia.

To investigate the mechanisms of Krox20 transcriptional regulation in bone, we have used a transgenic mice approach and identified a bone-specific enhancer in the 5′ flanking region of the mouse Krox20 gene which spans 860 bp and recapitulates Krox20 expression during bone development. Combining phylogenetic footprinting analyses and in vitro and in vivo experiments, we have defined three types of regulatory elements within the enhancer: Krox20 binding sites involved in a direct positive autoregulatory loop, Runx2 binding sites modulating Krox20 expression and a 13 bp A/T rich essential for Krox20 activation in both osteogenic and chondrogenic cells. This key regulatory element is conserved among vertebrates and contains partially overlapping canonical binding sites for two transcription factors: Srf (Serum Response Factor) and NFAT (Nuclear Factor of Activated T-cells) known to control several developmental processes. Gel-shift, transfection experiments and mutational analyses have shown that Srf and NFATl/c2 are both required for initiation of Krox20 expression in the two bone-forming cells. Our data are consistent with recent studies showing that NFAT1 controls endochondral bone formation (Koga et al, Nat Med 8, 880–5, 2005). We have pursued our studies by establishing Srf spatio-temporal expression pattern in the developing bone. We have found that Srf is expressed in hypertrophic chondrocytes and in differentiating osteoblasts and precedes Krox20 activation in these cell types. To genetically establish that Srf lies upstream of Krox20 in the regulatory cascade, we have created a Srf conditional mutant inactivating Srf in hypertrophic chondrocytes and differentiating osteoblasts. We have found that the Srf conditional mutant shows a reduced level of Krox20 expression in the developing bone.

Altogether, our studies demonstrate that Srf and NFAT1 constitute upstream regulators of Krox20 in bone and highlight a novel role for Srf in regulating skeletogenesis.

Disclosures: M. Frain, None.

S027

Role of Dlx3 in Bone: Study of Inducible Cre Mediated Dlx3 Inactivation in Mice.M. Islam*¹, X. Jiang*¹, H. Li*¹, M. S. Kronenberg*¹, D. J. Adams*¹, M. M. Morasso*², A. C. Lichtler¹¹University of Connecticut Health Center, Farmington, CT, USA, ²NIAMS, National Institute of Health, Bethesda, MD, USA.

Dlx3 is a member of Dlx family of transcription factors. Trichodento osseous dysplasia, a human genetic anomaly, is caused by mutation of Dlx3 gene. This condition has distinct bone findings, which suggests that Dlx3 has a role in bone formation and metabolism. Studies in our lab also show that Dlx3 is highly expressed in mature osteoblast and osteocytes. Universal knock out of the gene causes embryonic lethality, so we have created a bone specific Dlx3 null mice using cre-LoxP recombination. We have used a transgenic mouse model in which a 3.6 kb type I collagen promoter drives constitutive or tamoxifen inducible (CreERT2) cre. In the constitutive model, cre is active in multiple tissues including bone, skin and tendon, and sometime in early embryonic development. Because of this lack of specificity, we focused on inducible cre mediated floxed Dlx3 knockout mouse model. Here the cre transgene is fused to a mutated estrogen receptor gene. Although cre activity in this model should require tamoxifen; we found that inducible cre transgenic mice show strong bone specific cre activity without tamoxifen in vivo and in calvarial osteoblast (mCOB) and bone marrow stromal cell (BMSC) cultures. Dlx3 mutant mice show distinct trabecular and cortical bone changes. In a microCT study of femur, there was a trend of increase bone volume fraction (BV/TV) and trabecular number while trabecular spacing and thickness was decreased in the mutant mice compared to age matched controls. Femoral cortical bone in microCT showed a trend of age related increase in cortical porosity in the mutant mice starting at 3 weeks of age. In histological bone sections it appeared that there were more osteocytes in the femoral cortical bone. Dynamic histomorphometry did not show a difference in mineral apposition rate (MAR), but did show a significant decrease of mineralized surface in the trabecular compartment of mutant mice. Expression of bone marker genes is currently being studied. We believe that Dlx3 plays a role towards the end stages osteoblast differentiation pathway, hence downstream to Runx2 and osterix transcription factors.

Disclosures: M. Islam, None.

This study received funding from: National Institute of Health.

S029

OASIS, an Endoplasmic Reticulum Stress Transducer, Is Involved in Normal Bone Formation.T, Murakami¹, S. Kanemoto*¹, S. Kondo*¹, A. Wanaka*², K. Imaizumi*¹¹Division of Molecular and Cellular Biology, Department of Anatomy, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan, ²Department of Anatomy and Neuroscience, Nara Medical University, Nara, Japan.

OASIS (Old Astrocyte Specifically Induced Substance) is a basic leucine zipper (bZIP) transcription factor of the CREB/ATF family with a transmembrane domain that allows it to associate with the ER. In our previous report, we identified OASIS as a novel endoplasmic reticulum (ER) stress transducer in astrocytes. The molecule is cleaved at the membrane in response to ER stress, and its cleaved amino-terminal cytoplasmic domain, which contains the bZIP domain, translocates into the nucleus where it activates the transcription of target genes such as GRP78/BiP. To assess the role of OASIS in vivo, we generated OASIS knockout mice. OASIS-/- mice were viable, but exhibited growth retardation. In macroscopic observation on OASIS -/- mice, they exhibited deformity of limb bones and swellings in joint. Analysis of in situ hybridization showed that OASIS mRNA was detected not only in astrocytes but also in bones. From the results of histopathological examination of osseous tissues, OASIS deficiency resulted in abnormal bone formation with significant loss of bone mass. Electron microscopic analysis indicated the expansion of rough ER and accumulation of a large amount of secreted materials in the ER lumen in OASIS -/- osteoblast. In contrast, none of the structural changes were observed in osteocytes, osteoclasts and chondrocytes. These results suggested that abnormality of bone formation in OASIS -/- mice was caused by disturbance of production or secretion of bone matrix in osteoblasts. Next, we examined expression, production and secretion of bone matrix in primary osteoblast. OASIS proteins were highly expressed in primary osteoblasts. Secretion rate of osteopontin was decreased in -/- osteoblasts compared with WT, while that of osteocalcin was increased. These results showed that OASIS -/- osteoblasts were disturbed production and secretion of bone matrix. Taken together, these findings indicate that OASIS is required for normal bone formation and its deficiency causes osteogenesis imperfecta.

Disclosures: T. Murakami, None.

S031

Identification of a Novel Wnt/β-Catenin Response Element in the Osteoprotegrin Gene Promoter and its Regulation with Bone Morphogenetic Protein-2 Signaling.M. Sato*¹, A. Nakashima*¹, M. Nashimoto*², A. Ishisaki*¹, Y. Yavvaka*³, M. Tamura¹¹Biochemisrty and Molecular Biology, Grad. Sch. Dent. Med., Hokkaido University, Sapporo, Japan, ²Department of Applied Life Sciences, NUPALS, Niigata, Japan, ³Dentistry for Children and Disabled Person, Grad. Sch. Dent. Med., Hokkaido University, Sapporo, Japan.

Wnt/β-catenin signaling plays a role in the developing skeletal system. However, the exact mechanisms by which Wnt/β-catenin signaling regulates bone remodeling remain to be elucidated. Our previous studies demonstrated that an interaction between β-catenin and Smad was crucial for Wnt-mediated regulation of the BMP-2 responsive gene expression, and that BMP-2 up-regulated Wnt-induced lymphoid enhancing factor 1/T cell factor (LeflATcf)-dependent TopFlash transcriptional activity in the C2C12 cell line. Also, Wnt/β-catenin signaling regulates expression of osteoprotegerin (OPG) and receptor activator of NFkB ligand (RANKL) in osteoblasts. In this study, we investigated the mechanism of how OPG expression is induced by Wnt/β-catenin signaling in osteoblasts. OPG expression was induced by over-expression of Wnt3a or activated β-catenin in mesenchymal pluripotent C2C12 cells or osteoblastic MC3T3-E1 cells. In these cultures, BMP-2 synergistically enhanced OPG expression. Silencing of glycogen synthase kinase-3p by siRNA or sgRNA (tRNaseZL-utilizing gene silencing method) also increased OPG levels in the culture supernatant from C2C12 cells estimated by ELISA assay. To investigate the mechanisms of the Wnt/β-catenin signaling activated OPG gene transcription, we have cloned an approximately 1 5-kilobase pair genomic DNA fragment corresponding to the 5′-flanking promoter region of the murine OPG gene. Activated P-catenin results in a 10–15 fold increase in reporter gene transcription activity of the 1.5-kilobase fragment by transient transfection assay performed in C2C12 cells. Deletion analyses revealed that a proximal 253-base pair region in the promoter was required for Wnt/β-catenin responsiveness. In this region, we identified four putative Lefl/Tcf binding sites, which meet with a consensus sequence reported to be recognized by Lefl/Tcf proteins. Using site-directed mutation analyses, functional Lefl/Tcf binding site was identified in these regions. Smad protein was involved and interacted with the Wnt/β-catenin responsive element on the OPG promoter in response to BMP-2 stimulation. A chromatin IP assay indicated that β-catenin regulates transcription of OPG via a promoter region at their site. These results show that OPG is a target gene for Wnt/β-catenin signaling and that its induction is mediated by a novel Wnt/β-catenin response element of the OPG gene promoter.

Disclosures: M. Sato, None.

S033

Expression of a Hypomorphic Allele of FIAT (Factor Inhibiting ATF4-mediated Transcription) Improves Long Bone Mechanical Properties.V. W. C. Yu*, R. St-Arnaud. Genetics Unit, Shriners Hospital for Children, Montreal, PQ, Canada.

We have recently cloned and characterized FIAT, a 66 kDa leucine zipper protein that dimerizes with the basic domain-leucine zipper transcription factor ATF4 to form inactive dimers that cannot bind DNA. Overexpression of FIAT in osteoblasts of transgenic mice inhibited osteocalcin gene transcription and reduced osteoblastic activity, leading to osteopenia (J Cell Biol 169: 591; 2005). Reciprocally, inhibition of FIAT expression in osteoblasts, using siRNA technology, results in a higher transcriptional activity from the osteocalcin promoter, augmented collagen type I secretion, and enhanced mineralization. Together, these results support the role of FIAT in regulating ATF4 activity in osteoblasts and confirm the importance of FIAT in the modulation of osteoblast activity. To further assess the physiological role of FIAT, we have used the technique of homologous recombination in ES cells to engineer an allele of FIAT in which the first exon, containing the translational start site, was flanked by lox P sites. Mice carrying the floxed FIAT allele were crossed to transgenic mice expressing the Cre recombinase under the control of the collagen type I promoter (Col I-Cre) for osteoblast-specific inactivation. Since the FIAT gene maps to the × chromosome, initial characterization of the mutant phenotype was performed in Col I-Cre;FIAT^f/yl male mice. Mutant mice showed only 20% reduction of FIAT expression as measured by RT-qPCR, suggesting that we have engineered a hypomorphic allele and not a true null. The reduction in FIAT expression was statistically significant, however. Despite this moderate reduction in FIAT mRNA levels, the mutant mice showed significantly increased osteocalcin gene transcription and a trend towards increased bone volume. When the biomechanical properties of the mutant bones were tested using three-point bending, we measured significant increases in ultimate force and work to failure. These results show that even a small reduction in FIAT expression significantly improves long bone mechanical properties and suggest that FIAT represents an interesting target for therapeutic intervention to increase bone strength in osteoporotic patients.

Disclosures: R. St-Arnaud, None.

This study received'funding from: NIH-NIAMS.

S036

Mechanical Loading Induced Wnt/β-catenin Signaling Is Compromised in ERα-/- Mouse Tibia- In Vivo Microarray Analysis.G Zaman*, L. H Saxon, M. Muzylak*, J. S. Price, L. E. Lanvon. Basic Sciences, The Royal Veterinary College, London, United Kingdom.

Postmenopausal osteoporosis represents a failure of bone cells to adapt bone mass and architecture to withstand loading without fracture. A number of studies have shown that the adaptive response of bone to mechanical loading is deficient in the absence of estrogen receptor associated with estrogen deficiency. Wnt/β-catenin signalling is one of the pathways shown to regulate bone formation during growth and in response to mechanical load.

In order to identify the genes and signal pathways responsible for the loading-related osteogenic response, we used the RNG-MRC 25k mouse microarray analysis to compare differential gene expression in the right and left (loaded, 12N at 2Hz for 60 cycles and not loaded) tibiae of WT and ERα-/- mice after a single period of dynamic axial loading. This transient exposure to dynamic strain change has been shown to produce an osteogenic response in mice.

RNA was extracted at 3, 8, 12 and 24 hours after loading from control and loaded tibiae. In WT mice there were 663, 539, 173 and 33 differentially regulated genes at 3, 8, 12 and 24 hours respectively after loading. Lists of differentially regulated genes were loaded into Ingenuity Pathways Analysis software. Canonical pathway analysis of the sets of genes differentially regulated showed that a number of catabolic and anabolic pathways were affected. Six genes previously reported to be involved in the Wnt/β-catenin pathway (WNT2B, FZD2, GNAQ, PPP2R3A, CREBBP, RARγ) were differentially expressed in loaded versus control bones 3 hours after treatment (expression of 10 genes changing over a period of 24 hours). This differential expression was less evident at 8 and 12 hours after loading and returned to the levels observed in the contra-lateral control limb 24 hours after treatment

In ERα-/- mice the number of differentially regulated genes was much lower at each time point after loading (17, 122 and 8 genes at 3, 8 and 24 hours respectively). Only one Wnt/β-catenin related gene (RARγ) in ERα-/- mice showed a change in its expression between loaded and control bones at 3 hours and none at 24 hours. The gene expression profile in response to mechanical loading in WT mice confirms the regulation of a number of genes involved in Wnt/β-catenin signalling. The reduction in the number of Wnt/β-catenin-related genes differentially regulated by loading in the absence of ERα suggests a role for ERa in early loading-related activation of the Wnt/β-catenin signalling in bone cells. Diminished effectiveness of loading-related Wnt/β-catenin activity due to reduced ERα activity, as would occur with estrogen deficiency, may contribute to less effective control of structurally appropriate bone mass in osteoporosis in both men and women.

