ASBMR 29th Annual Meeting

1001

Identification of a Novel TGF-beta Dependent-Prometastatic Signature Mediating Bone Colonization. I. Garcia-Tunon*¹, S. Vicent*¹, D. Luis-Ravelo*¹, I. Anton*¹, C. Zandueta*¹, S. Martinez*¹, J. De Las Rivas*², F. Lecanda¹. ¹Division of Oncology, Center for Applied Medical Research, Pamplona, Spain, ²Bioinformatics, CIC, Salamanca, Spain.

Bone represents a frequent target organ of metastasis in a variety of solid tumors including lung. We have recently developed a rapid and selective model in which to identify genes implicated in bone metastasis of lung cancer. After intracardiac inoculation (i.c.) in nude mice, highly metastatic subpopulations (HMS) were selected with increase prometastatic activity. Microarray expression analysis revealed a novel metastatic gene signature of differentially expressed genes in HMS vs parental. Four overexpressed genes in all HMS were further validated by real time qPCR. These genes encode signaling molecules (such as TCF4 and PRKD3) and cell anchorage related proteins (MCAM, and putative adhesion molecule SUSD5). To delineate their functional contribution in metastatic activity we assessed metastatic area by X-ray image analysis and μCT scan of retrovirally transduced simple, double and triple gene combinations. I.c. of single overexpressors TCF4, PRKD3 or SUSD5 induced a significant increase in metastatic area as compared to mock cells (p<0.01). Furthermore, i.c. of triple overexpressors in nude mice synergistically induced a dramatic increase in prometastatic activity, with prominent osteolytic lesions and metastatic area (p<0.001), whereas cell homing activity to bone was unaltered. This multigenic signature was also ineffective promoting cell proliferation and subcutaneous tumor growth. By contrast, intratibial injection of triple overexpressors induced aggressive bone colonization leading to overt osteolytic lesions as compared to mock cells (p<0.001). These results were correlated with high osteoclastogenic activity induced by conditioned medium of triple overexpressors. Furthermore, TCF4 and SUSD5 were strongly up-regulated in a coculture system mimicking in vivo tumor-stroma interactions, an effect that was further abrogated by an anti-TGF-β peptide. Interestingly, after i.c. of HMS, in vivo treatment with a specific anti-TGF-β peptide severely reduced tumor burden (X-ray analysis and μCT scans) and osteolytic activity of tumor cells (osteoclast number) compared to an irrelevant peptide and vehicle treated animals. These studies identified a novel signature involving several genes that act cooperatively promoting strong osseous colonization, partially mediated by high osteoclastogenic activity. This multifunctional program is strongly enhanced by TGF-β, which plays a critical role in perpetuating a “vicious cycle” involved in metastatic progression.

Disclosures: 1. Garcia-Tunon, None.

This study received funding from: FIS, ISCIII-RETIC RD06/0020, Gov. Navarra.

1002

Osmosensing Receptor TRPV4 Mediates Cancerinduced Bone Pain. H. Wakabayashi*¹, L. Wang*², T. Matsubara*², A. Mizuno*³, M. Suzuki*³, A. Uchida*¹, T. Yoneda²¹Orthopaedic Surgery, Mie University Graduate School of Medicine, Tsu, Japan, ²Biochemistry, Osaka University Graduate School of Dentistry, Suita, Japan, ³Pharmacology, Jichi Medical School, Tochigi, Japan.

Bone pain is becoming a major clinical problem as survival of cancer patients is extended. Bone pain can be caused by noxious chemical and/or mechanical stimuli produced by cancer cells and associated inflammatory cells. H* is among such a noxious chemical stimulus. It has been shown that the acidic microenvironment caused by H* release by bone-resorbing osteoclasts induces bone pain through an activation of the acid sensing receptors such as transient receptor potential vanilloid 1 (TRPV1). TRPV4 is a transducer of hypo-osmotic stimuli in primary nociceptive afferents. Recent studies using TRPV4-/- mice suggest a critical role of TRPV4 in mechanosensing. Since TRPV4 has 45% homology with TRPV1, we reasoned that TRPV4 is also involved in the pathogenesis of cancer-induced bone pain. In the present study, we investigated the role of TRPV4 in the induction of bone pain associated with cancer colonization in bone. We established an animal model of cancer-induced bone pain in which cancer cells were directly inoculated into the marrow cavity of tibiae. Three behavioral assays including the measurement of paw withdrawal latency, grip force and hind-limb lifting (flinching) were employed to evaluate bone pain. Tumor-inoculated hind-limbs displayed hyperalgesia, decreased grip force and increased flinching as the tumor enlarged, whereas non-tumor-bearing hind-limbs showed no changes. Of note, there was significant reduction in paw withdrawal latency, grip force and flinching in tumor-inoculated hind-limbs in TRPV4-/- mice compared with WT mice, despite that tumor growth and area of osteolytic lesions did not differ between TRPV4-/- and WT mice.

The expression of immunoreactive neurons for pERK, a widely-used molecular marker for neural activation by noxious stimuli, was increased in the ipsi-lateral dorsal root ganglia (DRG) compared with the contra-lateral DRG in WT mice. p-ERK immunoreactive neurons were significantly decreased in the DRG in TRPV4-/- mice compared with WT mice. Hypo stimuli increased p-ERK expression in neuron-like F11 cells that expressed TRPV4 mRNA but not TRPV1 by RT-PCR. Ruthenium red, which is a non-selective TRPV4 inhibitor, decreased p-ERK expression induced by hypo stimuli. In conclusion, our results suggest that TRPV4 as well as TRPV1 plays an important role in inducing bone pain associated with cancer cell colonization in bone. TRPV4 might be a new molecular target for the development of novel analgesics in cancer-induced bone pain.

Disclosures: H. Wakabayashi, None.

1003

Role of Hypoxia in Cancers that Metastasize to Bone: HIF1α Enhances TGFβ Signaling and Expression of Prometastatic Factors CXCR4 and VEGF. L. A. Kingsley, P. G. J. Fournier, J. M. Chirgwin, T. A. Guise. Internal Medicine, University of Virginia, Charlottesville, VA, USA.

Several cancers frequently metastasize to bone, where the microenvironment is hypoxic and rich in growth factors, such as transforming growth factor (TGF)β. TGFβ stimulates tumor secretion of proteins that activate osteoclastic bone resorption, angiogenesis and tumor homing to bone. Many of the same proteins are also regulated by hypoxia-inducible factor (HIF)1α. We hypothesized that hypoxia-induced HIF1α enhances the effects of TGFβ on cancer metastases to bone.

First, we determined that bone metastases were hypoxic. Mice with MDA-MB-231 breast cancer bone metastases were injected prior to euthanasia with pimonidazole, which forms insoluble adducts in hypoxic cells. Immunohistochemical staining of decalcified sections showed that tumor cells adjacent to bone were hypoxic. Next, we determined by Western blot that hypoxia (1% O2) culture conditions induced the expression of HIF1α by TGFβ-responsive cancer cells which metastasize to bone: MDA-MB-231, PC-3 prostate cancer and 1205Lu melanoma.

Effects of hypoxia and TGFβ on promoters of bone metastatic factors VEGF and CXCR4 were studied using luciferase constructs. Both factors are known to be increased by hypoxia. MDA-MB-231 cells and HepG2 hepatocarcinoma cells were treated ± 5ng/ml TGFβ and ± 1%O₂ and lysates assayed for luciferase after 24hrs. Activity of both promoters was increased by TGFβ or 1%O₂. Combined treatment resulted in further promoter activation. Promoter activities correlated with increased mRNAs by quantitative real-time PCR (Q-PCR). These results suggest that TGFβ and HIF1α cooperatively regulate prometastatic gene expression.

We cotransfected cDNAs for HIF1α and the TGFβ signaling proteins Smads 2, 3, 4 and assayed for CXCR4 and VEGF promoter activity. Overexpression of HIF1α and Smads resulted in greater than 10-fold activation of the promoters by combined treatment with TGFβ and 1%O₂, providing further evidence for crosstalk between TGFβ and hypoxia signaling pathways. Finally, TGFβ target gene expression in MDA-MB-231 cells treated ± TGFβ and ± 1%O₂ was surveyed by Q-PCR. Hypoxia decreased two negative regulators of TGFβ signaling, Smad7 and SnoN, in addition to upregulating VEGF and CXCR4. Thus, hypoxia may potentiate TGFβ actions by down-regulating inhibitors of the Smad signaling pathway. Our results provide evidence for HIF1α and TGFβ crosstalk in vitro and suggest that these two pathways may cooperatively promote bone metastasis in vivo.

Disclosures: L.A. Kingsley, None.

1004

Osteoclasts in Myeloma Are Derived From Gr-1+CD11b+ Mononuclear Cells of the Bone Marrow Niche. J. Zhuang¹, L. Yang*², J. R. Edwards¹, C. M. Edwards¹, G. R. Mundy¹¹Vanderbilt Center for Bone Biology, Nashville, TN, USA, ²Cancer Biology, Vanderbilt University, Nashville, TN, USA.

When cancer cells are resident in bone, they initiate a vicious cycle with osteoclasts (OCs) which perpetuates their growth and aggressive behavior. OCs are critical for the maintenance of the vicious cycle, since they control not only bone destruction associated with cancer, but also the aggressive behavior of tumor cells. It has recently been recognized that tumor cells grow in distant sites because they induce non-malignant cells to establish a “pre-metastatic niche” for tumor cells to later engraft. But nothing is yet known for bone. Primitive bone marrow mononuclear cells, called myeloid immune suppressor cells (MISCs), which suppress immune reactivity, are important niche components. MISCs belong to the myelomonocytic lineage with surface markers of CD11b and Gr-1. We hypothesize that MISCs are precursors of OCs recruited by tumors to assist in the establishment of the vicious cycle. To test this hypothesis, we used the well-characterized 5TGM1 murine myeloma model. 5TGM1-GFP transfected myeloma cells were inoculated by tail vein injection. MISCs were assessed by FACS at weeks 1, 2, 3, 4 after tumor cell inoculation. The percentage of Gr-1+CD11b+ cells in marrow, spleen and blood in the tumor group began to increase significantly on week 2 compared with the control group. On week 4, this increase was much greater (60.9±7.8% vs 37.7±8.6% p<0.05 in marrow; 21.1±4.84% vs 2.4±0.85% p<0.05 in spleen; 23.1±4.27% vs 5.4±1.1% p<0.05 in blood) and paralleled the myeloma burden in bone and spleen. We sorted the Gr-1+CD11b+ cells from the spleen of tumor and non-tumor bearing mice. MISCs were induced into multinucleated TRAP positive OCs in medium containing M-CSF (25ng/ml) and RANKL (50ng/ml). The number of OCs derived from tumor MISCs was dramatically greater than those from control mice after 2 weeks of culture (13±2 per x200 field vs no more than 3). MISCs lost the Gr-1 and CD11b markers and gained CD51 and calcitonin receptor as they differentiated into mature OCs. These cells caused resorption pits on dentine discs, demonstrating they were functional OCs. We co-cultured sorted MISCs with 5TGM1 cells in a transwell plate (8um pore). MISCs markedly stimulated migration of GFP+ myeloma cells at 24h compared with non-MISCs (12.5±2.08 vs 3.75±0.83 per x200 field, p<0.05). Our data suggest that MISCs are increased significantly in marrow, spleen and blood of myeloma-bearing mice and parallel the appearance of lytic bone lesions. These MISCs differentiate avidly and rapidly into functional OCs. These results have a number of implications, including the possibility of reducing bone lesions in myeloma and other malignancies by depleting specific subpopulations of osteoclast precursors.

Disclosures: J. Zhuang, None.

1005

Collapse of the Vascular Bone Remodeling Compartment Is a Key Process in the Development of Myeloma-induced Osteolysis. T. L. Andersen*¹, T. E. Sondergaard*¹, K. Søe*¹, T. Plesner*², J. Delaisse¹¹Clinical Cell Biology (KBC), Vejle Hospital, Sourthern Denmark University, Vejle, Denmark, ²Department of Hematology, Vejle Hospital, Sourthern Denmark University, Vejle, Denmark.

Bone remodeling is a tightly coupled process where bone formation follows bone resorption to maintain the integrity of the bones throughout life. Bone remodeling occurs in a specialized vascular bone remodeling compartment (VBRC), separated from the marrow compartment by a cellular wall. In the case of myeloma-induced osteolysis, bone formation does not follow and compensate for the increased bone resorption. This uncoupling is a major hallmark of multiple myeloma (MM). Here we report that VBRCs often are disrupted in MM and that this destabilization correlates with: 1) an uncoupling of bone resorption and bone formation, 2) the presence of bone osteolysis as shown by x-rays, 3) direct cell contact between MM cells coming from the marrow compartment and osteoclasts, leading to the formation of myeloma-osteoclast hybrid cells.

We have established through bone histomorphometric analysis of the VBRC, eroded surfaces (ES), and osteoid surfaces (OS), that MM patients showing less than 75% ES in VBRC have a significantly increased ES/BS (20% vs 11%) and a significantly decreased OS/BS (15% vs 26%) compared to patients with more than 75% ES in VBRC. Interestingly, the collapse of VBRCs correlated significantly with the number of osteolytic lesions demonstrated by X-ray, as patients with no osteolytic lesions showed 80% of the ES in VBRC, while patients with more than 5 osteolytic lesions showed only 58% of the ES in VBRC.

We have previously reported that the myeloma-osteoclast hybrid cells account for more than 35% of the osteoclasts present in MM patients by identifying nuclei within osteoclasts having translocations specific for the MM cell clone. Here we show that the disruption of VBRC is a critical event in the generation of hybrid cells. This allows a direct contact between the MM cells of the marrow compartment and the resorbing osteoclast resulting in fusion of these two cell types.

In vitro, we mimicked the VBRC wall by establishing a confluent G₀-arested mono-layer of MC3T3 cells and tested its response to TGFβ or MM cells. Both addition of TGFβ and co-cultures with MM cells disrupted the confluent layer of MC3T3 cells mimicking the destabilization of the VBRC wall.

In conclusion the VBRC is a micro-anatomical structure that is important for the integrity of the bone remodeling process and its collapse is a key event in the development of MM bone disease. The VBRC is disrupted in MM, potentially both directly and indirectly by MM cells, favoring the development of osteolytic lesions and the formation of myelomaosteoclast hybrid cells.

Disclosures: T.L. Andersen, None.

1006

Accelerated Bone Resorption, Due to Dietary Calcium Deficiency, Promotes Tumor Growth in a Murine Model of Breast Cancer Bone Metastasis. Y. Zheng, H. Zhou, J. R. K. Modzelewski*, R. Kalak*, J. M. Blair*, M. J. Seibel, C. R. Dunstan. Bone Research Program, ANZAC research Institute, University of Sydney, Sydney, Australia.

The skeleton is a major site of breast cancer metastases. High bone turnover increases risk of disease progression and death. However, there is no direct evidence that high bone turnover is causally associated with the establishment and progression of metastases. In this study, we investigate the effects of high bone turnover on tumor growth in a rodent model of bone metastasis.

Female nude mice were fed a diet containing normal (0.6%) (‘Normal-Ca’) or low (0.1%) (‘Low-Ca’) calcium content starting on day −3. Mice were concurrently treated with vehicle or recombinant osteoprotegerin (OPG; 1mg/kg/day sc; n = 16/group). Three days later (day 0), 50,000 TxSA cells (a bone-seeking variant of MDA-MB-231 cells) were implanted by intra-tibial injection and mice were followed until day 17.

On day 0, mice receiving ‘Low-Ca’ had increased serum PTH and TRAcP5b levels, indicating secondary hyperparathyroidism and high bone turnover, which was maintained until day 17. Treatment with OPG increased serum PTH but profoundly reduced bone resorption. On day 17, in mice receiving ‘Low-Ca’ alone, lytic lesion area, tumor area and cancer cell proliferation increased by 43%, 24% and 24%, respectively compared to mice receiving ‘normal Ca’ (p < 0.01). In contrast, OPG treatment completely inhibited lytic lesions, reduced tumor area, decreased cancer cell proliferation and increased cancer cell apoptosis.

We conclude that increased bone turnover, due to dietary calcium deficiency, promotes tumor growth in bone, independent of the action of PTH. These findings have clinical implications as breast cancer patients, much like the older population in general, frequently have a low dietary calcium intake and high bone turnover.

Disclosures: C.R. Dunstan, Amgen 1.

This study received funding from: The National Health and Medical Research Council of Australia (NHMRC project grant #352332).

1007

ATF4 Is Required for the Anabolic Actions of PTH on Bone In Vivo. S. Yu¹, M. Luo*¹, R. T. Franceschi², D. Jiang*³, J. Zhang¹, K. Patrene*¹, K. D. Hankenson⁴, G. D. Roodman¹, G. Xiao¹¹Medicine, University of Pittsburgh, Pittsburgh, PA, USA, ²Periodintics and Oral Medicine, University of Michigan, Ann Arbor, MI, USA, ³Periodontics and Oral Medicine, University of Michigan, Ann Arbor, MI, USA, ⁴University of Pennsylvania, Philadelphia, PA, USA.

Parathyroid hormone (PTH) is a potent stimulator of bone formation and a proven anabolic agent for the treatment of osteoporosis. However, the mechanism whereby PTH increases bone formation remains poorly understood. Activating transcription factor 4 (ATF4) is a critical factor for bone formation during development and throughout postnatal life. This study examined if ATF4 is required for the anabolic actions of PTH on bone using an Atf4-/- mouse model. Five-day-old wt and Atf4-/- mice were given daily subcutaneous injections of vehicle (saline) or hPTH(1–34) (0.04 μg/g body weight) for 28 days. In wt mice, μCT analyses of femurs show that this PTH regimen significantly increased bone volume/tissue volume (BV/TV, 4.3-fold), trabecular thickness (Tb.Th, 50%), trabecualr numbers (Tb.N, 1.5-fold), cortical thickness (Cort. Th, 77%), and cross sectional area (CSA, 24%) and decreased trabecular spacing (Tb.Sp, 1.7-fold). These PTH effects were dramatically reduced or completely abolished in the absence of ATF4. Histological analyses show that PTH displayed potent anabolic effects on tibiae, vertebrae, and calvariae, which were significantly reduced in Atf4-/- mice. At the molecular level, PTH markedly increased levels of osteocalcin (Ocn) and bone sialoprotein (Bsp) mRNA of long bones as measured by quantitative real-time RT/PCR. This increase was completely abolished in the absence of ATF4. This study demonstrates that ATF4 is required for the anabolic actions of PTH on bone in vivo and also suggested that modulation of the levels and activity of ATF4 may have therapeutic significance for the treatment of metabolic bone diseases such as osteoporosis.

Disclosures: S. Yu, None.

1008

Rac2 Knockout Mice Have an Augmented Anabolic Response to PTH, T. Kawano*, B. Sun*, J. Wu*, I. Karl. Department of Medicine, Yale University, New Haven, CT, USA.

Rac2 is a small GTPase that is required for CSF1-dependent osteoclast chemotaxis. Rac2^−/− mice have increased bone mass, and osteoclast-like cells prepared from these animals have reduced basal resorptive capacity. Histomorphometric analysis has demonstrated a trend towards increased numbers of osteoclasts while osteoblast number is normal. Recent studies have suggested that cells of the osteoclast lineage are necessary for the full anabolic effect of PTH to be manifest. We hypothesized that since osteoclast precursors would be normal or increased in Rac2^−/− animals while mature osteoclast function was impaired, the anabolic effect of PTH might be enhanced in these animals. To test this idea, adult Rac2^−/− mice and wild-type C57B1/6 mice were treated with either a single daily sc injection of h(1–34) PTH (80ng/g body weight) or vehicle for 4 weeks, and BMD measured after 0, 2 and 4 weeks of treatment. At the end of 2 weeks the PTH-treated Rac2^−/− mice showed a greater increase in spine, femur and total body BMD compared to baseline than did the wild-type mice (3.9±6.8% vs. 1.6±6.5%, 9.0±5.7% vs. 5.8±5.3% and 7.2±3.1% vs. 4.9±2.5%; spine, femur, total body respectively; M±SD). After 4 weeks of treatment this difference was the same or greater (7.4±4.1% vs. 3.7±6.6%, 23.6±9.0% vs. 7.4±6.5% and 13.5±3.4% vs. 5.4±2.6%; spine, femur, total body respectively; M±SD). Further, when compared to vehicle-treated Rac2^−/− animals, the PTH-treated Rac2^−/− mice showed significantly greater mean increases in bone mass as compared to the increment in BMD observed in PTH-treated wild-type compared to vehicle-treated wild-type mice (6.7% vs. 1.8%; 16.2% vs. 3.8%; and 9.5% vs. 3.5% spine, femur and total body). Consistent with our hypothesis, serum CTX at 4 weeks was significantly lower in the PTH-treated Rac2-/- animals than in the PTH-treated wild-type mice (13.8±2.9 vs. 22.4±9.5 ng/ml; M±SD). While the detailed cellular mechanisms that underlie this effect are not yet clear, these data support the notion that subtley altering osteoclast function may be a feasible way to improve the anabolic response to PTH. Further, since Rac2 is restricted in its expression to hematopoietic tissue it could represent a reasonable target for drug discovery.

Disclosures: T. Kawano, None.

1009

RANK Ligand Inhibition by Denosumab Prevents Cortical Bone Loss in a Murine Model of Glucocorticoid-induced Osteoporosis, L. C. Hofbauer¹, U. Zeitz*², M. Schoppet*¹, M. Skalicky*², M. Stolina³, P. J. Kostenuik³, R. G. Erben²¹Philipps-University, Marburg, Germany, ²University of Veterinary Medicine, Vienna, Austria, ³Amgen Inc., Thousand Oaks, CA, USA.

Osteoporosis is a frequent and severe complication of systemic glucocorticoid therapy, and in vitro studies suggest that RANK ligand (RANKL) represents a potential mediator. We examined the ability of Denosumab (DMab), a fully human monoclonal antibody against RANKL, to prevent glucocorticoid-induced osteoporosis in mice. DMab does not inhibit murine RANKL, so knockin mice were created by replacing exon 5 of the murine RANKL gene with a chimeric 5th exon (murine non-coding and human coding regions). These mice exclusively express a chimeric RANKL protein that is inhibited by Denosumab. Male huRANKL mice (8 months old) received slow-release pellets containing either vehicle or prednisolone (PRED, 5 mg) which was released at a mean dose of 2.1 mg/kg/d over 4 weeks. HuRANKL mice were treated with denosumab (10 mg/kg, twice/week, s.c.) or vehicle (PBS) starting 3–4 days after pellet implantation. In addition, two groups of wild-type mice (Black Swiss/129) were treated with subcutaneous slow release pellets containing vehicle or PRED. Four weeks later all mice were killed after in vivo double fluorochrome labeling. Renal handling of sodium, potassium, and phosphate was unchanged by PRED or denosumab in huRANKL mice. However, PRED increased renal excretion of calcium in wild-type and huRANKL mice. PRED treatment also significantly increased urinary deoxypyridinoline (DPD) excretion as well as serum and bone TRAP5b activity, and denosumab treatment prevented these increases. For example, protein extracts of the whole humerus contained 11.7 ± 2.7, 17.3 ± 5.1, and 7.7 ± 7.5 U/g TRAP5b activity (mean ± SD) in huRANKL mice receiving vehicle, PRED, or PRED + denosumab, respectively (all P<0.05). Quantitative computed tomography (pQCT) revealed that PRED induced significant bone loss in the axial and appendicular skeleton mainly by thinning of cortical bone, consistent with increased endocortical bone resorption. Trabecular BMD and cancellous bone area remained unchanged in PRED-treated mice. Treatment with denosumab completely prevented PRED-induced cortical bone loss in the L4 vertebrae and in the distal femoral metaphysis without impairing biomechanical properties as determined by three-point bending tests in femurs.

In summary, PRED treatment caused cortical bone loss that was consistent with increased osteoclastic bone resorption. Treatment of huRANKL knock-in mice with denusomab, a fully human anti-RANKL antibody, prevented glucocorticoid-induced bone loss in association with suppression of bone resorption.

Disclosures: L.C. H of bauer, None.

1010

A Small Molecule Inhibitor of the Wnt Antagonist Secreted Frizzled-Related Protein (SFRP)-1 Stimulates Bone Formation. P. V. Bodine¹, B. Stauffer*¹, H. Ponce-de-Leon*¹, R. A. Bhat¹, A. Mangine*¹, L. M. Seestaller-Wehr*¹, R. A. Moran¹, J. Billiard¹, S. Fukayama², B. S. Komm¹, K. Pitts*³, G. Krishnamurthy*³, A. Gopalsamy*³, M. Shi*³, J. C. Kern*⁴, T. J. Commons*⁴, M. A. Wilson*⁴, G. S. Welmaker*⁴, E. J. Trybulski*⁴, W. J. Moore*⁴¹Women's Health & Musculoskeletal Biology, Wyeth Research, Collegeville, PA, USA, ²Women's Health & Musculoskeletal Biology, Wyeth Research, Cambridge, MA, USA, ³Chemical & Screening Sciences, Wyeth Research, Pearl River, NY, USA, ⁴Chemical & Screening Sciences, Wyeth Research, Collegeville, PA, USA.

Canonical Wnt signaling has been demonstrated to increase bone formation by enhancing osteoblast proliferation, differentiation and activity, while suppressing apoptosis. Consequently, Wnt pathway components have been pursued as potential drug targets for metabolic bone diseases like osteoporosis. Deletion of the Wnt antagonist SFRP-1 in mice activates canonical signaling in bone and increases trabecular bone formation in aged animals. We have developed small molecules that bind to and inhibit SFRP-1 in vitro and demonstrate robust anabolic activity in an ex vivo organ culture assay. A library of over 400,000 compounds was screened for inhibitors of human SFRP-1 using a cell-based functional assay that measured activation of canonical Wnt signaling with an optimized T cell factor (TCF)-luciferase reporter gene assay. One of the hits in this screen, a diarylsulfone sulfonamide, bound to human SFRP-1 with a Kd = 0.3 uM in a tryptophan fluorescence quenching assay. This compound inhibited human SFRP-1 with an EC50 = 2.2 uM in the cell-based functional assay, but did not impede other SFRP family members. Moreover, the compound did not antagonize the activity of a SFRP-1 construct that lacked the C-terminal netrin domain, suggesting that this region contains the binding site. The compound also increased total bone area in a murine calvarial ex vivo assay at a concentration of 1.0 uM. Optimization of this high throughput screening hit for binding and functional potency as well as metabolic stability and other pharmaceutical properties led to improved lead compounds. One of these leads bound to SFRP-1 with a Kd = 0.08 uM and inhibited SFRP-1 with an EC50 = 0.7 uM. Moreover, it increased total bone area in the murine calvarial ex vivo assay at concentrations as low as 0.001 uM. Low oral bioavailability of this compound limited its in vivo evaluation in mice and rats. This work demonstrates the feasibility of developing small molecules that inhibit SFRP-1 and stimulate canonical Wnt signaling to increase bone formation.

Disclosures: P.V. Bodine, Wyeth Research 1, 3.

This study received funding from: Wyeth Research

1011

A Bisphosphonate Analog that Lacks Anti-Remodeling Activity Prevents Osteocyte and Osteoblast Apoptosis In Vivo. L. I. Plotkin, J. Goellner, K. Vyas*, R. S. Shelton*, R. A. Wynne*, R. S. Weinstein, S. C. Manolagas, T. Bellido. Center for Osteoporosis and Metabolic Bone Diseases, Univ Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, AR, USA.

A major effect of bisphosphonates (BPs) on bone is inhibition of resorption resulting from their ability to induce osteoclast death or interfere with osteoclast function. Nonetheless, BPs also prevent osteocyte and osteoblast apoptosis in vitro and in vivo, but the contribution of the latter property to the overall beneficial skeletal effects of these agents remains unknown. We compared herein the effect of IG9402, a BP analog that preserves osteoblast and osteocyte viability but does not induce osteoclast apoptosis in vitro, with that of alendronate, a classic BP that affects osteoclasts as well as osteoblasts and osteocytes. Swiss Webster mice (7–8 per group) were implanted with placebo or prednisolone (GC) pellets (2.1 mg/kg/d) for 10 days, and were injected daily with saline or 2.3 μmol/kg/d alendronate or IG9402, starting 3 days prior to pellet implantation. Alendronate decreased the serum levels of C-telopeptide and osteocalcin, markers of bone resorption and formation, respectively, as well as the levels of osteocalcin and collagen 1A1 mRNA in vertebral bone. On the other hand, IG9402 did not affect serum levels of C-telopeptide or osteocalcin nor bone mRNA levels of osteocalcin or collagen1A1. Moreover, whereas alendronate decreased cancellous bone formation rate from 0.43±0.25 to 0.09±0.03 μm²/μm/d (p<0.05), IG9402 did not affected it significantly (0.39±0.04 μm²/μm/d). These findings are consistent with a decrease in bone remodeling resulting from inhibition of resorption by alendronate but not by IG9402. Furthermore, the increase in osteoblast and osteocyte apoptosis induced by GC (from 5.6±2.5 to 15.1±6.0% and 3.1±1.7 to 7.1±1.1%, respectively, p<0.05) was prevented not only by alendronate, as previously shown, but also by IG9402. In addition, GC induced a significant decrease in the load required to break the 6^th lumbar vertebra (68.1±11.6 to 47.1±8.5 N, p=0.006) and a decrease that was non-significant in the compression stress (27.8±5.3 to 21.2±4.9 in MPa p=0.095); and both alendronate and IG9402 prevented the decreased ability of the vertebrae to sustain load induced by GC. We conclude that preservation of osteoblast and osteocyte apoptosis by bisphosphonates is an important, but heretofore unappreciated, mechanism of the beneficial effect of these drugs on bone. Preservation of bone strength without inducing osteoclast apoptosis by IG9402 opens new possibilities for the treatment of bone fragility in conditions in which a decrease in bone remodeling is undesirable.

Disclosures: L.I. Plotkin, None.

This study received funding from: NIH and University of Arkansas for Medical Sciences.

1012

VDRM 1: A Tissue-Selective, Non-Secosteroidal Vitamin D Receptor Modulator with Improved Therapeutic Window of Bone Efficacy over Hypercalcemia, Y. L. Ma, Y. K. Yee*, Q. Zeng*, A. Schmidt*, D. E. Coutant*, R. J. Barr*, L. I. Boone*, S. R. Chintalacharuvu*, J. A. Dodge*, H. U. Bryant, M. Sato. Eli Lilly and Company, Indianapolis, IN, USA.

The active form of vitamin D3 and analogs have been shown to be beneficial for osteoporosis, autoimmune diseases and cancer treatment in animal models; however, the narrow therapeutic window between therapeutic efficacy and hypercalcemia has limited their clinical utility. We have identified an orally active, tissue selective, non-secosteriodal, VDR ligand to be a potent agonist in bone, while having modest calcemic effects in vivo. When compared to 1,25(OH)2D3 in vitro, VDRM 1 was 10 fold less potent in inducing RXR-VDR heterodimerization (3.9 and 38 nM, respectively) and 100 fold less potent in inducing osteocalcin expression in a reporter gene assay (0.2 and 20 nM, respectively), but VDRM 1 was >300 fold less active than 1,25 (OH)2D3 in inducing epithelial cell calcium channel 2 (CAT1) gene expression in Caco-2 cells (3.3 and 1062 nM, respectively). In 7 months old, one month post overiectomized (Ovx), osteopenic rats, two months of VDRM 1 treatment dose dependently restored Ovx induced loss of vertebral BMD to Sham levels at 0.73 ug/kg/d. Hypercalcemia was not observed until about 4.6ug/kg/d in these animals. Biomechanical analyses showed that VDRM 1 improved bone strength (ultimate load) of lumbar vertebra, femoral midshaft and the femoral neck to significantly above Sham control levels. Static and dynamic histomorphometry showed a dose dependent reduction of both resorption and formation activity in the proximal tibial metaphysis; however a dose-dependent stimulation of the periosteal bone formation rate of > 2 fold was observed on the cortical surface. Histopathology evaluation indicated a 17 fold separation between the bone efficacy and soft tissue calcification in these Ovx rats. Taken together, these animal data suggest that VDRM 1 may have promise to treat postmenopausal women with osteoporosis.

Disclosures: Y.L. Ma, Eli Lilly 3.

1013

Disruption of Hypoxia Inducible Factor-1α in Osteoblasts Exacerbates Ovariectomy Induced Bone Loss in Mice, L. Deng*¹, X. Liu*¹, C. Wan², J. Wang*¹, J. Qi*¹, T. Luo*¹, J. Wang*¹, Q. Zhou*¹, Y. Zhu*¹, L. Wei*¹, T. L. Clemens²¹Shanghai Institute of Traumatology and Orthopaedics, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China, ²Pathology, University of Alabama at Birmingham, Birmingham, AL, USA.

Postmenopausal and senile osteoporosis are associated with decreased skeletal blood flow, reduced capillary networks and impaired endothelial cell function. Vasoactive agents can attenuate oophorectomy-induced bone loss, suggesting that estrogen normally regulates the vascular supply to bone. Angiogenesis during bone development and repair is controlled by the hypoxia inducible factor-1α (HIF-1α) which transcriptionally activates VEGF and other proangiogenic proteins. In this study, we examined the contribution of the HIF-1α pathway on estrogen action in mouse bone in vitro and in vivo. Treatment of wild-type primary mouse osteoblasts with estradiol increased HIF-1α and VEGF protein expression in a time and concentration dependent manner; these effects were more pronounced under hypoxic conditions. To examine the role of HIF-1α on osteoblast differentiation, primary osteoblasts carrying floxed HIF-1α alleles were infected with adenovirus constructs expressing Cre to disrupt HIF-1α. Osteoblasts lacking HIF-1α had reduced the levels of VEGF, Runx2, OC and ALP, and decreased RANKL mRNA levels consistent with recent reports that hypoxia regulates osteoclast activity through HIF-1-RANKL interactions. To examine the role of HIF-1α in vivo, mice carrying an osteoblast specific mutation of HIF-1α were subjected to ovariectomy. At baseline, HIF-1α mutants had modestly reduced bone volume, mineral apposition rate, osteoblast numbers, and increased osteoclast numbers relative to control mice. Following ovariectomy, HIF-1α mutants exacerbated bone loss by histomorphometry and microCT (BV/TV −1.33 fold, trabecular thickness −1.27 fold, and mineral apposition rate −1.13 fold, n=4, P<0.05) and an increase in osteoclast numbers (+1.20 fold) versus controls. These results support the hypothesis that estrogen normally regulates HIF-1α and that loss of proangiogenic HIF-1α signaling exacerbates bone loss following ovariectomy.

Disclosures: L. Deng, None.

1014

Deletion of PYK2 Led to High Bone Mass Through a Positive Balance Between Bone Formation and Resorption in Aged Mice, M. Li, D. T. Crawford*, H. Qi*, H. A. Simmons, Y. Li*, D. R. Healy*, E. A. Smith, L. Buchbinder, T. A. Brown. Cardiovascular and Metabolic Diseases, Pfizer Global Research and Development, Groton, CT, USA.

We have recently found that PYK2, a tyrosine kinase, plays an inhibitory role in bone formation. Genetic deletion of PYK2 (PYK2^−/−) resulted in increased bone formation and bone mass in adult mice. However, it is not known if the PYK2^−/− phenotype persists with age and how the bone performs biomechanically. Therefore, we characterized the skeletal phenotype of female PYK2^−/− mice at 12 months of age. There were no differences in body weight and femur length between PYK2^+/+ and PYK2^−/− mice, demonstrating that PYK2 is not essential for bone growth. Three dimensional μCT analysis of trabecular bone of 2^nd lumbar vertebra showed that trabecular bone volume per total volume, trabecular number, thickness, and connectivity density were significantly increased in PYK2^−/− mice by +300%, +74%, +149%, and +80%, respectively. In contrast, trabecular separation was significantly decreased by −36% in PYK2^−/− mice. These data indicate that PYK2^−/− mice had higher cancellous bone mass and improved microarchitecture than PYK2^+/+ controls. Histomorphometric analysis was performed on the 5^th lumbar vertebral bodies. Consistent with μCT findings, cancellous bone volume, trabecular number and thickness were significantly higher whereas trabecular separation was lower in the PYK2^−/− mice than those of PYK2^+/+ controls. Osteoclast surface and number were significantly lower by −40% and −39%, respectively, suggesting a decrease in bone resorption in the PYK2^−/− mice. However, these mice had a +36% and +69% significant increase in tissue referent bone formation rate and ratio of mineralizing surface/osteoclast surface, respectively, which combined with the increased bone mass suggested a positive balance between bone formation and resorption in PYK2^−/− mice. Biomechanical properties were examined in the femoral diaphysis, a cortical bone site, and lumbar vertebra body, a cancellous bone site, by four point bending and compression tests, respectively. Maximum load and stiffness were significantly increased by +27% and +24%, respectively in the femurs of PYK2^−/− mice. Similarly, maximum load and energy were significantly higher in the lumbar vertebra of PYK2^−/− mice. These data indicate that PYK2^−/− mice not only had high bone mass but superior structural properties than PYK2^+/+ controls in both cancellous and cortical bone sites. In summary, deletion of PYK2 caused a positive balance between bone formation and resorption, resulting in augmentation of bone mass and biomechanical properties. Our findings in aged mice indicate that the positive impact of PYK2 inhibition persists with aging.

Disclosures: M. Li, Pfizer Inc. 3.

1015

β-adrenergic Signaling Requires Bone Matrix Protein OPN to Suppress Bone Formation and to Activate Bone Resorption. M. Nagao*¹, Y. Saita*¹, J. Nagata*¹, Y. Izu*¹, T. Hayata*¹, H. Hemmi*², Y. Ezura*¹, K. Nakashima*¹, H. Kurosawa*³, M. Noda*¹¹Dept Morecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo, Japan, ²MTT Program, Tokyo Medical and Dental University, Chiyoda-ku, Tokyo, Japan, ³Orthopedics, Juntendo University School of Medicine, Bunkyo-ku, Tokyo, Japan.

Sympathetic nervous system controls bone mass through the action of β-adrenergic signals that suppress bone formation and stimulate bone resorption. Sympathetic tone would exert its effects through the activation of osteoclast differentiation and in part facilitation of hematopoietic cell progenitor mobilization. Osteopontin (OPN), one of the major components of noncollagenous matrix proteins in bone, has been shown to promote cell adhesion and negatively regulate stem cell niche. However, the role of OPN in bone loss induced by sympathetic signaling via β-adrenergic receptors is unknown. We therefore investigated how signaling of a β-adrenergic agonist, isoproterenol (ISO) signaling, interacts with OPN signaling in bone. MC3T3E-1 cells were treated with 10uM isoproterenol and this treatment decreased OPN expression levels after 2 hours and it reached down to one-third of initial levels after 8 hours, whereas expression levels of RANKL were increased about 20 folds at 2 hours. These observations suggest that OPN may play a role in ISO-induced bone loss. Therefore, to explore the involvement of OPN in ISO-induced bone loss in vivo, ISO was administrated into 9 weeks old wild type (WT) and OPN-/- mice. Administration of ISO for 3 weeks reduced trabecular bone mass (BV/TV) in WT mice based on uCT analysis. In contrast, such reduction was not observed in OPN-/- mice. Histomorphometric analysis showed that Iso treatment deceased bone formation rate(BFR) and mineral apposition rate(MAR) in WT mice. In contrast, OPN deficiency suppressed ISO-induced reduction in BFR and MAR. Since OPN is involved in bone resorption, we examined osteoclasts in OPN-/- mice. Iso treatment increased the number and surface of TRAP positive cells in WT mice. In contrast, OPN deficiency suppressed ISO treatment-induced increase in these parameters of osteoclasts. These data indicated that deficiency of OPN blocks β-stimulant-induced bone loss. In conclusion, OPN plays a critical role in β-adrenergic signaling-induced bone loss in vivo via modulation of both bone formation and bone resorption.

Disclosures: M. Nagao, None.

1016

The Basis for Osteoporosis in the Aldehyde Reductase Knockout Mouse, K. H. Gabbay*¹, K. M. Bohren*¹, R. Morello², J. Song*¹¹Pediatrics, Baylor College of Medicine, Houston, TX, USA, ²Molecular & Human Genetics, Baylor College of Medicine, Houston, TX, USA.

The aldehyde reductase enzyme (AKR1A4 - GR) knockout (GRKO - GR^−/−) mouse develops an age-related (>1 year) osteoporosis phenotype with spontaneous fractures that preferentially affect females. There is a marked acceleration of the phenotype in GR^−/− males after castration (CX) and in females after ovariectomy (OVX), and upon induction of streptozotocin diabetes. Both GR and a related enzyme, aldose reductase (AR) are able to reduce D-glucuronate to L-gulonate which is converted to ascorbic acid (ASC) by gulonolactone oxidase (GULO). Our data identify GR as the main source of L-gulonate for ASC synthesis in the mouse. AR has a secondary role indicated by a double knockout model, AR/GRKO, which recapitulates the clinical features of the GULO knockout model (GULOKO) with rapid onset of scurvy in both genders and death at 8–10 weeks of age. While the AR/GRKO and GULOKO models have absolute deficiencies, the GR-/- mice have a moderate ASC deficiency. They show a marked decrease of trabecular (Tb) bone with hypertrophy of dysplastic mesenchymal cell masses, and absence of osteoblasts (Ob) at the growth plate. These cell masses (presumably dysplastic Ob) resolve and form disorganized osteoid after ASC feeding. Serial DEXA measurements in CX GR-/- mice placed on regular diet (ASC content =0) show a statistically significant (p<0.001) decrease in bone mineral density (BMD). The bone phenotype is prevented by dietary ASC. Quantitative bone histomorphometry shows statistically significant decreases in bone volume, trabecular thickness and number with a marked increase in osteoclast (Oc) activity and number in CX GR-/- mice after 3 weeks on regular diet. ASC administration after CX significantly suppresses Oc activity and number below those observed in intact GR-/- levels on regular diet. We propose that the limited ASC synthesis in the GR-/- mouse is adequate for normal skeletal development, but results in an uncoupling of bone homeostasis (formation and resorption) when the mice are subjected to stress. Thus, CX, OVX, or diabetes in the GR-/- mice increase bone resorption in the face of reduced ASC availability, resulting in severe osteoporosis and fractures. ASC suppresses Oc activity and number in addition to promoting the differentiation of Ob. We suggest that oxidative stress in CX, OVX and diabetes is the common effector pathway for osteoclastogenesis. The GRKO model permits differentiation between the pro-osteoblastic activity of ASC and its additional anti-oxidant properties. This mouse model is relevant to understanding the role of ASC in bone homeostasis and the development of osteoporosis in normal and diabetic populations.

Disclosures: K.H. Gabbay, None.

This study received funding from: The Harry & Aileen Gordon Foundation.

1017

Risedronate Prevents Bone Loss in Breast Cancer Survivors: A Two-Year, Randomized, Double-Blind, Placebo-Controlled Clinical Trial. S. L. Greenspan¹, K. T. Vujevich*¹, R. Bhattacharya², S. Perera*³, A. Brufsky*¹, B. C. Lembersky*¹, V. Vogel*¹¹Medicine, University of Pittsburgh, Pittsburgh, PA, USA, ²Medicine, University of Kansas, Kansas City, KS, USA, ³Medicine/Biostatistics, University of Pittsburgh, Pittsburgh, PA, USA.

Chemotherapy induced menopause and aromatase inhibitors (AI) are associated with bone loss and increased risk for osteoporotic fractures in breast cancer survivors. Little data are available on the efficacy of oral antiresorptive therapy to prevent this. The REBBeCA Trial was a single center, 2-year, randomized, double-blind, placebo-controlled trial to evaluate the efficacy of once weekly oral risedronate (RIS) 35mg, for the prevention of bone loss in newly postmenopausal women, following chemotherapy induced menopause for nonmetastatic breast cancer. All received calcium and vitamin D. Outcomes included changes in spine and hip bone mineral density (BMD, Hologic Discovery A) and bone resorption assessed by urinary NTX. 97% of the 87 women (mean age 50 years) randomized had normal or low bone mass by WHO criteria. At baseline, 71% were on tamoxifen and 16% were on an AI. Baseline clinical characteristics and BMD were similar between the 2 groups. During year 2, 49% of women were started on an AI by their oncologist. After 24 months, women in the PLB group had significant losses of BMD at the spine and hip sites (p<0.05) that were not observed in the RIS group (Table). At 24 months, BMD was significantly higher at the lateral spine and hip sites in women on RIS compared to PLB (2.3–2.4%, p<0.05; Table). In a secondary analysis we compared the response in women ever on an AI (+AI, n=47) and never on an AI (No AI, N=40) to their respective PLB group. Women on RIS No AI or RIS + AI had BMD 1.5 and 4.0% greater at the hip compared to women in the PLB No AI or PLB + AI groups respectively (p<0.05; Table). Women on RIS No AI had BMD 3.7% greater at the lateral spine compared to women in the PLB No AI group (p<0.05). Bone resorption was significantly lower in the women on RIS. We conclude that once weekly oral RIS improves BMD and prevents excess bone resorption in breast cancer survivors, even in women on an AI.

Table Table:. Mean percent change ± standard error in BMD or Marker at 24 months

*p<0.05, **p<0.01 from baseline, +p<0.05, ++p<0.01 RIS vs. PBO

Disclosures: S.L. Greenspan, Procter and Gamble 2. 5.

This study received funding from: Procter and Gamble/Investigator Initiated.

1018

XXY Mice Have an Osteoporotic Phenotype. P. Y. Liu*¹, R. Kalak*², Y. Lue*³, K. Erkkila*³, U. Simanainen*¹, H. Zhou², A. Hikim*³, D. Handelsman*¹, M. Seibel², C. Wang*³, R. Swerdloff*³, C. Dunstan²¹Andrology, ANZAC Research Institute, Concord, Australia, ²Bone Biology, ANZAC Research Institute, Concord, Australia, ³Endocrinology, Harbor UCLA Medical Center, Los Angeles, CA, USA.

Klinefelter's syndrome is the most common chromosomal aneuploidy in men (XXY karyotype, 1:600 live births) and is characterised by infertility and androgen deficiency, cognitive impairment and osteoporosis. The extent to which skeletal changes are due to sex hormone deficiency or arise directly from gene over-dosage cannot be easily determined in humans. To answer this question, we generated XXY mice through a complex 4-generation breeding scheme.

Eight intact XXY mice and 9 XY littermate controls, and 8 castrated XXY mice and 8 castrated XY littermate controls, were sacrificed at one year of age. Castration occurred 6 months prior to sacrifice. Tibiae were harvested and examined by mCT and histomorphometry. Blood testosterone was assayed by radioimmunoassay.

Compared to intact XY controls, XXY mice had a lower bone volume (6.8±1.2 vs 8.8±1.7%, mean±SD, P=0.01) and trabeculae were thinner (, 50±4 vs. 57±5mm, P=0.007). Trabecular separation (270±20 vs. 270±20mm), osteoclast number relative to bone surface (2.4±1.0 vs 2.7±1.5/ mm2) and blood testosterone concentrations (5.3±4.7 vs 2.5±3.9 ng/mL) did not differ significantly. These mCT, histomorphometric and blood parameters were similar between castrated XXY and castrated XY mice. As expected, castration drastically decreased relative bone volume (P<0.001), trabecular thickness (P=0.05), trabecular separation (P<0.01) and blood testosterone concentrations (P<0.001) with equivalent effects in XY and XXY mice.

In conclusion, XXY mice replicate many features of human Klinefelter's syndrome making them a useful model for studying bone, as well as testis and behaviour (1). Testosterone deficiency may not fully explain the bone phenotype in the non-castrate state as XXY mice show both reduced bone volume and similar blood testosterone levels. These data suggest that novel genes, that escape × inactivation, contribute to bone loss, and when identified, may provide unique molecular targets for the management of osteoporosis.

(1) Lue Y, et al 2005 XXY mice exhibit gonadal and behavioral phenotypes similar to Klinefelter syndrome. Endocrinology 146:4148–4154

Disclosures: P.Y. Liu, None.

