ASBMR 29th Annual Meeting

M092

Actin Binding Activity in the B Subunit Is Required for Sorting Vacuolar H⁺-ATPases (V-ATPase) in Osteoclasts.

L. S. Holliday¹, J. Zuo*¹, R. Ricofort*¹, E. Serrano*¹, A. Cooper*¹, S. S. Grieshaber*². ¹Orthodontics, University of Florida College of Dentistry, Gainesville, FL, USA, ²Oral Biology, University of Florida College of Dentistry, Gainesville, FL, USA.

Actin binding activity is required for transport of virally-expressed B1 subunit of the vacuolar H⁺-ATPase (V-ATPase) to the ruffled membranes of osteoclasts(1). We are seeking the mechanistic basis for this requirement. Gel filtration chromatography, immunoprecipitations, differential detergent extractions, differential centrifugations and Native Blue gel electrophoresis were used to determine the assembly state of V-ATPases that bound actin in osteoclasts. Cholera toxin beta chain, which binds ganglioside GM, (GM1), lysotracker probes and anti-V-ATPase subunit antibodies were used to probe the pathways responsible for ruffled membrane formation in murine osteoclasts. V-ATPase subunit B1 and B1 mut (which does not bind actin) were expressed using adeno-associated virus. LY294002 was used to block phosphatidylinositol-3 kinase activity. Our results were consistent with B subunit interacting with actin only as part of intact, membrane-associated V-AT Pases. This suggests that the actin binding activity is required for sorting V-AT Pases into a specific vesicular trafficking pathway. GM1 was identified as a “surprising” marker for V-AT Pase-rich vesicles in inactive osteoclasts and for ruffled membranes of resorbing osteoclasts. GM1 is normally endocytosed from the plasma membrane and sorted into a retrograde pathway through the golgi to the endoplasmic reticulum. Virally-expressed B1 subunit co-localized extensively with GM1, but B1 mut did not. Sorting along this retrograde pathway has been reported to require the activity of vps34p (class III PI 3-kinase) and phosphatidylinositol 3-phosphate 5-kinase (PIK fyve). When PI 3-kinases were blocked using LY294002, large vacuoles that were rich in GM1 and V-AT Pases quickly became apparent, a result consistent with disruption of the retrograde pathway. We also found that PIK fyve was upregulated 5-fold during osteoclastogenesis suggesting it plays an important role in osteoclasts. Our data indicate that the actin binding activity of subunit B of V-AT Pase enables sorting of V-AT Pases away from their default locations in the endocytic pathway into GM1-rich vesicles that then fuse with the plasma membrane as osteoclasts activate to form ruffled membranes. We are testing the hypothesis that this actin-dependent sorting targets V-AT Pases into the vps34p/PIK fyve-dependent retrograde pathway, which may have been adapted in osteoclasts for the formation of ruffled membranes.

1. Zuo J, Jiang J, Chen SH, Vergara S, Gong Y, Xue J, Huang H, Kaku M, Holliday LS 5/2006, J Bone Miner Res 21:714–721.

Disclosures: L.S. Holliday, None.

This study received funding from: NIH NIAMS R01 AR-47959.

M093

See Sunday Plenary Number S093

M094

Alterations in MicroRNAs and GW Bodies Are Associated with Osteoclastogenesis.

J. Zuo*¹, A. Jakymiw*², K. M. Pauley*², E. K. Chan*², L. S. Holliday¹. ¹Orthodontics, University of Florida College of Dentistry, Gainesville, FL, USA, ²Oral Biology, University of Florida College of Dentistry, Gainesville, FL, USA.

Understanding and manipulating osteoclastogenesis requires knowing the mechanisms that regulate gene expression during this process. Micro RNAs, small endogenous RNAs, about 22 nucleotides in length, are thought to use the elements of the RNA-interference pathway to post-transcriptionally down-regulate the expression of protein-coding genes. Recent reports have identified GW bodies (mammalian P bodies) as sites that are important for micro RNA-dependent regulation. We screened for differential expression of micro RNAs in RAW 264.7 cells grown with or without treatment with recombinant Receptor Activator of Nuclear Factor Kappa B-ligand (RANKL) using a micro RNA microarray. Some results were then confirmed by real time PCR. GW bodies, sites of micro RNA-based regulation were identified using specific antibodies. We identified 9 micro RNAs that were strongly downregulated (expressed to not expressed) and 6 micro RNAs that were strongly upregulated (not expressed to expressed) in response to RANKL during osteoclastogenesis. In addition, the members of the let-7 family were expressed at high levels in both stimulated and unstimulated RAW cells, and all increased from 2–3 fold in response to RANKL. Six of the twelve subunits of vacuolar H⁺-AT Pase as well as a number of other proteins known to be upregulated during osteoclast formation are predicted targets (MIRANDA and TargetScan 3.1, Sanger Institute) of one or more of the downregulated micro RNAs. MicroRNAs 146 and 155, which have been reported to be upregulated during inflammatory responses in macrophages, were strongly upregulated in response to RANKL. GW bodies increased in number in RAW 264.7 cells as they differentiated into osteoclast-like cells. In summary, our data support the hypothesis that regulation of gene expression by micro RNAs plays a role in osteoclastogenesis. Understanding this regulation may identify novel categories of pharmaceuticals for the treatment of osteoclast-mediated diseases.

Disclosures: L.S. Holliday. None.

This study received funding from: University of Florida College of Dentistry Seed Grant.

M095

Molecular Cloning and Characterization of Mouse RANK Gene Promoter Region.

J. Ishii*¹, R. Kitazawa¹, T. Kondo¹, K. Mori¹, K. P. McHugh², S. Kitazawa¹. ¹Division of Pathology, Kobe University Graduate School of Medicine, Kobe, Japan, ²Beth Israel Deaconess Medical Center, Orthopaedic Research, Boston, MA, USA.

Receptor Activator of NF-κB (RANK), expressed on osteoclasts and their precursors, is a receptor for RANK ligand (RANKL), a membrane-bound cytokine expressed in stromal/osteoblastic cells; signals transduced by RANK-RANKL interaction are prerequisite for differentiation, activation and maintenance of osteoclasts. To elucidate the mechanism regulating RANK gene expression during osteoclastogenesis, we cloned and characterized the 5′-flanking region of the mouse RANK gene. A 6-kb fragment containing this region was subcloned from a BAC clone and sequenced. The transcription start sites were determined by the 5′-RACE method. A 1-kb fragment upstream of the transcription start sites was ligated to the promoterless and enhancerless pGL3-basis vector (pGL3-RANK) and transfected into the mouse monocyte/macrophage cell line, RAW264.7, by the liposome mediated technique. The transfected cells were subjected to luciferase assay. Transient transfection studies showed significant pGL3-RANK promoter activity in RAW 264.7 cells. The basic promoter region of the mouse RANK gene lacked canonical TATA boxes but contained four Sp-1 binding sites located at −98, −79, −65, and −58, and four transcription start sites located at −54, −41, −27, and −23 upstream of the translation start site. Putative binding sites for PU.1 (-480), CRE/AP-1 (-240), E-box (-510, −260, −100), and NFAT (nuclear factor of activated T cells) (-720, −550, −370) were located within the 1-kb fragment of the transcription start sites. Co-transfection studies with the use of the expression vectors of MITF and PU.1 revealed that MITF and PU.1 increased RANK promoter activity three-fold and two-fold, respectively, and six-fold synergistically. Since lipopolysaccharide (LPS) is one of the factors that influences RANK gene expression in osteoclasts and their precursors, we examined the molecular mechanism whereby LPS affects RANK expression. Quantitative real-time RT-PCR showed that the expression of RANK as well as MITF and PU.1 mRNA was suppressed by treatment with LPS. Mirroring the decrease of MITF and PU.l mRNA to 70% by LPS treatment, transient transfection studies with pGL3-RANK revealed that short-term treatment with LPS also decreased the promoter activity to 70%. We therefore speculated that RANK gene expression is influenced by two transcription factors, MITF and PU.1, both of which are prerequisite for macrophage and osteoclast differentiation, and that LPS suppresses the RANK gene, at least in part, by downregulating the expression of MITF and PU.1 genes.

Disclosures: J. Ishii, None.

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

M096

See Sunday Plenary Number

M097

Iron Uptake Through the Transferrin Receptor 1 Promotes Osteoclast Differentiation and Function.

K. Ishii*¹, S. Takeshita¹, N. Shimohata*², M. Ito³, H. Aburatani*⁴, S. Taketani*⁵, K. Iwai*², K. Ikeda¹. ¹Department of Bone and Joint Disease, National Center for Geriatrics and Gerontology (NCGG), Obu, Japan, ²Department of Molecular Cell Biology, Osaka City University Graduate School of Medicine, Osaka, Japan, ³Department of Radiology, Nagasaki University School of Medicine, Nagasaki, Japan, ⁴Genomescience Division, University of Tokyo School of Medicine, Tokyo, Japan, ⁵Department of Biotechnology, Kyoto Institute of Technology, Kyoto, Japan.

Osteoclasts are acid-secreting polykaryons that are rich in mitochondria. However, how mitochondrial biogenesis is stimulated during osteoclast development remains unknown. Gene expression analysis with mouse genome arrays containing 39,000 transcripts revealed transferrin receptor (TfR) I mRNA, which was barely detectable in bone marrow macrophages (BMMs), was markedly induced along with osteoclast differentiation. Electrophoretic mobility shift assays revealed binding of iron regulatory protein (IRP)2 to iron-responsive element (IRE) in the TfR1 mRNA 3′UTR to be markedly increased during osteoclastogenesis. Thus, IRP2 senses a heme/iron deficient state in osteoclasts and, by binding to IRE of TfR1, leads to the upregulation of TfR1 through stabilization of its mRNA. Through the increased TfR1 on the cell surface, the addition of holo-Tf as well as apo-Tf markedly stimulated the formation of TRAP-positive osteoclasts from BMMs dose dependently, which effect was inhibited by iron chelation with desferrioxamine (DFO). The pit area resorbed by mature osteoclasts was also significantly increased by Tf and decreased by DFO, suggesting that TfR1 on osteoclasts is functional, and that iron uptake through TfR1 not only promotes osteoclast formation but also the bone-resorbing function of mature osteoclasts. Gene chip analysis disclosed that almost all of the mRNAs coding for respiratory complexes I-V of mitochondria were concomitantly upregulated during osteoclastogenesis. As many of these heme-containing proteins are involved in electron transport, the mitochondrial membrane potential was increased by RANKL, which was markedly enhanced by Tf. Finally, pre-treatment of OVX mice with DFO significantly inhibited bone loss and the level of a bone resorption marker, CTX, suggesting that iron is an important regulator of osteoclastic bone resorption in vivo. In conclusion, uptake of iron through TfR1 and subsequent incorporation of heme/iron into mitochondrial respiratory proteins are critical for osteoclast development and bone-resorbing function.

Disclosures: K. Ishii, None.

M098

Role of P-Rex1 in Osteoclast Differentiation.

H. Kang*¹, D. Wu*². ¹Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT, USA, ²Pharmacology, Yale University, New Haven, CT, USA.

Guanine nucleotide exchange factors (GEFs) directly activate Rho family GT Pases in response to various extracellular stimuli and play a crucial role in actin cytoskeleton rearrangement, an essential part for the differentiation and function of osteoclasts. Despite of presumed implication of GEFs in osteclast biology, in vivo role of GEFs in osteoclasts has been poorly demonstrated. Here, we test possible involvement of P-Rex1 in osteoclast formation and ultimately in bone remodeling. P-Rex1 was identified as a phosphatidyl inositol (3,4, 6) trisphosphate- and Gbetagamma-regulated GEF for Rac that is implicated in chemotactic response in neutrophils. In order to examine the role of P-Rex1, P-Rex1 knockout mice were generated by inactivating Db1 homology domain, which is a essential region for GEF function to activate target Rho GT Pases. Osteoclast differentiation of bone marrow-derived macrophages (BMMs) was induced by recombinant RANKL (receptor activator of NFκB ligand). The osteoclast formation was estimated using tartrate-resistant acid phosphatase (TRAP) staining. BMMs from P-Rex1 knockout mice showed a defect in forming TRAP-positive multinuclear cells (controls were syngenic wild-type mice). This result suggests that P-Rex1 is among the regulators for development of functional multinuclear TRAP-positive osteoclasts. Given the fact that nitrogen-containing bisphosphonates, which target small G proteins in osteoclasts, are used as antiresorptive agents in the treatment of metabolic bone diseases, identification of key GEFs in osteoclasts may contribute to more specific pharmaceutical targeting of small G proteins.

Disclosures: H. Kang, None.

This study received funding from: NIH.

M099

See Sunday Plenary Number S099

M100

BMP-2-Inducible Osterix Increases Osteoblastic CSF-1 and RANKL/OPG Ratio to Induce Osteoclast Maturation.

C. C. Mandal¹, G. Ghosh-Choudhury*², H. Drissi³, N. Ghosh-Choudhury⁴. ¹Pathology, UTHSCSA, San Antonio, TX, USA, ²Medicine, UTHSCSA, GRECC, STVHCS, San Antonio, TX, USA, ³Orthopaedics, University of Rochester Medical Center, Rochester, NY, USA, ⁴Pathology, UTHSCSA, STVHCS, San Antonio, TX, USA.

Bone remodeling results from balanced action of osteoblasts and osteoclasts. BMP-2-inducible master transcription factor osterix (Osx) is essential for osteoblast differentiation and mature bone formation. Through a paracrine cytokine signaling loop, osteoblasts contribute to differentiation and maturation of osteoclasts by producing two critical factors, CSF-1 and RANKL. Expression of Osx in two different mesenchymal progenitor cells, 2T3 and C2C12, resulted in increased expression of CSF-1 and RANKL mRNA. In contrast, Osx reduced mRNA expression of osteoprotegerin (OPG), a RANKL antagonist and negative regulator of osteoclast differentiation. To investigate the mechanism, we tested the effect of Osx on CSF-1, RANKL and OPG transcription using luciferase reporter constructs driven by these promoters. BMP-2 as well as Osx independently increased transcription of CSF-1 and RANKL, while OPG transcription was significantly reduced. However, expression of Osx additively increased CSF-1 and RANKL transcription and prevented transcription of OPG Thus osteoblast-specific Osx maintains significantly high RANKL-OPG ratio. In an effort to elucidate the mechanism, Genomatix analysis of the CSF-1 promoter revealed the presence of consensus Osx binding site between −287 bp and −246 bp (from transcription start site). Nuclear extracts prepared from BMP-2-stimulated C2C12 cells were tested in electrophoretic mobility shift assay (EMSA) using ³²P-labeled double stranded oligonucleotide representing this Osx site in the CSF-1 promoter. BMP-2 significantly increased protein-DNA complex formation, the specificity of which was determined by cold competition analysis. Use of Osx antibody in EMSA showed the presence of Osx in the DNA-protein complex. To further characterize the role of Osx in osteoclastogenesis, we stably transfected HA-tagged Osx into C2C12 and 2T3 cells respectively. Anti-H A immunoblotting of the cell lysates confirmed expression of Osx in both these cells. These cells were used in coculture assay to examine their osteoclastogenic property. Both C2C12 and 2T3 cells overexpressing Osx significantly increased TRAP positive multinucleated osteoclast formation. Together these data for the first time show a mechanism for the master transcription factor Osx action, which regulates expression of osteoblastic cytokines CSF-1, RANKL and OPG Furthermore, we provide the first direct evidence confirming the contribution of Osx in osteoclast maturation.

Disclosures: C.C. Mandal, None.

This study received funding from: NIAMS, VA.

M101

Use of Nanogels as a Drug Delivery System for Tumor Necrosis Factor-alpha Antagonist in Murine Bone Resorption Model.

C. N. R. Alles*¹, M. H. Mian*¹, N. S. Soysa*¹, H. Saito*², K. Aoki², H. Akiyoshi*¹, K. Ohya². ¹Center of Excellence for Frontier Research on Molecular Destruction and Reconstruction of Bone, Tokyo Medical and Dental University, Tokyo, Japan, ²Section of Pharmacology, Tokyo Medical and Dental University, Tokyo, Japan.

Our previous study has shown that a TNF antagonist peptide (WP9QY/W9) inhibits RANKL-induced bone resorption (J Clin Invest 116:1525–1534, 2006). Easy degradation in serum is a major drawback in clinical application of this peptide. On the other hand nanogels have emerged as a promising carrier in delivering proteins and peptides. Hence we hypothesized cholesterol group-bearing pullulan (CHP) nanogel as a candidate in delivering TNF-α antagonist in vivo. We reported previously (C.N.R Alles et al. ASBMR 2006) using C57BL/6J mice on low Ca diet injected with veh, CHP nanogel, W9 or CHP-W9 complex, that twice a day subcutaneous injections of the CHP-W9 complex prevented the reduction in BMD in mice on low Ca diet. From these BMD data it was not clear whether the formation was increased or the resorption is decreased. To this end histomorphometry was performed at the sites of metaphysis of tibiae. CHP-W9 complex prevented the decrease of bone volume parameters significantly: trabecular volume (p < 0.001), trabecular thickness (p < 0.05) and trabecular number (p < 0.001) and prevented the increase of trabecular separation (p < 0.001) compared to low Ca-veh group. CHP-W9 complex prevented the increase of osteoclast surface (p < 0.05) and number (p < 0.05) to bone surface brought by low Ca as well. Conversely the complex substantially mitigated the increase of osteoblast surface to bone surface (p < 0.005) and mineral apposition rate (MAR) (p < 0.005). Similarly the bone resorption parameter, urine deoxypyridinoline in low Ca-veh group (p < 0.05) was significantly increased compared to the normal Ca-veh and CHP-W9 complex groups. To further confirm the in vivo complexation of W9 with CHP nanogel, in vitro stability test was performed. W9 was easily dissolved in alkali whereas it tends to aggregate in low pH. By using Dynamic Light Scattering (DLS) analysis, we observed the association of peptide with nanogels. When peptide is added with CHP nanogel, even at pH 6.5, W9 could completely dissolve in nanogels. Taken together these results suggested that the twice a day subcutaneous injection of CHP-W9 complex was effective in preventing the bone resorption brought by low Ca feeding as agreeing with previous study which use 8x/day injections. This implied that the complex was stable in serum and achieved a constant release corroborating our in vitro data. Hence our study confirmed that nanogels could be used as an effective carrier to deliver W9 peptide.

Disclosures: C.N.R. Alles, None.

M102

Development of Cell-Based Assays for Identifying Antiresorptive Compounds Targeting RANK Signaling through High Throughput Screening.

J. Ashley*¹, Z. Shi*¹, E. Mills*¹, D. Clements*¹, T. Chen*², X. Feng¹. ¹Dept. of Pathology, University of Alabama at Birmingham, Birmingham, AL, USA, ²Dept. of Chemical Biology & Therapeutics, St. Jude Children's Research Hospital, Memphis, TN, USA.

Current antiresorptive drugs either lack satisfactory efficacy or cause serious side effects. The unraveling of the RANKL/RANK/OPG system has provided an opportunity to develop more effective antiresorptive drugs, but agents currently under development targeting RANK/RANKL interactions are likely to have side effects due to the role of the system in other biological processes. We recently identified three functional TRAF-binding motifs (Motif 1: PFQEP^369–373, Motif 2: PVQEET^559–564 and Motif 3: PVQEQG^604–609) in the RANK cytoplasmic domain. While Motif 2 and Motif 3 are very potent in mediating osteoclast formation and function, Motif 1 plays a minimal role in these processes, instead being the primary actor in RANKL-mediated immune function. Thus, Motifs 2 and 3 can serve as potent and specific antiresorptive targets.

We have developed cell-based high throughout screening (HTS) assays for identifying compounds inhibiting Motifs 2 and 3 signaling. The assays contain three key components: RAW264.7 (RAW) cells, an NF-κB-responsive luciferase reporter, and a chimeric receptor consisting of human Fas external domain linked to the transmembrane and cytoplasmic domains of normal or mutant RANK. First, we established a RAW cell line stably expressing the luciferase reporter, which showed a 6-fold induction of luciferase activity in response to RANKL treatment. We then prepared and expressed the following chimeric receptors in the stable RAW cell line: M2 (Motifs 1 and 3 in RANK domain are mutated), M3 (Motifs 1 and 2 in RANK domain are mutated). Cells expressing M2 or M3 can be used in HTS for identifying compounds inhibiting Motifs 2 and 3, respectively. For example, treatment of the cells expressing M2 with anti-human Fas antibody (hFas-AB) causes recruitment of TRAF3 to Motif 2, leading to the formation of a signaling complex and activation of NF-κB, which, in turn, activates the luciferase reporter. If a compound blocks the TRAF3-Motif 2 interaction or the formation of signaling complex, no NF-κB will be activated by the M2 receptor and subsequent reporter activation will be reduced. Preliminary data indicate that cells expressing M2 and M3 can induce ∼1.5-fold reporter activity following hFas-AB treatment. We have also developed additional assays for hit follow up and are now improving assay efficiency by optimizing hFas-AB concentration and treatment time as well as selecting subclones with higher levels of induction. In addition, we are seeking partnerships with groups interested in utilizing our assays.

Disclosures: J. Ashley, None.

M103

See Sunday Plenary Number S103

M104

Cytoplasmic Terminus of a V-ATPase Accessory Subunit Ac45 Is Required for Proper Interaction with VO Domain Subunits and Efficient Osteoclastic Bone Resorption.

T. Cheng*¹, H. Feng*¹, K. H. M. Yip*¹, N.J. Pavlos*¹, A. Carrello*¹, R. Seeber*², K. Eidne*², M. H. Zheng¹, J. Xu¹. ¹Centre for Orthopaedic Research, University of Western Australia, Perth, Australia, ²Western Australia Institute for Medical Research, University of Western Australia, Perth, Australia.

Solubilization of mineralized bone by osteoclasis is largely dependent on the acidification of the extracellular resorption lacuna driven by vacuolar type proton pump (V-AT Pases) polarized within the ruffled border membranes. V-AT Pases consist of two functionally and structurally distinct domains, V1 and V0. The peripheral cytoplasmically oriented V1 domain drives ATP hydrolysis which necessitates the translocation of protons across the integral membrane bound V0 domain. Here, we demonstrate that an accessory subunit, Ac45, interacts with the V0 domain and contributes to V-AT Pase-mediated function in osteoclasts. Consistent with its role in intracellular acidification, Ac45 was found to be localized to the ruffled border region of polarized resorbing osteoclasts and enriched in pH-dependent endosomal compartments which polarized to the ruffled border region of actively resorbing osteoclasts. Interestingly, truncation of the 26aa-residue cytoplasmic tail (aa437–463) of Ac45 (Ac45C-mutant) which encodes an autonomous internalization signal was found to impair bone resorption in vitro. Furthermore, immunoprecipitation and bioluminescence resonance energy transfer (BRET) analysis revealed that although both wild type Ac45 and Ac45C-mutant were capable of associating with V-ATPase subunits a3, c, c″, and d, deletion of the cytoplasmic tail altered its binding affinity with a3, c″ and d. In all, our data suggest that the cytoplasmic terminus of Ac45 contains essential elements necessary for its proper interaction with V-AT Pase and efficient osteoclastic bone resorption.

Disclosures: T. Cheng, None.

M105

Modeling Role of ER in Transcellular Calcium Flux in Bone Resorbing Osteoclasts.