Disclosures: G. Zaman, None.

This study received funding from: The Wellcome Trust.

S039

Prostaglandin E2 (PGE2) Increases a Specific Subset of Primitive Hematopoietic Cells In Vivo.B. J. Frisch*, B. J. Gigliotti*, J. M. Weber*, R. J. O'Keefe, C. T. Jordan*, L. M. Calvi. University of Rochester School of Medicine, Rochester, NY, USA.

Parathyroid Hormone (PTH) stimulates osteoblastic cells (OBs) in the bone marrow (BM) microenvironment, expanding hematopoietic stem cells (HSCs) through activation of the Notch receptor. Similarly, we previously demonstrated that PGE2 increases the Notch ligand Jaggedl in OBs, and does so through Protein Kinase A (PKA) activation. Therefore, we hypothesized that PGE2 could also expand HSC in vivo. To test whether PGE2 regulates HSC, we treated mice with intermittent intraperitoneal PGE2 or vehicle. Several schedules were tested to maximize the PGE2 effect on the HSC-enriched lineage-Sca-1+ c-kit+ (LSK) cells in BM. At the end of each treatment, peripheral blood (PB) was analyzed and mice were euthanized (n=8 mice per treatment group). BM was harvested from one of the hindlimbs, while the contralateral limb was fixed for microCT and histologic analysis. Flow cytometric analysis demonstrated a time and dose dependent increase in BM LSK, which was maximal at day 16 with bid PGE2 vs vehicle (0.11 vs 0.04% BM mononuclear cells, P=0.0061). At this dose, there was no significant PGE2-dependent bone anabolic effect either histologically or by micro-CT. Maximal LSK expansion was superior to PTH-dependent HSC stimulation (approximately 350 vs 100% increase in LSK). When the more mature hematopoietic progenitor population was quantified using the CFU-C assay in vitro, there was no difference between PGE2- and vehicle -treated animals. Similarly, there were no significant PGE2 effects on Hct, Pits or WBC counts compared to vehicle. Therefore PGE2-dependent cell expansion was not global across differentiated subsets, but was restricted to primitive hematopoietic cells, similar to the effects of PTH in vivo. Competitive transplantation of irradiated recipients was then performed to assess the engraftment, proliferation, and self-renewal properties which define HSC. Cells derived from PGE2-treated mice demonstrated superior contribution to all hematopoietic lineages at 3 weeks after transplantation (33.3 vs 23.5% PB mononuclear cells, P=0.0045), but this increase was not persistent at 12 weeks. This surprising result suggests that PGE2 selectively expands a subpopulation of HSC, the short-term HSCs (or ST-HSCs), which have highly proliferative properties, but limited self-renewal. Further studies will determine if this effect is mediated by osteoblastic Jaggedl or by other niche components. This finding implicates for the first time the existence of specialized niches regulating subsets of HSC. These novel data therefore increase our understanding of the HSC niche, which could be exploited clinically for specifically targeted ST-HSC expansion.

Disclosures: B.J. Frisch. None.

This study received funding from: N1DDK, Pew Foundation, Wilmot Cancer Research Fellowship.

S041

Cell Cooperation Between Human OsteoProgenitor Cells and Endothelial Cells in Tissue Engineering.M. Grellier*¹, C. Bourget*¹, R. Bareille*¹, P. L. Grania*², M. A. Barbosa*², J. Amedee¹¹INSERM U577 - University V. Segalen Bordeaux 2, Bordeaux, France, ²INEB - Instituto de Engenharia Biomédica - University of Porto, Porto, Portugal.

Bone is a dynamic tissue that constantly undergoes remodelling which requires interactions between bone cells. However, the intimate association of the vascular endothelium with osteogenic cells appears necessary to modulate bone formation and degradation balance. The purpose of this work is to study the cell communication between osteoblast and endothelial cells before to apply this strategy for bone tissue engineering.

To investigate that, Human OsteoProgenitor (HOP) and Human Umbilical Vein Endothelial Cells (HUVEC) were co-cultured in direct contact and compared to isolated cultures. We analysed the regulation of cell communication, studying the expression of Connexin 43 and VE-cadherin, by Quantitative-PCR and the localization of beta-catenin, by immunostaining. We investigated genes involved in extracellular matrix organisation (ECM) such as MMP-1, TIMP-1, VEGF and TGFbeta-1. We followed cell migration by time lapse. We used an extrusion system to encapsulate cells inside beads of alginate.

After 24 hours of co-culture, HOP and HUVEC form specific multicellular networks which suggest an ECM reorganisation and cell migration. The expression of Connexin 43 is up-regulated while VE-cadherin expression is down regulated in co-culture compared to HUVEC culture. We observed a modification of the beta-catenin distribution when cells are in direct contact: beta-catenin is cytoplasmic in HOP culture and localised to the membrane after 6 hours of co-culture while its expression, analysed by western blot, was unchanged. MMP1 and TIMP1 results suggest that the co-culture could enhance a production of an ECM and both VEGF and TGFbeta-1 are upregulated in co-culture. We observed by time lapse that HUVEC are able to migrate through HOP filopodes.

These results suggest that ECM remodelling occurred when endothelial and osteoblastic cells are in direct contact. This mechanism could be due either to a regulation of cell communication between HOP and HUVEC or/and to a production of growth factors involved in cell migration and ECM remodelling. A better knowledge of this cell to cell cross-talk will be helpful for the development of a vascularised bone tissue for tissue engineering. Preliminary data obtained using alginate microspheres revealed that these cells entrapped inside such beads are still able to proliferate after one week of culture and their implantation into a critical size bone defect allows us to evaluate this newly bone regeneration strategy.

Disclosures: M. Grellier, None.

S043

Progressive Ankylosis Gene (ank) Regulates Osteoblast Differentiation.T. Kirsch¹, H. Kim¹, J. A. Winkles*²¹Orthopaedics, University of Maryland School of Medicine, Baltimore, MD, USA, ²Surgery, University of Maryland School of Medicine, Baltimore, MD, USA.

The progressive ankylosis gene (ank) is a transmembrane protein that transports intracellular pyrophosphate (PP_i) to the extracellular milieu. Human mutations of ank lead to craniometaphyseal dysplasia (CMD), a disease, which is characterized by the overgrowth of craniofacial and long bones, suggesting that Ank plays a role in the regulation of osteoblast differentiation. To determine the role of Ank in osteoblast differentiation, we suppressed Ank expression in osteoblastic MC3T3 cells using siRNA or studied the osteoblastic differentiation of bone marrow stromal cells isolated from the bone marrow of ank/ank mice, which express a truncated, non-functional Ank protein, or wild type littermates. MC3T3 cells mineralized within 20 days after the addition of vitamin C and β-glycerophosphate. Ank gene and protein expression in these cells was the highest at 3 days and declined afterwards, whereas alkaline phosphatase (APase) expression and activity increased on day 8 and was the highest between day 17 and day 20. Suppression of Ank expression in MC3T3 cells using siRNA led to a decrease of APase expression and activity. Suppression of Ank expression in these cells also resulted in a decrease of the expression of bone-related genes, including type I collagen, bone sialoprotein, and osteocalcin. In addition, the expression of the transcription factor osterix was downregulated in Ank expression suppressed MC3T3 cells, whereas runx 2 expression in these cells was upregulated. Bone marrow stromal cells isolated from bone marrow of ank/ ank mice and wild type littermates were cultured in the presence of 100 μg/ml ascorbate phosphate for various days and analyzed by staining for APase enzyme activity and van Kossa to determine mineralized colonies. The number of APase activity positive and van Kossa stained colonies was markedly reduced in bone marrow stromal cell cultures from ank/ank mice compared to the number of APase activity and van Kossa positive colonies in bone marrow stromal cell cultures from wild type littermates. These findings suggest that Ank is a positive regulator of differentiation events towards a mature osteoblastic phenotype and Ank appears to act upstream of osterix. Loss of Ank function in osteoblastic precursor cells results in an arrest of cells in a premature osteoblastic stage, in which they express high levels of runx2 and low levels of osterix.

Disclosures: T. Kirsch, None.

This study received Junding from: National Institutes of Health.

S045

Wnt5A Signals Through Ror2 to Promote Osteoblastogenesis.Y. Liu, B. Rubin*, P. V. N. Bodine, J. Billiard. Women's Health and Musculoskeletal Biology, Wyeth Research, Collegeville, PA, USA.

Wnts are a large family of growth factors that regulate important cellular events by signaling through both canonical/beta-catenin pathway and non-canonical pathways. While activation of canonical Wnt signaling has been implicated in osteoblast physiology, very little is known about the role of non-canonical pathways in osteoblasts. Ror2 is an orphan receptor tyrosine kinase that induces osteogenic commitment and differentiation of pluripotent mesenchymal stem cells (MSCs). We and others have shown that both canonical and non-canonical Wnts bind Ror2, but their ability to activate Ror2 signaling has not been examined. Here we tested if canonical or non-canonical Wnts induce downstream signaling of the Ror2 receptor and lead to increased osteogenesis. We used purified recombinant non-canonical Wnt5A and canonical Wnt3A that bound Ror2 equally well in pull-down assays. We found that Wnt5A, but not Wnt3A, induced homo-dimerization of Ror2 and stimulated tyrosine phosphorylation of the Ror2 substrate 14–3–3β scaffold protein. Treatment of pluripotent human MSCs with Wnt5A dose-dependently induced formation of the mineralized extracellular matrix, which is the ultimate phenotype of an osteogenic tissue. Down-regulation of Ror2 expression by short hairpin RNA greatly inhibited Wnt5A-induced mineralization, indicating that Wnt5A effect is mediated at least in part through activation of the Ror2 receptor. Finally, using mouse calvarial ex vivo organ culture model, we demonstrated that treatment with Wnt5A results in significantly increased bone formation in this complex system.

In summary, we identify Wnt5A as the ligand that activates the Ror2 receptor tyrosine kinase signaling pathway and promotes osteogenic commitment and differentiation of pluripotent stem cells.

Disclosures: Y. Liu, Wyeth 3.

This study received funding from: Wyeth.

S047

Growth Inhibition of Osteoblast Progenitors by Runx2 Depends on Its C-Terminal Regulatory Domain and Is Accompanied by Changes in G-Protein Coupled Signaling Pathways.N. Teplyuk, M. Galindo*, V. Teplyuk*, D. W. Young*, D. Lapointe*, M. van der Deen*, J. Pratap, S. K. Zaidi*, A. Javed, J. L. Stein*, J. B. Lian, G. S. Stein, A. J. van Wiinen. Department of Cell Biology and Cancer Center, University of Massachusetts Medical School, Worcester, MA, USA.

The runt-related transcription factor Runx2 mediates osteogenic cell fate determination, as well as stimulates anabolic activities and inhibits the proliferative potential of osteoblasts. The levels of Runx2 change during the cell division cycle, and preferential expression of Runx2 in G1 may control the responsiveness of osteoblasts to extracellular signals that influence commitment to enter a new proliferative cycle. Runx2 elicits biological responses in osteoblasts by acting as the end-point effector of multiple signaling axes, including the Src/Yes-Yap, TGFβ/BMP-Smad and CDK-cyclin pathways, which each converge at nuclear matrix-associated transcriptional sites. Here we addressed what functions of Runx2 protein are required for cell growth suppression, and what genes are regulated by Runx2 to control osteoblast growth. We first examined the biological phenotypes of osteoblastic progenitors upon introduction of Runx2 proteins with mutations that affect interactions with co-factors that transduce cell signals and/or control transcriptional activation or repression. These Runx2 point mutants were introduced into Runx2 null mouse calvarial cells using adenoviral vectors that permit efficient transient expression of Runx2 proteins. Wild type Runx2 or point mutants that can not interact with Yap or Smad or the nuclear matrix each inhibit growth of Runx2 null cells. Thus, several key signaling pathways are dispensable for growth suppression by Runx2. A point mutation in the runt-homology DNA binding domain establishes that cell growth control requires direct regulation of putative Runx2 target genes. Deletion of the C-terminal VWRPY repressor domain reduces but does not abolish growth suppressive potential indicating that both gene activation and repression contribute to the anti-proliferative effect of Runx2. Affymetrix gene expression profiling shows that the C-terminus of Runx2 is required for the induction of its classical targets (e.g., osteocalcin, osteopontin, MMPI3), but also a number of genes involved in cell cycle related pathways, including multiple components of G-protein coupled receptor signaling. Because defects in Gs-signaling have been linked to fibrous dysplasia (FD), our data suggest that Runx2 may contribute to the hyper-proliferation of mesenchymal cells characteristic of FD.

Disclosures: A.J. van Wijnen, None.

S049

Regulation of Osteoblast Differentiation by MicroRNA.H. Inose¹, S. Takeda², A. Kirnura*², S. Sato*¹, K. Shinomiva*¹¹Department of Orthopaedics, Tokyo Medical and Dental University, Tokyo, Japan, ²21COE,Tokyo Medical and Dental University, Tokyo, Japan.

MicroRN As (miRNA) regulate the expression of genes by binding and modulating the translation of specific mRNAs. They have been shown to be critical in the development of organisms and cell proliferation. However, their role in osteoblast differentiation remains to be elucidated. In this study, we aimed to identify miRNAs expressed in osteoblasts and investigate their role in osteoblast differentiation.