1019

A Stone Mouse: Transgenic Expression of an Engineered Gs-coupled Receptor in Osteoblasts Produces Increased Bone Mass. E. C. Hsiao¹, B. Boudignon², P. W. Chang*¹, M. Benscik*², J. Peng*², C. Manalac*¹, B. Halloran², B. Conklin*¹, R. Nissenson²¹Gladstone Institutes, University of California, San Francisco, CA, USA, ²Endocrine Unit, VA Medical Center, San Francisco, CA, USA.

Activation of the G-protein coupled PTH/PTHrP receptor (PPR) in osteoblasts can elicit a marked increase in bone formation and bone mass. The PPR is capable of activating multiple G protein pathways; however the specific signals responsible for the anabolic effects of the PPR are not fully understood. To address this problem, we engineered a novel GPCR (“Rs1”) that activates the Gs/cyclic AMP pathway and displays constitutive Gs-linked signaling in transfection experiments of cultured cells. Rs1 was targeted to osteoblasts in vivo using a Coll 2.3kb promoter fragment combined with the TetO/tTA (TetOff) system. Expression of Rs1 in maturing osteoblasts from conception was sufficient to induce a dramatic anabolic skeletal response. Mutant mice displayed a 2–5 fold increase in femoral bone volume as compared to age and sex-matched littermate controls at 3 weeks. Whole body DEXA demonstrated a 3-fold increase in BMD in 9-week-old mice over controls (0.14 g/sq cm vs. 0.05 g/sq cm, respectively); microCT imaging confirmed a massive and generalized increase in mineralized bone by 9 weeks of age. Histomorphometry showed a marked increase in bone cellularity and osteoid with disorganized trabecular bone and loss of the cortical shell. Serum markers of bone metabolism were elevated in the mutant mice as compared to wildtype controls, including alkaline phosphatase at 3 weeks (970.6+/-306.8 vs 251.6+/-27.1 IU/L), and osteocalcin (507.2+/-86.6 vs 131.6+/-45.3 ng/ml) and pyridinoline cross links (2.45+/-0.64 vs 1.28+/-0.07 nmol/ml) at 9 weeks. No skeletal phenotype was observed in mice where Rs1 expression was suppressed by doxycycline administration from conception through the first 4 weeks of postnatal life. Our results indicate that Gs-GPCR signaling in osteoblasts during the first 4 weeks of life can produce a massive and progressive increase in bone formation and bone mass. The observed phenotype of these mice differs significantly from that of fibrous dysplasia and of transgenic mice expressing a constitutively-active form of the PPR, suggesting that G-protein signaling may interact with other crucial pathways to regulate bone formation. Our model provides a novel approach to activating the Gs pathway in osteoblasts and may indicate an anabolic pathway that could be used to maximize acquisition of peak bone mass for the treatment of osteoporosis.

Disclosures: E.C. Hsiao, None.

1020

Impaired Bone Formation in Mice Lacking the G Protein Subunit Gsα in Early Osteoblasts Leads to Marked Bone Fragility. J. Y. Wu¹, S. Rodda*², N. A. Sims³, I. J. Poulton*³, M. Chen*⁴, L. S. Weinstein*⁴, A. P. McMahon*², H. M. Kronenberg¹¹Endocrine Unit, Massachusetts General Hospital, Boston, MA, USA, ²Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA, USA, ³St. Vincent's Institute of Medical Research, Fitzroy, Australia, ⁴Metabolic Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD, USA.

Gsα is a ubiquitous heterotrimeric G protein subunit that mediates signaling downstream of numerous G protein-coupled receptors, including the parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PPR). We have deleted Gsα in osteoblast progenitors expressing osterix, a transcription factor expressed early in osteoblastogenesis. Osterix-Cre:Gsα(fl/fl) mice have markedly decreased trabecular and cortical bone mass, and experience fractures of the posterior ribs and long bones by the first day of life, leading to early postnatal lethality. Histomorphometric analysis reveals a significant (70%) reduction in trabecular bone volume, with reduced trabecular thickness and number, and osteoblast surface and numbers are dramatically diminished by 87% in mutant mice. We have previously shown that micro-computed tomography of cortical bones revealed increased cortical porosity in Gsα conditional knockout mice. Double calcein labeling demonstrates abnormal woven cortical bone in the absence of Gsα. Sirius red staining followed by analysis under polarized light confirms a striking absence of lamellar bone in cortical bones from mutant mice. Similarly, calvariae of conditional knockout mice are notable for markedly increased porosity and aberrant persistence of woven bone. TRAP staining of trabecular, cortical and calvarial bones do not demonstrate any increase in osteoclasts, and serum TRACP 5b levels are significantly reduced in mutant mice, suggesting that this phenotype cannot be attributed to enhanced bone resorption. Furthermore, osteoclast surface and numbers are not increased in mutant mice. Taken together, these results suggest that mice lacking Gsα early in the osteoblastic lineage have abnormal and/or inadequate bone formation affecting both intramembranous and endochondral bones. The resulting failure to remodel woven bone into lamellar bone may underlie the profound fragility of Gsα-deficient bones.

Disclosures: J. Y Wu, None.

This study received funding from: NIH.

1021

Designer G Protein Coupled Receptors Suggest Opposing Roles for Gs and Gi Signaling in Osteoblasts. S. M. Millard¹, J. Peng*¹, M. Bencsik*¹, A, Storer*¹, W. Lu*¹, T. J. Wronski*², B. Conklin*³, R. A. Nissenson¹, ¹Endocrine Research Unit, Veteran's Affairs Medical Center, San Francisco, CA, USA, ²Department of Physiological Sciences, University of Florida, Gainsville, FL, USA, ³Department of Medicine, University of California, San Francisco, San Francisco, CA, USA.

Designer GPCRs are powerful tools for dissecting the functional roles of specific G protein signaling pathways. We utilized two such receptors, Ro1 which signals through the Gi pathway and Rsl which signals through the Gs pathway, to examine the role of these pathways in osteoblasts. Transgenic expression of Ro1 in osteoblasts using the 2.3 kb-Col I promoter resulted in a dramatic osteopenic phenotype. Histomorphometric analysis of the proximal tibia revealed no change in the numbers of osteoblasts or osteoclasts on the bone surface, but decreased mineral apposition and bone formation rates were evident in Ro1 mice compared to littermate controls (Male BFR: 23.98±3.83 vs 36.46±1.44, Female BFR: 31.88±2.47 vs 44.32±4.57, p values <0.001). Osteoblast specific Ro1 transgene expression was tet-regulatable (tet-off system), allowing for the controlled expression of Ro1 in primary bone marrow stromal cell (BMSC) cultures. Induction of Ro1 expression in BMSC failed to influence osteoblast specific gene expression or formation of mineralized colonies, even in the presence of spiradoline, a synthetic agonist that activates Gi signaling by Ro1. These findings indicate that Gi signaling in osteoblasts in vivo has a marked negative effect on bone formation, apparently independent of cell autonomous inhibition of osteoblast function. To examine the role of Gs/cAMP signaling in osteoblasts, we have employed Rsl, a modified 5HT4 receptor that signals through Gs/cAMP in response to the synthetic drug RS23597, but not in response to 5HT (serotonin). The ability of Rsl signaling to mimic cAMP dependent PTH responses was assessed in the osteoblast-like cell line Ros17/2.8. RS23597 induced an increase in intracellular cAMP, increased mRNA expression of the cAMP early response gene, ICER, and repressed Dickkopfl mRNA expression in cells transiently transfected with Rsl. Neither 5HT treatment nor treatment with RS23597 in the absence of Rsl expression produced these effects. This demonstrates that Rsl is a suitable designer GPCR for assessing the role of Gs signaling in osteoblasts. Transgenic expression of Rsl in osteoblasts in vivo produces a marked increase in bone mass, implying that the Gs/cAMP pathway is a major positive anabolic signaling pathway in osteoblasts. The contrasting phenotypes of the osteoblast-specific Ro1 and Rsl transgenic mice are suggestive of critical and opposing roles for Gi and Gs signaling in osteoblast biology, the mechanisms of which are yet to be clearly delineated.

Disclosures: S.M. Millard, None.

This study received funding from: NIH grant DK072071, VA Merit Review award (to R.A. Nissenson).

1022

Severe Osteopenia in PERK-Knockout Mice Is Due to Impaired Osteoblast Differentiation Associated with Reduction of Type II RUNX2 Expression. J. Wei*, X. Sheng*, B. McGrath*, D. R. Cavener, Biology, Penn State University, University Park, PA, USA.

PERK deficiency in human (Walcott-Rallison Syndrome) causes multiple skeletal dysplasias and severe osteopenia. As revealed by Micro-CT analysis, PERK deficient mice show a remarkable reduction in trabecular bone mineralization and cortical bone thickness as early as postnatal day 2. The expression of osteoblast markers (Alkaline phosphatase, Osteocalcin and Type I Collagen) was found to be significantly down-regulated in Perk KO mice by −60-70% compared to wild-type littermates, and osteoclast markers were also reduced. To aid in the in situ identification of mature osteoblasts, the Col2.3GFP transgene was introduced into Perk KO mice. We found that the reduction in the expression of the osteoblast markers in Perk KO mice was the result of fewer mature osteoblasts and lower expression of osteoblast-specific genes per osteoblast. No increase in cell death was observed in KO mice, thus indicating that major defect in Perk-/- osteoblasts was abnormal or incomplete differentiation. A reduction of osteoblast markers was also seen in osteoblast-specific Co12.3 Perk KO mice, suggesting that PERK intrinsically regulates osteoblast differentiation. PERK is well known as a positive translational regulator of certain genes, including the transcription factor ATF4, which contain 5' upstream open reading frame (uORF) in their mRNAs. ATF4 was recently shown to be important in bone development and in regulating amino acid metabolism genes in osteoblasts. However, we found that in bone tissue of Perk KO mice, ATF4 mRNA and protein are normally expressed as well as its downstream amino acid metabolism genes. Another potential candidate for PERK-dependent regulation is RUNX2-type II, which contains five uORFs in its 5' UTR. In Perk-/- primary osteoblast culture, protein level of RUNX2-II is 50-30% of WT, while mRNA level is 80-90% of WT. Furthermore, DTT, a ER stress inducer, can strongly up-regulate RUNX2-II expression in both MC3T3-E1 and WT primary osteoblast, but only mild induction is seen in Perk-/- osteoblast, suggesting translation of RUNX2-II is PERK dependent. In summary our results illustrate that PERK is essential to normal osteoblast differentiation and reduction of type II RUNX2 possibly accounts for the osteoblast defects observed in Perk-/- mice. Supported by NIH AR49816 (D.R.C.).

Disclosures: J. Wei, None.

1023

Bone Cell Autonomous Effects of Osteoactivin/Gpnmb In Vivo. J. Y. Belcher¹, M. C. Rico¹, Arango-Hisijara¹, S. Salihoglu*¹, K. B. Buck*¹, S Abdelmagid¹, A. Sanjay¹, M. C. Nakamura², S. N. Popoff¹, F. F. Safadi¹, ¹Anatomy and Cell Biology, Temple University, Philadelphia, PA, USA, ²University of California, San Francisco, San Francisco, CA, USA.

Osteoactivin/Glycoprotein nmb (OA/gpnmb) is a transmembrane glycoprotein. The protein is synthesized, processed and heavily glycosylated by osteoblasts. Its expression is associated with increased osteoblast differentiation and matrix mineralization. We have previously shown that OA/gpnmb expression in osteoblasts is regulated by BMP-2 through the Smad-1 signaling pathway. In this study, we used a mouse model with a naturally occurring mutation in the OA/gpnmb gene resulting from a premature stop codon that leads to the production of a truncated OA/gpnmb protein with no biological functions. OA/ gpnmb mutant mice develop osteoporosis with age when compared to normal, wild type (WT) littermates. Histological and micro-CT measurements of femurs in mutant mice revealed a decrease in bone volume (BV/TV), trabecular number (Tb.N), and trabecular thickness in OA/gpnmb mutants compared to WT controls. Primary osteoblasts were generated from newborn OA/gpnmb and WT mice and examined for their differentiation ex vivo. All markers for early (alkaline phosphatase activity and collagen type I expression) and late (nodule formation, matrix mineralization and osteocalcin production) osteoblast differentiation were significantly reduced in the OA/gpnmb mutant osteoblasts compared to controls. We also examined bone marrow stromal cells isolated from OA/ gpnmb and WT mice and testing their ability to differentiate into osteoblasts. Colony forming unit-fibroblasts (CFU-F) and CFU-osteoblasts (OB) (determined by alkaline phosphatase staining) were significantly reduced in mutant compared to WT mice. These data suggest that OA acts as positive regulator of osteoblast differentiation and function in vivo. We next examined osteoclast differentiation using a co-culture system established using normal osteoblasts as feeder cells and bone marrow (monocyte/macrophage) obtained from either OA/gpnmb mutant or WT mice in the presence of 1,25(OH)2 vitamin D3 and PGE2. Osteoclast formation/differentiation was determined by TRAP-staining and actin ring formation. Co-culture of bone marrow cells isolated from OA/gpnmb mutant mice and WT osteoclasts showed marked increase in osteoclast numbers and size when compared to osteoclasts generated from normal bone marrow cells and normal osteoblasts. These data suggest the OA/gpnmb acts as a negative regulator of osteoclast formation in vivo. Collectively, these data suggest that OA/gpnmb acts to regulate bone remodeling by positively affecting osteoblastogenesis and negatively regulating osteoclastogenesis in vivo.

Disclosures: J. Y. Belcher, None.

This study received funding from: NIA MS/NIK

1024

Characterization of Osteoblast-Specific capn4 Knockout Mice. M. Shimada¹, P. A. Greer*², A. P. McMahon*³, E. Schipani¹, ¹Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA, ²Pathology, Queen's University, Kingston, ON, Canada, ³Molecular & Cellular Biology, Harvard University, Cambridge, MA, USA.

We have previously demonstrated that the calpain small subunit directly binds the intracellular C-terminal tail (C-tail) of the PTH1R at a site near its N-terminus, and it cleaves PTH1R at its C-tail in a calcium- and ligand- dependent manner in cell lysates and in intact cells. Moreover, the absence or reduction of calpain activity reduces PTH-mediated intracellular cAMP accumulation in both embryonic fibroblasts expressing PTHlRandMC3T3-Elcells.

The universal knockout mice of capn4, a gene encoding the calpain small subunit, are embryonic lethal. Thus, to investigate calpain's role in cells of osteoblast lineage which express PTH1R, in vivo, we established osteoblast-specific capn4 knockout mice by mating capn4fl/fl mice with mice expressing Cre under the control of osterix promoter (osx-cre). Thereafter, capn4fl and capn4+ indicate floxed and wild-type alleles of capn4, respectively. Efficiency of Cre recombinase, as assessed by real-time PCR of genomic DNA, was approximately 85%. Osx-cre;capn4fl/fl mice were phenotypically normal at birth and fertile. Whole skeletal analysis of newborn mice showed that osx-cre;capn4fl/fl mice had no patterning defect, but they displayed smaller body size and a delay in mineralization of calvaria compared to either control or osx-cre;capn4fi/+ littermates. Body weight of osx-cre;capn4fl/fl stayed significantly lower than those of osx-cre;capn4+/ + and capn4fl/fl mice at least up to 12 weeks of age. Detailed histological analyses showed that tibias of osx-cre;capn4fl/fl had reduced trabecular bone and lower expression of both osteoblast-specific markers including type I collagen, collagenase 3, osteopontin and osteocalcin, and osteoclast makers such as tartrate-resistant acid phosphatase in comparison to control littermates. Consistent with these data, bone mineral density, as measured by dual-energy X-ray absorptiometry, was also significantly lower in femurs of osx-cre;capn4fl/fl male mice than in controls at 12 weeks old. Preliminary histomorphometric analysis confirmed that osx-cre;capn4fl/fl mice had significantly lower bone volume/total volume ratio and trabecular number with concomitant increase in trabecular spacing when compared to control littermates. Collectively, these results suggest that capn4 plays a critical role in bone development and remodeling in vivo.

Disclosures: M. Shimada. None.

This study received funding from: N1H/N1DDK.

1025

Overexpression of Human VDR in the Intestine Rescues the Hypocalcaemia-Related Phenotype of VDR Knockout Mice. Y. Xue*, J. C. Fleet, Dept. of Foods and Nutrition, Purdue University, West Lafayette, IN, USA.

Vitamin D Receptor (VDR) knockout mice have reduced intestinal calcium absorption (Ca Abs) and develop severe hypocalcaemia and rickets when fed a standard 0.72% Ca chow diet. A highly bioavailable, 2% Ca, 20% lactose rescue diet that bypasses the transcellular Ca Abs pathway partially rescues this phenotype and reveals the importance of vitamin D-regulated intestinal Ca Abs in whole body Ca metabolism. To further study the role of VDR-mediated intestinal Ca absorption in Ca and bone metabolism, we generated transgenic (TG) mice with intestine-specific expression of hemaglutinin-tagged human VDR (HA-hVDR) driven by a 12.4 kb fragment of the villin promoter. Two TG lines, HV1 and HV2, were characterized. HA-hVDR mRNA and protein expression was detected from duodenum (Dd) to colon in TG mice; intestinal VDR protein was increased by 2- and 8- fold in HV1 and HV2, respectively, compared to the wild-type (WT) mice. In contrast a small level of HA-hVDR mRNA was seen in kidney of HV1 and HV2 and no HA-hVDR mRNA was seen in any other tissue. The HV2 line was used to examine whether TG expression could recover the VDR knockout (-/-) mouse phenotype. VDR +/-, TG -/- (control), VDR -/-, TG +/- (recovered KO or TG/KO) and VDR-/-, TG-/- (KO) mice were fed a standard chow diet for 8 or 12 weeks. At the 12 weeks of age, KO mice were growth arrested, developed alopecia, and had severe rickets. TG/KO mice also developed alopecia but growth and bone density were completely recovered. Serum Ca and PTH in TG/KO mice were completely restored to control levels. Serum 1,25(OH)₂D₃, and renal CYP27B1 mRNA levels were dramatically elevated in KO (100X and 400X above control, respectively). In TG/KO, these endpoints were restored to normal (CYP27B1 mRNA) or near normal (serum l,25(OH)₂D₃, 7X above control) levels. Intestinal expression of calbindin Dw and TPRV6 mRNA levels were reduced in KO but recovered in TG/KO mice. In contrast, renal CYP24 mRNA levels were only partially recovered (25% control levels) in TG/KO. At 8 wks, basal Ca Abs in the Dd of TG/KO was 26% higher than controls and both lines increased Ca Abs after treatment with 1,25 (OH)₂D₃ (25 ng/100g body, 9 h; 29.94% vs. 51.46% in controls; 37.81% vs. 52.36% in TG/KO). Surprisingly, while 1,25 (OH)₂D₃ treatment significantly induced CYP24 (Dd, kidney), calbindin D_9k (Dd), and TPRV6 (Dd) mRNA in both control and TG/KO, the response in TG/KO was blunted. Our data demonstrate that restoration of active VDR-mediated Ca Abs throughout the intestine is necessary to recover the phenotype of VDR KO mice. This further shows the critical importance of the intestine in controlling whole body Ca metabolism.

Disclosures: Y Xue, None.

This study received funding from: NIH awards DK54UI and CA 101113 to JCF

1026

Studies Using Nullmutant Mice Reveal Active Intestinal Calcium Transport in the Absence of Calbindin-D_9k or TRPV6. B. S. Benn*¹, P. Dhawan*¹, A. Porta*¹, X. Peng*¹, M. Hediger*², J. Peng*³, G Oh*⁴, S. Christakos¹, ¹Biochemistry and Molecular Biology, UMDNJ- New Jersey Medical School, Newark, NJ, USA, ²Institute of Biochemistry and Molecular Medicine, University of Berne, Berne, Switzerland, ³Department of Medicine, University of Alabama, Birmingham, AL, USA, ⁴Laboratory of Cardiovascular Genomics, Ewha Woman's University, Seoul, Republic of Korea.

To study the role of the calcium binding protein calbindin-D_9k and the epithelial calcium channel TRPV6 in intestinal calcium absorption, calbindin-D_9k and TRPV6 nullmutant mice were generated. Nullmutation was verified by RT-PCR and Western blot analyses. When fed a standard rodent chow diet, TRPV6 kockout (KO) mice and calbindin-D_9k KO mice have serum calcium levels similar to those of wild type (WT) mice (∼ 9 mg Ca⁺+/dl). In the TRPV6 KO mice, however, there is a 3 fold increase in serum PTH and 2.4 fold increase in serum 1,25(OH)²D³ levels. Levels of calbindin-D_9k mRNA and protein in intestine were not significantly different in TRPV6 KO mice compared to WT nor were levels of intestinal TRPV6 mRNA significantly different in calbindin-D_9k KO mice compared to WT. However, in calbindin-D_9k KO mice there is a significant 2-3 fold increase in renal calbindin-D_28k mRNA and protein (vs. WT), suggesting that renal calbindin-D2!k is compensating for the lack of renal calbindin-D_9k. Active intestinal calcium absorption was measured using the everted gut sac method and the first 5 cm of the duodenum of WT or nullmutant mice. Under low dietary calcium conditions [mice were fed a low calcium (0.02%) diet from weaning for 4 weeks] there was a 4.4, 4.1 and 2.8 fold increase in calcium absorption in the duodenum of WT, calbindin-D_9k and TRPV6 KO mice respectively [n=5-18/group; p>0.1 WT vs. calbindin-D_9k KO and p<0.05 WT vs. TRPV6 KO on the low calcium diet]. Duodenal calcium absorption was increased 2.2 +/-0.1 fold in the calbindin-D_9k/TRPV6 double knock out mice fed the low calcium diet (p<0.05 WT vs. double KO). Calcium absorption was not stimulated by low dietary calcium in the ileum of the WT or KO mice. In addition to low dietary calcium, l,25(OH)₂D₃ administration (three injections of l,25(OH)₂D₃ 100ng/100g body weight; 48, 24 and 12h prior to sacrifice) to vitamin D deficient mice also resulted in a significant increase in duodenal calcium absorption (1.8 – 2 4 fold; p<0.05 compared to vitamin D deficient mice and p>0.1 among all groups) in WT, calbindin-D_9k, and TRPV6 KO mice. This study provides evidence for the first time using nullmutant mice that significant aclive intestinal calcium transport occurs in the absence of calbindin-D_9k or TRPV6, thus challenging the dogma for the need for calbindin-D_9k and TRPV6 for vitamin D induced active intestinal calcium transport.

Disclosures: B.S. Benn, None.

1027

Novel Mechanism of Vitamin D Receptor (VDR) Activation: Histone H3 Lysine 9 Methyltransferase Is a Transcriptional Coactivator for VDR. Y. Zhong*, S. Christakos, Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School, Newark, NJ, USA.

Acetylation of core histone tails plays an essential role in transcriptional activation by nuclear hormone receptors. In addition to aceylation, methylation also occurs on core histones. The H3 specific lysine 9 methyltransferase G9a has been reported to function as a co-repressor of transcription by associating with transcriptional repressors. Here we report a novel mechanism of VDR receptor activation where G9a functions as a transcriptional activator in the l,25(OH)₂D₃ regulation of 25-hydroxyvitamin D₃ 24 hydroxylase (24(OH)ase). Using COS-7 cells transfected with VDR and the rat 24(OH)ase promoter (-298/+74) and treatment with suboptimal l,25(OH)₂D₃ increasing concentrations of G9a (0.02 – 0.2 ug) alone or in combination with the H3 specific arginine methyltransferase CARM1/PRMT4 (0.2 ug) had little effect on VDR mediated transcription. Expression of GRIP-1 (a pi60 coactivator) alone (0.05 ug) or in combination with CARM1 (0.2 ug) or G9a (0.2 ug) stimulated transcription a maximum of 3 fold above the 1,25(OH),D3 induced response. However transfection of G9a (0.2 ug) in combination with GRIP-1 (0.05 ug) and CARM1 (0.2 ug) resulted in a significantly greater enhancement of l,25(OH)₂D₃ induced 24(OH)ase transcription 7- 8 fold above the l,25(OH)₂D₃ induced response (p<0.01 compared to each enzyme alone or in combination or compared to activation with GRIP-1; maximal l,25(OH)₂D₃ induced transcription was 60-70 fold). The synergy was dependent on l,25(OH)₂D₃ and VDR. When either a mutant of CARM1, E267Q, or a mutant of G9a, that lacked enzymatic activity, was transfected, synergistic coactivator function was reduced. In vitro translation and co-immunoprecipitation studies have indicated that GRIP-1 binds directly to CARM1 and indirectly to G9a. When the GRIP-1 mutant δAD2 (which lacks the binding site for CARM1) was used, synergy was not observed, indicating a critical role of GRIP-1 in recruiting the secondary coactivators for VDR mediated transcriptional activation. Results of ChIP assays indicated that G9a is associated with the 24(OH)ase VDRE (-151/-137). Using the mouse la hydroxylase promoter and transfection in AOK-B50 kidney cells, we found that G9a can act as a repressor of PKA activation. These findings suggest that the histone modification induced by GRIP-1 can result in G9a acting as a transcriptional activator and that activation or repression depends on the context of transcription factors at a specific promoter. Our findings also suggest, for the first time, that cooperativity between histone methyltransferases and pi60 coactivators may play a fundamental role in VDR mediated transcriptional activation.

Disclosures: Y Zhong, None.

1028

Intracrine Synthesis and Action of 1,25-dihydroxyvitamin D Rescues Tolllike Receptor Suppression of Innate Immunity. S. Ren¹, L. Nguyen*¹, P. Liu*², R. L. Modlin*², J. S. Adams¹, M. Hewison¹¹Endocrinology, Diabetes and Metabolism, Cedars-Sinai Medical Center, Los Angeles, CA, USA, ²Division of Dermatology, UCLA, Los Angeles, CA, USA.

Vitamin D can act as a versatile modulator of the immune system by suppressing adaptive T-cell responses whilst stimulating macrophage innate immunity. We have shown recently that the latter involves toll-like receptor (TLR)-mediated induction of the enzyme lα-hydroxylase (CYP27b1) and concomitant autocrine metabolism of 25-hydroxyvitamin D (25D₃) to 1,25-dihydroxyvitamin D₃ (1,25D₃) by macrophages. This enhances innate immunity by stimulating expression of the antibacterial protein cathelicidin (LL37). Here we have used human macrophages cultured in the presence of autologous human serum to assess the effects of circulating 25D₃ levels on LL37 and beta-defensin 4 (DEFB4), another antibacterial peptide associated with innate immunity. Peripheral blood mononuclear cells were isolated from the patients as part of an established IRB protocol. Adherent monocytic cells were then isolated from non-adherent lymphocytes in serum-free medium and re-cultured in medium containing 10% of the patient's own serum. Under these conditions treatment with ligand to either TLR2 (19 kDa lipoprotein [19 kDa], 1ng/ml) or TLR4 (LPS, 100 ng/ml) suppressed expression of LL37 20-fold (p<0.001, n=9) and 32-fold (p<0.001, n=6) respectively. Analysis of patient vitamin D metabolites showed that suppression of LL37 by 19 kDa was inversely correlated with the patient's serum 25D₃ level (R=0.801, p=0.009), indicating that intracrine conversion of patient 25D₃ to 1,25D₃ protected against inhibition of LL37. Monocytes cultured in autologous serum showed relatively high levels of CYP27bl expression, and addition of exogenous 25D₃ (100 nM) alone increased expression of mRNA for LL37 (17-fold, p<0.001, n=10). Furthermore, co-treatment with exogenous 25D₃ (100 nM) rescued the suppressive effects of 19 kDa and LPS on LL37 expression (68-fold and 75-fold respectively for 19 kDa and LPS relative to cells treated with TLR ligand alone). Parallel analysis of DEFB4 showed that, in contrast to LL37, expression of this innate immunity gene was induced by either 19 kDa or LPS (349- and 86-fold respectively), and this response was unaffected by co-treatment with 25D₃. In summary, we have shown that high concentrations of TLR ligands act to suppress expression of antibiotic LL37, possibly as a mechanism by which pathogens can evade innate immunity. However, increased serum levels of 25D₃ protect against this pathogenic evasion mechanism. These data emphasize further the importance of intracrine metabolism of 25D₃ as a pivotal mechanism for normal human innate immunity.

Disclosures: S. Ren, None.

1029

Barrier Site Metabolism of Vitamin D: A Mechanism for Protection Against Inflammatory Bowel Disease. S. Wu, L. Nguyen*, S. Ren, R. F. Chun, J. S. Adams, M. Hewison. Endocrinology, Diabetes and Metabolism, Cedars-Sinai Medical Center, Los Angeles, CA, USA.

The active form of vitamin D, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) is a versatile immunomodulator. In addition to suppression of adaptive T-cell responses, 1,25(OH)₂D₃ is also a potent inducer of innate antibacterial activity in macrophages. Both of these mechanisms are dependent on localized synthesis of 1,25(OH)₂D₃ via the enzyme lα-hydroxylase (CYP27bl). We have postulated that this autocrine/paracrine response is crucial to innate and adaptive immune responses at ‘barrier sites’ where cells have routine contact with pathogen-associated molecular patterns. To test this hypothesis we have used dextran sodium sulfate (DSS) to induce a gastrointestinal barrier challenge in mice, thereby generating symptoms of inflammatory bowel disease (1BD). Wild type (+/+) BL6 mice treated with 2.5% DSS in water for 7 days followed by 2 days recovery showed a decrease in weight of 8.0 ± 4.4% compared to weight at day 0. Analysis of colonic tissue from these animals showed no change in expression of the Th2 cytokine interleukin-10 (IL-10), but there were increased levels of mRNA for the inflammatory cytokine, IL-1 (14.4-fold, p<0.001). This was associated with a parallel rise in CYP27bl expression, at the level of mRNA (2.1-fold, p=0.003) and protein (the latter defined by immunohistochemistry). Analysis of sera from the DSS-treated animals showed no change in circulating levels of 25-hydroxyvitamin D₃ but these mice exhibited significantly lower levels of 1,25(OH)₂D₃ (42 pg/ml) when compared to untreated mice (58 pg/ml) (p= 0.013). DSS-treatment of mice +/- for the CYP27b1 gene resulted in a similar loss of weight to WT mice (7.1 ± 5.2% compared to 6.5 ± 0.6% weight gain in control mice). By contrast, mice with ablation of the CYP27bl gene (-/-) exhibited a striking weight loss of 23.8 ± 3.9% when treated with DSS, compared to 2.1 ± 2.0% weight gain in untreated -/- mice. The enhanced sensitivity of CYP27b1 -/- mice to DSS treatment was associated with increased colonic expression of mRNA for both IL-1 (20-fold compared to DSS-treated +/- mice) and IL-10 (8-fold compared to DSS-treated +/- mice). Increased expression of IL-1 and IL-10 in DSS-treated CYP27bl -/-mice relative to +/- mice was also observed in spleens from these animals but not kidneys. This study provides further evidence of a role for vitamin D in protecting against the onset of IBD. In particular, our data show that a crucial determinant of this protective effect is the expression of CYP27b1 in affected tissues, including both the colonic epithelium and immune system.

Disclosures: M. Hewison, None.

1030

Molecular Mechanisms of lα,25(OH)₂ vitamin D₃-induced ATP Release in Osteoblasts. P. Biswas*, L. P. Zanello*. Biochemistry, UCRiverside, Riverside, CA, USA.

Steroid hormones including lα,25(OH)₂ vitamin D₃ (1,25D) are known to modulate bone cell populations and their activities. Changes in osteoblast to osteoclast ratios have been identified as a possible cause of some bone diseases. Some therapeutic approaches aim at reduction of osteoclast generation or activity, which to a large extent is governed by factors released by osteoblasts. One such factor is ATP. ATP released by osteoblasts at localized sites, exerts autocrine/paracrine signaling and modulates osteoclast functions. However, molecular mechanisms of ATP release and its regulation are not fully known. Our study describes rapid exocytotic release of ATP induced by the steroid hormone 1,25D in osteoblastic rat ROS 17/2.8 and human Saos-2 cells. Single exocytotic events were monitored in real time with time-lapse videomicroscopy on live osteoblasts stained with quinacrine, which has high affinity for ATP stores. Physiological concentrations of 1,25D (1–10 nM) caused a 3 fold and 5 fold increase of ATP release respect to vehicle in ROS 17/ 2.8 cells and Saos-2 cells respectively. We found that 1,25D-induced ATP release is vesicular. Pretreatment of cells with 1 mM NEM and 100 μM monensin, both agents known to interfere with vesicular transport and release, almost completely abolished 1,25D-induced ATP exocytosis. By means of siRNA silencing of the vitamin D receptor (VDR), we demonstrated that a classic VDR is required for 1,25D potentiation of Ca²⁺ and CI channels coupled to ATP exocytosis. In addition, we found that an increase in intracellular Ca²⁺ induced by 1,25D is also required for 1,25D-mediated ATP secretion. The L-type Ca²⁺ channel blocker nifedipine (2 μM) reduced 1,25D induction of ATP exocytosis by about 80%, while the L-type Ca²⁺ channel agonist S(-) BayK8644 (0.5 μM) and depletion of internal calcium stores with thapsigargin (3μM) caused a significant increase in ATP release. Involvement of 1,25D-induced increase in intracellular Cl⁻ in 1,25D-mediated ATP release was determined by pretreatment of cells with the Cl⁻ channel blocker DIDS (200 μM). We found that DIDS almost completely inhibited the 1,25D-induced ATP secretion. Furthermore, siRNA silencing of the ClC-3 voltage-gated chloride channel in ROS 17/2.8 cells significantly reduced 1,25D-stimulation of ATP release. Our findings provide for the first time, a signaling mechanism for the rapid release and increase of localized ATP concentrations in bone, and its regulation by the steroid hormone 1,25D.

Disclosures: P. Biswas, None.

This study received funding from: USPHS grant DK-071115–01.

1031

Dominant-Negative GCMB Mutations Cause Hypoparathyroidism. M. Mannstadt¹, G. Bertrand*², B. Grandchamp*², H. Jueppner¹, C. Silve²¹Endocrine Unit, Massachusetts General Hospital, Boston, MA, USA, ²Faculté de Médecine, Hǒpital Xavier Bichat, Paris, France.

Glial cells missing (GCM), which was first discovered in the fruit fly, belongs to a small family of transcription factors that are key developmental regulators. The rodent GCM ortholog, Gcm2, is exclusively expressed in parathyroid glands and was shown to be a key regulator of parathyroid gland development since mice homozygous for ablation of Gcm2 fail to form parathyroid glands and consequently develop hypocalcemia and hyperphosphatemia; heterozygous animals are phenotypically normal. Consistent with these findings, homozygous mutations in GMCB, the human homolog of Gcm2, provided a plausible explanation for several cases of familial autosomal recessive hypoparathyroidism; importantly, heterozygous carriers of these mutations were healthy. We now report the identification of heterozygous GCMB mutations as the cause of autosomal dominant hypoparathyroidism in two unrelated families with several affected members; in both families, mutations in the genes encoding parathyroid hormone and the calcium-sensing receptor were excluded. Because of the dominant mode of inheritance, we therefore considered the possibility of dominant negative GCMB mutations. Indeed, direct nucleotide sequence analysis of GCMB led to the identification of two novel, heterozygous mutations in genomic DNA of the affected, but not in the unaffected individuals in each family. Both heterozygous GCMB mutations are single nucleotide deletions within GCMB exon 5 (cl389delT and cl399delC) that introduce a frame-shift resulting in the truncation of the C-terminal GCMB region, which contains the putative transactivation domain. To further elucidate the mechanism through which these heterozygous GCMB mutations cause autosomal dominant hypoparathyroidism, we tested in HEK and DF-1 cells whether the mutant GCMB inhibits the actions of the wild-type transcription factor using a GCMB-activated luciferase reporter. These experiments demonstrated that both GCMB mutants impaired dose-dependently the transactivation capacity and thus the biological activity of wild-type GCMB; when expressed alone, the mutants exhibited a reduced capacity to activate the reporter. These findings strongly indicate that the newly discovered GCMB mutants have dominant-negative properties.

Disclosures: M. Mannstadt, None.

1032

TßRI Inhibitor Rescues Uncoupled Bone Resoprtion and Formation Caused by TGF-ß1 Mutations in Camurati-Engelmann Disease. X. Wu¹, Y. Tang¹, W. Lei*¹, W. Van Hul², T. R. Nagy*³, M. Wan¹, X. Cao¹¹Pathology, University of Alabama at Birmingham, Birmingham, AL, USA, ²Medical Genetics, University of Antwerp, Antwerp, Belgium, ³Nutrition Sciences, University of Alabama at Birmingham, Birmingham, AL, USA.

TGFß1, the most abundant cytokine deposited in bone matrix, plays a critical role in bone, but its exact function in the bone milieu is still unclear. Previously, we found that TGF-ß1 is activated during bone resorption to induce migration of osteoprogenitors for bone formation as a coupling factor. Camurati-Engelmann Disease (CED) is an inherited bone disease associated with amino acid substitutions in the latency-associated peptide (LAP), but not in the mature TGFß1 peptide. In examining cellular function of six different CED-derived TGFß1, we found that mutations in LAP cause premature release of mature TGFß1 upon secretion. As a result, TGFß1 mutants exhibit much more potent effects on migration of osteoprogenitors than WT TGFß1, suggesting its potential disturbance of coupling of bone resorption and formation. Indeed, CED TGFß1 mutants are hyperactive for inducing phosphorylation of Smad2 and luciferase transcription activity.

To examine whether premature release of active TGFß1 uncouples bone resorption and formation, we generated a CED mutant and WT TGFß1 transgenic mice driven by 2.3 kb Col I osteoblast-specific promoter. High levels of active TGFß1 were detected only in bone marrow and matrix of the TGFß1 mutant mice. A typical progressive diaphyseal dysplasia was observed in the tibia of one month-old TGFß1 mutant mice. More than 95% mice had tibial fractures by 3 months of age. X-ray and μCT analysis revealed that very high bone density was present at certain areas whereas severe bone resorption was shown at other regions, indicating that bone resorption and formation is uncoupled. No significant differences were observed in bone mineral density (BMD), osteoclast and osteoblast activity between WT and CED transgenic mice, suggesting that premature release of active TGFß1 interrupts coupling in TGFß1 mutant mice. Most importantly, treatment of TGFß1 mutant mice with the inhibitor of TGFß type 1 receptor (TßR1) for 6 weeks rescued the uncoupled bone resorption and bone formation and completely prevented tibial fractures in TGFß1 mutant mice. Taken together, CED-derived TGFß1 mutations cause premature release of active TGFß1 as well as uncoupling bone resorption and bone formation. TßRI inhibitor could be used as a potential agent for the treatment of CED patients.

Disclosures: X. Wu, None.

1033

RUNX2 Trinucleotide Repeat Mutations Are Associated with Decreased Bone Density and Altered Protein Function. A. Stephens*¹, J. Doecke*¹, S. Ralston², R. Prince³, G. Nicholson⁴, P. Sambrook⁵, M. Osato*⁶, N. Morrison¹¹School of Medical Science, Griffith University, Gold Coast, Australia, ²Rheumatic Diseases Unit, University of Edinburgh, Edinburgh, United Kingdom, ³Department of Endocrinology and Diabetes, University of Western Australia, Perth, Australia, ⁴Department of Medicine, Geelong Hospital, University of Melbourne, Geelong, Australia, ⁵Department of Rheumatology, Royal North Shore Hospital, St Leonards, Australia, ⁶Institute of Molecular and Cell Biology, Proteos, Singapore.

The RUNX2 transcription factor is essential for osteoblast differentiation and chondrocyte maturation. Rare mutations within RUNX2 cause the dominantly inherited skeletal disorder cleidocranial dysplasia which is charaterized by gross dysgenesis of the skeleton. A unique feature of RUNX2 is the polyglutamine and polyalanine (poly Q/A) domain encoded by a trinucleotide repeat sequence. Such repeat sequences are prone to strand slippage resulting in high mutation rates. We hypothesized that mutations within the RUNX2 poly Q/A domain would exist and these variants would be associated with altered bone density and protein function. 4361 DNA samples were obtained from multiple epidemiological studies of bone density. Bone density was measured by DEXA and ultrasound and expressed as Z-scores around the appropriate age-adjusted mean. A total of 21 subjects were identified as being heterozygous for a wild type 23Q/17A allele and a poly Q/A repeat variant allele. Deletions (15Q and 16Q) and insertions (30Q and 23A) were identified. Collectively Q/A repeat variants presented with significantly lower Femoral Neck BMD (DEXA) displaying a 0.65 SD decrease (n = 21, p = 0.0004) and lower bone density as measured by quantitative ultrasound of similar magnitude (-0.79 SD, n = 8, p = 0.031). To understand the mechanism via which the rare mutations were decreasing bone density, functional analyses were carried out on 23Q (WT), 16Q and 30Q RUNX2. The ability of the proteins to bind to the RUNX2 DNA binding site was assessed by EMSA. The analysis did not reveal any obvious changes in the DNA binding capacities of the mutant proteins. However, reporter gene analysis using the mouse osteocalcin promoter revealed significant decreases in the transactivation function of 16Q and 30Q. Finally, the ability of the proteins to localise to the nucleus was assessed. Subcellular analysis revealed the 16Q and 30Q proteins displayed defective nuclear localisation compared to WT. We have identified a new class of functionally relevant RUNX2 variants that occur at collective frequency of ∼0.5%. These mutations are associated with significantly lower bone density and altered protein function.

Disclosures: A. Stephens, None.

1034

A Constitutively Activated BMP Receptor, ALK2, Induces Heterotopic Bone Formation in Patients with Fibrodysplasia Ossificans Progressiva (FOP). T. Fukuda¹, M. Kohda*², K. Kanomata*¹, J. Nojima¹, J. Kamizono*³, H. Oda*⁴, K. Nakayama*⁵, A. Ohtake*⁶, K. Miyazono*⁷, E. Jimi⁸, L. Owan*⁹, Y. Okazaki*², T. Katagiri¹¹Division of Pathophysiology, Saitama Medical Univ., RCGM, Saitama, Japan, ²Division of Translational Research, Saitama Medical Univ., RCGM, Saitama, Japan, ³Department of Pediatric Emergency, Kitakyusyu city Yahata Hospital, Fukuoka, Japan, ⁴Department of Orthopedic Surgery, Saitama Medical Univ., Saitama, Japan, ⁵Department of Endocrinology and Diabetes, Saitama Medical Univ., Saitama, Japan, ⁶Department of Pediatrics, Saitama Medical Univ., Saitama, Japan, ⁷Department of Molecular Pathology, Univ. of Tokyo, Tokyo, Japan, ⁸Department of Bioscience, Kyushu Dental Collage, Fukuoka, Japan, ⁹Department of Orthopedic Surgery, Univ. of Ryukyus, Okinawa, Japan.

Fibrodysplasia Ossificans Progressiva (FOP) is a rare autosomal dominant disorder of skeletal malformations and progressive heterotopic bone formation in muscle. Particularly, heterotopic bones were markedly induced in traumatic injury and inflamated area. Recently, the 617G>A and its related mutations were identified in ALK2, one of the BMP type I receptors, in patients with FOP. However, the functional changes of the mutant ALK2 proteins were not elucidated. In the present study, we examined genetic mutations of the ALK2 gene in Japanese patients with FOP and analyzed biochemical alterations of the mutant ALK2. The identical 617G>A heterozygous mutation, which causes a substitution mutation of Arg206 to His, was found in the ALK2 gene in all of the patients examined. Transient overexpression of ALK2(R206H) in C2Cl2 myoblasts induced phosphorylation and nuclear accumulation of Smad1/5/8 and transactivation of their target gene, Idl. ALK2(R206H) inhibited maturation of myoblasts to myosin heavy chain-positive myocytes. In contrasat, ALP activity was synergistically induced by co-expression of ALK2(R206H) and Smadl or Smad5 in C2Cl2 myoblasts. Treatment of C2C12 cells overexpressing ALK2(R206H) with BMP-4 or BMP-7 further increased the ALP activity. Co-transfection of Smad7, but not Smad6, suppressed the induction of ALP activity by ALK2(R206H). Moreover, we found that expression of the BMP signaling molecules were up-regulated during muscle regeneration in vivo. Taken together, these results clearly indicated that ALK2(R206H) is an active BMP receptor and constitutively transduces intracellular signaling of BMPs. It was also suggested that ALK2(R206H) cooperatively induces the heterotopic bone formation with BMP ligands and/or BMP signaling molecules induced at the sites of muscle regeneration. Smad7 might be a therapeutic target of FOP to inhibit BMP signaling induced by ALK2(R206H).

Disclosures: T. Fukuda, None.

1035

Regulation of the Transcription Factor Pitx1 and its Role in Osteoarthritis Pathogenesis. C. Picard*¹, B. Azeddine*¹, D. Wang*¹, J. Martel-Pelletier*², J. Fernandes*³, F. Moldovan*⁴, A. Moreau⁵¹Research Center CHU Sainte-Justine, Montreal, PQ, Canada, ²Osteoarthtitis Research Unit, CHUM Hǒpital Notre-Dame, Montreal, PQ, Canada, ³Research Center, Hǒpital du Sacré-Coeur, Montreal, PQ, Canada, ⁴Department of Stomatology, Université de Montréal, Research Center CHU Sainte-Justine, Montreal, PQ, Canada, ⁵Department of Stomatology & Biochemistry, Université de Montréal, Research Center CHU Sainte-Justine, Montreal, PQ, Canada.

The presence of Pitx1, a homeodomain transcription factor, during development of periarticular regions giving rise to the hip and knee joints prompted us to investigate its putative role in Osteoarthritis (OA) pathogenesis. Our previous study demonstrated that partial inactivation of Pitx1 gene led to a progressive formation of OA-like lesions in aging Pitx1+/- mice. Indeed, a loss of Pitx1 expression was observed only in cartilage of OA patients when compared to controls. The purpose of this study is to identify the mechanisms responsible for the down regulation of Pitx1 expression in primary OA. Search for mechanisms turning off Pitx1 expression led us to identify a mutation (-3727 C→T) present only in OA patients and localized into an E2F-response element within the human pitx1 promoter. Electrophoretic mobility shift assays were performed with nuclear extracts prepared from OA and matched control articular chondrocytes and allowed the detection of a protein complex bound to either the wild-type probe or the probe containing the mutated E2F-like site. Off-rate analysis revealed that such mutation increased the stability of the bound complex. To identify proteins part of this complex, we performed DNA pull down assays followed by peptide digestion coupled to mass spectrometry analysis, which allowed the detection of Prohibitin (PHB-1), Prohibitone (PHB-2) and BCL-6 interacting co-repressor (BCoR). PHB-1 plays a role in the maintenance of mitochondrial function and localizes as well as in the nucleus where it facilitates cellular senescence by recruiting specific co-repressors to inhibit E2F target genes. The pathophysiological relevance of PHB-1 was further supported by the detection of an aberrant nuclear accumulation of PHB-1 by immunohistochemistry methods only in OA articular chondrocytes, while the co-localisation in vivo of PHB-1 and BCoR on this E2F site was confirmed in OA articular chondrocytes by ChIP assays. The recruitment of this repressor complex was independent of the presence of the mutation suggesting that nuclear accumulation of PHB-1 could be a primary mechanism leading to OA onset. Collectively, these data define an unrecognized and crucial role for Pitx1 in OA pathogenesis and the recruitment of a novel repressor complex switching off its expression in OA.

Disclosures: C. Picard, None.

This study received funding from: Canadian Institutes of Health Research.

1036

Mice with a Truncation Mutation Affecting the UBA Domain of SQSTM1 Develop Several Phenotypic Features Paget's Including Focal Lytic Lesions, and Increased Bone Turnover In Vivo. J. A. Rojas*¹, A. Daroszewska*¹, R. Layfield*², M. Helfrich³, R. J. Van't Hof*¹, S. H. Ralston¹¹Rheumatic Diseases Unit, University of Edinburgh, Edinburgh, United Kingdom, ²Institute of Neuroscience, University of Nottingham, Nottingham, United Kingdom, ³Bone Research Group, University of Aberdeen, Edinburgh, United Kingdom.

Introduction: Paget's disease of bone (PDB) is characterized by focal increases in bone turnover and mutations affecting the Sequestosome 1 gene (SQSTM1) are an important cause of this condition.