H. K. Datta. Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, United Kingdom.

It has been shown that Ca²⁺ produced in the osteoclast (OC) hemivacuole by the action of secreted acid is continually transported to the basolateral surface; thus, preventing accumulation of the cation in the resorptive lacuna. Ca²⁺ may be taken up through store-operated Ca²⁺ channels in the basal membrane, the site of the resorptive hemivacuole, and is pumped into the basal endoplasmic reticulum (ER) by Ca²⁺-ATPases. Ca²⁺ then diffuses through the lumen of the ER from the base to the apical region, where it is released into the cytosol via specific Ca²⁺ channels i.e. inositol 1,4,5-triphosphate (IP₃) and ryanodine receptors. Ca²⁺ is then pumped out of the cell by plasma membrane Ca²⁺-AT Pases which are located in the apical membrane. The route through the lumen of the ER is attractive because of the abundance of calreticulin, a high capacity Ca²⁺-binding protein which is involved in Ca²⁺ homeostasis. In view of these observations, we attempted to define the role of ER and have modeled the calcium disposal through bone resorbing osteoclast. The modeling was performed using Virtual Cell Software, available from National Resource for Cell Analysis and Modeling (NRCAM), at the website: http://www.nrcam.uchc.edu. The model is comprised of four physiological structures, namely hemivacuole, cytosol, ER and extracellular fluid space, giving four respective resolved geometric subdomains. For these resolved structure mixed boundary conditions Neuman and and Dirichlet were chosen. The kinetics of calcium pumps and channels are given by linear equation; SERCA pump uptake is modelled by a Hill type equation and channels influx is catalyzed by reaction described by the Michaelis-Menton equation. 2-D geometry involves extensive network, two exact mirror image of the osteoclast-hemivacule; the line of symmetry of two opposing actively resorbing osteoclast therefore passes through the resorptive hemivacuole. Other relevant parameters, such as constants, functions, and volumes were selected based on published literature. The results of simulations experiments were compared with the experimental observations, and were found to successfully mimic in vivo observations, such as elevated [Ca²⁺]_ER and maintenance of [Ca²⁺]_i within know physiological range but with high localized [Ca²⁺]_i (so-called calcium puffs) near the hemivacuole site. Perturbation experiments performed established a critical role of the ER and its key components in the transcellular Ca²⁺-transport and buffering. The model has ER which is functionally continuous unit, where Ca²⁺ released from the apical ER is quickly replenished from the base. In conclusion, OC provides an excellent model to test recent suggestion that ER is an important conduit for the transcellular transport of Ca²⁺ in polarized cells.

Disclosures: H.K. Datta, None

M106

Tetracyclines [Doxycycline(Dox) and Minocycline(Mino)] Effectively Inhibit Osteoclast Differentiation and Function.

S. Kinugawa*¹, M. Koide², T. Ninomiva*², H. Nakamura³, Y. Kobayashi², N. Takahashi², N. Udagawa⁴. ¹Graduate School of Oral Medicine, Matsumoto Dental University, Siojiri, Japan, ²Institute for Oral Science, Matsumoto Dental University, Siojiri, Japan, ³Department of Oral Histology, Matsumoto Dental University, Siojiri, Japan, ⁴Department of Biochemistry, Matsumoto Dental University, Siojiri, Japan.

Tetracyclines (Dox and Mino) are widely used in the treatment of periodontal disease to suppress the growth of bacteria. Tetracyclines also have been shown to prevent bone loss, but the mechanism of action of tetracyclines to inhibit bone resorption is not elucidated. Here, we examined how tetracyclines inhibit bone resorption. We found that both Dox and Mino directly acted on osteoclast (OC) precursors to inhibit their differentiation into OCs, and on mature OCs to inhibit their function.

(1) Increasing concentrations of Dox and Mino were added to co-cultures of mouse bone marrow cells (BMCs) and osteoblasts (OBs) in the presence of PGE₂ and 1α,25(OH)₂D₃. Both Dox and Mino dose-dependently inhibited OC formation induced by PGE₂ and 1α,25(OH)₂D₃. (2) Effects of Dox and Mino on RANKL and COX-2 mRNA expression in OB cultures were examined by RT-PCR. Treatment of OBs with 1α,25(OH)₂D₃ up-regulated RANKL mRNA expression. Dox and Mino added to OB cultures failed to affect 1α,25(OH)₂D₃-induced RANKL mRNA expression. Neither Dox nor Mino induced OB COX-2 mRNA expression. (3) Mouse bone marrow macrophages (BMMs) and human CD14-positve monocytes were cultured as OC precursors in the presence of RANKL and M-CSF with increasing concentrations of Dox and Mino. RANKL and M-CSF stimulated mouse and human OC formation. Dox and Mino dose-dependently inhibited mouse and human OC formation with the complete inhibition at 20 μg/ml of either compound. (4) When mouse OCs were cultured on dentine slices, they formed many resorption pits on the slices in the presence of OBs. Addition of Dox and Mino at 20 μg/ml also inhibited the pit-formation activity of OCs. (5) Western blot analysis demonstrated that the treatment of BMMs with lipopolysaccharide stimulated phosphorylation of ERK, a MAP kinase, which was completely inhibited by pretreatment with Dox and Mino. Thus, both Dox and Mino directly inhibited differentiation and function of not only mouse but also human OCs, These inhibitory effects on bone resorption are proposed to be associated with the suppression of MAP kinases. These results suggest that Dox and Mino have great therapeutic potential for the periodontal therapy to suppress alveolar bone loss.

Disclosures: S. Kinugawa, None.

M107

M-CSF Receptor C-fms Antibody Inhibits Mechanical Stress-Induced Root Resorption.

H. Kitaura, Y. Fuiimura*, M. Yoshimatsu*, T. Eguchi*, H. Kohara*, N. Yoshida*. Division of Orthodontics and Dentofacial Orthopedics, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan.

Root resorption is occurred during orthodontic treatment, and lead to serious problem. However, because a suitable experimental animal model has been lacking, the biological mechanism is not clearly understood. We established mechanical stress-induced osteoclastogenesis model in mice using orthodontic tooth movement in which an Ni-Ti coil spring was inserted between the upper incisors and the upper first molar. And we found root resorption was occurred in this model during mechanical loading. The formation of osteoclasts depends on macrophage-colony-stimulating factor (M-CSF) and a ligand for the receptor activator of necrosis factor κB (RANKL). It has recently been reported that administration of an antibody of the M-CSF receptor c-fms completely blocked pathological osteoclastogenesis and bone erosion induced by TNF administration or inflammatory arthritis. This study aimed to examine the effect of monoclonal anti-c-fms antibody to a root resorption in mechanical stress-induced osteoclastogenesis model. We injected 10 micro gram anti-c-fms antibody in PBS daily into a local site for 12 days during mechanical loading. We assessed the root resorption of upper first molar after mechanical loading and carried out histological observations. The amount of the root resorption was evaluated by measuring the resorption area at disto-buccal root of the first molar by an electron microscope. Anti-c-fms antibody significantly inhibited root resorption after mechanical loading (p < 01). Further, the number of osteoclasts stained for TRAP on the root surface in the anti-c-fms antibody injected group was markedly reduced compared with controls. The results suggested that M-CSF and/or its receptor are potential therapeutic targets in mechanical stress-induced osteoclastogenesis, and that injection of an anti-c-fms antibody might be useful for inhibition of mechanical stress-induced root resorption.

Disclosures: H. Kitaura, None.

M108

A Possible Suppressive Role of Galectin-3 in Osteoclastic Bone Destruction Accompanying Adjuvant-induced Arthritis in Rats.

Y. Li*¹, J. Teramachi*¹, K. Nagata*¹, Z. Wu*¹, A. Kukita², A. Akamine¹, T. Kukita¹. ¹Faculty of Dental Science, Kyushu University, Fukuoka, Japan, ²Faculty of Medicine, Saga University, Saga, Japan.

Galectin-3 is a β-galactoside-binding animal lectin having pleiotropic effects on cell growth, differentiation and apoptosis. This lectin has shown to be involved in phagocytosis by macrophages and inflammation. Here we investigated an involvement of galectin-3 in the regulatory process of inflammatory bone resorption in rats with adjuvant-induced arthritis accompanying severe bone destruction in the ankle joints. The protein level of galectin-3 in the ankle-joint-extracts was markedly augmented at week 3 after adjuvant injection, at the time when severe bone destruction was observed. Immunohistochemical analysis revealed an extremely high expression of galectin-3 in macrophages and granulocytes infiltrated in the area of severe bone destruction in the distal tibia. To estimate a role of galectin-3 in osteoclastogenesis and ostoclastic bone resorption, recombinant galectin-3 was added to in vitro systems. Exogenously added recombinant galectin-3 markedly inhibited the formation of osteoclasts in cultures of murine osteoclast precursor cell line (RAW-D cells) as well as in rat bone marrow culture systems for evaluating osteoclastogenesis in a carbohydrate-independent manner. This inhibition was not observed by heat-inactivated galectin-3 and by galectin-7. Although recombinant galectin-3 did not affect signaling through MAP-kinases and NF-κB, it specifically suppressed the induction of NFATc1, a key transcription factor required for osteoclastogenesis. Furthermore, galectin-3 significantly inhibited dentin resorption by mature osteoclasts. Thus, abundant galectin-3 observed in the area of severe bone destruction could act as a negative regulator for the up-regulated osteoclastogenesis accompanying inflammation, that would contribute to preventation of excess bone destruction.

Disclosures: T. Kukita, None.

M109

P2×7 Nucleotide Receptors Couple to Isoform-Specific Activation of PKC in Osteoclasts: Spatio-Temporal Patterning of PKC Translocation Revealed by Live-Cell Imaging.

S. Armstrong, A. Pereverzey*, S. M. Sims*, S. J. Dixon. Physiology and Pharmacology, University of Western Ontario, London, ON, Canada.

Nucleotides, released from cells in response to mechanical or inflammatory stimuli, signal through P2 nucleotide receptors in many cell types including osteoclasts. Osteoclasts express functional P2×7 receptors – calcium-permeable channels that are activated by high concentrations of extracellular ATP. Targeted disruption of the gene encoding the P2×7 receptor leads to increased resorption and reduced skeletal response to mechanical stimuli. However, little is known about the signaling pathways stimulated by P2×7 receptors in osteoclasts. Our purpose was to investigate whether P2×7 receptors couple to activation of protein kinase C (PKC). An early step in activation of PKC involves translocation, with many PKC isoforms moving from the cytosol to the plasma membrane. RAW264.7 mouse monocytic cells were differentiated with RANKL and transiently transfected with plasmids encoding PKC-EGFP fusion proteins or the fluorescent protein EGFP alone. Live-cell confocal imaging was used to monitor changes in the distribution of PKC isoforms in the resulting multinucleated osteoclast-like cells. Within 20–40 s, benzoylbenzoyl-ATP (BzATP, 150 μM, a relatively potent P2×7 receptor agonist) induced translocation of PKCα to the basolateral membrane. Translocation was transient with duration of 5–10 min. In contrast to BzATP, the PKC activator phorbol myristate acetate (PMA) induced sustained translocation of PKCα. UTP (150 μM) or ATP (10 μM), which activate several P2 receptors other than P2×7, failed to induce translocation of PKCα; whereas, ATP (3 mM, a concentration sufficient to activate P2×7) caused translocation. Translocation was inhibited by the selective P2×7 antagonist Brilliant Blue G These data are consistent with involvement of P2×7 receptors. To investigate the role of cytosolic calcium, we simultaneously monitored PKCα location and calcium concentration. BzATP induced transient rise in cytosolic calcium that was temporally correlated with translocation of PKCα. Moreover, removal of extracellular calcium prevented translocation induced by BzATP, but not by PMA. BzATP induced translocation of PKCα and PKCβ1, which are both calcium-dependent PKC isoforms. In contrast, BzATP did not induce translocation of the calcium-independent isoform PKC8. These findings establish that activation of P2×7 receptors induces calcium-dependent translocation of PKC to the basolateral membrane domain of osteoclasts, an aspect of spatio-temporal signaling not previously recognized. Thus, ATP – released in response to inflammatory or mechanical stimuli – may signal through specific PKC isoforms to regulate osteoclast functions in vivo.

Disclosures: S. Armstrong. None.

This study received funding from: The Canadian Arthritis Network & Canadian Institutes of Health Research.

M110

See Sunday Plenary Number S110

M111

Novel Pro-Survival Functions of the Kruppel-Like Transcription Factor Egr2 in Promotion of M-CSF-Mediated Osteoclast Survival Downstream of the MEK/ERK Pathway.

E. W. Bradley*¹, M. M. Ruan*², M. J. Oursler². ¹Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN, USA, ²Endocrine Research Unit, Mayo Clinic, Rochester, MN, USA.

M-CSF promotes differentiation and survival of cells within the monocyte/macrphage cell lineage, including bone resorbing multinucleated osteoclasts. As osteoclast survival is in part mediated by M-CSF, determining the molecular mechanisms of M-CSF-promoted osteoclast survival is crucial in treating pathological bone loss. However, the signaling mechanism M-CSF utilizes to support osteoclast survival is poorly characterized. This study addressed activation of MEK/ERK, a signaling pathway implicated in controlling osteoclast survival, in response to M-CSF and identified a novel pro-survival downstream effector of this pathway. M-CSF transiently activated the MEK/ERK pathway, an effect blocked by chemical inhibition of MEK1/2 or adenoviral expression of dnMEK1. M-CSF also induced expression of two kruppel-like transcription factors, Egr1 and Egr2. Induction of both Egr1 and Egr2 expression was blocked through chemical inhibition of MEK1/2. To determine if one of these Egr transcription factors was a potential downstream mediator of the M-CSF-activated MEK/ERK pathway, the nuclear Egr1 and Egr2 corepressor Nab2 was employed to inhibit the function of both Egr1 and Egr2. In addition, the difference between Nab2 expression alone or Nab2 expression in combination with MEK inhibition by UO126 was examined. While both Nab2 and MEK inhibition increased osteoclast apoptosis, a concomitant increase in osteoclast apoptosis with Nab2 expression in combination with MEK1/2 inhibition treatment was not evident. These data suggest a role for Egr1 and/or Egr2 in M-CSF-promoted osteoclast survival and demonstrate the necessity of Egr family members in the promotion of MEK-dependant osteoclast survival. Both Egr1 and Egr2 were therefore pursued as downstream effectors of MEK/ERK meditated osteoclast survival downstream of M-CSF. Two forms, a wild-type (wt) and a mutant form (ca) lacking the Nab corepressors binding site, were utilized. Expression of wtEgr2 and caEgr2, not wtEgr1 or caEgr1, reduced the basal rate of osteoclast apoptosis. In addition, expression caEgr2 bypassed the block of M-CSF-promoted osteoclast survival meditated through MEK1/2 inhibition. M-CSF therefore promotes osteoclast survival through activation of the MEK/ERK pathway and induction of Egr2, providing a novel function of Egr2 in the promotion of cell survival.

Disclosures: E. W. Bradley, None.

M112

See Sunday Plenary Number S112

M113

Disruption of WASP-Associated Signaling Complex Formation Leads to Defects in Sealing Ring Formation and Bone Resorption in Osteoclasts.

T. Ma*, M. A. Chellaiah. Department of BMS Rm 7207 South Dental School, University of Maryland, Baltimore, MD, USA.

Wiscott-Aldrich Syndrome protein (WASP) has a unique regulatory role in sealing ring formation and bone resorption in osteoclasts. The activities of different kinases have been correlated to the phosphorylation of WASP and osteoclast bone resorption. The purpose of this study was to identify the WASP domain that regulates osteoclast function in vitro. TAT-mediated delivery of different WASP domains and full-length WASP into osteoclasts was performed. Transduction of TAT-fused full-length WASP peptide induced Arp2/3 complex formation, F-actin content, sealing ring formation and bone resorption largely as compared with other peptides containing different WASP domains. Transduction of WASP peptides containing basic, verpolin-central, pTyr294, and proline-rich regions exhibited inhibitory effects at various levels on the processes listed-above. The ability to resorb bone by WASP peptides containing basic, verpolin-central, and proline-rich regions was reduced and the resorbed area matched the size of the sealing ring. However, osteoclasts transduced with WASP peptide containing pTyr294aa demonstrated total loss of sealing ring-like structures but displayed actin-rich clusters at the peripheral edge that contains filopodia-like projections. Abnormal cytoskeletal changes have been coupled with the reduced capacity for bone resorption in vitro. The resorption pits were small, simple, and superficial. Taken together, these finding suggest that modulation of phosphorylation state of Tyr294aa assists in integrating multiple signaling molecule and pathways that partake in the assembly of sealing ring

Disclosures: M.A. Chellaiah, None.

This study received funding from: NIH-NIAMS RO1AR46292 to MAC.

M114

See Sunday Plenary Number S114

M115

Regulation of RANKL-induced MAP Kinase Activation by Reactive Oxygen Species.

H. Choi¹, H. Yu¹, J. Shin¹, S. Lee². ¹Life and Pharmaceutical Sciences, Ewha Womans University, Seoul, Republic of Korea, ²Division of Life and Pharmaceutical Sciences, Center for Cell Signaling & Drug Discovery Research, Ewha Womans University, Seoul, Republic of Korea.

Osteoclasts (OCs) are multinucleated hematopoietic cells that resorb bone and are essential for bone homeostasis. Signaling between receptor activator of NF-κB ligand (RANKL) and TNF (tumor necrosis factor) receptor-associated factor (TRAF) 6 is essential for differentiation of bone marrow monocyte/macrophage (BMM) lineage cells into osteoclasts. Here, we show that enhanced intracellular reactive oxygen species (ROS) levels by RANKL in BMM are dependent on the binding strength between RANK and TRAF6. ROS generation was decreased in overexpression of modified RANK (E342/449A and E342/375/449A) in TRAF6-binding sites, but not in reconstitution of wild-type RANK which has normal interaction between RANK and TRAF6. Consistently, MAPKs activation including ERK, JNK and p38 were attenuated by the modified RANK expression. To examine whether MAPK phosphatases (MKPs) play a role in the negative regulation of MAPKs through inactivation by ROS, we first investigated expression of MKPs in BMM through quantitative real-time PCR. The mRNA expression levels of MKP1 and MKP3 were much higher than other MKPs. We found MKP1 regulates JNK activation and MKP3 controls JNK and p38 activation in response to RANKL in 293-TR cells that express Flag epitope-tagged RANK inducibly in 293 cells. Moreover overexpression of catalytic mutant of MKP1 and MKP3 prompted osteoclast differentiation. These results suggest that MKPs including MKP1 and MKP3 might be an intracellular signal modulator for osteoclast differentiation between ROS and MAPKs.

Disclosures: H. Choi, None.

This study received funding from: BK 21 fellowship.

M116

Mice Overexpressing Nucleolar PTHrP Driven by Type II Collagen Promoter Show Disordered Distribution of Chondrocytes in the Epiphyseal Cartilage.

N. Amizuka¹, T. Kornori², K. Oda*³, D. Goltzman⁴, A. Karaplis⁴, M. Li¹. ¹Center for Transdisciplinary Research, Niigata University, Niigata, Japan, ²Division of Cell Biology, Nagasaki University, Nagasaki, Japan, ³Division of Biochemistry, Niigata University, Niigata, Japan, ⁴Faculty of Medicine, McGill University, Montreal, PQ, Canada.

The main pathway for parathyroid hormone-related peptide (PTHrP) action is that of extracellular secretion and subsequent activation of the cell membrane-bound PTH/PTHrP receptor. PTHrP has also been reported to translocate into nucleoli, and modulate cellular functions through its nuclear/nucleolar actions. PTHrP molecule, lacking the signal sequence but encompassing the nucleolar targeting signal, was shown to successfully translocate into nuclei and nucleoli, without following the secretory pathway in transfected chondrocytic cell line, CFK2. When this nuclear form of PTHrP cDNA tagged with the myc epitope was inserted into a type II collagen promoter/enhancer cassette and transfected in primary cultured chondrocytes from mouse epiphyseal cartilage, PTHrP and myc immunoreactivity was observed in their nuclei and nucleoli, but not in the cytoplasmic region. Based on these experiments, we generated mice overexpressing nucleolar PTHrP, which is driven by the type II collagen promoter. Transgenic mice at 18–19 day-old fetal stages were sacrificed, and the transgene was examined by PCR using specific primers recognizing the DNA sequence from the C-terminal region of myc-tagged PTHrP. Immunohistochemistry for PTHrP and myc demonstrated their localization to be restricted to nuclei and nucleoli of resting and proliferative chondrocytes, but not in hypertrophic cells. The width of the resting, proliferative and hypertrophic zones of the tibial and femoral epiphyses, as well as cell proliferation estimated by statistical analysis for PCNA (proliferating cell nuclear antigen)-immunopositive cells were not different between transgenic and wild-type fetuses. In addition, the width of the type × collagen-positive hypertrophic zone in transgenic mice was similar to that of wild type mice. Therefore, unlike PTHrP's receptor-mediated pathway, nucleolar PTHrP appeared not to dynamically affect proliferation or differentiation of chondrocytes. However, chondrocytes of various sizes were present in the lower region of proliferative zone were irregularly distributed. In summary, overexpression of the nuclear form of PTHrP appears to influence the distribution of chondrocytes rather than their proliferation or differentiation in mouse epiphyseal cartilage.

Disclosures: N. Amizuka. None.

M117

Induction of kinin B₁ Receptor in Osteoarthritis Is Performed by Vasoactive Peptide Endothelin-1.

H. L. Attalah*¹, D. Deblois*², C. A. Manacu*³, N. Newman*⁴, A. Moreau⁵, F. Moldovan*⁵. ¹Faculty of Dentistry of Montreal University, Research Center CHU Ste Justine Hospital, Canada, PQ, Canada, ²Pharmacology, University of Montreal, Canada, PQ, Canada, ³Research Center CHU Ste Justine Hospital, Canada, PQ, Canada, ⁴University of Montreal, Canada, PQ, Canada, ⁵Dentistry Faculty of Montreal University, Research Center CHU Ste Justine Hospital, Canada, PQ, Canada.

Endothelin-1 (ET-1) and kinin B1 receptors are involved in pain. ET-1 and B1 receptor activate similar signalling pathways, and their expression is under the control of the proinflammatory mediators that are key molecules for Osteoarthritis (OA) physiopathology. Kinins are potent agonists that participate in inflammatory and pain responses to insults by acting through the inducible B1 receptor. We hypothesize that ET-1 has a specific role in OA physiopathology in the early phase by promoting cartilage degradation and late phase by inducing B1R expression leading to thereby exacerbating inflammation and pain induction in late stage OA.

Analysis of B1R expression in synovial tissues of OA patients by immunohistochemistery and in situ hybridization revealed that its presence locally dependent on the level of tissue inflammation since its expression appears to be directly regulated by inflammatory mediators such as ET-1 and IL1β. We also observed that B1R co-localizes with ET-1 in both endothelial cells and type A synoviocytes (macrophage like cells). In OA chondrocytes, western blotting revealed p70S6 and p85S6 kinases and the p70S6 kinase is phosphorylated by ET-1, and that this phosphorylation is inhibited by rapamycin. Both of which act on mitogen-activated signalling pathways downstream of phosphoinositide 3-kinase (P13K), and are targets of rapamycin FRAPm/TOR. Activation of P13K-Akt and Ras-Raf MEK-ERK signalling cascades by ET-1 is thought to play an important role in cartilage matrix degradation via the activation of MMP-1 and MMP-13 which specifically cleave OA cartilage collagen.

These results prompted us to propose that ET-1 over expression in OA synovial tissues contributes to B1R up-regulation, although it remains unclear whether such on effect is solely due to elevated ET-1 levels. These data point toward a possible role of ET-1 in inflammatory pain. Thus, ET-1 is potentially attractive therapeutic target for OA pathology characterised by a chronical pain.

Disclosures: H.L. Attalah. None.

This study received funding from: The Arthritis Society.

M118

See Sunday Plenary Number S118

M119

Hydrostatic Loading of Growth Plate Chondrocytes in Vitro.

Y. Y. Shao*¹, J. Welter², L. Wang*³, R. T. Ballock⁴. ¹Department of Biomedial Engineering, Cleveland Clinic, Cleveland, OH, USA, ²Department of Biology, Case Western Reserve University, Cleveland, OH, USA, ³Department of Biomedical Engineering, Cleveland Clinic, Cleveland, OH, USA, ⁴Departments of Orthopaedics and Biomedical Engineering, Cleveland Clinic, Cleveland, OH, USA.

Objective: It is well-established that mechanical forces can influence the behavior of cartilage chondrocytes, however little is known about the response of growth plate chondrocytes to these mechanical forces. The objective of this study was to examine the effects of hydrostatic compression on the behavior of growth plate chondrocytes by testing the hypothesis that hydrostatic loading would inhibit proliferation and differentiation of these cells through modulation of the Ihh-PTHrP feedback loop.