C2C12 cells were induced to differentiate into osteoblasts by BMP2 treatment. Using miRNA microarray, we performed comprehensive analysis of miRNAs. Most of the known miRNAs were expressed in C2C12 cells. Among these, we focused on several miRNAs whose expression was reduced during the course of osteoblast differentiation as potential negative regulators for osteoblast differentiation. We confirmed that they were also expressed in primary osteoblasts and their expression was downregulated along differentiation. To address their physiological role, we manipulated their expression in osteoblasts and examined the effect. Indeed, forced expression of those miRNAs in primary osteoblasts significantly repressed osteoblast differentiation as demonstrated by reduced alkaline phosphatase activity and expression of osteoblastic genes. Conversely, inhibition of the activity of those miRNAs by antisense miRNA oligonucleotide promoted osteoblast differentiation. We further examined their potential target using in silico approach and verified that those candidates are, bona fide, target genes of the miRNAs by in vitro experiments. Taken together, our results suggest that miRNA is a physiological regulator of osteoblast differentiation and manipulating their activity would lead to a development of a novel therapy for increasing bone formation.

Disclosures: H. Inose, None.

S052

Mesenchymal Stem Cells from Aging C57BL/6 Exhibit Increased Oxidative Stress and Defective Replication: Correction of Both by Provision of an Extracellular Matrix from Young Mice.X. Chen, C. M. Skinner*, E. Ambrogini*, L. Han, M. Almeida, S. C. Manolagas, R. L. Jilka. Center for Osteoporosis and Metabolic Bone Diseases, Central Arkansas Veterans Healthcare System, Univ. of Arkansas for Medical Sciences, Little Rock, AR, USA.

The supply of osteoblasts depends on the replication of mesenchymal stem cells (MSCs) and their commitment to the osteoblast lineage. It has been shown previously that a cell-free extracellular matrix (ECM) made by marrow stromal cells greatly increases the frequency of MSCs by promoting their replication and restraining their differentiation. And, it has been reported that age-related bone loss in C57BL/6 mice is associated with decreased osteoblast number and bone formation, as well as increased oxidative stress in the bone marrow. Here, we have examined whether aging and oxidative stress negatively impact the number and ex vivo replication of MSCs using femoral marrow cells from 3-month old (young) and 18-month old (old) female C57BL/6 mice. We report that the number of MSCs in marrow from old mice, measured by their ability to form a colony of osteoblastic cells (CFU-OB), was 5–10% lower as compared to young mice. Moreover, MSCs from old mice exhibited a replication defect during 6 days of culture on plastic, as measured subsequently upon re-plating and determination of CFU-OBs. Thus, whereas CFU-OB from young mice increased during this period by 1.8 fold, MSCs from old mice did not. As a result, the frequency of CFU-OB in the expanded population of cells from old mice was only 30% of that of young mice. In parallel cultures of marrow cells maintained for 6 days on an ECM made by marrow stromal cells from 2 month old mice, the number of CFU-OB from both young and old mice increased indistinguishably (14±2, and 16±2 fold, respectively). Further, the intracellular level of reactive oxygen species (ROS), quantified using dichlorodihydrofluorescein diacetate, was 20% higher in cultures from old mice, as compared to young, when the cultures were performed on plastic. Fascinatingly, ROS levels in cultures from young and old mice were reduced by 30% and 50%, respectively, when cells were maintained on the ECM. Consistent with these ex vivo results, oxidative stress induced by administration of buthionine sulfoxide to 6-month old C57BL/6 mice for 4 weeks reduced femoral CFU-OB number by 30–50%. Based on these findings, we hypothesize that the increased oxidative stress associated with old age exhausts a limited pool of MSC osteoblast progenitors, and that the old ECM itself, and/or the factors embedded in it, may contribute to the increased oxidative stress. Moreover, we suggest that culture of aging MSCs on an ECM may improve their number and quality, thereby optimizing the effectiveness of autologous MSC administration for therapeutic applications.

Disclosures: X. Chen, None.

S056

AMP-kinase Activator (AICAR) and Rho-kinase Inhibitor (hydroxyfasudil) Induce the Differentiation and Mineralization of Osteoblastic MC3T3-E1 Cells via Inhibiting the Mevalonate Pathway and Enhancing BMP-2 Expression.I. Kanazawa*, T. Yamaguchi, S. Yano, M. Yamauchi, M. Yamamoto, T. Sugimoto. Internal Medicine 1, Shimane University Faculty of Medicine, Izumo, Japan.

AMP-activated protein kinase (AMPK) and Rho kinase (ROK) have recently attracted widespread attention due to their beneficial anti-diabetic and anti-atherosclerotic effects. AMPK is known to modulate the mevalonate pathway via suppressing HMG-CoA reductase, and ROK comprises this pathway and acts in its downstream. Although the inhibition of the mevalonate pathway by statins is well known to stimulate bone formation via enhancing BMP-2 expression, the effects of AMPK activator and ROK inhibitor on bone metabolism by modulating the pathway are still unclear. We investigated the effects of pharmacological AMPK activator (5-amino-imidazole-4-carboxamide-riboside; AICAR) and ROK inhibitor (hydroxyfasudil; HF) on the differentiation and mineralization of osteoblastic MC3T3-E1 cells. AMPK activation by AICAR (0.1 −0.5mM) was confirmed by Western blot. AICAR significantly stimulated ALP activity, and promoted mRNA expressions of both collagen-I and osteocalcin (OC) by real-time PCR as well as mineralization by von Kossa and Alizarin Red stainings. Moreover, AICAR significantly stimulated BMP-2 mRNA expression in dose- and time-dependent manners. Simultaneous addition of either mevalonate or geranyl-geranyl pyrophosphate (GGPP), which also comprise the mevalonate pathway and act in the downstream of HMG-CoA reductase, significantly antagonized BMP-2 and OC mRNA expressions and mineralization enhanced by AICAR. On the other hand, inhibition of ROK by HF (10–5– 10–4M) also stimulated OC mRNA expression and mineralization through enhancing BMP-2 mRNA expression in dose- and time-dependent manners. In conclusion, the suppression of mevalonate pathway by AICAR and HF promoted the differentiation and mineralization of osteoblastic MC3T3-E1 cells via enhancing BMP-2 expression. Our present findings suggest that modulation of the mevalonate pathway might be useful for not only creating anti-diabetic or anti-atherosclerotic drugs but also developing new drugs promoting bone formation for the treatment of osteoporosis.

Disclosures: I. Kanazawa, None.

S058

Molecular Mechanisms of Lysophosphatidic Acid-Stimulated Osteoblastic Cell Chemotaxis.S. A. Karagiosis*, L. K. Opresko*, W. B. Chrisler*, N. J. Karin. Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA, USA.

Our focus is the understanding of molecular mechanisms of lysophosphatidic acid (LPA)-induced osteoblastic cell chemotaxis. We hypothesize that LPA exerts anabolic effects on bone mass through the stimulation of pre-osteoblast migration, and that LPA is therefore a factor that supports bone repair. Osteoblast progenitor cells must migrate to injury sites where they elaborate mineralized matrix in the compromised region. LPA is released by activated platelets at the site of injury, and we have shown that this lipid factor is a potent chemotactic agent for MC3T3-E1 pre-osteoblasts [Masiello et al. (2006) Bone 39:72–82]. LPA-stimulated MC3T3-E1 cells exhibit dramatic changes in morphology and F-actin architecture, including robust stress fiber formation. We investigated the role of phosphatidylinositol-3 kinase (PI3K), which is essential in many other cells for driving the initial cytoskeletal rearrangements and cell polarization required for cell locomotion. We found a dose-dependent reduction in LPA-induced migration upon treatment with the PI3K inhibitor, LY294002. The distribution of two actin regulatory proteins, zyxin and arp2/3, was also consistent with our hypothesis. Zyxin was enriched at the newly-formed stress fibers in LPA-treated pre-osteoblasts and to focal adhesions and filopodia tips. The arp2/3 complex immunolocalized to sites of dynamic F-actin assembly in lamellipodia and filopodia. The propagation of ERK1/2 activation is required for the coordinated-migration of cells during epithelial wound healing [Matsubayashi et al. (2004) Curr. Biol. 12:731–735]. MC3T3–E1 cell ERK1/2 exhibited two waves of phosphorylation upon LPA stimulation: a rapid transient wave and a more sustained wave. Using live cell imaging of osteoblasts expressing low levels of ERK1-GFP, we observed ERK1 nuclear translocation upon LPA addition. LPA-induced cell motility was not blocked by an inhibitor of ERK1/2 activation. Additionally, we demonstrate that LPA-induced ERK1/2 activation and migration are independent of EGF receptor transactivation in osteoblasts. We conclude that PI3K-associated cytoskeletal changes, but not ERK1/2 phosphorylation or EGF receptor transactivation, are essential for LPA-stimulated MC3T3-E1 cell migration.

Disclosures: S.A. Karagiosis, None.

This study received funding from: U.S. Department of Energy.

S060

Proliferation and Differentiation of Periosteal Osteobalst Progenitors Are Differentially Regulated by Sex Steroids and Intermittent PTH Administration.M. Ogita, E. Dworakowski*, J. P. Bilezikian, S. Kousteni. Division of Endocrinology College of Physicians & Surgeons, Department of Medicine, Columbia University Medical Center, New York, NY, USA.

The periosteum is recognized as a homeostatic and therapeutic target for actions of sex steroids and intermittent parathyroid hormone (PTH) administration. However, the mechanisms by which estrogens suppress but androgens and PTH promote periosteal expansion are not known. In this report, we show that intermittent PTH(1–34), within 16 days of treatment, upregulates the expression and activity of alkaline phosphatase (AP) in periosteal cells isolated from the surface of murine or rat long bones. Similar to PTH, dihydrotestosterone (DHT) dose-dependently stimulates AP activity. The stimulatory action of PTH or DHT on AP activity is suppressed by inhibitors of ERKs, BMP-2 and Wnt signaling. In contrast, l7β-estradiol (E₂) has no effect by itself; but dose-dependently attenuates PTH- or BMP-2-induced stimulation of AP expression or activity. E₂ also attenuates the stimulatory effect of PTH or BMP-2 on AP, osteocalcin, as well as Smad6 and axin2 expression in primary periosteal cells. In agreement with these in vitro observations, administration of intermittent PTH (40 ng/g) or a bone protective dose of DHT (300 ng/g) to ovariectomized adult C57B1/6 mice, induces, within 1 hour, phosphorylation of Smad 1/5/8 in the periosteum as assessed by western blot analysis of femoral periosteal lysates. A replacement dose of E₂ (30 ng/g) has no effect by itself but suppresses PTH-induced phosphorylation of Smads. None of the 3 hormones affect Smad phosphorylation in trabecular bone from the femurs of the same experimental animals. In contrast to its pro-differentiating effects, PTH suppresses proliferation of primary periosteal osteoblast progenitors. E₂, not only promotes proliferation, but attenuates the anti-proliferative effect of PTH. Finally, all 3 hormones protect periosteal osteoblasts from apoptosis induced by etoposide, TNFα or dexamethasone. These observations suggest that the different effects of sex steroids and PTH on the periosteum result from opposing actions on the recruitment of early periosteal osteoblast progenitors. Intermittent PTH and androgens promote osteoblast differentiation from periosteum-derived mesenchymal progenitors by actions that require ERK, BMP and Wnt signaling. Estrogens promote the expansion of early osteoblast progenitors but inhibit their differentiation by osteogenic agents such as PTH or BMP-2. The property of estrogens to increase periosteal osteoblast precursor number, yet retain them in an undifferentiated state may reflect actions of the hormone on a cell population distinct from that affected by PTH.

Disclosures: S. Kousteni. Radius 1.

S062

P2×7 Receptors on Osteoblasts Couple to Production of Lysophosphatidic Acid: A Novel Signaling Axis Promoting Osteogenesis.N. Panupinthu*¹, J. T. Rogers*¹, L. Zhao*², F. Possmaver*², S. M. Sims*¹, S. J. Dixon¹¹CIHR Group in Skeletal Development and Remodeling, The University of Western Ontario, London, ON, Canada, ²Obstetrics & Gynaecology, The University of Western Ontario, London, ON, Canada.

Nucleotides are released in response to mechanical stimuli and signal through P2 receptors. P2×7 receptors are ATP-gated cation channels that, under certain conditions, induce formation of large membrane pores. Targeted disruption of the gene encoding the P2×7 receptor diminishes periosteal bone formation and impairs response of the skeleton to mechanical loading. Our goal was to investigate the pathways underlying the actions of P2×7 receptors on osteogenesis. Benzoylbenzoyl-ATP (BzATP, an agonist more potent than ATP at P2×7 receptors) induced pore formation in live cells on the surface of calvariae from wild-type (WT), but not P2×7 receptor knockout (KO) mice. When rat calvarial cells were differentiated to form bone-like nodules in vitro, BzATP induced pore formation in cells associated with these nodules. These data indicate that functional P2×7 receptors are present in cells of the osteoblast lineage. BzATP, but not equivalent concentrations of UTP (P2Y agonist) or inorganic phosphate, accelerated the appearance of alkaline phosphatase activity and promoted mineralization in rat calvarial cell cultures. Moreover, expression of genes encoding osterix and osteocalcin was significantly enhanced in BzATP-treated cultures. Next, calvarial cells from WT and KO mice were differentiated in the absence of exogenous nucleotides. Alkaline phosphatase activity and mineralization were markedly reduced in KO compared to WT cultures, without significant difference in cell numbers. Transcript levels of osterix, bone sialoprotein and osteocalcin were also suppressed in KO compared to WT cultures. These changes were accompanied by increased levels of the adipocyte markers PPARγ and lipoprotein lipase. Using thin layer chromatography, we found that BzATP activates production of the potent lipid mediator lysophosphatidic acid (LPA) through activation of phospholipases D and A₂. The specific LPA receptor antagonist VPC-32183 suppressed LPA-induced elevation of cytosolic free calcium in rat calvarial cells. Importantly, the same concentration of VPC-32183 abolished the stimulatory effects of BzATP on mineralization and expression of osterix and osteocalcin. We conclude that P2×7 receptors on osteoblasts enhance bone formation through a cell autonomous mechanism. Furthermore, a novel signaling axis links P2×7 receptors to production of LPA, which in turn stimulates osteogenesis. These findings explain the role of P2×7 receptors in skeletal development and mechanotransduction.