Methods: Here we report upon our preliminary analysis of the skeletal phenotype of mice with a truncating mutation at codon 409 (S409X) of the SQSTM1 gene which deletes most of the UBA domain.

Results: We studied 6 wild type and 11 mutant mice carrying a S409X mutation of SQSTM1. Five were heterozygous and 6 were homozygous for the mutation. The age range of animals was 13.5 ± 0.25 months (range 12–15 months). Analysis of the femur and tibia by microCT examination ex-vivo showed large focal lytic lesions in 3/5 heterozygotes and 2/6 homozygotes, although smaller lytic lesions were observed in other mutants such that 9/11 of the animals which carried mutations (81.8%) had lytic lesions of the lower limbs. Three animals had more than one lytic lesion. Lesions of the type seen in the S409X mutant mice were not observed in control littermates. Histological analysis of lesions revealed evidence of increased bone turnover. Moreover bone histomorphometry of the tibial metaphysis showed a dramatic increase in bone turnover in homozygous mutants. There was a 3-fold increase in osteoclast number from 1.2 ± 0.5 /mm² in WT to 4.5 ± 0.8 / mm² in mutant (p<0.01) and a two fold increase in eroded surface from 6.1 ± 2.0 % to 13.2 ± 1.8 %(p<0.01). Osteoblast numbers were also increased from 15.2 ± 4.0/mm² in WT to 37.5 ± 5.0 / mm² in mutant (p<0.01). Studies in vitro showed a 8.6 ± 14.4% increase in RANKL-induced osteoclast formation in mutant mice compared with WT (p=0.03), but there was no difference in osteoblast growth between genotypes.

Conclusions: Mice carrying a S409X truncating mutation of the SQSTM1 gene exhibit several features that are reminiscent of PDB including increased bone turnover, focal lytic lesions in the lower limbs, and show increased osteoclast formation in vitro. Further studies are in progress to conduct more detailed characterisation of these mice but our data suggest that the S409X mutant mice may represent an animal model of PDB.

Disclosures: J.A. Rojas, None.

1037

Is Bone Strength a Simple Function of Muscle, or Does Other Mechanical Loading Play a Role?. J. N. Dowthwaite¹, P. P. E. Flowers*², J. A. Kanaley*³, R. M. Hickman*¹, J. A. Spadaro¹, T. A. Scerpella¹¹Orthopedic Surgery, SUNY Upstate Medical University, Syracuse, NY, USA, ²Engineering, Syracuse University, Syracuse, NY, USA, ³Exercise Science, Syracuse University, Syracuse, NY, USA.

Many researchers tout muscular forces as the primary impetus for bone formation, but weight-bearing and impact-loading may also yield bone adaptations. We hypothesize that childhood gymnastic loading is linked to high arm BMC, bone diameter and bone strength, independent of arm lean mass (FFM), a surrogate for muscle size.

The Institutional Review Board of SUNY Upstate Medical University approved the project, and subjects/guardians assented/consented to participation. Total body DXA and pQCT radius scans were performed on 33 post-menarcheal girls. Non-dominant arm FFM and BMC were measured by DXA (Hologic QDR 4500W). Non-dominant polar strength-strain indices (SSI) and total bone cross sectional areas (CSA) were measured by pQCT at 4% and 33% distal radius sites. Forearm length was measured with a ruler. Calcium intake was assessed by food frequency questionnaire. Gynecological age was calculated as time from menarche to scan date. Focal outcomes were: non-dominant total arm DXA BMC; pQCT SSIs, total bone CSAs and fall SSIs (SSI/ (weight & forearm length)) at 4% and 33% radius sites. Gymnasts and ex-gymnasts (ex/gymnasts) had spent at least 5 hours per week in gymnastic training for at least 2 years out of the past 10 years. ANOVA compared means for ex/gymnasts (n= 19) vs. non-gymnasts (n= 14); ANCOVA adjusted for gynecological age, calcium intake and arm FFM.

Age, maturity, body size and calcium intake did not differ by gymnastic group. Arm FFM was a strong, consistent correlate of bone outcomes (R> 0.53–0.79, p<0.003). All bone outcomes were greater in ex/gymnasts than non-gymnasts with large effect sizes (all p<0.02, Cohen's d> 0.98). Gymnastics effects were large regardless of ANCOVA adjustment (p<0.05, Cohen's d> 0.78), but arm FFM adjustment tempered 4% CSA significance (p=0.08, large d= 0.67). Muscle and bone properties are closely related, and arm FFM is a useful gauge of upper extremity bone characteristics. However, gymnastic exposure acts as an independent factor in bone growth, linked to elevated bone accrual, geometry and strength. Thus, mechanical loading from non-muscular sources is a distinct and important determinant of human skeletal structure.

Disclosures: J.N. Dowthwaite, None.

This study received funding from: NIH NIAMS; OREF.

1038

Negative Effect of Dietary Protein Intake on Bone Mass Accretion in Chinese Pubertal Girls with Low Calcium Intake. O. Zhang*¹, H. Greenfield*², G. S. Ma*¹, K. Zhu², X. O. Du*², L. H. Foo*², X. O. Hu*¹, C. T. Cowell*³, D. R. Fraser*²¹Chinese Center for Disease Control and Prevention, Beijing, China, ²University of Sydney, Sydney, Australia, ³Children's Hospital at Westmead, Sydney, Australia.

The aim of this study was to assess the relationship between background nutrient intakes and bone mass accrual in a longitudinal study including two-year milk supplementation trial and three-year follow-up study in Chinese pubertal girls. Total 757 girls (aged 10.1 yr) were divided into three groups, supplied with either 330 ml calcium fortified milk (n=238) or milk fortified with calcium and vitamin D (n=260) on each school day for 24 mo, or consuming habitual diet without milk supplementation as controls (n=259). Over 24 mo, each subject consumed 144 ml/d supplementary milk on average, containing calcium 245 mg/d with/without vitamin D 3.33μg/d. Of 698 subjects completing the supplementation trial, 504 subjects agreed to be re-examined at 36 mo after supplement cessation.

All indices were measured at 0, 12, 24, 48 and 60 mo from the baseline. Bone mass of the forearm and whole body was measured with dual energy X-ray absorptiometry (DXA, Norland XR-36). Nutrient intakes were assessed by a three-day food record (including two weekdays and one weekend day).

Continuous variables were logarithm-transformed to observe the proportional association after adjustment for corresponding baseline value, pubertal development at baseline, age at each survey, physical activity percentage distribution at each survey, and clustering by schools. A linear mixed model in SAS program was used with p<0.1 as the standard for retention in backward elimination regression. Subjects from all groups and subjects from the control groups (representing typical urban girls in China at puberty) were analyzed separately to identify the influential components.

Most positive effects of milk supplementation on total body and proximal forearm became significant after 24 mo supplementation, and disappeared at two or three yr after supplementation withdrawal. During the five-year study, the background calcium intake (440 mg/d on average, independently of that from the milk supplement) positively influenced bone mineral content (BMC) at total body (TB), proximal forearm (PF) and distal forearm (DF), bone area (BA) at PF and DF, as well as bone mineral density (BMD) at DF. However, negative associations were observed between protein intake (55 mg/d on average) and BMC at TB, PF and DF, BA at PF, as well as BMD at DF.

In summary, besides the effects of milk supplementation, a higher background calcium intake had a positive effect on bone mass accrual, while higher protein intakes appeared to diminish bone mass accrual in Chinese pubertal girls perhaps in association with their low calcium intakes.

Disclosures: Q. Zhang, None.

This study received funding from: Nestlé Foundation and Danone China.

1039

Sexual Dimorphism in Radial and Longitudinal Bone Growth Differ by Tempo and Magnitude: A Study in Male-Female Co-twins Pairs. S. Iuliano-Burns¹, J. Hopper*², R. Zebaze¹, E. Seeman¹¹Endocrinology, Austin Health / University of Melbourne, West Heidleberg, Australia, ²Epidemiology, University of Melbourne, Parkville, Australia.

An individual's bone trait position on the sample distribution influences fracture risk. Fracture risk is lower in males than females, in part due to sex differences in bone achieved during growth. We studied within pair differences in bone structure and mass in 61 dizygotic (DZ) boy-girl twin pairs aged 7–18 years to test the hypothesis that sexual dimorphism in bone traits emerge at puberty and are negligible prior to puberty. Total body and posterior anterior lumbar spine (LS) BMC and mid-femoral shaft (FS) dimensions were measured using DEXA. Regional BMC was acquired for the total body scan. Height was measured using a Holtain Stadiometer, and limb lengths using a Harpenden anthropometer. 41 twin pairs were pre- and 20 pairs were or peri/post-pubertal based on Tanner staging (pubic hair, genital development). Within pair comparisons were performed using unpaired t-tests. Contrary to the hypothesis, sexual dimorphism in bone structure and BMC was evident before puberty. BMC was 2–8% greater in the male than female depending on the site measured (p < 0.05). LS width (not height) was greater in boys than girls before puberty (3.3 ű 0.03 v 3.0 ű 0.03 mm, p < 0.001). Similarly, mid-FS periosteal widths (not femoral length) were higher in boys than girls (15.6 ű 0.3 v 14.5 ű 0.3 mm, p < 0.01), resulting in greater slenderness (bone width / length) at the femur in females than males (p < 0.1, NS). These existing differences remained during and after puberty. Sexual dimorphism in BMC and structure is detectable prior to puberty challenging the notion that the dimorphism is driven only by differences in sex hormones.

Disclosures: S. luliano-Burns, None.

1040

A School-based Physical Activity Intervention Positively Affects Change in Imax in Pre- and Early Pubertal Boys. H. M. Macdonald*¹, D. M. L. Cooper¹, S. A. Kontulainen², H. A. McKay¹¹Orthopaedics, University of British Columbia, Vancouver, BC, Canada, ²University of Saskatchewan, Saskatoon, SK, Canada.

Bone structural adaptations to physical activity during growth are not well understood. We previously reported a strong trend for greater gain in peripheral QCT (pQCT)-derived bone strength (density-weighted polar section modulus, SSI) at the tibial midshaft in boys who participated in a school-based physical activity intervention compared with control boys.¹ The objective of the present study was to further explore the structural adaptations underpinning the trend for bone strength gains. Participants were 202 boys aged 9–11 years from 10 schools that were randomly assigned to control (CON, 3 schools, n = 63 boys) and intervention (INT, 7 schools, n = 139 boys) groups. Boys in INT schools participated in 60 minutes of additional classroom physical activity each week, which included a simple, progressive, high-impact bone-loading program (Bounce at the Bell), over 11 months. We used ImageJ (1.37v) with a threshold of 480 mg/cm³ to process the grayscale pQCT images (XCT-2000) from the tibial midshaft (50% site) and determine the maximum and minimum second moments of area (Imax, Imin, mm⁴) and the cortical area (CoA, mm²) by quadrant (anterior, medial, lateral and posterior). The quadrants were defined according to the pixel coordinates about the cross-sectional centroid. We used a mixed linear model (group = fixed effect, school = random effect) to compare change in Imax, Imin and CoA (by quadrant) between CON and INT boys after adjusting for baseline bone value, change in tibial length and maturity at followup. Boys were 10.2 ± 0.6 years at baseline and the majority (60%) were prepubertal (Tanner stage 1). Boys in the INT group had a significantly greater gain in Imax than CON boys (+ 365.7 mm⁴; 95% CI: 83.5 to 647.9). Boys in the INT group also tended to have a greater gain in Imin, but the difference between groups was not statistically significant (+ 109.3 mm⁴; 95% CI: −25.4 to 243.9). Underpinning the greater gain in Imax in INT boys was a slightly greater gain (1–1.5%) in CoA in the anterior, medial and posterior quadrants although the differences between groups were not statistically significant. Change in CoA in the lateral quadrant was similar between CON and INT boys. Our school-based intervention effectively increased bending strength (Imax) at the tibial midshaft in young boys. Together with the tendency for quadrant-specific changes in CoA, these data suggest regional variation in the bone response to AS! BC. Our findings are consistent with expected patterns of bone formation induced by bending loads in the anterior-posterior direction.

I. Macdonald HM et al. JBMR 2007; 22:434–46.

Disclosures: H.M. Macdonald, None.

1041

High Protein Intake Enhances the Positive Influence of Physical Activity on Bone Mineral Content in Pre-Pubertal Boys, T. Chevallev, S. Ferrari, J. P. Bonjour, R. Rizzoli. Service of Bone Diseases, Department of Rehabilitation and Geriatrics, Geneva University Hospitals, Switzerland.

Physical activity is an important lifestyle determinant of bone mineral mass acquisition. Its influence during childhood can be modulated by nutrition, particularly by protein and calcium intakes. We analyzed the relationship between physical activity levels and protein intake, as compared to calcium intake, on bone mineral mass in 232 healthy pre-pubertal boys (age: 7.4±0.4 yrs; standing height: 125.7±5.9 cm; body weight: 25.3±4.6 kg, mean ±SD). Bone mineral content (BMC) was measured by dual X-Ray aborptiometry (DXA) at 6 skeletal sites: radial metaphysis; radial diaphysis; femoral neck; trochanter; femoral diaphysis and L2-L4 vertebrae. Physical activity, protein and calcium intakes were 242±94 kcal.d⁻¹, 1.79 g.kg BW,d⁻¹ (a value about twice the usual recommended dietary allowance), and 752±263 mg.d⁻¹, respectively. In univariate analysis, correlation coefficients r between physical activity, protein and calcium intakes, and the BMC sum of the 6 skeletal sites were 0.33 (p=0.0001), 0.26 (p=0.0001), and 0.16 (p=0.013), respectively. By multiple regression analysis, physical activity (β adjusted = 0.292, p <0.0001) and protein intake (β adjusted = 0.224, p=0.007) remained correlated to BMC, whereas calcium intake was not (β adjusted = −0.027 p=0.740). Very similar results were found at the 6 individual skeletal sites. Within the group with protein intake above the median (2.0 g.kg BW.d⁻¹), increased physical activity from 168 to 321 kcal.d⁻¹ was associated with greater total BMC Z-score (+0.6, p=0.0005). In contrast, in the group with protein intake below the median (1.5 g.kg BW.d⁻¹) increased physical activity from 167 to 312 kcal.d⁻¹ was not associated with a significantly greater total BMC Z-score (+0.2, p=0.371). The interaction between physical activity and protein intake was close to statistical significance for total BMC Z-score (p=0.055) and total radius BMC (p=0.054), and significant for femoral neck BMC (p=0.012). In keeping with the results derived from multiple regression analysis, the increased physical activity on total BMC Z-score was not influenced by difference in calcium intake (above the median: i.e. 945 mg d⁻¹; below the median: i.e. 555 mg d⁻¹). No significant interaction (p=0.754) was detectable for total BMC Z-score as well as for any of the 6 skeletal sites between physical activity and calcium intake. In conclusion, in healthy pre-pubertal boys the influence in increased physical activity on bone mineral content appears to be enhanced by protein intake within a range above the usual recommended allowance.

Disclosures: T. Chevalley, None.

1042

Determinants of Bone Mass and Fracture in Adolescent Children: Early Life, Prepubertal and Current Factors. G. Jones, K. L. Hynes*, S. Foley, J. Flynn*. Menzies Research Institute, Hobart, Australia.

Fractures are common in children while variation in peak bone mass contributes approximately two thirds of fracture risk in later life. Reports in prepubertal children suggested that maternal smoking during pregnancy, breastfeeding and birth weight were all determinants of early bone mass and/or fracture but it is unclear if this persists through puberty. The aim of this study was to examine the role of early life, prepubertal and current factors in the determination of bone mass and fracture in adolescent male and female children.

During 2004/5, 415 16 year old Southern Tasmanian children were studied. Early life factors were available from a 1988 study of risk factors for SIDS (smoking during pregnancy, weight gain, age at birth, birthweight, duration of breastfeeding, period of gestation, anthropometrics and socioeconomic variables). Data were also available from age 8 (bone mass and body composition by DXA, physical activity, sun exposure). Bone mass was assessed at age 16 at the total body, femoral neck and spine using an Hologic Delphi densitometer and at the heel by a Sahara ultrasound densitometer. Fractures were collected by questionnaire (site, age, circumstances) with confirmation from medical records.

Of the 415 children, 160 (39%) had a fracture. In multivariate analysis, bone mass at age 16 was predicted by breastfeeding (3% higher at hip and total body), birth weight (hip and total body), lean mass, sports participation, shuttle run and standing long jump at age 8 (hip and total body) as well as bone mass at age 8. Fracture (particularly those involving the upper limb) was predicted by breastfeeding (HR yes v no 0.69, p=0.02) (but not birth weight) and bone mass at age 8 (total body and spine, HR 1.5–2.5/SD). No significant associations with either bone mass or fracture were present for maternal smoking during pregnancy or sun exposure at age 8. Both DXA and heel ultrasound measures at age 16 were also able to discriminate those with and without prevalent fracture (especially those involving the upper limb, OR 1.3–1.8/SD).

These unique data provide strong support to indicate that bone mass in adolescence can be influenced by environmental factors both breastfeeding in early life as well as during childhood where physical activity is most important. In addition, both breastfeeding during early life and bone mass assessed by DXA at age 8 were strong predictors of fracture while heel ultrasound is also reduced in those with fracture. These results suggest that osteoporosis and fracture prevention programs should commence very early in the life cycle.

Disclosures: G. Jones, None.

This study received funding from: NHMRC.

1043

Targeted Deletion of E11/gp38 in Osteocytes Results in Increased Skeletal Size and Bone Mineral Density. D. Guo*¹, Y. Mishina*², J. Feng¹, M. Ray*², G. Scott*², M. Harris³, L. Bonewald¹¹Oral Biology, UMKC, Kansas City, MO, USA, ²Molecular Developmental Biology, NIEHS, Research Triangle Park, NC, USA, ³Department of Periodontics, UTHSCSA, San Antonio, TX, USA.

As the osteoblast differentiates into an osteocyte, a dramatic change in morphology occurs from polygonal to dendritic. E11/gp38 is a membrane glycoprotein highly expressed in the embedding osteocyte that appears to be responsible for dendrite formation and elongation due to fluid flow shear stress. Therefore, we hypothesized that a reduction of E11 in the osteocyte would be detrimental as the cell would have less capacity for communication and viability. Conventional deletion results in lethality at birth due to a lung defect, therefore to determine function in osteocytes, postnatal targeted deletion of the E11 gene was performed by generation of E11 flx/flx mice (5'loxp site in Sacl of exon 1 non-coding region, 3' loxp site in BstE11 of intron 1) crossed with mice expressing the osteocalcin promoter driving CRE. The first cKO mouse showed approximately 10% greater body weight than its control littermate at 1 to 3 months of age. Bone mineral density was also 10∼20% higher in the E11cKO mouse as determined by PIXIMUS analysis, confirmed by microCT and radiography. Histomorphometric analysis showed a 10% reduction in osteocyte density in ulnar cortical bone compared to control. To validate that E11 was deleted in bone, immunostaining was performed showing dramatic reduction in expression in bone. Furthermore, scanning electron microscopy of acid-etched ulnae embedded in resin revealed an irregular lacuno-canalicular network dramatically different from the uniformly distributed lacunae in control cortical bone. In addition to fewer lacunae, fewer canaliculi per lacunae were observed in the E11 cKO. These observations further support the role of E11 in dendrite formation, however suggest that a reduction in dendrite formation may actually maintain the osteoblast in the matrix producing stage for an extended time before embedment in matrix resulting in greater bone mass.

Disclosures: D. Guo, None.

This study received funding from: NIH.

1044

Dynamic Imaging of Fluorescently Tagged Osteoblast and Osteocyte Populations Integrates Mineralization Dynamics with Osteoblast to Osteocyte Transition, S. L. Dallas¹, P. A. Veno*¹, L. F. Bonewald¹, D. W. Rowe², I. Kalajzic*²¹Univ. of Missouri Kansas City, Kansas City, MO, USA, ²Univ. of Connecticut Health Ctr, Farmington, CT, USA.

A well accepted model of osteocyte differentiation proposes that the osteoblast becomes trapped within an osteoid matrix, followed by mineralization of the matrix to entomb the osteocyte within its lacuna. Thus, osteocyte differentiation and bone mineralization occur simultaneously and may be interrelated. As a model to understand the dynamic process of osteocyte to osteoblast transition and how this integrates with mineralization, we have performed automated time lapse imaging in mineralizing bone cell cultures from transgenic mice with fluorescently tagged osteoblast and osteocyte populations.

Calvarial cells from neonatal mice expressing a Dmpl-GFP transgene as a marker for osteocytes, a 3.6kb Collal-DsRed transgene as a marker for osteoblasts or both transgenes together, were cultured with ascorbate and β-glycerol phosphate (ßGP) to promote mineralization. Mineral deposition was monitored using differential interference contrast optics or using alizarin red as a vital stain for calcium. Mineral deposition was found to occur exclusively at sites where small clusters of 4–20 cells expressed the Dmp1-GFP transgene and the boundary of the mineralized area followed the boundary of the GFP+ve cell clusters. The kinetics of mineralization were quantified by time lapse imaging in conjunction with thresholding of image stacks. After addition of ßGP there was a lag phase of 10–20h followed by a rapid period of mineral deposition that lasted 10–14h before reaching a plateau. A large amount of membrane ruffling and mitosis within the nodule preceded mineral deposition. There was also a high degree of cell motility within the nodule, with a subpopulation of Dmp1-GFP+ve cells, presumably representing embedded cells, remaining stationary throughout the imaging period. A large number of cells switched on Dmp1-GFP expression just prior to or during mineralization, suggesting transition from an osteoblast to an osteocyte-like phenotype. The osteocytic nature of these cells was confirmed by immunostaining for the early osteocyte marker, E11. Interestingly, the Colla1-DsRed and Dmpl-GFP transgenes identified two distinct cell populations with little overlap. The majority of cells that turned on the Dmpl-GFP osteocyte marker during mineralization were derived from a cell type that did not express high amounts of the Colla1-DsRed transgene. Together, these data suggest that mineralization is directed by a cell type that is already transitioning into an osteocyte and suggest that mineralization and osteocyte differentiation are simultaneous, interrelated and possibly non-extricable processes.

Disclosures: S.L. Dallas, None.

1045

Dynamic Imaging in Living Calvaria Reveals the Motile Properties of Osteoblasts and Osteocytes and Suggests Heterogeneity of Osteoblasts in Bone. P. A. Veno¹, D. P. Nicolella², I. Kalajzic³, D. W. Rowe³, L. F. Bonewald¹, S. L. Dallas¹¹Univ. of Missouri Kansas City, Kansas City, MO, USA, ²Southwest Res. Inst., San Antonio, TX, USA, ³Univ. of Connecticut Health Ctr, Farmington, CT, USA.

Osteocytes, once thought to be quiescent cells, are now known to be mechanoresponsive, capable of modifying their microenvironment and play a key role in phosphate homeostasis. We have shown that osteocytes within their lacunae may be highly dynamic and show motions of their cell body and dendrites. To determine the dynamic interactions between osteoblasts and osteocytes and understand the role of cell motility in their function we have performed dynamic imaging using mice with fluorescently tagged osteocyte and osteoblast populations.

Calvarial explants from 5–12 day old mice expressing a Dentin matrix protein 1-GFP (Dmpl-GFP) transgene in osteocytes, a 3.6kb Collal-DsRed transgene in osteoblasts or both together, were imaged by time lapse fluorescent microscopy. Osteoblasts on the bone surface were highly motile and continually moved randomly with a mean velocity of 5.3 μm/h. We have previously shown that this motility drives assembly of extracellular matrix proteins. Three distinct motile cell populations were observed on the bone surface. The majority of motile cells were red only (CollalDsRed+ve) with smaller populations that were green only (Dmp1 GFP+ve) or red and green (GFP/DsRed dual expressing). 65% of the osteocytes showed motions of their cell bodies within their lacunae, with deformations between 4–12% and 98% of the osteocytes extended and retracted their dendrites. Alizarin red staining confirmed that these motions occurred in osteocytes within mineralized lacunae. Transient dendritic connections appeared to be made between adjacent osteocytes and between osteocytes and surface cells. Osteocytes also extended dendrites beyond their canaliculi into vascular channels and resorption pits and appeared to contact cells in these spaces. Osteocytes exposed during bone resorption underwent cell death or migrated out of their lacunae into the resorbed area. Dynamic imaging was next combined with immunostaining to correlate expression of specific markers with the motile history of the cell. In the case of E11, an early osteocyte marker, this revealed distinct cell populations. 75% of Dmpl-GFP+ve surface motile cells and 100% of the partially embedded osteocytes with dendrite motion expressed E11. In contrast, 14% of embedded osteocytes with dendrite motion were E11+ve.

These data suggest that osteocytes and osteoblasts are more motile than previously known and suggest heterogeneity of osteoblasts on the bone surface. A surface motile cell expressing Dmpl-GFP and E11 appears to represent a precursor cell that is destined to become an osteocyte.

Disclosures: P.A. Veno, None.

1046

Control of the SOST Bone Enhancer by PTH Via MEF2 Transcription Factors, O. Leupin*¹, I. Kramer*¹, N. M. Colette*², G. G. Loots*², F. Natt*¹, M. Kneissel¹, H. Keller¹¹Musculoskeletal Disease Area, Novartis Institutes for BioMedical Research, Basel, Switzerland, ²Chemistry, Materials and Life Sciences Directorate, Lawrence Livermore Laboratory, Livermore, CA, USA.

The SOST gene encodes for sclerostin, an osteocyte-secreted key inhibitor of bone formation. SOST expression is restricted to osteocytes in adult bone and requires the presence of a distant enhancer that is deleted in Van Buchem disease patients, which are charaterized by a generalized progressive bone overgrowth. Parathyroid hormone (PTH) has been shown to suppress endogenous SOST expression in UMR-106 bone cells and in adult bone in vivo. Recently, we have reported that PTH completely inhibited the transcriptional activity of the SOST bone enhancer, but only marginally affected the proximal promoter using transient transfection reporter gene assays in UMR-106 cells. Footprint and functional mutation analysis revealed a myocyte enhancer factor 2 (MEF2) response element that is essential for enhancer activity. Furthermore, gel retardation assays and antibody-induced supershifts demonstrated direct binding of MEF2 transcription factors. Here, we show that the MEF2 response element is sufficient for enhancer activity. It conferred comparable enhancer activity as the full length enhancer sequence. Moreover, its activity was fully suppressed by PTH in a manner similar to the full length enhancer sequence. Expression of MEF2 transcription factors in bone was detected by quantitative RT-PCR and in situ hybridization. Expression levels of MEF2A, B, C and D in femur and UMR-106 cells were similar to those in heart and brain, two known MEF2-expressing tissues. Furthermore, immunohistochemistry demonstrated co-expression of MEF2s and sclerostin in UMR-106 cells and in osteocytes of adult bone in vivo. Overexpression of MEF2C in reporter gene assays had no effect on the activity of the proximal SOST promoter, but stimulated the SOST enhancer activity about 3-fold. Conversely, co-expression of a dominant negative MEF2C mutant protein inhibited enhancer activity by 55% in UMR-106 cells. Finally, siRNA-mediated knockdown of MEF2A, C and D suppressed endogenous SOST expression by 65, 59 and 84%, respectively, in UMR-106 bone cells. Combinations of siRNAs against MEF2A&C, A&D, C&D led to increased inhibition of SOST expression reaching levels similar to those observed with SOST-specific siRNAs. These data suggest that MEF2 transcription factors control SOST expression in osteocytes and mediate PTH responsiveness. Hence, MEF2 transcription factors are implicated in the regulation of adult bone mass.

Disclosures: H. Keller, Novartis 1, 3.

This study received funding from: Novartis.

1047

Target Ablation of PTH/PTHrP Receptor in Osteocytes.. P. P. Divieti¹, W. F. Powell*¹, T. Kobayashi¹, S. E. Harris², F. Bringhurst¹¹Endocrine Unit, Mass General Hospital, Harvard Medical School, Boston, MA, USA, ²Department of Periodontic, University of Texas Health Center at San Antonio, San Antonio, TX, USA.

Osteocytes are the most abundant cells in bone, but their relative inaccessibility and the lack of good in vitro cell models have impeded progress in understanding their functional roles. Parathyroid hormone (PTH) is known to act upon osteocytes via both PTH/PTHrP receptors (PPRs), specific for the amino-terminal PTH (and PTHrP) sequence, and CPTHRs, which recognize amino-truncated forms of the hormone.

To study the role of PTH and PTHrP activation of PPRs in osteocytes we have conditionally ablated PPRs in osteocytes using the Cre-LoxP technique. We first generated three independent lines of transgenic mice in which the tamoxifen inducible Cre-recombinase (Cre-ERT2) is driven by an osteocyte-specific promoter (the 10Kb region of DMP1 promoter). The function, induction and specificity of the DMP1-Cre-ERT2 transgene was evaluated by mating male and female transgenics with homozygous Rosa26R Cre-reporter mice. The dams were injected with tamoxifen (2 mg) at day 17.5 dpc, and X-Gal staining was performed in newborn pups. All osteocytes were positive for the X-Gal staining demonstrating that the promoter was functional and and Cre-dependent recombination could be fully induced upon tamoxifen injection. Cre-recombinase mRNA expression was confirmed by RT-PCR analysis of RNA from newborn calvarial bones. Two 10Kb-DMP1-Cre-ERT2 lines were mated with PPR(fl/fl) animals to create double heterozygotes, which then were mated with PPR(fl/fl) mice to generate DMP1-Cre/PPR(fl/fl) animals. To assess for PPR deletion in osteocytes we assessed down regulation of SOST expression in 4 and 8 week old mice (WT and PPRKO) fed a low calcium diet (0.2% Ca) for 1 or 2 weeks to induce secondary hyperparathyroidism. All animals were injected with tamoxifen (0.5 mg every 2 days for up to 2 weeks), and ionized and total calcium were measured at the end of the study period. Long bones and calvaria were fixed, decalcified and analyzed by immunohistochemistry for SOST expression. As expected, SOST expression remained readily detectable in KO mice but was greatly suppressed in WT animals, consistent with successful deletion of the PPR in osteocytes. Further studies to evaluate message levels of SOST and other PTH-regulated genes are currently underway. In summary, we have established a mouse model in which PPR expression can be specifically ablated in osteocytes. These mice will provide an excellent model to dissect the roles of PPRs in osteocytes. Furthermore, the 10Kb-DMP1-Cre-ERT2 mice can be used to delete additional genes of interest from this specific bone cell population

Disclosures: P.P. Divieti, None.

1048

Mutations in LRP5 β-Propeller 1 Block Sclerostin (SOST) Mediated Inhibition of the LRP5-Wnt-TCF Signals in U2OS Osteoblast-like Cells.. B. M. Bhat, V. E. Coleburn*, P. J. Yaworsky*, P. V. N. Bodine, S. Harada. Women's Health & Musculoskeletal Biology, Wyeth Research, Collegeville, PA, USA.

Loss of function mutations in the Sclerostin (SOST) gene are linked to Sclerosteosis and van Buchem disease which exhibit progressive growth of dense bone that can lead to clinical complications. However, heterozygous SOST mutations are clinically normal despite having high bone mass (HBM). The specific effect of SOST on bone formation was further demonstrated by increased bone mineral density in SOST knockout mice, osteopenia in SOST transgenics and complete reversal of overiectomy induced bone loss in rats by SOST antibodies. Osteocyte secreted SOST was considered to be an antagonist of BMP-signaling, but recent studies indicate that this effect is preceded by the inhibition of LRP5-Wnt signaling. It is now well established that an HBM trait in humans is associated with mutations in LRP5 β-propeller 1, among its four β-propellers. Additionally, by in-vitro studies we have shown along with others that the HBM-like mutations in LRP5 confer resistance to Dkk1, a secreted inhibitor of LRP5-Wnt-β-catenin signaling. Interestingly, Dkk1 interacts with LRP5 propellers 3 and 4 region while SOST binds to propellers 1 and 2. To investigate the LRP5-SOST interaction further, we studied several LRP5 mutants generated within all four β- propellers. These included the HBM-mutant LRP5G171V, and its structurally equivalent site mutants created within and outside the propeller 1. The analysis involved transient co-transfection assays using a Wnt-β-catenin responsive TCF-reporter and LRP5 or its mutant cDNAs in U2OS bone cells. In addition, we compared the potential role of Kremen2 in modulating Dkk1 and SOST function. Our results indicate that Kremen2 is essential for the inhibition of the LRP5-mediated TCF signal by Dkk1 while SOST mediated inhibition is Kremen independent. It is interesting to observe that Dkk1 and Kremen2 inhibited TCF activity of all three Wnts (1, 3a & 10b) tested while SOST inhibited only Wnt1 and Wnt10b. Out of the eight LRP5 mutants, only those in β-propeller 1 showed resistance to SOST and further supports the specificity of the SOST interaction with β-Propeller 1. In conclusion, the study demonstrates that inhibition of LRP5-Wnt-β-catenin signaling can be dependent on the co-expression of Kremens and the antagonists Dkk1 or SOST. Moreover, the analysis shows the importance of LRP5 β-propeller 1 and specific Wnts in SOST function. Continued investigations would lead to better understanding of LRP5 signaling and the role of different antagonists in maintaining bone homeostasis.

Disclosures: B.M. Bhat, None.

1049

Increased Mortality in Patients with a Hip Fracture - Effect of Pre-morbid Conditions and Post-fracture Complications., P. Vestergaard*¹, L. Reinmark², L. Mosekilde²¹The Osteoporosis Clinic, Aarhus Amtssygehus, Aarhus, Denmark, ²Department of Endocrinology and Metabolism C, Aarhus Amtssygehus, Aarhus, Denmark.

Background: Patients with a hip fracture have a significant excess mortality. However, it remains unclear if the mortality is linked to the pre-morbid conditions or to complications to the fracture.

Aim: To investigate the causes of mortality after a hip fracture.

Material and methods: All subjects with a hip fracture in Denmark between 1977 and 2001 compared with three age- and gender-matched subjects from the general population. Co-morbidity at the time of fracture and causes of death were evaluated.

Design: Historic cohort study.

Results: A total of 169,145 fracture cases were compared to 524,010 controls. The cases had a much higher prevalence of co-morbidity than the controls. The mortality rate was twice as high in fracture cases compared with controls (HR=2.26, 95% CI: 2.24–2.27). Adjustments for confounders only changed the excess mortality risk little. The mortality after the hip fracture was divided into two categories: an excess mortality of 19% within the first year following the fracture (relative survival=0.81 compared to controls), and an excess mortality of 1.8% per year (relative survival 0.982) for every additional year following the fracture. The major causes of the excess mortality were due to complications to the fracture event (70.8% within the first 30 days). Elderly hip fracture patients were more likely to die from trauma related factors than younger patients.

Conclusions: Patients with a hip fracture have a pronounced excess mortality risk. The major cause was linked to the fracture event and not to pre-existing co-morbidity.

Disclosures: P. Vestergaard, None.

This study received funding from: Helga and Peter Kornings Foundation.

1050

Risk of Subsequent Fracture Depends on Bone Mineral Density and Fracture Type: A 15-Year Prospective Study., D. Bliuc, N. Nguyen, T. V. Nguyen, J. A. Eisman, J. R. Center. Bone and Mineral Program, Garvan Institute of Medical Reasearch, Sydney, Australia.

Half of all fractures occur in people with non-osteoporotic BMD (T-score > −2.5). Any osteoporotic fracture increases re-fracture risk, yet, there is no information on re-fracture risk for different T-scores and how normal or osteopenic BMD affects that risk. This study therefore aimed to examine re-fracture risk according to BMD for all types of osteoporotic fractures in 1374 women and 871 men aged 60+ from the Dubbo Osteoporosis Epidemiology Study (April 1989 - May 2005).

BMD was measured at baseline at spine and hip, and all osteoporotic fractures and deaths were recorded. Fractures were classified into hip, vertebral, major (proximal humerus, distal femur, proximal tibia, pelvis, multiple rib) and minor (all other peripheral fractures excluding digits). Subsequent fracture rates were calculated according to gender, fracture type, and BMD T-score and compared with initial fracture rate of a BMD-matched group. There were 520 incident fractures in women (35% osteopenia and 57% osteoporosis) and 198 in men (44% osteopenia and 25% osteoporosis) during 14,203 and 9,709 person-years, respectively. Following initial fracture 176 women and 48 men had subsequent fractures (2683 and 808 person-years, respectively).

Absolute re-fracture risks were highest for women and men with osteoporosis [86 (72–103) and 135 (87– 209); RR: 1.4 (1.1– 1.7) and 2.9 (1.7– 4.9), respectively] and still increased for osteopenia [40 (30– 55) and 52 (32– 83): RR: 1.5 (1.1– 2.1) and 2.5 (1.5– 4.3) respectively], but for those with normal BMD there was no significant increase in re-fracture risk [RR: 2.1 (0.9– 4.6) and 1.7 (0.8– 3.7) respectively]. As for overall re-fracture risk the increase in relative risk was 2-fold higher for men than women.

In women and men with hip and vertebral fracture¹, re-fracture risk was increased irrespective of BMD, except in the few men with normal BMD where over 50% died within 3 years.

There was an overall increased re-fracture risk following initial fracture in women and men with osteopenia and osteoporosis. Hip and vertebral fractures were associated with the highest re-fracture risk. Neither women nor men with normal BMD had an increased re-fracture risk following minor fractures. These results should help guide physicians in treatment decisions following initial fracture.

¹ Center et al (2007) JAMA 4:387–94.

Disclosures: D. Bliuc, None.

1051

Decline in Age Adjusted Hip Fracture Incidence but a Drastic Increase in Hip Fractures Among the Very Oldest. Hip Fracture Curve Is Right-shifting.. U. Bergström¹, Y. Gustafson*², H. Jonsson*¹, U. Pettersson*¹, O. Svensson¹¹Dept of Orthopeadics, Umeå, Sweden, ²Dept of Community Med. and Rehab. Geriatric Medicine, Umeå, Sweden.

Introduction: Hip fracture incidence decreases in Europe and N. America. Is there a decreasing trend in all age groups or is just the fracture curve drifting?

Material and methods: This population-based material includes all hip fracture patients 1993–2005, ≥50 years, admitted to Umeå University Hospital: a total of 2,919 (31% men). The absolute numbers of fractures and incidence were mean value over the time periods 93–96 and 01–05.

Results: The overall hip fracture incidence was showing a declining trend, for the periods 93–96, 97–00 and 01–05: for women (men) 706 (390), 709 (314), and 625 (317) hip factures per 100,000. However, the absolute numbers of hip fractures per year did not change over time. A 50% increase in absolute fracture rate was noted in women age 90 or older (11,5 hip fractures/year (93–96) and 24,6 hip fractures/year(01–05). The latter fracture rate can be compared with the absolute number of hip fractures in women age 75–79 (27.2 fractures/ year 01–05) (Table 1). The incidence was also increasing during this period of time, from 2700/100,000 to 3900/100,000 among the women ≥ 90 years (Table 2). In men there were a declining trend in both incidence and absolute numbers, only a small increase of absolute fractures were noted among men ≥ 90 years.

Conclusion: Though the absolute incidence shows a declining secular trend, the absolute facture rate and incidence are increasing among the very oldest. Women ≥ 90 now account for the same number of hip fractures every year as women 75–79 years. There seems to be a right shift and skewing of the hip fracture distribution towards the very oldest. This is probably due to the increased number of the octo- and nonagenarians, perhaps also to a better general- and bone health among the septuagenarians. This changed pattern will further strain the scarce orthopaedic and geriatric resources- surgery is more technically demanding, and local and general complications are much more common than in younger.

1052

Ethnic Variations in Hip Geometry Among Women at Baseline from the Women's Health Initiative.. D. A. Nelson¹, T. J. Beck², C. E. Lewis³, T. Bassford*⁴, J. A. Cauley⁵, M. S. LeBoff⁶, S. B. Going*⁴, Z. Chen⁴¹Wayne State University, Detroit, MI, USA, ²Johns Hopkins University, Baltimore, MD, USA, ³University of Alabama, Birmingham, AL, USA, ⁴University of Arizona, Tucson, AZ, USA, ⁵University of Pittsburgh, Pittsburgh, PA, USA, ⁶Brigham and Women's, Boston, MA, USA.

The risk of osteoporotic fracture varies among ethnic groups in the U.S., presumably due to differences in bone mechanical strength. African Americans suffer fewer osteoporotic fractures than whites although fracture susceptibility is less well studied in other groups. Our objective was to describe ethnic differences in bone geometry, and possible contributing factors, using hip structure analysis (HSA) of the femoral neck (FN), shaft (FS) and intertrochanteric (IT) regions based on data from the Women's Health Initiative (WHI). Baseline data included DXA of bone and soft tissue, body size, lifestyle, and medical history in 10,516 women representing four ethnic groups (non-Hispanic White-NHW, African-American -AA, Mexican-American-AA, Native-American -NA), ages 50 to 79. Geometry was adjusted for age, height, weight and % lean mass (see FN results in Table).

Table  .  

*P<0.001 vs. NHW

Compared to NHW, AA women had consistently narrower femora with smaller buckling ratios at all regions. Geometric strength differences were variable: bending resistance (section modulus) was higher in FN but lower in IT and FS, while axial resistance (bone cross-sectional area) was higher in the FN and IT and lower in the FS. Neither MA nor NA women differed from NHW at the FN, but both had higher axial and bending resistance at the IT. MA women had slightly higher bending resistance at the FS. There appear to be ethnic differences in size adjusted femur strength parameters but the clearest advantage for AA women is in their lower buckling ratio. This suggests that they may be less susceptible to loss of strength due to local buckling compared to NHW. Geometric strength differences in MA and NA compared with NHW are less clear and are reduced by adjustment for selected skeletal dimensions in the hip.

Disclosures: D.A. Nelson, None.

This study received funding from: NIAMS.

1053

Risk Factors for Falls in Older Community-dwelling Women., K. A. Faulkner¹, J. A. Cauley¹, J. M. Zmuda¹, D. P. Landsittel*², S. R. Cummings³, S. A. Studenski*⁴, M. C. Nevitt*³¹Epidemiology, University of Pittsburgh, Pittsburgh, PA, USA, ²Duquesne University, Pittsburgh, PA, USA, ³University of California San Francisco, San Francisco, San Francisco, CA, USA, ⁴University of Pittsburgh, Pittsburgh, PA, USA.

Identifying risk factors for a single fall is difficult due to accidents. The purpose of the study is to identify risk factors for more frequent incident falls among women with no recent history of falling. Study participants included 9,704 older community-dwelling women who were enrolled in the Study of Osteoporotic Fractures. The analysis sample consisted of 6,750 women (70%) of median age (IQR) =72 years (67–75) who had no history of falls in past 12 months. A wide range of potential risk factors were assessed at baseline including 1) age, body mass index (BMI), height and education, 2) medical conditions, 3) medications, 4) lifestyle and 5) neuromuscular function. Data on the number of falls were self-reported every four months using post-card mailings over four years (97% complete) and fall rates were calculated (# falls/woman-years). Poisson regression models with Generalized Estimating Equations were used to calculate the Risk Ratio (RR), or ratio of fall rates. Step-wise building of a multivariate model was performed from 46 potential risk factors that all were minimally adjusted for age, clinic, height, BMI, and education. In the analysis sample including non-fallers at baseline, the average annual fall rates were 344 falls per every 1,000 woman-years. We identified the several risk factors for more frequent falls (p≤0.05 for all), including having a high school education (RR= 1.03, vs. no high school education), higher number of Instrumental Activities of Daily Living (IADLs) with difficulty (RR=1.14, per 1 IADL), faster usual-paced walking speed (RR=1.07, per 1 SD), self-reported physician diagnosis of arthritis (RR=1.11), self-reported fear of falling (RR=1.22), and the current use of antidepressants (RR=1.25) and anticonvulsants (RR=1.46). Protective factors for more frequent falls (p≤0.05 for all) included taller height (RR= .95, per 2 inches) and a higher number of step-ups completed in 10 seconds (RR=.93, per 1 SD). The RR indicates the increased risk (or decreased risk) in the average rate of falling that is associated with a given factor. Hence, the use of antidepressant and anticonvulsant medications is associated with fall rates 22% and 46% higher, respectively, compared to not using the medications. In conclusion, among older women with no recent history of falls, potentially modifiable risk factors for more frequent falls include the use of antidepressant and anticonvulsant medications, fear of falling, weaker muscle power and poorer functional status.

Disclosures: K.A. Faulkner, None.

1054

Age Effects on Hip Structure: Results in 636 WOMEN AGed 20 to 97.. J. K. Brown¹, C. E. Cann*¹, R. L. Prince²¹Mindways Software Inc., Austin, TX, USA, ²Sir Charles Gairdner Hospital, Perth, Australia.

The effects of age on three dimensional bone structure remains uncertain. 636 women, recruited from ten centres across the USA, had a standard, clinical, wholebody CT scan to develop reference data for QCT for regulatory purposes. Slices were taken at 2.5 – 3.0 mm intervals, pixel size 0.75mm and results normalized using a proprietary phantom. Images were analysed using Mindways CTXA Hip Version 3 software with partial volume correction.

The CV error for the various measures was under 3%. The data relating bone structure to age is shown in the table. 

Table  .  

**P < 0.01. P ***0.001

Bone mass in the various compartments decreased with age as a quadratic by about 40% from age 20. The external bone volume and trabecular (marrow) volume increased with age linearly from age 20 by about 30%, while the cortical volume decreased with age as a quadratic. Volumetric density decreased with age in the total and trabecular compartment, but the cortical volumetric density data increased slightly with age. The changes at the total hip were representative of the changes at the femoral neck, trochanter and intertrochanter sites except for trochanter trabecular mass which was constant with age.

Bone mass at the hip falls from age 20 but the rate increases with age probably reflecting an effect of estrogen deficiency at menopause. However the external size of the hip increases from age 20 likely independent of changes in sex hormone levels. The cortical mass and volume decrease with age in such a way that there is little apparent change in cortical density consistent with thinning of the cortex with age.

Thus age effects on hip bone mass differ in critical respects to age effects on bone volume. Increases in external volume throughout life may increase bone strength and delay the onset of hip fracture.

Disclosures: J.K. Brown, Mindways Software Inc. 3, 4.

1055

Efficacy and Safety of Zoledronic Acid 5 mg in Preventing Fractures in Men and Women With Prevalent Hip Fracture: The HORIZON-Recurrent Fracture Trial.. K. Lyles¹, C. Colon-Emeric*¹, J. Magaziner², J. Adachi³, C. Pieper*¹, L. Hyldstrup*⁴, C. Mautalen⁵, C. Recknor⁶, K. Moore*¹, C. Lavecchia*⁷, J. Zhang*⁷, P. Mesenbrink*⁷, E. Eriksen⁸, S. Boonen⁹¹Duke Univ Med Ctr, Durham, NC, USA, ²Univ of Maryland, Baltimore, MD, USA, ³McMaster Univ, Hamilton, ON, Canada, ⁴Hvidovre Hospital, Univ of Copenhagen, Copenhagen, Denmark, ⁵Centro de Osteopatias Medicas, Buenos Aires, Argentina, ⁶United Osteoporosis Centers, Gainesville, GA, USA, ⁷Novartis Pharmaceuticals Corp, East Hanover, NJ, USA, ⁸Novartis Pharma AG, Basel, Switzerland, ⁹Univ of Leuven, Leuven, Belgium.