Methods and Results: Epiphyseal chondrocytes were isolated from the distal femoral growth regions of two day old Sprague-Dawley rats and cultured as three-dimensional cell pellets (3×10⁵ cells/pellet) for five days. The pellets were then either subjected to hydrostatic compression or cultured as unloaded controls. A custom-designed mechanical loading system was used apply an intermittent 1MPa hydrostatic compression force (1 hour on, 1 hour off) for 1 or 2 days at 37°C and 5% CO₂. After the compression period, AQ 96 proliferation assays were carried out immediately for analysis of cell viability, while others were frozen for later testing. The growth plate chondrocytes subjected to hydrostatic compression increased proliferation more than the control cells after one day of loading, but were identical to control samples after two days. Real-time quantitative RT-PCR analysis demonstrated that type × collagen (CoIX) expression was increased by 12 fold in the loaded samples after one day of compression, while Ihh mRNA was up-regulated more than 6-fold and expression of patched (Ptc) and runt-related transcription factor (RunX2) were both increased two-fold. In addition, Ihh expression was also examined after shorter periods of compression (0.5, 1, 2, and 4 hours), Ihh expression increased 1.5 fold in response to the compression as early 30 minutes following application of the hydrostatic load. Measurement of caspase 3/7 activity and immunoblotting of Bcl₂ protein failed to reveal any increase in apoptotic activity in the hydrostatically loaded pellets compared to the unloaded controls.

Conclusions: Our results show that in vitro hydrostatic compression can stimulate Ihh expression, chondrocyte proliferation and terminal differentiation in growth plate chondrocytes cultured as three-dimensional cell pellets.

Disclosures: R.T. Ballock, None.

M120

Establishment of a Rat Culture System for Studying the Morphological Diversity of Hypertrophic Chondrocytes.

K. Chen*, L. Tatarczuch*, Y. Ahmed*, M. Mirams*, C. N. Pagel*, E. J. Mackie.. School of Veterinary Science, The University of Melbourne, Parkville, Australia.

During endochondral ossification, hypertrophic chondrocytes exist in two forms, “dark” and “light” cells. We have recently observed that these cells undergo two different forms of non-apoptotic physiological cell death (PCD) in horse growth cartilage, and have established a culture system for studying hypertrophy and death of equine chondrocytes. The aim of the current study was to develop a culture system using rodent chondrocytes in order to study differences between dark and light cells and their modes of death. Chondrocytes were isolated from femoral epiphyseal cartilage from neonatal rats and cultured as pellets in the presence of triiodothyronine (T3) in 0.1% FCS. After 14 days, the pellets were examined by light and electron microscopy. The proportion of hypertrophic light chondrocytes was double that of hypertrophic dark chondrocytes. Hypertrophic light chondrocytes from the T3-treated pellets could be purified through centrifugation in a gradient concentration of Ficoll-Paque™ Plus. The microarray technique was performed to compare the difference in gene expression between the purified hypertrophic light chondrocytes and the mixed hypertrophic dark and light cells in the T3-treated pellets. A number of genes of interest have been selected for validation by real-time PCR. These include genes encoding Egr2 and tissue plasminogen activator, which appear to be more highly expressed in light cells than dark cells, and periostin and osteoglycin, which appear to be more highly expressed in dark cells than light cells. Staurosporine induces apoptosis in many cell types, but when staurosporine was added to pellets cultured in 10% FCS, apoptotic chondrocytes were rarely observed. Instead, there was an increase in the proportion of dark cells dying by the process observed for these cells in vivo. Staurosporine significantly down-regulated mRNA expression of type × collagen, type II collagen, runx 2 and aggrecan. Fewer dark cells were seen in chondrocyte cultures from rats than in those from horses, which is probably due to the fact that very few dark cells are observed in neonatal growth cartilage from rats. This will be a useful system for studying molecular mechanisms of chondrocyte hypertrophy and death.

Disclosures: K. Chen, None.

M121

See Sunday Plenary Number S121

M122

Role of the Transcription Factor Lbh in Endochondral Bone Formation.

K. L, Conen¹, S. Nishimori*¹, S. Provot², H. M. Kronenberg¹. ¹Endocrine Unit, Massachusetts General Hospital, Harvard Medical Shool, Boston, MA, USA, ²Department of Anatomy, University of California, San Francisco, CA, USA.

In endochondral bone formation, mesenchymal condensations develop into chondrocytes that proliferate and form the fetal growth plate. As round, flat and hypertrophic chondrocytes they undergo progressive, well-ordered stages of differentiation. All differentiation steps are characterized by the synthesis of stage-specific matrix proteins and the expression of different transcription factors (TF). In order to study the transcriptional control of endochondral bone formation we previously analyzed genes expressed in discrete layers of the mouse newborn (NB) growth plate using microarray analyzes. There the TF limb-bud-and-heart (Lbh) appeared to be expressed predominantly in hypertrophic chondrocytes. Lbh is thought to act as a transcriptional co-activator and is highly conserved among species. It has a to date uncharacterized role in endochondral bone formation. In situ hybridization (ISH) of sections of NB mouse tibia confirmed that Lbh is expressed in hypertrophic chondrocytes. We therefore investigated whether Lbh has a key regulating role in the induction of the differentiation steps from flat to hypertrophic chondrocytes in an experimental model, providing advantages for a relatively easy and fast testing: The fetal growth plate of chicken embryos at E10, a time when growth cartilage and bone become visible. Our hypothesis is that Lbh plays a key role in hypertrophic chondrocyte differentiation. We have begun to use the avian Replication-Competent ASLV long terminal repeat with Splice acceptor (RCAS) retroviral vector strategy. Lbh cDNA samples have been cloned into this RCAS virus. We infect chicken limb buds at E3-E3.5 in ovo and then analyze the effect of Lbh by comparing the gross aspect of E10 infected limbs compared to the uninfected contralateral limb using skeletal preparations and ISH. Whole-mount ISH of Lbh in the chicken system revealed an early signal in the entire outgrowing limb buds of E4.5 chickens. This signal subsequently remains visible only in the fetal growth plate area between E6 and E10. These data suggest a possible role of Lbh in chondrogenesis of chicken and mouse endochondral bone formation.

Disclosures: K.L. Conen, None.

M123

See Sunday Plenary Number S123

M124

Effect of Removal of Nucleus Pulposus Cells on the Annulus Fibrosis of the Intervertebral Disc.

C. Dahia*¹, A. Durrani¹, E. Mahoney*¹, C. Wylie*². ¹Orthopaedic Surgery, Cincinnati Children's Hospital and Medical Center, Cincinnati, OH, USA, ²Developmental Biology, Cincinnati Children's Hospital and Medical Center, Cincinnati, OH, USA.

The relationship between the nucleus pulposus and the Annulus fibrosis in normal differentiation and maintenance of the IVD is not clearly understood. We studied the effect of removal of NP cells on the AF.

The NP's were surgically aspirated from L2-L3 and L3–4 discs of 2-week old male mice (during the period of rapid postnatal vertebral growth), using a 27-gauge syringe. L4–5 discs in the same mice were sham-operated (needle insertion without aspiration) as controls. The effects on the IVD were assayed 2–8 weeks after the surgery. 8 μm cryosections were collected in the coronal plane and histological analysis was carried out using H&E staining. Cell proliferation and cell death was determined by phospho Histone H3 (PH3) and active caspase-3 staining respectively.

5-weeks following removal of the NP cells there was significant collapse of the IVDs (Fig-B) from which the NP had been removed, compared with those of sham-operated controls (Fig-A). By 7-weeks, fibrocartilage cells derived from the AF were invading the disc space, which became completely filled by 8 weeks. Growth of the disc was reduced in both cranio-caudal and transverse diameters by 30%, compared to control discs. Mitotic cells were absent in the AF indicating that AF cells stopped proliferating in the absence of NP cells. A large number of activated caspase-3 positive cells were found in AF cells 8 weeks after removal of NP cells (Fig-D), compared to sham controls (Fig-C).

Removal of NP cells leads to invasion of the disc space, and its fibrosis by AF cells, decreased proliferation and increased cell death of the AF cells, and decrease in disc growth. These data suggest a requirement for the NP cells in normal differentiation and growth of the AF cells.

Disclosures: C. Dahia, None.

This study received finding from: Children s Hospital Research Foundation.

M125

Spatial and Temporal Localization of Components of the TGF-beta, BMP, IHH and FGF Signaling Pathways in the Postnatal Mouse Lumbar Vertebral Growth Plate (LVGP).

C. Dahia*¹, E. Mahoney*¹, A. Durrani¹, C. Wylie*². ¹Orthopaedic Surgery, Cincinnati Children's Hospital and Medical Center, Cincinnati, OH, USA, ²Developmental Biology, Cincinnati Children's Hospital and Medical Center, Cincinnati, OH, USA.

Little is known about postnatal spine growth. A number of signaling pathways have been shown by gene targeting in the mouse to be important in the growth of fetal long bones. The objective of this study was to find out if these signaling pathways are present in postnatal LVGP, and their expression pattern during growth and aging.

8 μm cryosections in the coronal plane from decalcified LVGP of 1–12 weeks old male FVB mice were analyzed by H&E and alkaline phosphatase (AP) staining. Cell proliferation & cell death was determined by phospho-histone H3 (PH3) & TUNEL assay, respectively. Immunolocalization of components of the TGFβl&2, BMP2&4, IHH & FGF2 pathways was carried out using confocal microscopy.

PH3 positive staining disappeared from the proliferative zone chondrocytes (PZc) of LVGP at 3-weeks indicating cessation of cell proliferation. Membrane localization of IHH (Fig-A) & its receptor PTC-1 was observed on the PZc of LVGP only until 2-weeks of age. Between 2–12 weeks of age, active TGFβl&2 signaling was present at the junction of PZc and early hypertrophic zone chondrocytes (HZc) of the LVGP, as shown by staining for its activated downstream intermediate (p)Smad2/3 (Fig-B). BMP2&4 pathways were active only in the HZc (Fig-C) of the LVGP at all ages. Staining for its active downstream molecule, pSmad1/5/8 (Fig-D), decreased from 3-weeks onwards, but did not disappear. FGF2 signaling was observed to be active only in the HZ of the LVGP of 2-week old mice and decreased with age. Intensity of AP staining increased in the HZc from 2-week onwards.

Our data suggests that IHH regulates the proliferation of PZc in the LVGP. TGFβ signaling regulates the transition of PZc to HZc. FGF signaling is seen only in the HZ indicating it may play a role in regulating HZc. Active BMP & AP signaling seen only in the HZc in all ages suggests its role in the maintenance of the extracellular matrix.

Disclosures: C Dahia, None.

This study received funding from: Children's Hospital Research Foundation

M126

Histological and Molecular Analysis of the Growth and Differentiation of the Mouse Lumbar Interverterbral Disc.

C. Dahia*¹, E. Mahoney*¹, A. Durrani¹, C. Wylie*². ¹Orthopaedic Surgery, Cincinnati Children's Hospital and Medical Center, Cincinnati, OH, USA, ²Developmental Biology, Cincinnati Children's Hospital and Medical Center, Cincinnati, OH, USA.

Intervertebral disc (IVD) degeneration is the most common cause of back pain. We wish to understand the cellular and molecular mechanisms of disc growth and development in mouse.

8μm thick coronal and transverse cryosections, from lumbar vertebrae (LV) of 1 −48 weeks old male mice, were analyzed by H&E and alkaline phosphatase (AP) staining. Physical growth of the discs was measured using a 1mm graticule. The number and thickness of the layers of the annulus fibrosus (AF), was measured using DIC optics. Cell proliferation and death was determined by phospho Histone H3 (PH3) and TUNEL staining, respectively. Immunolocalization of components of the TGFβ, BMP and Shh pathways was carried out using confocal microscopy.

Growth of the IVD paralled that of the vertebral column, being most rapid between birth and 3 weeks, and plateauing off by 9 weeks of age. Proliferating cells were found until 3 weeks of age in both the AF and NP (Fig-A). During the first week, the AF became divided into a mineralized component over the vertebral bodies, which stained positively for AP, and non-mineralized component between the vertebrae (Fig-B). Cells in the mineralized component also became hypertrophied. Apoptosis was only found in the NP. DIC imaging revealed that the number of layers of AF increased from 1–2 weeks of age, after which each layer becomes very thick due to the extracellular matrix secreted by them. The number of AF layers decreased in the IVDs of older mice. TGFβ as well as BMP signaling pathway was active in the NP as well as the AF cells, while Shh was secreted by the NP and fibrous AF cells but acted on the mineralized AF cells.

The IVDs grow coordinately with the vertebrae. Both components of the disc grow, and then involute with age. The AF becomes divided into a mineralized and non-mineralized component during growth. Active cell signaling pathways like BMP, TGFβ and Shh suggests that these cells are actively involved in disc maintenance.

Disclosures: C. Dahia, None.

This study received funding from: Children's Hospital Research Foundation.

M127

See Sunday Plenary Number S127

M128

Non-covalent Interaction of MMP13 and LTBP1 in a Unique TGFβ Large Latent Complex Produced by Hypertrophic Chondrocytes.

B. N. Dragann*¹, V. L. Scheinfeld*¹, S. M. Routson*¹, B. M. Mentzer*¹, P. M. Mattioli*¹, A. H. Selim², D. M. Appelt*¹, M. D'Angelo¹. ¹Center for Chronic Disorders of Aging, PCOM, Philadelphia, PA, USA, ²Department of Biology and Physics, KFUPM, Dhahran, Saudi Arabia.

Our lab has shown that hypertrophic chondrocytes produce a unique TGFβ2 large latent complex that contains collagenase 3 (MMP13) in non-covalent association with latent TGFβ binding protein (LTBP1). In this study, we investigate hypertrophic chondrocyte production of the elements of this unique TGFβ2 large latent complex. Hypertrophic chondrocytes from the avian sterna were cultured 5 days in serum-free alginate. RNA, conditioned media, cell-associated matrix (isolated after release of the chondrocytes from alginate), and cell extracts were analyzed for TGFβ2, MMP13 and LTBP1. Immunoblot analysis revealed the presence of immunoreactive bands for TGFβ2, MMP13 and LTBP1 in the cellular extracts. Assembly into the extracellular matrix and secretion of the components was indicated by the presence of immunoreactive bands for all three proteins in the cell-associated matrix and conditioned media fraction. Immunocytochemistry demonstrated the presence of TGFβ2, MMP13 and LTBP1 in association with the cells and in a pericellular staining pattern. In order to determine whether MMP13 binds non-covalently to LTBP1 of the TGFβ large latent complex, primary monolayer cultures of hypertrophic chondrocytes were double-labeled with antibodies against MMP13 and LTBP1. Optical serial sections were collected from these cells and the images deconvoluted into three-dimensional micrographs. LTBP1 and MMP13 were present both intracellularly and in association with the extracellular matrix between cells. In addition, the fluorescent signal indicated substantial co-localization of the two proteins within the extracellular matrix of the cultures. For additional confirmation, we produced a biotin-labeled peptide corresponding to the C-terminal hemopexin domain of MMP13 that bioinformatics indicated could interact non-covalentty with the C-termina! EGF-calcium domains of LTBP1. Protein from hypertrophic chondrocyte tissue was extracted from day 17 avian upper sternum and immunoprecipitated with antibody to LTBP1. Dot blot analysis of the immunoprecipitate showed binding of the biotin-labeled MMP13 peptide with proteins in the tissue extracts. These data demonstrate that the components of the unique TGFβ2 large latent complex are produced and secreted by hypertrophic chondrocytes and are present in compartments that are characteristic of extracellular matrix assembly. In addition, these data confirm that MMP13 can interact with LTBP1 and underscores the candidacy of MMP13 in the unique mechanism of activation of TGFβ by hypertrophic chondrocytes.

Disclosures: M. D'Angelo, None.

M129

Twistl Stage-specifically Regulates Chondrocyte Differentiation Downstream of TGF-β and Wnt Signaling.

Y. Dong, Y. Chang*, D. Y. Soung, R. O'Keefe, E. Schwarz, H. Drissi.. Orthopaedics, University of Rochester, Rochester, NY, USA.

We investigated the molecular mechanisms underlying the transition between immature and mature chondrocytes downstream of TGF-β and canonical Wnt signals. We used two developmentally distinct chondrocyte models isolated from the caudal portion of embryonic chick sternum or chick growth plates. Lower sternal chondrocytes exhibited immature phenotypic features, while growth plate extracted cells display a hypertrophic phenotype. TGF-β significantly induced β-catenin in immature chondrocytes, while it repressed it in mature chondrocytes. TGF-β further enhanced canonical Wnt-mediated transactivation of the Topflash reporter expression in lower sternal chondrocytes. However, it time-dependently inhibited Topflash activity in growth plate chondrocytes. Our immunoprecipitation experiments showed that TGF-β induced Smad3 interaction with TCF-4 in immature chondrocytes, while it inhibited this interaction in mature chondrocytes. Similar results were observed by chromatin IP showing that TGF-β differentially shifts TCF-4 occupancy on the Runx2 promoter in lower sternal chondrocytes versus growth plate chondrocytes. To further determine the molecular switch between immature and hypertrophic chondrocytes, we assessed the expression and regulation of Twist1 and Runx2 in both cell models upon treatment with TGF-β and Wnt3a. We show that Runx2 and Twist1 are differentially regulated during chondrocyte maturation. Furthermore, while TGF-β induced Twistl in mature chondrocytes, it inhibits Runx2 expression in these cells. Opposite effects were observed upon Wnt3a treatment which predominates over TGF-β effects on these cells. Finally, over-expression of chick Twistl in mature chondrocytes dramatically inhibited their hypertrophy. Together, our findings show that Twist1 may be an important regulator of chondrocyte progression towards terminal maturation in response to TGF-β and canonical Wnt signaling.

Disclosures: H. Drissi, None.

M130

See Sunday Plenary Number S130

M131

Role of Fe³⁺ Ion and Ferritin on Differentiation of Chondrocytes at the Stage of Calcification.

H. Hagiwara¹, N. Hashimoto*², T. Ohno*², K. Yamazaki*¹, H. Okumura*³, T. lto*³. ¹Dept. of Biomedical Engineering, Toin University of Yokohama, Yokohama, Japan, ²Dept. of Bioscience and Biotechnology, Tokyo Institute of Technology, Yokohama, Japan, ³Health Effects Research, Energy and Environment Research, Japan Automobile Research Institute, Tsukuba, Japan.

Fe ions are major contents of metal in our body. However, there is a little information about the effect of Fe ions on bone metabolism. In the present study, we examined the effects of Fe³⁺ ion and ferritin, a storage protein of Fe³⁺ ion in cells, on the differentiation and mineralization of cultured chondrocytes, ATDC5 cells. At first, we monitored the expression pattern of ferritin mRNA with RT-PCR during the culture period of ATDC5 cells. Ferritin mRNA was expressed in ATDC5 cells, from day 21, at the late stage of culture. We used ferric ammonium citrate (FAC) as a donor of Fe³⁺ ion and desferrioxamine (DFO) as a chelator of Fe³⁺ ion. Both chemicals did not affect the production of proteoglycan by ATDC5 cells. By contrast, FAC inhibited the deposition of calcium by ATDC5 cells and DFO accelerated it. Furthermore, FAC inhibited the expression of MMPI3 mRNA, a differentiation marker at a late stage of differentiation of chondrocytes. In addition, DNA microarray methods revealed that Fe³⁺ ion regulated the expression of collagen mRNAs in ATDC5 cells. These results suggested that Fe³⁺ ion might play an important role on chondrocyte differentiation and mineralization

Disclosures: H. Hagiwara, None.

This study received funding from: Grants-in-Aid for Scientific Research from the Ministry of Education, Science. Sports, and Culture of Japan.

M132

A Novel Tumor Necrosis Factor-α Responsive CCAAT/Enhancer-binding Protein Site Regulates Cartilage Cd-Rap Expression.

T. Imamura*¹, A. McAlinden*², K. Terada*¹, H. Mivahara*², Y. twamoto*³, L. J. Sandell*². ¹Orthopaedic Surgery, Kyushu Medical Center, Fukuoka, Japan, ²Orthopaedic Surgery, Washington Univ., St. Louis, MO, USA, ³Orthopaedic Surgery, Kyushu Univ., Fukuoka, Japan.

Objective. Rheumatoid arthritis is characterized by an inflammatory process in the synovium resulting in progressive destruction of the affected joints. Cytokines such as Tumor Necrosis Factor (TNF)-α and Interleukin 1 (IL-1)-β regulate inflammation, causing functional impairment. Previous studies in our laboratory have shown that expression of cartilage characteristic genes, such as cartilage-derived retinoic acid sensitive protein (Cd-rap) and Type II collagen (Col2a1), are repressed by IL-1β by a process involving a CCAAT/Enhancer binding protein (C/EBP) site. The aim of this study is to investigate the mechanism of TNF-α induced inhibition of cartilage gene expression by studying regulation of the Cd-rap gene.

Methods. Rat chondrosarcoma (RCS) cells were transiently transfected with cDNA constructs encoding Cd-rap in the presence of TNF-α. Expression of C/EBPβ, Sox9 and p300 after treatment with TNF-α were examined by RT-PCR and Western blot in RCS cells and primary human articular chondrocytes. The effect of TNF-α on endogenous binding of C/EBPβ or Sox9 to the Cd-rap promoter was examined by chromatin immunoprecipitation (ChIP) assays.

Results. We identified a new C/EBP binding site in the Cd-rap promoter (position −1059 to −1046). Binding of C/EBP to this site was regulated by TNF-α, but not IL-1β, resulting in down-regulation of Cd-rap expression. This effect was reversed by mutational inactivation of the C/EBP motif. In addition, the activation potential of Sox9 and CBP/p300 on the Cd-rap promoter was enhanced after mutation of the new C/EBP binding site, indicating that blockage of this site would increase transcription

Conclusion. TNF-α regulates the expression and/or DNA binding potential of key positive and negative-acting transcription factors that control the expression of the cartilage matrix gene, Cd-rap.

Disclosures: T. Imamura, None.

This study received funding from: N1H.

M133

See Sunday Plenary Number S133

M134

OPG Inhibits Cartilage Degradation in a Knee Instability Mice Model.

A. Kadri*, H. Ea*, B. Uzan*, C. Bazille*, M. Ah Kioon*, F. Liote*, M. E. Cohen-Solal. Hopital Lariboisiere, INSERM U606, Paris, France.

Osteoarthritis (OA) is a focal rheumatic disease characterised by cartilage destruction. Mechanical stress and several other local factors are responsible for cellular and molecular changes that contribute to matrix proteolysis. Proteases as aggrecanases (ADAMTS-4 and ADAMTS-5) participate to chondrolysis in OA. Subchondral bone (SCB) may be involved in the pathogenesis of OA. We used a meniscectomy-induced OA model in mice in order to assess to the changes of SCB and the effects of inhibition of cytokines involved in bone and cartilage remodeling (IL1ra and OPG). Ten week-old C57/BL6 male mice underwent medial meniscectomy of the right knees (Mnx) and the left knees were sham-operated (sham). Mice were separated in 3 groups and received intraperitoneous injections of PBS, OPG (10mg/kg)or IL1ra (100mg/kg) twice a week for 6 weeks. At sacrifice, serial saggital sections of decalcified knees were performed to assess OA score, ADAMTS expressions and micro-architecture of SCB. At the tibia, OA score was increased in Mnx compared to sham-operated mice in PBS group (0.5 ± 0.27 vs 1.9 ± 0.41, p<0.05). Compared to PBS-treated mice with Mnx, OA score was not altered with IL1ra (1.9 ± 0.41 vs 2.29 ± 0.84), but was decreased by OPG (1.9 ± 0.41 vs 0.17 ± 0.17, p<0.05). In PBS-treated mice, the number of ADAMTS-4+ cells was significantly enhanced in Mnx compared to sham (64.6 ± 2.1 vs 35.9 ± 1.6% of total cells, p<0.0001) as well as ADAMTS-5+ cells (47.3 ± 1,9 vs 78.9 ± 2.7 p<0.0001). The number of positive cells in Mnx was then expressed using sham as referent (Δ ADAMTS). Compared to PBS, Δ ADAMTS-4+ cells were lower with IL1ra and OPG (0.81 ± 0.78 vs 0.51 ± 0.12, vs 0.46 ± 0.29 respectively). Δ ADAMTS-5+ cells were also lower with IL1ra (0.67 ± 0.08 vs 0.42 ± 0.1) and further decreased with OPG (0.67 ± 0.08 vs 0.26 ± 0.09, p < 0.05). In PBS treated-mice, BV/TV was decreased in Mnx vs sham knees (39 ± 036% vs 49 ± 048%) along with an increased Marrow Star Volume (Ma.Sv: 8.9.10⁵ ± 3.4.10⁵ vs 3,7.10⁵ ± 1.5.10⁵) suggesting increased bone resorption. In Mnx knees, IL1ra did not change BV/TV (39 ± 036% vs 42 ± 046%), whereas it was increased with OPG (39 ± 036% vs 53 ± 059%). Only OPG decreased MaSv compared to PBS treated-mice (1.3 ± 0.37 10⁵ vs 8.9 ± 3.4 10⁵, p<0.05).