Disclosures: N. Panupinthu, None.

This study received funding from: The Canadian Institutes of Health Research (CIHR).

S064

Estrogen Receptor α Is Required for Strain-Related (β-Catenin Signaling in Osteoblasts.V. J. Armstrong*, M. Muzylak*, A. Sunters*, G. Zaman*, L. K. Saxon, J. S. Price, L. E. Lanvon. Veterinary Basic Sciences, The Royal Veterinary College, London, United Kingdom.

Wnt/β-catenin signaling has been implicated in the regulation of bone mass through its involvement in bone cells' response to their mechanical environment [1]. Since Estrogen Receptor a (ERα) is also involved in bones' response to loading [2], we investigated whether strain induced signaling through ERα uses the same pathway as β-catenin.

Western blot and immuno-cytochemical analysis showed that in ROS 17/2.8 cells a short period of dynamic strain in vitro increased the levels of activated β-catenin (^aβcat) in the cytoplasm and within 1 hour stimulated its translocation to the nucleus. These changes in ^aβcat were paralleled by inhibitory phosphorylation of GSK-3β. Strain, and the GSK-3β inhibitor LiCI, also induced a significant increase in TCF/LEF transcriptional activity. In contrast, estrogen had no influence on the level or distribution of ^aβcat, nor any effect on TCF/LEF activation. Nuclear translocation of ^aβcat and TCF/LEF activation stimulated by both strain and LiCI were inhibited by the ER modulator ICI 182,780, which also reduced strain-induced nuclear accumulation of ERα. The ER modulator Tamoxifen also inhibited LiCI stimulated nuclear translocation of ^aβcat.

In primary cultures of osteoblast-like cells derived from the long bones of Wild Type mice and those lacking ERβ, β-catenin was similarly activated and translocated to the nucleus in response to strain and LiCI. In these cells this response was blocked by ICI 182,780. In contrast, in cultures of osteoblast-like cells from mice lacking ERα neither strain nor LiCI stimulated nuclear accumulation of ^aβcat. ICI 182,780 had no effect in these cells.

These data show that in osteoblastic cells exposure to strain causes similar activation of β-catenin, its translocation to the nucleus, and regulation of transcription as GSK-3β inhibition by LiCI. These changes require ERα but not ERβ. To our knowledge these are the first data to demonstrate that in osteoblasts ERα is required for these cells' responses to strain involving Wnt/β-catenin. Reduced effectiveness of bone cells' responses to bone loading, associated with decline in bio-available estrogen and ERα, may contribute to the failure to maintain structurally appropriate bone mass in osteoporosis in both men and women. This failure may in part be due to reduced effectiveness of Wnt/β-catenin signaling.

1. Sawakami K, et al (2006) J Biol Chem 281(33):23698

2. Lee K, et al (2003) Nature 424:389

Disclosures: V.J. Armstrong, None.

This study received funding from: BBSRC.

S068

Effects of PTH(1–34) on Primary Osteocytes.S. Baneriee*¹, S. Shaheen*¹, S. E. Harris², F. R. Bringhurst¹, P. Divieti¹¹Endocrine Unit, Massachusetts General Hospital, Boston, MA, USA, ²Department of Periodontic, University of Texas Health Center, San Antonio, TX, USA.

Parathyroid hormone (PTH) induces a plethora of effects on bone cells, depending on the mode and timing of administration. While continuous PTH in vivo induces catabolic effects, including rapid bone turnover, intermittent treatment increases bone mass (anabolic effect). At the cellular level, continuous treatment with PTH inhibits osteoblast differentiation while intermittent treatment promotes it. Osteocytes play a major “mechanosensory” role and PTH is known to influence the output of this mechanosensoiy response and to delay osteocyte apoptosis. PTH exerts its classical effects via activation of a G-protein coupled receptor, the PTH/PTHrP receptor (PPR) that recognizes and is fully activated by the first 34 amino acids of the hormone. Osteocytes also express CPTHRs, which recognize amino-truncated forms of the hormone.

In this study we compared the effects of continuous vs intermittent activaton of PPRs in osteocytes in vitro. Cells were isolated enzymatically from calvariae of 4-day-old transgenic mice in which the green fluorescent protein (GFP) is driven by the 8Kb promoter of dentin matrix protein 1 (DMP1). Primary bone cells cultured for 10 days before sorting them for GFP+ (osteocytes) and GFP- (predominantly osteoblasts) populations.

We have previously shown that both PPRs and CPTHRs were expressed in GFP+ cells and that their expression was 2–3 fold higher than in GFP- cells, suggesting that both PPRs and CPTHRs might play an important role in osteocyte functions. Cyclic AMP accumulation in response to PTH(1–34) was also present in these cells. The expression of selected osteocytic marker proteins - Sclerostin (SOST), DMP1 and the membrane mucoprotein Podoplanin (Ell) was analyzed following intermittent vs continuous PTH treatment in vitro. Unsorted calvarial cells were treated with lOnM PTH(1–34) for 2 hr every second day (intermittent) or continuously (refed with fresh PTH every 2 days) for 10 days before FACS analysis. Continuous PTH reduced the percentage of GFP+ cells, increased expression of SOST and reduced that of DMP1 and E11 in the GFP+ population. Intermittent PTH decreased SOST and Ell but increased DMP1. We conclude that terminal differentiation of osteoblasts is inhibited by continuous PTH exposure in vitro and that the osteocyte phenotype is sensitive to different temporal patterns of PTH exposure.

Disclosures: P. Divieti, None.

This study received funding from: NIH.

S073

Rho GTPase Signaling Pathways in Osteoclasts: Wrchl/rhou Role In Cell Adhesion and Osteolysis.A. Blangy, H. Brazier*, S. Orv*, S. Stephens*, G Pawlak*. CNRS UMR 5237 CRBM, CNRS Montpellier France

Rho GTPase signaling pathways regulate essential apects of osteoclast biology through the regulation of actin cytoskeleton dynamics. In particular they control osteoclast adhesion by regulating podosome organization and sealing zone architecture, thereby modulating osteolysis. We established the expression pattern of all Rho GTPases and Rho GTPase activator during osteoclast differentiation, identified novel genes induced during osteoclastgenesis and confirmed their importance in osteoclast by retrovirus-mediated RNA interference (Brazier et al., 2006, JBMR, 21(9) 1387–1398).

In particular we reported that the expression of Wrch1/RhoU is higly induced during osteoclastogenesis but absent in macrophages, suggesting an specific role for the GTPase in osteoclasts. Using retrovirus-mediated infection of osteoclast precursors, we now report that Wrch1/RhoU localizes to osteoclast adhesion structures: it colocalized with vinculin around isolated podosomes and the podosome belt. Furthermore, when osteoclasts were seeded on mineralized matrices, we observed Wrch1/RhoU association with the sealing zone. The distribution of Wrch1/RhoU was identical in RAW264.7 cell or mouse bone marrow derived osteoclasts. We expressed constitutively active (Q107L) and inactive (T63N) mutants of Wrch1/RhoU in osteoclasts and showed that the GTPase associates with the podosomes and the sealing zone requires in its active GTP bound form. We next examined the influence of Wrch1/RhoU on adhesion structure and sealing zone formation. Using dynamic imaging on live cells, we have shown that Wrch1/RhoU influences the distribution and amount of adhesion structures, that it modifies integrin linked signaling and affects cell migration. We will also present how the modification of Wrch1/RhoU activity by mutant expression or lentivirus-mediated gene silencing in osteoclasts affects their migration and osteolytic activity on mineralized matrix.

Our results show that Wrch1/RhoU associates with osteoclasts adhesion structures and the sealing zone and affect their ability to migrate and to resorb mineralized matrix. To establish its importance in the maintainance of bone homeostasis, we are currently generating mouse strains bearing a conditional knock out of Wrch1/RhoU gene in the osteoclast lineage.

Disclosures: A. Blangy, None.

This study received funding from: ARC, CNRS, ARE

S082

An Osteoclast Niche Is Composed of 5-FU-insensitive Osteoclast Precursors In Vivo.T. Mizoguchi*¹, A. Muto*², A. Hosova*³, T. Yamashita¹, Y. Kobayashi*¹, T. Ninomiva*¹, Y. Nakamichi¹, N. Udagawa⁴, M. Ito*¹, N. Takahashi¹¹Institute for Oral Science, Matsumoto Dental Univ., Shiojiri, Japan, ²Department of Periodontology, School of Dentistry, Aichi Gakuin Univ., Nagoya, Japan, ³Department of Oral Histology, Matsumoto Dental Univ., Shiojiri, Japan, ⁴Department of Biochemistry, Matsumoto Dental Univ., Shiojiri, Japan.

We have shown that cell cycle progression and subsequent cell cycle arrest in osteoclast precursors are required for their differentiation into osteoclasts. The quiescent osteoclast precursors were named “postmitotic osteoclast precursors (pOCPs)” (JBMR 21 Suppl 1: F259,2006). We also showed that osteoblasts prepare the osteoclast niche, which supports a long-term survival of pOCPs. pOCPs in the osteoclast niche differentiated into osteoclasts without cell cycle progression in response to several stimuli including RANKL administration. These findings suggest that, like hematopoietic stem cells, pOCPs should be resistance to 5-fluorouracil (5-FU), which induces apoptosis of cells having high proliferative potential. Here, we examined effects of 5-FU administration in mice on the osteoclast differentiation from pOCPs in bone. 5-FU was administered intravenously to mice for 6 days. Mice received intraperitoneal injections of 2MD (a highly potent analog of lα, 25(OH)₂D₃) for additional 2 days. To discriminate between proliferating and quiescent cells, mice were given 5′-bromo-2′-deoxyuridine (BrdU) in drinking water during the period of 2MD administration. Tibiae and serum samples were collected from mice. Bone marrow cells were recovered from tibiae and subjected to cellular analysis. Sections of tibiae were subjected to histological analysis using TRAP and BrdU staining. TRAP5b activity, a marker of osteoclastic activity, and calcium concentrations in serum were measured using the respective assay kits. (1) The number of monocytes and lymphocytes in the bone marrow was markedly decreased by 5FU administration. (2) 2MD administration scarcely affected the bone marrow cellularity, but significantly increased the number of TRAP-positive osteoclasts in tibiae even after treatment with 5FU. None of the nuclei in osteoclasts incorporated BrdU. In contrast, BrdU-positive nuclei in chondrocytes were detected in growth plates. (3) Serum TRAP5b activity was significantly increased in mice injected with 2MD even after administration of 5-FU. Serum calcium levels were also increased in response to 2MD in the 5-FU-treated mice. These results suggest that osteoclasts, induced by administration of 2MD, differentiated from 5-FU-insensitive pOCPs. Our observations support the hypothesis that quiescent pOCPs exist in the osteoclast niche.

Disclosures: T. Mizoguchi, None.

S084

The RANK Cytoplasmic Motif, IVVY^535–538, Plays an Essential Role in TNFα- and IL-1-induced Osteoclastogenesis.J. Jules, Z. Shi*, W. Liu*, S. Wang*, X. Feng. Pathology, University of Alabama at Birmingham, Birmingham, AL, USA.

RANKL and its cognate receptor RANK play a pivotal role in osteoclastogenesis. RANK, a member of the TNFR family, possesses three cytoplasmic motifs that recruit various TNFR-associated factors (TRAF) to transmit downstream signaling pathways to regulate osteoclastogenesis. Recently, a TRAF-independent RANK cytoplasmic motif (IVVY^535–538) was shown to play an essential role in osteoclastogenesis by committing bone marrow macrophages (BMMs) to the osteoclast lineage. Notably, both TNFα and IL-1 utilize TRAFs to activate signaling, but they are unable to mediate osteoclastogenesis in the presence of M-CSF. However, TNFα and IL-1 can induce osteoclastogenesis from BMMs exposed to permissive levels of RANKL in the presence of M-CSF, indicating that TNFα-/IL-l-mediated osteoclastogenesis requires prior priming of BMMs by RANKL. Therefore, we hypothesize that it is the RANK motif IVVY^535–538 that plays a role in priming BMMs in TNFα-/IL-l-mediated osteoclastogenesis. To address the issue, we used a previously established chimeric receptor approach. The receptor comprises human Fas external domain linked to the transmembrane and cytoplasmic domains of mouse RANK. When the chimera was expressed in BMMs from C3H mice, the clustering of the chimera induced by a human Fas activating antibody (hFas-AB) activates the RANK intracellular domain of the chimera without affecting its endogenous RANK on the BMMs. Since hFas-AB recognizes only human Fas, it does not activate endogenous mouse Fas on BMMs. We developed two chimeric receptors: Ch-1 and Ch-2. While Ch-1 comprises the human Fas external domain linked to the transmembrane and intracellular domains of normal RANK, Ch-2 contains an inactivating mutation in the RANK motif. BMMs expressing Ch-1 or Ch-2 were pre-treated with hFas-AB for 18 hours in the presence of M-CSF, followed by treatments with TNFα or IL-1 in the presence of M-CSF for 96 hours. The assays showed that only the chimeric receptor containing the normal RANK motif (Ch-1) was able to incite osteoclastogenesis, indicating that the RANK motif IVVY^535–538 plays an important role in priming BMMs in TNFα-/IL-l-mediated osteoclastogenesis. To further address the issue, we are currently inserting a RANK fragment containing IVVY^535–538 to TNFR1. If the addition of the RANK fragment renders TNFR1 to be able to mediate osteoclastogenesis, the data will provide firm proof that IVVY^535–538 is involved in TNFα-/IL-l-mediated osteoclastogenesis. Significantly, these studies may establish IVVY^535–538 as a potent therapeutic target for bone loss in rheumatoid arthritis.