Hip fractures put patients at risk for future fracture and increased mortality. HORIZON-RFT is an international, multicenter, randomized, double-blind, placebo-controlled, parallel group trial assessing whether zoledronic acid (ZOL) 5 mg reduces subsequent clinical fractures in men and women ≥50 yrs of age with a prevalent hip fracture. Inclusion/exclusion criteria as previously described (Colon-Emeric et al. CMRO 2004;20:903). Patients received loading dose vitamin D2 or D3 and daily supplements of 800–1200 IU vitamin D3 and 1000–1500 mg elemental calcium. Patients were followed until 211 had experienced confirmed new clinical fractures (the primary efficacy endpoint). Of 2127 patients randomized, 2111 were treated with once-yearly IV infusions of ZOL 5 mg (N=1054) or placebo (PBO; N=1057). Baseline characteristics were similar between groups. Median age was 76 yrs (range, 50–98); 76% were women. Clinical fractures occurred in 231 patients (92 ZOL, 139 PBO) for a 2-yr cumulative event rate of 8.59% (ZOL) and 13.88% (PBO), based on Kaplan-Meier estimates, and a relative risk reduction of 35% (HR=0.65; 95% CI: 0.50–0.84; P = .0012). ZOL reduced risk for clinical vertebral and non-vertebral fractures relative to PBO by 46% (HR=0.54; 95% CI: 0.32–0.92; P =.0210) and 27% (HR=0.73; 95% CI: 0.55–0.98; P =.0338), respectively. ZOL reduced risk of hip fractures by 30% relative to PBO (HR=0.70; 95% CI: 0.41–1.19; P =.1815 [NS]). Overall incidence of AEs and SAEs was comparable between treatment groups. No significant differences were seen in any cardiovascular parameters or long-term renal function. No cases of ONJ were reported. Death occurred in 101/1054 ZOL patients (9.58%) vs 141/1057 PBO patients (13.34%), yielding a 28% lower mortality risk in ZOL relative to PBO (HR=0.72; 95% CI: 0.56–0.93, P =.0117). In conclusion, this trial showed that subjects with a prevalent hip fracture treated with an annual IV infusion of ZOL experienced significantly fewer clinical fractures compared with placebo. In addition, ZOL had a favorable safety profile and was well tolerated. Finally, this is the first trial to demonstrate a mortality benefit for an antiresorptive agent.

Disclosures: K. Lyles, Novartis, Procter & Gamble Sanofi-Aventis, Amger 2: Novartis, Procter & Gamble Sanofi-Aventis, Merck, Amgen, Bone Medical LTD 5: Novartis, Dr. Lyles is co-inventor with Novartis Pharmaceuticals on a use patent for zoledronic acid US Provisional Patent Application No. 60–411,067. 9.

This study received funding from: Novartis Pharma AG Basel Switzerland.

1056

Risk Factors for Serious Adverse Events (SAEs) of Atrial Fibrillation in the HORIZON-PFT Trial of Zoledronic Acid.. S. R. Cummings¹, P. Mesenbrink*², E. F. Eriksen³, R. Eastell⁴, D. M. Black⁵¹San Francisco Coordinating Center, C.P.M.C. Research Institute and University of California, San Francisco, San Francisco, CA, USA, ²Novartis Pharmaceuticals Corp, East Hanover, NJ, USA, ³Novartis Pharma AG, Basel, Switzerland, ⁴University of Sheffield, Sheffield, United Kingdom, ⁵San Francisco Coordinating Center, University of California San Francisco, San Francisco, CA, USA.

Findings from 2301 HORIZON-PFT Trial of zoledronic acid in postmenopausal women with osteoporosis (but not the 2310 Trial in elderly women with hip fracture) and from the Fracture Intervention Trial (FIT) of alendronate raise a possibility that use of bisphosphonates (BPs) might be associated with an increased risk of atrial fibrillation SAEs (defined as events resulting in hospitalization, disability, or judged to be life-threatening).

The 2301 Trial included 7714 women with osteoporosis treated with annual infusions of 5 mg of zoledronic acid or placebo for 3 years. Women who had taken a bisphosphonate for ≥1 year must have stopped for at least 2 years before enrollment. Previous bisphosphonate use was reported by 1161 or 14.5% of participants (type, dose and duration were not recorded). Arrhythmias were adjudicated by a panel of cardiologists. In an exploratory analysis using this dataset, we selected the best set of risk factors for atrial fibrillation by building stepwise proportional hazards models from age, baseline cardiovascular history, prior BP use and other potential risk factors after fixing treatment and study stratum into the model.

Significant risk factors for atrial fibrillation SAEs included congestive heart failure (CHF), tachyarrhythmia, age and previous use of bisphosphonates. The incidence of atrial fibrillation SAEs was 2.2% with prior BP use vs. 1.6% for no use. In an additional analysis, prior bisphosphonate use was not significantly associated with all (serious and non-serious) atrial fibrillation AEs. 

Table  .  

*Assignment to zoledronic acid vs. placebo

Older women with CHF and tachyarrhythmias have an increased risk of atrial fibrillation. This observational analysis suggests that women who have previously used bisphosphonates might have an increased risk for atrial fibrillation SAEs. This possibility needs additional investigation.

Disclosures: S.R. Cummings, Amgen, Novartis, Lilly, and Pfizer 2; Lilly, Merck, Zelos, Organon, and Amgen 5.

This study received funding from: Novartis.

1057

Efficacy of Continued Alendronate for Fractures in Women without Prevalent Vertebral Fracture: The FLEX Trial.. A. V. Schwartz¹, D. C. Bauer¹, J. A. Cauley², K. E. Ensrud³, L. Palermo¹, R. B. Wallace*⁴, M. C. Hochberg⁵, A. C. Feldstein⁶, A. Lombardi⁷, S. R. Cummings⁸, D. M. Black¹¹University of California, San Francisco, San Francisco, CA, USA, ²University of Pittsburgh, Pittsburgh, PA, USA, ³VA Medical Center & University of Minnesota, Minneapolis, MN, USA, ⁴University of Iowa, Iowa City, IA, USA, ⁵University of Maryland, Baltimore, MD, USA, ⁶Kaiser Permanente Northwest Center for Health Research, Portland, OR, USA, ⁷Merck & Co. Inc., Rahway, NJ, USA, ⁸California Pacific Medical Center, San Francisco, CA, USA.

The Fracture Intervention Trial (FIT) found that 4 years of alendronate (ALN) in women without prevalent vertebral fracture reduced the risk of non-vertebral fractures (NVF) in those with femoral neck (FN) T-score ≤ −2.5 but not in those with higher BMD. In the main results of the FIT Long Term Extension trial (FLEX), 10 years of ALN did not significantly reduce the risk of NVFs, compared with 5 years of ALN. Continuing ALN reduced the risk of clinical vertebral fractures but not the risk of x-ray defined vertebral fractures. We tested whether the long term effect of ALN on fracture among women without a prevalent vertebral fracture differs by FLEX baseline FN T-score.

In FLEX, 1099 women randomized to ALN in FIT (mean previous duration 5 years) were re-randomized to placebo (40%) or ALN 5 (30%) or 10 (30%) mg/d for an additional 5 years. The ALN groups were combined in these analyses. At FLEX baseline, 723 women (66%) did not have a prevalent vertebral fracture. We analyzed fracture results in this group, excluding 3 women without baseline hip BMD. Interaction models used continuous FN T-score.

Among women without vertebral fracture at FLEX baseline, we found significant interactions between FLEX baseline FN T-score and treatment for NVF (table). Continuation of ALN reduced NVF in women with FLEX baseline FN T-score ≤ −2.5 but not in women with T-score > −2. Results for clinical vertebral or morphometric vertebral fracture did not differ significantly by FN T-score at FLEX baseline.

Table  .  

*p<0.05

This post hoc analysis suggests that continuing alendronate for 10 years instead of stopping after 5 years reduces the risk of non-vertebral fracture in women without prevalent vertebral fracture whose FN T-score, achieved after 5 years of ALN, is ≤ −2.5, but does not reduce risk of NVF in women whose T-score is > −2. These results are similar to those previously reported for 4 years of ALN use.

Disclosures: A.V. Schwartz, GSK 5.

This study received funding from: Merck.

1058

Giant Osteoclast Formation After Long-Term Oral Aminobisphosphonate Therapy for Postmenopausal Osteoporosis.. R. S. Weinstein, T. M. Chambers*, E. A. Hogan*, W. W. Webb*, C. A. Wicker*, S. C. Manolagas. Center for Osteoporosis, Central Arkansas Veterans Healthcare System, Univ of Ark for Med Sci, Little Rock, AR, USA.

Treatment of postmenopausal osteoporosis with aminobisphosphonates leads to progressive increases in bone density and a reduction in the incidence of new fractures. The efficacy of these drugs is believed to result from their ability to promote osteoclast apoptosis. However, bone specimens taken from non-human primates and patients treated with long-term aminobisphosphonates often show little or no effect of the drugs on the number of osteoclasts. We examined bone biopsies from 26 women with postmenopausal osteoporosis after a 3-yr double-blind, randomized, placebo-controlled trial of 10 mg/d of oral alendronate (ALN) for the prevention of postmenopausal bone loss. We report that in the subjects receiving ALN, osteoid perimeter, bone formation rate and biochemical markers of bone remodeling decreased whereas bone density increased as expected. Strikingly, osteoclast number was significantly increased in the subjects receiving ALN compared with those receiving placebo: 0.114 osteoclasts per mm cancellous perimeter ± 0.078 SD, n = 9 vs. 0.059 ± 0.038, n = 17, p = 0.012. In the ALN group, most of the osteoclasts were in superficial resorption cavities. Unexpectedly, giant osteoclasts were present in 4 out of the 9 (44%) of the ALN-treated subjects. Specifically, in these 4 subjects, 19% (13/68) of the osteoclasts observed were unusually large and had as many as 15 to 30 nuclei, as compared to 3 to 8 nuclei in the normal-sized osteoclasts of the placebo group. None of the subjects receiving placebo exhibited such cells (0/91). The atypical osteoclasts were partially detached from bone and they were clearly separated from the cancellous bone by unidentified mononuclear cells interspersed between them and the bone surface. In addition, the ruffled borders of the giant cells were distorted or missing. Further, the giant osteoclasts demonstrated positive nuclear in situ end labeling (ISEL) indicative of apoptosis, as well as nuclear condensation, fragmentation and peripheral beading. We conclude that long-term treatment with ALN decreases osteoclast resorptive ability rather than number, possibly by disrupting the ruffled border. Moreover, such treatment gives rise to a previously unrecognized cell phenotype: an apoptotic, excessively nucleated, colossal osteoclast, with apparent resistance to the phagocytosis that normally results from the detachment of cells from the bone matrix. Although these abnormal cells may well be dysfunctional and of no direct danger to the patient, awareness of the condition is crucial because giant osteoclasts could lead to a mistaken diagnosis of Paget's disease or hyperparathyroidism.

Disclosures: R.S. Weinstein, Merck 8.

1059

Effect of Daily Oral Minodronate on Vertebral Fractures in Japanese Postmenopausal Women with Established Osteoporosis: A Randomized Placebo-Controlled Double-Blind Study.. T. Matsumoto¹, H. Hagino², M. Shiraki³, M. Fukunaga⁴, T. Nakano⁵, K. Takaoka⁶, H. Morii⁶, Y. Ohashi⁷, T. Nakamura⁸¹University of Tokushima Graduate School of Health Biosciences, Tokushima, Japan, ²Tottori University, Yonago, Japan, ³Research Institute and Practice for Involutional Diseases, Nagano, Japan, ⁴Kawasaki Medical School, Okayama, Japan, ⁵Tamana Central Hospital, Kumamoto, Japan, ⁶Osaka City University, Osaka, Japan, ⁷University of Tokyo, Tokyo, Japan, ⁸University of Occupational and Environmental Health, Kitakyushu, Japan.

Minodronate (ONO-5920/YM529), a potent bisphosphonate, has been shown to increase the lumbar bone mineral density (BMD) of postmenopausal women with established osteoporosis. However, its efficacy in reducing osteoporotic fractures has not been evaluated. The present study was undertaken to examine the efficacy and safety of daily oral minodronate on the risk of vertebral and other fractures in subjects with established osteoporosis.

Seven hundred and four postmenopausal Japanese women at 55 to 80 years of age with one to five vertebral fractures and BMD below 80% of the young adult mean were enrolled in this study. The subjects were randomly assigned to receive either daily 1 mg minodronate (n=359) or placebo (n=345) for 26 months. All the participants received daily supplements of 600 mg calcium and 200 IU vitamin D₃.

Daily oral 1 mg minodronate for 26 months, compared with placebo, reduced the cumulative incidence of new vertebral fractures (10.4 vs 24.0%; p<0.0001) by 58.9% (95% CI, 36.6–73.3%) over 26 months. Because a large number of vertebral fractures occurred during the first 6 months in both groups (20 and 27 in minodronate and placebo groups, respectively), the cumulative incidence of new vertebral fractures from 6 to 26 months was reduced by 74.1% (4.7% vs 16.6%; p<0.0001) by minodronate treatment. A nonsignificant reduction in the incidence of overall clinical fractures was observed by 26 months of minodronate treatment (9.4 vs 13.8%). Minodronate treatment also reduced height loss both after 12 months (1.2 mm vs 3.4 mm in placebo) and 26 months (3.7 mm vs 6.8 mm). Bone turnover markers were suppressed by about 50% after 6 months of minodronate treatment, and remained suppressed thereafter. The overall safety profile including gastrointestinal safety was similar between the two groups.

These results demonstrate that daily oral minodronate is safe, well-tolerated, and is effective in reducing vertebral fracture incidence in postmenopausal women with established osteoporosis. Because the dose of minodronate in reducing fracture incidence was low, further studies are warranted to evaluate the efficacy of intermittent administration of higher oral doses of minodronate on osteporotic fractures.

Disclosures: T. Matsumoto, ONO Pharmaceutical Co.,ltd. 5; Astellas Pharma Inc. 5; Chugai Pharmaceutical Co., Ltd. 5; Eli Lilly Japan K.K. 5.

1060

Efficacy of Clodronate on Fracture Risk in Women Selected by 10-year Fracture Probability., E. McCloskey¹, H. Johansson*¹, A. Oden*¹, S. Aropuu*², T. Jalava*², J. Kanis¹¹University of Sheffield, Sheffield, United Kingdom, ²Bayer Schering Pharma, Berlin, Germany.

Treatments for osteoporosis are commonly targeted to patients with low bone mineral density. The WHO tool for fracture prediction estimates an individual's probability of fracture in the next 10 years from clinical risk factors, with or without BMD measurement. To determine if patients identified at high risk by the algorithm are responsive to anti-resorptive treatment, we examined the effects of the bisphosphonate, clodronate, on fracture incidence during 3 years of treatment across the range of fracture probabilities estimated by the WHO tool.

5212 women aged 75 years and older, randomly selected from the local population were randomised, regardless of the presence or absence of osteoporosis, to receive 3 years of therapy with clodronate (BONEFOS®) 800mg daily p.o. or an identical placebo. The 10-year probability of fracture in each woman was computed using baseline clinical risk factors including BMI, prior fracture, glucocorticoid use, parental hip fracture, smoking, alcohol and secondary osteoporosis. Femoral neck BMD measurements were also obtained at baseline. The interaction between probability of fracture and treatment efficacy to reduce the incidence of all osteoporosis-related fractures was determined using Poisson regression analysis.

Complete clinical risk factor data were available in 3974 women (76.2% of the cohort). The 10-year probability for osteoporotic fracture without BMD was 20.0±7.3% (mean±SD) with the range 7.3–72.8%. The probability for osteoporotic fracture with BMD was 18.1±8.6% (mean±SD) with the range 1.4–73.0%. The interaction between fracture probability and treatment efficacy was not significant when probability was assessed either with or without BMD (p=0.098 and p=0.11 respectively). A non-significant trend was observed for greater fracture reduction at higher fracture probability, with or without the use of BMD. For example, women lying at the 75^th percentile of fracture probability in the absence of BMD had a fracture probability of 24% and treatment reduced the risk of fracture by 23% over 3 years (HR 0.77, 95%CI 0.63–0.95). In those with a fracture probability of 30% (90^th percentile), the fracture risk reduction was 31% (HR 0.69, 0.53–0.90). We conclude that the estimation of an individual's 10-year probability of fracture identifies patients at high risk of fracture who will respond to bisphosphonate therapy.

Disclosures: E. McCloskey, Bayer Schering Pharma 2, 8.

This study received funding from: Bayer Scheringn Pharma.

1061

Central Control of Bone Remodelling by Neuromedin U: A Mediator of the Leptin-dependent Regulation of Bone Formation., S. Sato*¹, S. Takeda¹, R. Hanada*², M. Iwasaki*¹, H. Inose¹, A. Kimura*¹, K. Kangawa*³, M. Kojima*², K. Shinomiya*¹¹Orthopaedic Surgery & 21COE Program, Tokyo Medical and Dental University, Tokyo, Japan, ²Molecular Genetics, Institute of Life Science, Kurume University, Kurume, Japan, ³Biochemistry, National Cardiovascular Center Research Institute, Osaka, Japan.

Leptin inhibits bone formation through a hypothalamic relay and the sympathetic nervous system (SNS). However, hypothalamic target molecule of leptin remains to be elucidated. Neuromedin U (NMU) is an anorexigenic neuropeptide whose expression is induced by leptin. In this study, we investigated the role of NMU on bone remodelling and the possible crosstalk between NMU signalling pathway and leptin-SNS pathway.

NMU deficient mice (NMU^−/− mice) exhibited a 30% increase in bone mass compared to wild-type (wt) mice. Bone formation in NMU^−/− mice was increased as revealed by higher bone formation rate and osteoblast surface. Indeed, in vivo, osteoblast mitotic index analyzed by BrdU incorporation was significantly increased in NMU^−/− mice. In contrast, bone resorption parameters of NMU^−/− mice were comparable to those of wt mice. In vitro, differentiation or proliferation of primary osteoblasts isolated from NMU^−/− mice was similar to those from wt mice. In addition, treatment with varying concentrations of NMU did not affect osteoblast function, suggesting the lack of direct effect of NMU on osteoblast. In contrast, continuous intracerebroventricular infusion (ICV) of NMU to NMU^−/− mice decreased bone volume significantly, indicating the central nature of NMU's action on bone formation. Leptin ICV decreased bone volume and bone formation in wt mice, as previously reported. However, and surprisingly, leptin ICV paradoxically increased bone formation and osteoblast number in NMU^−/− mice. Likewise, injection of isoproterenol, a beta agonist, reduced bone mass in wt mice. However, NMU^−/− mice were resistant to the antiosteogenic activity of isoproterenol. Furthermore, NMU/Adrb2 (gene encoding beta2 adrenergic receptor) double heterozygote mice had higher bone mass than Adrb2 single heterozygote mice or NMU single heterozygote mice.

Collectively, our study shows that NMU inhibits osteoblast proliferation and bone formation through the central nervous system. Moreover, it suggests that NMU mediates the antiosteogenic action of the leptin-SNS pathway.

Disclosures: S. Sato, None.

1062

ECM1, a Direct Targeting Molecule of PTHrP, Is a Novel Potent Mediator of Chondrogenesis.. L. Kong*¹, Y. Zhang*¹, B. Jiang*², Y. Xie*², J. O. Feng², T. Kobayashi*³, H. M. Kronenberg³, C. Liu¹¹Department of Orthopaedic Surgery, New York University, New York, NY, USA, ²Department of Biomedical Sciences, Baylor College of Dentistry, Texas A&M University System Health Science Center, Dallas, TX, USA, ³Endocrine Unit, Massachusetts General Hospital, Boston, MA, USA.

Chondrogenesis is controlled by cellular interactions with surrounding matrix proteins and growth factors that mediate cellular signaling pathways. In a functional genetic screen for proteins associating with cartilage oligomeric matrix protein, we identified a novel extracellular matrix protein, ECM1 (extracellular matrix protein 1), a molecule that had previously been linked to lipoid proteinosis and lichen sclerosus, a common chronic inflammatory condition. We verified this novel interaction using in vitro pulldown and in vivo coimmunoprecipitation assays and demonstrated that these two proteins colocalized on the cellular surface of primary human chondrocytes. Using RT-PCR and western blotting assays, we showed that both mRNA and protein levels of ECM1 were upregulated during differentiation of chondrocytes. ECM1 significantly inhibited mRNA levels of both early and later genes critical for chondrogenesis such as Sox9 (78% reduction), collagen type II (82% reduction), and collagen × (90% reduction) and repression of ECM1 via the siRNA silencing markedly enhanced the expression of Collagen × in the course of chondrogenesis (22 and 5 fold in ADTC5 and 10T1/2 mesenchymal stem cells respectively). To determine the potential mechanisms by which ECM1 negatively regulates chondrogenesis, we studied the effects of ECM1 on PTHrP (an essential negative regulator for later chondrogenesis) and IHH (a positive regulator of chondrogenesis). ECM1 induced PTHrP 5- to 18-fold and inhibited IHH (50% reduction) during chondrogenesis. We further showed that PTHrP induced mRNA levels of ECM1 (28-fold) in chondrocytes. Importantly, knocking down ECM1 mRNA levels via the siRNA silencing or blocking ECM1 protein activity via anti-ECM1 antibodies completely abolished the effects of PTHrP on chondrogenesis in vitro. Using an immunohistochemistry assay, we showed that ECM1 was expressed throughout the chondrocyte zone of growth plates and articular cartilage and that ECM was no longer detectable in PTHrP null growth plates at day E18.5 in vivo. Last, we demonstrated that both mRNA and protein levels of ECM1 were upregulated in arthritis using microarray, real-time PCR and western blotting assays. Our findings demonstrate for the first time that ECM1, a direct downstream molecule of PTHrP in cartilage, is a novel negative regulator of chondrocyte differentiation and suggest that ECM1 may also plays an important role in the pathology of arthritis.

Disclosures: L. Kong, None.

This study received funding from: NIH.

1063

Regulation of Chondrocyte Hypertrophy and Bone Mineral Density by Matrilin-3 Through Modulating Bone Morphogenetic Protein Signaling Pathways.. X. Yang*¹, L. van der Weyden*², L. Wei*¹, Z. Wang*¹, A. Bradley*², O. Chen¹¹Brown University, Providence, RI, USA, ²Wellcome Trust Sanger Institute, Cambridge, United Kingdom.

Regulation of the entrance to and the exit from the hypertrophic stage of chondrocyte differentiation during endochondral ossification is crucial to the normal development and mineralization of a long bone. Mis-regulation of hypertrophy leads to chondrodysplasia. Important regulators of chondrocyte hypertrophy include growth factors such as bone morphogenetic proteins (BMPs) and extracellular matrix (ECM) proteins. Matrilin-3 (MATN3) is a member of the matrilin family, a group of the adhesive ECM proteins. Mutations in the human gene of MATN3 result in a variety of skeletal diseases including multiple epiphyseal dysplasia, spondylo-epi-metaphyseal dysplasia, and hand osteoarthritis. To determine the function of matrilin-3 in endochondral ossification, we examined the consequences of both knocking out MATN3 and over-expressing MATN3 in hypertrophic chondrocytes. In MATN3 KO mice, the hypertrophic zone of the embryonic tibial growth plate was expanded in comparison to its wildtype littermates. Thus, the lack of matrilin-3 leads to mis-regulation of chondrocyte differentiation in the developing epiphyseal growth plate. By 18 weeks of age MATN3 null mice had a significantly higher total body and knee joint bone mineral density (BMD) than wild-type littermates. Aged MATN3 null mice were much more predisposed to develop severe osteoarthritis than their wild-type littermates. Conversely, Over-expression of MATN3 in embryonic chondrocytes during their transition to hypertrophy led to reduced expression of Col × mRNA, a marker of chondrocyte hypertrophy. Deletion of the cis-acting element in the Col × promoter indicated that the 5' distal region containing BMP responsive elements was necessary and sufficient to mediate the inhibitory effect of MATN3 on Col X. This suggests that MATN3 inhibition of Col × expression is mediated by the BMP pathways. Immunoprecipitation pull-down assay detected BMP-2 in a complex with MATN3 in the conditioned medium of MATN3 transfected cells, suggesting MATN3 may inhibit BMP signaling by interacting and sequestering BMP-2 extracellularly. Interestingly, increased BMP signaling in MATN3 KO mice was indicated by an increase of phosphorylated Smad1 in the growth plate. Our data revealed that MATN3 plays a negative role in regulating chondrocyte hypertrophy by modulating BMP signaling. It suggest a novel pathological mechanism by which matrix defects lead to abnormal BMP signaling that ultimately affects ossification, bone mineral density, and skeletal development and aging.

Disclosures: Q. Chen, None.

This study received funding from: NIH/NIA.

1064

Perlecan Modulates FGF and VEGF Signaling and Is Essential for Vascularization in the Development of the Cartilage Growth Plate.. M. Ishijima¹, E. Arikawa-Hirasawa*², K. Hozumi*¹, N. Suzuki*¹, K. Kosaki*¹, T. Matsunobu*¹, Y. Yamada*¹¹Laboratory of Cell and Developmental Biology, National Institute of Dental and Craniofacial Research, National Institute of Health (NIH), Bethesda, MD, USA, ²Research Institute for Diseases of Old Age, Juntendo University School of Medicine, Tokyo, Japan.

Perlecan (Perl) is a heparan sulfate proteoglycan that is expressed in all basement membranes and in cartilage. Perl-deficiency in mice and humans causes lethal chondrodysplasia, indicating that Perl is essential for cartilage development. However, the precise function of Perl in cartilage development is unknown. The purpose of this study is to investigate the mechanism of Perl functions in growth plate development. Here, we describe the critical role of Perl in FGF/VEGF signaling and angiogenesis in growth plate formation. The Perl-/- growth plate is short with severely impaired endochondral bone formation. Chondrocyte proliferation is reduced, and the hypertrophic matrix is disorganized. Although chondrocytes are differentiated, the expression of FGF downstream genes is dysregulated. We found that both FGFR3 and FGFR1 were activated in the Perl-/- growth plate. Expression of VEGF, a FGF/FGFR target gene, was upregulated by Perl-/- hypertrophic chondrocytes, suggesting that the lack of vascularization into the hypertrophic zone is not due to the reduced VEGF expression. We demonstrated that Perl bound VEGFR but not VEGF. Expression of the Perl transgene specifically in cartilage of Perl-/- mice rescued the perinatal lethality of Perl-/- mice, and vascularization into the growth plate was restored, indicating that perlecan in the growth plate is critical in this process. These results suggest that Perl in the growth plates activates VEGFR signaling of endothelial cells for vascularization into the growth plate. Thus, we conclude that perlecan plays a critical role in endochondral bone formation via modulating FGF/VEGF signaling and promoting angiogenesis.

Disclosures: M. Ishijima, None.

1065

Genetic and Pharmacologic Activation of the Hypoxia Inducible Factor Pathway Promotes Angiogenesis and Accelerates Bone Regeneration.. C. Wan¹, S. R. Gilbert², Y. Wang¹, X. Cao*¹, X. Shen*¹, G. Ramaswamy*³, K. A. Jacobsen*⁴, Z. S. Alaql*⁴, L. C. Gerstenfeld⁴, T. A. Einhorn⁴, A. W. Eberhardt*³, L. Deng*¹, T. L. Clemens¹¹Pathology, University of Alabama at Birmingham, Birmingham, AL, USA, ²Orthopaedic Surgery, University of Alabama at Birmingham, Birmingham, AL, USA, ³Biomedical Engineering, University of Alabama at Birmingham, Birmingham, AL, USA, ⁴Boston University School of Medicine, Boston, MA, USA.

Angiogenesis is critical for bone formation and occurs in close spatial and temporal association with osteogenesis during skeletal development and regeneration. Osteoblasts sense fluctuations in oxygen tension and respond to hypoxia by activating the hypoxia-inducible factor - 1α (HIF-1 α) pathway, which in turn activates vascular endothelial growth factor (VEGF) and other angiogenic gene programs. In this study, we applied genetic and pharmacologic strategies to manipulate HIF-1 α levels in osteoblasts and determine its role in bone regeneration. Overexpression of HIF-1 α was achieved by deleting VHL, a key component of the E3 ubiquitin degradation pathway. Mice overexpressing HIF-1 α (Δ VHL) or lacking HIF-1 α (Δ HIF-1 α) in osteoblasts were subjected to distraction osteogenesis (DO) and bone vascularity and regeneration were measured by angiography and CT. HIF overexpressing Δ VHL mice showed increased vascularity (2.4 fold in vessel volume/total volume, VV/TV), increased VEGF expression and CD31 staining and markedly increased (1.6 fold) BV/TV after consolidation compared with control littermates. Three point bending testing showed increased peak load to failure (3.5 fold) of the Δ VHL bones compared with controls. Concomitant treatment of the Δ VHL mice with VEGF receptor 1 and 2 neutralizing antibodies during distraction markedly attenuated both the angiogenic and osteogenic phases of the repair process. By contrast, Δ HIF-1 α mice exhibited decreased vascularity (2 fold in VV/TV) and decreased bone volume (1.7 fold) in the distraction gap after consolidation compared to control littermates. To pharmacologically induce HIF-1 α, we screened a series of prolyl hydroxylase inhibitors for their ability to activate a HIF-responsive reporter gene. Two compounds, desferrioxamine (DFO) and L-mimosine were then evaluated for their bone regenerative efficacy by local injection in the DO model. Wild type mice receiving DFO, but not L-mimosine, exhibited significantly increased vascularity (2.3 fold in VV/TV) and increased bone (1.5 fold in BV/TV) in the distraction gap compared to vehicle treated mice. We conclude that activation of the HIF pathway accelerates bone healing by stimulating both angiogenesis and osteogenesis. Our results demonstrate the feasibility of developing HIF-1 α activating agents to facilitate bone regeneration.

Disclosures: C. Wan, None.

This study received funding from: NIH grant R01 AR049410(TLC) and P30AR046031(SRG).

1066

Over-expression of Runx2 in Mesenchymal Cells Enhanced Bone Formation but Inhibited Cartilage Formation and Limb Development., C. A. Yoshida*, T. Maeno*, N. Kanatani*, T. Fujita*, S. Izumi*, T. Komori*. Department of Cell Biology, Unit of Basic Medical Sciences, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.

Runx2 is an essential transcription factor for skeletal development. Although the roles of Runx2 in osteoblast differentiation and chondrocyte maturation have been extensively studied, the functions of Runx2 at an early developmental stage, when multipotent mesenchymal cells commit to the osteoblastic or the chondrocytic cell lineage, still remain to be investigated. To clarify this issue, we prepared Runx2 transgenic mice using a 2.5kb Prx 1 promoter, which drove Runx2 expression to mesenchymal cells in calvarium, limb primordia, maxilla, mandible, and neck.

At embryonic day (E) 13.5 skeletal preparations of Runx2 transgenic mice, but not wild-type mice, displayed calcification of calvarial tissues. At E15.5 Runx2 transgenic mice showed brain hernia due to a fusion of the frontal, parietal, and occipital bones. Furthermore, at this stage, the absence of the sternum, resulted in heart and liver hernia. Histological examination of transgenic mice at E12.5, revealed abnormal Collal expression and mineralization in the calvarium and neck.

Development of the limbs in Runx2 transgenic mice was clearly inhibited, and the level of inhibition ranged from mild to a complete abrogation of cartilage formation. Limb buds of Runx2 transgenic embryos at E10.5 showed reduced or undetectable expression levels of Fgf8, Shh, and Fgf4 but augmented apoptotic cells in the ectoderm compared to those of wild type mice, indicating that apical ectodermal ridge formation was inhibited.

These data demonstrate that Runx2 is able to induce differentiation of mesenchymal cells into osteoblasts in presumptive regions of bone formation and even in ectopic areas but to inhibit the differentiation of mesenchymal cells into chondrocytes. Further, our findings indicate that Runx2 can disturb limb development by inhibiting the formation of apical ectodermal ridge.

Disclosures: C.A. Yoshida, None.

This study received funding from: Ministry of Education, Culture, Sports, Science, and Technology, Japan.

1067

Targeted Deletion of a Distant Transcriptional Enhancer of the RANKL Gene Reduces Bone Remodeling and Increases Bone Mass., C. Galli¹, P. E. Cazer*¹, L. A. Zella², J. A. Fretz*², J. W. Pike², R. S. Weinstein¹, S. C. Manolagas¹, C. A. O'Brien¹¹Center for Osteoporosis and Metabolic Bone Diseases, University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, AR, USA, ²Department of Biochemistry, University of Wisconsin, Madison, WI, USA.

Previous in vitro studies, using BAC-based transcriptional reporter constructs and ChIP-on-chip assays, indicated that PTH and 1,25(OH)₂D₃ stimulate murine RANKL expression in stromal/osteoblastic cells via an evolutionarily-conserved transcriptional enhancer. This enhancer is located 76 kb upstream from the transcription start site and is designated the RANKL distal control region (DCR). To determine the significance of this enhancer for control of RANKL expression and bone homeostasis, we created knock-out (KO) mice in which the 2 kb DCR was eliminated by gene targeting. DCR null mice were viable, fertile, and, in contrast to RANKL KO mice, were not osteopetrotic. Nonetheless, deletion of the DCR reduced PTH- and 1,25(OH)₂D₃-stimulation of RANKL mRNA and osteoclast formation in primary bone marrow cultures. Consistent with this, stimulation of RANKL mRNA in bone by a single injection of either PTH or 1,25(OH)₂D₃ was blunted in DCR KO mice. DCR deletion also reduced basal RANKL mRNA levels in bone, thymus, and spleen. Spinal and femoral bone mineral density (BMD) was elevated in both male and female mice lacking the DCR beginning at 2 months of age and remained elevated up to at least 6 months of age, with the difference in BMD between genotypes remaining relatively constant during this time period. The increase in spinal BMD was approximately 9% in 5-month-old mice and was associated with a 28% increase in vertebral compression strength. Histomorphometric analysis of lumbar vertebra revealed reduced osteoclast and osteoblast formation leading to a low rate of bone remodeling similar to that observed in humans and mice with hypoparathyroidism. Moreover, deletion of the DCR increased trabecular number and thickness and decreased trabecular separation, suggesting that reduced remodeling of the primary spongiosa during growth may be responsible for the increase in bone volume. Consistent with this finding, DPD excretion in urine was reduced in DCR KO mice. However, circulating calcium and PTH levels were similar in wild-type and DCR-deficient mice, demonstrating that PTH control of RANKL is not required for calcium homeostasis under basal conditions. Taken together, these findings demonstrate that PTH control of RANKL expression via the DCR is a critical determinant of the rate of bone remodeling and provide new insights into the relationship, or lack thereof, between the calcium regulating actions of PTH and its effects on the skeleton.

Disclosures: C. Galli, None.

1068

Oncostatin M Is an Essential Stimulus of Bone Formation and Osteoclastogenesis.. N. A. Sims, E. C. Walker*, I. J. Poulton*, N. E. McGregor*, M. T. Gillespie, T. J. Martin. St. Vincent's Institute of Medical Research, Melbourne, Australia.

The gpl30 receptor is common to many cytokines, including IL-6, IL-11, LIF and cardiotrophin-1 (CT-1) which are all important for regulating bone mass in vivo. Murine oncostatin M (OsM) signals through gp130 bound to a specific receptor, OsMR. Osteoblasts express OsMR, and OsM has been reported to increase osteoclast formation by dramatically increasing their RANKL production. The effects of OsM on bone formation however, are not clearly defined.

To determine whether OsM action is essential for bone formation in vivo, we studied the bone phenotype of 10 week-old OsMR deficient mice. While cortical dimensions and density were not altered, male and female OsMR null mice demonstrated a 75% increase in trabecular bone volume in the distal tibia and 4th lumbar vertebrae. Femoral trabecular BMD was also elevated (by 40%) in both males and females. This was associated with a significant (30%) reduction in osteoclast surface and reduced bone resorption, consistent with the known effect of OsM on osteoclast generation in co-cultures. Bone formation was also significantly reduced, evidenced by low osteoid thickness (reduced to 30%), osteoblast surface (reduced to 60%) and bone formation rate (halved). In contrast, the number of marrow adipocytes was elevated ∼4 fold.

To determine the effects of OsM on bone formation, Kusa 4b10 murine stromal cells were treated with OsM (1.25–10ng/ml) for 14 to 21 days. Under mineralising conditions, OsM treatment dose-dependently increased mineralisation; doses as low as 1.25 ng/ml more than doubled alkaline phosphatase activity and alizarin red staining compared to untreated controls. In contrast, under adipogenic conditions adipocyte formation was more than halved. Both the reduced adipogenesis and increased bone formation with OsM treatment are consistent with the opposite observations in OsM-deficient animals.

To identify targets of OsM, we studied its effects on gene expression by undifferentiated Kusa4b10 cells and Kusa4b10 cells differentiated along the osteoblast lineage for 17 days. In both systems, there was a 90% reduction in PPARgamma at 24 hours and a rapid 20-fold increase in C/EBPdelta (but no change in C/EBPbeta) expression within 1 hour of OsM administration. Since C/EBPdelta has been shown to synergise with runx2 to enhance osteocalcin transcription we investigated the effect of OsM on a 6xOSE-luciferase reporter construct in UMR106–06 cells. We detected a dose-dependent increase in reporter activity in response to OsM that was maximal by 6 hours and equivalent to levels observed with 1,25D3 and CT-1.

This reveals a critical role for OsMR in maintaining normal levels of bone resorption and bone formation in vivo, as well as an inhibitory role in adipocyte differentiation.

Disclosures: N.A. Sims, None.

1069

Zfp521, a D2DFosB-interacting Protein, Regulates Chondrocyte Differentiation Downstream of PTHrP.. D. Correa, R. Kiviranta, L. Neff*, W. C. Horne, R. Baron. Orthopaedics, Yale University, New Haven, CT, USA.

Chondrocytes are derived from mesenchymal precursor cells through a tightly regulated differentiation process. The paracrine factor PTHrP and the transcription factor Runx2 are key regulators in this program and have opposing actions in prehypertrophic chondrocytes, with PTHrP inhibiting and Runx2 favoring chondrocyte differentiation.

We identified the zinc finger protein Zfp521, a 180 kDa protein consisting of 30 kruppel-like zinc fingers, as a Δ2ΔFosB-interacting protein in a yeast-two hybrid screen of an osteoblast cDNA library. In situ hybridization with a Zfp521-specific probe showed that Zfp521 is expressed in mesenchymal condensations as early as day E12.5 and in the developing growth plates of long bones, as well as in osteoblasts. Immunostaining detected Zfp521 mainly in PTHrP receptor (PTH1R)-expressing prehypertrophic chondrocytes. In the well-established ATDC5 cell model of in vitro chondrogenesis, the endogenous expression of Zfp521 was observed primarily at the stage of transition from prehypertrophic to hypertrophic chondrocytes, with a peak around the third week of culture, sharing again a similar pattern of expression with the PTH1R. Transient overexpression of Zfp521 in the ATDC5 cells inhibited chondrogenic differentiation, with decreased Alcian Blue staining and impaired cartilage nodule formation at day 12 of culture. This effect was comparable to the inhibitory effect of PTHrP(1–34). Treatment with PTHrP (1–34) did not increase the Zfp521-induced inhibition of nodule formation in these cells, suggesting that PTHrP and Zfp521 might share the same signaling pathway. Consistent with this hypothesis, stimulation of ATDC5 cells with PTHrP(1–34) and Forskolin promoted Zfp521 expression, regardless of the stage of differentiation, and inhibited chondrocyte progression from prehypertrophic to hypertrophic stages. In contrast, PTHrP (7–34), a known competitive antagonist of PTHrP (1–34), downregulated Zfp521 expression suggesting that it blocked the auto/paracrine effect of PTHrP (1–34), including its positive influence on Zfp521 expression. Additionally, PTHrP (1–34) but not PTHrP (7–34) had a negative effect on Runx2 expression during the differentiation of the ATDC5 cells. Therefore, based on Zfp521's negative effect on chondrogenesis, its pattern of expression in the growth plate and in the ATDC5 model, and its upregulation by PTHrP (1–34), we propose that Zfp521, which antagonizes the transcriptional activity of Runx2, lies downstream of PTHrP at the control point of the progression from prehypertrophic to hypertrophic chondrocytes in the growth plate.

Disclosures: D. Correa, None.

1070

Insulin Promotes Osteoblast Differentiation Independent from IGF-1 Signaling and Its Receptor Is Required for Normal Post Natal Bone Acquisition.. K. Fulzele*¹, Z. Liu*¹, C. Wan¹, W. Zhang*¹, J. Bruening*², T. L. Clemens¹¹Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, USA, ²Department of Mouse Genetics and Metabolism, Institute for Genetics, Center of Molecular Medicine, University of Cologne, Cologne, Germany.

Defective bone formation is common in patients with insulin-dependent diabetes mellitus, suggesting that insulin normally exerts anabolic actions in bone. Previous studies suggested that insulin might exert direct effects on osteoblast proliferation and apoptosis. However, because insulin can cross-activate the IGF-1 receptor (IGF-1R), which also functions in bone, it has been difficult to establish direct (IGF-1 independent) actions of insulin in osteoblasts. In this study, we first compared the effects of insulin and IGF-1 on differentiation of primary osteoblasts in vitro. Primary calvarial mouse osteoblasts expressing normal levels of IGF-1R (WT) or deficient in IGF-1R (IGF-1R KO) were differentiated in osteogenic medium with or without insulin or IGF-1. In WT osteoblasts, expression of IGF-1R mRNA declined with differentiation whereas insulin receptor (IR) mRNA remained constant. WT osteoblasts treated with insulin differentiated more rapidly than those treated with IGF-1, as evidenced by increased ALP activity and von Kossa staining. Insulin induced robust activation of Akt and GSK3beta that were not dependent on IGF-1R since they were also observed in IGF-1R KO osteoblasts. Moreover, insulin, but not IGF-1, upregulated expression of Runx2 in WT and KO cells suggesting that insulin promotes differentiation of the primary osteoblasts via the Akt-Runx2 pathway. To investigate the effects of insulin in bone in vivo, we selectively disrupted the IR in osteoblasts using Cre-loxP recombination. Mice carrying floxed IR alleles were mated with a mouse expressing the Cre recombinase driven by the human osteocalcin promoter to generate IR osteoblast-specific knock out mice. Micro-CT analysis of femurs from 3-week-old male OC-Cre IR floxed mice (N=4) showed that bone volume was significantly decreased in IR KO mice compared with IR-floxed littermates. Taken together, our results suggest that insulin exerts direct actions on osteoblasts and signaling through its receptor is required for normal post-natal bone acquisition in vivo. We predict that the loss of insulin action in osteoblasts during states of insulin resistance may contribute to defective bone formation seen in patients with diabetes.

Disclosures: K. Fulzele, None.

This study received funding from: Veterans Administration Merit Review Grant.

1071

Osteogenic Stem Cell Homing to Sites of Endochondral Bone Formation., C. M. Fouletier-Dilling*¹. A. M. Wada*², Z. Lazard*¹, M. E. Dickinson*², A. R. Davis¹, F. H. Gannon³, M. H. Heggeness*⁴, E. A. Olmsted-Davis¹¹Cell and Gene Therapy, Baylor College of Medicine, Houston, TX, USA, ²Molecular Physiology, Baylor College of Medicine, Houston, TX, USA, ³Orthopedic Surgery and Pathology, Baylor College of Medicine, Houston, TX, USA, ⁴Orthopedic Surgery, Baylor College of Medicine, Houston, TX, USA.

Using a model of de novo bone, we recently demonstrated the recruitment of bone marrow derived cells of myeloid lineage to undergo chondrogenesis and osteogenesis. Their differentiation program appears to be dictated by the local micro-environment established in the tissues prior to stem cell engraftment. However, these cells must first mobilize from the bone marrow into the circulation and target the site of new bone formation. This process requires the cells to pass through the vessel wall, known as extravasation, into the target tissue. Receptor/integrins on the stem cell surface specifically bind corresponding molecules on specialized endothelial cells. We induce endochondral bone formation in the mouse hind limb by delivery of cells transduced with adenovirus to express BMP2. Immunohistochemical analysis of tissues isolated from the model prior to bone/cartilage formation, revealed the synthesis of new vessels simultaneous with specific temporal expression of several molecules known to be involved in extravasation. Microarray analysis of the tissue surrounding the bone induction site confirmed a specific enhancement in these genes including SDF-1, CXCR4, CD44, Mac 1, and E-selectin. To further confirm the formation of new vessels, we utilized mice possessing a cassette consisting of the VEGF receptor (flk-1) promoter driving the expression of nuclear yellow fluorescent protein (YFP). Previous reports suggest that flk1 expression is somewhat restricted to endothelial cells present on smaller newly forming vessels. Our initial characterization of these mice confirmed YFP expression on subpopulations of CD31⁺ endothelial cells. Quantification of the flk-H2B:YFP⁺ in tissues isolated from either the region receiving cells transduced with either AdBMP2 or a control adenovirus, showed a significant increase in YFP expression as compared to the control. We observed this increase as early as two days after induction prior to the appearance of stem cells within the tissues. The results collectively suggest the ability of BMP2 to both recruit the necessary chondro-osseous stem cells but also establish the essential vasculature for extravasation and engraftment into the tissues. This is the first report demonstrating a specific mechanism for stem cell homing for the production of endochondral bone, a process that is essential for tissue engineering, but also provides novel targets for treatment of heterotopic bone formation, and bone metastases.

Disclosures: C.M. Fouletier-Dilling, None.

This study received funding from: NIBIB: RO1EB005173 and DOD:PRO33169.

1072

TGFβ1 Induces Migration of Mesenchymal Stem Cells in Coupling Bone Resorption and Formation., Y. Tang, X. Wu, S. Xie*, L. Zhao*, C. Wan*, Z. Shi*, X. Feng, X. Peng*, M. Wan, X. Cao. Pathology, University of Alabama at Birmingham, Birmingham, AL, USA.

Bone is continuously remodeled by the balanced processes of bone resorption and concomitant bone formation. The recruitment of osteoprogenitors to the site of bone resorption is the initial event necessary for coupling of bone resorption and formation. However, such factor(s) for the coupling process is not clear. Here we demonstrate that TGFβ1 released from bone matrix during osteoclastic bone resorption induces migration of stro-1* primary human bone marrow mesenchymal cells (MSCs). Migration of MSCs was examined in coupling with in vitro osteoclastic bone resorption. Osteoclastic bone resorption-conditioned medium (CM) stimulates migration of MSCs, whereas CM from monocytes/macrophage or osteoclasts without bone slices had no effect. Importantly, addition of an antibody against TGFβ1 inhibited the migration, but antibodies against TGFβ2, TGFβ3, FGF-I, FGF-II, PDGF or norggin did not affect the migration. Western blot analysis revealed that only bone resorption-CM contains active TGFβ1. In addition, bone resorption-CM prepared from TGFβ1^−/− mice did not induce migration of MSCs. The results indicate that TGF-β1 released and activated during osteoclastic bone resorption is a key factor in inducing MSCs migration.

In characterization of the mechanism of TGFβ1-induced migration of MSCs, we found that Smad signaling promotes cell migration through TGFβ1 type I receptor (TβRI). TβRI specific inhibitor (SB-431542) blocks the migration. Importantly, injection of the TβRI inhibitor in the rat tibia reduced bone formation dose-dependently as demonstrated by X-ray, μCT and histomorphometry. TGFβ1 activates RhoA which induces formation of focal adhesion and results in reduction of migration of MSCs. Injection of RhoA inhibitor (Y-27632) in the rat tibia stimulated bone formation. The TβRI-mediated migration mechanisms were also confirmed in MSCs isolated from either TβRII or Smad4 conditional knockout mice. Finally, the TGFβ1^−/− mice was crossed with immuno-deficient Rag2^−/− mice to prevent the early death of the TGFβ1^−/− mice due to organ failure of with inflammatory disease. The adult TGFβ1^−/− mice exhibited smaller trabecular bone volume and thickness, and larger trabecular bone space. Histology staining showed lack of preosteoblasts at the surface of trabecular bone. Moreover, TRAP staining of mature osteoclasts confirmed an absence of preosteoblasts around the mature osteoclasts on the surface of trabecular bone. Thus, TGFβ1^−/− mice exhibit significant bone loss with features characteristic of uncoupled bone resorption and formation, which indicates that TGFβ1 functions as a primary coupling factor for bone formation and resorption.

Disclosures: Y. Tang, None.

1073

Bone Turnover Markers and Prediction of Fracture: Nine-Year Follow-Up Study of 1040 Elderly Women., K. K. Ivaska, P. Gerdhem, K. Åkesson, K. J. Obrant. Department of Orthopaedics, Lund University, Malmö, Sweden.

High level of bone turnover has been suggested to be a risk factor for future fracture. The aim of this study was to investigate if biochemical markers of bone turnover are associated with long-term incidence of fracture in elderly women followed prospectively for a mean of nine years.