In conclusion, meniscectomy induces cartilage degradation and increases aggrecanase expressions. This was associated with an increased bone resorption. Inhibition of bone resorption by OPG prevents matrix cartilage degradation and the increase of protease expressions. These data suggested that increased bone resorption at SCB could be implicated in the pathogenesis of OA at early stage. Targeting specifically the inhibition of bone resorption might lead to the prevention of OA.

Disclosures: A. Kadri, None.

M135

Therapeutic Effect of KHBJ9 on Cartilage Degradation and Inflammation in Collagenase-induced Rabbit Osteoarthritis.

D. Kim¹, J. Huh*², Y. Baek*², J. Lee*², D. Choi*², D. Park*². ¹Internal Medicine, Kyung Hee University Hospital, Seoul, Republic of Korea, ²Acupuncture & Moxibustion, College of Oriental Medicine, Kyung Hee University, Seoul, Republic of Korea.

The aim of this study was to determine the therapeutic effects of an herb medicine, KHBJ9 on osteoarthritis. We investigated whether KHBJ9 could suppress the disease progression of collagenase-induced osteoarthritis (CIA) in rabbits.

The right knees of rabbits were injected intra-articularly with collagenase, and rabbits were orally administrated with distilled water, KHBJ9 (50, 100, 400 mg/kg) or celecoxib (100 mg/kg) once a day for 28 days after the initiation of the CIA induction. The knee joints were evaluated by gross morphology and histology. Target gene expression was analyzed by RT-PCR such as proteoglycan, MMPS, and cyclooxygenases (COX) in knee joints. Also, we measured glycosaminoglycan (GAG) release, MMPs activity, PGE₂ production by colorimetric analysis. COX-1 and COX-2 protein expression were confirmed by immunohistochemistry.

Histological evaluation showed that KHBJ9 dose-dependently suppressed damage in cartilage and synovium as compared with the control. The mRNA expression of proteoglycan, MMP-1, MMP-3, and COX-2 was dose-dependently decreased, whereas COX-1 expression not affected. As well, GAG release and matrix degradation enzymes including MMP-1 and MMP-3 activities, and PGE-2 production significantly reduced at a dose-dependent manner.

This study indicates that KHBJ9 play a protective role against cartilage degradation and anti-inflammatory effect in rabbits with OA. The role may be achieved through the down regulation of the MMP-1, MMP-3, PGE2 and COX-2 activity, and expression in CIA rabbit model.

Disclosures: D. Kim, None.

This study received funding from: A grant of the Oriental Medicine R&D Project, Ministry of Health & Welfare, Republic of Korea.

M136

See Sunday Plenary Number S136

M137

Gene-Trap Mutagenesis Revealed the Involvement of Vinculin in Chondrogenic Differentiation in ATDC5 Cells.

T. Koshimizu*¹, M. Kimata*¹, K. Tachikawa*¹, K. Ozono², T. Michigami¹. ¹Department of Bone and Mineral Research, Osaka Medical Center and Research Institute for Maternal and Child Health, Osaka, Japan, ²Department of Pediatrics, Osaka University Graduate School of Medicine, Osaka, Japan.

Gene-trap mutagenesis is based on the idea that the random insertion of a trapping vector may disturb the function of inserted genes. In the current study, to identify the genes involved in chondrocytic differentiation, we have applied this method to a chondrogenic mesenchymal cell line, ATDC5. As the trap vector, we used pPT1-geo, which lacks its own promoter and enhancer, but contains a lacZ-neo fusion gene geo as a reporter and selection marker. The reporter activity reflects the expression level of the trapped gene. We screened the isolated clones for high β-galactosidase activity, and the selected clones were subjected to the chondrogenic induction to evaluate the phenotypical changes. 5'-RACE was performed to identify the gene trapped in each clone. In clone #4–17, the trap vector pPT1-geo was integrated in intron 13 in the gene encoding vinculin. Vinculin is a cytoskeletal protein and occurs in multimolecular complexes that are thought to function in adhesion and/or signaling between the extracellular milieu and the cell, via integrins and cadherins. Since vinculin-knockout mice are embryonic lethal, the roles of vinculin in skeletogenesis remain unclear. As the result of integration of pPT1-geo, a truncated vinculin lacking carboxyl-terminal paxillin-binding domain fused to geo product was produced from the trapped allele, and Western blotting confirmed that both wild-type and the truncated vinculin were expressed in #4–17. There was no apparent morphological change in the clone. When cultured in the differentiation medium, #4–17 exhibited impaired nodule formation and less accumulation of cartilaginous matrices. RT-PCR analyses revealed that the expression of chondrocytic marker genes including Col2al and PTHrP was reduced in the clone, although that of SOX9 was retained. The expression of wild-type vinculin in the parental ATDC5 and that of lacZ in #4–17 were elevated during the culture in differentiation medium, suggesting the involvement of vinculin in chondrocytic differentiation. Integrin-stimulated FAK autophosphorylation was markedly enhanced in the clone #4–17, demonstrating the aberrant signaling transduction induced by adhesion to extracellular matrix, while the phosphorylation state of paxillin was not obviously changed. Taken together, the phenotypical changes caused by trapping of vinculin gene indicated its critical roles in chondrocytic differentiation, and enhanced the indispensability of the signals from extracellular matrix in the process.

Disclosures: T. Koshimizu, None.

M138

Effect of Vertebroplasty Filler Materials on Viability and Gene Expression of Mouse and Human Nucleus Pulposus Cells.

Á. Lazáry¹, P. Varga*¹, G. Speer*², K. Bácsi*², B. Balla*², J. Kósa*², Z. Nagy², I. Takács², P. Lakatos². ¹Center of Spinal Disorders, Buda Health Center, Budapest, Hungary, ²1st Department of Internal Medicine, Semmelweis University, Budapest, Hungary.

Consequences of intradiscal cement leakage — often occur after vertebral cement augmentation for the treatment of vertebral compression fractures — are still unknown. In this study, we have investigated the influences of vertebroplasty filler materials (polymethilmetacrylate-, calcium phosphate- and calcium sulfate based bone cement) on isolated nucleus pulposus cells. Cell viability of cultured human nucleus pulposus cells were measured after treatment with vertebroplasty filler materials. Gene expression profile of selected genes was determined with quantitative real-time PCR. The widely used polymethilmetacrylate and calcium phosphate cement significantly decreased cell number in a dose- and time-dependent manner (e.g. cell number was reduced by 79% in human cultures in case of 2 day treatment of 0.1% v/v polymethilmetacrylate) while calcium sulfate cement affected cell viability less. Expression of genes involved in matrix metabolism of nucleus pulposus — aggrecan, collagens, small proteoglycans -, as well as important transcription factors have also significantly changed due to treatment (e.g. 2.5-fold decrease in aggrecan expression was determined in human cultures due to polymethilmetacrylate treatment). Our results suggest that intradiscally leaked bone cement — depending on the type of applied material — can accelerate the degeneration of nucleus pulposus resulting in a less flexible disc. This process may increase the risk of a subsequent new vertebral fracture, the main complication of vertebral augmentation. In conclusion, avoiding of cement leakage and development of vertebroplasty filler material that does not interfere with vital gene expression and cell viability would be desirable.

Disclosures: A. Lazáry. None.

This study received funding from: NKFP-1A/002/2004, NKFP-1A/007/2004. ETT-55059.

M139

Regulation of Matrix Metalloproteinase (MMP)-9 Expression by MMP-12 in Interleukin-1β-treated Articular Chondrocytes.

H. Oh, M. Lee*, J. Chun*. Department of Life Science, Gwangju Institute of Science and Technology, Gwangju, Republic of Korea.

Interleukin (IL)-1β is a major pro-inflammatory cytokine that causes destruction of articular cartilage via induction of several matrix-metalloproteinases (MMPs). MMPs play an essential role in pathological extracellular matrix (ECM) breakdown in arthritic cartilage. In primary culture articular chondrocytes and cartilage explants, IL-1β caused induction of several MMPs such as MMP-1, −3, −9, −12, and −13, whereas expression of other MMPs including MMP-2, −14, and −15 was not affected. Because the role of MMP-12 in chondrocytes remains largely unknown, we examined the signaling mechanisms and the role of IL-1β-induced expression of MMP-12 in primary culture articular chondrocytes. Among the mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase and p38 kinase mediated expression and activation of MMP-12, whereas c-Jun N-terminal kinase regulated MMP-12 activation without effects on its expression. MMP-12 expression by IL-1β was also mediated by nuclear factor kappa enhancer binding protein (NFκB). Because ECM fragments produced by MMP action were known to regulate expression of several MMPs, we next examined the role of MMP-12 in expression of other MMPs in chondrocytes. Exogenous MMP-12 in IIL-1β-treated chondrocytes caused both expression and secretion of MMP-9, which is known to degrade proteoglycan and denatured type II collagen. However, MMP-12 did not affect of MMP-9 expression in absence of IL-1β. Taken together, our results suggest that IL-1β causes expression and activation of MMP-12 through signaling pathway involving MAP kinases and NFκB, and activated MMP-12 may contribute to cartilage destruction by increasing MMP-9 expression.

Disclosures: H. Oh. None.

M140

BMP4 in Postnatal Alveolar Bone Formation and Tooth Cytodifferentiation: Conditional Deletion of BMP4 with the 3.6 Collagen type 1a-Cre Model.

J. Gluhak-Heinrich¹, L. Martineze*¹, W. Yang¹, J. Zhang²., J. Feng³, D. Guo³, A. Lichtler⁴, B. Cream⁴, M. Harris¹, S. Harris¹. ¹UTHSCSA, San Antonio, TX, USA, ²Univ.of Vanderbilt, Nashville, TN, USA, ³UKMC, Kansas City, MO, USA, ⁴UCONN, Farmington, CT, USA.

Bone morphogenetic protein 4 (BMP4) is essential for early tooth and bone development in the postnatal animal. Smad 1/5/8 plays a central role in BMP4 signaling, with other transcription factors like: Runx2, Dlx5 and Osx. BMP4 conditional knockout (Bmp4 cKO) leads to mice with reduced bone and osteopenia, as well as reduction in dentin formation during tooth cytodifferentiation. The mechanisms by which BMP4 deficiency leads to defects during bone and tooth cytodifferentiation have not been explored. The objective of this study was to determine the biologic effect of postnatal BMP4 deficiency on tooth and alveolar bone formation and mineralization.

To obtain 3.6collagenla-Cre/BMP4cKO mice, we crossed 3.6 collagen la-Cre mice with BMP4 floxed mice. Deletion of BMP4 (by excision of exons 3 and 4) occurs in collagen producing osteoblasts and odontoblasts of progeny cells. BMP4cKO mice and their sibling controls were selected for phenotype characterization using analytical methods including X-ray, histological techniques, in situ hybridization, and immunocytochemistry. Our results show that by day 12, BMP4cKO mice display reduced size of calvarias and osteopenia in alveolar bone of the mandible, a 99% decrease in immunoreactivity of phospho-Smad1/5/8 and reduced DMPI mRNA in the odontoblasts. By day 18, BMP4cKO mice show 82% reduction of BMP4 mRNA with changed odontoblast morphology and dental tubule formation and 45% decrease in D1×5 mRNA. Osterix expression was reduced 84% and DSPP 67% in odontoblasts. Amelogenin was reduced 60–80% in ameloblasts in one month old BMP4cKO mice. Dentin thickness was reduced 40% and enamel formation was delayed, although the 3.6 Collal-Cre shows no activity in ameloblasts at any stage. In conclusion, our results indicate that the absence of BMP4 in osteoblasts and odontoblasts impairs mineralization of the calvaria and mandible, greatly delays the processes of dentinogenesis and amelogenesis. Delayed enamel formation was associated with BMP4 deficiency in odontoblasts and reduced dentin formation, suggesting that disruption in odontoblast differentiation by lack of BMP4 leads to disruption in amelogenesis or enamel formation. These results support the concept that odontogenesis is tightly linked to amelogenesis and is controlled by BMP4. Elucidation of downstream targets for BMP4 signaling using our BMP4cKO mice may provide novel strategies for enhancing bone and tooth integrity.

Disclosures: J. Gluhak-Heinrich, None.

This study received funding from: AR46798 and DE16949.

M141

Angiotensin II Suppresses Bone Mass via AT1 receptor in Long Bones of Adult Mice.

Y. Izu, T. Hayata, H. Hemmi*, J. Nagata, Y. Ezura, K. Nakashima*, M. Noda. Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan.

Angiotensin II (Ang II), a major effector peptide of the renin angiotensin system, controls blood pressure and body fluid homeostasis. Ang II, derived from Angiotensin I by angiotensin converting enzyme (ACE), binds to its receptors comprised of two major subtypes, angiotensin II type 1 (AT1) and type 2 (AT2). Ang II induces vasoconstriction and sodium retention through AT1 activation, whereas it induces vasodilation and tissue regeneration through AT2 activation. In rodents, two type 1 receptors (AT1a and AT1b) are encoded by distinct genes, although both receptors are pharmacologically identical. In human, clinical and epidemiologic studies suggested that Ang II might have functions to regulate bone metabolism. Indeed, Ang II inhibits differentiation of osteoblasts and matrix calcification in vitro through AT1 activation. In addition, Ang II stimulates osteoclastic bone resorption in a bone marrow cell culture system. However, physiological roles and actions of Ang II in bone metabolism in vivo remain unclear. In order to examine whether Ang II signaling is involved in bone metabolism in vivo, we first examined if its specific receptors, AT1 and AT2, are expressed in the bone of adult mice (9 weeks old). By immunohistochemical approach, we identified AT1 and AT2 expressions in both osteoblasts and osteoclasts in adult long bones. These observations suggest that Ang II may directly regulate osteoblast and osteoclast function in vivo. Additionally, to distinguish physiological roles of AT1 and AT2 in bone in vivo, specific receptor antagonist for AT1, losartan (peroral administration of 10 mg/kg/day) or for AT2, PD123319 (intraperitoneal injection of 10 mg/kg/day) was administrated into adult mice (9 weeks old). 3D-micro-CT analysis further revealed that losartan inhibited of AT1 enhanced the levels of bone mineral density (BMD) and cancellous bone volume (BV/TV) in hind limbs. In contrast, blockade of AT2 by PD1233I9 tended to reduce the levels of both BMD and cancellous BV/TV. These data suggest that Ang II signalings through AT1 and AT2 exhibit opposite roles in bone metabolism in the adult mice.

Disclosures: Y. Izu. None.

M142

See Sunday Plenary Number S142

M143

Identification of Translationally Controlled Tumor Protein (TCTP) as a Nuclear Matrix Protein in Osteoblasts.

Y. Jung*¹, J. Jeong*¹, N. Park*¹, K. Lee*¹, A. J. van Wijnen², J. B. Lian², J. L. Stein², G. S. Stein², J. Choi¹. ¹Biochemistry & Cell Biology, Kyungpook Natl. Univ. School of Medicine, Skeletal Diseases Genome Research Center, Daegu, Republic of Korea, ²Cell Biology, UMASS Medical School, Worcester, MA, USA.

The composition of nuclear matrix proteins changes as the osteoblast differentiates. To isolate nuclear matrix proteins unique to the bone phenotype, we analyzed nuclear matrix proteins from the primary cultures of rat calvarial osteoblasts by two-dimensional gel electrophoresis at two different stages:proliferation (day3) and differentiation (day 18). We characterized a protein (20 KDa; pI 4.76) that was detected only in the nuclear matrix of differentiated osteoblasts. By MALDI-TOF mass spectrometry and microsequencing, this protein was identified as the TCTP. Expression of TCTP was detected both in vitro bone cell cultures and in vivo bone tissues. In Northern blot analysis, TCTP mRNA expression was detected from early stage to late differentiation stage with no change of its level during the differentiation of human or rat primary cultured osteoblasts. In Western blot analysis, TCTP expression was not changed during osteoblast or chondrocyte differentiation. However, TCTP was detected only in the nuclear matrix fractions of differentiated osteoblasts. In immunohistochemical staining, TCTP was detected in both osteoblasts and chondrocytes in long bone of 17.5 days mouse embryo. These results suggest that TCTP may play an important role in the nucleus during osteoblast differentiation.

Disclosures: Y. Jung, None.

M144

Expression of Splice Variants of Collagen XII in Human Osteoblast-like Cells.

A. Kamada¹, T. Ikeo¹, Y. Yoshikawa¹, I. Tamura*¹, S. Goda*¹, E. Domae*¹, Y, Takaishi¹, T. Miki². ¹Biochemistry, Osaka Dental University, Hirakata, Japan, ²Geriatric Medicine, Osaka City University, Osaka, Japan.

Type XII collagen, which is a fibril-associated collagen (FACIT), is characterized by a short triple-helical domain with three extended noncollagenous NC3 domains, and may contribute to the stability of collagen I fibrils. Little is known about the role of this FACIT collagen in bone metabolism. In this study, we demonstrated collagen XII gene expression in human osteoblast-like cells, and the influence of beta-glycerophosphate (BGP) as a biomineralization-inducing agent on the expression of splice variants, which have a long or short NC3 domain.

Two sets of primers and probe for a large variant specific site and a common site in collagen XII NC3 domain were designed based on cDNA sequences in the Gene Bank database. RNA was extracted from cells cultured with or without BGP for 0,2,7 days. The expression levels of mRNA were assessed using real-time quantitative RT-PCR technique. The mRNA expression of alpha1(XII) in intact normal human osteoblasts (NHOB) and human osteoblast-like osteosarcoma cells (SaOS2) was confirmed by RT-PCR and agarose gel electrophoresis. Western blot analysis also showed the protein expression of both the long and short form of alpha1(XII) in osteoblast-like cells. When cells were cultured with BGP, mRNA expression levels of alpha1(I), osteocalcin and ALP increased compared with controls (p<0.05). In contrast, the level for the long variant of alpha1(XII) was significantly lower than that in controls two days after BGP stimulation (p<0.05). It is known that the long form NC3 domain of collagen XII can carry glycosaminoglycans and bind to tenascin, while collagenous domains attach to collagen I. Our results clarify that collagen XII gene is transcripted in human osteoblast-like cells, and indicate that the transcription control of splice variants may be concerned with bone formation.

Disclosures: A. Kamada, None.

M145

Murine Bone Loss Following Local Irradiation.

E. R. Bandstra¹, S. Judex², M. E. Vazquez*³, T. A. Bateman¹. ¹Bioengineering, Clemson University, Clemson, SC, USA, ²Biomedical Engineering, SUNY Stony Brook, Stony Brook, NY, USA, ³Medical Department, Brookhaven National Laboratory, Upton, NY, USA.

Spaceflight presents a complex set of challenges to the skeletal system. Unloading is a well documented stressor that results in bone loss. The impacts of radiation on bone have not been well studied. On long-duration exploratory missions, astronauts will be exposed to higher doses of ionizing radiation from both solar and cosmic sources, ranging from protons to iron ions. We have recently identified that multiple spaceflight-relevant types of radiation, approaching doses astronauts will be exposed to on exploratory missions, cause profound trabecular bone loss. This study aims to investigate the effects of local (head only) irradiation on regional and systemic bone. Male C57BL/6 mice were irradiated with IGeV/n HZE ⁵⁶Fe at 16 weeks of age at the NASA Space Radiation National Laboratory. The radiation was collimated such that the head received 2.4 Gy and the rest of the body, including humerus and tibia, received 14% (33.6 cGy) of the total dose delivered. The mice were sacrificed 8 weeks after irradiation. MicroCT analysis was performed on the trabecular and cortical bone of the proximal humerus and tibia. Within the humerus, a significant 15.1% decline in trabecular volume fraction was observed compared to the control group. However, no differences were observed within the tibia compared to control (insignificant decline in trabecular volume fraction of 4%). Analysis of the cortical bone revealed a 4.2% decrease in bone volume with a 6.5% increase in cortical porosity in the proximal humerus, with no significant cortical changes in the proximal tibia. The bone loss in the humerus, without corresponding loss in the tibia, indicates either a differential response of these bones or a greater loss in bones anatomically closer to higher doses of radiation. Previous work has not found changes in cortical bone following similar doses of low-LET radiation, possibly indicating cortical bone changes are LET-dependent. In contrast, previous studies of radiation at these doses have not shown an LET-dependent response in trabecular bone. Another important contribution of this study is the demonstration of bone loss in mice that are near peak bone mass. Our previous studies examined mice irradiated at 9 weeks of age when the skeletal system is rapidly growing. Radiation-induced bone loss in the mature skeleton further demonstrates that this challenge represents a potential concern for astronauts on long-duration exploratory missions, and should be studied to further characterize the bone loss following irradiation, identify causal mechanisms, and develop countermeasures.

Disclosures: E.R. Bandstra. None.

This study received funding from: National Space Biomedical Research Institute.

M146

See Sunday Plenary Number S146

M147

Fracture Threshold in Breast Cancer Patients Treated with Adjuvant Aromatase Inhibitors.

F. Bertoldo*, L. Dalle Carbonare*, S. Zenari*, S. Pancheri*, M. Zanatta*, B. Giovanazzi*, M. T. Valenti*, V. Lo Cascio.. Biomedical and Surgical Sciences, Medicina D, University of Verona, Verona, Italy.

Aromatase inhibitors (AI) in the adjuvant treatment of breast cancer patients is notoriously associated with an increased risk of fractures as opposed to tamoxifene therapy (ATAC trial). Prospective studies evaluating the long-term effects of AI treatment on bone mineral density (BMD) showed a relatively modest decrease in BMD after 2 years of treatment and no patients with normal BMD at baseline became osteoporotic at the end of observation. Actually it is not clear which patients should receive preventive measures. The latest ASCO guidelines identified BMD as a valid tool in treatment decision making. As the value of BMD T-score < −2.5 for fracture threshold is applicable only for postmenopausal osteoporosis (PMO), and pathophysiology underlying AI induced bone loss may be different from PMO, we planned a pilot cross-sectional study to assess the BMD fracture threshold in AIs treated women. We compared lumbar and femoral neck BMD (Hologic Discovery A), vertebral fracture (vFX) prevalent number, Spine deformity Index (SDI), bone turnover markers among 21 breast cancer patients treated for one year with AIs (AI group), 96 PMO patients (PMO group) and 42 breast cancer patient not treated with AIs (BC group). The groups were comparable for age (52+ 5 y.o. 52+6 and 51.8+4 respectively) height and BMI. The three group did not differ for the number of prevalent vFX and SDI. Lumbar spine BMD T-score was −2.21 ± 0.5 in AI group, −2.53 ± 0.3 in PMO group and −2.49 ± 0.5 in BC group (p 0.042 ANOVA). Among marker of bone turnover, CTX was significantly higher in AI group than in PMO and BC groups (0.684 ± 0.123, 0.427+0.185 and 0.408 ± 0.210 respectively; p 0.036 ANOVA). A weak inverse correlation between CTX and spine BMD T-score was found only in PMO and BC group (r 0.48 and r 0.32 respectively). These preliminary data suggest that independent predictive factors of fracture risk, i.e high bone turnover, could increase BMD fracture threshold in AIs treated patients compared to PMO patients or BC patients not treated with AI.