Disclosures: J. Jules, None.

S086

Dok-1 and Dok-2 Deficiency Enhances Bone Resorption and Modulates the Geometry of Bone In Vivo.A. Kawamata¹, A. Inoue*², Y. Ezura*¹, K. Nakashima*¹, T. Amagasa*³, Y. Yamanashi*², M. Noda¹¹Department of Molecular Pharmacology, 21st Century COE Program, MRI, Tokyo Medical and Dental University, Tokyo, Japan, ²Department of Cell Regulation, MRI, Tokyo Medical and Dental University, Tokyo, Japan, ³Maxillofacial Surgery, Tokyo Medical and Dental University, Tokyo, Japan.

Dok-1 and Dok-2 are adaptor proteins acting downstream of protein tyrosine kinases (Yamanashi et al. Cell 1997). Both Dok-1 and Dok-2 are mainly expressed in the cells of hematopoietic lineage (DiCristofano et al. JBC 1998) and suppress Ras-Erk signaling (Yasuda et al. JEM 2004). Dok-1 and Dok-2 also suppress TLR4 signaling in innate immunity (Shinohara et al. JEM 2005). Recently, defects in Dok-7, another family member were shown to be involved in congenital myasthenic syndromes (Beeson et al. Science 2006). However, the functions of Dok-1 and Dok-2 in bone are not understood. The aim of this paper is to elucidate the role of Dok-1 and Dok-2 in bone metabolism. Dok-1 or Dok-2 single deficient mice did not show overt phenotypes possibly because of overlapping functions of Dok-1 and its closest family member, Dok-2. Thus, we analyzed bone in Dok-1 and Dok-2 double-deficient (dKO) mice. Dok-1/2 double deficient mice revealed reduction in the levels of cortical bone thickness and cortical bone volume in the femora of 9-week-old mice compared to wild type (WT). In addition, Dok-1/2 deficiency increased periosteal perimeters and endosteal perimeters in the mid shaft of femora. Possibly as a result of these changes, bone mineral density levels in femur of dKO mice were similar to those in WT. For cancellous bone envelope, bone volume and mineralized nodule formation in ex vivo bone marrow cultures were similar between dKO and WT. Histomorphometrical analysis of the dynamic bone parameters indicated that the levels of mineral apposition rate (MAR) and bone formation rate (BFR) in cortical bone of Dok-1/2 dKO mice were similar between dKO and WT. To understand the role of Dok-1/2 in bone resorption, we measured urine deoxypyridinoline (Dpyr), a marker of bone resorption. Double Dok-1/2 deficiency significantly increased Dpyr levels in urine. Intriguingly, the levels of the number of TRAP positive cells per bone surface were similar between dKO and WT, suggesting that Dok-1 and Dok-2 would be involved in the suppression of osteoclastic function specifically without altering osteoclast number. In conclusion, we identified that the novel signaling molecules Dok-1 and Dok-2 suppress bone resorption and are involved in the maintenance of bone geometry.

Disclosures: A. Kawamata, None.

S090

RNA Interference Machinery Is Essential for Osteoclastogenesis.T. Sugatani, K. A. Hruska. Department of Pediatrics, Washington University Medical School, St. Louis, MO, USA.

RNA interference (RNAi) is an evolutionarily conserved pathway, central to a broad spectrum of biological phenomena, including development, stem cell differentiation, transcriptional gene silencing, heterochromatin formation, and transposon silencing. RNAi is triggered by small RNAs such as microRNAs and small interfering RNAs (siRNAs) that regulate gene expression by translational inhibition and mRNA degradation. However, the biological function of the RNAi-related pathway in osteoclastogenesis is not completely understood. Here we show that RNAi machinery is essential for osteoclastogenesis. Moreover, microRNA-223 expression in bone marrow macrophages (BMMs) is critical for osteoclastogenesis. To investigate whether RNAi machinery is a key function in osteoclastogenesis, we focused on the function of Dicer and Argonaute 2 in the RNAi-related pathway. Endogenous small non-coding microRNAs are processed by Dicer from precursor microRNAs. Then, these small double-stranded RNAs assemble into the RNA-induced silencing complex (RISC), which contains Ago 2, Dicer and other cellular factors. Activated RISC finds its target mRNA and affects mRNA translation or stability. We employed a retroviral system for delivery of Dicer or Ago 2 siRNA into primary BMMs, and osteoclasts were induced by RANKL. The knockdown of Dicer or Ago 2 expression attenuated cell proliferation in BMMs. Moreover, the knockdown of Dicer or Ago 2 expression in BMMs suppressed the number of TRAP-positive multinucleated osteoclasts formed compared with scrambled siRNA as a control. However, TRAP expression was not blocked by the knockdown of Dicer or Ago 2 expression. These results indicate that RNAi machinery regulates TRAP-positive multinucleated osteoclast formation. We have reported that microRNA-223 is expressed in RAW264.7 cells, and the overexpression of precursor microRNA-223 in cells blocks TRAP-positive multinucleated osteoclast formation and bone resorption. Here, we examined the effect of the overexpression of microRNA-223 in primary BMMs using a retroviral system. The overexpression of precursor microRNA-223 in BMMs suppressed the number of TRAP-positive multinucleated osteoclasts formed, demonstrating that the continuous expression of microRNA-223 in BMMs results in the inhibition of TRAP-positive multinucleated osteoclast formation. Thus, we provide the first evidence that the Dicer and Ago 2-related RNAi machinery is important for the formation of TRAP-positive multinucleated osteoclasts. In particular, the regulation of microRNA-223 expression in osteoclast precursors during osteoclastogenesis may be critical for osteoclast differentiation and function.

Disclosures: T. Sugatani, None.

S093

The Mechanism of RANKL-evoked [Ca²⁺]i Oscillations Mediated by RGS10 in Osteoclast Differentiation.S. Yang, Y. Li. Cytokine Biology, The Forsyth Institute, Harvard School of Dental Medicine, Boston, MA, USA.

Increased osteoclastic resorption leads to many bone diseases, including osteoporosis and rheumatoid arthritis. To better understand the mechanisms underlying osteoclast-based diseases and design relevant therapies, it is necessary to unveil the molecular basis of osteoclast differentiation and function as well as the regulatory mechanisms of osteoclast signaling. In our previous work, we have characterized RANKL-induced signaling protein, Regulator of G-protein Signaling 10 (RGS10) and found it is predominantly expressed in osteoclasts. Impaired osteoclast differentiation in RGSIO-deficient (RGS10^−/−) mice with severe osteopetrosis indicates the essential role of RGS10 for RANKL-evoked [Ca²⁺]i oscillations in osteoclast differentiation. However, the mechanism underlying how RGS10 regulates the RANKL-evoked [Ca²⁺]i oscillations and NFATc1 expression in osteoclast differentiation still remains unclear. In this study, we focused on the mechanism of RANKL-evoked [Ca²⁺]i oscillations mediated by RGS 10 in osteoclast differentiation. Our data demonstrated that RGS 10 interacts with calmodulin in a Ca²⁺-dependent manner. Ca²⁺/calmodulin complexes are formed at high [Ca²⁺]i and compete for the PIP³ binding site on the RGS 10 protein to regulate [Ca²⁺]i oscillations and activity of PLCγ. Calcineurin inhibitor, FK506, inhibited the rescue effect of RGS10 reintroduction in RANKL-induced RGS10^−/− BMMs and the formation of TRAP⁺ cells from RGS10^+/+ BMMs driven by RGS1O ectopic expression without RANKL induction. Ectopic expression of NFATc1 rescues impaired osteoclast differentiation in RGS10^−/− mice, indicating that RGS10 acts upstream of Calcinuerin and NFATc1. Our results revealed a working model: PIP³ specifically binds RGS10 at low [Ca²⁺]i to inhibit RGS10 activity. When RANKL activates ITAM and PLCγ, which hydrolyzes PIP² to generate inositol 3-phosphate (IP³), Ca²⁺/calmodulin complexes are formed at high [Ca²⁺]i and the complexes compete for the PIP³ binding site on the RGS 10 protein to restore RGS 10 activity, which in turn regulates PLCγ activity. The dual regulation of RGS 10 activity can cause [Ca²⁺]i oscillations, implying the mechanism for RGSlO-mediated modification of intracellular [Ca²⁺]i oscillations that activate calcineurin (CN) and NFATc1 for osteoclast terminal differentiation. To our knowledge, this is the first in vivo model revealing the mechanism of [Ca²⁺]i oscillation regulation by RGS10. The essential role of RGS 10 selectively involved in RANKL-induced [Ca²⁺]i oscillations in osteoclast differentiation may offer a very specific and powerful therapeutic target for treatment of bone diseases caused by excessive bone resorption.

Disclosures: S. Yang, None.

S096

A Dramatic Increase in Hematopoiesis and Angiogenesis in Bone Marrow of Rescued RANKL-Deficient Mice by Expression of Soluble RANKL Transgene.H. Yasuda¹, A. Minamida*², J. M. Penninger*³, Y. Iwakura*²¹Nagahama Institute for Biochemical Science, Oriental Yeast Co., Ltd., Shiga, Japan, ²Institute of Medical Science, University of Tokyo, Tokyo, Japan, ³Insitute of Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria.

Receptor activator of NF-kB ligand (RANKL) is a key regulator of osteoclastogenesis, lymphocyte development and lymph node (LN) organogenesis. RANKL is a membrane-bound ligand but its soluble form is released from different type of cells by shedding. In humans increases of serum sRANKL level were reported in patients with rheumatoid arthritis and juvenile Paget's disease. RANKL knockout (KO) mice showed severe osteopetrosis, with no osteoclasts, marrow spaces, or tooth eruption, and exhibited profound growth retardation. These mice also showed defects in early differentiation of T and B cells, and lacked all LNs. We previously reported that most of all abnormalities in RANKL KO mice were rescued by expression of soluble RANKL (sRANKL) transgene by mating the KO mice with sRANKL transgenic (TG) mice. The purpose of this study is to analyze the bone marrow cells appeared in the rescued mice (hereafter called TG/KO mice). The histological analysis of the TG/KO mice revealed that a dramatic increase in hematopoiesis and angiogenesis in their bone marrow cavities. The ratio of Terl 19+ cells (erythroid cells), Mac1+ cells (macrophages), Gr-1+ cells (granyurocytes), and Thy1.2+ cells (T cells) increased in the flow cytometry, respectively. In contrast, the ratio of B220+ cells (B cells) decreased. These results suggested the possibility of a lineage shift from B cells to others. The increase in hematopoiesis and angiogenesis was observed only in TG/ KO mice, but not in TG mice, suggesting that the phenotypic changes were not directly caused by overexpression of sRANKL. RT-PCR analysis revealed that the sRANKL transgene was expressed in T-cells, B-cells, and macrophages, but not in osteoblasts in the TG/KO and TG mice. Numerous TRAP+ multinucleated cells were formed in bone marrow macrophage culture of TG/KO and TG mice in the presence of M-CSF without addition of sRANKL. Addition of OPG inhibited the osteoclast formation. Together, these results suggest that sRANKL acts on bone marrow macrophages in an autocrine fashion. TRAP+ cells were observed only in the bone surface in TG/KO mice, suggesting that the hypothetical factors besides RANKL regulated the localization of osteoclasts. In conclusion, the TG/KO mice showed a marked increase in hematopoiesis and angiogenesis accompanied with a lineage shift from B cells to other cell populations in the resuced bone marrow cavities and these mice would represent a good animal model studying the formation of bone marrow.

Disclosures: H. Yasuda, Oriental Yeast Co., Ltd. 3.

S099

Expression of OCZF Directed by the Cathepsin K Promoter Affects Bone Mass and Osteoclast Formation in Transgenic Mice.T. Shobuike*¹, T. Kukita², K. Nagata*², J. Teramachi*², M. Asagiri³, H. Takavanagi³, A. Kukita¹¹Saga University, Saga, Japan, ²Oral Biological Science, Kyushu University, Fukuoka, Japan, ³Cell Signaling and COE program, Tokyo Medical and Dental University, Tokyo, Japan.

We previously isolated the OCZF cDNA encoding Kat-6 antigen specifically expressed in rat osteoclast. The OCZF gene product is a member of POK protein family that contains two domains of POZ/BTB and zinc finger in their N- and C-terminal region respectively, and that is shown to be involved in various cellular processes. Suppression of OCZF by antisense RNA inhibits osteoclast differentiation in vitro, indicating that OCZF is involved in osteoclastogenesis, but in vivo role in the process is not clear. To this end we generated transgenic mice, which expressed OCZF under the control of mouse cathepsin K promoter. Nine transgenic mice were identified among the offspring by PCR and Southern blot analysis of mouse tail genomic DNA. Transgene expression was verified by RT-PCR using osteoclasts differentiated in vitro from bone marrow at 10 weeks of age. Two lines of the transgenic animals exhibited relatively high transgene expression, and were further investigated. The c-fos and NFATc1 protein exhibit higher levels during differentiation into osteoclasts in vitro from bone marrow of the transgenic mice than that of normal littermates. Micro-CT and peripheral quantitative CT (pQCT) analyses and histomorphometry of femur were performed in mice at 6 weeks of age. Micro-CT imaging demonstrates a reduction in trabecular bone volume in the transgenic mice relative to normal littermates. pQCT analyses also demonstrate a reduction in bone mineral density and bone mineral content especially in trabecular bone with a decrease in strength strain index, whereas those in cortical bone were slightly affected. Consistently, von Kossa staining shows a smaller mineralized area in the transgenic mice than in normal littermates. Histological analyses with tartrate-resistant acid phosphatase staining and calcein labeling indicate an increase in the number of osteoclast, whereas bone formation rate did not significantly change. These results suggest that OCZF has an important role in osteoclastogenesis in vivo.

Disclosures: T. Shobuike, None.