The ability of bone turnover markers to predict future fracture was studied in a population-based sample of 1040 randomly recruited elderly women, all 75–years of age at inclusion (the Malmö OPRA study). Six bone turnover markers (S-TRACP5b, S-CTX-I, S-OC[1–49], S-TotalOC, S-boneALP, urinary osteocalcin (U-OC)) were measured at baseline and after a new sampling one year later, at the age of 76. Prospective fractures were recorded and verified.

During the mean follow-up of 9.0 years (range 7.4–10.9), a total of 364 women sustained prospectively at least one fracture, including 116 hip fractures and 103 clinical vertebral fractures. Women in the highest tertile for S-TRACP5b, S-CTX-I or U-OC had significantly greater risk for sustaining fracture when compared to women in the lowest tertile. The hazard ratio for any prospective fracture was 1.3–1.4 (p<0.05), depending on marker. The results were consistent when we analyzed resorption markers measured at one-year follow-up visit and the incidence of any prospective fracture during the remaining follow-up time of mean 7.9 years (range 6.3 – 9.9). Similar tendency for increased risk was observed when hip fractures or vertebral fractures were analyzed separately, but it did not reach statistical significance. The associations to hip or vertebral fracture became, however, significant with hazard ratios up to 2.0 (p<0.05), when we used the average value of two marker measurements (baseline and one-year visit). Bone formation markers S-OC[1–49], S-TotalOC and S-boneALP were not associated with prospective fractures.

We conclude that elevated levels of bone resorption markers S-TRACP5b, S-CTX-1 and U-OC are consistently associated with increased fracture risk for up to a decade in elderly women.

Disclosures: K.K. Ivaska, None.

1074

Biochemical Markers of Bone Turnover, Hip Bone Loss and Non-spine Fracture in Men: A Prospective Study.. D. C. Bauer¹, P. Garnero², S. L. Harrison³, J. A. Cauley⁴, R. Eastell⁵, K. E. Ensrud⁶, E. Barrett-Connor⁷, E. S. Orwoll⁸¹UCSF, San Francisco, CA, USA, ²Synarc, Lyon, France, ³CPMC Research Institute, San Francisco, CA, USA, ⁴University of Pittburgh, Pittsburgh, PA, USA, ⁵University of Sheffield, Sheffield, United Kingdom, ⁶University of Minnesota, Minneapolis, MN, USA, ⁷UCSD, San Diego, CA, USA, ⁸OHSU, Portland, OR, USA.

Previous studies have found that biochemical markers of bone turnover are independently associated with fracture risk in older women, but few prospective data exist in men.

We performed a nested case-cohort analysis within the Osteoporotic Fractures in Men (MrOS) study to determine if men with higher levels of bone turnover had accelerated bone loss or an elevated risk of fracture. Among the 5,995 MrOS subjects enrolled at baseline (mean age: 72.2 +/- 6.1), 440 men had documented non-spine fractures during a mean follow-up of 5.1 yr. Hip BMD (Hologic QDR4500) was measured at baseline and after a mean follow-up of 4.6 yr. Using fasting baseline serum stored at −190C, bone turnover (PINP, Roche Diagnostics; TRACP 5b, SBA-Sciences; and beta CTX, Roche Diagnostics) was measured on 428 fracture cases (including 82 hip fractures) and 943 randomly selected men. Bone loss (in the randomly selected men) and fracture outcomes were examined in multivariate models adjusted for age and clinic.

Hip bone loss was greater among men in the highest quartile of PINP (>45.6 ng/mL), TRACP 5b (>3.7 U/L) or CTX (>0.494 ng/mL). For example, total hip bone loss was 0.6% per year among those in the highest quartile of PINP and 0.4% per year among those in the lower 3 quartiles (p=0.002). Compared to men without fracture, those with one or more non-spine fracture during follow-up were older (75.5 yr. vs. 73.7) and had lower total hip BMD (0.89 gm/cm² vs. 0.96), but had similar BMI (27.2 kg/m² vs. 27.3). Elevated PINP, but not TRACP 5b or CTX, was associated with an increased risk of fracture, particularly in the highest quartile (Table). Further adjustment for baseline hip BMD attenuated the associations between PINP and fracture, and they were no longer statistically significant.

We conclude that higher levels of bone turnover are associated with greater hip bone loss in older men, but increased turnover is not independently associated with the risk of hip or non-spine fracture. Our findings suggest that a single serum measurement of bone turnover may not be useful to predict hip or non-spine fracture risk in this population.

Table  .  

Disclosures: D.C. Bauer, None.

This study received funding from: NIAMS.

1075

Quantitative Ultrasound Predicts Incident Vertebral and Hip Fractures at Least as Strongly as DXA: The OPUS Study.. C. C. Glüer¹, R. Barkmann¹, T. Blenk*², A. Stewart*³, S. Kolta*⁴, J. Finigan*³, C. Graeff¹, J. v. d. Gablentz*², R. Eastell⁵, D. M. Reid³, C. Roux⁴, D. Felsenberg²¹Medical Physics, Diagnostic Radiology, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany, ²Charité Universitätsmedizin, Berlin, Germany, ³University of Aberdeen, Aberdeen, United Kingdom, ⁴René Descartes University, Paris, France, ⁵University of Sheffield, Sheffield, United Kingdom.

In the Osteoporosis & Ultrasound (OPUS) study we recruited a population-based sample of 2409 women (ages 55 to 81 years) from 5 European cities. Baseline examinations included dual-x-ray absorptiometry (DXA) of the lumbar spine and the total hip and Quantitative Ultrasound (QUS) of the heel using 4 devices (Lunar Achilles+, Osteometer DTU-one, Quidel/Metra QUS-2, and DMS UBIS 5000) and of the finger using one device (IGEA DBM Sonic BP). At baseline and 6 years later 1566 women underwent repeat spinal radiography for assessment of incident vertebral fractures and reported hip fractures since the baseline visit. Incidence of osteoporotic vertebral fractures was centrally assessed in Berlin according to standard radiological and semiquantitative assessment (vertebral height reduction >20%). We examined the association of the baseline measurements with incident fractures by calculating standardized odds ratios (sOR, per one SD decrease of population variance, adjusted for age) from logistic regression models. There were 67 incident vertebral osteoporotic fractures and 11 incident hip fractures (no major trauma). Prediction based on Speed of Sound (SOS) results was somewhat stronger than for Broadband Ultrasound Attenuation (BUA) or DXA but the differences were not significant (ROC analysis). Finger measurements did not show significant associations with incident fractures. In multivariate age-adjusted vertebral fracture models including both SOS and BMD, only spinal BMD but not total hip BMD contributed independently of SOS (e.g. sOR=1.6, p<.003 for DTU-one SOS and sOR=1.3, p<.04 for DXA spine BMD). 

Table  .  

****;p<.05, **;p<.01, ***;p<.001, ****;p<.0001

Conclusion: Calcaneal QUS methods, best evaluated based on SOS, predict incident vertebral and hip fractures at least as well as central DXA approaches.

Disclosures: C.C. Glüer, GE Medical 5; IGEA 2, 5.

This study received funding from: Sanofi-Aventis, Demetech, DMS, Elil Lilly, GE Medical, IGEA, Novartis, OSI/Osteometer Meditech, Procter & Gamble Pharmaceuticals, Pfizer, Quidel/Metra, Roche.

1076

Abdominal Aortic Calcification (AAC) on Vertebral Fracture Assessment (VFA) Images Predicts Incident Myocardial Infarction and Stroke Independent of Clinical Cardiovascular Disease Risk Factors.. J. T. Schousboe¹, B. C. Taylor², D. P. Kiel³, K. E. Ensrud², K. E. Wilson⁴, E. V. McCloskey⁵¹Park Nicollet Health Services, Minneapolis, MN, USA, ²Minneapolis VAMC, Minneapolis, MN, USA, ³Hebrew SeniorLife, Harvard Medical School, Boston, MA, USA, ⁴Hologic, Inc, Bedford, MA, USA, ⁵University of Sheffield, Sheffield, United Kingdom.

The risk for cardiovascular disease (CVD) among older women is not adequately captured by traditional clinical CVD risk factors. Lateral spine images obtained on bone densitometers intended for VFA can detect abdominal aortic calcification, an important additional subclinical marker of CVD.

Objective: To estimate the association between AAC scored on VFA images and subsequent MI and stroke in women age 75 years or older participating in a randomized trial of clodronate versus placebo after adjustment for traditional risk factors.

Methods: 408 women sustaining either a fatal or non-fatal MI or stroke during the median 4-year study follow-up period were selected as cases, and an equal number of controls were randomly selected from the remainder of the study population. 732 (89.7%) had a baseline VFA with adequate space visualized anterior to the lumbar spine such that AAC could be scored by a single reader using a previously validated 24-point scale and a simplified 8-point scale, blinded to case status and covariate data. Covariates were systolic BP, LDL and HDL cholesterol, triglycerides, smoking, presence of diabetes, renal function, self-reported diagnoses of hypertension, angina, prior stroke, and health status. Missing covariate data was statistically imputed. 

Results: Using logistic regression models, the multivariate-adjusted odds ratios of incident MI/stroke for the top and middle tertiles compared to the bottom tertile of AAC score were:

Table  .  

Results for the subset with complete covariate data (n=420) were very similar.

Conclusion: AAC scored on lateral VFA images is a predictor of incident MI and stroke independent of other clinical risk factors. Since bone densitometry is indicated for all women age 65 and older, VFA imaging offers an opportunity to measure this subclinical indicator of cardiovascular disease in the female population at very little marginal cost.

Disclosures: J.T. Schousboe, Hologic, Inc. 2; Amgen, Inc. 5; Merck, Inc. 5.

This study received funding from: Hologic, Inc.

1077

Clinical Risk Factors for Incident Vertebral Fractures: Data from the Six-Year Prospective OPUS Study.. K. Briot¹, C. Roux¹, S. Kolta¹, R. Eastell², D. M. Reid³, D. Felsenberg⁴, C. C. Gluer⁵¹Rheumatology, Cochin Hospital, Paris, France, ²University of Sheffield clinical sciences centre, Sheffield, United Kingdom, ³Departement of Medicine and Therapeutics, University of Aberdeen, Aberdeen, United Kingdom, ⁴Diagnostic Radiology, Free University Berlin, Berlin, Germany, ⁵Medical Physics, Department of Diagnostic Radiology, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany.

Fractures related to osteoporosis represent a strong public health burden. Low bone mineral density explains only a part of the fracture risk; clinical risk factors play an important role. Many clinical risk factors are identified, but responsibility of some of them needs to be clarified. The objective of the study was to identify clinical risk factors for incident vertebral fractures. Using the 6-years data from the Osteoporosis and Ultrasound study (OPUS), a multi-centre wide study, 2409 European postmenopausal ambulatory women 55–81 years old with baseline clinical risk factors assessment (age, weight, current smoking, personal or familial previous fracture, corticosteroids, medical diseases, physical activity), and bone mineral density (BMD) measurements, were included in the analysis. Spine X-rays were performed using standardized procedures and assessed in a central facility. Incident vertebral fractures (defined as >20% vertebral height reduction and radiological signs of osteoporotic fracture) occurred in 67 women during the six years of follow-up. We performed logistic regression analysis on each of the potential risk factors, with adjustment for age. We chose for multivariate analysis those risk factors that were statistically significant (p ≤ 0.05). In the age-adjusted multivariate analysis, several clinical factors were significantly associated with incident vertebral fractures, independently of BMD value: age (per 10 years) (sOR=1.7; 95% CI, 1.0–2.7; p<0.04), previous fall (sOR=1.4; 95% CI, 1.0–1.9; p<0.04), previous paternal hip fracture (sOR=3.0; 95% CI, 1.5–5.9; p<0.002), and current intake of proton pump inhibitor therapy (omeprazole) (sOR=1.9; 95% CI, 1.2–2.9; p<0.006). There was a trend for an association with previous low traumatic fracture (sOR=1.3; 95% CI, 1.0–1.8; p<0.08). Using data from a cohort of ambulatory postmenopausal European women, we showed that few clinical risk factors are associated with an increase risk of incident vertebral fracture over 6 years; two of them (paternal hip fracture and current intake of omeprazole) have not been observed in prospective studies before. This 6-year prospective study results confirm recent data from case-control studies: proton pump inhibitor therapy appeared to be associated with an increase risk of vertebral fractures.

Disclosures: K. Briot, None.

1078

Validation of Ten-Year Fracture Risk Prediction in a Large Clinical Cohort., W. D. Leslie¹. J. F. Tsang*², L. M. Lix*³¹Dept of Medicine, University of Manitoba, Winnipeg, MB, Canada, ²University of Manitoba, Winnipeg, MB, Canada, ³Manitoba Centre for Health Policy, University of Manitoba, Winnipeg, MB, Canada.

Absolute 10-year fracture risk is the preferred method for fracture risk assessment. The validity of applying published fracture rates from one population to another population is uncertain.

Women age 47.5 y or older at the time of baseline femoral neck BMD (N=20579, 1990–2002) were identified in a database containing all clinical DXA test results for the Province of Manitoba, Canada. Individual 10 y fracture risk was predicted from age-only and age+BMD (NHANES-III femoral neck T-score) using published 10 y fracture risk for Swedish women. Health service records were assessed for the presence of non-trauma ‘osteoporotic’ fracture codes (hip, clinical spine, wrist, humerus) subsequent to BMD testing (86447 person-y follow up, 1173 patients with osteoporotic fractures). Fracture rates were derived for subgroups stratified by age (5 y strata) and estimated risk (5% strata). 10 y fracture rates were computed directly by the Kaplan-Meier method (10 y continuous data) and by the actuarial method (two 5 y periods with adjustments for aging, death and expected BMD loss). Direct and actuarial methods gave nearly identical point estimates, but the latter were more precise.

There was a strong linear correlation between predicted and observed fracture rates based upon age-only (weighted r=0.95) and age+BMD (weighted r=0.99). For age strata 50 to 75, and for estimated risk strata from 0–5% to 20–25%, the confidence intervals overlapped the line of identity. For women age >77.5 or estimated risk >25%, observed exceeded estimated fracture rates. This is explained by healthy selection bias whereby elderly women referred for BMD testing have lower mortality than expected, hence more years at risk for fracture. Corrected for survival bias, women age >77.5 had observed fracture rates no different than predicted.

In conclusion, Swedish 10-year fracture risk data are generally applicable to the Canadian female population referred for BMD testing, though fracture rates may be underestimated in the oldest and highest risk subgroups due to healthy selection bias. 

Figure Figure:. Predicted 10-year fracture risk versus observed fracture rates (actuarial method) for age-only (left, 5 y age strata from 50 to 85) and age+BMD (right, 5% risk strata for estimated risk, from 0–5% to >30%). Dotted line is the line of identity.

Disclosures: W.D. Leslie, Merck Frosst Canada Ltd: Sanofi-Aventis; Proctor & Gamble Pharmaceuticals Canada, Inc. 2, 5, 8.

1079

Bone Mineral Density and Hip Fracture: A Revisit with Time-varying Effects.. T. V. Nguyen, N. D. Nguyen, S. A. Frost*, J. R. Center, J. A. Eisman. Bone and Mineral Research Program, Garvan Institute of Medical Research, Sydney, Australia.

All previous studies of association between BMD and fracture were based on a single measurement, and assumed that BMD do not change. The assumption is clearly untenable since BMD is known to decrease with advancing age. The present study sought to estimate the magnitude of association between BMD and hip fracture risk taking into account the time-dependent bone loss.

A cohort of 782 women and 566 men aged 60+ years who have had at least two BMD measurements preceding a hip fracture was studied. The individuals have been followed-up for a median period of 15 years with an average of 5 BMD measurements (GE Lunar DPX) per subject. During the follow-up period, 77 women and 23 men had sustained at least one hip fracture. The Cox's proportional hazards model with time-varying covariates was used to evaluate the absolute risk of fracture for an individual conditioned on the individual's age, history of prior fracture and fall, and BMD measurements.

For a given risk profile, in addition to baseline BMD, bone loss further increased the risk of hip fracture in an individual. For example, a 70-year old woman with baseline BMD T-score of −2.0, and with a prior fracture and a fall is estimated to have the 5-year and 10-year risk of hip fracture of 6.8% and 10.6%, respectively. However, conditional on an annual bone loss is 1%, her 5-year and 10-year risks of hip fracture increase to 7.7% and 14.3%, respectively. Using this time-varying approach, it is possible to construct a full profile of BMD and fracture risk.

These data suggest that to accurately assess the risk of hip fracture in an individual, the dynamic nature of BMD should be considered. The present model takes into account the time-varying effect of BMD, and can therefore be used for individualizing the risk of hip fracture for any given unique risk profile 

Figure Figure:. Cumulative incidence of fracture stratified by baseline BMD and time-dependent BMD

Disclosures: T. V. Nguyen, None.

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

1080

Clinical Validation of a Simplified Absolute Fracture Risk Algorithm in Canadian Women.. W. D. Leslie1¹, J. F. Tsang*², L. M. Lix*³¹Dept of Medicine, University of Manitoba, Winnipeg, MB, Canada, ²University of Manitoba, Winnipeg, MB, Canada, ³Manitoba Centre for Health Policy, University of Manitoba, Winnipeg, MB, Canada.

Absolute 10 y fracture risk based upon multiple factors is the preferred method for assessment. A simplified risk assessment system from gender, age, minimum T-score, and two clinical risk factors (CRFs) - prior fracture and systemic corticosteroid (CS) use - has been used in Canada since 2005. The current study was undertaken to validate this system in the Canadian female population.

16,205 women of white ethnicity age 50 y or older at the time of baseline BMD (1998–2002) were identified in a population-based database containing all clinical DXA test results for the Province of Manitoba, Canada. Individual basal 10 y fracture risk (from age and minimum T-score lumbar spine, femur neck, trochanter, total hip) was categorized as low (<10%), moderate (10–20%) or high (>20%). Health service records since 1987 were assessed for non-craniofacial non-trauma fracture codes prior to BMD (N=5,224). CS use was defined as possession for 90d or more during the year prior to BMD testing at a prednisone-equivalent mean daily dose of 7.5mg or greater (N=616). Fracture rates (per 1,000 person-y) after BMD testing were calculated from non-trauma fracture codes for the hip, clinical spine, wrist, or humerus (collectively designated ‘osteoporotic’, N=757).

In women without CRFs, fracture risk predicted from age and BMD alone showed a significant gradient in observed fracture rates (low 4.1±0.6, moderate 8.4±0.8, high 17.1±1.3 per 1,000 person-y; p-for-trend <.0001). There was an incremental increase in incident fracture rates from a prior fracture (13.9 per 1,000 person-y, p<.0001) or recent CS use (8.4 per 1,000 person-y, p=.0077). Site of prior fracture further substratified fracture risk (‘osteoporotic’ 25.9 vs all other sites 5.5 per 1,000 person-y, p<.0001). Lower dose CS use (<7.5mg daily) had no observed effect on fracture rates. A Cox proportional hazards model gave: HR basal risk moderate 1.94 (1.61–2.34) and high 4.51 (3.73–5.45) vs low (ref); CS use 1.54 (1.16–2.05); prior ‘osteoporotic’ fracture 2.68 (2.26–3.17) vs other fracture 1.41 (1.17–1.71).

This simplified fracture risk assessment system provides an assessment of fracture risk that is broadly consistent with observed fracture rates. 

Figure Figure:. Observed fracture rates (SE bars) in relation to age, BMD and two clinical risk factors.

Disclosures: W.D. Leslie, Merck Frosst Canada Ltd; Sanofi-Aventis; Proctor & Gamble Pharmaceuticals Canada, Inc. 2, 5, 8.

1081

Leptin-deficient (ob/ob) Mice Exhibit Increased Bone Mechanosensitivity and Corresponding Osteoblasts Show Increased Anabolic Shear Stress Responses In Vitro.. K. H. W. Lau¹, S. Kapur*¹, M. Amoui*¹, X. Wang*¹, C. Kesavan*¹, S. Mohan¹, D. J. Baylink²¹Loma Linda VAMC, Loma Linda, CA, USA, ²Loma Linda Univ., Loma Linda, CA, USA.

We sought to test the hypothesis that the leptin receptor (Lepr) pathway plays an important role in bone mechanosensitivity based on the rationales that Lepr is located within one of the mouse genetic loci that showed mechanosensitivity modulating effects and that Lepr signaling is essential for skeletal maturation and metabolism. To test this hypothesis, the osteogenic response to loading (in the form of 2-week four-point bending) in tibia of adult female ob/ob mice [in C57BL/6J (B6) background] was compared with those in adult female B6 tibia. To adjust for the 14% greater bone size in ob/ob mice, the load was adjusted to produce similar levels of mechanical strain (2129 με at 9N for ob/ob mice vs. 2500 με at 6N for B6 mice). This mechanical strain was insufficient to produce a bone anabolic response in B6 mice; whereas in ob/ob mice this strain increased total BMC (16%), cortical area (22%), content (29%), and thickness (28%), and BMD (8%) [p<0.05 for each]. To further test if leptin deficiency would enhance osteogenic response to mechanical stimuli, the effects of a 30-min fluid shear (20 dynes/cm²) on [³H]thymidine incorporation (TdR) and Erk1/2 phosphorylation in ob/ob osteoblasts were compared to those of wild-type (WT) littermates and B6 mice. While the shear stress increased TdR and Erk1/2 in osteoblasts of B6 and WT littermates (each by ∼2-fold, p<0.05), the stimulation in ob/ob osteoblasts was greater (> 3-fold, p<0.05 vs. B6 osteoblasts). In addition, 2-hr pretreatment of ob/ob osteoblasts with 100 ng/ml of leptin completely abrogated the enhanced mitogenic response. Because it has been reported that the mechanism whereby mechanical stimuli act to stimulate proliferation involves upregulation of genes of the IGF-I, Wnt, BMP/TGFβ, and estrogen receptor pathways, we next determined whether the Lepr pathway acts upstream to these 4 pathways by assessing the effects of the fluid shear on the expression levels of representative genes of these pathways in ob/ob and B6 osteoblasts (by real-time PCR). The upregulation of each test gene of the 4 pathways in ob/ob osteoblasts was much greater (p<0.05) than those in B6 osteoblasts. The 2-hr leptin pretreatment also abrogated the shear stress-induced upregulation of these genes in ob/ob osteoblasts. Conclusions: 1) In vivo, the Lepr pathway inhibits the anabolic responses to mechanical loading which is consistent with increased basal values for bone size in the ob/ob mouse, 2) In vitro, the Lepr pathway also has a negative modulating role in the mechanosensitivity of mouse osteoblasts, and 3) The Lepr pathway acts upstream of 4 selected major anabolic pathways to modulate mechanosensitivity.

Disclosures: K.H.W. Lau, None.

This study received funding from: Department of Defense.

1082

Denosumab (AMG 162), a Fully Human RANKL Antibody, Improves Cortical and Cancellous Bone Mass and Bone Strength in Ovariectomized Cynomologus Monkeys.. M. S. Ominsky¹, J. Schroeder*¹, 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, a fully human monoclonal antibody that inhibits RANKL, decreases bone resorption and increases bone mineral density (BMD) in postmenopausal women participating in clinical studies. Denosumab was shown to cause similar responses in adult ovariectomized (OVX) cynomolgus monkeys (cynos). We now describe the consequences of these changes on bone strength.

One month after OVX or sham surgery, OVX cynos (9 to 16 years old) were treated with vehicle (OVX-Veh) or denosumab (25 or 50 mg/kg, SC, once/month) for 16 months (n=14 to 20/group). Sham controls were treated with vehicle (n=17). After sacrifice, ex vivo DXA and pQCT scans were taken of the intact right femur, L3-L4 vertebral bodies, and L5-L6 vertebral 5mm cancellous cores. Destructive testing was performed by 3-point bending of the femur diaphysis and humeral cortical beams, shearing of the femur neck, and compression of the lumbar vertebral specimens. Statistically significant differences between the denosumab and OVX-Veh groups are reported as p<0.05 by ANOVA.

Compared with OVX-Veh, both doses of denosumab were associated with greater BMC at the femur neck (+19% to 22%), L3-L4 bodies (+23% to 25%), L5-L6 cores (+29% to 30%), and femur diaphysis (+10% to 11%). Both doses of denosumab were associated with significantly greater peak load for femur neck (19% to 34%), L3-L4 bodies (54% to 55%), and L5-L6 cores (69% to 82%), compared with OVX-Veh. Both doses of denosumab were associated with significantly greater stiffness for femur neck (21% to 26%), L3-L4 bodies (39% to 46%), L5-L6 cores (61% to 62%), and femur diaphysis (15% to 16%), compared with OVX controls. Intrinsic (material) properties for cortical sites were derived from geometric data and extrinsic (whole bone) strength parameters. Intrinsic strength parameters of femur diaphysis and humerus cortical beams were similar in OVX-Veh and treatment groups, and the relationship between femur diaphysis vBMC and peak load was strong and similar among all groups (r²=0.79 to 0.91). For the lumbar vertebral bodies and cancellous cores, significant positive correlations were found between vBMC and yield load for all groups (r²=0.57 to 0.84).

In summary, long term (16-month) denosumab therapy increased bone mass and density at both cortical and cancellous sites in ovariectomized cynomolgus monkeys. These increases were highly correlated with improvements in the extrinsic strength of cortical and cancellous sites. The results suggest that in this study, denosumab improves bone strength primarily by increasing bone mass.

Disclosures: M.S. Ominsky, Amgen Inc. 1, 3.

This study received funding from: Amgen, Inc.

1083

Concerted Growth of Bone Length and Width - Influence of Sex Hormones, IGF-1 and Muscle Size.. S. Cheng¹, O. Wang¹, P. Rahkila*¹, M Alen*¹, A. Mahonen*², H. Kröger², H. Suominen¹, A. Lyytikäinen¹, E. Völgyi*¹, U. M. Kujala*¹, F. Tylavsky³, E. Seeman⁴¹Univ. of Jyväskylä, Jyväskylä, Finland, ²Univ. of Kuopio, Kuopio, Finland, ³Univ. of Tennessee, Memphis, TN, USA, ⁴Univ. of Melbourne, Melbourne, Australia.

Bone growth is determined by genetic and environmental factors. The effect of mechanical loading on growth in bone width and length has not been specifically addressed. We hypothesized that 1) sex hormones and IGF-I correlate with bone growth prior to puberty, since hormone levels are changing rapidly, but correlate less well after puberty when levels vary less; and 2) loading, as reflected by muscle size, correlates with bone width and length throughout growth. 258 girls aged 11.2 yrs at baseline were followed on average for 6.5 years. Left tibial shaft was scanned using pQCT (XCT2000). Tibial length (TL) was determined from whole body DXA scans. Serum 17β-estradiol (E2), testosterone (Te) and IGF-I concentrations were assessed using immunofluorometry and serum sex hormone binding globulin (SHBG) using time-resolved fluoroimmunoassays.

In hierarchical multiple regression models the growth of tibial length, total cross-sectional area (tCSA), bone marrow cavity area (bmCSA) and cortical thickness (cTh) were explained by E2, Te, IGF-I, SHBG, muscle cross-sectional area (mCSA), and time relative to menarche (TRM). The results of these models are summarized in the Fig. There was no correlation between E2 and bone size measures either before or after menarche. mCSA correlated positively with IGF-I and negatively with SHBG both before and after menarche. Te was positively associated with TL, tCSA and cTh before menarche. Of the hormones studied none were associated with bone variables after menarche. Interestingly, TRM was negatively associated with the bmCSA but was not associated with tCSA after menarche. This implies increased periosteal apposition in the long bone shaft as the girls age. The moderate correlation found between TL and tCSA (r²=0.44) was partly due to growth asynchrony between the two.

The results support our hypothesis that before and after menarche bone growth in length and width are influenced differently by hormones and mechanical loading. Mechanical loading is the dominant factor throughout the pubertal period. Hormones may have an indirect influence on bone growth via their regulatory role on muscle mass based on our results.

Disclosures: S. Cheng, None.

This study received funding from: ASBMR Bridge Funding, Academy of Finland and Finnish Ministry of Education.

1084

Role for Estrogen Receptor-Beta in Trabecular Bone Mechanotransduction., A. B. Castillo¹. J. M. Doyle*¹, M. R. Allen², C. H. Turner¹¹Biomedical Engineering, Indiana University Purdue University, Indianapolis, IN, USA, ²Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN, USA.

Recent studies suggest that estrogen receptor beta (ERb) plays a role in bone mechanotransduction at cortical bone sites. To determine whether ERb plays a role in trabecular bone mechanotransduction, 16-wk-old wildtype and ERb-/- female mice were subjected to 28 days of tail suspension (TS). Age-matched, normally-loaded (NL) mice served as controls. Trabecular bone microarchitecture in the distal femur was evaluated using high-resolution computed tomography (vivaCT40, Scanco Medical), and the following parameters were calculated: bone volume/total volume (BV/TV), connectivity density (Conn.D), trabecular number (Tb.N, 1/mm), trabecular thickness (Tb.Th, mm), trabecular spacing (Tb.Sp, mm) and structure model index (SMI). Data were checked for normality and constancy of variance and analyzed using a 2-factor ANOVA with a Fisher's PLSD post-hoc analysis. TS had a significant factor effect on all variables (p<0.001) and resulted in significantly different BV/TV, Conn.D, Tb.N and SMI in TS wildtype and ERb-/- when compared to NL controls (Table 1). Genotype had a significant factor effect on BV/TV (p=0.029), Tb.Sp (p=0.029) and SMI (p<0.001). TS wildtype and ERb-/- mice exhibited a 38% and 60% decrease in BV/TV, and an 8% and 24% increase in Tb.Sp, respectively, when compared with NL controls. Disuse results in a decrease in osteoblast number and activity with no change in osteoclastic activity in most cases. Estrogen inhibits bone turnover by suppressing both osteoblastogenesis and osteoclastogenesis. The fact that TS ERb-/- mice showed a greater loss in BV/TV compared to wildtype mice implies that signaling via ERb may normally inhibit disuse-related osteoclastic activity in the trabecular bone compartment. The role of ERb in disuse-related osteoblastic activity remains unclear. Taken together, these data implicate a role for ERb-/- in disuse bone loss and suggest that ERb plays a role in trabecular bone mechanotransduction.

Table  .  

Data are presented as mean±standard error

NL= normally loaded; TS = tail suspension

(a) vs. NL wildtype, p<0.05

(b) vs. NL ERb-/-, p<0.05

(c) vs. TS wildtype, p<0.05

Disclosures: A.B. Castillo, None.

This study received funding from: NIH.

1085

Effects of One Year Treatment with PTH (1–34) on Bone Microstructure at the Ultradistal Radius.. S. Kirmani, M. Holets*, S. Khosla. Mayo Clinic, Rochester, MN, USA.

Previous studies have shown a beneficial effect of PTH (1–34) on bone density and fracture rate reduction in women with osteoporosis. Compared to placebo, a 13% and 6% increase in areal BMD (aBMD) using DXA was seen at the spine and hip, respectively (NEJM 344: 1434, 2001). At the distal radius, there was no significant effect on aBMD with the 20 μg/d dose, but at the 40 μg/d dose, there was a 2% decrease as compared to placebo. Using pQCT, a subset of women from the same trial had a 5–7% increase in cortical bone area, but no significant change in cortical vBMD or cortical thickness (JBMR 18:539, 2003). Despite these data on changes in BMD, there are sparse data on possible effects of PTH therapy on bone microstructure, and the available data are limited to studies using bone biopsies from the iliac crest. Thus, to further define effects of PTH on bone microstructure, we used high resolution 3D-pQCT (XtremeCT, Scanco AG, voxel size ∼90 microns) to measure bone microstructural variables at the ultradistal radius in 7 women with osteoporosis (mean age, 74 yrs) at baseline and 1 yr after initiation of 20 μg/d of PTH (1–34) (Forteo) for osteoporosis. None of the women suffered a fracture during the 1 year of treatment. Compared to baseline, we noted a 6.9% increase in bone volume/tissue volume (BV/TV) (Table, P = 0.035). This was similar in magnitude to the 5.7% change noted at the iliac crest in women treated for 3 years with PTH (JBMR 16:1846–1853). Two-thirds of this increase in BV/TV was due to an increase in trabecular thickness (Tb.Th), and only one-third due to an increase in trabecular number (TbN). There was a trend towards a decrease in cortical thickness (CTh) and cortical volumetric BMD (vBMD). In summary, these data represent the first assessment of trabecular microstructure at the wrist following PTH therapy, and they demonstrate that (1) changes in trabecular parameters seen at the radius are very similar to those previously reported at the iliac crest; and (2) the beneficial effects of PTH (1–34) on trabecular bone seem to result primarily from increased trabecular thickness, with the minor apparent increase in trabecular number seen likely due to coalescence of the thickened trabeculae.

Table  .  

Disclosures: S. Kirmani, None.

1086

Glucocorticoids, Aging and Bone Hydration: New Insights into Qualitative Aspects of Bone Strength.. R. S. Weinstein, J. Goellner, T. M. Chambers*, E. A. Hogan*, S. B. Berryhill*, R. Shelton*, W. W. Webb*, C. A. Wicker*, S. C. Manolagas. Central Arkansas Veterans Healthcare System, Center for Osteoporosis, University of Arkansas for Medical Sciences, Little Rock, AR, USA.

Both aging and glucocorticoid excess are associated with a decline in bone strength that is greater than the decline in bone mass. In addition, both conditions may decrease the water content of bone, as determined by the intensity of an inert aqueous fluorescent tracer (Procion red), which provides a visible outline of the lacunocanalicular network, following injection into the tail vein. Prompted by these lines of evidence, we obtained femora (including bone marrow) from 15 C57BL/6 mice at 4 months of age and placed them in a vacuum chamber for 50 hours at 4° C. Contralateral femora from the same animals were maintained in normal saline at the same temperature. Compared to zero time, there was a 58.7 ± 3.0% (SD) reduction of total weight in the femora, as early as 24 hours, and weights remained stable thereafter, indicating that maximum water removal was accomplished by this time point. We next determined the biomechanical properties of the femora during 3-point bending of the wet versus the vacuum dried femora taken from the same animals. Loss of wet weight reduced the average breaking stress by 33.7% and toughness (i.e. the work required to break the bone) by 59%. In agreement with this line of evidence, prednisolone administration to 8 month old C57BL/6 mice (n = 3) for 7 days (a time period insufficient to affect bone mass) decreased Procion fluorescence by 71%, as compared to placebo control. This effect was prevented in transgenic mice overexpressing the enzyme 11β-HSD2 in the context of an OG2 promoter, a model for deflecting glucocorticoid action from osteoblasts. In a second experiment with C57BL/6 mice (n = 16), crystallinity determined by Fourier transformation infrared microscopy (FTIRM) as well as strength decreased in mice receiving prednisolone for 28 days; and the two measures were directly correlated (r = 0.53; p < 0.03). Moreover, both the decrease in crystallinity and strength were prevented in the 11β-HSD2 transgenic animals. Lastly, Procion red epifluorescence was decreased by more than 40% in 31 month old as compared to 4 month old female or male C57BL/6 animals (p < 0.001). In addition, the mRNA levels in bone of 11β-HSD1 - the enzyme that locally amplifies glucocorticoid action, was increased by 1.8 fold between 8 and 31 month old mice and were inversely related to strength (r = −0.67, p < 0.05). These results lend strong support to our working hypothesis that skeletal water volume may protect against mechanical failure by means of hydraulic stiffening; and that both aging and glucocorticoid excess compromise bone strength, in part by decreasing skeletal hydration.

Disclosures: R.S. Weinstein, Merck 8.

1087

Effects of Mechanical Loading and Estrogen Are Structurally Distinct.. J. Jokihaara¹, I. Pajamäki¹, H. Sievänen*², P. Kannus*², T. Vuohelainen*¹, T. L. N. Järvinen*¹¹University of Tampere, Tampere, Finland, ²UKK-Institute, Tampere, Finland.

The objective was to study the effects of effects of estrogen and locomotion on trabecular bone structure. We hypothesized that the independent and potentially interactive effects of these two factors should become distinct after separate or simultaneous inclusion of their influence; i.e. using the classic 2 × 2 factorial study design.

Accordingly, 30 three-week old littermates of female rats were first randomly assigned into bilateral ovariectomy (E-) or sham surgery (E+), after which the left hindlimb of each rat was cast immobilized (L-) while the contralateral (right) hindlimb served as normally loaded control (L+). After eight-week study period, trabecular structure of the distal femoral metaphysis was analyzed with micro-computed tomography. To eliminate the inherent bias arising from comparisons between animals differing in body weight and size, all data pertaining to bone mechanical competence was equalized in terms of the animal's apparent loading environment by using the body weight and femoral length as covariates. Loading increased bone size (P=0.002), while estrogen was not associated with changes in bone size (P=0.280). Both factors displayed a significant stimulatory effect on trabecular bone volume (P=0.001 and P=0.003, respectively). Further, both factors increased trabecular number (P=0.006 and P=0.007, respectively) while only loading increased trabecular thickness (p<0.001 and P=0.244, respectively) and only estrogen decreased trabecular separation (P=0.775 and P=0.004, respectively).

The anabolic effects of loading and estrogen were shown to be independent and the mechanisms of action of these two factors were distinct: loading alone (E-L+) resulted in thickening of individual trabeculae and estrogen effect (E+L-) was discernible as a denser (less spacing) trabecular meshwork. Moreover, when the factors were combined (E+L+), the effects add up. In conclusion, our results show that there is a structural dimorphism in the skeletal actions of estrogen and loading so that the actions of these two factors are independent and additive in nature.

Disclosures: J. Jokihaara, None.

1088

Transgenic Overexpression of OPG Results in Increased Bone Mass and Strength and Decreased Bone Turnover in One-Year-Old Female Rats.. M. S. Ominsky, F. J. Asuncion, M. Barrero, O. Niu*, T. J. Corbin*, D. Dwyer*, K. S. Warmington*, M. Grisanti, H. Tan*, X. Li, M. Stolina, P. J. Kostenuik. Metabolic Disorders and Laboratory Animal Research, Amgen Inc., Thousand Oaks, CA, USA.

RANK ligand (RANKL) is an essential mediator of bone resorption, and its activity is inhibited by osteoprotegerin (OPG). We created transgenic (Tg) rats to study the long-term impact of increased circulating levels of OPG on bone turnover, volume, density, and strength. CD rats were engineered to overexpress full-length soluble rat OPG via a liver promoter. Serum OPG levels in female Tg rats (n=32) were up to 260-fold greater than the mean value (257±16 pg/ml) found in wildtype (WT) controls (n=23). At 1 year of age, rats were injected with fluorochrome labels for dynamic histomorphometry, bled for biomarker analyses, scanned by DXA, and sacrificed. Lumbar vertebra 2 (L2) was used for histomorphometry, and L5 was scanned by microCT and subjected to destructive mechanical testing.

In normal WT rats, there were no significant correlations between log serum OPG levels and lumbar BMD, serum TRAP-5b, serum osteocalcin, bone formation rate, or bone strength parameters. In contrast, log serum OPG in OPG-Tg rats had significant (p<0.01) and positive linear correlations with lumbar BMD (r²=0.53), BV/TV (r²=0.77), trabecular number (r²=0.93), and peak load (r²=0.54). Log serum OPG in OPG-Tg rats had significant (p<0.01) and negative linear correlations with serum TRAP-5b (r²=0.64), serum osteocalcin (r²=0.35), and bone formation rate (r²=0.51).

Tg rats with the highest levels of serum OPG (OPG-hi, 50- to 260-fold greater than the mean value for WT controls; n=15) had 70% lower osteoclast surface and 91% lower bone formation rates in 1.2 compared with WT controls (p<0.001). Despite the profound suppression of bone turnover, the vertebra of the OPG-hi rats had 56% greater peak load, 62% greater stiffness, and 53% greater energy absorption compared with WT controls (p<0.01). To approximate material properties, apparent strength parameters were derived by normalizing to bone volume. Apparent toughness was similar between WT and Tg groups, while apparent strength was significantly higher in the OPG-hi group versus WT (p<0.01).

In summary, 1 year of OPG overexpression led to marked changes in bone turnover, volume, density and strength that were consistent with the suppression of bone resorption. The dose-dependent suppression of resorption in OPG-Tg rats was accompanied by a secondary coupling-related suppression of bone formation. Rats with the highest levels of OPG overexpression had the greatest reductions in bone turnover parameters, and the greatest improvements in extrinsic bone strength with no evidence of impairment of material properties.

Disclosures: M.S. Ominsky, Amgen Inc. l, 3.

This study received funding from: Amgen, Inc.

1089

Comparison of the Effects of Teriparatide and Alendronate on Parameters of Total Hip Strength as Assessed by Finite Element Analysis: Results from the Forteo and Alendronate Comparison Trial.. T. M. Keaveny¹, P. F. Hoffman*², D. L. Kopperdahl², D. W. Donley*³, K. Krohn³, E. V. Glass*³, B. H. Mitlak³¹University of California, Berkeley, CA, USA, ²O.N. Diagnostics, Berkeley, CA, USA, ³Eli Lilly and Company, Indianapolis, IN, USA.

Biomechanical computed tomography (BCT) uses finite element analysis of QCT scans to provide non-invasive measures of femoral strength and density plus a strength:density ratio that can be considered a measure of bone “quality”. Teriparatide [rhPTH (1–34), TPTD] 20 μ/d and alendronate 10 mg/d (ALN) were previously shown to have positive effects on vertebral strength as assessed by BCT during a randomized, double-blind, 18-month study in postmenopausal women with osteoporosis. The purpose of the present analysis was to extend these studies to analysis of proximal femoral strength for a simulated sideways fall. Using the QCT scans, volumetric density from QCT and strength from BCT were determined for the total hip and for the trabecular and cortical compartments, and a strength:density ratio was calculated. In the TPTD group, whole total hip density was not significantly different from baseline at any time point, however, at 18 months trabecular density significantly increased 5.1% and cortical density significantly decreased 1.0% from baseline. In the ALN group, density was not significantly different from baseline at any time point for any compartment. Total hip strength significantly increased 5.9% from baseline at 18 months in the TPTD group, and strength did not significantly change from baseline at any time point in the ALN group No significant changes were seen in the strength measures associated with isolated changes in the cortical or trabecular compartments. The strength:density ratio significantly increased 4.1% from baseline at 18 months in the TPTD group, whereas no significant changes were seen for ALN. In conclusion, total hip strength at 18 months significantly increased in the TPTD group and did not significantly change in the ALN group. This significant biomechanical effect for TPTD was associated with a significant decrease in cortical density and a somewhat larger increase in trabecular density. 

Table Table:. Median percent change from baseline (Interquartile range) at 6 and 18 months

*p<0.05, **p<0.005 within treatment from baseline; ‡p<0.005 between treatments

Disclosures: T.M. Keaveny, Merck, Amgen, GSK, Novartis, Pfizer 5; P & G, Merck, Pfizer, Novartis, Lilly, NPS, Teijin 2; O.N. Diagnostics 4.

This study received funding from: Eli Lilly and Company.

1090

Prediction of 24 Month Change in BMD on PTH Followed by Alendronate: The PaTH Study.. D. M. Black¹, D. C. Bauer¹, L. Palermo¹, C. J. Rosen², S. L. Greenspan³, J. P. Bilezikian⁴¹UC San Francisco, San Francisco, CA, USA, ²MECORE, St. Joseph Hospital, Bangor, ME, USA, ³University of Pittsburgh, Pittsburgh, PA, USA, ⁴Columbia University, New York, NY, USA.

We previously reported the PaTH study results for postmenopausal women on 2 years of parathyroid hormone (PTH 1–84, 100 μg daily) used either alone or in combination with alendronate (ALN, Black et al., NEJM, 2005). The treatment group that received 1 year of PTH (1–84) followed by 1 year of ALN had the largest mean spine BMD change of 12%, but response varied, with 17% of women having BMD increases over 20%. Previous PaTH analyses have examined predictors of 1 year BMD change while on PTH therapy. This analysis aimed to identify early predictors of response over 2 years in women taking 1 year of PTH followed by 1 year of ALN.

This analysis included 53 women assigned to 1 year of PTH 1–84 followed by 1 year of ALN. The main endpoint was 2 year change in (DXA) spine BMD. Potential predictors included baseline variables (age, BMI, bone marker levels (serum CTX, PINP, Bone ALP), serum chemistries) and early 1- and 3-month on-treatment bone marker values. We used univariable analyses (logistic regression) with change in BMD (>20%) as the outcome adjusted for significant covariates. Bone markers were categorized into highest quartile vs. lower 3 quartiles combined.

The following baseline factors had weak, but significant relationships to the 24-month change in BMD: age, BMI and serum calcium. Baseline bone markers were not significantly associated, but bone marker values at 1 and 3 months after start of treatment were very strongly related to BMD change at 24 months. While all 3 bone markers were associated with 24 month BMD change, PINP was most strongly correlated. The figure compares 24 month % change in spine BMD in those with 1 month PINP levels in the highest quartile (>142ng/ml) vs. those in the lower 3 quartiles (≤142ng/ml):

The data show that those in the highest quartile of PINP at 1 month were 10 times more likely to show an increase in DXA BMD of ≥20% at 24 months. This relationship was confirmed with multivariable adjustment. In addition to 1-month bone marker values, the 3-month values and change from baseline to 1 and 3 months were similarly predictive of 2 year BMD change. In conclusion, a single measurement of PINP as early as 1 month after initiation of PTH therapy strongly predicts 2 year spine BMD response to 1 year of PTH followed by 1 year of ALN.

Disclosures: D.M. Black, NPS Pharmaceuticals 5: Novartis Pharmaceuticals 2; Merck & Co., Inc. 8; Roche/GSK 2, 5.

This study received funding from: N01 AR92245; NIAMS-045.

1091

Patients Previously Treated with Risedronate Demonstrate Greater Responsiveness to Teriparatide than Those Previously Treated with Alendronate: The OPTAMISE Study.. P. Miller¹, R. Lindsa², N. Watts³, S. Meeves*⁴, T. Lang*⁵, P. Delmas⁶, J. Bilezikian⁷¹Colorado Center for Bone Research, Lakewood, CO, USA, ²Helen Hayes Hospital and Columbia University, West Haverstraw, NY, USA, ³University of Cincinnati College of Medicine, Cincinnati, OH, USA, ⁴sanofi-aventis US Inc., Bridgewater, NJ, USA, ⁵Center for Molecular and Functional Imaging, University of California, San Francisco, CA, USA, ⁶Inserm Research Unit 831 and Université de Lyon, Lyon, France, ⁷Columbia University College of Physicians and Surgeons, New York, NY, USA.

Previous or simultaneous treatment with alendronate (ALN) has been shown to blunt or delay the anabolic response to teriparatide (TPTD). It is not known if the effect is the same with other bisphosphonates. OPTAMISE evaluated the anabolic effect of TPTD in postmenopausal women previously treated for ≥24 months with risedronate (RIS) or ALN.

Post-RIS (n=146) and post-ALN (n=146) subjects were stratified by duration of prior therapy, discontinued their bisphosphonate and were treated with TPTD (20 μg/d SQ) for 12 months. We measured bone turnover markers (BTM) and bone mineral density (BMD) by DXA and QCT.

At baseline, BTMs were higher in the post-RIS group (p<0.05); otherwise, the groups were comparable for key characteristics including BMD by DXA and QCT. Compared with post-ALN subjects, post-RIS changes in BTMs were significantly greater at Months 1–5 (p<0.05, including primary endpoint of PINP change at Month 3), and for BMD by DXA at Month 12 at the spine (p<0.05) and hip (p<0.01). Results were not related to duration of prior bisphosphonate therapy, baseline BTMs, or baseline BMD. Post-RIS subjects showed a 76% greater increase in QCT of trabecular bone at the spine (24.1% vs 13.7%, P=0.0230; Figure). Spine QCT changes correlated with PINP changes at 3 months (r=0.47). TPTD was well-tolerated with a similar incidence of adverse events between groups.

When switched to teriparatide, subjects previously treated with risedronate showed a greater QCT response that correlated with a more pronounced, early increase in PINP as compared with those previously treated with alendronate. Our findings support differences between these bisphosphonates that affect subsequent response to the anabolic effects of teriparatide.

Disclosures: P. Miller, Alliance for Better Bone Health 2, 5, 8.