Disclosures: E Bertoldo. None.

M148

See Sunday Plenary Number S148

M149

Identification of Proteins that Interact with the Collagen Type XI (α1) Amino Terminal Domain.

R. J. Brown*, J. R. T. Oxford.. Biomolecular Research Center and Department of Biology, Boise State University, Boise, ID, USA.

The extracellular matrix of articular cartilage is composed of collagens, proteoglycans and a variety of noncollagenous, nonproteoglycan molecules which interact to ultimately contribute to the structural integrity, compressibility and tensile strength of the tissue. The ultimate goal of our investigation was to examine the potential interactions of the amino terminal domain (NTD) of collagen. Utilizing the recombinant aminopropeptide common to each of the collagen α1(XI) isoforms, proteins extracted from bovine articular cartilage were separated by affinity chromatography. Molecules extracted from the articular cartilage and enriched by their association with the surface of the collagen fibril were identified by tandem mass spectrometry. Proteins enriched by the association with the surface of the α1(XI) collagen fibril were predominantly: the collagen types IX, XII and XIV; the extracellular matrix proteins, COMP (thrombospodin-5), thrombospondin-1, matrilin-1 (CMP), chondroadherin, PARP, matrilin-3, chondrocalcin and proteoglycans; biglycan and fibromodulin. The results presented here provide evidence that the NTD of collagen α1(XI) may associate, either directly or indirectly, with a variety of cartilage matrix constituents in the connective tissue and suggest the possibility that this region of collagen α1(XI) may add structural support bridging the environment between chondrocytes and the surrouding extracellular matrix or play a role in fibrillogenesis.

Disclosures: R.J. Brown, None.

M150

See Sunday Plenary Number S150

M151

Bioreactor Design for Osteochondral Tissue.

S. Cartmell¹, J. Gittings*², L. Turner*², J. Chaudhuri*³, M. Ellis*³, S. Waters*⁴, L. Cummings*⁴, N. Kuiper*¹, V. Michael*¹. ¹Institute of Science and Technology in Medicine, University of Keele, Stoke on Trent, United Kingdom, ²Department of Mechanical Engineering, University of Bath, Bath, United Kingdom, ³Department of Chemical Engineering, University of Bath, Bath, United Kingdom, ⁴Division of Applied Mathematics, University of Nottingham, Nottingham, United Kingdom.

Bioreactor design for the culture of tissue-engineered constructs is essential for the production of functional tissue. Co-cultures of cartilage and bone tissue together have been shown to be beneficial compared to monocell cultures.

We have developed a bioreactor for culturing osteoblasts/chondrocytes together (A). Two separate bone and cartilage culture chambers are assembled together with the bone/cartilage separated by a silicone membrane. This system allows nutrient administration via two separate methods to allow scale up of larger tissues. Media is perfused to the chambers whilst a PLGA porous fibre runs through the bone scaffold delivering extra nutrients via diffusion. Through this fibre, a temperature/oxygen sensor will be introduced to the samples, allowing monitoring of cellular activity. Non-invasive lactate and glucose culture media analysis also allows further quantification. Porous calcium-phosphate scaffolds (15mm × 10mm) and Chitosan hydrogel substrates (15mm × 6mm) are used to support the bone/cartilage cells. The bone scaffold (B) mimics the arrangement in an articulating joint where the porous trabecular bone is covered with a shell of dense cortical bone which, in turn, interfaces with the cartilage component. It is hypothesised that the cap to the porous scaffold will restrict mixing between the bone and cartilage media. Static experiments have been performed by seeding bone scaffolds with MG-63 cells. Mathematical modelling of the bone chamber and porous scaffold has been performed.

Preliminary seeding data reveal 59% and 76% of cells were adherent on the scaffold after 2 hours and 4 days in culture respectively (C). Numerical simulations were able to visualise the fluid distribution through the bone chamber, and determine the optimum chamber size and inlet/outlet configuration to achieve minimal regions of stagnation, while maintaining shear stress below the threshold (5–15 dyne/cm2).

This sophisticated co-culture bioreactor has the potential to significantly improve the functionality of tissue-engineered osteochondral plugs.

Disclosures: S. Cartmell. None.

M152

See Sunday Plenary Number S152

M153

A Novel Model of Impaired Fracture Healing.

T. S. Gross¹, S. L. Poliachik*¹, S. E. Nork*¹, S. D. Bain². ¹Orthopaedics and Sports Medicine, University of Washington, Seattle, WA, USA, ²MDS Pharma Services, Bothell, WA, USA.

The biological pathways responsible for normal fracture repair are well understood. However, the causal factors leading to unsuccessful fracture healing are ill defined and poorly understood. Based on recent data indicating that transient muscle paralysis rapidly induces bone loss in mice, we hypothesized that a transient loss of muscle function superimposed upon a closed femur fracture in rats would lead to delayed fracture healing. To test this hypothesis, Sprague-Dawley female rats (age: 4 months, n = 14) underwent a standard closed femur fracture with a Kirschner wire insetted through the right femur diaphysis prior to blunt guillotine impact. Following radiographic confirmation of the location and disposition of all fractures, 7 rats were given a single injection of Botox in their right quadriceps (2 Units/100g, 1/2 of dose 5 mm proximal, ½ dose 5 mm distal to the fracture site). The remaining 7 rats received identical volume Saline injections. All rats were allowed to ambulate freely, with 10 rats killed at 4 wk (n=5/grp) and 4 rats killed at 6 wk (n=2/grp). Following sacrifice, right femurs were radiographed and micro-CT scans were obtained of all right and left femurs (1.6 cm region centered at the mid-diaphysis, 21 μm voxel). For each animal, callus volume and the polar moment of inertia (pMOI) of the callus were determined by contrast with the contralateral intact left femur. Botox induced quadriceps paralysis significantly diminished fracture callus volume compared to Saline treated rats at 4 wk (-42.1%, p=0.003) and 6 wk (-39.9%, p=0.05). Similarly, callus pMOI was profoundly reduced in Botox treated rats compared to Saline treated rats (4 wk: −60.0%, p<0.001; 6 wk: −49.8%, p=0.03). Radiographic examination indicated an absence of bone union in 6 of 7 Botox treated rats, while all Saline treated rats demonstrated clear evidence of bone union across the fracture gap. These data clearly demonstrate that transient muscle paralysis of a single muscle group adjacent to the femur leads to impaired fracture healing. Further, the lack of callus formation in the Botox rats was not spatially confined to the anterior cortex adjacent to the quadriceps. As diminished muscle function following trauma or due to aging have both been associated with poor fracture healing, this novel model holds potential to directly impact clinical care by exploring why normal muscle function is required for successful fracture healing.

Disclosures: T.S. Gross. None.

M154

Formation of Haversian-Type Bone and Blood Vessels Guided by the Artificial ECM of Vasculature-Inducing Geometry.

T. Kaku¹, H. Kobavashi*¹, S. Iku*², K. Nemoto*², T. Mivata*², Y. Kuboki*³. ¹Oral Pathology, Health Sciences University of Hokkaido, Hokkaido, Japan, ²Koken Bioscience Institute, Hokkaido, Japan, ³Koken Bioscience Institute & Hokkaido University, Hokkaido, Japan.

We have proposed that geometrical property of the artificial extracellular matrices (ECM) must be taken into consideration for successful development of tissue engineering (JBJS 83A:S1–105–115, 2001). Geometry is defined in this case as the three dimentional (3D) structure of artificial ECM at the order of micrometer, which can direct growth of tissues and organs in vivo and in vitro. Previously we have reported that honeycomb-shaped hydroxyapatite (HC-HAP) with the pores less than 100-um induced endochondral ossification, while one with 350-um pores induced direct bone formation. By the fact that a single large blood vessels was always noticed in the center of the 350-um pores of HC-HAP), we attemped to direct the haversian-type bone formation by an artificial ECM with similar geometry to HC-HAP but with biodegradable property. We chose a honeycomb-shaped collagen product (HC-COL) (Honeycomb sponge, Koken, Japan). HC-COL was cut into block-form, combined with the purified BMP cocktail (500 ug) or rhBMP-2 (5 ug) and implanted into rat skin. HC-COL was calcified with a series of the solutions, which contained calcium and phosphate to obtain the hydroxyapatite-coated honeycomb collagen(HAP-HC-COL), which was also implanted into the skin. Two weeks after implantation, it was found that the concentric layered osteogenesis occurred within the each tunnels of HC-COL/BMP, the centers fo which, there was a single large blood vessel. The structure was reminded us the haversian system. More interestingly, HAP-HC-COL itself(without BMP) induced the concentric fibrous tissue formation with a blood vessel in the center of the tunnel. Although bone formation was not observed. It was concluded that the characteristic 3D-structure (vasculature-inducing structure) of HC-COL or HAP-HC-COL induced vasculature first, irrespective of the presence of BMP. Then bone formation will occurs if the mesenchymal cells were differentiated into osteoblasts by BMP.

Disclosures: T. Kaku. None.

M155

Mineral and Matrix Characteristics in Mice with LRP5 G171V Mutation.

N. P. Camacho¹, M. P. Akhter². ¹Imaging & Spectroscopy Lab, The Hospital for Special Surgery, New York, NY, USA, ²Medicine, Creighton University, Omaha, NE, USA.

Greater bone density and strength has been reported in mice with the LRP5 G171V mutation. We characterized femoral cortical bone properties to determine if the LRP5 G171V mutation differences in mineral and matrix properties as compared to wild type (WT). Sixteen female mice (4 mo.) representing two genotypes (WT=wild type, HBM=High bone mass with LRP5 G171V mutation) were used. Femoral mineral and matrix properties were analyzed by Fourier transform infrared imaging spectroscopy (FT-IRIS). This allows information on mineral, collagen, and carbonate content and distribution to be obtained at ∼ 7 μm spatial resolution from PMMA-embedded bone sections. Infrared vibrations of both the mineral (a poorly crystalline carbonated apatite (HA)) and matrix phases (primarily Type I collagen) were monitored simultaneously. The ratio of the area of the HA phosphate v₁, v₃ absorbance from 900 to 1200 cm⁻¹, to the area of the protein amide I absorbance from 1590 to 1720 cm⁻¹ was calculated to obtain the relative amounts of mineral and protein present (min:matrix), a parameter previously shown to be correlated to ash weight. The carbonate-to-mineral ratio (carb:min), an indicator of carbonate content of the mineral phase, was calculated as the ratio of the area of the carbonate n₂ absorbance from 850 to 890 cm⁻¹ to the area of the phosphate n₁, n₃ absorbance. Crystallinity of the mineral phase was calculated by ratioing the intensity of the absorbance at 1030cm⁻¹ to that at 1020 cm⁻¹, a parameter previously shown to be related to the apatite crystal length in the c-axis direction. The crosslink (Xlink) parameter, related to the ratio of mature to immature collagen crosslinks, was obtained by ratioing the intensity of the absorbance at 1660cm⁻¹ to that at 1686 cm⁻¹, We analyzed the data using the Student's t test for differences due to genotype. There were no differences in FT-IRIS determined bone parameters, except for the Xlink parameter, which tended to be greater in the HBM mice compared to the WT mice ( p = 0.08). An increase in this collagen parameter likely reflects increased mature crosslinks, a finding which could be consistent with the greater bone strength previously reported in the HBM mice. Further investigations will focus on how the relatively increased crosslinks in HBM mice influence their bone intrinsic material properties as measured by nano-indentation at ultrastructural level. 

Table  .  

Disclosures: M.P. Akhter, None.

M156

Secreted Phosphoprotein-24 Is Converted from an Inhibitor to a Functional Enhancer During BMP-Directed Osteoblastogenesis.

M. J. Beckman¹, S. C. Ramage¹, A. Maiti², O. Korchvnskvi². ¹Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA, USA, ²Orthopaedic Surgery, Virginia Commonwealth University, Richmond, VA, USA.

Bone morphogenetic proteins (BMPs) are members of the TGFβ family and key regulators of osteogenesis. BMPs signal through their cognate receptors to SMAD proteins that transduce the signals to activate genes crucially important to osteogenesis. Secreted phosphoprotein-24 (Spp24) is a 24 kDa bone matrix protein isolated from demineralized bovine bone matrix. Spp24 has known homology domains to both cystatin and TGFβ receptor type II (TβRII), and like many bone matrix proteins has numerous sites for glycosylation as well as serine and threonine phosphorylation. The TβRII domain of Spp24 has shown a low affinity binding to BMP-2. A truncated form of Spp24 has been shown to possess osteoinductive character and enhance BMP activity. Overexpression of mouse Spp24 (mSpp24) inhibited various BMPs' signaling at early time points. We hypothesize that Spp24 is converted to a more active factor that can enhance later stages of BMP-mediated osteoblastogenesis. This concept was tested using an adult human bone marrow mesenchymal stem cell model (MSC). A mouse Spp24 adenovirus (Adv5CMV-mSpp24) construct was used to transduce MSCs with 100% efficiency for long duration. MSC differentiation to osteoblasts (OB) was induced and maintained by controlled treatment with BMP-2/7 heterodimer dosed over a 14 day period, resulting in fully differentiated OB cells. The experimental design was a 2 × 2 factorial of AdvSCMV-LacZ control, LacZ plus BMP-2/7, mSpp24, and mSpp24plus BMP-2/7. All groups were also co-transduced with a BMP-specific response element reporter to assay BMP-Smad-induced signaling and β-galactosidase for normalization. Results demonstrated marked repression of BMP signaling by mSpp24 after 3 days in culture. By day 7, the mSpp24 repression of BMP signaling was not evident. However, at the subsequent time points, particularly on day 10, the effect of mSpp24 was to significantly enhance BMP signaling. Western blots were performed with the HA-tagged mSpp24 protein product at 3, 7, 10 and 14 days. The results of this analysis revealed a truncated 18 kDa fragment of mSpp24, representing the putative active form, appearing by day 7 and increased at day 10. In conclusion, these data indicate that Spp24 plays and antagonistic role to osteogenic signaling until it is modified in a yet unspecified manner to create this smaller 18 kDa form that is capable of enhancing BMP activity. Our study confirms earlier work by Wozney that described a slow release factor capable of regulating BMP-mediated osteogenesis and later by Murray that Spp18 is the active fragment.

Disclosures: M.J. Beckman, None.

M157

See Sunday Plenary Number S157

M158

Cross-Talk Between CTGF and TGF-β1 in Mesenchymal Stem Cell Condensation.

F. Del Carpio-Cano*¹, J. Y. Belcher¹, K. B. Buck*¹, J. A. Arnott¹, R. A. DeLa Cadena*², S. N. Popoff¹, F. F. Safadi¹. ¹Anatomy and Cell Biology, Temple University, Philadelphia, PA, USA, ²Physiology, Temple University, Philadelphia, PA, USA.

Condensation or the aggregation of mesenchymal stem cells (MSCs) precedes chondrocyte differentiation and is required for cartilage formation. CTGF is a matricellular protein that has been found to be expressed during MSC condensations in vivo. It has been shown that TGF-β1 regulates CTGF expression and that CTGF acts as a downstream mediator of TGF-β1 effects on extracellular matrix production. It has also been reported that CTGF has the ability to bind TGF-β1 and modulates its effects. Using C3H10T1/2 MSCs as a model for mesenchymal condensation, we have shown previously that TGF-β1 induces MSC condensation in vitro associated with increased matrix production, proliferation and migration and this induction is mediated by CTGF. In this study, we were interested to examine whether CTGF overexpression can mediate MSC condensation in the absence or presence of TGF-β1. C3H10T1/2 MSCs were infected with adenovirus over-expressing CTGF tagged with GFP achieving a 6–7 fold increase in CTGF mRNA and protein expression. Adenovirus expressing only GFP was used as control. Cells overexpressing CTGF did not show any MSC condensation. Surprisingly, TGF-β1 induced MSC condensation was inhibited in cells overexpressing CTGF. These results suggest that a fine equilibrium of CTGF expression is required for TGF-β1-induced MSC condensation. We next examined the effect of CTGF overexpression on MSC adhesion and spreading associated with vinculin localization at focal adhesion and actin cytoskeletal reorganization. Cells overexpressing CTGF spread more robustly with an increased punctuated signal of vinculin at sites of focal adhesion with the formation of lamelopodia when compared to cells infected with GFP virus. We next examined the signaling pathway associated with MAP Kinase family to evaluate differences between TGF-β1-induced MSC condensation and the inhibitory effect of CTGF overexpression on MSC condensation. Phosphorylated P38, Jnk and Erk were increased in the GFP-infected MSCs treated with TGF-β1. However, MSCs infected with GFP-CTGF and treated with TGF-β1 showed only an increase in phosphorylated Jnk and Erk but not P38. These findings indicate that p38 MAPK may mediate MSC condensation by TGF-β1. Further studies are warranted to modulate P38 expression to elucidate the interaction between CTGF and TGF-β1 in regulating MSC condensation.

Disclosures: F. Del Carpio-Cano, None.

This study received funding from: Pennsylvania Dept. of Health and NIAMS/NIH.

M159

The Three Dimensional ¹H NMR Structure of Bovine Lead Ion-Bound Osteocalcin and Implications for Lead Toxicity.

L. Li*¹, C. M. Gundberg², T. L. Dowd*³. ¹National Institute of Environmental Health Science, Research Triangle Park, NC, USA, ²Orthopedics, Yale University School of Medicine, New Haven, CT, USA, ¹Chemistry, Brooklyn College of the City University of New York, Brooklyn, NY, USA.

Structural information on the effect of Pb^2* on proteins under physiologically relevant conditions is largely unknown. We have solved the three dimensional structure of bovine lead coordinated osteocalcin, a bone protein from a target tissue, using ¹H 2D NMR techniques and suggested a molecular mechanism for lead toxicity. The protons in the 49 amino acid sequence were assigned using 2D homonuclear NMR experiments. Lead, at a stoichiometry of only 1:1, was shown to induce a tertiary structure in the protein similiar to that induced by Ca²⁺ at a stoichiometry of 3:1. The structure of Pb²⁺-osteocalcin consists of an unstructured N terminus and a C-terminal hydrophobic core (residues 16–49) formed by long range hydrophobic interactions. Elements of secondary structure within residues 16–49 include a type III turn (residues 27–30) and a 3₁₀ helix (residues 31–34). Residues 21–26 formed loose turns with helical characteristics. The 3 Gla residues project from the same face of the helical turns and are surface exposed. The genetic algorithm -molecular dynamics simulation approach was used to place 1 lead ion on the NMR derived structure. The lead ion was coordinated by side chain oxygen ions of OE3 and OE4 of Gla 24 and the side chain oxygen ions of OE1 and OE2 of Gla 21. The best correlation of the distances between the uncoordinated Gla oxygen ions is with the intercalcium distance of 9.43 Å in hydroxyapatite. This mineral binding is similiar to what was proposed from the structure of Ca²⁺-osteocalcin. A comparison of Pb²⁺- and Ca²⁺-osteocalcin indicates Pb²⁺ may induce conformational changes and subsequent molecular processes in proteins prematurely. Lead exposure may alter the amount of mineral bound osteocalcin contributing to abnormal bone remodeling. This may play a role in the increased bone resorption and decreased bone density observed in bones from lead exposed animals.

Disclosures: T.L. Dowd, None.

This study received funding from: NIH.

M160

See Sunday Plenary Number S160

M161

The Biological Trials of Hyaluronic Acid on Osteoclast Differentiation Induced by IL-1.

M. Hirata¹, M. Kobavashi*¹, K. Mivazaki*², C. Mivaura¹, M. Inada¹. ¹Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Tokyo, Japan, ²Seikagaku Corporation, Tokyo, Japan.

Hyaluronic acid (HA) is a component of extra cellular matrix (ECM) in cartilage, polymer of a glycosaminoglycan organized the network with majority of agrican. In human bone marrow, HA produced in both stromal and hematopoietic lineage of cells, the capacities could be counted on regulating cellular response for bone cells. In bone ECM, HA present as a high molecular size, however, the functional effect of HA on osteoclast differentiation is still unclear. Here we demonstrate that the roles of HA in osteoclast differentiation induced by IL-1. When the co-culture of primary osteoblasts from newborn calvaria and bone marrow cells from tibiae in the presence of IL-1, TRAP (Tartrate Resistant Acid Phosphatase) positive multinucleated cells were formed 7 days of culture. HA single treatment did not alter the proliferation of osteoblast/stromal cells. In the presence of IL-1 (2 ng/ml) and HA (900 KDa, 1 mg/ml), however, the number of TRAP positive cells was decreased. RT-PCR was performed using primer sets of RANKL (as an inducible ligand on osteoblast) and NFATcl (inducible transcriptional factor on osteoclast differentiation). The result showed RANKL expression induced by IL-1 was suppressed in the presence of HA. NFATcl expression slightly decreased in the culture. To identify further mechanisms of the HA, ex vivo calvarial organ culture was performed. Calvarial bone from day 4 old mice were dissected into 2 portions (control vs. challenge group) of culture, bone resorbing activity was measured by cultured medium calcium. When the presence of IL-1 (2ng/ml) in culture, bone resorbing activity was increased and the activity was suppressed by the simultaneous treatment with HA (900 KDa, 1 mg/ml). These results suggested HA attenuated IL-1 induced RANKL production in osteoblast, following NFATcl expression was suppressed in osteoclast precursors sequentially decrease the number of mature osteoclast. The potentials shown in the presented study suggested that HA could apply various inflammatory diseases as an inhibitor of bone loss.

Disclosures: M. Hirata. None.

M162

The Inhibitor of Metalloproteinases TIMP-1 Rescues the Strong Bone Loss Induced by Runx2 Overexpression in Mice.

C. Schiltz*, C. Prouillet*, C. Marty*, M. De Vernejoul, V. Geoffroy.. Hǒpital Lariboisière, INSERM U606, Paris Cedex 10, France.

The Runx2 gene is essential for osteoblast differentiation and function. Runx2 deficiency results in complete lack bone formation and overexpression of Runx2 in cells of the osteoblastic lineage to severe osteoporosis. Osteoblastic matrix metalloproteinases (MMPs) have been reported to be required for normal bone resorption. Several lines of evidence suggest that osteoblastic MMPs could take part of the increased bone resorption observed in mice after overexpression of Runx2. The goal of our study was to define using transgenic approach whether inhibition of osteoblastic MMPs can reduce the bone loss induced by Runx2.

We examined the rescue of the severe osteopenic phenotype in Runx2 mice by overexpressing the MMPs inhibitor TIMP-1 (tissue inhibitor of metalloproteinase 1) specifically in osteoblasts. 1-, 4- and 8-month-old females with the different genotypes (WT, Runx2, TIMP-1 and TIMP-1/Runx2) were generated. Bone density was measured by DXA and by pQCT for all mice. Microarchitecture and formation and resorption parameters were evaluated in 1- and 4-month-old females by histomorphometry. Primary osteoclasts were differentiated ex vivo from bone marrow or co-cultured with primary osteoblasts isolated from calvaria. Primary osteoblasts were also used to evaluate gene expression by qPCR.

Bone density analysis indicated that TIMP-1 overexpression is efficient at reducing bone loss observed in Runx2 mice but only in 4- and 8-month-old females. The rescue and prevention of bone loss in TIMP-1/Runx2 mice were visible at the long bones and the caudal vertebrae. Histomorphometry analysis on trabecular bone indicated that this rescue in 4-month-old TIMP-1/Runx2 females was due to a decrease of the resorption activity (decreased trabecular separation and osteoclastic surfaces) and a sustained osteoformation (increased trabecular thickness and maintained BFR) compared to Runx2 mice. In addition, we also reported a partial rescue of the cortical bone loss (increased cortical thickness and outer bone diameter) in TIMP-1/Runx2 mice compared to Runx2 mice. Our ex vivo study showed that the ability of osteoclastic cell to differentiated is reduced in TIMP-1/Runx2 mice compared to Runx2 mice due to a reduction in the expression of RANK-L by TIMP-1/Runx2 osteoblastic cells.