S103

Potential Role of Rab3D-Calmodulin Interaction in Osteoclastic Bone Resorption.J. Xu. T. Cheng*, N. Pavlos*, E. Ang*, M. H. Zheng. Orthopaedic Surgery, The University of Western Australia, Nedlands, WA, Australia.

Osteoclastic bone resorption is a highly dynamic process that requires the tight ordering of intracellular trafficking events in order to maintain the structural and functional polarization of the ruffled border and basolateral domains. Rab3 proteins are a subfamily of GTPases, known to mediate membrane transport in eukaryotic cells and play a role in exocytosis. Our recent data indicates that Rab3D modulates a post-TGN trafficking step that is required for osteoclastic bone resorption (1). Here, to identify down-stream regulatory molecules of Rab3D, we have performed a yeast two-hybrid screen. Amongst several candidate Rab3D-interacting proteins identified, Rab3D was found to associate with calmodulin, an established regulator of osteoclastic bone resorption. As an initial effort to better define the interaction between Rab3D and calmodulin, we generated several mutants of Rab3D which interfere with the GDP/GTP nucleotide exchange (Rab3DQ81L, Rab3DN135I) and/or membrane attachment of Rab3D (Rab3D-CXC). By in vivo bioluminescence resonance energy transfer (BRET) assay, Calmodulin was found to associate equivalently with wild-type Rab3D as well as Rab3DN135I and Rab3DCXC variants. Overexpression of constitutively-active Rab3D (Rab3DQ81L) enhanced this interaction suggesting that the active form of Rab3D (i.e. GTP-bound) might recruit additional effector molecules which further potentiate its binding to calmodulin. In an attempt to address the impact of calmodulin activity on Rab3D-catmodulin interaction and osteoclastic bone resorption, we performed complementary BRET and in vitro bone resorption assays in the presence of the calmodulin inhibitor, calmidazolium chloride. Interestingly, we show that suppression of calmodulin activity via calmidazolium chloride impairs the association of Rab3D with calmodulin, an affect that correlates with a disruption in osteoclastic bone resorption. We propose that the recruitment of calmodulin by Rab3D might be an important requirement for osteoclast-mediated bone resorption.

I. Pavlos, N.J., Xu, J, Riedel, D., Yeoh, J.S.G, Teitelbaum, S.L., Papadimitriou, J.M., Jahn, R., Ross, F.P., Zheng, M.H. (2005) Mol. Cell. Biol 25, 5253-5269

Disclosures: J. Xu, None.

This study received funding from: NHMRC.

S110

TNFR1 and 2 Differentially Regulates Inflammatory Bone Resorption Induced by LPS In Vivo.M. H. Mian*¹, K. Aoki², N. Alles*¹, H. Saito*², K. Ohya^2,. ¹Center of Excellence program for Frontier Research on Molecular Destruct. and Reconst. of Bone, Tokyo Medical and Dental University, Tokyo, Japan, ²Department of Hard tissue Engineering, Section of Pharmacology, Tokyo Medical and Dental University, Tokyo, Japan.

Chronic bone infection is often complicated by severe osteolysis. It has been reported that TNF receptors differentially regulate osteoclastogenesis, where TNFR1 accelerate and TNFR2 inhibit osteoclastogenesis (Abu-Amer et al, J Biol Chem, 2000). But whether these receptors influence bone resorbing activity of osteoclast differentially is yet to be elucidated. The purpose of this study was to investigate bone resorbing activity through TNFR1 and TNFR2 in vitro as well as to clarify the role of these receptors on inflammatory bone resorption in vivo. We examined the roles of TNF receptors using bone marrow cells in vitro as well as in LPS induced local bone resorption model in vivo. For in vitro study, pit formation assay in dentin slices using a confocal microscope was performed to check the functional osteoclast activity of TNFR1 and TNFR2 upon stimulation of TNF-α. Average pit depth in Wild type (WT) and TNFR1 deficient (KO) culture influenced by TNF-α were not significantly changed, but significant increase in average pit depth were observed in TNFR2 KO culture. For in vivo study, 10 mg/kg LPS was injected subcutaneously on calvaria of TNFR1KO, TNFR2 KO, and their WT mice, sacrificed on day 5 after the LPS injections. Calvariae, tibiae and femurs were dissected. The calvarial BMD measured by DXA in LPS-injected group was significantly decreased in WT and TNFR2 KO mice compared to the control. In contrast, there was no significant decrease of calvarial BMD in TNFR1 KO mice. On the other hand, the significant decrease on the BMD of tibiae and femurs measured by peripheral quantitative computed tomography (pQCT) was observed in TNFR2 KO mice, but no significant decrease was observed in WT and TNFR1 KO mice. The results of urinarydeoxypyridinoline reflected these resorption and histomorphometric analyses of tibia shows the significant increase of osteoclast number in TNFR2 KO mice by LPS injection. These BMD data were confirmed by n-CT analyses. The present data indicate that TNFR I plays the pivotal role for inducing local bone resorption induced by LPS and TNFR2 might have a protecting role in inflammatory bone resorption process.

Disclosures: M.H. Mian, None.

S112

Transgenic Overexpression of Osteoclastic Protein-tyrosine Phosphatase, PTP-oc, in Cells of Osteoclastic Lineage Led to Increased Bone Resorption and Marked Reduction in Trabecular Bone Mass and Density in Adult Mice.M. H. C. Sheng, M. Amoui*, A. K. Srivastava, J. E. Wergedal, K. H. W. Lau. Loma Linda VAMC, Loma Linda, CA, USA.

Past in vitro studies have suggested that the structurally unique osteoclastic transmembrane PTP-oc acts as a positive regulator of osteoclast activity. This study sought to determine whether transgenic (TG) overexpression of PTP-oc in cells of osteoclastic lineage would increase (↑) bone resorption and decrease (↓) bone mass or density (BMD) in adult mice. To generate TG mice, a TG construct containing the rabbit PTP-oc cDNA driven by a tartrate-resistant acid phosphatase (TRACP)-1C promoter was injected into the pronucleus of fertilized ova, which were implanted into pseudo-pregnant B6D2F1 mice. TG founders, identified by a PCR-based genotyping assay using primers that span unique regions of TRACP-1C promoter and rabbit PTP-oc sequence, were bred with C57BL/6J mice to produce F2 mice. Ten-week-old male F2 progenies were extensively followed for pQCT, histological, and serum resorption marker analyses. The body weight of TG mice was 10% less (p<0.05, n=8 each) than that of wild-type (WT) littermates. pQCT analyses of the femur revealed that TG mice had 30% ↓ in trabecular BMD (p<0.002). Histologic analyses at the secondary spongiosa in the femur of 9 TG and 7 WT littermates confirmed that TG mice showed 35% ↓ in trabecular surface (Tb.Pm, p<0.01), 27% ↓ in % trabecular area (Tb.Ar, p<0.05), 11% ↓ in trabecular number (Tb.N, p<0.05), and 36% ↑ in trabecular separation (Tb.Sp, p<0.005). The lumbar vertebra of 12 TG mice, when compared to 8 WT littermates, also showed 20% ↓ in Tb.Pm (p<0.005), 17% ↓ in Tb.N (p<0.003) and 35% ↓ in Tb.Sp (p<0.004). Consistent with an ↓ in bone resorption, the serum c-telopeptide level of TG mice (n=14) was 25% (p<0.05) higher than that of littermates (n=ll). That the number of osteoclasts and the length of TRACP-labeled bone surface per total bone surface were not different suggested that TG overexpression of PTP-oc increased osteoclast activity rather than osteoclast differentiation. The same phenotype was confirmed in a second TG line. In congruent with an ↓ in osteoclastic activity, the average pit area created by osteoclasts derived from bone marrow cells of 10-week-old male TG mice in response to RANKL and mCSF in the pit formation assay was −50% greater (p=0.008) than those by osteoclasts of WT littermates (n=8 each). However, the TRACP activity per cellular protein in osteoclasts derived from the two groups of mice was not different. In summary, TG overexpression of PTP-oc in cells of osteoclastic lineage led to a marked reduction in trabecular bone mass in adult mice due to an ↓ in osteoclastic activity. These findings provide compelling in vivo evidence that PTP-oc is a positive regulator of osteoclastic activity.

Disclosures: M.H.C. Sheng, None.

This study received funding from: Veterans Administration.

S114

The CaMK-CREB Pathway Regulates Osteoclast Differentiation and Function.A. Suematsu¹, K. Sato*², T Nakashima*¹, S. Takemoto-Kimura*³, K. Aoki⁴, K. Ohva⁴, A. Yamaguchi⁵, T. A. Chatila*⁶, H. Bito*³, H. Takavanagi¹¹Department of Cell Signaling, Tokyo Medical and Dental University, Tokyo, Japan, ²Division of Rheumatology and Applied Immunology, Saitama Medical University, Saitama, Japan, ³Department of Neurochemistry, University of Tokyo, Tokyo, Japan, ⁴Department of Hard Tissue Engineering, Tokyo Medical and Dental University, Tokyo, Japan, ⁵Department of Oral Pathology, Tokyo Medical and Dental University, Tokyo, Japan, ⁶The David Geffen School of Medicine at the University of California at Los Angeles, Los Angeles, CA, USA.

Calcium (Ca²⁺) signaling is critical for the regulation of various cellular events. Intracellular Ca²⁺ binds to calmodulin, and the Ca²⁺/calmodulin complex activates Ca²⁺/calmodulin-dependent kinases (CaMKs) as well as phosphatases such as calcineurin. We revealed the importance of calcineurin-nuclear factor of activated T cells (NFAT) pathway in osteoclastogenesis. However, it remains unclear whether CaMKs play a crucial role in bone homeostasis. Here we examined the role of CaMKs in osteoclast differentiation and function. Because knockdown of CaMKIV had a suppressive effect on RANKL-induced osteoclastogenesis, we analyzed the bone phenotype of CaMKIV-deficient mice. We observed an increase in bone volume in CaMKIV-deficient mice, which resulted from the reduction in osteoclast number. In vitro osteoclastogenesis in CaMKIV-deficient cells stimulated with RANKL was also reduced. We focused on cAMP response element (CRE)-binding protein (CREB) as a physiological substrate of CaMK in the osteoclast differentiation. CREB was phosphorylated and activated through CaMKIV after RANKL stimulation. The pharmacological inhibition of CaMKs suppressed osteoclast differentiation and downregulated the expression levels of c-fos, which is required for the induction of NFATc1. Furthermore, CREB together with NFATc1 induced the expression of osteoclast-specific genes. Finally, we explored the possibility whether modulation of CaMK activity can be used as therapeutic target in an ovariectomy (OVX) mice. The reduction in bone volume associated with an increase in osteoclast number in OVX mice was ameliorated by systemic administration of CaMK inhibitor. These results indicate that both kinases and phosphatases downstream of Ca²⁺ signaling cooperatively regulate the transcriptional program of osteoclast differentiation and function. Thus, inhibition of the CREB-CaMK pathway may be a new therapeutic target for various bone diseases.

Disclosures: A. Suematsu, None.

S118

Thyroid Hormone-induced Terminal Differentiation of Growth Plate Chondrocytes Involves IGF-1 Modulation of Canonical Wnt Signaling.L. Wang*¹, Y. Y. Shao*¹, R. T. Ballock²¹Department of Biomedical Engineering, Cleveland Clinic, Cleveland, OH, USA, departments of Orthopaedics and Biomedical Engineering, Cleveland Clinic, Cleveland, OH, USA.

Thyroid hormone is a potent regulator of skeletal maturation in the growth plate. Wnt signaling has also been recognized as an important signal transduction pathway in regulating terminal differentiation of growth plate chondrocytes into hypertrophic cells. Wnt ligands and β-catenin have been shown to promote chondrocyte maturation in the chick growth plate. Since thyroid hormone is a known stimulator of IGF-1 receptor expression, and IGF-1 has been described as a stabilizer of β-cetenin in other cell types, we hypothesize that thyroid hormone may regulate terminal differentiation of growth plate chondrocytes in part by stimulating Wnt/β-catenin signaling pathway through the modulation of 1GF-1/1GF-1R signaling.

Three-dimensional pellet cultures of 2-day old rat growth plate chondrocytes were maintained in serum-free medium. Thyroid hormone treatment upregulated Wnt-4 mRNA expression, increased the accumulation of β-catenin and upregulated β-catenin target gene Runx2/cbfal. Both Wnt4 conditioned medium and adenovirus expressing stabilized β-catenin increased the markers of hypertrophy alkaline phosphatase (ALP) activity and Coll0al mRNA expression in growth plate chondrocytes. Blocking Wnt ligand/receptor interactions with Wnt inhibitors Frzb/sFRP3 or DKK1 decreased the T3-induced increases of β-catenin accumulation and inhibited the maturation-promoting effects of T3 in growth plate cells. IGF-1R mRNA expression was upregulated by thyroid hormone treatment in growth plate cells. Incubation of the cells with recombinant IGF-1 protein or IGF-1R adenovirus not only resulted in increases of β-catenin accumulation and upregulation of Wnt4 and Runx2 mRNA expression, but also increased ALP activity and Coll0al mRNA expression in growth plate chondrocytes. The IGF-1 receptor inhibitor picropodophyllin (PPP) inhibited T3-induced increases of Wnt4 mRNA as well as T3-mediated hypertrophy of growth plate chondrocytes.

These data demonstrate that thyroid hormone promotes terminal differentiation of growth plate chondrocytes in part through IGF-1 modulation of the canonical Wnt signaling pathway.

Disclosures: R. T. Ballock, None.

S121

Proteasome Inhibition Induces Permanent Growth Retardation and Apoptosis in Growth Plate Chondrocytes.E. E. Eriksson, F. Zaman*, A. S. Chagin*, D. Chrysis*, L. Sävendahl*. Women and Child Health, Pediatric Endocrinology, Stockholm, Sweden.