This study received funding from: The Alliance for Better Bone Health.

1092

Bone Turnover Markers Demonstrate Greater Earlier Responsiveness to Teriparatide Following Treatment with Risedronate Compared with Alendronate: The OPTAMISE Study.. P. Delmas¹, N. Watts², P. Miller³, D. Cahall⁴, J. Bilezikian⁵, R. Lindsay⁶¹Inserm Research Unit 831 and Université de Lyon, Lyon, France, ²University of Cincinnati College of Medicine, Cincinnati, OH, USA, ³Colorado Center for Bone Research, Lakewood, CO, USA, ⁴sanofi-aventis, Bridgewater, NJ, USA, ⁵Columbia University College of Physicians & Surgeons, New York, NY, USA, ⁶Helen Hayes Hospital and Columbia University, West Haverstraw, NY, USA.

Previous treatment with alendronate (ALN) has been shown to blunt or delay the anabolic response to teriparatide (TPTD). It is not known if the effect is the same with other bisphosphonates. OPTAMISE evaluated the anabolic effect of TPTD on bone turnover markers (BTM) in postmenopausal women previously treated for ≥24 months with risedronate (RIS) or ALN.

Post-RIS (n=146) or post-ALN (n=146) patients were stratified by duration of prior bisphosphonate therapy, and after discontinuation were treated with TPTD (20 μg/d SQ) for 12 months. We measured N-terminal propeptide (PINP, primary endpoint change at Month 3), bone-specific alkaline phosphatase (BAP), osteocalcin (OC), serum CTX and urine NTX in both groups.

Baseline BTMs were significantly higher in the post-RIS group (p<0.05); otherwise the groups were comparable for key characteristics, including duration of prior therapy. Absolute changes in PINP were significantly greater for post-RIS than post-ALN groups during Months 1–5 (Figure 2). Comparable results were observed for BAP, OC, CTX and NTX (Figure 2). Results were consistent regardless of duration of prior therapy or baseline BTMs. TPTD was well-tolerated with a similar incidence of adverse events between groups.

In response to treatment with TPTD, subjects previously treated with RIS showed greater increases in BTMs compared with those previously treated with ALN. The increase in the resorption markers occurred as early as the increase in formation markers, but to a lesser extent. Our findings suggest differences between these bisphosphonates in which RIS allows the skeleton to be more responsive to the anabolic properties of TPTD.

Disclosures: P.Delmas, Alliance for Better Bone Health 2, 5, 8.

This study received funding from: The Alliance for Better Bone Health.

1093

Teriparatide Reduces Bone Microdamage Accumulation in Postmenopausal Women Previously Treated with Alendronate.. H. Dobnig¹, J. J. Stepan², D. B. Burr³, A. F. Fahrleitner-Pammer¹, J. Li³, A. Sipos*⁴, M. Sato*⁴, I. Pavo⁴¹Endocrinology and Nuclear Medicine, Medical University of Graz, Graz, Austria, ²Institute of Rheumatology, Charles University Faculty of Medicine, Prague, Czech Republic, ³Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN, USA, ⁴Lilly Research Laboratories, Indianapolis, IN, USA.

Suppression of bone turnover by bisphosphonates is associated with increased bone microdamage accumulation in animal models. It has been demonstrated at the baseline analysis of the present study, that after adjustment for potential confounders, crack density (Cr.D) was elevated in alendronate (ALN) treated patients compared with treatment naïve (TN) patients. In addition, an association of microdamage accumulation with low bone mineral density was only present in ALN treated patients. To assess effects of bone formation on bone quality, our objective was to investigate the change in microdamage accumulation in iliac crest biopsies by teriparatide (TPTD) in these previously TN patients and in those switched from ALN to TPTD. Sixty-six postmenopausal women with osteoporosis (mean age of 68.0 years and mean BMD T-score of −1.7 at total hip and −2.8 at lumbar spine; 62% with prevalent fractures) entered this prospective, non-randomized study and started with 24-month 20 μg/day subcutaneous TPTD treatment in monotherapy: thirty-eight stopped previous ALN treatment (10 mg/day or 70 mg/week for a mean duration of 63.6 months) and switched to TPTD while twenty-eight were treatment naive (TN) previously. Paired biopsies with two intact cortices were collected and analyzed for microdamage accumulation at baseline and after 24-month TPTD administration in 13 TN and 18 ALN treated patients. A within-group t-test comparing the mean percent change was carried out on log-transformed data. Following 24 months of teriparatide treatment, Cr.D, crack surface density (Cr.S.D) and crack length (Cr.Le) were decreased in previously ALN treated patients while only Cr.Le was reduced in former TN patients (Table). The changes in microdamage accumulation parameters were not statistically different between the two groups. Our data indicate that TPTD reduces microdamage accumulation in iliac crest biopsies of patients previously treated with ALN.

Table  .  

Disclosures: H. Dobnig, Eli Lilly 8; Sanofi-Aventis 2, 8.

This study received funding from: Eli Lilly.

1094

Year 3 Effects on Bone Mass in Women using Bisphosphonates after PTH Therapy: Follow-up Results from the PaTH II Study.. T. F. Hue¹, J. P. Bilezikian², K. E. Ensrud³, S. L. Greenspan⁴, C. J. Rosen⁵, L. Palermo¹, T. F. Lang¹, D. M. Black¹¹University of California, San Francisco, San Francisco, CA, USA, ²Columbia University, New York, NY, USA, ³University of Minnesota, Minneapolis, MN, USA, ⁴University of Pittsburgh, Pittsburgh, PA, USA, ⁵Maine Center for Osteoporosis Research & Education, St. Joseph Hospital, Bangor, ME, USA.

We previously reported the results of the 2-year PaTH trial of 238 postmenopausal women comparing 1 year of therapy with either parathyroid hormone (PTH 1–84, 100 μg daily) alone, the bisphosphonate, alendronate (ALN), alone or the two in combination, followed by 1 year of ALN or placebo (Black et al., NEJM, 2004; NEJM, 2005). At 24 months, 1 year of PTH followed by 1 year of ALN resulted in the largest increases in aerial bone mineral density (BMD) at the spine and hip, by dual-energy x-ray absorptiometry (DXA). This group also showed the greatest BMD increase in trabecular spine BMD by quantitative computed tomography (QCT).

PaTH II was a 1 -year observational study designed for a 3^rd year of follow-up in women originally randomized into PaTH (from 4 sites: ME, MN, NY, and PA) to assess if BMD continues to increase or is maintained in those treated with PTH followed by a bisphosphonate. After completion of PaTH, 60% (n=109) of the 182 women in follow-up were prescribed a bisphosphonate (ALN or residronate). In addition, 90% of the 182 remained on calcium/vitamin D supplements. Results are presented for the 109 participants who used a bisphosphonate (BIS) during year 3 (months 25–36).

Women who used Bisphosphonates in year 3 (n=109): BMD Mean Percent Changes by DXA and QCT† 

Table  .  

* Mean change within group, p-value < 0.05 † QCT scans were not measured in participants at the New York site. PaTH (months 0–24), n=95; PaTH II (months 25–36), n=76.

The results suggest that BMD increases were maintained with BIS use in year 3 (for groups B & C), after 1 year of PTH followed by 1 year of BIS or the two in combination, followed by 1 year of BIS. However, women (in group A) who received PTH for 1 year, had no treatment the following year, and then took BIS in year 3, lost approximately 50% of their previous gain (from months 0–24) in trabecular spine BMD. Group A also had a loss of all gains in trabecular hip BMD and reverted to below the mean baseline level. We conclude that treatment of up to 2 years with bisphosphonate, immediately following 1 year of daily PTH, appears to maintain bone mass gains from the initial anabolic therapy in postmenopausal osteoporotic women.

Disclosures: T.F. Hue, None.

This study received funding from: NIAMS-NIH and NPS Pharmaceuticals.

1095

Teriparatide Improves Bone Microarchitecture in Postmenopausal Women Previously Treated with Alendronate.. J. Li¹, D. B. Burr¹, J. J. Stepan², H. Dobnig³, A. Fahrleitner-Pammer³, A. Sipos⁴, T. Mullarney*⁴, M. Westmore⁴, M. Sato⁴, I. Pavo⁴¹Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN, USA, ²Institute of Rheumatology, Charles University Faculty of Medicine, Prague, Czech Republic, ³Department of Internal Medicine, Medical University of Graz, Graz, Austria, ⁴Lilly Research Laboratories, Indianapolis, IN, USA.

Teriparatide stimulates mineral apposition resulting in increased trabecular bone volume and cortical thickness in postmenopausal women with osteoporosis who were not treated previously with antiresorptive agents. Patients previously on alendronate (ALN) may have a delayed response to teriparatide (TPTD) as measured by an increase in serum levels of biochemical markers of bone formation activity and bone mineral density (BMD). Our aim was to investigate the effect of teriparatide on bone microarchitecture in patients after long-term ALN treatment. Sixty-six postmenopausal women with osteoporosis (mean age of 68.0 years and mean BMD T-score of −1.7 at total hip and −2.8 at lumbar spine; 62% with prevalent fractures) entered a prospective, non-randomized study. Thirty-eight had been switched from previous ALN treatment (10 mg/day or 70 mg/week for a mean duration of 63.6 months) while twenty-eight were treatment naive (TN). All patients were subsequently treated with TPTD (20 μg/day, subcutaneously) as monotherapy for 24 months. Iliac crest bone biopsies were collected at baseline and endpoint and were analyzed by two-dimensional (2D) histomorphometry (N=35) and three-dimensional (3D) microcomputed tomography (μCT, N=45). Mean activation frequency, an indicator of bone turnover was increased from baseline in both TN and ALN treated patients (mean change, mean % change, within-group p-value: 0.165 cycles/year, 138%, p=0.002 and 0.212 cycles/year, 359%, p<0.001 for TN and ALN respectively). The more accurate μCT 3D indices demonstrated an increase in both trabecular and cortical thickness (Table). There was no difference in 3D changes between the two patient groups. Our results show that after 24 months of treatment, TPTD therapy improves bone microstructure in patients, irrespective of whether they had received prior ALN antiresorptive therapy.

Table  .  

* mean (standard deviation) # p-value from within-group t-test comparing mean percent change to zero (untransformed or log-transformed data depending on observed data distribution)

Disclosures: J. Li, Eli Lilly & Co. 2. This study received funding from: Eli Lilly and Company.

1096

Unusual Osteoclast Morphology in Teriparatide-treated Patients Who Have Been Pretreated with Alendronate.. D. W. Dempster¹, H. Zhou¹, J. W. Nieves¹, N. Barbuto*¹, M. Bostrom*², F. Cosman¹, R. Lindsay¹¹Regional Bone Center, Helen Hayes Hospital, West Haverstraw, NY, USA, ²Hospital for Special Surgery, New York, NY, USA.

It has long been established that osteoblasts signal to osteoclasts. Recent observations suggest that signaling also occurs in the opposite direction, from osteoclasts to osteoblasts (Karsdal et al, JBMR 2007). Such signaling almost certainly plays an important role in mediating the stimulatory effect of anabolic agents, such as teriparatide (TPTD) on bone formation and may also be influenced by prior treatment with antiresorptive agents such as bisphosphonates. The purpose of the present study is to define better the effects of TPTD and prior treatment with the bisphosphonate alendronate on osteoclasts. We recruited 37 women with osteoporosis (16 treatment naïve; 21 pretreated with alendronate for at least one year. The two groups were randomized to control or TPTD (20 mcg/day). Iliac crest biopsies were taken either prior to TPTD treatment (n=11), after 7.5 weeks (n=13), or after 7.5 months (n=13). Biopsies were analyzed without knowledge of treatment assignment. During the morphometric analysis, osteoclasts with unusual morphology were observed in a number of subjects. These cells were approximately twice the size of normal osteoclasts, and contained many more nuclei (up to 27 per cell profiles). They were frequently not attached to the bone surface or were separated from it by lining cells. They were often not associated with Howship's lacunae, or the lacunae were shallow. We refer to them as large, inactive osteoclasts (LI.Oc). LI.Oc were observed in only 1 of 13 subjects at 7.5 weeks but were commonly seen on cancellous, endocortical or both surfaces at 7.5 months. 

Table  .  

LI.Oc were observed in 33% of ALN treated patients prior to TPTD treatment, in 100% of patients in whom ALN was followed by TPTD, and in only 20% of Naïve patients both before or after TPTD treatment. We conclude that treatment with ALN followed by TPTD results in an increase in LI.Oc. We hypothesize that, by 7.5 months, TPTD enhances osteoclast recruitment, but the resorptive capacity of these osteoclasts is inhibited by ALN. Osteoclast nuclear number and size increase as a compensatory response to inhibition of activity. While resorptive function is inhibited, signaling to osteoblasts remains intact as bone formation is markedly increased by TPTD in ALN-treated subjects (see abstract from our group, this meeting)

Disclosures: D. W. Dempster, None.

1097

The Aging Associated Gene SIRT-1 Regulates Osteoclast Formation and Bone Mass In Vivo.. J. R. Edwards¹, K. Zainabadi*², F. Elefteriou¹, L. Connelly³, F. Yull*³, T. S. Blackwell*³, F. Alt*², L. Guarente*², G. R. Mundy¹¹Center for Bone Biology, Vanderbilt University Medical Center, Nashville, TN, USA, ²MIT, Cambridge, MA, USA, ³VUMC, Nashville, TN, USA.

Progressive bone loss is a universal accompaniment of aging and involves multiple mechanisms. As the aging process has a powerful genetic component we questioned if genes associated with aging had a direct role in controlling bone mass. We examined the role of the longevity gene Sir2 which directly regulates lifespan in S. cerevisiae, C. elegans and Drosophila, in the control of vertebrate bone remodeling. The mammalian homolog of Sir2 is the highly conserved histone deacetylase Silent Information Regulator T-l (SIRT-1). We examined the phenotype of SIRT-1 null mice using Piximus, μCT scanning, histomorphometric analysis, bone resorption and cell proliferation assays of primary bone cells from SIRT-1 null and WT mice. In addition, the effect of SIRT-1 agonists and inhibitors on normal osteoclast formation and NfkB activity were studied using a GFP-tagged NfkB-reporter (NGL) mouse model, and q-PCR used to assess SIRT-1 expression in bone extracts from the SAM P6 osteoporotic mouse model. The phenotype of these mice was striking. Skeletal tissues from 1,4 and 11 mth SIRT-1 null mice showed up to 40% decrease in BV/TV by μCT analysis (p<0.001) and 46% decrease in BMD (p<0.01) compared to WT. Histomorphometric analysis of undecalcified SIRT-1 null L3–4 vertebrae supported μCT data showing up to 44% decrease in BV/TV (p<0.001). TRAP staining demonstrated a 27% increase in osteoclast surface/bone surface compared to WT animals (p<0.05). When cells extracted from normal bone marrow were treated with M-CSF (30ng/ml), RANKL (50ng/ml) and the SIRT-1 inhibitor Nicotinamide (10mM) the number and size of VNR+, TRAP+ multinucleated cells formed and resorption of dentine slices was significantly increased. The SIRT-1 agonist Resveratrol (1uM) demonstrated a decrease in osteoclast formation (p<0.05). In agreement with pharmacological data, primary bone marrow cells isolated from SIRT-1 null mice also formed TRAP+ multinucleated cells at significantly increased rates compared with WT cells. Isolated bone marrow cells from NGL mice treated with M-CSF, RANKL and Nicotinamide showed a x10 increase in fluorescence (p<0.05). Resveratrol treatment reduced GFP expression to a level lower than that of RANKL treatment alone (p<0.05) suggesting that NfkB is a downstream target of SIRT-1. In addition, bone extracts from SAM P6 mice showed a 7.5 fold decrease in SIRT-1 expression compared to SAM Rl controls (p<0.001). This data suggests that SIRT-1 activity regulates osteoclast formation and bone mass through the release of NfkB from SIRT-1 inhibition. These findings identify a new molecular link between aging and osteoporosis.

Disclosures: J.R. Edwards, None.

1098

Functional Analysis of Bcl-xL in Osteoclasts by Osteoclast-Specific Deletion of bcl-x Gene in Mice.. M. Iwasawa¹, Y. Nagase¹, T. Miyazaki¹, T. Akiyama¹, Y. Kadono¹, M. Nakamura¹, Y. Oshima¹, T. Yasui*¹, T. Nakamura*², S. Kato*², K. Nakamura¹, S. Tanaka¹¹Department of Orthopaedic Surgery, The University of Tokyo, Tokyo, Japan, ²The Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan.

Osteoclasts undergo rapid apoptosis without trophic factors, such as macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kB ligand (RANKL). However, the molecular events implicated in these processes still remain elusive. Bcl-2 family proteins are central regulators of apoptosis, and include pro- and anti-apoptotic members. Bcl-xL is an anti-apoptotic member of the Bcl-2 family, which is generated by alternative splicing of bcl-x gene, and suppresses apoptosis in many cell types. To investigate the role of Bcl-xL in osteoclasts, we generated osteoclast-specific Bcl-xL knock-out (cKO) mice by mating bcl-x flox/flox mice with Cathepsin K-Cre mice, in which Cre recombinase gene was knocked into a single allele of the cathepsin K gene locus. Expression of Bcl-xL was specifically reduced in osteoclasts derived from cKO mice (-/-OCs) compared to those from wild-type littermates (+/+OCs), but not in osteoblasts or other tissues. Osteoclasts differentiated normally from cKO mouse bone marrow cells in the presence of RANKL and M-CSF. However, -/-OCs exhibited increased cell death and Caspase-3 activity after cytokine withdrawal. In spite of their susceptibility to apoptosis, -/-OCs showed higher bone-resorbing activity than +/+ OCs as determined by pit formation assay. These phenotypes of -/-OCs were restored by adenoviral introduction of Bcl-xL. We further analyzed the effect of Bcl-xL deficiency in osteoclast differentiation using RNA interference technique, and found that osteoclast differentiation was not affected by Bcl-xL knockdown. cKO mice were alive at birth and were obtained at predicted Mendelian frequencies. Although they grew normally with no apparent morphological abnormalities, they exhibited a decrease in trabecular bone volume after 8 weeks, and developed significant osteopenia at 1 year of age as determined by radiological (soft X-ray and bone densitometry) and histological analysis. Histomorphometric analysis of distal femur revealed that the eroded surface/bone surface (ES/BS) was significantly increased in the cKO mice, while bone formation markers were equivalent to those of wild-type mice. From these observations, we conclude that Bcl-xL deficiency in OCs leads to decrease of bone volume through increased bone resorbing activity, despite of increased apoptosis. Bcl-xL plays a pivotal role in both survival and bone-resorbing activity of osteoclasts, thus creating a novel link between anti-apoptotic molecule and skeletal homeostasis.

Disclosures: M. Iwasawa, None.

1099

ADAM8 Binds α₉β₁ to Increase OCL Formation and Function by Interacting with Pyk2 and Activating Paxillin.. G. Lu¹, K. Patrene*¹, V. Garcia-Palacios¹, J. Anderson*¹, C. Boykin*², D. Del Prete*³, J. J. Windle*², G. D. Roodman⁴¹Medicine/Hem-Onc, Univeristy of Pittsburgh, Pittsburgh, PA, USA, ²Human Genetics, Virginia Commonwealth University, Richmond, VA, USA, ³Human Anatomy & Histology, University of Bari Medical School, Bari, Italy, ⁴Medicine/Hem-Onc, Univeristy of Pittsburgh and VA Pittsburgh Healthcare System, Pittsburgh, PA, USA.

We have previously identified ADAM8 (A Disintegrin and Metalloproteinase 8) as a novel osteoclast (OCL) stimulator whose disintegrin domain mediates its stimulatory effects. Further, we identified integrin α₉β₁, which is expressed both on OCLs and their precursors, as a receptor of ADAM8. Since α₉ integrin only forms heterodimers with β₁, and β₁ knockout (KO) mice are embryonic lethal, we determined whether there are other receptors for ADAM8 by assessing osteoclastogenesis in α₉ -/- mice, as well as in human bone marrow cells infected with an α₉ integrin shRNA lentivirus. Bone marrow cells from α₉ -/- mice and human OCL precursors infected with α₉ shRNA formed decreased numbers of OCL which were small and had disrupted actin rings. When bone marrow non-adherent cells and spleen cells from 7-day-old α₉ -/- mice were infected with a full length human α₉ construct, normal numbers of OCLs formed. Further, bone marrow cells from α₉ -/- mice failed to respond to treatment with soluble ADAM8, whereas, wild type bone marrow cells formed OCLs in the presence of soluble ADAM8. These data suggest that expression of full length α₉ integrin can rescue the α₉ KO phenotype in OCL formation. To determine the mechanisms responsible for the stimulatory effects of ADAM8 on osteoclastogenesis, we used an ADAM8 disintegrin domain GST fusion protein to identify the proteins that interact with ADAM8. This GST fusion protein can stimulate osteoclast formation to the same level as the full length ADAM8. GST ADAM8 protein pull-down assays with OCL precursor lysates demonstrated that protein tyrosine kinase 2 (Pyk2) formed a complex with the ADAM8 disintegrin domain. The identity of Pyk2 in the complex was confirmed by peptide mass fingerprinting (MS/MS). Further, we found that treatment of wild type OCL cultures with ADAM8 for 5 to 10 minutes induced transient tyrosine phosphorylation (pY118) of paxillin. Paxillin phosphorylation was not detected in α₉ -/- OCL cultures. These results demonstrate that α₉ is the only receptor for ADAM8, and suggest that ADAM8/α₉β₁ increases OCL formation and function through interacting with Pyk2 and signaling through paxillin.

Disclosures: G. Lu, None.

This study received funding from: NIH.

1100

RANKL Stimulates Osteoclasts to Release the Lymphatic Growth Factor, VEGF-C, and Enhances Osteoclastic Bone Resorption Through an Autocrine Mechanism.. R. Guo, O. Zhang, Y. Lu*, E. M. Schwarz, B. F. Bovce, L. Xing. University of Rochester, Rochester, NY, USA.

Osteoclasts (OCs) are bone-resorbing cells, which mediate normal bone remodeling and pathologic bone loss, such as in rheumatoid arthritis (RA). Recent studies indicate that OCs also function as immunomodulators and secrete cytokines that contribute to inflammation and autoimmunity. To investigate if OCs produce angiogenic factors in RA, we performed microarray analysis using purified CD11b⁺/Gr-1^−/0 osteoclast precursors (OCPs) from blood and bone marrow of TNF transgenic (TNF-Tg) arthritic mice and wt littermates. Among 50 angiogenic factors examined, VEGF-C expression was significantly increased in TNF-Tg mice (6-fold vs wt cells). VEGF-C is a recently identified lymphatic growth factor, but little is known about its role in bone. Here, we examined if osteoclastogenic cytokines regulate VEGF-C production by OCs and if VEGF-C affects OC function. We cultured wt spleen cells with M-CSF for 3 days to generate OCPs, treated them with RANKL or TNF, and examined expression of VEGF members by real-time RTPCR. Both RANKL (12.1+/-0.5 fold) and TNF (3.5+/-0.2 fold) stimulated OCP expression of VEGF-C, but not of other VEGFs. RANKL also stimulated mature OCs to produce VEGF-C (40+/-1 fold). RANKL failed to induce VEGF-C expression in NF-κB p50/p52 double knockout OCPs. In an EMSA assay, RANKL promoted nuclear protein binding to a putative NF-κB binding sequence from the mouse VEGF-C promoter. To determine if VEGF-C affects OC function, we treated OCPs with VEGF-C or over-expressed VEGF-C in them by retrovirus infection and cultured them on bone slices. VEGF-C increased OC resorption pit area, but not OC numbers. To test if VEGF-C works as an autocrine factor, OCPs were treated with RANKL plus the VEGF-C specific receptor inhibitor, VEGFR3-Fc, or VEGFR-2-Fc, which inhibits other VEGFs. VEGFR3-Fc, but not VEGFR2-Fc, reduced RANKL-mediated OC pit formation (pit area (mm2)/slice: IgG 2.1 +/- 0.25; VEGFR3-Fc 0.8+/- 0.1; VEGFR2-Fc 2.1+/-0.22) and had no effect on OC numbers. Furthermore, immunostaining of joint sections from RA patients or TNF-Tg mice showed that OCs expressed high levels of VEGF-C and that lymphatic vessel numbers were increased in joints of the TNF-Tg mice. In summary, RANKL stimulates OCs and OCPs to release large amounts of VEGF-C, which enhances OC bone resorption by binding to the VEGF-C receptor on OCPs and OCs. Thus, VEGF-C is a RANKL-induced autocrine factor for OC function. Importantly, our study indicates that RANKL and OCs may directly affect lymphangiogenesis through VEGF-C signaling, which will require further investigation given the importance of the lymphatic system in inflammation and tumor metastasis.

Disclosures: R. Guo, None.

This study received funding from: NIH.

1101

TRPV4 Affects Bone Remodeling by Regulating Calcium Signaling Required for Osteoclast Activity.. R. Masuyama¹, J. Vriens*², S. Torrekens*¹, K. Moermans*¹, A. Vanden Bosch*¹, R. Bouillon¹, B. Nilius*², G. Carmeliet¹¹Laboratory of Experimental Medicine & Endocrinology, Katholieke Universiteit Leuven, Leuven, Belgium, ²Department of Cell Biology, Division of Physiology, Katholieke Universiteit Leuven, Leuven, Belgium.

Intracellular calcium [Ca²⁺]_i signaling is necessary for osteoblast and osteoclast function implying that calcium permeable channels are implicated in local bone remodeling rather than in systemic calcium homeostasis. TRPV4 is a widely expressed Ca²⁺ entry channel of the “Transient Receptor Potential” (TRP) family of cation channels. We detected TRPV4 expression in both osteoblasts and osteoclasts. To elucidate whether TRPV4-mediated [Ca²⁺]_i signaling is important for bone, TRPV4 deficient mice were investigated.

Lack of TRPV4 did neither affect systemic calcium homeostasis nor endochondral bone development. However, an increased bone mass (+60%) was observed by histomorphometry in 12-week-old TRPV4^−/− tibiae (p<0.01), which was associated by a decreased osteoclast surface (-40%) compared to WT mice (p<0.05). In addition, calcitonin receptor mRNA levels were decreased in TRPV4^−/− femora (p<0.05), consistent with decreased serum CTX levels (p<0.05). At the other hand, osteoblast function was not different between genotypes as evidenced by unaltered serum osteocalcin levels and dynamic bone histology. Also, RANKL/OPG mRNA expression in the femur was not altered. These data suggest that TRPV4 promotes osteoclastogenesis cell-autonomously. Accordingly, multinuclearity evaluated by the number of mature osteoclasts containing more than 10 nuclei was decreased in cultures of TRPV4^−/− bone marrow-derived hematopoetic cells (p<0.01) treated with RANKL and MCSF. This was associated with decreased resorption activity (-60%) in TRPV4^−/− osteoclasts compared to WT osteoclasts (p<0.05) assessed by culturing osteoclasts on apatite-coated disks. In these polarized osteoclasts on apatite disks, TRPV4 localized at the apical membrane as shown by vertical reconstruction of confocal microscope scanning images. Furthermore, the TRPV4-specific agonist, 4α-PDD, evoked [Ca²⁺]_i influx specifically in large mature WT osteoclasts, but not in small WT osteoclasts containing less than 5 nuclei or TRPV4^−/− osteoclasts. Noteworthy, TRPV4 inactivation resulted in lower basal [Ca²⁺]_i in osteoclasts measured at 37°C. In addition, treatment with TRPV4 agonists induced NFATc1 mRNA expression accompanied by increased calcitonin receptor mRNA expression in cultured WT osteoclasts.

In summary, our findings provide evidence that TRPV4 maintains [Ca²⁺]_i homeostasis, osteoclast maturation and function, and therefore contributes to normal bone remodeling.

Disclosures: R. Masuyama, None.

1102

Cell-cell Fusion of Osteoclasts Is Stimulated in DC-STAMP Transgenic Mice.. R. Iwasaki*¹, T. Miyamoto², H. Kawana*³, T. Nakagawa*³, T. Suda*⁴¹Department of Cell Differentiation, Dentistry and Oral Surgery, Keio University School of Medicine, Tokyo, Japan, ²Department of Cell Differentiation, Musuculoskeletal Reconstruction and Regeneration Surgery, Keio University School of Medicine, Tokyo, Japan, ³Department Dentistry and Oral Surgery, Keio University School of Medicine, Tokyo, Japan, ⁴Department of Cell Differentiation, Keio University School of Medicine, Tokyo, Japan.

Osteoclasts are multinuclear giant cells derived from hematopoietic stem cells in the presence of M-CSF and RANKL. The multinucleation of osteoclasts is induced by a cell-cell fusion of mononuclear osteoclasts. Recently, we have isolated DC-STAMP (Dendritic Cell Specific Transmenbrane Protein), a seven transmembrane protein, as an essential molecule for osteoclast cell-cell fusion. Osteoclasts in DC-STAMP deficient mice show complete lack of cell-cell fusion in osteoclasts (J. Exp Med, 2005). Since the osteoclast differentiation molecules such as TRAP and CathepsinK, and the transcriptional factors such as c-fos and NFATc1 were not suppressed in DC-STAMP deficient compared with wild-type osteoclasts, DC-STAMP is specifically required for osteoclast cell-cell fusion. However, it is not yet characterized the mechanisms of cell-cell fusion via DC-STAMP. Here we generated DC-STAMP transgenic mice (Tg) under the control of an actin(CAG) promoter to express DC-STAMP ubiquitously in vivo. 95 lines of offspring were generated by the embryo microinjection, and the presence of the transgene in the offsprings was tested by PCR of tail DNA using transgene specific primers. 10 transgenic founder lines were established, and DC-STAMP expression was detected in various tissues of DC-STAMP Tg mice such as liver, muscle and brain, all of which do not express DC-STAMP physiologically. Interestingly, ectopic cell-cell fusion was not observed in liver and muscle, and the multinucleation of myotube was not stimulated by the forced expression of DC-STAMP in Tg mice.

Indeed defects of osteoclast cell-cell fusion in DC-STAMP deficient mice was rescued by crossing with DC-STAMP Tg mice in vivo and in vitro. In vitro osteoclast formation assay, the number of multinuclear cells as well as the number of nuclei in each multinuclear cell were significantly upregulated in osteoclasts of Tg mice compared with that of wild-type littermates. The cell-cell fusion was not induced even in the cells derived from Tg bone marrow in the presence of M-CSF alone, while the addition of RANKL in turn induced hyper-multinucleation of osteoclasts. Taken together our results suggest that DC-STAMP promotes cell-cell fusion in a tissue specific manner, and DC-STAMP induces a cell-cell fusion downstream of RANKL-RANK cascade in osteoclasts.

Disclosures: R. Iwasaki, None.

1103

Characterization of Osteoclast Inhibitory Peptide-1 (OIP-1/hSca) Binding to Fc Gamma Receptor II B (FcRγ) on Osteoclast Precursor Cells.. S. Shanmugarajan¹, C. Beeson*², S. V. Reddy¹¹Charles P. Darby Children's Research Institute, Charleston, SC, USA, ²Pharamaceutical Sciences, Medical University of South Carolina, Charleston, SC, USA.

We have previously identified and characterized the osteoclast inhibitory peptide-1 (OIP-1/hSca) as an autocrine/paracrine inhibitor of osteoclast differentiation. OIP-1 is also known as RIG-E or TSA-1, a member of Ly-6 gene family. We have recently demonstrated that mice targeted with the OIP-1/hSca expression to the osteoclast lineage develop osteopetrosis bone phenotype. We have also shown that OIP-1 c-peptide region is critical for osteoclast (OCL) inhibitory activity; however a cognate receptor/membrane protein which interacts with OIP-1 in osteoclast precursor cells is unknown. Evidence suggests a functional physical association between TSA-1 and Fc gamma receptor II B (FcRγ) on the surface of activated B-cells. Immunoreceptor tyrosine-based activation motif (ITAM) bearing adapter proteins such as FcRγ and DAP12 play a critical role in OCL development. We therefore, hypothesized that OIP-1 binding to FcRγ on osteoclast precursor cells inhibit OCL differentiation. We examined binding of the OIP-1 c-peptide to RAW 264.7 osteoclast progenitor cells using FACS analysis. Fluorescein conjugated OIP-1 c-peptide (10 uM) binds to these cells indicating the presence of a surface receptor or membrane protein partner in these cells. Co-immune precipitation and subsequent mass spectrometric analysis identified OIP-1 associated to FcRγ expressed in RAW264.7 cells. Confocal microscopy analysis demonstrated co-localization of fluorescein conjugated OIP-1 c-peptide with FcRγ expressed on the cell membrane in osteoclasts formed in RAW 264.7 and OIP-1 mouse bone marrow cultures. An ELISA binding assay confirmed that the OIP-1 c-peptide forms a 1:1 complex with recombinant FcRγ protein characterized by an equilibrium dissociation constant of K_d = 5 ± 1 uM. We further examined if OIP-1 signals through FcRγ to inhibit OCL differentiation. siRNA suppression of FcRγ expression in RAW 264.7 cells rescues OIP-1 c-peptide inhibition of RANKL stimulated OCL differentiation in vitro. We further show that OIP-1 c-peptide (100 ng/ml) treatment of RAW 264.7 and OIP-1 transgenic mouse bone marrow derived preosteoclast cells suppresses (6-fold) RANKL induced ITAM phosphorylation. Taken together, our results suggest OIP-1 binding to FcRγ in preosteoclasts inhibits osteoclast differentiation through suppression of ITAM phosphorylation. Thus, OIP-1 is an important physiologic regulator of osteoclast development and may have therapeutic utility for bone diseases with high bone turnover such as osteoporosis and Paget's disease of bone.

Disclosures: S. Shanmugarajan, None.

This study received funding from: NIH.

1104

Calcium and Phospholipase Cγ2 Signaling Regulates RANKL-induced Osteoclastogenesis.. Y. Chen¹, X. Wang*², L. Di*³, G. Fu*³, Y. Chen*³, L. Bai*³, J. Liu¹, H. Liu*¹, L. Ma*¹, X. Feng¹, Y. He*³, R. Wen*³, J. M. McDonald⁴, D. Wang*³, H. Wu*²¹Pathology, MCP, University of Alabama at Birmingham, Birmingham, AL, USA, ²Pediatric Dentistry, University of Alabama at Birmingham, Birmingham, AL, USA, ³Blood Research Institute of Wisconsin, Milwaukee, WI, USA, ⁴Pathology and VA medical center, University of Alabama at Birmingham, Birmingham, AL, USA.

Activation of calcium and phospolipase γ (PLCγ) signaling has been demonstrated in osteoclastogenesis stimulated by the receptor activator of NF B ligand (RANKL). Inhibition of PLCγ blocks RANKL-induced Ca²⁺ release from intracellular stores. PLCγ1 is ubiquitously expressed, whereas its highly homologous PLCγ2 is primarily expressed in hematopoietic cell lineages, including osteoclasts. With the use of the PLCγ2 knock out (PLCγ2^−/−) mice, we investigated the role of PLCγ2 on bone homeostasis in vivo and RANKL-induced osteoclastogenesis in vitro.

Bone morphology of mice was characterized by dual-energy X-ray absorptiometry, MicroCT and histomorphometry analyses. Increased bone mineral density was demonstrated in PLCγ2^−/− relative to wild-type mice. Quantitative measurements of the ratio of trabecular bone volume to tissue volume showed a 2-fold increase in PLCγ2^−/− mice. Bone perimeter and trabecular numbers were increased and trabecular space was decreased in PLCγ2^−/− mice. The increased bone volume was associated with a decreased number of osteoclasts and decreased relative osteoclast surface. Moreover, upon stimulation by RANKL in vitro, the PLCγ2^−/− bone marrow macrophage precursors showed increased expression of early osteoclast markers tartrate-resistant acid phosphatase (TRAP) and Cathepsin K (Cath K), but failed to differentiate into mature multinucleated osteoclasts with expression of the later osteoclast marker calcitonin receptor (CTR). Moreover, lack of PLCγ2 severely impaired RANKL-induced activation of NF-κB, AP-1, and NFATc1, the transcription factors involved in osteoclastogenesis. Tharpsigargin that is known to increase calcium induced the expression of TRAP, CathK and CTR, and partially restored the RANKL-induced osteoclastogenesis, indicating that PLCγ related calcium signaling is critical for the late stages of RANKL-induced osteoclastogenesis. Further, a retrovirus-mediated gene transduction of PLCγ2 but not PLCγ1 restored RANKL-induced osteoclastogenesis of PLCγ2^−/− cells. Therefore, PLCγ2 plays an important and unique role in RANKL-mediated osteoclastogenesis.

Taken together, PLCγ2 is essential for RANKL signaling, and its deficiency leads to defective osteoclastogenesis. Characterization of the role of PLCγ2 in RANKL-induced osteoclastogenesis is important for osteoporosis prevention and therapy.

Disclosures: Y. Chen, None.

1105

Nell-1, a Key Functional Mediator of Runx2, Partially Rescues Craniofacial Defects in Runx2 Haploinsufficient Mice.. X. Zhang¹, K. Ting¹, C. M. Cowan*², H. Lee*¹, J. Wong*¹, T. Hsu*¹, D. Pathmanathan*¹, S. Kuroda*³, C. Soo*⁴¹Dental&Craniofacial Research Institute, University of California, Los Angeles, Los Angeles, CA, USA, ²Department of Bioengineering, University of California, Los Angeles, Los Angeles, CA, USA, ³Department of Structural Molecular Biology, Institute of Scientific and Industrial Research, Osaka University, Osaka, Japan, ⁴Department of Plastic and Reconstructive Surgery, University of Southern California, Los Angeles, CA, USA.

Mesenchymal stem cell commitment to an osteochondroprogenitor lineage requires Runx2. Heterozygous Runx2 mice (Runx2^+/−) manifest delayed membranous bone development/ossification, causing open anterior and posterior fontanelles, a phenotype similar to cleidocranial dysplasia (CCD) in humans. We have recently demonstrated that craniosynostosis associated protein, Nell-1, is a direct transcriptional target of Runx2. The purpose of this study was to investigate the functional relationship of Nell-1 and Runx2 in craniofacial bone development via in vitro, ex vivo and in vivo experiments. To determine if Nell-1 could functionally compensate for Runx2 absence, Runx2^−/− calvarial cells were infected with adenoviral (Ad) Nell-1 (AdNell-1). To determine if Nell-1 was required for Runx2 activity, Nell-1 siRNA were applied on wild-type calvarial cells infected with AdRunx2. Osteoblastic differentiation was assessed by real-time PCR, ALP activity, and von Kossa. To determine if Nell-1 could stimulate mineralization on explanted Runx2^+/− mice calvaria, in situ calcein deposition was monitored after recombinant Nell-1 (rNell-1) application. Lastly, to determine if Nell-1 could compensate for Runx2 haploinsufficiency, Nell-1 overexpression mice (Nell-1^overexp) were mated to Runx2^+/− mice and the progenies were evaluated by skeletal staining, micro-CT, histology, and immunohistochemistry. The results show that Nell-1 induced delayed Alp and Opn upregulation, but no mineralization in Runx2^+/− calvarial cells. Meanwhile, Nell-1 siRNA inhibited Alp and Ocn upregulation, ALP activity, and mineralization in AdRunx2 infected wild-type calvarial cells. Furthermore, rNell-1 significantly increased ossification in calvarial explants from Runx2^+/− mice. Finally, increased calvarial bone growth as evidenced by smaller anterior fontanelle, narrower sagittal suture width and significantly thicker parietal bone plate were apparent in the Nell-1 ^overexp/Runx2^+/− progenies. In conclusion, Nell-1 serves as a key functional mediator of downstream Runx2 effects in supporting full preosteoblastic calvarial cell differentiation and inducing craniofacial bone development.

Disclosures: X. Zhang, co-founder of Bone Biologics, Inc. 4.

This study received funding from: NIH/NIDCR, March of Domes Birth Defect Foundation, Thomas R. Bales Endowed Chair.

1106

Role of BMP2 in Postnatal Bone Biology: Conditional Knock-Out (cKO) of BMP2 Using the 3.6 Collagen Type IA1 - Cre Model., W. Yang¹, D. Guo², J. Gluhak¹, M. A. Harris¹, A. Lichtler³, B. Kream*³, J. Edwards⁴, G. R. Mundy⁴, Y. Mishina⁵, S. E. Harris¹¹UTHSCSA, San Antonio, TX, USA, ²UMKC, Kansas City, MO, USA, ³UCONN Health Center, Farmington, CT, USA, ⁴Vanderbilt U, Nashville, TN, USA, ⁵LRDT, NIEHS/NIH, Research Triangle Park, NC, USA.

BMP2 is expressed in early and late osteoblasts and has been shown to accelerate bone regeneration in adults. Genetic data have demonstrated an association of polymorphisms in the BMP2 gene with increased fracture risk and osteoporosis. However, the postnatal function and mechanism of endogenous BMP2 in bone formation has not been established due to the embryonic lethality of BMP2-null mice at 10.5dpp. Thus to define the function of BMP2 in skeleton postnatally, we created a BMP2 floxed mouse model with loxP sites flanking exon 3. Mice embryos with the BMP2 floxed homozygous allele and with the Mox2-Cre allele, which is active at the blastula stage, have the same phenotype as the global BMP2-null model, lethal around 10.5 dpc along with the absence of chorion and defective heart development. This BMP2 floxed model now allows us to remove BMP2 spatiotemporally with various Cre-Loxp recombination models. With 3.6ColIa1-Cre recombinase that target early collagen type 1 expressing osteoblasts, BMP2 expression is not detected in osteoblasts and osteocytes in the BMP2 cKO mice. Removal of BMP2 from osteoblasts postnatally results in skeletal defects in vertebrae, ribs, craniofacial bones, and long bones. BMP2 cKO mice show a pronounced osteopenia with reduced radio-opacity and a 12–25% reduced BMD in bones from 2 weeks to 6 months of age. The vertebrae and humeri are revealed to be the most sensitive bones to the removal of BMP2 in osteoblasts. Quantitation μCT analysis on tibia of 1-month BMP2 cKOs has shown a 33% reduction in the vBV/TV and a 24% reduction in the trabecular number. Furthermore by histology evaluation, we observed the reduction of the trabecular bone and decreased amounts of mineralized bone matrix. Reduced osteoclast activity and number were also detected in BMP2 cKO mice at 1 month. This suggests that deletion of BMP2 in osteoblasts is linked to disruption of bone remodeling. Deletion of BMP2 and a 30% reduction of Osterix expression were also determined in bone extracts from calvaria, humeri, and femurs by Northern analysis. By in situ hybridization, we detected reduced mRNA expression levels of BMP4, Osterix and DLX5 in bone associated osteoblasts. Further investigation on dynamic bone formation rates and detailed histomorphometric analysis is underway. Collectively, our results suggest BMP2 action is upstream of BMP4 in these collagen producing osteoblasts and BMP2 is necessary but not sufficient for the continued differentiation of osteoblasts to stages required for production of mineralized bone postnatally.

Disclosures: W. Yang, None.

This study received funding from: NIH.

1107

Targeted Disruption of Ephrin B1 in Osteoblasts Reduces Bone Size in Mice.. W. Xing, K. Govoni, A. Kapoor*, S. Mohan. JL Pettis VA Medical Center and Loma Linda Univ, Loma Linda, CA, USA.

Mutations of Ephrin B1 (Efnb1), a ligand for Ephrin B tyrosine kinase receptors, in humans caused craniofrontonasal syndrome while deletion of Efnb1 gene in mice resulted in perinatal lethality and defects in skeletal patterning. Based on these findings that Efnb1 is required for survival of mice and bone development, we hypothesized that Efnb1 produced by osteoblasts plays a key role in the attainment of peak bone mass. To test this hypothesis, we used a Cre-loxP approach to disrupt Efnb1 specifically in osteoblasts (Obs). Transgenic mice expressing Cre recombinase under the control of the promoter/enhancer unit of Collα2 gene were crossed with Efnb1 loxP mice to generate Cre⁺ (Efnb1 disruption) and Cre- (control) loxP homozygous mice. Cre expression was limited to type I collagen producing cells of mesenchymal origin. Western blot confirmed that Efnb1 was expressed in Obs isolated from calvariae of Cre- mice, but not Cre⁺ mice. The skeletal phenotypes of mice (n = 10 to 19) were characterized by PIXImus measurements at 3 & 8 weeks of age as shown below. An asterisk indicates a significant difference between Cre⁺ and Cre- mice (P < 0.05).

pQCT analysis of femurs at 8 weeks of age revealed that periosteal and endosteal circumferences were reduced by 22 and 16%, respectively, in the femurs of Cre⁺ mice vs. corresponding littermate control mice (P < 0.01). In contrast, total volumetric BMD was not affected indicating that the reduction in aBMD by PIXImus was caused by reduced bone size. Because ephrin ligands are known to regulate tissue renewal and tumor cell progression, we tested the prediction that Efnb1 regulates bone size by influencing cell proliferation. Accordingly, overexpression of Efnb1 in MC3T3-E1 cells increased cell number by 32% compared to GFP control (P < 0.05). In summary: 1) Targeted disruption of ephb1 in Obs results in decreased peak bone mass caused predominantly by reduced bone size; 2) Overexpression of Efnb1 in Obs increased cell number. In conclusion, our data provide the first experimental evidence that ephb1 is a critical regulator of bone size in mice and may be an important regulator of osteoblast proliferation.

Disclosures: W. Xing, None.

This study received funding from: US Army DAMD17–03–2–0021.

1108

mTOR Signaling: A Novel Molecular Mechanism Underlying Wnt's Anabolic Effects on Osteogenesis., H. Ouyang¹, K. Inoki*², X. Zhang*¹, T. Zhu*², C. Bennett*³, C. Lindvall⁴, B. O. Williams⁴, O. A. MacDougald³, K. Guan*²¹Cariology, Restorative Sciences and Endodontics (CRSE), University of Michigan, Ann Arbor, MI, USA, ²Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA, ³Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI, USA, ⁴Laboratory of Cell Signaling and Carcinogenesis, Van Andel Research Institute, Grand Rapids, MI, USA.

The canonical Wnt signaling plays a critical role in regulating osteogenesis. It inhibits GSK3, resulting in stabilization of β-catenin, a transcription factor important for cell growth-related gene expression. Little knowledge exists as to whether Wnt signaling also regulates protein synthesis, in addition to its role in gene transcription. The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that controls cell growth and protein translation via phosphorylating the molecular components of translation apparatus, such as S6K, S6 and 4EBP. TSC2 is an upstream inhibitor for mTOR; mutation in TSC2 causes tuberous sclerosis complex, an autosomal dominant genetic disorder featured by hamartoma formation in multiple tissues. Here, we reported that Wnt signaling stimulated the mTOR signaling in osteoblasts, both in vivo and in vitro. Wnt3a increased phosphorylation of S6K and S6 in a time- and dose-dependent manner in osteoblasts. Wnt10b stable expression in mouse bone marrow stromal cells also activated mTOR. Consistently, Wnt10b transgenic mice displayed greater mTOR activities than the wild type animals, in vivo. These inductions occurred in a β-catenin-independent manner. Overexpression of a constitutively active form of β-catenin failed to induce mTOR activities, and Wnt3a stimulated mTOR signaling in MEF cells null for β-catenin. Moreover, GSK3 phosphorylated TSC2 in a manner dependent on AMPK, a serine/threonine kinase that acts as a sensor for intracellular energy level. Such phosphorylation enhanced TSC2 inhibition on mTOR. Wnt stimulated the mTOR signaling via inhibiting GSK3 phosphorylation of TSC2. Importantly, inhibition of mTOR by rapamycin, a specific inhibitor for mTOR, blocked Wnt signaling-induced osteoblastic proliferation and biomineralization, in vitro. Taken together, these results show that Wnt stimulates protein translation machinery and cell growth by activating mTOR signaling. Furthermore, they reveal a role of TSC2/mTOR signaling in Wnt-induced osteogenesis, therefore suggesting a therapeutic value of rapamycin in treating osteosclerosing dysplasia associated with activated Wnt signaling. (This work is supported by grants from NIH (Guan, KL, Ouyang, HJ), and American Association of Endodontists (Ouyang, HJ).)