In conclusion, we showed that TIMP-1 overexpression in the runx2 background mainly reduces bone resorption (decrease osteoclasts differentiation and activity) and maintains bone formation in trabecular and cortical bone. Our results indicate that osteoblastic MMPs are partly responsible for the bone loss in mice overexpressing Runx2.

Disclosures: V. Geoffroy, None.

This study received funding from: ANABONOS consortious (Sixth Framework Programme of the european community).

M163

See Sunday Plenary Number S163

M164

Identification of Novel Transcription Factors Necessary for Chondrocyte Hypertrophy.

A. M. Ionescu, A. B. Lassar*. BCMP, Harvard Medical School, Boston, MA, USA.

To date, only two families of transcription factors are known to regulate chondrogenesis, Sox and Runx family members. The early steps of chondrogenesis, including mesenchymal condensation and expression of chondrocyte-specific extracellular matrix proteins are critically regulated by Sox9, Sox5, and Sox6. In contrast, the latter steps of chondrogenesis appear to be regulated by Runx2 and/or Runx3. Runx2 is expressed in chondrocytes as they initiate chondrocyte hypertrophy, and loss of this factor in genetically engineered mice severely delays chondrocyte maturation in a number of developing bones.

Interestingly, recent work in the Lassar lab has demonstrated that expression of exogenous Runx2 fails to activate expression of endogenous collagen × in naïve somites, but can activate expression of this gene in somites that have been induced to initiate the chondrocyte differentiation program. Consistent with this result, I have found out that Runx2 and Smad1 (which transduces BMP signals) are only able to induce the expression of a Col X-luciferase reporter in upper sternal chondrocytes (USCs) but not in fibroblasts (CEFs) isolated from chicken embryos, in the presence of exogenous BMP signals. This suggests that hypertrophic chondrocytes express another factor that is required for the expression of Col X.

To identify a chondrocyte-specific DNA binding factor(s), a series of overlapping 40 bp oligomers that cover the Col × enhancer were used in an EMSA to detect DNA binding activities that are specifically present in primary chondrocytes but are absent from fibroblasts. This analysis led to the identification of a Fast Mobility Complex (FMC) that binds to 4 sites in the collagen × regulatory sequences, a Slow Mobility Complex (SMC) that binds to 2 sites and Sox S which binds in multiple places along the collagen × regulatory sequences. Mutational analysis of these binding sites indicated that the FMC sites are necessary for efficient induction of a Col X-luciferase reporter by co-transfected Runx2 and Smad1 in chondrocytes. Moreover, 4-fold reiteration of one of the Fast Mobility Complex binding sites (FMC3) placed upstream of Col X-promoter proximal sequences (4xFMC3-Col X-luciferase), led to chondrocyte-specific induction of the downstream luciferase reporter by co-transfected Runx2 and Smad1. Thus, our immediate focus consists in identification and characterization of the proteins that comprise FMC3. The successful identification of FMC3 will lead to the identification of new hypertrophy-specific factors which, if inhibited, could reverse chondrocyte hypertrophy which is pathologically induced during osteoarthritis.

Disclosures: A.M. Ionescu, Arthritis Foundation postdoctoral fellowship 2.

This study received funding from: Arthritis Foundation.

M165

See Sunday Plenary Number S165

M166

Alpha-2-Macroglobulin Is a Novel Substrate for ADAMTS-7 and ADAMTS-12 and Inhibits Their Degradation of Cartilage Oligomeric Matrix Protein.

Y. Luan*¹, D. R. Howell*², L. Kong*², C. Liu². ¹Orthopaedic Surgery, New York University, New York, NY, USA, ²New York University, New York, NY, USA.

Degradative fragments of cartilage oligomeric matrix protein (COMP) have been observed in both osteoarthritis and rheumatoid arthritis. We previously reported that ADAMTS-7 and ADAMTS-12, two members of ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family, sharing the similar domain organization in the family, degraded COMP in vitro and were significantly induced in the cartilage and synovium of arthritic patients (Liu, et al, FASEB J. 2006; 20(7):988 and Liu, et al, J. Biol. Chem. 2006; 281(23): 15800). In this study we further demonstrated the importance of COMP degradation by ADAMTS-7 and ADAMTS-12 in vivo, since i) the size of the COMP fragments produced by either ADAMTS-7 or ADAMTS-12 is similar to that of COMP-degradative fragments seen in the patients with osteoarthritis; ii) antibody blocking assay indicated that specific antibodies against ADAMTS-7 or ADAMTS-12 dramatically inhibits TNF-α-induced COMP degradation in the cultured cartilage explants and iii) suppression of ADAMTS-7 or ADAMTS-12 expression using siRNA silencing approach in the human chondrocytes also markedly prevents COMP degradation. Furthermore, we revealed that α₂-Macroglobulin (α₂M) is a novel substrate for ADAMTS-7 and ADAMTS-12 based on the observations that both metalloproteinases were able to cleave α₂M in a dose-dependent manner, which gave rise to the cleavage products with the molecular weights of 180 and 105 kDa respectively. More significantly, α₂M inhibits both ADAMTS-7- and ADAMTS-12-mediated COMP degradation in a does-dependent manner; thus α₂M represents the first endogenous inhibitor of ADAMTS-7 and ADAMTS-12.

Disclosures: Y. Luan. None.

This study received funding from: NIH.

M167

MT3-MMP Is a Major Mesenchymal Collagenase Essential for Skeletal Development.

J. Shi*, M. Son*, S. Yamada*, L. Szabova*, S. Kahan*, K. Chrysovergis*, L. Wolf*, A. Surmak*, K. Holmbeck*. CSDB/MMPU, NIDCR, Bethesda, MD, USA.

Peri-cellular remodeling of mesenchymal extracellular matrices is considered a prerequisite for mesenchymal cell proliferation, motility and development. Here we explore the molecular mechanisms responsible for collagen remodeling in the skeleton and peri-skeletal tissues of the mouse. We demonstrate that membrane-type 3 MMP, MT3-MMP, is expressed in mesenchymal tissues of the skeleton and in peri-skeletal soft connective tissue. Consistent with these observations MT3-MMP-deficient mice display growth inhibition tied to a decreased viability of mesenchymal cells in skeletal tissues. We document that MT3-MMP works as a major collagenolytic enzyme, enabling cartilage and bone cells to cleave high-density fibrillar collagen and modulate their resident matrix to make it permissive. Collectively, these data uncover a novel extracellular matrix remodeling mechanism required for proper function of mesenchymal cells. The physiological significance of MT3-MMP is highlighted in mice double deficient for MT1-MMP and MT3-MMP. Double deficiency transcends the combined effects of the individual single deficiencies and leads to severe embryonic defects in palatogenesis and bone formation incompatible with life. These defects are directly tied to loss of indispensable collagenase activities required in mesenchymal tissues for extracellular matrix remodeling and cell proliferation during embryogenesis. Based on these observations we conclude that peri-cellular collagen remodeling mediated by the MT1-MMP/MT3-MMP synergizing duo of collagenases is essential for skeletogenesis and palatogenesis in the mouse and demonstrates the requirement for embryonic remodeling of collagen.

Disclosures: J. Shi. None.

M168

BMP-2 Induces Sustained Expression of COX-2 in MC3T3-E1 Osteoblasts.

K. A. Blackwell*¹, P. Hortschansky*², L. G. Raisz³, C. C. Pilbeam³. ¹Endocrine Division and Musculoskeletal Institute, University of Connecticut Health Center, Farmington, CT, USA, ²Leibniz Institute for Natural Product Research and Infection Biology, Hans-Knöll-Institute (HKI), Jena, Germany, ³Endocrine Division and Musculoskeletal Institute, University of Connecticute Health Center, Farmington, CT, USA.

BMP-2 potently stimulates osteoblastic differentiation. We have shown that BMP-2 induces cyclooxygenase-2 (COX-2) expression in murine primary calvarial osteoblasts via a Runx2 binding site at −267/-261 bp in the COX-2 promoter and that the full anabolic response to BMP-2 requires the induction of COX-2 and production prostaglandin (PG) E₂ in osteoblasts. To study further the mechanisms by which BMP-2 regulates COX-2 mRNA and by which PGE₂ may contribute to effects of BMP-2, we treated MC3T3-E1 osteoblastic cells with vehicle, BMP-2 (300 ng/ml), PGE₂ (1 μM), or BMP-2 + PGE₂ and measured COX-2 mRNA by real time PCR. To prevent effects of endogenous PGE₂ induced by BMP-2, all experiments were performed in the presence of the COX-2 selective inhibitor, NS-398 (0.1 μM). Although the induction of COX-2 is generally transient, BMP-2 induced elevation of COX-2 mRNA was more sustained: 2.9-fold at 4 h, 3.0-fold at 8 h and 2.6-fold at 24 h. PGE₂ alone had no significant effect on COX-2 mRNA levels. However, the addition of PGE₂ to BMP-2 increased the elevation of COX-2 mRNA, relative to BMP-2 treatment alone, by 1.3-fold at 4 h, 1.5-fold at 8 h, and 2.5-fold at 24 h. To assess the role of promoter regulation by BMP-2 in these effects, we used MC3T3-E1 cells stably transfected with 371 bp of the COX-2 promoter fused to the luciferase gene. BMP-2 transiently stimulated luciferase activity by 2.7-fold at 4 h, with levels returning to baseline at 24 h. At 4 h, PGE₂ alone had a small effect on luciferase activity (1.6-fold) and addition of PGE₂ to BMP-2 further amplified luciferase levels by 1.6-fold over BMP-2 alone. Similar to BMP-2 treatment alone, levels returned to baseline by 24 h. Similar results were obtained with MC3T3-E1 cells stably transfected with 3 kb of the COX-2 promoter fused to the luciferase gene. Measurement of mRNA degradation after treatment with inhibitors of transcription indicated that neither BMP-2 or BMP-2 + PGE₂ stabilized COX-2 mRNA compared with controls or PGE₂ alone. These results suggest that BMP-2 may transcriptionally elevate COX-2 expression not only via the proximal promoter region (-300 bp), which stimulates the transient expression of COX-2, but also via distal elements (> −3 kb), which stimulate more sustained expression of COX-2. Additionally, PGE₂ enhances both the transient and sustained induction of COX-2 mRNA by BMP-2.

Disclosures: K.A. Blackwell. None.

This study received funding from: NIH DK-483611.

M169

See Sunday Plenary Number S169

M170

Craniofacial Reconstruction with Bone Morphogenetic Protein-2.

Docherty Skogh, C. Amander*, T. Engstrand*. Plastic and Reconstructive Surgery, Karolinska University Hospital, Stockholm, Sweden.

The purpose of this study is to report the effectiveness and safety of using titanium mesh combined with chitosan-heparin sponge and rhBMP-2(InductOs) for the reconstruction of large through-and-through cranial defects in humans. Previous to this, a study of boneformation in Sprague Dawley rats was performed that showed very good results with the above mentioned carrier system.

Methods: Three patients were operated on and subsequently followed up with CT scans, clinical examination and histological samples. The patients had frontotemporal or parietal defects ranging from 54 to 117 cm(2) resulting from postoperative infection and necrosis of the bone following removal of neoplasms, cerebral aneurysms or haemorrhage. The first patient had received radiation to the operating field. The patients were reconstructed with (1) titanium mesh, chitosan-heparin sponge on the dura mater with 4 mg rhBMP-2, chitosan-heparin sponge with 8 mg rhBMP-2 on the titanium mesh, (2) titanium mesh, chitosan-heparin sponge with 12 mg rhBMP-2 on the dura mater, (3) titanium mesh, chitosan-heparin sponge with 10 mg rhBMP-2 on the titanium mesh. All the patients were followed up by clinical examination and repeated CT scans for a minimum of 8 months. Histological samples were taken from two of the patients.

Results: All the patients had signs of postoperative infection and were given antibiotics. One patient experienced hair loss in the operating field two months postoperatively. The hair grew back uneventfully a month later. Two of the patients had exposure of the titanium mesh and were reoperated once and five times respectively. The latter patient required free flap surgery to get the wound to finally heal. CT scans showed calcification of the dura mater in the operating field, but no signs of boneformation in two of the patients. The third patient showed ongoing boneformation after eight months and CT scans are to be repeated thirteen months postoperatively.

The histological samples taken six and nine months postoperatively showed no sign of boneformation or angioneogenesis.

Conclusions: Titanium mesh combined with chitosan-heparin sponge and rhBMP-2 (InductOs) appears to be a potential way to reconstruct significant craniofacial defects. The patients did have quite a lot of complications postoperatively, in particular the patient with the chitosan-heparin sponge and rhBMP-2 directly on the dura mater. The chitosan seems to cause an inflammatory response that impairs the healing of the wound. In one patient local hair loss was seen, most likely due to the effect of BMP-2. This study illustrates the difficulties of translating the results from studies on rodents to humans, and also the need for a more inert carrier system.

Disclosures: A. Docherty Skogh. None.

M171

See Sunday Plenary Number S171

M172

Acid Swelling Overcomes Osteogenesis Inhibition of Xenogeneic Collagenous Matrix Delivery System used for Naturally-Derived Bone Morphogenetic Protein Complex.

B. Rothman*¹, E. Olivier*², N. Duneas³. ¹Altis Biologics (Pty) Ltd, Pretoria, South Africa, ²School of Pharmacy, Tshwane University of Technology, Pretoria, South Africa, ³Biomedical Sciences, Tshwane University of Technology, Pretoria, South Africa.

The bone morphogenetic proteins (BMPs) are pleiotropic morphogens belonging to the greater transforming growth factor-ß (TGF-ß) superfamily of growth factors responsible for embryonic patterning as well as post-natal tissue regeneration.

BMPs require a safe and effective delivery system to bring about osteogenesis in clinical settings. Native porcine bone matrix-derived insoluble collagenous bone matrix (ICBM) was treated with various protocols involving organic acid and proteases (pepsin) to determine whether the chemical swelling of the matrix and telopeptide reduction would reduce the inflammatory response in the xenogeneic host, which has been associated with inhibition of the biologic activity of BMPs.

Native and treated porcine ICBM samples were combined with various doses of naturally derived porcine BMP complex and implanted intra-muscularly and subcutaneously in rodents in order to study bone formation. Histological examination of tissue specimens retrieved 12 days post implantation showed that the degree of inflammation induced by native porcine ICBM is high, and bone induction was inhibited at high doses of up to 50mg porcine BMP complex per gram delivery system. In contrast, acetic acid treated porcine ICBM was found to be an effective delivery system for BMPs, dose dependently stimulating bone formation and alkaline phosphatase activity, a specific enzyme marker for bone formation, at dose ranges of between 0 to 12mg porcine BMP complex per gram delivery system.

Porcine ICBM that was treated successively with acetic acid followed by enzyme digestion using porcine gastric mucosa-derived pepsin showed only a slight improvement in bringing about osteoinduction (not significant p>0.05). A striking observation was that the inflammatory response to chemically and/or enzymatically treated ICBM was dose dependently reduced by increasing concentrations of porcine BMP complex. The data demonstrate that chemical swelling plays a major part in improving xenogeneic ICBM for use as a BMP delivery system. This work will assist in the development of xenogeneic derived collagenous matrices with improved host immuno-compatibility which may function as safe and effective delivery systems for BMPs to be used in the clinical repair and regeneration of bone defects in humans.

Disclosures: N. Duneas, Altis Biologics 1, 2, 4.

This study received funding from: National Research Foundation RSA.

M173

Xenogeneic Bone Morphogenetic Protein Complex Enhances Allogeneic Demineralised Bone Matrix Osteoinductiviy in Rats.

N. Duneas¹, B. Rothman*², E. Olivier*³, G. U. Mohangi*⁴. ¹Biomedical Sciences, Tshwane University of Technology, Pretoria, South Africa, ²Altis Biologics, Pretoria, South Africa, ³School of Pharmacy, Tshwane University of Technology, Pretoria, South Africa, ⁴School of Dentistry, University of Pretoria, Pretoria, South Africa.

Demineralised Bone Matrix (DBM) is a widely used allograft material in a variety of clinical settings, including bone voids, as bulking agent for autogenous bone graft material, and orthopaedic and periodontal defects. DBM promotes osteogenesis due to its content of bone morphogenetic proteins (BMPs), a family of morphogens classified under the larger transforming growth factor-ß superfamily of growth and differentiation factors. The BMPs induce and regulate bone formation during embryogenesis and in postnatal life during regeneration and healing of traumatic bone defects. We present data that show in animal models that allogeneic DBM can be fortified with xenogeneically sourced BMP complex to improve DBM performance. Rat DBM was fortified with BMP complex purified from porcine diaphyseal bone. BMP complex contains a number of BMPs, and is standardized with respect to hBMP-2 content determined by commercial ELISA kit. Implantation of 25 mg rat allogeneic DBM fortified with 0, 3, 6 and 12 mg BMP complex per gram of DBM resulted in dose dependant upregulation of bone formation on day 12 as scored histologically and against alkaline phosphatase activity, an enzyme marker specific for bone formation. The data may assist in developing tissue banked DBM that is fortified by the adsorption of xenogeneically sourced BMP complex, thereby improving the performance of human sourced DBM.

Disclosures: N. Duneas, Altis Biologics 2, 4.

This study received funding from: National Research Foundation. RSA.

M174

Oxidative Stress Suppresses Osteoblastogenesis by Antagonizing Wnt/β-catenin and BMP Signaling.

M. Almeida, E. Ambrogini, L. Han, M. Martin-Millan, V. Lowe*, A. Warren*, R. L. Jilka, S. C. Manolagas. Center for Osteoporosis and Metabolic Bone Diseases, University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, AR, USA.

Increased levels of reactive oxygen species (ROS) in aging female or male C57BL/6 mice are temporally associated with progressive loss of bone strength and mass, decreased osteoblast (Ob) number and bone formation rate and increased Ob and osteocyte apoptosis. Moreover Axin2 and OPG mRNA expression is decreased and GADD45 is increased in the bone of old as compared to young C57BL/6 female mice, consistent with compelling in vitro evidence that ROS attenuate Wnt/β-catenin signaling by diverting β-catenin from TCF- to FOXO-mediated transcription. To test directly the hypothesis that increased oxidative stress decreases osteoblastogenesis, 5 month-old C57BL/6 mice were injected intraperitoneally twice daily (5 days a week) for a total of 6 weeks with 2 mM / Kg of L-buthionine-(S,R)-sulphoximine (BSO), a specific inhibitor of glutathione synthesis. Additionally, 20 mM of BSO was provided in their drinking water. At the end of the experiment, several markers of oxidative stress were determined along with osteoblastogenesis. ROS levels increased and glutathione reductase activity decreased in the bone marrow, while p66^shc phosphorylation increased in vertebral lysates, in mice treated with BSO as compared to saline treated controls. Moreover, BSO treatment promoted a decrease in osteoblastogenesis as measured by the number of colony forming units-osteoblasts in ex vivo bone marrow cultures. In addition, treatment of C2C12 cell with H₂O₂ for 24 h inhibited osteoblastogenesis induced by Wnt3a, as measured by alkaline phosphatase (AP) activity. Furthermore, overexpression of a plasmid encoding FOX03a abrogated AP activity induced by Wnt3a. Conversely, silencing FOXOs using siRNA for FOXO 1, 3a, and 4, up-regulated the basal levels of AP activity. Activation of osteoblastogenesis induced by BMP-2 was also blocked by exposure of C2C12 cells to H₂O₂ for 24 h. This effect was associated with abrogation of BMP-2 induced Smadl/5/8 phosphorylation by H₂O₂ as determined by Western blotting. Taken together with evidence that the replication of mesenchymal stem cell progenitors of osteoblasts is decreased in old as compared to young mice, presented elsewhere in this meeting, these results strongly support the hypothesis that aging, and specifically oxidative stress, decreases osteoblastogenesis and thereby bone formation by antagonizing Wnt/β-catenin and BMP-signaling.

Disclosures: M. Almeida, None.

M175

Evaluation of Bone Phenotype in Mice Carrying Adenomatous Polyposis Coli (APC^min) Gene Mutation.

Q. Su*¹, S. Pun*¹, B. Pennypacker¹, C. Winkelmann*², O. A. Flores*¹, H. Glantschnig*, D. B. Kimmel¹, F. Chen*¹. ¹Molecular Endocrinology, Merck Research Laboratories, West Point, PA, USA, ²Imaging, Merck Research Laboratories, West Point, PA, USA.

The identification of LRP5 mutations that are associated with high or low bone mass in humans has triggered great interest in studying Wnt signaling in bone formation. Current information clearly demonstrates that the canonical Wnt/β-catenin pathway plays a crucial role in regulating bone mass. Reducing the inhibitory effects of Wnt-signaling antagonists like Dkk1, SFRP1 or SOST which indirectly increase β-catenin in the nucleus has been shown to increase bone mineral density (BMD). LiC1 inhibition of glycogen synthase kinase-3β which stabilizes β-catenin was also reported to increase BMD in mice and humans by a mechanism that is likely independent of membrane Wnt signaling. Heterozygous mice with APC loss of function gene mutation (Apc^min/+) also have high levels of nuclear β-catenin. In this study, we investigated the bone phenotype in Apc^min/+ mice by evaluating bone mass and genes related to bone function in comparison to wild type (wt) controls.

BMD of wt and Apc^min/+ mice femurs at 9 weeks and 16 weeks were measured using PIXIMUS. Tibia RNA samples from the above mice were analyzed for gene expression via Taqman.

At 9 weeks of age, no difference in BMD between wt and Apc^min/+ mice was detectable in the distal or central femur. However, at 16 weeks of age, BMD in the distal femur was significantly increased by 5% in Apc^min/+ mice vs. the wt controls (P-value of 0.0107). Gene expression studies of bone (tibia) samples from 9-week old mice showed that both Dkkl and SOST expression levels were ∼7 folds higher in Apc^min/+ mice compared to wt-control, This could indicate negative regulatory feedback mechanism to counter a constitutively active Apc mutation. In addition, markers for osteoblast function COL1A1, ALP, OC and osteoclast function OPG, TRACP5, CatK were all ∼ 2–4 folds increased in tibia of Apc^min/+ mice. This effect was transient as at 16 weeks of age differences in the above genes between wt and Apc^min/+ were insignificant, with the exception of SOST transcript levels which remained slightly elevated (∼2 folds).

In conclusion, increased β-catenin activity derived from Apc^min/+ locus results in subtle but measurable change in BMD at discrete skeletal sites. Constitutive cytoplasmic Wnt pathway activation resulted in a transient increase in bone cell activities.

Disclosures: F. Chen. Merck Research Laboratories 1, 3.

M176

See Sunday Plenary Number S176

M177

Is Erythropoietin a Regulator of Bone Density and Serum Calcium in Elderly Men? — Mr Os Sweden.

M. Ethnersson*¹, U. Lerner², H. Nilsson-Ehle*³, H. Wadenvik*³, M. Lorentzon¹, M. Karlsson*⁴, Ö. Ljunggren⁵, E. Orwall⁶, U. Smith*³, C. Ohlsson¹, D. Mellström¹. ¹Center for Bone research at the Sahlgrenska Academy, Goteborg University, Goteborg, Sweden, ²Oral Cell Biology, Umeå University, Umeå, Sweden, ³Medicine, Goteborg University, Goteborg, Sweden, ⁴Orthopedics, Lund-Malmoe University, Malmoe, Sweden, ⁵Medical Scienes, Uppsala University, Uppsala, Sweden, ⁶Bone and mineral Unit, Oregon Health and Sciences University, Portland, OR, USA.