Proteasome inhibitors, a new class of anti-cancer drugs has recently been approved for multiple myeloma treatment in adults and is under clinical trial in pediatric cancers. However, any specific side effects on normal bystander tissues in children are unknown. The objective of this research was to study the effects of the proteasome inhibitors, MG262 and the clinically used proteasome inhibitor, Velcade on linear bone growth in young mice (male, 5 weeks old) and in fetal rat metatarsal bones. To address this question, C57B and NMRI mice were treated with the proteasome inhibitors, Velcade and MG262, in a clinical relevant dose. We followed the linear bone growth of the mice longitudinally and observed permanent growth retardation. To elucidate the underlying mechanisms, we used a transgenic mouse model expressing a green fluorescent protein reporter allowing monitoring the ubiquitin/proteasome system. MG262 treatment results in accumulation of the green fluorescent protein in the growth plate and increased apoptosis in stem-cell like chondrocytes. To further prove local effect of proteasome inhibition on linear growth, fetal rat metatarsal bones were cultured in presence of either Velcade or MG262. These experiments also showed bone growth retardation and increased chondrocyte apoptosis. To understand the apoptotic pathways induced by proteasome inhibitors in chondrocytes, we used rat- and human chondrocytic cell lines that both were found to undergo apoptosis upon proteasome inhibitor treatment. A pan-caspase inhibitor (Z-VAD-fmk) suppressed MG262-mediated apoptosis by 32% in rat and 35% in human chondrocytes. Together with early up-regulation of p53 and apoptosis inducing factor (AIF) these observations suggest involvement of both caspase-dependent and independent apoptosis. Indeed, apoptosis was suppressed by 30% employing p53-sequence specific siRNA. In short, we conclude that impairment of the ubiquitin/proteasome system causes permanent growth retardation by inducing massive apoptosis in chondrocytes in vivo and in vitro, an effect which is both dependent and independent of caspases. Our findings suggests that proteasome inhibitors have to be cautiously used in growing children and that involvement of p53 in chondrocyte apoptosis opens targets for new potential ways to rescue growth.

Disclosures: E.E. Eriksson. None.

S123

The Regulation of Chondrocyte Proliferation, Function, and Differentiation by Akt Signaling Pathways., T. Fujita*, S. Rokutanda*, Hi Kanatani*, C. Yoshida*, T. Komori*. Cell Biology, Nagasaki university, Nagasaki, Japan.

Akt is a broadly expressed multifunctional protein, but its roles in chondrocytes still remain to be clarified. We investigated chondrocyte proliferation, function, and differentiation in transgenic mice expressing myristoylated Akt (myrAkt) by Col2al promoter. Chondrocyte proliferation was enhanced in the reservoir zone, but it was reduced in the proliferative zone, in which an increase in matrix production was observed. Chondrocyte maturation was enhanced in the spine and sphenoid but inhibited in the long bones. Accelerated chondrocyte proliferation by IGF-I was inhibited by Fox03a, and an inhibitory effect of Akt on chondrocyte proliferation was abolished by rapamycin, a mTOR specific inhibitor, suggesting that Akt-FoxO and Akt-mTOR axes functioned as a proliferative pathway in the reservoir zone and as an anti-proliferative pathway in the proliferative zone, respectively, in myrAkt transgenic mice. The enhanced matrix synthesis by myrAkt was blocked by rapamycin, indicating that Akt-mTOR pathway plays an important role in matrix synthesis. Fox03a overexpression mildly enhanced chondrocyte maturation, and lithium, a GSK3 inhibitor, inhibited it in long bone of wild-type mice. Further, the stimulatory effect of Akt on chondrocyte maturation in spine was abolished by rapamycin. These results indicate that and Akt-FoxO and -GSK3β pathways are involved in the negative regulation of chondrocyte maturation in long bone but Akt-mTOR pathway is involved in the positive regulation of chondrocyte maturation in spine. These results demonstrate that Akt regulates chondrocyte proliferation, matrix synthesis, and chondrocyte maturation depending on the skeletal elements and the maturational stages of chondrocytes.

Disclosures: T. Fujita, None.

S127

Transcriptional Regulation of Type × Collagen by Runx2: Molecular Network Underlying Chondrocyte Hypertrophy Causing Osteoarthritis., A. Hipashikawa, T. Saito, S. Kamekura*, S. Ohba, T. Ikeda, K. Nakamura, U. Chung, H. Kawaguchi. Sensory & Motor System Medicine, University of Tokyo, Tokyo, Japan.

Chondrocyte hypertrophy is essential not only for physiological skeletal development and growth, but also for pathological disorders like osteoarthritis (OA). When we created an experimental mouse OA model by producing instability in the knee joint through surgical transection of the medial collateral ligament and resection of the medial meniscus, Runx2 expression determined by immunostaining and real-time RT-PCR was induced in chondrocytes at the superficial and middle layers of joint cartilage as early as 2 weeks, which was not observed in the sham-operated joint. Most of the Runx2-positive chondrocytes then expressed type × collagen (COL 10), the representative marker of hypertrophic differentiation, at 2–4 weeks, and thereafter produced collagenases causing degradation of the cartilage matrix. In heterozygous Runx2-deficient mice, however, COL 10 expression, as well as the subsequent collagenase expression and matrix degradation, was suppressed under the OA induction, indicating that Runx2 contributes to the pathogenesis of OA through chondrocyte hypertrophy. COL10 mRNA level, as well as Alizarin-red and von Kossa stainings, were markedly increased by the Runx2 overexpression, whereas they were decreased by the dominant negative Runx2 overexpression, in the cultures of respective stable lines of mouse chondrogenic ATDC5 cells. In HeLa cells transfected with a luciferase-reporter gene construct containing a 4.5 kb fragment of the human COL 10 promoter region, the transcriptional activity was enhanced by co-transfection with Runx2. Deletion analysis using a series of 5′-deletion constructs of the COL 10 promoter identified the core responsive element to Runx2 to be within 10 bp around the −80 bp region (HY-box). The transcriptional activity of Runx2 was suppressed by a site-directed mutagenesis in the HY-box. The tandem-repeat constructs responded to Runx2 depending on its repeat number. Electrophoretic mobility shift assay showed specific binding of nuclear extracts from Runx2-overexpressed COS7 cells with the wild-type HY-box oligonucleotide probe, but not with the mutated probe. Cold competition with an excess of unlabelled wild-type probe, but not with the unlabelled mutated probe, suppressed formation of the complex. In addition, the complex underwent a supershift by the antibody to Runx2, confirming the specific binding of the HY-box and Runx2. In conclusion, we identified the Runx2-responsive region HY-box in the COL 10 promoter. Studies on molecules related to Runx2 / HY-box will lead to elucidation of the molecular network underlying chondrocyte hypertrophy and OA pathogenesis.

Disclosures: A. Higashifcawa, None.

S130

Inhibitory Smad6 and Smad7 Differently Regulate Cartilage Formation.T. Iwai¹, J. Murai*¹, H. Yoshikawa¹, N. Tsumaki². Orthopaedic Surgery, Osaka University Graduate School of Medicine, Osaka, Japan, 2Cartilage and Bone Biology, Osaka University Graduate School of Medicine, Osaka, Japan.

TGF-b / BMP signals are mediated by Smad proteins. Biochemical studies have revealed that Smad6 inhibits BMP signaling, whereas Smad7 inhibits both TGF-b and BMP signaling. Transgenic / knockout mouse studies showed that BMP siganals are definitely necessary for cartilage formation, whereas importance of TGF-b signals in cartilage formation is controversial. In this annual meeting last year, we reported generation of conditional transgenic mice overexpressing Smad7 in cartilage. Smad7 overexpression inhibited cartilage formation and delayed chondrocyte hypertrophy. In the present study, we further clarified roles of Smads 6 and 7. In situ hybridization analysis revealed that the expression levels of both Smad6 and Smad7 were low in cartilage compared with those in surrounding soft tissues and bone. To investigate the importance of this Smad7 down-regulation in cartilage during endochondral bone formation, we analyzed trangenic mice (Tg) overexpressing Smad 6 or Smad7. In situ hybridization analysis showed elevated expression of Smad 6 in cartilage of Smad6 Tg and elevated expression of Smad7 in cartilage of Smad7 Tg. Cartilage size was normal in Smad6 Tg, but significantly reduced in Smad7 Tg. We next cultured mesenchymal cells derived from 12.5 d.p.c. limb buds and induced cartilaginous nodule formation in the presence of rhBMP2 in micromass system. Cartilaginous nodule formation induced by exogenous BMP2 was prohibited in mesenchymal cells prepared from Smad7 Tg, but not in those from Smad6 Tg. Real-time RT-PCR analysis of the cells in micromass culture showed that the Smad6 expression level in Smad6 transgenic mesenchymal cells was 1.7 times higher than wild-type mesenchymal cells and that the Smad7 expression level in Smad7 Tg was 1.8 times higher. The expression level of Sox9 was normal in Smad6 transgenic cells, but dramatically reduced in Smad7 transgenic cells. Cartilaginous nodule formation induced by rhBMP2 in wild-type mesenchymal cells was inhibited by addition of the SB203580 (inhibitor of p38 MAPK pathway), but not by addition of the SB431542 (inhibitor of TGF-b pathway). Immunoblot analysis of ATDC5 cells showed that BMP-induced phosphorylation of ATF2 was repressed by overexpression of Smad7 but not by overexpression of Smad6. In general, BMP stimulate cartilage formation and chondrocyte hypertrophy. Smad6 Tg showed that BMP signals for chondrocyte hypertrophy is mediated by Smad 1/5/8 that Smad6 blocks. The present results suggest that BMP signals for cartilage formation are mediated by Smad-independent pathways. This pathways could be inhibited by Smad7 but not Smad6. The p38 MAPK pathways may be one of candidates for this pathway.

Disclosures: T. Iwai, None.

S133

Analysis of the Role of Runxl in Chondrocyte Differentiation using Chondrocyte-specific Runxl Deficient Mice, A. Kimura*¹, S. Takeda*¹, S. Sato*², M. Iwasaki*², H. Inose*¹, K. Sinomiva*¹¹21COE, Dept. of Orthopedics, Tokyo Medical and Dental University, Tokyo, Japan, 2Dept. of Orthopedics, Tokyo Medical and Dental University, Tokyo, Japan.

Chondrocyte differentiation is a multistep process essential for endochondral bone formation.

The transcription factor Runx family is involved in chondrocyte differentiation. Namely, Runx2 accelerates chondrocyte differentiation and Runx3 also regulates chondrocyte maturation in collaboration with Runx2. However, the physiological role of the other member Runxl in chondrocyte differentiation remains to be elucidated. We previously reported that all Runx genes are expressed in cells of chondrocyte linage in mouse embryo. In this study, we tried to address the role of Runxl in chondrocyte differentiation, in vivo, by generating mice deficient for Runxl specifically in chondrocyte, since mice lacking Runxl die in the midgestation stage.

We generated Runxl-floxed mice (Runx 1^flo/flo) harboring loxP sites surrounding exon4 which encodes DNA binding domain of Runxl by gene targeting. By crossing them with Colllal-Cre transgenic mice that drive ere expression by chondrocyte-specific ColIIal promoter, we obtained chondrocyte-specific Runxl deficient (ColIIal-Cre/Runx1^flo/flo) mice.

Successful deletion of Runxl in ColIIal-Cre/Runxl^flo/flo) mice was confirmed by southern blot analysis of genomic DNA. Unexpectedly, Comal-Cre/Runx1^flo/flo mice were grossly normal. By skeletal preparation at birth, ColIIal-Cre/Runxl^flo/flo mice were indistinguishable from wild-type mice, namely the size of the body and calcification of the skeleton were similar. Histologically, ColIIal-Cre/Runxl^flo/flo embryos showed no overt abnormality. We also analyzed them by In situ hybridization analysis using molecular markers expressed in chondrocytes such as ColIIal, ColXal and Indian hedgehog, but we did not detect any abnormality.

Taken together, this study show that Runxl is not necessary for chondrocyte differentiation and the absence of Runx 1 may be compensated by Runx2 and Runx3.

Disclosures: A. Kimura, None.

S136

Osteoprotegerin Negatively Regulates Chondroclast Formation and Recruitment in Endochondral Ossification.N. Ota¹, H. Takaishi*¹, J. Takito*¹, T. Kimura*², Y. Okada*², K. Matsuzaki¹, H. Yasuda*³, H. Kawaguchi⁴, Y. Toyama*¹¹Orthopaedic Surgery, Keio University School of Medicine, Tokyo, Japan, ²Pathology, Keio University School of Medicine, Tokyo, Japan, ³Nagahama Institute for Biochemical Science, Oriental Yeast, Shiga, Japan, ⁴Orthopaedic Surgery, University of Tokyo, Tokyo, Japan.