Disclosures: H. Ouyang, None.

This study received funding from: NIH, American Association of Endodontists.

1109

IGF-I Signaling Is Required for Postnatal Growth Plate Development., Y. Wang¹, H. Z. ElAlieh*¹, E. Nakamura*², M. Nguyen*², S. Mackem*², D. D. Bikle¹, W. Chang¹¹Endocrine Unit, University of California, San Francisco/VA Medical Center, San Francisco, CA, USA, ²Laboratory of Pathology, Center for Cancer Research, NCI, NIH, Bethesda, MD, USA.

Studies of mice with global knockouts genes encoding the insulin-like growth factor-I (IGF-I) and IGF-I receptor (IGF-IR) support a critical role for IGF-I signaling in skeletal development. These mice are born dwarf with severely deformed skeletons and with a high perinatal mortality rate. It is assumed that the lack of local IGF-I/IGF-IR signaling is the main cause for much of the skeletal phenotype in these mice, but systemic derangements are likely to contribute as well. To investigate the role of IGF-I signaling in postnatal GP development, we generated mice (^TamCarIGF-IR^−/−) with tamoxifen (Tam)-inducible, cartilage-specific knockout of IGF-IR gene by breeding floxed IGF-IR mice with mice carrying a cDNA encoding Cre recombinase fused to a mutated estrogen responsive element. The transcription of this cDNA is controlled by a type II collagen promoter. ^TamCarIGF-IR^−/− mice were viable and fertile without injection of tamoxifen. To knockout the IGF-IR postnatally, we injected the ^TamCarIGF-IR^−/− mice with tamoxifen (0.2 mg/mouse) at day 5, day 7, day 9 and day 11 after birth. Bones from the ^TamCarIGF-IR^−/− mice and their wild-type littermates (WT) were harvested at day 14 for analysis. Tamoxifen-injected ^TamCarIGF-IR^−/− mice were smaller in size (70% of WT) at day 14. Histology showed disorganized proliferating zone, shortened hypertrophic zone (62% of WT), and reduced proliferation (by 60%) in the proliferating zone in tibial GPs of the ^TamCarIGF-IR^−/−. The protein levels of chondrocyte differentiation markers type II collagen and type × collagen, and the levels of chondrocyte differentiation regulator Indian Hedgehog (Ihh) were also decreased in these mice as assessed by immunohistochemistry. Our data indicate that IGF-I/IGF-IR signaling pathway regulates postnatal GP development by stimulating chondrocytes proliferation and differentiation, support the idea that local IGF-I/IGF-IR signaling is essential for the growth and maturation of chondrocytes in the postnatal periods, and is required for the orderly progression of GP maturation.

Disclosures: Y. Wang, None.

1110

Beta Blockade Mitigates Bone Loss During Energy Restriction Oartially via Leptin.. K. Baek*, J. Stallone*, S. Bloomfield. Department of Health & Kinesiology and Department of Nutrition, Texas A&M University, College Station, TX, USA.

Leptin is a candidate for responsible for linking energy metabolism to bone mass. In previous work, we demonstrated that energy restriction (ER) is a major contributor to the bone loss during global food restriction. Also, we observed decreased serum leptin during mild food restriction, which may be due to reduced adipocyte number/size and/or sympathetic nervous system (SNS) activation of beta-adrenoreceptors during ER, inhibiting release of leptin from adipocytes. In the present study, we tested whether beta-adrenoreceptor blockade attenuates bone loss during energy restriction and whether such an effect is associated with changes in serum leptin level and leptin localization in bone tissues. Female, 4-mo-old Sprague-Dawley rats were acclimatized to new AIN-93M purified diet for 8 weeks, then assigned into four groups (n=10 each): 2 groups of 40% energy restriction treated with vehicle (ERVEH; saline) or beta-blocker (ERβB; DL-propranolol, Sigma; 250μg/kg*hr) via drinking water during 12 weeks, and 2 groups of ad-lib fed controls treated with the same 2 agents (CONVEH, CCβB, respectively). On days 0 and 84, peripheral computed tomography (pQCT) assessed proximal tibial volumetric bone mineral density (vBMD) and geometry, and dual energy X-ray absorptiometry (DXA) assessed total body fat, lean mass, and total body and spine BMD. Sagittal sections of proximal femur were analyzed by standard immunohistochemistry for assessing leptin expression in marrow adipocytes and leptin localization in bone cells. All animal procedures were approved by Texas A&M University Laboratory Animal Care Committee. Over 84 days, CONVEH and CCβB rats gained, but ERVEH and ERβB rats lost body fat mass (+ 4.8%, + 10.08% vs −14.4% and −11.9%, respectively), but lean mass did not change in any group. Reduction in serum leptin (by ELISA) in ERVEH rats was mitigated in ERβBrats (-5.32 vs −1.15ng/ml, respectively). The decline in proximal tibia cancellous vBMD observed in ERVEH rats was attenuated in ERβB rats (-85.24 vs −53.94 mg/cm³ respectively). Spine BMD (by DXA) did not change in any group over 84 days. More leptin expression was observed in bone marrow adipocytes in groups showing higher serum leptin level and higher cancellous vBMD. In the same groups, more bone lining cells, osteocytes and chondrocytes in cartilage also stained positive for leptin. In conclusion, beta blockade mitigated metaphyseal bone loss during energy restriction and also attenuated reductions in serum levels and bone tissue-specific localization of leptin. These data suggest a contributory role for beta-adrenoreceptor signaling via adipocytes in the bone response to energy restriction.

Disclosures: K. Baek, None.

This study received funding from: DOD.

1111

Collagen/Annexin V Interactions Regulate Growth Plate Chondroyte Mineralization.. H. Kim, T. Kirsch. Orthopaedics, University of Maryland School of Medicine, Baltimore, MD, USA.

Mineralization of growth plate cartilage is crucial for normal bone formation. Therefore, an understanding of the mechanisms regulating mineralization is important. We have shown that annexins (II, V and VI) form Ca²⁺ channels in growth plate chondrocytes enabling the initiation of mineralization. In addition, binding of annexin V to types II and × collagen stimulates annexin V Ca²⁺ channel activities. In this study we hypothesized that type II or type × collagen matrix stimulates mineralization of growth plate chondrocytes via binding and activating annexin V channel properties resulting in increased intracellular Ca²⁺ concentration, [Ca²⁺]_i. Increased [Ca²⁺]_i then stimulates mineralization related events. To test this hypothesis, we treated growth plate chondrocytes with vitamin C to stimulate collagen synthesis in the absence or presence of 3,4-dehydro-L-proline (inhibitor of collagen fibril formation and secretion). On the other hand we cultured growth plate chondrocytes on type I or type II collagen coated dishes in the absence or presence of an annexin V specific Ca²⁺ channel blocker (K-201). We measured [Ca²⁺]_i, expression of mineralization-related genes, alkaline phosphatase (APase) activity and the degree of mineralization. Vitamin C stimulated type II and × collagen synthesis, APase activity and mineralization of growth plate chondrocytes, whereas 3,4-dehydro-L-proline inhibited the stimulation of these events in vitamin C-treated cells. Vitamin C also led to an increase of [Ca²⁺]^l in growth plate chondrocytes, whereas 3,4-dehydro-L-proline inhibited this increase. Flow cytometric and co-immunoprecipitation experiments revealed that annexin V is cell surface exposed and interacts with types II and × collagen. Culturing growth plate chondrocytes on type I or type II collagen coated dishes increased [Ca²⁺]_i, APase activity and the degree of mineralization compared to the levels of cells cultured on non-coated dishes. K-201, a specific annexin V channel blocker, inhibited increase of [Ca²⁺]_i of growth plate chondrocytes cultured on type II collagen coated dishes but not on type I collagen coated dishes. Furthermore, overexpression of full length annexin V, which binds to types II and × collagen, was more effective in stimulating APase activity, MMP-13 expression and mineralization of growth plate chondrocytes than overexpression of N-terminus deleted mutant annexin V, which binds to type × collagen but not to type II collagen. In conclusion, our findings reveal that types II and × collagen via binding to annexin V stimulate annexin-mediated Ca²⁺ influx, thereby regulating growth plate chondrocyte Ca²⁺ homeostasis and ultimately mineralization events.

Disclosures: H. Kim, None.

This study received funding from: NIH.

1112

Runx1, Co-activator of Sox5, Sox6 and Sox9 (the Sox trio) Regulates Chondrogenic Differentiation.. F. Yano¹, T. Ikeda¹, T. Saito², N. Ogata¹, S. Takeda³, A. Kimura³, S. Ohba¹, F. Kugimiya², K. Nakamura², T. Takato¹, H. Kawaguchi², U. Chung¹¹Division of Tissue Engineering, Faculty of Medicine, University of Tokyo, Tokyo, Japan, ²Department of Sensory and Motor System Medicine, Faculty of Medicine, University of Tokyo, Tokyo, Japan, ³Department of Orthopedics, Tokyo Medical and Dental University, Tokyo, Japan.

Aiming at clinical applications to cartilage regeneration, we previously screened natural and synthetic compound libraries and found that a thienoindazole derivative small compound T-198946 (TM) strongly induced chondrogenic differentiation without inducing hypertrophy. To clarify its transcriptional targets and signal transduction mechanism, we screened for the target molecules of TM by a microarray analysis using undifferentiated mesenchymal C3H10T1/2 cells treated with TM for 48 hours. Compared to the control, 581 genes were up-regulated by TM, among which Runx1, Sox5, and Sox6 were especially markedly induced. Real-time RT-PCR analysis confirmed that expressions of Runx1, Sox5, and Sox6 were strongly induced by TM treatment. To investigate the functional contribution of Runx1 to chondrocyte differentiation, Runx1 was adenovirally overexpressed, but it did not induce endogenous expression of chondrogenic markers in C3H10T1/2 cells. However, when Runx1 was combined with Sox5, Sox6 and Sox9 (the Sox trio), it enhanced induction of chondrogenic differentiation and cartilage matrix synthesis without inducing hypertrophy, mimicking effects of TM treatment. On the other hand, silencing of Runx1 reduced effects of TM on chondrogenic differentiation. We analysed the effects of Runx1 on the human type II collagen promoter containing putative binding motif for Runx family transcriptional factors. Luciferase-reporter assay using several deletion constructs identified the region containing the binding site of Runx1 at −293∼ −288bp, which is highly conserved among species in the proximal promoter and distinct from the Sox9 responsive element. Immunoprecipitation analysis showed that Runx1 physically associated with Sox 5, 6 and 9 proteins. Immunohistochemistry revealed that Runx1, Sox5, 6, and 9 were co-localized in the proliferative and pre-hypertrophic chondrocytes of the mouse growth plate. These data suggest that Runx1 cooperatively works with the Sox trio to induce chondrogenic differentiation. Elucidation of the molecular network of TM targets may help realize to the realization of the regenerative medicine of permanent cartilage.

Disclosures: F. Yano, None.

1113

A Homozygous Missense Mutation in Human KLOTHO Causes Severe Tumoral Calcinosis, but not Premature Aging.. S. Ichikawa*¹, E. A. Imel¹, M. L. Kreiter*², X. Yu¹, D. S. Mackenzie*¹, A. H. Sorenson*¹, R. Goetz*³, M. Mohammadi*³, K. E. White¹, M. J. Econs¹¹Indiana University School of Medicine, Indianapolis, IN, USA, ²Children's Memorial Hospital and Feinberg School of Medicine, Northwestern University, Chicago, IL, USA, ³New York University School of Medicine, New York, NY, USA.

Familial tumoral calcinosis is a rare metabolic disorder characterized by ectopic calcifications and hyperphosphatemia. The known genetic causes of tumoral calcinosis are inactivating mutations in the genes encoding fibroblast growth factor 23 (FGF23) and GalNAc transferase 3 (GALNT3). Klotho (Kl)-deficient mice were originally reported to model early aging; however, these mice manifest a biochemical phenotype similar to Fgf23-null mice and tumoral calcinosis patients.

We examined a 13-year-old girl with severe tumoral calcinosis with significant dural and carotid artery calcifications. This patient had defects in mineral ion homeostasis with marked hyperphosphatemia, hypercalcemia, and elevated parathyroid hormone (PTH). Serum FGF23 concentrations were markedly elevated [16,140 pg/mL intact (NL<71 pg/mL) and 10,900 RU/mL C-terminal (NL<149 RU/mL)], suggesting resistance to the actions of FGF23 as the cause of the observed hyperphosphatemia. However, there were no features of premature aging.

Mutational analysis was performed for the calcium sensing receptor (CASR), FGF23, GALNT3, and KL. We identified a homozygous missense mutation (His193Arg) in the KL gene of the patient. Analysis of the mutation in the context of published glycosidase structures revealed that the mutated histidine 193 is at the base of the catalytic site. Substitution of the histidine residue to arginine should destabilize the tertiary folding of the KL1 domain, attenuating the production of membrane-bound and secreted KL. Indeed, compared to wild-type KL, cell surface expression and secretion of mutant KL was drastically reduced in vitro. Immunoprecipitation analysis of the FGF23-KL-FGFR1 ternary complex revealed that only minute amounts of mutant KL bound to FGF23. Furthermore, stimulation of HEK293 cells expressing wild-type KL with FGF23 resulted in a 56-fold increase in mRNA of early growth response-1 (EGR1), a downstream mediator of FGF23 signaling, whereas EGR1 expression was reduced by −80% in cells expressing mutant KL (p<0.0001).

In summary, our clinical and molecular findings demonstrate that human loss-of-function mutations in KL impair the bioactivity of FGF23 to signal via its cognate FGF receptors and lead to severe tumoral calcinosis, rather than premature aging as previously reported in the Kl-deficient mice. Thus, KL plays an essential role in FGF23-mediated phosphate and vitamin D metabolism in humans.

Disclosures: S. Ichikawa, None.

1114

Phosphate-Independent Effects of Fgf-23 on Skeletogenesis.. D. Sitara¹, C. Bergwitz², S. Kim*¹, R. Erben³, H. Jüppner², B. Lanske¹¹Developmental Biology, Harvard School of Dental Medicine, Boston, MA, USA, ²Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA, ³Natural Sciences, University of Veterinary Medicine, Vienna, Austria.

Phosphate homeostasis is regulated by hormones such as parathyroid hormone (PTH), 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) and fibroblast growth factor-23 (Fgf23). Each of these factors can modulate directly or indirectly the activity of sodium-dependent phosphate co-transporter proteins (NaPi) located in kidney and intestine, affecting both renal and intestinal phosphate handling and thereby influencing overall mineral ion homeostasis. Fgf23 ablation leads to a complex phenotype, including hyperphosphatemia, elevated 1,25(OH)₂D₃ and suppressed PTH serum levels, accompanied by enhanced renal tubular phosphate reabsorption through NaPi2a and NaPi2c, leading to abnormal bone mineralization and soft tissue calcifications.

Using genetically modified mice we have now demonstrated an inverse correlation between Fgf23 and NaPi2a; homozygous ablation of NaPi2a from Fgf23^−/− mice to generate Fgf-23^−/−/NaPi2a^−/− double mutants resulted in urinary phosphate-wasting and hypophosphatemia at 6 weeks of age but normal phosphate levels at 3 weeks, despite significantly elevated 1,25(OH)₂D₃ serum levels. This data suggest that altered phosphate homeostasis in Fgf23^−/− mice is a NaPi2a-dependent process. Histological analysis of tibial bone sections from Fgf23^−/−/NaPi2a^−/− double mutants revealed a close resemblance to the findings in Fgf23^−/− bones, i.e. hypermineralization of the primary spongiosa together with severe osteoidosis in secondary spongiosa and cortical bone. To further investigate these findings, we examined the expression of genes associated with skeletal development. In Fgf23^−/− bones, we observed an up-regulated expression of osteopontin and dental matrix protein-1 (Dmp-1), two sibling proteins known to affect bone mineralization; expression of these genes in Fgf23^−/−/NaPi2a^−/− double mutants is currently underway.

We are furthermore in the process of evaluating NaPi2c expression, which may be up-regulated to compensate for the loss of NaPi2a in the double mutants, leading to normal serum phosphate levels in Fgf23^−/−/NaPi2a^−/− double mutants at 3 weeks of age. Moreover, since PTH is known to decrease NaPi2a expression, we are studying the in vivo role of PTH in Fgf23-mediated phosphate homeostasis and skeletogenesis.

In conclusion, our current findings suggest that Fgf23 not only exhibits endocrine functions by regulating phosphate homeostasis through its actions on the kidney, but it also affects skeletogenesis through yet undefined autocrine/paracrine actions.

Disclosures: D. Sitara, None.

1115

The Role of the Type IIc Sodium-dependent Phosphate Transporter (Npt2c), which Is Involved in Hereditary Hypophosphatemic Rickets with Hypercalciuria (HHRH).. H. Segawa*¹, A. Onitsuka*¹, M. Kuwahata*¹, E. Aranami*¹, J. Furutani*¹, I. Kaneko*¹, Y. Tomoe*¹, S. Kuwahara*¹, N. Amizuka², M. Matsumoto*³, K. Miyamoto¹¹Molecular Nutrition, Health Biosciences, The Univ. of Tokushima Graduate School, Tokushima, Japan, ²Center for Transdisciplinary Research, Niigata University, Niigata, Japan, ³Molecular Immunology, Enzyme Research, University of Tokushima, Tokushima, Japan.

Primary loss of inorganic phosphate (Pi) from the kidneys results in hypophosphatemia, the severity of which is directly related to the degree of secondary bone mineralization defects. Two transporters (Npt2a and Npt2c) are expressed exclusively in the proximal convoluted tubule and are regulated by Pi intake, PTH, and FGF23 levels. The type IIa Na/Pi cotransporter (Npt2a) play a major role in reabsorption (70–80%), while type IIc Na/Pi transporter (Npt2c) is more important in weaning animals and less so in adult animals. While the renal Npt2a play a major role in renal Pi reabsorption, Npt2a null (Npt2a^−/−) mice do not exhibit rickets/osteomalacia. More recently, linkage analyses have suggested that hereditary hypophosphatemic rickets with hypercalciuria (HHRH) may arise from mutation(s) in the NPT2c/SLC34A3 gene. The goal of the present study was to characterize the role of renal Pi wasting in bone disorders by studying mice deficient in the Npt2c gene, which is the pathogenic protein involved in HHRH. Homozygous mutants (Npt2c^−/−) mice are viable, fertile and do not display any gross physical or behavioral abnormalities. Npt2c^−/− mice exhibit hypercalcemia, hypercalciuria, and elevation of plasma 1,25-dihydroxyvitamin D3 levels. Interestingly, the levels of plasma fibroblast growth factor 23 (FGF23) levels were significantly reduced in Npt2c^−/− mice. However, histologic examination of bone samples from Npt2c^−/− mice failed to identify typical features of advanced rickets. Since Npt2c KO mice exhibited abnormally phosphate/calcium homeostasis, our results support that Npt2c may be involved in phosphate/calcium homeostasis.

The further studies required to clarify the mechanism of rickets/osteomalacia which caused by Npt2c-genetic abnormality.

Disclosures: H. Segawa, None.

1116

Molecular Analyses of DMP1 Mutants Causing Autosomal Recessive Hypophosphatemic Rickets (ARHR).. E. G. Farrow¹, S. I. Davis*¹, X. Yu¹, L. M. Ward², K. E. White¹¹Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA, ²Pediatrics, Children's Hospital of Eastern Ontario, Ottawa, ON, Canada.

Heritable disorders of hypophosphatemia involving FGF23 result from mutations in several genes. We previously demonstrated that the recessive mutations M1V (A1>G) and 1484–1490 deletion (del) in Dentin Matrix Protein-1 (DMP1) cause the novel disorder autosomal recessive hypophosphatemic rickets (ARHR), associated with elevated serum FGF23. The goals of the current studies were to understand the effects of the ARHR mutations on DMP1 and to test the regulation of DMP1 expression. Western analyses of wild type DMP1 following transient transfection into cells demonstrated that DMP1 is normally expressed equally in the cellular lysates and the media. In contrast, the ARHR 1484–1490del mutant was primarily detected in the media whereas the M1V mutant was only detected in the cellular lysates. In order to investigate the ARHR mutations on DMP1 intracellular trafficking, transfected cells were fluorescently co-stained for DMP1 in parallel with staining for the trans-golgi network (TGN), ER, and nucleus. Wild type and 1484–1490del mutant DMP1 localized to the TGN, consistent with their cellular secretion. The M1V mutant did not localize to the TGN but filled the entire cell due to loss of the highly conserved DMP1 signal peptide. We obtained the same results with these constructs produced as fusion proteins with V5 tags or untagged, in HEK293, UMR106, and MC3T3 cells. The last 18 amino acids of DMP1 are highly conserved and are replaced by 33 novel residues due to a frameshift caused by the 1484–1490del mutation. We deleted the last 18 DMP1 residues to determine the role of the C-terminus in processing. This truncated DMP1 acted the same as the wild type species, and had balanced intra- and extracellular expression. Therefore, the unbalanced expression of 1484–1490del DMP1 is a function of the 33 novel residues, and not a consequence of deleting the last 18 native residues. UMR-106 cells were treated with excess phosphate or with 1,25(OH)₂ vitamin D (1×10⁻⁷M) for 4 h to test endogenous DMP1 response. qRT-PCR showed a 2–3 fold increase in DMP1 mRNA compared to control cells with both treatments. In summary, our studies of the ARHR DMP1 mutants illustrate that modifications to the signal peptide and to the C-terminus of DMP1 affect cell processing, and that DMP1 is responsive to metabolites that also control FGF23 production. The alterations in DMP1 secretion due to the ARHR mutations provide unique insight into domains of DMP1 critical for normal biological function.

Disclosures: E.G. Farrow, None.

1117

Vascular Calcification and Aortic Gene Expression Are Consistent with Hyperphosphatemia in OPG-Deficient Mice.. S. Morony¹, Z. Zhang*², Y. Tintut², P. J. Kostenuik¹, L. L. Demer²¹Amgen Inc., Thousand Oaks, CA, USA, ²Medicine, UCLA, Los Angeles, CA, USA.

Osteoprotegerin (OPG) is a cytokine that suppresses bone resorption by inhibiting the catabolic effects of receptor activator of NF-kappaB ligand (RANKL). OPG-deficient mice [opg^(−/−) are osteoporotic and exhibit medial calcification of the aorta. This vascular phenotype in opg^(−/−) mice was the first evidence suggesting a protective role for OPG in the vasculature. However, the mechanism of this effect has not been determined and could be an indirect result of high turnover, or the direct result of RANKL or OPG actions within the vasculature. We evaluated these possibilities by assessing vascular calcification, serum calcium/phosphate levels, and aortic gene expression in opg^(−/−) mice and wildtype (WT) controls.

We quantified vascular calcification in opg^(−/−) mice by analyzing aortas ex vivo, using a high-resolution digital radiographic system. Radiographs were scored semi-quantitatively (0 to 5) based on the extent of mineralization, by an investigator blinded to genotype, sex, or age of the mice. No evidence of aortic mineralization was noted in any of the 24 WT aortas; whereas all 27 aortas were calcified in opg^(−/−) mice (mean calcification score = 2.7 ± 1.2). Aortic calcium content was quantified biochemically from a subset of mice, and was elevated 12-fold in opg^(−/−) mice compared to WT controls (p < 0.05).

We found increased serum phosphate (11.5 ± 1.4 mg/dL vs. 9.6 ± 1.1 mg/dL; p < 0.01) and calcium/phosphate product (114 ± 16 mg/dL vs. 94 ± 13 mg/dL; p < 0.01), but normal serum calcium levels (9.9 ± 0.5 mg/dL vs. 9.7 ± 0.4 mg/dL; p = 0.12) in opg^(−/−) mice compared to WT controls. The hyperphosphatemia in opg^(−/−) mice is likely related to increased bone resorption, as RANKL (926 ± 397 vs. 21 ± 22 pg/ml; p < 0.01), TRAP5b (0.10 ± 0.02 vs. 0.05 ± 0.02 AU; p < 0.001), and osteopontin (1736 ± 312 vs. 374 ± 91 pg/ml; p < 0.01) levels were elevated relative to WT controls.

Quantitative PCR was used to evaluate aortic gene expression between opg^(−/−) and WT mice and no difference was noted in expression of bone/cartilage transcription factors, BMP/TGF signaling molecules, or mineral binding/matrix factors. However, consistent with hyperphosphatemia, expression of matrix GLA protein (MGP) in opg^(−/−) mice was increased 90% (p < 0.01) while mRNA for alkaline phosphatase (akp2) was decreased 94% (p < 0.02). Interestingly, mRNA for ectonucleotide pyrophosphatase phosphodiesterase 1 (enppl), which generates inorganic pyrophosphate (PPi), a mineralization inhibitor, was not increased in opg^(−/−) mice. Thus, the lack of enppl induction, together with remodeling-related hyperphosphatemia, may be important etiological factors in the development of vascular calcification in opg^(−/−) mice.

Disclosures: S. Morony, Amgen Inc. 1, 3.

This study received funding from: Amgen, Inc.

1118

Reduced Kidney-stone Formation and Altered Renal Phosphate-homeostasis in Mice Over-expressing Murine-MEPE.. A. Martin¹, V. David¹, L. W. Fisher², A. Hedge*¹, P. S. N. Rowe¹¹Internal Medicine, Kansas University Medical Center, Kansas City, KS, USA, ²NIDCR-NIH, Bethesda, MD, USA.

Transgenic-mice constitutively over-expressing recombinant murine-MEPE (ColIa1 2.3kb promoter) were used to study the role of MEPE in phosphate mineral-metabolism. Ten 19 week-old transgenic (TGC) male C57B16 mice were compared to ten age-matched wild-type (WT) male mice on a high 1% phosphate diet. Biochemical measurements were performed on sera and urine. RT-PCR, western-blots and immunohistochemistry were performed on kidneys to analyse the expression of different markers involved in phosphate-regulation. Computed 3D-microtomography (uCT) was used to assess kidney-stone formation.

Remarkably, despite a low bone-mass phenotype induced by MEPE over-expression, an increase in serum phosphate (WT: 8.1 mg/dL vs. TGC: 11.4 mg/dL, p<0.01) and a mild hypocalcemia (WT: 9.95 mg/dL vs. TGC: 8.96 mg/dL, p<0.05) occurred. Consistent with the increased serum-PO4, the fractional excretion of phosphate decreased by 66% (p<0.01) and serum PTH decreased by 33% (170 pg/mL to 120 pg/mL, p<0.05). Interestingly, despite a mild hypocalcemia, serum 1,25-(OH)2-Vit-D3 was significantly increased (18 pg/mL to 40 pg/mL, p<0.05), Also, increased renal NPT2a Na-dependent co-transporter mRNA levels (+267%, p<0.01) and protein (as measured by western-blots and immunohistochemistry) occurred in transgenic mice, again consistent with the hyperphosphatemia. In contrast transgenic-mice had significantly decreased renal NTP2b and NPT2c mRNA levels (75% and 80% reduction respectively, p<0.05). Phex expression dramatically increased by 4X (p<0.05) in TGC mice. Compared with WT, TGC mice displayed a 700% increase in MEPE expression (protein and mRNA) in the kidney (p<0.05) and a reciprocal decrease in DMP-1 expression (76% decrease, p<0.05), This in combination resulted in a decrease in kidney stones induced by the high-phosphate diet as assessed by 3D-uCT (Figure 1; 97% decrease, p<0.05).

In summary, over-expression of MEPE under a ColIa1 2.3kb promoter markedly alters renal-phosphate homeostasis and dramatically influences susceptibility to renal-calcification. Also, these data show an association between MEPE, renal sodium-phosphate co-transporters and susceptibility to kidney-stone formation.

Disclosures: A. Martin, None.

This study received funding from: R01-AR51598–01 & R03-DE015900–01.

1119

Phospholipase C Signaling via the PTH/PTHrP Receptor Is Essential for Normal Full Response to PTH in both Bone and Kidney.. J. Guo¹, M. Liu*¹, C. C. Thomas*¹, D. Yang¹, M. L. Bouxsein², E. Schipani¹, F. R. Bringhurst¹, H. M. Kronenberg¹¹Endocrine Unit, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA, ²Orthopedic Biomechanics Laboratory, Beth Israel Hospital and Harvard Medical School, Boston, MA, USA.

To study role of signaling by distinct second message systems in vivo, we have constructed “knock-in” mice with mutant PTH/PTHrP receptors (PTHR) that can activate adenyl cyclase normally but cannot activate phospholipase C (PLC). We have previously shown that hypertrophic differentiation of chondrocytes is delayed in these so-called DSEL mice. To better understand role of PLC signaling via the PTHR in bone modeling and remodeling we examined the bone phenotype in DSEL mice that were fed either a standard diet or a calcium-deficient diet. Levels of serum PTH, phosphate and total calcium in DSEL mice fed a standard diet were not significantly different from those observed in wild type (WT) mice. Histomorphometric and uCT analysis demonstrate reduced trabecular bone (decreased by 15–20%) in DSEL mice fed a standard diet, and this decrease is associated with a decrease in trabecular number and an increase in trabecular spacing. The mechanisms underlying the low bone mass is not fully understood, since histomorphometric parameters of cell number and bone formation rate are normal in the secondary spongiosa of DSEL tibiae. The decreased bone mass may reflect abnormal modeling, as suggested by indices of increased osteoclast activity in the primary spongiosa, such as increased TRAP activity and mRNAs encoding TRAP and MMP9.

Interestingly, DSEL mice fed a low calcium diet was able to develop secondary hyperparathyroidism including elevated serum PTH and substantial bone loss but exhibited a strikingly curtailed peritrabecular stromal cell response (peritrabecular bone marrow fibrosis), whereas WT mice exhibited extensive peritrabecular stromal cell responses to the low calcium diet. Such stromal cells express mRNAs encoding al(I) collagen, osteocalcin, and osteopontin, suggesting that they are preosteoblasts. In addition, the mutant mice fed low calcium diet were hyperphosphatemic, in contrast to the fall in blood phosphate in the WT control mice. The DSEL mice also exhibited an attenuated elevation of la-hydroxylase mRNA, despite dramatic elevation of serum PTH in both groups. No significant change in mRNA levels of NaPi2a and NaPi2c was observed in the mutant mice in response to low calcium diet. Levels of FGF23 fell in response to the low calcium diet in both WT and DSEL mice, but on both diets FGF23 levels were not affected by the DSEL mutation. Our findings show that in both kidney and bone, PLC signaling through the PTHR in response to PTH are needed for the normal full responses; molecular analysis of the abnormalities is in progress.

Disclosures: J. Guo, None.

This study received funding from: NIH.

1120

Deletion of the Calcitonin/CGRP Gene Causes a Profound Cortical Resorption Phenotype in Mice.. R. F. Gagel, A. O. Hoff, S. E. Huang*, G. J. Cote*. Endocrine Neoplasia & Hormonal Disorders, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA.

There has been confusion regarding the long-term biological effects of calcitonin (CT) deficiency. Prior studies describing CT/CGRP gene or CT Receptor knockout models have provided contradictory phenotypes. To address these inconsistencies we have bred the knockout model (J Clin Invest 2002; F3 generation) into a pure C57/B6 mouse background (F10 generation). In this mouse model exons 2–5 of the mouse CT/CGRP were deleted in the germline by targeted disruption. Analysis of C57/B6 CT/CGRP KO mice (F10 generation) demonstrated a striking phenotype characterized by increased trabecular remodeling, trabecular & cortical thinning, and cortical porosity. The cortical thinning leads to morphophological changes of vertebral bodies characterized by a 5% reduction of the height/width ratio (p< 0.05) in 12 and 18 month-old animals. The earliest indication of increased remodeling is the presence of a trabecular thinning in 3 & 6 month-old animals.

By 12 months of age a striking phenotype has emerged characterized by trabecular and cortical thinning (25%) with evidence of cortical tunneling (see Figure), collections of osteoclasts, and a 1.5–2-fold increase of serum C-telopeptide. The phenotype is most striking in 12 and 18 month females. Analysis of expressed genes (Affymetrix chip) derived from flushing of marrow with extraction buffer showed > 2-fold increases in CSF2Rα, CSF2Rβ2, CCR2, Flotilin 2, Vimentin and > 1.5-fold increases of Prokineticin 2 and NFATc1, all genes involved in monocyte or osteoclast differentiation.

Conclusions: CT/CGRP deficient C57/B6 F10 mice have a profound and progressive phenotype characterized by increased trabecular thinning and cortical remodeling. These mice retain the greater calcemic response to PTH described earlier. Although there is evidence of these effects in 3 & 6 month-old animals, the major histologic effects are observed in older animals, possibly accelerated by gonadal steroid deficiency. The emergence of this phenotype argues persuasively for an important role of the CT/CGRP in the prevention of age-related bone loss and regulation of trabecular remodeling. The modulation of genes important in monocyte and osteoclast differentiation in the CT/CGRP KO indicates a previously unknown regulatory function of calcitonin.

Disclosures: R.F. Gagel, None.

This study received funding from: National Institutes of Health.

1121

PTH-Regulated Gene Expression in the Mouse Skeleton Suggest a Role for Ephrins in PTH Anabolism versus Catabolism.. E. N. Bianchi*, R. Rizzoli, S. L. Ferrari. Div. of Bone Diseases, Geneva University Hospital, Geneva, Switzerland.

Intermittent PTH (i.PTH) induces a positive bone mineral balance while continuous PTH (c.PTH) does the opposite, particularly in the cortical bone compartment. The molecular mechanisms for PTH anabolism versus catabolism however remain poorly understood. Bidirectional ephrin signaling mediated by the binding of ephrinB2 ligand (EfnB2) to the ephrinB4 receptor (EphB4) was recently reported to induce osteoblast differentiation and reciprocal inhibition of osteoclast differentiation. This led us to hypothesize that ephrins expression could be up- and down-regulated by, respectively, i.PTH and c.PTH. Using gene expression profiling (Affymetrix Mouse 430A 2.0 Array) of PTH-stimulated primary osteoblasts from neonatal mouse calvariae, we first delineated a set of ephrin ligands (Efn) and receptors (Eph) that was expressed in bone cells. Then, we investigated PTH effects on skeletal gene expression of ephrins in both adult C57/B6J mice and beta-arrestin2 (Arrb2) KO mice, which lack a regulatory protein for PTH-mediated signaling. For this purpose, mice were treated with either daily (s.c.) or continuous (Alzet pump) PTH (80 μg/kg/day) or vehicle (n=4/gr) for 1 week. RNA was extracted separately from the femur cortical diaphysis (Dia) after flushing of the bone marrow and trabecular metaphysis-epiphysis (Met) region. Targeted gene expression levels of Efn B1 and B2, and Eph A3, A4, B2, B3 and B4, first identified by gene chips analysis in vitro, were analyzed by qRT-PCR (TaqMan MGB probes). Intermittent PTH up-regulated EfnB1, EphB2 and EphB3 1.4 to 1.5 fold in both trabecular and cortical bone compartments, whereas c.PTH did not. In contrast, in Arrb2 KO mice, i.PTH down-regulated the expression of EphA3 and EphA4 in Dia (-1.5x). Moreover, in these mice, c.PTH down-regulated expression of all Efn and Eph (except EphB4) in Dia (-1.4 to −2.5x.) and of EphA3, EphA4 and EphB3 in Met (-1.4 to −1.6x). Interestingly, the profile of EfnB1, EphB2 and EphB3 gene expression induced by PTH paralleled the profile of osteoprotegerin gene (OPG) expression, namely an up-regulation of OPG by i.PTH (2x in Met), but not by c.PTH, in WT mice and a down-regulation (-1.4x in Met and −1.7x in Dia) by c.PTH in KO mice. In summary PTH regulates skeletal gene expression of ephrins and their receptors, which depends on the mode of PTH administration and signaling regulation by beta-arrestins. In turn, up-regulation of ephrins/ephrins receptors, such as EphB3, and OPG by i.PTH should promote osteoblast differentiation and inhibition of osteoclastogenesis. Thereby ephrins could play a role in i.PTH anabolism on both trabecular and cortical bone compartments, while down-regulation of the ephrins system by c.PTH could favor catabolism.

Disclosures: E.N. Bianchi, None.

1122

Treatment with an Anti-Sclerostin Antibody Increased Bone Mass by Stimulating Bone Formation Without Increasing Bone Resorption in Aged Male Rats.. X. Li, K. S. Warmington*, O. Niu*, M. Grisanti, H. Tan, D. Dwyer*, M. Stolina, M. S. Ominsky, W. S. Simonet, P. J. Kostenuik, C. Paszty, H. Z. Ke. Amgen Inc., Thousand Oaks, CA, USA.

Male osteoporosis is becoming more common as the population ages, and decreased bone formation appears to be one of the characteristics of this condition. We previously reported that treatment with an anti-sclerostin antibody (Scl-Ab) stimulated bone formation, restored bone mass and bone strength in aged ovariectomized rats. We now describe the effects of Scl-Ab treatment on bone formation and bone mass in aged gonad-intact male rats. Sixteen-month-old male SD rats were treated s.c. with vehicle or Scl-Ab at 5 or 25 mg/kg, twice a week, for 5 weeks (9–10/group). In vivo bone mineral density (BMD) measurements by DXA showed that rats treated with Scl-Ab at 5 or 25 mg/kg had significantly higher BMD at lumbar vertebrae (+16%; +27%), distal femur (+15%; +20%) and femur-tibia (+9%; +11%) compared with vehicle control. Serum osteocalcin, but not serum CTX-I, was significantly increased in Scl-Ab treated rats as compared with control. Histomorphometric analysis at the proximal tibial metaphysis showed that rats treated with 5 or 25 mg/kg Scl-Ab had significantly greater trabecular bone volume (BV/TV, +133% and +166%, respectively), trabecular thickness, and a significant decrease in trabecular separation compared with vehicle controls. Scl-Ab treated rats had significantly greater osteoblast surface but not osteoclast surface compared with vehicle control. Furthermore, Scl-Ab treated rats had significantly greater mineralizing surface (MS/BS), mineral apposition rate (MAR) and bone formation rate (BFR/BS, +252% and +374% for 5 and 25 mg/kg, respectively) in trabecular bone of proximal tibia. At the tibial shaft increases in endocortical and periosteal bone formation were seen at both the 5 and 25 mg/kg doses, with greater increases seen at the endocortical surface (for 25 mg/kg dose: MS/BS +383% vs +130%, BFR/BS +852% vs +285%). In summary, inhibition of sclerostin by a Scl-Ab increased bone mass and increased bone formation on trabecular, periosteal and endocortical surfaces without evidence of increased bone resorption in aged male rats. These results suggest that anti-sclerostin antibody represents a novel anabolic therapy for age related bone loss in males.

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

This study received funding from: Amgen, Inc.

1123

Gαq Signaling in Osteoblasts Inhibits Bone Formation.. N. Ogata¹, Y. Shinoda¹, F. Yano¹, N. Wettschureck*², S. Offermanns*², G. V. Segre³, K. Nakamura¹, H. Kawaguchi¹¹Sensory and Motor System Medicine, University of Tokyo, Tokyo, Japan, ²Phalmakologisches Institut, University of Heidelberg, Heidelberg, Germany, ³Endocrine Unit, MGH, Boston, MA, USA.

Gαs/cAMP/PKA and Gαq/PLC/PKC are known to be the two major signal pathways of PTH. Although accumulated evidence has revealed the anabolic role of the Gαs signaling in bone formation, little is known about that of Gαq. To study the role of the Gαq signaling, we initially produced transgenic (Tg) mice that overexpressed the constitutively active Gαq gene exclusively in osteoblasts under the control of 2.3-kb type I collagen α1 chain (COLIA1) promoter. The Tg mice exhibited osteopenia in trabecular and cortical bones with decreased bone formation parameters. Cultured calvarial osteoblasts from the Tg mice showed impairment of differentiation and matrix formation, which was restored by addition of a PKC inhibitor GF109203X. To further explore the role of endogenous Gαq-mediated signaling in bone, we established double knockout (DKO) mice with osteoblast-specific ablation of Gαq and global ablation of Gα11, a Gαq subfamily member that also activates PLC; we did this by crossing Cre transgenic mice under the control of the COLIA1 promoter above (COLI-Cre^+/−) and mice with the Gαq gene flanked with loxP / global ablation of Gα11(Gαq^fl/fl;Gα11^−/−). The DKO (COLI-Cre^+/−;Gαq^fl/fl;Gαll^−/−) mice developed and grew normally without abnormality of major organs. In addition, bone densitometry, 3D-μCT, and histomorphometric analyses showed no significant difference of bone volume or formation in DKO as compared with hetero-knockout of either Gαq (COLI-Cre^+/−;Gαq^fl/fl;Gαll^−/−) or Gαll (COLI-Cre^fl/fl;Gαq^fl/fl;Gαll^+/−), or the wild-type (COL-Cre^+/−;Gαq^fl/fl;Gαll⁺⁺) littermates at 4–20 weeks of age under physiological condition. However, when rhPTH (1–34) (0.08 mg/kg × 5 times × 4 weeks) or the vehicle was subcutaneously injected to 8-week-old mice of all genotypes (n=10/group), the bone densities of the femur, tibia, and L2–5 vertebrae of the PTH group were 12.2, 11.3, and 5.0% higher than the vehicle group (p<0.01 PTH vs. vehicle) in wild-type mice. Although the PTH effect in the Gαll hetero-knockout mice (12.4, 11.7, and 5.5%) was comparable to WT, it was moderately enhanced by the Gαq hetero-knockout (13.7, 13.1, and 6.7%, p<0.10 vs. wild-type) and significantly by the DKO (15.3, 14.9, and 8.5 %, p<0.05 vs. wild-type), indicating dose-dependent stimulation by the Gαq deficiency, but not by Gαll. Taken together, both the gain- and the loss-of-functions of Gαq in osteoblasts clearly demonstrated the inhibitory role of the Gαq signaling in bone formation. Suppression of the Gαq signaling to enhance the PTH anabolic action may lead to a novel treatment of osteoporosis.

Disclosures: N. Ogata, None.

1124

Adenosine and Osteoporosis: Adenosine A₁ Receptor Blockade Reverses Bone Loss in Ovariectomized Mice and Deletion of Adenosine A_2A Receptors Leads to Diminished Bone Density., F. M. Kara*¹, S. B. Doty*², A. Boskey*², B. Fredholm*³, B. N. Cronstein¹¹Medicine, NYU School of Medicine, NYC, NY, USA, ²Hospital for Special Surgery, NYC, NY, USA, ³Karolinska Institute, Stockholm, Sweden.

Adenosine regulates a wide variety of physiological processes via interaction with one or more of four G protein-coupled receptors. Because we have previously reported that adenosine A₁ receptors promote fusion of stimulated human monocytes to form giant cells whereas A_2A receptor activation inhibits in vitro giant cell formation we determined whether there was a similar requirement for A₁ & A_2A receptor occupancy in osteoclast formation.

Spleens were harvested from C57Bl/6 female mice (or A₁R knockout mice and their wild type littermates) and splenocytes were cultured in the presence of M-CSF (30 ng/ml) and RANKL (30 ng/ml) with or without various concentrations of the A₁R antagonist DPCPX for 7 day. Cells were stained for tartrate-resistant acid phosphatase (TRAP). The number of TRAP-positive multinucleated cells/well was then enumerated. Histological examination of bones from A₁, A_2A knockout and wild type mice was carried out with both standard (H&E) and immunohistologic (TRAP & Cathepsin K) stains and electron microscopy of bone sections was also carried out. A₁KO and A_2A KO mice were analyzed by DEXA (PIXImus) to measure the BMD and by MicroCT. Four week old C57BL/6 mice underwent ovariectomy (OVX) or SHAM surgery; those mice were treated with DPCPX (50 mg/kg/day) or vehicle (VEH) for 5 weeks.

The A₁R antagonist DPCPX inhibits osteoclast formation by as much as 93% in a dose-dependent fashion (P=0.0012) splenocytes from A₁R KO mice formed almost no osteoclasts in response to M-CSF and RANKL. Histological, pQCT and PIXImus examination from A₁R KO mice shows osteopetrosis, where A_2A KO mice shows Osteoporosis. Immunohistological demonstrates that although osteoclasts are present in the A₁R KO mice the osteoclasts are smaller and often not associated with bone. Electron microscopy from A₁KO femur shows an absence of ruffled borders of osteoclasts and resorption of bone. Where A_2A mice shows that the osteoclasts are pretty actively in resorbing bone, pQCT and PIXImus scan measurements indicated that by 5 weeks of DPCPX treatment trabecular bone density (TbBD) in the proximal femur increased in both OVX and SHAM compared to VEH. Ex vivo analysis of whole mice by DXA (PIXImus) scan confirmed increased BMD in DPCPX treated mice.

These results indicate that endogenously-released adenosine plays a critical role in regulating bone turnover via interaction with adenosine A₁ & A_2A receptors on osteoclasts and their precursor cells. Moreover the A₁ & A_2A receptors may be useful targets in treating diseases characterized by excessive bone turnover such as osteoporosis.

Disclosures: F.M. Kara, None.

1125

Effects of Cyclic and Daily PTH in Combination with OPG.. A. Iida-Klein¹, S. Johnston*¹, V. Shen², F. Cosman³, R. Lindsay³, D. W. Dempster¹¹Regional Bone Center, Helen Hayes Hospital, West Haverstraw, NY, USA, ²MDS Services Inc., Bothell, WA, USA, ³Clinical Research Center, Helen Hayes Hospital, West Haverstraw, NY, USA.

We recently demonstrated that combination of PTH with the anti-resorptive agent alendronate markedly enhances bone strength as well as bone density in mice (Iida-Klein et al, NOF, 2007) Accordingly, we address whether another anti-resorptive agent osteoprotegerin (OPG) has similar effects to alendronate on bone strength as well as density when added to daily or cyclical PTH regimens. We treated 20-week-old, female C57BL/J6 mice (n=10/group) for 7 weeks with the following: 1) vehicle [control], 2) OPG-Fc 10 mg/kg/day 2 days/week [OPG], 3) hPTH (1–34) 40 mcg/kg/day [daily PTH], 4) daily PTH in addition to OPG [daily PTH + OPG], 5) PTH and vehicle alternating weekly [cyclic PTH], 6) cyclic PTH in addition to OPG [cyclic PTH + OPG]. We examined the effects of these regimens on femoral and vertebral BMD, bone markers and cortical bone structure of the femur assessed by DXA, biochemical assays and pQCT, respectively. At 7 weeks, all treatment groups markedly increased BMD, and the combination of PTH and OPG additively increased BMD (Table). The positive effect of OPG on vertebral BMD was more marked in the cyclic PTH + OPG group. PTH significantly increased osteocalcin (OC) and mTRAP, while OPG significantly decreased OC and mTRAP. Furthermore, OPG blunted the stimulatory effects of PTH on OC and mTRAP. PTH significantly increased periosteal circumference while OPG tended to decrease endosteal circumference. This effect of OPG at the endocortical site was further enhanced in the presence of PTH as shown by significant decreases in endosteal circumference in the combined groups. At the periosteal site, OPG slightly blunted the PTH-stimulated periosteal expansion. Cortical thickness significantly increased in all groups.

The current study suggests that combination of PTH and OPG may work in a complementary fashion. Combination of these 2 agents in an appropriate protocol in humans may potentially produce more beneficial effects on bone than treatment with either agent alone.

Table Table 1. Percent increase from control in femoral and vertebral BMD, femoral bone structure and bone markers

^a p<0.05 vs. control, ^b p<0.01 vs. control.

Disclosures: A Iida-Klein, Amgen 2.

This study received funding from: Amgen.

1126

A Comparison of Denosumab and Alendronate in Combination with PTH in Ovariectomized Knockin Mice Expressing Humanized RANKL.. S. L. Ferrari¹, D. Pierroz¹, N. Bonnet¹, D. Dwyer*², M. Stolina*², P. Kostenuik²¹Div. of Bone Diseases, WHO Collaborating Center for Osteoporosis Prevention, Geneva University Hospital, Geneva, Switzerland, ²AMGEN Inc., Metabolic Disorders Research, Thousand Oaks, CA, USA.