Erythropoietin (EPO) is the main regulator of the erythropoiesis. Erythrocytes and osteoclasts are derived from the same hematopoetic stem cell. With increasing age there is a decline in haemoglobin resulting in an increasing EPO. The aim of the present study is to investigate if erythropoietin, EPO, is related to bone density and serum calcium in elderly men in the Göteborg part of the Mr Os Sweden cohort (n=1010, age 69–80). Haemoglobin (Hb), EVF and white blood cell count were analyzed in 1010 men between 69–80 years of age. Blood samples were drawn after 10 hours of fasting and non-smoking in the morning. Serum and plasma were placed in a 80°C freezer. Plasma erythropoietin was analyzed with an ELISA method Quantikine IVD, R&D system, Inc, Minneapolis in 980 men. Bone density was measured with a Hologic 4500A.

Mean plasma EPO was 11,51 (9,1) IU/L. We found that EPO inversely correlated to Hb r = −0,324 (p < 0,0001) and EPO increased with age, r = 0,11 p < 0,001. EPO directly correlated to all bone mineral density (BMD)-sites (p < 0,001) and to PTH r = 0,18 (p = 0,0001) and indirectly to serum calcium r = −0,13 (p < 0,001). EPO correlated indirectly to lung function (FEV1,0) and renal function (cystatin C) but also directly to inflammatory parameters IL-6 and CRP (p < 0,001) but not to vitamin D, phosphate or sex hormones. Heigth, weight, age, Hb, kidney function, PTH, vitamin D, phosphate were included in regression models for bone mineral density (BMD). EPO was an independent predictor of Hip total BMD ß = 0,123, p < 0,001, trochanter BMD ß = 0, 108, p < 0,001, femoral neck ß = 0,143 p < 0,0001 and lumbar spine BMD ß = 0,0701 p < 0,05.

A multiple regression model with EPO, PTH, age, phosphate, albumin and cystatin C showed that EPO was an independent predictor of serum calcium ß = −0,08 p < 0,01. A multiple regression model with EPO, serum calcium, albumin, phosphate, age, cystatin C showed that EPO was an independent predictor of PTH β = 0,119 (p < 0,0001). In conclusion we found an inverse correlation between EPO and Hb and EPO increases with age. EPO correlated directly to bone mineral density at all sites and to PTH and indirectly to serum calcium even after multiple regression models taking in consideration several possible covariates. These novel findings indicate that EPO might be involved in the regulation of BMD and calcium metabolism in elderly men.

Disclosures: M. Ethnersson, None.

M178

FGF2 and TGFβ1 Have Opposite Effects on the Mural Cell Phenotype of CD146* BMSCs.

A. Funari*¹, S. Michienzi*², B. Sacchetti*³, S. Cersosimo*³, M. Riminucci*¹, P. Bianco². ¹Experimental Medicine, University of L'Aquila, L'Aquila, Italy, ²Experimental Medicine and Pathology, University of La Sapienza, Rome, Italy, ³Fondazione Parco Biomedico S.Raffaele, Rome, Italy.

CD146+ Bone Marrow Stromal Cells (BMSC) include skeletal stem cells and are closely associated with sinusoids either in human bone marrow (hBM) and in heterotopic ossicles. Factors regulating proliferation and differentiation of CD146+ BMSC are unknown. In this work, we analyzed the effects of FGF2 and TGFβ1 on CD146+ BMSC morphology, proliferation and gene expression. Furthermore, we investigated the pattern of expression of FGF2 and TGFβ1 in the ossicle as compared to human bone. FGF2 stimulated BMSC proliferation and downregulated most mRNAs characteristic of mural cell phenotype, including: α-SMA; caldesmon 1; calponins; desmin; PDGF-R beta; basement membrane proteins such as SMOC1 and COL4A1. Of note, FGF2 treatment upregulated VEGF, that promotes cell migration and inhibits apoptosis and a group of sphingosine-1-phosphate receptors including EDG1 that is involved in endothelial cell differentiation and in the proliferative and migratory response of mural cells. All these genes were oppositely regulated by TGFβ1, which stabilizes mural cells and nascent vessels. In the hBM, FGF2 and TGFβ1 are produced by connective tissue cells, embedded in the matrix and either released upon matrix degradation, or activated by proteases (TGFβ1). To assess the expression of the two factors during ectopic osteogenesis, heterotopic ossicles were generated by BMSC transplantation in SCID mice and harvested at different time points. Immunohistochemistry was performed by using antibodies against FGF2, TGFβ1, activated TGFβ1 and human mithocondria antigen. As in the hBM in vivo, in the ossicle either FGF2 and TGFβ1 were expressed in mesenchymal and osteoblastic cells in the developing marrow space. High levels of immunoreactivity for TGFβ1 were also observed in marrow adipocytes which derive from adventitial reticular cells. Active TGFβ1 was not observed in the cell types immunoreactive for the latent form and was specifically detected in a peri-sinusoidal location and in the nascent bone. This result is consistent with its extracellular activation. In conclusion, we have shown that CD146+ BMSCs have a mural phenotype in vivo and in vitro and that is strengthened by TGFβ1 and dowregulated by FGF2. Both factors are expressed in the ossicle with the same spatial layout observed in post-natal human trabecular bone. The activation of TGFβ1 is detected at the sites of pericyte-endothelial contact where active TGFβ1 induces the expression of SMC/pericyte markers by mesenchymal cells and inhibits proliferation of ECs.

Disclosures: A. Funari, None.

M179

See Sunday Plenary Number S179

M180

Demonstration of an Anabolic Effect of Prostaglandin E₂ on Bone in CD-1 Mice.

O. Gao*, M. Xu, P. Zhan*, C. B. Alander*, C. C. Pilbeam, L. G. Raisz. Endocrine Division and Musculoskeletal Institute, University of Connecticut Health Center, Farmington, CT, USA.

Previous studies have shown that treatment with prostaglandin E₂ (PGE₂) can lead to increased bone resorption and bone loss in C57BL/6 mice. However, dynamic histomorphometric analyses in these mice showed that osteoblast function was also stimulated, as indicated by increased mineral apposition rate (MAR) and bone formation rate (BFR/BS). To determine if there can be a true anabolic effect of PGE₂ on bone in mice with an appropriate dose and duration of treatment, 9 week old CD-1 male and female mice were injected intraperitoneally with vehicle or PGE₂ (3 mg/kg, 2 times a week for 4 weeks) and euthanized 5 days after the last injection. Body weight, serum calcium and total protein were not significantly changed between vehicle and treatment groups. By dynamic histomorphometric analysis of distal femurs, PGE₂ treatment in both gender groups showed an increase in MAR compared to vehicle treated animals (1.44 ± 0.01 vs. 1.03 ± 0.07 μ/d in males and 2.04 ± 0.04 vs. 1.31 ± 0.27 μ/d in females, n=5–6, p<0.05). BFR/BS was also increased in both males and females (0.52 ± 0.06 vs. 0.27 ± 0.05 μ³/μ²/d in males and 0.74 ± 0.05 vs. 0.38 ± 0.10 μ³/μ²/d in females, p<0.01). Double labeled surface was similar in vehicle treated males and females, and increased significantly from 13.7% to 26.6% with PGE₂ treatment. In contrast to previous experiments, there was no loss of trabecular bone volume (BV/TV) in males treated with PGE₂ and there was a trend for an increase in females (3.4 vs. 6.2 % p=0.06). Tibial mRNA was collected at the end of the experiment. Samples were reverse transcribed and RQ values for mRNA calculated by real time PCR. There was an increase in BMP-2 expression in the PGE₂ treated group compared to vehicle (1.45 ± 0.16 vs. 0.99 ± 0.12, p<0.05, n=11–12, pooled male and female data). RUNX-2 expression was also increased in the PGE₂ treated group (1.19 ± 0.11 vs. 0.91 ± 0.06, p<0.05). These results indicate that an anabolic response to PGE₂ treatment can be obtained in mice. This model can be used to study the effects of PGE₂ or selective agonists, as well as the effects of deletion of COX-2 or prostaglandin receptors, on the murine skeleton.

Disclosures: Q. Gao, None.

This study received funding from: 5R01AR018063–31.

M181

TNF-alpha/CCL3–5/CCR5 Cascade Mediates RANKL+ Cells Migration in Inflammatory Bone Resorption.

G. P. Garlet*¹, C. R. B. Cardoso*², S. B. Ferreira*¹, C. E. P. Repeke*¹, M. J. Avila-Campos*³, A. P. Campanelli*¹, J. S. Silva*⁴. ¹Department of Biological Sciences, School of Dentistry of Bauru, São Paulo University -FOB/USP, Bauru — SP, Brazil, ²Department of Biochemistry and Immunology, School of Medicine of Ribeirão Preto, São Paulo University — FMRP/USP, Ribeirão Preto — SP, Brazil, ³Department of Biological Sciences, Institute of Biomedical Sciences – São Paulo University — ICB/USP, Bauru — SP, Brazil, ⁴Department of Biochemistry and Immunology, School of Medicine of Ribeirão Preto – FMRP/USP, Ribeirão Preto — SP, Brazil.

Inflammatory immune reactions in response to periodontopathogens are thought to protect the host against infection, but may trigger inflammatory bone resorption in periodontium, in a milieu characterized by high levels of the pro-inflammatory cytokine TNF-α and inflammatory cells expressing the osteoclastogenic factor RANKL. Thus, we examined the mechanisms by which TNF-α drives RANKL+ cells migration to bone resorption focus, and therefore modulates the outcome of A. actinomycetemcomitans-induced periodontal disease in WT and genetically modified C57B1/6 mice. Our results showed that TNF-αp55 deficiency results in significantly decreased levels of chemokines CCL3, 4 and 5, and RANKL (evaluated by ELISA), associated with a lower inflammatory infiltrate and less alveolar bone loss. Flow cytometry analysis demonstrate that majority (72%) of RANKL+ leukocytes in inflammatory infiltrate are T cells (CD3+), and that RANKL+ cells are also highly positive (61%) to CCR5, a receptor whose ligands include CCL3–5. Interestingly, CCL3-knockout mice presented a minor decrease in RANKL+CCR5+ cells and in bone loss. However, CCL3 knockout mice presented similar levels of CCL4–5 in the periodontal tissues than WT mice, suggesting a redundancy of these chemokines in determining CCR5+ cell migration. Conversely, the absence of CCR5 resulted in pronounced reduction in the number of leukocytes and RANKL+ cells, and decreased RANKL levels and bone loss, suggesting an important role for this chemokine receptor in the migration of RANKL+ leukocytes to inflammatory focus surrounding alveolar bone. Our results demonstrate a cytokine-chemokine(s)-chemokine receptor cascade involved in inflammatory bone resorption: TNF-α induces the expression of the chemokines CCR3–5, which mediates the chemoattraction of CCR5+RANKL+ cells to periodontal tissues, and consequently leads to bone resorption. Therefore, chemokines and their receptors are potential targets to therapeutical intervention in inflammatory bone diseases.

Disclosures: G.P. Garlet, None.

This study received funding from: FAPESP #06/00534–1.

M182

See Sunday Plenary Number S182

M183

Relationship of Serum Cytokines and Monocyte Gene Expression to Bone Mineral Density and Content in Postmenopausal Women.

E. R. Gertz*¹, N. Silverman*¹, C. P. Kirschke*², J. W. Stewart*³, L. N. Hanson*³, L. Huang*², D. L. Alekel³, M. D. Van Loan². ¹Dept Nutrition, UC Davis, Davis, CA, USA, ²USDA, ARS, WHNRC, Davis, CA, USA, ³Dept Food Science and Human Nutrition, ISU, Ames, IA, USA.

The dynamic balance of bone formation and resorption can be influenced by cytokine and gene interactions. The purposes of our study were to 1) examine the relationships among inflammatory markers, monocyte gene expression, and bone mineral density (BMD) and content (BMC) and 2) determine the contributions of inflammatory markers and gene expression to BMD and BMC in healthy postmenopausal women not taking hormones. Serum samples were obtained from a subsample of 81 women who were enrolled in a 3-yr clinical trial. BMD and BMC from the lumbar spine, total hip, and femoral neck were assessed by dual energy x-ray absorptiometry. Inflammatory markers were: Cortisol, IL-1, IL-4, IL-6, IL-7, IL-8, INF-γ, G-CSF, GM-CSF, and TNF-α. Serum cytokines were measured by a cytokine multiplex immunoassay (LINCOplex; Millipore). Quantitative PCR was performed on a PRISM ABI 7900HT Sequence Detection System using TaqMan Assay-on-Demand kits for IFN-β, c-Fos, and β-Actin (Applied Biosystems). Genes were quantitated by an absolute standard curve method and normalized to the expression of β-Actin. Cytokine and gene expression data were transformed for normality. We observed a negative association between femoral neck BMC with TNF-α (p<0.05) and a positive association with Cortisol (p<0.05). Total hip and femoral neck BMDs were associated with c-Fos gene expression (p<0.05 and p<0.01, respectively). Gene expression of IFN-β was found to be significantly and negatively associated with femoral neck BMC (p<0.05). The contribution of inflammatory markers and gene expression to BMD and BMC was examined using stepwise multiple regression:

Total hip BMC=16.294 + 0.446(weight) — 0.879(BMI) + 1.137(c-Fos)

Total hip BMD=0.61 + 0.002(weight) + 0.021(c-Fos)

Femoral neck BMC=4.075 + 0.013(weight) — 0.181(IFN-β) — 0.384(TNF-α)

Femoral neck BMD=0.312 + 0.002(weight) + O.027(c-Fos) + 0.006(Cortisol) + 0.007(IL-6)

In conclusion, c-Fos, IFN-β, TNF-α, Cortisol, and IL-6 were significant contributors to hip and femoral neck BMC and BMD and may be important factors in bone health for postmenopausal women. Supported by funds from NIAMS/NIH (AR046922) and USDA, ARS, WHNRC.

Disclosures: E.R. Gertz, None.

M184

Epigenetic Mechanisms Are Involved in the Cytokine-induced de novo Expression of IL-1 beta in Human Articular Chondrocytes In Vitro.

K. Hashimoto*¹, S. Kokubun*¹, E. Itoi¹, H. I. Roach². ¹Department of Orthopaedics, Tohoku University, Sendai, Japan, ²Bone & Joint Research Group, University of Southampton, Southampton, United Kingdom.

Inflammatory cytokines, such as IL-1β, TNF-α and oncostatin M (OSM), induce abnormal expression of proteases in articular chondrocytes in vitro, analogous to the cytokine-induced abnormal expression in OA chondrocytes in vivo. Previous studies had shown that the latter was associated with loss of DNA methylation (Arthritis & Rheumatism 52:3110–3124) at specific CpG sites in the relevant promoters. DNA methylation at crucial CpG sites usually silences the gene and is replicated during cell division by the maintenance methyl transferase DNMT1. Since loss of methylation is a pre-requisite for gene induction, we hypothesized that the cytokines-induced IL-1β expression also included DNA de-methylation. Human chondrocytes, isolated from the femoral articular cartilage of six non-OA patients, did not express proteases, IL-1β or TNF-α, but did express collagen II, aggrecan and DNMT1. The cells from each patient were divided into 5 groups: non-culture; no additions (cultured control); + IL-1β (10ng/ml); + 5-aza-deoxycytidine (5-aza-dC, 2μM); + TNF-α (10ng/ml) combined with OSM (10ng/ml). 5-aza-dC is a non-specific de-methylation agent, which was used for comparison with the effects of cytokines. After 4–5 weeks of culture, mRNA expression and the % of DNA methylation at −289bp were quantified, after initial studies had identified this CpG site to be of crucial importance.

Before culture, 60–66% of cells were methylated, i.e. only 40% of the cells could theoretically respond to inductive factors. However, complete absence of IL-1β expression suggested that the right inductive factors were not present in these non-OA chondrocytes. Culture per se induced slight expression (set to 1) and some loss of methylation (30–40% still methylated). As expected, 5-aza-dC caused a further 20% loss of methylation, but this corresponded only to a 4–8 fold induction of expression. Remarkably, IL-1β induced its own expression with a 15–190 fold increase and only 4–25% of cells remaining methylated. However, the greatest effect was seen with the combined TNF-α/OSM treatment, which increased IL-1β expression 300–1700 fold and abolished methylation. Expression of DNMT1 was reduced considerably by culture alone, but further by cytokine treatment. This study is the first to correlate quantitatively mRNA expression and loss of DNA methylation. The results suggest that inflammatory cytokines are able to cause demethylation, which leads to increased transcription. The challenge for the future will be to determine the mechanisms of the cytokine-induced loss of DNA methylation.

Disclosures: K. Hashimoto, None.

M185

See Sunday Plenary Number S185

M186

Identification of TNF-α Shedding Enzyme in Macrophages.

A. Hikita¹, R. Suzuki*¹, S. Tohma*¹, S. Tanaka², N. Fukui*¹. ¹Clinical Research Center, National Hospital Organization Sagamihara Hospital, Kanagawa, Japan, ²Orthopaedic surgery, The University of Tokyo, Tokyo, Japan.

Tumor necrosis factor-α (TNF-α) is a cytokine that plays pivotal roles in the pathologies of various inflammatory diseases such as rheumatoid arthritis and Crohn's disease. TNF-α is first synthesized as a membrane-bound protein, and converted into a soluble form by proteolytic cleavage. These two forms are shown to bind to two TNF receptors with different affinities to exert different biological activities. A disintegrin and metalloproteinase 17 (ADAM 17), which is also known as TNF-α converting enzyme (TACE), is considered to be a primary sheddase for TNF-α. However, the significance of this enzyme in macrophages is not fully investigated while macrophages are the primary cells for TNF-α production in rheumatoid arthritis. Considering that the activity of TNF-α differs between the membrane-bound form and the soluble form, it is important to identify the sheddase(s) for TNF-α in macrophages.

In order to identify the sheddase(s), we developed an assay system using a plasmid containing cDNA for secreted placental alkaline phosphatase (SEAP) fused with the cDNA coding the stalk region of mouse TNF-α. The TNF-α-SEAP plasmid was transfected into HEK293 cells together with the expression vectors for various matrix metalloproteinases (MMPs) (MMP-1, 2, 3, 7, 9, 11, 13, 19, 23, 28, MT1, 2, 3, 4, 5, 6-MMP) and ADAMs (ADAM9, 10, 17, 19), and the TNF-α shedding activity of these proteinases was detected as alkaline phosphatase activity in the culture media. In this assay, ADAM9, 10, 17, MMP7, MT1, 2, and 3-MMP showed shedding activities for TNF-α. Among these proteinases, the expression of ADAM9, 10, 17 and MT1-MMP was detected at substantial levels in mouse macrophages by real-time PCR. Then the plasmids for these proteinases were transfected to 293 cells together with a full-length TNF-α expression vector, and the occurrence of shedding was evaluated by the appearance of soluble TNF-α in the culture media, which was detected by Western blotting. As predicted by the TNF-α-SEAP system, the occurrence of shedding was observed with all four proteinases with the full length TNF-α. Among these proteinases, suppression of ADAM10 by siRNA resulted in a significant reduction in the TNF-α shedding.

These results suggest that, besides ADAM17, ADAM10 also works as a dominant sheddase for TNF-α in macrophages. The results also suggest that other proteinases such as ADAM9 and MT1-MMP can be the sheddases for TNF-α, depending on the biological situations. It may be notable that several other proteinases can take the place of ADAM 17 as a shaddase for TNF-α.

Disclosures: A. Hikita, None.

M187

Lysophosphatidic Acid Promotes Maturation and Survival in Rat Growth Plate Chondrocytes.

J. L. Hurst-Kennedy*¹, J. Greene*², Z. Schwartz², B. D. Boyan². ¹School of Biology, Georgia Institute of Technology, Atlanta, GA, USA, ²Department of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA, USA.

The bioactive phospholipid metabolite lysophosphatidic acid (LPA) has been implicated in a number of cellular processes including wound healing, smooth muscle contraction, cell proliferation, survival, and migration. Recent studies have shown that LPA is a potent stimulator of osteoblast maturation as well. LPA is derived from phosphatidic acid (PA), which is produced by the action of phospholipase D (PLD). One physiological regulator of PLD in growth plate cartilage cells is the vitamin D metabolite 24,25-dihydroxyvitamin D3 [24R,25(OH)₂D₃]. When resting zone chondrocyte cultures are treated with 24R,25(OH)₂D₃, PLD activity is increased, resulting production of diacylglycerol (DAG), as well as increased PKC activity, increased cellular maturation and increased cell survival. DAG is a metabolite of LPA, suggesting that LPA acts as a second messenger during the promotion of chondrocyte differentiation and survival in growth plate cartilage cells. The purpose of this study was to determine if chondrocytes produce LPA and if so, whether LPA signaling mediates chondrocyte maturation and survival and the mechanisms involved. To investigate this, we used resting zone chondrocytes isolated from the rat costochondral cartilage growth plate as a model system. 24R,25(OH)₂D₃ treatment regulated LPA production in a dose-dependent manner. These cells were shown to express the LPA receptors LPA1, LPA3, LPA5, and peroxisome proliferator-activated receptor gamma (PPAR-γ). LPA treatment also promoted an increase in two markers of chondrocyte differentiation: alkaline phosphatase activity and proteoglycan sulfation. Furthermore, apoptosis induced by phosphate and the protein kinase C (PKC) inhibitor chelerythrine was attenuated by LPA. Western Blot analysis indicated that LPA decreased the abundance of the tumor-suppressor protein p53. LPA treatment regulated the expression of the p53-target genes Bcl-2 and Bax to enhance cell survival. Collectively, these data suggest that LPA signaling promotes cellular maturation and survival in resting zone chondrocytes demonstrating a novel function of LPA signaling in 24R,25(OH), D,-mediated chondrogenesis.

Disclosures: J.L. Hurst-Kennedy, None.

This study received funding from: Children s Healthcare of Atlanta Pediatric Hospital.

M188

Continuous Infusion of Insulin-like Growth Factor-I into the Epiphysis of the Tibia.

A. Abbaspour, S. Takata, Y. Matsui, M. Takahashi*, S. Katoh*, N. Yasui*. The Deparment Of Orthopedics, The University of Tokushima, Tokushima, Japan.

We have developed a method to promote longitudinal bone growth at the level of a specific growth-plate in young rabbits. Twenty-four male young rabbits weighing about 500 grams were divided into three groups, one-week treatment of IGF-I (n=5), two-week treatment of IGF-I (n=5), and four-week treatment of IGF-I (n=14). Insulin-like growth factor-1 was continuously infused by means of an osmotic pump into the bone marrow cavity of the proximal epiphysis of the left tibia. In the epiphysis of right tibia, normal saline was infused continuously as a control. Longitudinal growth of the tibia was monitored by weekly-taken soft X-ray. After 4 weeks of treatment, the animals were sacrificed for histological examination and peripheral OCT (pQCT) analysis. Radiological measurement showed a 2-mm overgrowth of the tibia after 4 weeks of treatment, while histological analysis demonstrated a 15% increase in the thickness of the selected growth-plate. The local infusion of IGF-I increased the numbers of both proliferative and hypertrophic chondrocytes and promoted hyperplasia of bony trabeculae within the epiphysis. Bone mineral density (BMD) at the proximal metaphysis of the tibia was also significantly higher in both 2 weeks and 4 weeks treatment of IGF than in control sides. The distribution of material infused locally into the epiphysis was simulated by the infusion of Indian ink using the same methodology (osmotic pump) as that for IGF-I. Most of the dye remained within the bone marrow cavity of the epiphysis, but a portion infiltrated into the growth-plate, reaching the deep layer of the physeal chondrocytes and primary spongiosa of the metaphysis. These results suggest that the method reported here is a valid one for delivering cytokines or growth factors to the selected growth-plate and for controlling the growth and differentiation of physeal chondrocytes.

Disclosures: A. Abbaspour, None.

M189

IGF-Binding Protein 3, 4 and 5 Triple Knock-out Mice Have Larger Bones.

B. M. Boudignon*¹, M. Qin*², J. Pintar*², B. P. Halloran¹. ¹Endocrine unit, Veteran Affairs Medical Center San Francisco, San Francisco, CA, USA, ²Department of Neuroscience and Cell Biology, University of Medicine and Dentistry of New Jersey, Piscataway, NJ, USA.