Osteoprotegerin (OPG) is a decoy receptor for receptor activator of NF-B ligand (RANKL). It has been suggested that RANKL expression in hypertrophic chondrocytes is involved in the formation and activation of chondroclasts, which function in resorption of cartilaginous matrix at the growth plates. The fracture repair process closely resembles normal development of the skeleton during embryonic bone formation. We previously reported the accelerated fracture healing in OPG deficient mice (OPG KO) via the rapid resorption of cartilaginous callus due to the enhanced chondroclast recruitment. The purpose of the present study was to further investigate the role of OPG in endochondral ossification using the chondrocytes obtained from OPG KOand wild type (WT). Newborn rib chondrocytes were disseminated as feeder cells in the presence of 1,25-dihydroxy vitamin D3 and PGE2, and cocultured with spleen cells, in order to examine inducing effects of chondrocytes on the chondroclast formation. Strikingly, OPG KO-derived chondrocytes induced numerous TRAP positive multinucleated cells in a coculture system with spleen cells isolated from WT. In contrast, WT-derived chondrocytes could not induce any TRAP positive cells. Addition of recombinant OPG to the coculture system decreased the chondroclastogenesis in a concentration dependent manner. RT-PCR analysis showed that WT chondrocytes increased RANKL mRNA in response to PTHrP, FGF-18, PGE2, IL-1 and TNF-a. On the other hand, TGF-b increased the expression of OPG Next, we examined whether the chondrocytes fully differentiated in atellocollagen pellets could induce chondroclastogenesis. Carriage pellets from OPG KO cocultured with RAW 264.7 cells induced TRAP positive multinucleated cells in the presence of 10 ng/ml RANKL, while WT cartilage pellets did not. ELISA measurements indicated that the OPG KO-derived cartilage pellets produced the significant level of soluble RANKL. Finally, the transplantation assay of the cartilage pellets under the kidney capsules of adult OPG KO revealed the remarkable recruitment of TRAP positive cells around the OPG KO-derived cartilage pellets, which were subsequently replaced by osseous tissues. These observations suggest that RANKL and OPG expression in chondrocytes is regulated by various cytokines and inflammatory mediators, and OPG might controls the process of enchondral ossification as a negative regulator of chondrocyte dependent chondroclastogenesis.

Disclosures: N. Ota, None.

S142

CITED 1 Ablation Impairs Endochondral Bone Formation During Embryonic Development.D. Yang, J. Guo*, R. Bringhurst*. Endocrine Unit, Massachusetts General Hospital, Boston, MA, USA.

CITED1 (CBP/P300 interacting Transactivator with Glutamic Acid [E]/ Aspartic Acid [D]-Rich C-terminal domain) is a transcriptional cofactor located on the × chromosome. We previously observed that CITED 1 expression is rapidly and transiently induced by PTH1R activation in osteoblasts and that cultured osteoblasts from mice lacking CITED1 display abnormal differentiation in vitro. To determine the possible role of CITED1 in early bone development, we have analyzed endochondral bone formation in tibiae from CITED1-knockout (KO) embryonic mice in which the CITED1 gene is replaced with a functional LacZ/Neo cassette and bred into the 129Sv background for five generations. The tibiae of CITED1 KO mice were shorter than those of wild type (WT) littermates, and bone volume and the extent of trabeculae in CITED1 KO tibiae at E18.5 and post-natal day 1 (P1) were significantly reduced, as shown by von Kossa staining and histology. At E15.5, the regions of overall chondrogenesis and of chondrocyte hypertrophy, demonstrated by Col II and Col × expression respectively, were not different in WTs and KOs; but bone formation, shown by Col I in situ analysis, appeared slightly earlier inside the tibia and expanded wider in perichondral region in the WT. Osteopontin (OPN) is expressed by late hypertrophic chondrocytes and osteoblasts within the primary and secondary spongiosa. Relative to WT, the length of the central region of OPN expression was reduced in all KO bones from E15.5 through P1, accounting for the foreshortening of the overall bone length. In situ hybridyzation and LacZ staining analysis showed strong expression of CITED1 in this same central region of the developing bones but not in adjacent chondrocytes. We conclude that CITED1 expression, probably by cells of the osteoblast lineage, is important for normal endochondral bone formation and that loss of functional CITED1 expression leads to a reduction in the rate of bone formation. Whether this defect reflects impaired actvity of osteoblasts that normally express CITED1 and/or loss of an indirect effect on chondrocyte proliferation and/or differentiation, which could involve PTHrP, remains to be investigated.

Disclosures: D. Yang, None.

S146

A Spontaneous Mutation (Dhe) in the Mouse Lmna Gene Results in Hypoplastic Cranial Sutures and Under-mineralization of the Skeleton.L. R. Donahue¹, D. Liu*¹, M. Curtain*¹, J. Hurd*¹, C. Marden*¹, C. A. MacKav*², P. R. Odgren*²¹The Jackson Laboratory, Bar Harbor, ME, USA, ²University of Massachusetts Medical School, Worcester, MA, USA.

The Lmna gene encodes the nuclear lamins A and C, which, along with lamin B, constitute the proteinaceous nuclear envelope. Lmna mutations are associated with connective tissue disorders, including cardiomyopathies, muscular dystrophies, lipodystrophies, progeria and bone abnormalities. We discovered a new mutation in mouse Lmna that demonstrates a critical role for Lmna in cranial suture development and skeletal mineralization. Dhe, (Dominant hair and ears) is a semi-dominant mutation in exon I of Lmna (1.52R). Dhe/+ mice exhibit a sparse coat, small ear pinnae, hypoplastic cranial sutures, and under-mineralization of the skeleton.

To assess suture formation we used X-ray, histological, and in situ analyses. Skeletal mineralization was measured by DEXA and X-ray.

DEXA showed that areal BMD is lower in Dhe/+ than in controls (Dhe/+ 0.037; +/+ 0.045 g/cm²). Under-mineralization can be seen in radiographs of axial and appendicular skeleton. In X-rays of Dhe/+ skulls, the premaxillary/maxillary and cranial vault sutures, and the entire parietal bone, are clearly visible and radiolucent, indicating severe hypomineralization.

Microscopic examination revealed severely hypomorphic sutures in Dhe/+ skulls. The bony plates fail to overlap, the cells and connective tissue connecting the bones are loosely organized and hypoplastic, and suture material is poorly attached.

Extracellular matrix gene expression by in situ hybridization assessed with probes for expression of type I collagen mRNA in bone matrix and type III collagen mRNA in the fibrous suture matrix revealed gross deficiency of collagen gene expression. In controls, rows of osteoblasts on bone surfaces were intensely stained for col 1. Bone synthesis was highly polarized, with active bone synthesis confined to specific surfaces, indicating a high degree of site specificity to the production of bone in the cranium. In contrast, Dhe/+ mice had extremely low levels of col I mRNA at the cranial sutures, consistent with a pronounced deficiency of bone synthesis. In +/+ mice, the fibroblastic cells inhabiting the suture expressed high levels of col III, consistent with the production of dense connective tissue in the suture proper. In Dhe/+, the level was extremely low, consistent with the loose strands of connective tissue connecting the cranial bones. These observations provide evidence that there is a severe loss of extracellular matrix synthesis at the cellular level in osteoblasts and sutural fibroblasts. Thus, Lmna is critical to normal cranial bone and suture formation and to skeletal mineralization.

Disclosures: L.R. Donahue, None.

This study received funding from: NIHROI grant: EYO15073 to Dr. Donahue.

S148

Body Mass Influences Bone Mass Independent of Leptin Signaling.U. T. Iwaniec¹, S. Boghossian*², R. T. Turner¹, S. P. Kalra*²¹Nutrition and Exercise Sciences, Oregon State University, Corvallis, OR, USA, ²Neuroscience, University of Florida, Gainesville, FL, USA.

Skeletal growth and turnover are coupled to energy balance via incompletely understood mechanisms. Anorexia is associated with low bone mass, whereas obesity correlates with increased bone mass. Leptin, a hormone produced by fat cells, functions as a sentinel of energy balance and may be a potential mediator of the effects of fat mass on bone mass. The objective of this study was to determine whether increased weight due to increased fat intake increases bone mass, and, if so, whether this requires central leptin signaling. Leptin-deficient ob/ob mice were used in the experiment. In order to replete hypothalamic leptin, 8–10 week-old ob/ob mice were injected in the hypothalamus with a recombinant adeno-associated virus containing the leptin gene (rAAV-lep) or a control vector, rAAV-GFP (green fluorescent protein). The mice were maintained on a regular control diet (RCD) for 7 weeks and then subdivided into groups (n=8–9/group) and either continued on the RCD or fed a high fat diet (HFD, 45 kcal% fat; D12451, Research Diets) for 8 weeks. Bone mass and architecture were evaluated by μCT. Administration of rAAV-lep resulted in decreased body weight, increased femur length and total femur volume, and reduced cancellous bone volumes in the distal femur and lumbar vertebra, indicating that leptin affects multiple aspects of bone growth and architecture. Body weight increased (p<0.003) with high fat intake (+18%), irrespective of rAAV treatment; mice administered rAAV-GFP weighed 56±2g on RCD and 66±2g on HFD, while mice administered rAAV-lep weighed 28±3g on RCD and 33±2g on HFD. Total femur bone volume increased with HFD in leptin-deficient rAAV-GFP mice (p<0.001, 14.3±0.6 mm³ vs. 18.2±0.4 mm³) and there was a tendency for an increase in total femur bone volume in HFD-fed rAAV-lep-treated mice (p<0.06, 16.2±0.2 mm³ vs. 17.1±0.4 mm³). Distal femur and vertebral cancellous bone volume were not altered by diet. There was a significant correlation between body weight and total femur bone volume in wildtype mice fed normal diets (data not shown). Taken together, these findings suggest that increased body weight has a positive effect on total femur bone mass that is independent of dietary fat and leptin signaling.

Disclosures: V. T. Iwaniec. None.

This study received funding from: N1H.

S150

Phenotypic Analysis of the Crtap-/- Mice: The First Animal Model for Recessive Osteogenesis Imperfecta.R. Morello¹, J. N. Lennington*², S, Kakuru*1. M. Jiang*³, Y. Chen*³, D. R. Keene*⁴, B. Lee³¹Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, USA, ²Pediatrics, Baylor College of Medicine, Houston, TX, USA, ³Howard Hughes Medical Institute, Houston, TX, USA, 4Shriners Hospital for Children, Portland, OR, USA.

We recently described the phenotype of the Crtap-/- mice. These mice exhibit a severe osteochondrodysplasia, with low bone mass, kyphosis and shortening of the long bones. Crtap interacts with prolyl 3-hydroxylase 1 (P3h1) and is essential for proper type I and II collagens post-translational modification. Importantly, the phenotype of Crtap-/- mice closely resembles that of OI patients and mutations in the CRTAP gene cause recessive osteogenesis imperfecta (OI). In this study we extended the phenotypic analysis of our mutant mice in order to obtain a better understanding of the Crtap role during development and adulthood. Adult skeletal preparations showed marked craniofacial abnormalities in the Crtap null mice, consisting of shortening and compression of the anterior portion of the cranium with occasional asymmetry causing malocclusion and overgrowth of the incisor teeth. The skin showed increased laxity compared to WT littermates: a histological analysis revealed a decreased thickness of the dermis while at the ultrastructural level, areas of decreased matrix density as well as collagen fibrils with increased diameter were visible. Histological analysis of visceral organs showed evidence of abnormal lung morphology, with alveolar dilatation associated with thinning of the alveolar walls. Moreover, the kidney glomerular basement membrane appeared to have an expanded lamina lucida at the expenses of the lamina densa, suggesting perhaps an additional Crtap role in the regulation of type IV collagen modification. Finally, loss of Crtap causes a chondrodysplasia with rhizomelic shortening of long bones. Interestingly, BrdU incorporation studies of the growth plate point to a slight increase in chondrocyte proliferation in the zeugopodal growth plates of E15.5 Crtap null mice compared to controls. Our studies point to a broad spectrum of connective tissue disease that can arise from loss of CRTAP and altered collagen 3-prolyl-hydroxylation.

Disclosures: R. Morello, None.

S152

Female, But Not Male, TIEG-Null Mice Display Severe Osteopenia and Abnormal Cancellous Bone Microarchitecture.M. Subramaniam¹, J. R. Hawse¹, U. T. Iwaniec², S. F. Bensamoun*³, K. D. Peters*¹, N. M. Raiamannan⁴, M. J. Oursler¹, R. T. Turner², T. C. Spelsberg¹¹Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA, ²Department of Nutrition and Exercise Sciences, Oregon State University, Corvallis, OR, USA, ³Genie Biologique, Universite de Technologie de Compiegne, Compiegne, France, ⁴Department of Cardiology, Northwestern University Medical School, Chicago, IL, USA.

TGFβ Inducible Early Gene-1 (TIEG), also classified as KrÜppel-like transcription factor-10 (KLF-10), was originally cloned from human osteoblasts and has been shown to play an important role in osteoblast growth and differentiation. To better understand the biological role of TIEG in the skeleton, we generated TIEG-null (TIEG^−/−) mice. Calvarial osteoblasts isolated from TIEG^−/− mice express reduced levels of Runx2 and other important osteoblast marker genes and have significant defects in their mineralization capacity relative to wild-type (WT) controls. Through the use of PIXImus and pQCT analysis, we have demonstrated that the femurs and tibias of two-month-old C57Black6 female TIEG^−/− mice display significant decreases in numerous bone parameters including total bone mineral content, density and area, relative to WT littermates. However, no differences were observed for any of these bone parameters in male mice. In order to further characterize the bone phenotype of female TIEG^−/− mice, we performed mechanical 3-point bending tests. These studies revealed that the femurs of TIEG^−/− mice are much weaker when compared to WT littermates. Micro-CT analysis of femurs of two-month-old TIEG^−/− and WT mice revealed a highly significant 9% decrease in total femur bone mass (cortical + cancellous bone) in the TIEG^−/− mice compared to WT littermates. Histomorphometric evaluation of the distal femur revealed that TIEG^−/− mice also display a 31% decrease in cancellous bone area, which is primarily due to a 22% decrease in trabeculae number. At the cellular level, TIEG^−/− mice exhibit a 42% reduction in bone formation rate which is almost entirely due to a reduction in double labeled perimeter. Differences in the mineral apposition rate were not detected between WT and TIEG^−/− mice. Interestingly, we have demonstrated that TIEG is regulated by estrogen in an ERβ isoform-specific manner in osteoblasts. Since ERβ is primarily expressed in cancellous bone, and since TIEG^−/− mice display significant defects in cancellous bone in a gender specific manner, we speculate that the estrogen regulation of TIEG may play a vital role in this phenomenon. Taken together, these findings suggest that TIEG^−/− mice are osteopenic mainly due to a decrease in total osteoblast number and support a critical role for TIEG in skeletal development and/or homeostasis.

Disclosures: M. Subramaniam, None.