Denosumab (DMab, a human Ab versus RANKL), was shown to improve bone volume, density and strength in ovariectomy (OVX) models. The antiresorptive action of DMab typically leads to a secondary increase in serum PTH, which might contribute to Dmab effects on bone. We explored this possibility by examining the endogenous PTH response to DMab or alendronate (ALN), the effects of those therapies on bone density and architecture, and the effects of adding daily PTH to those regimens.

DMab does not inhibit murine RANKL, so studies were performed in knockin mice that express a humanized RANKL gene wherein murine exon 5 was replaced by a human sequence. Adult huRANKL mice (4 months) were sham-operated or OVX, and treated twice weekly (sc) with ALN (100 ug/kg) or Dmab (10 mg/kg) or vehicle (VEH) (n=14–16/group) for 8 weeks. After 4 weeks, half the animals in each group were co-treated with PTH (80 ug/kg/d) or VEH for the remaining 4 weeks. BMD was assessed by DXA at weeks 4 and 8, and trabecular (Tb) and cortical (Ct) microarchitecture of the distal femur was assessed by ex vivo micro-CT at week 8.

Compared to sham, OVX induced a significant loss of whole body (WB) BMD, Tb BV/TV (-74%), number (TbN, −43%), thickness (TbTh, −12%), connectivity (ConnD, −90%) and Ct width (-18%). ALN significantly improved WB BMD, BV/TV and TbN compared to VEH, whereas ALN plus PTH was significantly better than ALN alone on these parameters as well as on TbTh and ConnD. DMab was statistically superior to ALN alone and equivalent to ALN plus PTH on all Tb bone parameters. DMab also significantly increased Ct width (+17%), which ALN +/- PTH did not.

The addition of daily PTH to DMab at week 4 did not significantly change any DXA/microCT parameters compared to 8 weeks of DMab alone. The lack of additive effects may have been related to the pronounced effects of DMab monotherapy, which increased WB BMD, BV/TV, TbN and ConnD to levels significantly greater than sham controls. Moreover, DMab caused a significantly greater increase in serum PTH(1–84) (+250%) and lower serum phosphate at week 4 vs ALN. Circulating PTH levels correlated positively with WB BMD (r=0.62, p<0.001) and Tb BV/TV (r=0.59, p<0.001).

In summary, Dmab was superior to ALN and similar to ALN plus PTH in preventing OVX-related changes of WB BMD, Tb and Ct microarchitecture in huRANKL mice. Intermittent PTH did not further improve Dmab effects, while Dmab induced a significant increase in PTH (1–84) levels and activity. These results support the possibility that endogenous PTH response to DMab may contribute to its ability to improve bone mass and architecture.

Disclosures: S.L. Ferrari, AMGEN 2, 5; MSD 5, 8.

This study received funding from: Amgen.

1127

GLP-2 Significantly Increases Hip BMD in Postmenopausal Women: A 120-Day study.. D. Henriksen¹, P. Alexandersen², B. Hartmann¹, I. Byrjalsen*³, J. J. Holst*⁴, C. Christiansen³¹Sanos Bioscience A/S, Roedovre, Denmark, ²Center for Clinical & Basic Research, Ballerup, Denmark, ³Nordic Bioscience A/S, Herlev, Denmark, ⁴Department of Medical Physiology, University of Copenhagen, Copenhagen, Denmark.

Glucagon-like peptide-2 (GLP-2) is an intestinal hormone released in response to food intake. We have earlier shown that treatment with GLP-2 for 14 days caused a significant post-administration reduction of bone resorption. This effect was not followed by a change in bone formation and s-osteocalcin levels were unaffected by treatment.

We now present a randomized, placebo-controlled 120-day phase II trial with 160 postmenopausal women. The aim was to test the sustainability of above findings over a longer treatment period and to evaluate the potential for long-term treatment with GLP-2. The study subjects were osteopenic (BMD T-score, −2.5 < T ≤ −1.0) at one or more of the regions: lumbar spine, femoral neck, or total hip. The objectives were relative change from baseline of total hip and spine BMD, as well as pharmaco-dynamic changes in bone resorption (s-CTX) and formation (s-osteocalcin) over the 120 days. Subjects received daily bedtime injections of 0.4, 1.6, or 3.2 mg GLP-2, or placebo. All subjects received supplements of calcium and vitamin D. Treatment with GLP-2 resulted in a dose dependent increase in total hip BMD from baseline, which reached statistical significance (p=0.006) in the 3.2 mg GLP-2 group compared to placebo. This dose dependent relationship in BMD was also seen at the trochanter, (p=0.01, 3.2 mg group). All GLP-2 doses levels elicited an acute and sustained reduction of the nocturnal rise in s-CTX seen in the placebo group. The reduction in the s-CTX level was greater at Day 120 than at Day 1 indicating an overall improved suppression of bone resorption during the 120 days treatment. At Treatment Day 120, all GLP-2 groups had a greater, and highly statistically significant, (p<0.0001) reduction in AUC_0.6H for s-CTX compared to placebo.

Injections of GLP-2 exerted no acute stimulatory or inhibitory effect on bone formation, and s-osteocalcin levels was unaffected by treatment at both Treatment Day 1 and 120.

None of the study participants developed any detectable antibodies against GLP-2 and no tachyphylaxis was observed. Overall, it was concluded that the treatment with GLP-2 for 120 consecutive days was effective, well-tolerated and safe. In conclusion, the results suggest that GLP-2 acutely disassociates the two processes of bone remodelling and thereby increases the bone calcium (ossification) balance, particularly at the hip. The favourable effect of GLP-2 on the BMD of the hip suggests that an agent with this mode of action could prove a valuable new treatment for osteoporosis.

Disclosures: D. Henriksen, Sanos Bioscience A/S 3.

1128

A Randomized, Double-Blind, Placebo-Controlled Study of a Cathepsin-K Inhibitor in the Treatment of Postmenopausal Women with Low BMD: One Year Results., H. G. Bone¹, M. McClung², N. Verbruggen*³, A. Rybak-Feiglin*⁴, C. DaSilva*⁴, A. C. Santora⁴, A. Ince⁴¹Michigan Bone and Mineral Center, Detroit, MI, USA, ²Oregon Osteoporosis Center, Portland, OR, USA, ³Merck Research Laboratories, Brussels, Belgium, ⁴Merck Research Laboratories, Rahway, NJ, USA.

Cathepsin K, a cysteine protease abundantly expressed in osteoclasts, is necessary for bone collagen degradation. MK-0822, a selective inhibitor of cathepsin K, has been shown to rapidly and reversibly decrease bone resorption in both preclinical and short-term clinical studies. In order to evaluate the safety, tolerability and efficacy of weekly doses of placebo, 3, 10, 25 or 50 mg of MK-0822 on BMD and biochemical indices of bone turnover, a 1+1 year dose-ranging trial is ongoing in postmenopausal women with low BMD. We present 12-month data here. Postmenopausal women (N=399) with BMD T-scores ≤-2.0 and ≥-3.5 at lumbar spine (LS), femoral neck (FN), trochanter, or total proximal femur were randomized to receive placebo or 1 of 4 doses of MK-0822. Mean age was 64.2 ± 7.8 years. The mean LS T-score was −2.2 ± 0.8, the FN T-score was −1.9 ± 0.7, and the urinary N-telopeptide/creatinine ratio was 44.1 ± 21.2 nmol/mol. Treatment produced dose-related increases in BMD from baseline. The highest dose of MK-0822 tested (50 mg) resulted in a 3.4% increase in LS BMD (Figure), a 2.5% increase in femoral neck BMD, and a 58% reduction in uNTx/Cr. The safety profile of MK-0822 was generally favorable. There were no dose-related trends in any adverse experiences (AEs). Rash was reported in 8.4% and 3.8% of those in the placebo and 50 mg arms, respectively. Among participants in the placebo arm, 9 discontinued the study due to AEs, and 4 discontinued due to AEs considered drug-related. By contrast, 4 and 0 participants in the 50 mg arm were in each of these AE categories, respectively. In summary, 12 months of MK-0822 treatment was generally safe and well-tolerated and increased LS and FN BMD in postmenopausal women with low BMD.

Disclosures: H.G. Bone, Merck & Co., Inc. 2, 8.

This study received funding from: Merck & Co., Inc.

1129

Anti-Sclerostin Antibody Increases Markers of Bone Formation in Healthy Postmenopausal Women.. D. Padhi*¹, B. Stouch*¹, G. Jang¹, L. Fang*¹, M. Darling*¹, H. Glise*², M. Robinson*³, S. Harris*⁴, E. Posvar*¹¹Amgen Inc, Thousand Oaks, CA, USA, ²UCB SA (Pharma), Braine-l'Alleud, Belgium, ³UCB-Celltech, Slough, United Kingdom, ⁴Seaview Research, Miami, FL, USA.

Genetic evidence, along with gene expression, cell biology and pharmacology data, demonstrate that the osteocyte-secreted protein, sclerostin, negatively regulates osteoblasts and plays a key role in controlling bone formation and bone mass throughout life.

In this blinded, placebo-controlled, rising single dose study, 48 healthy postmenopausal women were randomized (3:1) to receive a single SC dose (0.1, 0.3, 1, 3, 5, or 10 mg/kg) of a sclerostin neutralizing monoclonal antibody (scl-mAb) or placebo. Blood samples were collected up to 85 days postdose for determination of serum scl-mAb, BSAP, osteocalcin, P1NP and CTx concentrations.

Scl-mAb pharmacokinetics (PK) were non-linear with dose (eg. for the 30-fold increase in dose from 0.1 to 3 mg/kg, mean exposure based on the maximum serum scl-mAb concentrations or area under the serum concentration-time curve increased approximately 60- to 100-fold). Preliminary pharmacodynamic (PD) data for doses of 0.1 to 10 mg/kg scl-mAb are available. Dose-related increases in the bone formation markers P1NP, osteocalcin and BSAP were observed (relative to placebo), with mean percentage change from baseline for these markers of approximately 60 to 100% at 3 mg/kg by 21 days. A trend of dose-related decreases in serum CTx was also observed.

In conclusion, scl-mAb PK following SC administration in healthy postmenopausal women was nonlinear with dose, as observed with other mAbs. Single doses of scl-mAb resulted in significant increases in bone formation markers relative to placebo and a trend of dose related decreases in serum CTx. Scl-mAb was well tolerated following administration of single SC doses. These data support continued clinical investigation of sclerostin inhibition as a potential therapeutic strategy for conditions that will benefit from increased bone formation.

Disclosures: D. Padhi, Amgen 1, 3.

1130

Ostabolin-C™ Increases Lumbar Spine and Hip BMD after 1 Year of Therapy: Results of a Phase II Clinical Trial.. A. Hodsman¹, H. Bone*², C. Gallagher³, D. Kendler*⁴, J. P. Brown*⁵, S. Greenspan*⁶, C. L. Barclay*⁷, P. Morley*⁷, R. Anderson*⁷, B. R. MacDonald⁸¹St. Joseph's Hospital, London, ON, Canada, ²Michigan Bone and Mineral Clinic, Detroit, MI, USA, ³Creighton University, Omaha, NE, USA, ⁴ProHealth Clinical Research Center, Vancouver, BC, Canada, ⁵Laval University, Quebec, PQ, Canada, ⁶University of Pittsburgh, Pittsburgh, PA, USA, ⁷Zelos Therapeutics, Ottawa, ON, Canada, ⁸Zelos Therapeutics Inc., West Conshohocken, PA, USA.

Ostabolin-C (OC) is a cyclic analogue of parathyroid hormone (PTH) (1–31) that has demonstrated marked increases in bone mineral density (BMD) and bone strength in long term preclinical studies, without inducing hypercalcemia. 261 postmenopausal women aged 45–75 years with a BMD T score at lumbar spine (LS) or femoral neck ≤-2 were randomized into a double blind, placebo controlled dose ranging study investigating the safety and efficacy of 4 months of treatment with OC. Subjects received placebo, 7.5, 15, 30 or 45 μg/day by subcutaneous injection. 198 study subjects elected to continue into a blinded extension study of 8 additional months. The primary endpoint was change in LS-BMD at 1 year. Secondary measures included change in BMD at the hip, biomarkers of bone turnover, and safety measures, including the occurrence of hypercalcemia.

OC caused a striking, dose-dependent increase in LS-BMD that was evident by month 4.79% of subjects in the 45 μg group had an early BMD response (defined as ≥3% increase after 4 months). BMD measurements at all proximal femur sites were also increased at 4 months in the 45 μg group. At month 12, LS-BMD was further increased in all active treatment groups with a maximum mean % increase of 11.0 % in the 45 μg group compared to a 0.17 % increase in placebo (p<0.001). At month 12, 97% of subjects in the 45 μg group had an increase in LS-BMD of ≥ 3%. At month 12, total hip BMD increased by 2.4% in the 45 μg group compared to a 1.2% decrease in placebo (p < 0.001). The BMD effects at all doses were accompanied by commensurate significant increases in biomarkers of bone formation that were detectable at the earliest measured timepoint (4 weeks). Mean % change in P1NP was > 120%, and osteocalcin was > 100%, above baseline throughout the study. Compliance with study drug was > 93% in all dose groups. The most commonly reported adverse event was mild nausea, as seen with other PTH analogues. This adverse event usually occurred early in the treatment period. Few serious adverse events occurred. Most episodes of hypercalcemia were mild. Hypercalcemia > 11.0 mg/dL was infrequent, and only observed with higher incidence than placebo in the 45 μg group. The observed clinical profile, including a marked early BMD response at both hip and spine, indicates that OC has the potential to be a highly effective treatment for osteoporosis.

Disclosures: A. Hodsman, Zelos Therapeutics Inc. 1, 5.

This study received funding from: Zelos Therapeutics Inc.

1131

Dose-dependent Increases in Endogenous Parathyroid Hormone Concentrations After Administration of a Calcium Sensing Receptor Antagonist to Normal Volunteers: Potential for an Oral Bone Forming Agent.. D. Ethgen¹, J. Phillips*¹, C. Baidoo*¹, C. Matheny*¹, A. Roshak*¹, R. Marquis*¹, G. Stroup², S. Kumar¹, P. Bhatnagar*¹, W. F. Huffman*¹, V. S. Kitchen*¹, M. Gowen², L. A. Fitzpatrick¹¹GlaxoSmithKline, Collegeville, PA, USA, ²GlaxoSmithKline, King of Prussia, PA, USA.

Brief antagonism of the calcium-sensing receptor (CaR) in the parathyroid gland results in transient stimulation of endogenous parathyroid hormone (PTH) release, a profile known to stimulate bone formation. SB-423557 is an ethyl ester precursor of SB-423562. Upon administration, SB-423557 is converted into its corresponding acid, SB-423562. Both molecules are potent calcium-sensing receptor antagonists that are being investigated for the treatment of osteoporosis as a novel oral bone forming agent.

This first clinical study administering SB-423557 orally to healthy male volunteers was designed as a single-blind, placebo-controlled, crossover, randomized (with respect to placebo), dose-rising study. Fifty-four subjects participated in two study sessions separated by ≥7 days. In random order, subjects received placebo in one study session, and single oral dose SB-423557 in the other. Dose levels were 5, 10, 20, 50, 100, 200, 400 and 500 mg (n=5–6, except n=12 for 50 mg group). Blood samples for pharmacokinetic and pharmacodynamic analyses were obtained over a 24-h period after each dose.

Elevations in plasma PTH closely followed the time-course of SB-423562 concentrations in blood (SB-423557 generally was undetectable). The duration of PTH elevation (defined as PTH levels ≥ twice the pre-dose level) was usually under 8 hours. At SB-423557 doses of 100 mg and above, PTH exposure (AUC_{0.24 h}) was on average, 10% to 48% higher compared to placebo. The mean maximum post-dose PTH concentration ranged from 38% to 285% higher relative to placebo; median time to maximum concentration (Tmax) ranged from 1.01 h to 2.26 h. At doses of 50 mg and higher, relative to placebo, a 1.8- to 4.2-fold increase in PTH levels compared to baseline was noted at the Tmax. There were no significant effects on serum calcium levels.

SB-423557 was generally well tolerated at all doses and no serious adverse events were reported. There were no notable differences among regimens with regard to adverse events frequency, safety laboratory values, vital signs or 12-lead ECG interval values outside the threshold ranges.

In conclusion, the observed transient rises in PTH after single oral doses of a CaR antagonist, SB-423557, constitutes an early proof-of-concept in man, and provides the basis for further development of this class of compounds for delivering an oral bone forming treatment to osteoporotic patients.

Disclosures: D. Ethgen, GlaxoSmithKline 3.

1132

A Single Dose of ACE-011 Is Associated with Increases in Bone Formation and Decreases in Bone Resorption Markers in Healthy, Postmenopausal Women.. J. Ruckle*¹, M. Jacobs*¹, W. Kramer*², R. Kumar*³, K. Underwood*³, R. S. Pearsall³, A. Pearsall*³, J. Seehra³, C. Condon*³, M. L. Sherman³¹Covance Clinical Research, Honolulu, HI, USA, ²Kramer Consulting, LLC, North Potomac, MD, USA, ³Acceleron Pharma, Inc., Cambridge, MA, USA.

ACE-011 is a fusion protein consisting of the extracellular domain of the human type II activin receptor IIA (ActRIIA) linked to the Fc portion of human IgG1. ACE-011 binds to activin with a Kd of 50 pM. Nonclinical studies in ovariectomized mice treated with ActRIIA fused to the Fc region of a murine IgG have shown improvements in bone architecture and strength. Histomorphometry studies have demonstrated an anabolic effect. A randomized, double-blind, placebo-controlled study was conducted to evaluate ACE-011 administered to healthy, postmenopausal women. Forty-eight subjects were randomized in cohorts of 6 to receive either a single dose of ACE-011 or placebo (5 active-1 placebo). Dose levels ranged from 0.01 to 3.0 mg/kg intravenously and 0.03 to 0.1 mg/kg subcutaneously. All subjects were followed for 120 days. Subjects were excluded from study participation if they took medications affecting bone metabolism within 6 months of study entry. Safety evaluations were conducted following each cohort to determine dose escalation. In addition to pharmacokinetic (PK) analyses, the biologic activity of ACE-011 was also assessed by measurement of biochemical markers of bone formation and resorption, and FSH levels.

No serious adverse events were reported in this study. Adverse events (AEs) were generally mild and transient. Preliminary analysis of AEs included headache, elevated laboratory values, cold symptoms, emesis or vomiting, intravenous infiltration, and hematoma at injection site. PK analysis of ACE-011 displayed a linear profile with dose, and a mean half-life of approximately 25–32 days. The absorption after SC dosing was essentially complete. ACE-011 caused a rapid, sustained dose-dependent increase in serum levels of bone-specific alkaline phosphatase, and a dose-dependent decrease in C-terminal type 1 collagen telopeptide and tartrate-resistant acid phosphatase 5b levels. These bone biomarker changes were sustained for at least 29 days at the highest dose levels tested. There was also a dose-dependent decrease in serum FSH levels consistent with inhibition of activin.

A single dose of ACE-011 given to healthy postmenopausal women was safe and well-tolerated for the range of dose levels tested. The prolonged PK and pharmacodynamic effects suggest that intermittent dosing would be appropriate for future studies. ACE-011 is a first-in-class novel osteoanabolic agent with biological evidence of both an increase in bone formation and a decrease in bone resorption.

Disclosures: J. Ruckle, None.

This study received funding from: Acceleron Pharma, Inc.

1133

Compromised Bone Marrow Perfusion in Osteoporosis.. J. F. Griffith*¹, D. K. W. Yeung*¹, P. K. Tsang*¹, K. C. Choi*², T. C. Y. Kwok*³, A. T. Ahuja*¹, K. S. Leung*⁴, P. C. Leung*⁵¹Diagnostic Radiology and Organ Imaging, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin “Hong Kong, ²School of Public Health, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin” Hong Kong, ³Community and Family Medicine, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin “Hong Kong, ⁴Orthopaedics and Traumatology, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin” Hong Kong, ⁵Jockey Club Centre for Osteoporosis Care and Control, The Chinese University of Hong Kong, Prince of Wales Hospital, Shatin, Hong Kong.

Background: There may be a link between bone blood flow and osteoporosis. This study of the proximal femur investigates the relationship between bone mineral density (BMD), bone fat content, bone and muscle perfusion.

Methods: 120 healthy female subjects (mean age, 74 years) underwent DXA, proton (¹H) MR spectroscopy, and dynamic contrast-enhanced MRI. Marrow fat content was measured at the femoral head, neck and shaft. Perfusion indices (maximum enhancement, E_max and enhancement slope, E_slope) in the proximal femur (head, nack, shaft and acetabulum) and adductor muscle were measured. Correlation between T-score, fat content and perfusion indices was examined.

Results: Perfusion indices were significantly reduced in subjects with osteoporosis (Table) compared to subjects with osteopenia or normal BMD (Figure).

Adductor muscle perfusion was not affected by change in BMD (E_max, r = −0.061; E_slope, r = −0.057). As marrow perfusion decreased in the proximal femur, marrow fat increased. For normal BMD subjects, perfusion parameters in the femoral head were 1/3 those in the femoral neck or shaft and 1/5 those in the acetabulum.

Conclusion: Perfusion in the proximal femur is reduced in osteoporotic subjects compared to osteopenic and normal subjects. This reduction in perfusion occurs only within the bone and not in the extraosseous tissues.

Table  .  

Disclosures: J.F. Griffith, None.

1134

Neuropeptide Y Protects the Skeleton from Stress-induced Bone Loss.. P. A. Baldock*¹, S. Allison*¹, T. Karl*², D. Pierroz³, D. Lin*², A. Sainsbury*², R. Enriquez*¹, S. Ferrari³, H. Herzog*², J. Eisman¹¹Bone Program, Garvan Institue of Medical Research, Sydney, NSW, Australia, ²Neuroscience Program, Garvan Institue of Medical Research, Sydney, NSW, Australia, ³Service and Laboratory of Bone Diseases, Geneva University Hospital, Geneva, Switzerland.

Bone responses to stress are not well studied beyond corticosteroid-related effects. Neuropeptide Y has anxiolytic actions, attenuating the psychological effects of stress. Interestingly, the major Y-receptor mediating these anti-stress actions is the Y2 receptor, also known to control bone mass. We therefore investigated how the NPY pathway may modulate skeletal responses to physiological stress.

Behavioral stress responses in NPY^−/− mice were examined using established models. Skeletal response to stress was examined at 14 weeks in male NPY^−/− and wild type mice after 4 week low and high stress protocols. All mice were kept in a low stress environment until the age of 9 weeks. Following this, the “stress” groups were involved in a protocol that necessitated regular handling and other known stressors in mice, including rectal temperature, glucose tolerance test, 24h fasting and metabolic cage studies. Control mice remained in the lower stress environment.

Anxiety-like behaviours in the open field and light-dark tests, both standard methods for determination of explorative and anxiety-related parameters, were much greater in NPY^−/− mice.

As expected, whole body, femoral (53 ± 2 mg/mm² vs 65 ± 2, p<0.05) and tibial BMD was greater in NPY^−/− mice compared to wild type in low stressed group, coincident with greater cortical volume and thickness. Greater cancellous bone volume in distal femoral (16.7 ± 1.5% vs 11.6 ± 0.9, p<0.01), and lumbar vertebral metaphyses was coincident greater mineral apposition rate in NPY^−/− mice (1.9 ± 0.1 μm/d vs 2.8 ± 0.1, p<0.0005).

In the “stressed” groups, body weight was reduced in NPY^−/− mice (30.1 ± 1g vs 25.5 ± 1g, p<0.0001), with decrements in lean and fat mass, with no decrease in wild type. A mild cancellous response was evident in wild type mice, with trabecular number reduced but no significant loss of cancellous bone volume. However, in “stressed” NPY^−/− mice there was a marked decrease in cancellous bone volume compared to low stressed NPY^−/− mice (16.7 ± 1.5% vs 13.2 ± 0.7, p<0.01), again associated with reduced trabecular number. Importantly, mineral apposition rate was also reduced in “stressed” NPY^−/− mice, but still remained greater than wild type (1.6 ± 0.1 μm/d vs 2.4 ± 0.1, p<0.0001).

In summary, these data suggest that NPY plays a powerful role in regulating the effects of stress. Diminishing levels of NPY may enhance the bone loss induced by stress. This may explain a mechanism for bone loss associated with stress in otherwise healthy individuals.

Disclosures: P.A. Baldock, None.

This study received funding from: NHMRC.

1135

Fat-1 Gene Prevents Ovariectomy Induced Bone Loss by Modulating Osteoclastogenesis.. M. M. Rahman, A. Bhattacharya*, J. Banu, G. Halade*, G. Fernandes*. Medicine/CI, UTHSCSA, San Antonio, TX, USA.

Consumption of n-3 fatty acids (FA) positively influences on calcium and bone metabolism. Beneficial effects of n-3 FA on bone mineral density (BMD) have been reported in mice, rats and humans, but the precise mechanisms involved have not been described. In this study, we investigated the effect of fat-1 gene on ovariectomy induced bone loss, fat-1 gene encodes for an n-3 desaturase enzyme that can synthesize n-3 FA from n-6 FA. Ovariectomized (OVX) and sham operated wild type (WT) C57BL6 (B6) mice and fat-1 transgenic B6 mice were fed a diet containing 10% CO. After 24 weeks on diets, BMD was analyzed by dual energy x-ray absorptiometry, fat-1 OVX mice had significantly higher BMD in distal femoral metaphysis, proximal tibial metaphysis, femoral diaphysis, and lumbar vertebra as compared to WT OVX mice. Pro-inflammatory cytokines TNF-alpha, IL-6 and IL-1 beta and anti-inflammatory cytokine IL-10 were analyzed in LPS stimulated bone marrow (BM) culture supernatant by ELISA. Significantly lower level of IL-1 beta, TNF-alpha and IL-6, and higher level of IL-10 expression was found in fat-1 OVX mice compared to WT OVX mice. LPS stimulated NF-kappaB activation was also analyzed in BM cells. NF-kappaB activation was found significantly less in fat-1 OVX mice compared to WT OVX mice. We also examined the LPS stimulated inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in BM cells. Interestingly, LPS treated NO production in BM culture supernatant and iNOS protein expression in BM cells was significantly higher in fat-1 OVX mice as compared to WT OVX mice. Moreover, fat-1 transgenic, nontransgenic and WT BM cells were cultured in the presence of n-6 fatty acid (linoleic acid) with RANKL to differentiate into TRAP +ve cells. Interestingly, fat-1 transgenic BM cells generated significantly less TRAP +ve osteoclast like cells compared to fat-1 nontransgenic and WT B6 BM cells. We offer further the mechanisms involved in preventing the bone loss in OVX mice by n-3 FA using a fat-1 transgenic mouse model.

Disclosures: M.M. Rahman, None.

This study received funding from: NIH.

1136

A CD40L Mediated Cross-talk Between T Cells and Stromal Cells Is Required for Estrogen Deficiency and PTH to Induce Bone Loss.. Y. Gao, M. Terauchi, K. Page*, X. Yang*, F. Grassi, S. Galley, E. Shah*, M. R. Ryan, M. N. Weitzmann, R. Pacifici. Division of Endocrinology, Metabolism and Lipids, Emory University School of Medicine, Atlanta, GA, USA.

Activated T cells induce bone loss in inflammation and estrogen (E) deficiency by secreting RANKL, TNF, and IFNγ. T cells also regulate bone marrow (BM) stromal cells (SCs) via surface receptors, but the relevance of T cell membrane signals in bone diseases is unknown. Herein we investigated a) if T cells regulate the capacity of SCs to support osteoclast (OC) formation through CD40L, a T cell costimulatory molecule which binds to SC expressed CD40 receptors, and b) the role of CD40L/CD40 signaling in the bone loss induced by ovariectomy (ovx) and in vivo continuous PTH treatment (cPTH). As compared to T cell replete controls, SCs from nude mice and WT mice immunodepleted of T cells in vivo produce less M-CSF, RANKL, and TNF and supported the formation of fewer OCs when cocultured with BM macrophages (BMMs) from WT mice. Moreover, ovx and cPTH upregulate the osteoclastogenic activity of SCs from control, but not T cell deficient mice, demonstrating that E and PTH regulate SC function through T cells. Ovx and cPTH upregulate the T cell expression of CD40L and the SC expression of CD40 by 2–3 fold. CD40 expression is equally increased by IFNγ and TNF, cytokines produced in large amounts by T cells of ovx mice, suggesting that ovx upregulates CD40 expression through these factors. PTH induced OC formation in cocultures of BMMs and SCs is equally enhanced by soluble CD40L and WT T cells, while CD40L -/- T cells are ineffective. Moreover, neutralizing anti CD40L mAb blocks the T cell potentiation of PTH induced OC formation, and the capacity of T cells from ovx mice to increase the formation of OCs in cocultures of SCs and BMMs from intact mice, demonstrating a role for CD40L in PTH and ovx induced OC formation. Analysis by μCT reveals that CD40L-/- mice are completely protected against the loss of trabecular and cortical bone induced by ovx and cPTH, respectively. Thus, CD40L plays a key role in both ovx and PTH induced bone loss. When T cells from WT and CD40L -/- mice were adoptively transferred into TCRβ -/- mice, a strain lacking αβ T cells, cPTH stimulated bone resorption leading to cortical bone loss in hosts reconstituted with WT T cells, but not in those reconstituted with CD40L -/- T cells, thus demonstrating the specific role of T cell expressed CD40L. In summary, CD40L/CD40 costimulation is a novel pathway by which T cells regulate SC function and OC formation. Upregulation of the osteoclastogenic activity of SCs by T cell expressed CD40L is pivotal for the osteoclastogenic response to PTH and E deficiency. Understanding the cross-talk between T cells and SCs may thus yield novel therapeutic strategies for postmenopausal and PTH induced bone loss.

Disclosures: Y. Gao, None.

1137

Ovariectomy Decreases Bone Mass in Young and Old Female Athymic Mice.. W. M. Kozlow, K. Mohammad, C. R. McKenna*, H. Walton*, M. Niewolna*, T. A. Guise. Internal Medicine, University of Virginia, Charlottesville, VA, USA.

Ovariectomy (OVX) has been reported to have no effect on trabecular bone mass in female athymic (nude) mice because these mice lack T cells⁽¹⁾. However, recent data has demonstrated trabecular, but not cortical, bone loss 4 weeks after OVX in 6-week-old female nude mice⁽²⁾. The effect of OVX on bone mass in female nude mice may be related to mouse age at the time of surgery.

To determine the effect of mouse age (at the time of OVX) on bone mass, 4-week-old (young) and 16-week-old (old) female BALB-c nude mice were randomized to OVX or sham surgery (sham). Bone mineral density (BMD), as assessed by Lunar PIXImus, was assessed at baseline and every 2 weeks thereafter. At 20 weeks, the young OVX mice had decreased BMD at the total body (p=0.0056), spine (p<0.0001) and proximal tibia (p<0.0001) compared to the sham mice. Decreased BMD was noted as early as 2 weeks after OVX in the total body and proximal tibia, and by 4 weeks after OVX in the lumbar spine. Although there was no difference in BMD at the distal femur, BMD was surprisingly increased at the mid femur (p<0.0001) in the OVX mice compared to the sham mice. However, histomorphometry demonstrated no difference in trabecular bone volume at the distal femur or proximal tibia between the OVX mice and sham mice.

Twenty weeks after surgery, the old OVX mice had decreased BMD at the total body (p=0.0048), spine (p<0.0001), mid femur (p=0.0409) and distal femur (p<0.0001) as compared to the sham mice. Decreased BMD was noted as early as 2 weeks after OVX in the total body and distal femur, and by 4 weeks after OVX in the lumbar spine and mid femur. There was no difference in BMD at the proximal tibia.

At 20 weeks, differences between the OVX and sham mice were greater in the young mice versus the old mice: 3.4% versus 1.5% total body; 18.3% versus 9% spine; 9.2% versus 1.1% mid femur; 3.8% versus 6.1% distal femur; 20.1% versus 6.6% proximal tibia.

Bone marrow cultures from OVX mice exhibited a greater number of colony forming unit (CFU)-fibroblasts (p<0.0001 for young and old), CFU-osteoblasts (p<0.0001 young, p=0.0001 old) and TRAP-positive osteoclasts (p=0.0005 young) compared to sham mice. These experiments show that OVX does have an effect on bone mass at multiple sites in female nude mice. OVX-induced decreases in bone mass were seen in both young and old female nude mice, but the differences between the OVX and sham mice were more profound in the younger mice as compared to the older mice. Bone marrow cultures revealed that the lower bone mass was associated with increased bone turnover.

⁽¹⁾ Cenci S et al. J Clin Invest. 2000;106: 1229–37.

⁽²⁾ Lee S-K et al. J Bone Miner Res. 2006; 21:1704–12.

Disclosures: W.M. Kozlow, None.

This study received funding from: Department of Defense.

1138

IGF-1 Carrier Components Determine Skeletal Integrity Via Direct and Indirect Mechanisms.. S. Yakar¹, W. Mejia*¹, Y. Kawashima*¹, H. Sun*¹, M. L. Bouxsein², C. J. Rosen³¹Mount Sinai School of Medicine, New York, NY, USA, ²Beth Israel Deaconess Medical Center, Boston, MA, USA, ³The Jackson Laboratory, Bar Harbor, ME, USA.

Serum IGF-1 is secreted mainly by the liver and circulates bound to IGF-binding proteins (IGFBPs, predominantly IGFBP-3) in binary complexes and bound to IGFBP-3 and the acid labile subunit (ALS) in a ternary complex. To delineate the role of IGF-1 circulatory complexes in skeletal integrity, we employed three mouse lines that have reduced circulating IGF-1; liver-specific IGF-1 deficient (LID) mice; ALS knockout (ALSKO) mice, and IGFBP-3 knockout (BP3KO) mice. Bone micro-architecture is profoundly affected in the different IGF-1 mutants; LID mice exhibit minor reductions in trabecular BV/TV% (∼10%), but significantly lower areal BMD and mid-diaphyseal cortical thickness. In contrast, gene inactivation of the ALS, leads to a marked decrease in trabecular BV/TV% (∼20%), with no changes in cortical thickness and reduced cross-sectional area. In contrast to both LID and ALSKO mice, BP3KO mice have ∼30% decreases in trabecular bone volume fraction with no changes in cortical bone thickness or diaphyseal cross-sectional geometry. To determine what mechanisms may be operative in the skeleton of the IGF-1 deficient mice we studied marrow cells and their ability to form osteoblasts(OB) and osteoclasts (OC). ALSKO and BP3KO exhibited an increase in OB differentiation as assessed by alkaline phosphatase staining, whereas marrow stromal cells from LID mice did not differ from controls. OC differentiation was enhanced in BP3KO mice as reflected by increased TRAP positivity compared to control mice. In contrast, ALSKO mice exhibited reductions in OC number and activity as assessed by pit formation assay. In support of the latter, real time PCR for OC markers revealed a decrease in TRAP and cathepsin K expression in ALSKO cultures consistent with impaired differentiation of macrophage precursors. Taken together, these results suggest direct yet diverse effects of ALS and IGFBP-3 on OB and OC differentiation and activity. Alternatively it is conceivable that ALS and IGFBP-3 may have indirect effects on bone via alterations in the hematopoietic-monocytic lineage that are modulated by marrow stromal cells.

Disclosures: S. Yakar, None.

1139

Evidence for Lineage Dependent Non-canonical Wnt Signal Transduction by Bone Cells.. G. J. Spencer*, P. G. Genever*. Department of Biology, University of York, York, United Kingdom.

Wnt signaling has emerged as an important determinant of bone mass, regulating osteoblast and osteoclast formation and activity. Most studies have focused on the transcriptional effects of canonical Wnt signaling although Wnts are also known to signal through non-canonical pathways, evoking changes in intracellular calcium and activation of protein kinase signaling cascades. Using a combination of Affymetrix microarray analysis and RT-PCR we have identified expression of Wnt ligands (Wnt5a), Fzd receptors (Fz 1, 3 and 7) and PKC isoforms required for non-canonical Wnt signal transduction in human mesenchymal stem cells (MSCs). Exposure of MSCs to the protypical non-canonical Wnt ligand Wnt5a (conditioned medium, 10–50%) during osteogenic differentiation induced by dexamethasone, ascorbate and β-glycerol phosphate, significantly reduced cell numbers (p<0.001) and increased alkaline phosphatase activity (p<0.01), supporting a role for non-canonical signaling in stimulating osteogenesis. In contrast, exposure to the canonical ligand Wnt3a stimulated proliferation and inhibited osteogenesis. By confocal imaging, western blotting and immunolocalization we demonstrated that exposure of MSCs to Wnt5a (10–500ng/ml) caused a rapid, thapsigargin-sensitive increase in intracellular calcium and potently stimulated the phosphorylation of PKC isoforms PKCθ, PKCδ, PKCα/β and PKD/PKCμ. Wnt5a also stimulated the phosphorylation and nuclear translocation of the transcription factor CREB without affecting p42/44 MAPK activity. Significantly, using cultures of rat primary calvarial osteoprogenitors we were unable to demonstrate any stimulatory effect of Wnt5a on intracellular calcium, proliferation or osteogenic differentiation, although Wnt3a remained inhibitory. These data strongly suggest that uncommitted MSCs respond to non-canonical signals to promote osteogenesis with loss of responsiveness soon after the onset of differentiation, while anti-osteogenic effects of Wnt3a are independent of lineage commitment state. As potential anabolic therapies depend on their specificity for osteogenesis we determined whether cells of the osteoclast lineage also respond to non-canonical Wnts. Using RAW264.7 cells we showed that exposure to Wnt5a (10–500ng/ml) potently increased p42/44 MAPK activity and phosphorylation of JNK, PKCΔ, PKCα/β and PKD/PKCμ, without affecting CREB. These data have wide implications for our understanding of Wnt signaling in bone; non-canonical Wnts promote osteogenesis but also have potential to affect osteoclastogenesis and bone resorption through common activation of PKC isoforms but differential effects on the activity of CREB, MAPK and JNK.

Disclosures: G.J. Spencer, None.

This study received funding from: The Wellcome Trust.

1140

Reciprocal Regulation of Dkk1 Gene Transcription by Msx2 and c/EBPα.. S. Cheng, O. L. Sierra, D. A. Towler. Division of Bone and Mineral Diseases, Washington University School of Medicine, St. Louis, MO, USA.

Msx2 plays an important role in craniofacial bone formation, osteoblast gene transcription, and osteogenic-adipogenic differentiation. We previously demonstrated that Msx2 inhibits gene transcription via 3 mechanisms: (a) antagonistic protein-protein interactions with a multiprotein complex containing Runx2, Ku antigen, and MINT, (b) inhibitory interactions with TFII-F, a PolII-dependent transcription initiation and elongation factor, and (c) inhibitory protein-protein actions with c/EBPα that preclude PPARγ2 expression and adipogenic differentiation. We also demonstrated that Msx2 promotes calvarial osteogenesis in part via suppression of Dkk1, with concomitant activation of canonical Wnt signaling. Msx2 pro-osteogenic actions are abrogated by treatment with Dkk1 protein or transduction with a Dkk1-expressing retrovirus. We wished to examine the mechanisms that mediate Msx2 inhibition of Dkk1. Using 10T1/2 cells transduced with Msx2 retrovirus or primary calvarial osteoblasts from CMV-Msx2 transgenic mice, we find that Msx2 suppresses Dkk1 gene expression. Western blot analyses confirm reduction of Dkk1 levels in Msx2-expressing cells (30% of control). Conversely, “knock-down” of endogenous Msx2 levels in 10T1/2 cells with Msx2 siRNA enhances Dkk1 transcription. Msx2 variants with either increased (P148H) or absent (T147A) intrinsic DNA binding activity demonstrate that Msx2 inhibits Dkk1 transcription via antagonistic protein-protein interactions. Systematic 5′- and 3′-deletion analyses of the Dkk1 promoter (LUC reporter) mapped Msx2-dependent suppression to nucleotides +165 to +234 relative to the transcription initiation site. Increased expression of the adipogenic transcription factor c/EBPα sequesters Msx2, and upregulates Dkk1 gene transcription. Chromatin immunoprecipitation (ChIP) assays demonstrate that Msx2 reduces the stable interaction of PolII with Dkk1 gene. Inhibition is distinctly enhanced with distance downstream of the transcription initiation site, indicating that both initiation and elongation must be affected by Msx2. Thus, Msx2 promotes osteogenesis and prevents adipogenic gene expression in part via paracrine Dkk1 signals. Msx2 suppression of Dkk1 promotes canonical Wnt signaling and osteogenic differentiation. Conversely, inhibition of Msx2 by c/EBPα upregulates Dkk1, inhibits canonical Wnt signaling, and promotes adipogenic differentiation. Stable association of PolII with the Dkk1 gene is negatively regulated by Msx2, and represents an important transcriptional regulatory step during osteogenic differentiation of calvarial mesenchymal progenitors.

Disclosures: S. Cheng, None.

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ERK1 and ERK2 Are Essential for Osteoblast Differentiation. T. Matsushita*¹, G. Landreth*², S. Murakami¹¹Orthopaedics, Case Western Reserve University, Cleveland, OH, USA, ²Neurosciences, Case Western Reserve University, Cleveland, OH, USA.

To examine the roles of ERK MAPK in bone formation, we inactivated ERK2 using the Cre-loxP system. Inactivation of ERK2 in mesenchymal cells using the Prx 1-Cre transgene in the ERK1-null background caused severe bone defects in the limbs and calvaria. In severely affected long bones, no distinct cortical bone was formed. The lambdoid suture in calvaria failed to close postnatally. While normal levels of Runx2, Osterix, Atf4, and Colla1 expression were observed in in situ hybridization, Osteocalcin expression was abolished. These observations indicate that osteoblast differentiation was blocked after Osterix expression and before differentiation into fully mature osteoblasts. Furthermore, loss of ERK1 and ERK2 caused ectopic cartilage formation in the perichondrium, strongly suggesting that osteo-chondroprogenitor cells in the perichondrium were arrested in their differentiation into osteoblasts and instead differentiated into chondrocytes. Immunohistochemical analysis showed decreased beta-catenin protein levels in the perichondrium, suggesting reduced Wnt signaling. Loss of ERK1 and ERK2 in chondrocytes also resulted in severe disorganization of the epiphyseal cartilage. While the cartilage anlage was normally formed, chondrocytes failed to form columnar structures in the growth plate. There was a striking widening of the zone of hypertrophic chondrocytes. Furthermore, TRAP-positive osteoclasts were decreased, suggesting MAPK-mediated regulation of osteoclastogenesis by mesenchymal cells. Similar cartilage phenotype was observed when ERK2 was inactivated using the Col2a1-Cre transgene in the ERK1-null background. As a complementary experiment, we generated transgenic mice that express a constitutively active mutant of MEK1 under the control of a prx1 promoter. The transgene was expressed in the developing limb bud, the perichondrium and periosteum of the long bones, and the lambdoid suture in the cranium. These mice showed a delay in the formation of cartilage anlage, synostosis of long bones, fusion of carpal and tarsal bones, and accelerated closure of the lambdoid suture. These mice showed a thickening of Runx2-expressing periosteum and a dramatic increase in the cortical bone formation. Our observations indicate that ERK1 and ERK2 are essential for osteoblast differentiation, and increased MAPK signaling promotes bone formation. ERK1 and ERK2 inhibit ectopic cartilage formation in the perichondrium, and increased MAPK signaling inhibits the formation of cartilage anlage. Based on these observations, we propose that ERK1 and ERK2 play essential roles in the lineage specification of mesenchymal cells.

Disclosures: T. Matsushita, None.

1142

Polycystin-1 Selective Activation of Runx2-II Isoform Transcription Is Mediated Through the Calcium-PI3K-Akt Pathway. Z. S. Xiao, S. O. Zhang*, B. S. Magenheimer*, J. P. Calvet*, L. D. Quarles. The Kidney Institute, University of Kansas Medical Center, Kansas City, KS, USA.

The presence of primary cilia and polycystins 1 (PC1) and 2 (PC2) in osteoblasts and osteocytes, suggest that these molecules may form a mechanosensing complex in bone. At present, data supporting this hypothesis is limited to the observation that PC1-deficient (Pkd1 mutant) mice have osteopenia that is associated with a selective decrease in the expression of the Runx2-II isoform in bone. To further establish the role of primary cilia and polycystins in regulating bone cell function, we investigated PC1 and PC2 expression in primary cilia and examined the signal transduction pathways linking PC1 to Runx2-II expression in osteoblasts. Using immunofluorescent staining with antibodies to PC1, PC2, and the primary cilium marker alpha tubulin, we found that PC1 and PC2 co-localize to primary cilia in MC3T3-E1 osteoblasts. To further establish the role of Runx2-II in mediating PC1 effects on bone, we crossed heterozygous Pkd1 and Runx2-II mutant mice to create double heterozygous mice that are deficient in both PC1 and Runx2-II. Double heterozygous Pkd1 and Runx2-II mice exhibited a significantly greater reduction in bone mineral density than their heterozygous littermates, supporting the functional role of Runx2-II as a downstream signal for PC1 regulation of osteoblast function in vivo. To investigate the intracellular pathways linking PC1 to Runx2-II, we examined the effects of over-expressed PC1 C-tail constructs on P1 and P2 promoter-luciferase activity, intracellular calcium levels and PI3K/Akt pathways in MC3T3E1 osteoblasts. We found that PC1 C-tail constructs resulted in a significant increase in intracellular calcium and Runx2 P1 but not P2 promoter activity. Site-directed mutagenesis of critical amino acids in the coil-coil domain of the PC1 C-tail construct necessary for coupling to PC2 completely abolished PC1 C-tail-stimulated Runx2-II P1 promoter activity. Moreover, both PI3K (LY294002) and Akt inhibitors blocked the ability of PC1 C-tail-constructs to increase Runx2-II P1 promoter activity. Additional promoter analysis identified a PC1 responsive region in an area between positions −420 to −350 that contains both putative NF1 and AP-1 binding sites. These findings indicate that a primary cilia-polycystin complex is present and is selectively coupled to Runx2-II gene expression in osteoblasts through intracellular calcium and PI3K-AKT signaling pathways.

Disclosures: Z.S. Xiao, NIAMS (RO1-AR049712) and NIDDK (P50-DK057301) 2. This study received funding from: NIAMS (RO1-AR049712) and NIDDK (P50-DK057301).

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Identification of the Homeobox Protein, Prx1 (MHox), as a Regulator of Osterix Expression and Mediator of TNF Action. X. Lu*¹, G. R. Beck², L. C. Gilbert*¹, T. P. Conrads*³, M. J. Kern*⁴, M. S. Nanes¹¹Div Endocrinology, Dept Medicine, Emory Univ / VAMC, Atlanta, GA, USA, ²Div Endocrinology, Dept Medicine, Emory Univ, Atlanta, GA, USA, ³Div Clin Pharm, Hillman Cancer Inst, Univ Pittsburgh, Pittsburgh, PA, USA, ⁴Dept Cell Biology and Anatomy, MUSC, Charleston, SC, USA.

A network of transcription factors (TF) regulates the commitment of pluripotent mesenchymal cells to the osteoblast lineage. Identifying these factors is key to understanding skeletal development and lifelong renewal of bone forming cells. Osterix (Osx, SP7) is an important TF required for late chondrocyte and osteoblast differentiation as deletion results in a cartilaginous skeleton. We recently described the transcriptional regulation of Osx by defining 2 upstream promoters that direct synthesis of 2 mRNA species. A potent TNF/MAPK sensitive suppressor region was identified within the Osx promoter that was highly protein bound, inactivated by mutation, and functionally transferable to another promoter. We considered that identification of the proteins bound to the suppressor would reveal early TF regulators of osteoblast differentiation. Nuclear extracts from TNF-treated and untreated mesenchymal cells (C3H) were incubated with a DNA-Sepharose probe containing the 18 bp suppressor sequence. Tryptic fragments of the bound proteins were analyzed using tandem mass spectroscopy. Candidate TF were identified by subtraction analysis of identified protein fragments from the 2 treatment groups. 7 transcription factors were found among 62 unique nuclear proteins in the murine genome database. ChIP assay confirmed specific binding of some but not all of these to the Osx promoter suppressor region. One TF, the paired related homeobox protein (Prx1, MHox), had prev