IGF-I, the most abundant growth factor in the bone microenvironment, plays an important role in maintaining normal bone structure and mass. The activity of IGF-I is regulated by a family of IGF-binding proteins (IGF-BP 1–6). All 6 BPs have crucial roles in association with IGF-I and in some cases can modulate cell metabolism independent of IGF-I. Animals (C57BL6/129ReJ mixed background) harboring targeted defects in IGF-BP-3,4,5 have been developed and provide a unique opportunity to study the role of these BPs in metabolism and whole animal physiology (Ning et.al, Mol. Endo., 20:2173, 2005). Triple KO animals are slightly smaller and have reductions in serum total and bioactive IGF-I of 55% and 63%, respectively. To study the role of BP-3,4,5 in bone we examined bone structure and density in wild type and triple KO mice using microCT analysis. Femurs from triple KO animals were longer, and tissue volume of the secondary spongiosa in the distal femoral metaphysis was increased by more than 30%. Fractional bone volume and segmented bone mineral density remained unchanged. Tissue, bone and marrow volumes of the femoral midshaft were all significantly increased by nearly 20% whereas bone fraction remained unchanged. Whereas the endocortical surface of the femoral diaphysis in wild type animals displayed a smooth curvature, the surface in the triple KO mice was irregular and wavy (see fig.) These results suggest that the combined global loss of BP-3,4,5 results in an increase in bone size (length, volume and diaphyseal diameter) but proportionately with normal bone volume despite low serum IGF-I and reduced body weight.

Disclosures: B.M. Boudignon, None.

M190

In Vivo Models To Study IGF-1 Receptor Dynamics Of Bone Stem Cells.

B. C. Bragdon*¹, K. Young*¹, L. G. Horton*², R. Gundersen*¹, C. J. Rosen*², A. Nohe¹. ¹Univeristy of Maine, Orono, ME, USA, ²Jackson Laboratory, Bar Harbor, ME, USA.

Insulin-like Growth Factor-1 (IGF-I) is a critical peptide in skeletal growth and consolidation. In detail IGF-I is key in the differentiation of bone marrow stromal cells (BMSCs) into osteoblasts. Current research indicates that localization of the IGF-I receptor in specific regions of the membrane does effect the specificity of signaling. However research is lacking the correlation between membrane domain dynamics, osteoblast differentiation and osteoporosis. Using the Family of Image Correlation Spectroscopy (FICS) we showed that membrane domain dynamics are altered in bone marrow stromal cells derived from mice exhibiting an age related osteoporotic phenotype, B6.C3H-6T (6T). 6T contains the wild type C57BL/6J (B6) background with an inversion of a segment of chromosome 6. This inversion includes Ppar gamma. Ppar gamma is a key mediator of adipogenesis. The mouse also has low serum IGF-I and reduced bone density which is characteristic of age related osteoporosis. Using FICS we observed an increase in cluster density (number of clusters per unit area) of both the membrane domains and IGF-I receptors, while the percent of co-localization was unchanged in the BMSCs. Our data indicated that the receptors as well as the membrane domains reshuttle on the membrane. This shuttling is crucial for their function. Especially caveolae flask-shaped invaginations of the plasma membrane are effected. Utilizing reporter gene assays with reporters for major down stream signaling pathways we showed significant differences in IGF-I signaling in 6T mice versus control. Further our data showed that cholesterol content and lipid composition are altered in 6T mice measured by HPLC and cholesterol assays. These results point to the importance of lipid balance and cholesterol in IGF-I signaling. Our data indicated that membrane, receptor dynamics are affected in mice exhibiting an osteoporotic phenotype.

Disclosures: B.C. Bragdon, None.

M191

See Sunday Plenary Number S191

M192

Systemic IGF-I Shows Concerted Anabolic Actions on Bone and Muscle: Evidence from Male Mice with Liver-Specific Overexpression of IGF-I.

F. Callewaert*¹, K. Venken*¹, J. Ophoff*¹, L. Liao*², S. Boonen¹, R. Bouillon¹, J. Xu*², D. Vanderschueren¹. ¹Katholieke Universiteit Leuven, Leuven, Belgium, ²Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA.

Serum IGF-I is principally derived from the liver and stimulates not only bone but also muscle during growth. We questioned to what extent overexpression of IGF-I by the liver affects bone and muscle mass and how the changes in bone and muscle are interrelated. To answer this question, transthyretin (TTR)-IGF-I transgenic mice and controls were evaluated longitudinally. Tibial bone mineral density and whole-body composition were assessed weekly by in vivo pQCT and DEXA, respectively, until 16 weeks of age. Transgenic mice showed 21% higher serum IGF-I during the entire experiment (p < 0.01). The increased serum IGF-I level was accompanied by enhanced lean body mass (+7% vs. controls, p < 0.01) and muscle cross-sectional area (+10% vs. controls, p < 0.01). In addition, cortical bone mass (but not trabecular bone mass) and strength strain index were 7% and 12% greater in transgenic mice, respectively (p < 0.01). Regression analysis was performed to assess the impact of hepatic IGF-I overexpression on cortical bone mass and bone strength in relation to changes in muscle mass. Cortical bone mass and bone strength were very strongly related to changes in muscle cross-sectional area in both transgenic and control mice (p=0.0001), as represented by the figure, which shows the relationship between changes (Δ in figure, week 16 — week4) in cortical content and muscle cross-sectional area. Moreover, regression analysis showed that the difference in cortical bone mass and bone strength between transgenic and control mice did not persist after correction for changes in muscle mass so that changes in bone and muscle mass during growth were proportionate in transgenic mice versus control mice. In conclusion, our data indicate that systemic IGF-I shows significant and concerted anabolic actions on both bone and muscle.

Disclosures: F. Callewaert, None.

M193

Local Application of GH and IGF-I Have a Similar Effect on Intramembranous Bone Healing in a Rat Femur Osteotomy Model — A Comparative Study.

M. Huening¹, T. Lindner*¹, A. Flyybierg*², W. Weichert*³, M. Raschke*¹, H. Bail*¹. ¹Dept. of Trauma and Orthopaedic Surgery, Charité, Berlin, Germany, ²Medical Department (Diabetes and Endocrinology), Kommunehospital, University Aarhus, Aarhus, Denmark, ³Dept. of Pathology, Charité, Berlin, Germany.

Previous studies showed that systemic application of growth hormone GH stimulates bone and fracture healing. According to the “somatomedin-hypothesis” liver-derived IGF-I exerts this effect as the main endocrine transmitter of GH on physiological bone formation whilst the “dual-effector-theory” hypothesizes an additional direct effect of GH on longitudinal bone growth. To test these theories we performed an in-vivo study to elucidate the local effects of GH and IGF-I on callus formation.

A monolateral external fixator was mounted to the left femurs of 72 adult SD-rats followed by a standardized osteotomy creating a 0,3 mm gap. Three groups were created that received either GH, IGF-I or vehicle via mini-osmotic pumps with a tube routed to the osteotomy.

group I: phosphate buffer

group II: 100 μg/kg bodyweight/day IGF-I

group III: 100 μg/kg bodyweight/day rh-GH

Femurs of each group (n=8) were harvested after 7, 14 and 21 days and processed for histological and histomorphometrical analysis. Sections were stained and evaluated with a semi-quantitative histological score regarding bone formation and gap bridging at all time-points. In addition histomorphometrical measurement were performed after 21 days with an image analysis system.

While no difference for bone formation and gap bridging between groups was detectable after 7 days, scores appeared to be higher for groups II and III after 14 and 21 days with a significant higher score for group II compared to group I after 21 days. Histomorphometry displayed a significant increase of callus area and mineralized callus area in groups II and III compared to group I. The cartilage area and the cartilaginous share were nearly doubled in the vehicle group compared to groups II and III. The structure of the callus, represented by the callus bone density and the callus diameter were not different between the three groups after 21 days.

In conclusion our study demonstrates that both, local application of GH and IGF-1 increase hard callus formation in the process of bone healing. Moreover both agents change the callus composition towards a lower share of cartilage. The results also strongly suggest that GH exerts a direct, non-liver mediated effect on bone healing which is comparable to the effect induced by IGF-I. Our findings clearly support the hypothesis that the “dual-effector-theory” is not only applicable for longitudinal bone growth but also for intramembranous bone healing.

Disclosures: M. Huening, None.

This study received funding from: Deutsche Forschungsgemeinschaft (DFG Hu 876/1–1)

M194

Cortical Bone Is Positively Associated with Serum IGF-1 and Testosterone and Negatively with Fibrinogen in Men and Women.

K. L. L. Landin-Wilhelmsen¹, P. Trimpou*², S. Lindstrand*¹, A. G. Nilsson¹, A. Odén*², L. W. Wilhelmsen*². ¹Section for Endocrinology, Sahlgrenska University Hospital, Göteborg, Sweden, ²Sahlgrenska University Hospital, Institution of Medicine, Göteborg, Sweden.

Lower calcaneal ultrasound values predict fractures during follow-up. The purpose was to analyse factors of importance for the ultrasound variables in a random population sample of men and women aged 25–64 years, 54% women, from the 1995 World Health Organisation (WHO) MONItoring of trends and determinants in CArdiovascular disease (MONICA) study in Gothenburg, Sweden.

The baseline examination in 1995 included history of fractures, physical activity at work and during leisure time, psychological stress, smoking habits, coffee consumption, BMI, blood pressure, total cholesterol, HDL-cholesterol, ApoA, ApoB, triglycerides, fibrinogen, CRP, IGF-1, IGFBP-1, IGFBP-3, osteocalcin, procollagen, estradiol, free estradiol, testosterone, free testosterone, PTH, procollagen, SHBG, and insulin.

The calcaneal ultrasound (LUNAR Achilles) variables were: Stiffness, Speed Of Sound (SOS) and Broadband Ultrasound Attenuation (BUA).

We have previously shown that leisure time physical activity was positively associated with calcaneal cortical bone. In the present analysis we analysed the additional influence of selected blood variables and hormones. The table shows number of individuals with results available (n), partial correlation coefficients [r] and p-values. 

Table  .  

All calcaneal ultrasound variables were associated with IGF-1, SOS and stiffness with testosterone, whereas SOS was negatively associated with fibrinogen. A more detailed analysis of relations to gender showed that testosterone was important for the ultrasound variables in both men and women.

In summary, out of a large series of hormones IGF-1 and testosterone were positively associated with calcaneal cortical bone. Fibrinogen, indicating chronic inflammation, was negatively associated with cortical bone.

Disclosures: K.L.L. Landin-Wilhelmsen, None.

M195

The Role of IGF-I and IGFBP-1 Status and Secondary Hyperparathyroidism in Relation to Osteoporosis in Elderly Swedish Women.

H. S. Salminen¹, M. Sääf*², H. Ringertz*³, L. Strender*¹. ¹Centre for Family and Community Medicine, Karolinska Institutet, Huddinge, Sweden, ²Department of Endocrinology, Metabolism and Diabetes, Karolinska Institutet, Stockholm Sweden, Sweden, ³Department of Surgical Sciences, Section of Diagnostic Radiology, Karolinska Institutet, Stockholm, Sweden.

Our aim was to investigate among elderly women the relationship to osteoporosis of calcium-regulating hormones and anabolic growth factors.

A population-based cross-sectional study of 350 elderly women (mean age 73 years). Measurements of bone mineral density (BMD) of the left hip, lumbar spine and heel and risk markers for osteoporosis were studied.

The BMD values showed significant inverse relationship with the values of insulin-like growth factor binding protein (IGFBP-1) at all sites of measurement and significant positive relationship with the values of insulin-like growth factor-I (IGF-I) at all sites with the exception of the lumbar spine. There was no significant association between the values of BMD and the values of 25-hydroxy vitamin D (25(OH)D). The use of loop diuretics was strongly and significantly associated with elevated levels of PTH (OR 4.4, P<0.001). The anabolic growth factors IGF-I and IGFBP-1 showed a stronger association with the BMD values than the calcium regulating hormones 25(OH)D and PTH. In this study the use of loop diuretics was a more important cause of secondary hyperparathyroidism than vitamin D status.

Analysis of regression between BMD, vitamin D, PTH and IGF-I and IGFBP-1 

Table  .  

Disclosures: H.S. Salminen, None.

M196

Opposing Effects of Growth Hormone and Alcohol on Bone Marrow Adiposity and Cancellous Bone Mass and Turnover Reveal a Critical Role for the Local Action of Growth Hormone in the Reciprocal Relationship between Adipocyte and Osteoblast Differentiation.

R. T. Turner, P. J. Menagh*, G. Maddalozzo*, U. T. Iwaniec. Nutrition and Exercise Sciences, Oregon State University, Corvallis, OR, USA.

Cancellous bone loss is accompanied by an increase in bone marrow adiposity. Adipocytes and osteoblasts are derived from a common progenitor cell, and adipocytes produce cytokines (e.g., IGF-1) and adipokines (e.g., leptin) which affect bone turnover. Thus, it is possible that the formation of fat and reduction in bone are regulated by a common factor. Excessive alcohol consumption is known to result in bone loss and marrow adiposity. We evaluated the effects of alcohol in a rat model in which the caloric intake of the animals fed the alcohol and control diets was the same. Alcohol consumption decreased % body fat and bone mass but increased bone marrow adiposity. Alcohol consumption reduced IGF-I mRNA levels in liver and bone, suggesting that growth hormone (GH) signaling may play a causal role in the observed reciprocal relationship between bone marrow adiposity and bone mass. We further investigated this relationship in hypophysectomized (HYPOX) rats. HYPOX of rapidly growing male rats reduced bone formation and led to cancellous osteopenia in tibiae. Despite a large reduction in body weight, there was a dramatic increase in bone marrow adiposity (from <1% to >50% marrow volume). Similar changes were observed in sexually mature male and female HYPOX rats. GH (0.8 mg/kg/d) normalized bone formation and bone mass, skeletal expression of IGF-I, and bone marrow adiposity in HYPOX rats. In contrast, administration of IGF-I (200 ug/kg/d) to HYPOX rats had no effect on either bone formation or adiposity, and administration of PTH (80 ug/kg/d) to HYPOX rats increased bone formation and increased the skeletal expression of IGF-I but had no effect on adiposity. To rule out estrogen deficiency as a contributing factor, we administered GH to: 1) HYPOX, 2) ovariectomized (OVX) + HYPOX and 3) estrogen-replaced OVX + HYPOX rats. In each case, GH was effective in reversing the effects of HYPOX on bone and adiposity. Taken together, these findings demonstrate that GH, a key hormone in the integration of energy production and expenditure, regulates the balance between bone and adiposity by enhancing bone formation and inhibiting adipocyte differentiation. Furthermore, the absence of a positive response to IGF-I treatment suggest that GH acts directly on osteoblasts and adipocytes.

Disclosures: R. T. Turner, None.

This study received funding from: NIH.

M197

Transforming Growth Factor β Stimulates New Bone Formation in Neonatal Mouse Calvariae via Suppression of Dickkopf-1.

L. M. Bevelock*, K. L. Clines*, J. M. Chirgwin, T. A. Guise, G. A. Clines. Internal Medicine, University of Virginia, Charlottesville, VA, USA.

Wnt signaling is important in normal bone remodeling and the pathogenesis of bone metastases. The secreted Wnt inhibitor dickkopf-1 (DKK1) contributes to the dysregulation of bone formation in cancer metastases. Increased DKK1 in multiple myeloma is associated with osteolytic bone disease and suppressed bone formation, while tumor-produced endothelin-1 (ET-1) suppresses secretion of DKK1 from osteoblasts, contributing to the abnormal new bone formation associated with osteoblastic metastases. However, little is known about the local control of DKK1 expression. We hypothesized that the Dkk1 promoter in osteoblasts is regulated by TGFβ, a factor abundant in the bone microenvironment.

We first examined regulation of Dkk1 transcription by TGFβ. C3H10T1/2 pre-osteoblastic cells were transfected with a Dkk1 promoter/luciferase construct and treated with 5ng/ml TGFβ, which decreased promoter activity threefold (p=0.04). TGFβ significantly inhibited DKK1 protein secretion from neonatal mouse calvariae at 2d and 4d, as determined by ELISA. This was associated with an increase in new bone formation, osteoblast number and osteoblast nuclear phosphoSmad2 staining compared to control (p<0.001). Recombinant DKKI protein (50 ng/ml) blocked TGFβ-stimulated new bone formation (p<0.001). TGFβ treatment of neonatal calvarial mouse osteoblasts increased β-catenin nuclear staining, indicating activation of canonical Wnt signaling.

TGFβ can signal via Smad-dependent and -independent pathways. We mutated two putative Smad-binding elements (SBEs) within the Dkk1 promoter and transfected mutant and wild-type promoters into C3H10T1/2 cells. Mutation of the individual SBEs did not reverse TGFβ repression of Dkk1 transcription, suggesting a possible Smad-independent mechanism.

Our data from cells and calvarial organ cultures contrast with previous studies in whole animals, where inhibition of TGFβ signaling increases osteoblast activity and bone mass. In solid tumor metastases to bone, TGFβ is released from the bone matrix by osteoclastic bone resorption, and promotes metastases by stimulating tumor cell production of prometastatic factors. The paradoxical stimulation of calvarial organ cultures suggests that effects of TGFβ on bone are context-dependent. In calvariae ex vivo TGFβ can stimulate bone formation by suppressing DKK1 and activating Wnt signaling through Smad-dependent and -independent mechanisms. Further studies are needed to elucidate the context-dependent effects of TGFβ on osteoblast function and the role of DKK1.

Disclosures: L.M. Bevelock, None.

M198

NF-κB-Regulated Gene Expression and Kinase Phosphorylation Oscillate Rapidly Over Time.

J. Iqbal*, L. Sun, M. Zaidi. Mount Sinai Bone Program, Department of Medicine, Mount Sinai School of Medicine, New York, NY, USA.

For the first time, we show that the expression of any eukaryotic gene, in our case NF-κB regulated genes, and kinase phosphorylation oscillate with time. The closest to this paradigm is gene expression phasicity noted over days, not over hours, as we describe. Our fundamental observation is that TNFα-induced mRNA expression is dynamic, undergoing rapid oscillations over hours. Expression of the “early genes” c-fos and IL-6 was enhanced transiently within 30 minutes of TNF-α application. Expression of “late genes”, namely Mn-SOD and RANTES genes was oscillatory beginning 3 hours post-TNFα. A third group, “continual genes”, namely IκBα, A-20 and MIP-2α showed robust induction, but unlike c-fos and IL-6, continued to display oscillations over 8 hours. We also show that, whereas the early oscillatory profiles induced by TNFα and RANK-L can almost be superimposed, “continual genes” failed to oscillate after 4 hours of RANK-L. These results indicate that TNF superfamily members not only trigger a distinct pattern of oscillations for different gene groups, but that oscillatory behavior is ligand-specific. Second, we found that the phosphorylation of signalling molecules was dynamic, with parallel, but distinct, waves of activation. Intracellular phospho-flow cytometry showed that p65, JNK and p38 phosphorylation oscillated for up to 8 hours, while ERK1/2 displayed a single wave. The only period where all four molecules displayed an overlapping phosphorylation was during the first signaling wave. Furthermore, p38 and p65 phosphorylation were in unison. However, their oscillations were slower than JNK1/2 phosphorylation within the first 3 hours, but at 4 hours, the three became aligned. Finally, we showed that a super-repressor of Iκ-B altered the amplitude and frequency of kinase phosphorylation. The amplitude of JNK phosphorylation was increased and oscillation frequency altered. ERK1/2 phosphorylation was converted from a monophasic to oscillatory frequency in unison with JNK for the first 3 hours. ERK1/2 did not show any appreciable phosphorylation after 4 hours, while JNK1/2 phosphorylation continued to oscillate. These latter results suggest that NF-κB negatively modulates both the amplitude and frequency of JNK and ERK1/2 phosphorylation. In conclusion, we show that TNF superfamily members induce robust ligand-specific oscillations in NF-κB regulated genes and phosphorylation of downstream kinases. This constitutes a novel paradigm through which cells can use multiple signaling molecules to coordinate gene expression and so regulate time-resolved physiological processes, such as chemokine and cytokine secretion.

Disclosures: J. Iqbal. None.

M199

New Insights into the Determination of Smads Signaling by TGFβ Superfamily.

B. Kim*¹, J. Lee*¹, H. Park*¹, S. Lee*¹, M. Lee*¹, H. Koo*¹, W. Yoon*², J. Kim*¹, H. Ryoo², J. Cho¹. ¹Biochemistry, School of Dentistry, Kyungpook National University, Daegu, Republic of Korea, ²Department of Cell and Developmental Biology, School of Dentistry, Seoul National University, Seoul, Republic of Korea.

A considerable number of studies have been done on TGFβ superfamily / Smad pathway. But, the mechanisms how to specify Smads in response to TGFβ and BMP stimuli is still unknown. In this study, we have used a combined method of the enrichment of phosphoproteins with proteomics strategy to discover those mechanisms. In early stage (at 30 min) after stimuli, we purified phosphoproteins from premyoblasts C2C12 cells using antibody-based phosphoaffinity column for the identification of the phosphoproteins regulated by BMP-2 or TGFβ1. We have identified about 1600 potential phosphoproteins using by GeLC-MS/MS. Interestingly, we detected that both Smad1 and Smad2 are temporarily phosphorylated by opposite signals (that is, Smad2 by BMP-2, Smad1 by TGFβ1). This phosphorylation level, however, rapidly decreased (from 30 min to 1hr), and dramatically restored by proteosome inhibitor MG132 pretreatment. These data suggest that ubiquitination-proteosomal degradation system would play a key role in the early events of TGFβ signal transduction. In the proteome data analysis, several ubiquitination / deubiquitiantion enzymes were identified. HECT-domain E3 ligases Nedd4 and Nedd4L, deubiquitin enzymes UCHL3/4 and USP4 respectively. Nedd4L was identified as a unique phospho-protein by BMP-2, but Nedd4 was detected commonly in both BMP-2 and TGFβ1 stimulation. The Smads ubiqutination by Nedd4 has not been elucidated yet. So, we hypothesized that temporarily phosphorylated-Smads would be polyubiquitinated by Nedd4 family (that is, p-Smad1 by Nedd4, p-Smad2 by Nedd4L) in TGFβ signaling pathway and degraded by proteosome. When coexpressed together with Nedd4, wild type Smadl was polyubiqutinated and constitutive phospho-Smadl mutant (DVD) is more polyubiqutination than non-phospho Smadl mutant (AVA). In addition, ALP(Alkaline phosphoatase) staining confirmed that overexpressed Nedd4 suppress transdifferentiation of BMP-2 induced C2C12. Overall, our results suggested that TGFβ1 induced phospho-Smad1 was selectively ubiqutinated by Nedd4 and followed by proteosomal degradation. We argue that Nedd4 ubiquitin E3-ligase family would serve as ‘modulators’ of the TGFβ activated Smad signaling pathway determination by ubiqutination-proteosomal degradation system.

Disclosures: B. Kim, None.

M200

NELL-1 Promotes Bone Formation in a Sheep Spinal Fusion Model.

S. S. Lu*¹, J. Whang*¹, X. Zhang¹, B. Wu*¹, S. Turner*², H. B. Seim*², K. Ting¹, J. C. Wang*¹, C. Soo*¹. ¹School of Dentistry and Medicine, University of California-Los Angeles, Los Angeles, CA, USA, ²Veterinary Science, Colorado State University, Fort Collins, CO, USA.

NELL-1 is a Runx2 regulated secretory protein with marked osteochondroprogenitor cell specificity. NELL-1 exhibited comparable osteoinductivity to bone morphogenetic protein 2 (BMP-2) in a rodent calvarial defect model when placed on poly(lactic-coglycolic acid) scaffolds. The purpose of this study was to test NELL-1 the osteoinductivity in a sheep interbody fusion model and to establish preliminary dose ranges. Eight sheep divided into four groups underwent interbody fusion at L4-L5 and at L5-L6 with a radiolucent vertebral spacer (2 animals/group; 4 sites/group). The control group was filled with sheep demineralized bone matrix (DBX) and saline within and around each vertebral spacer. The other three groups received sheep DBX mixed with NELL-1 at final concentrations of 0.3, 0.6, and 1.5 mg/ml within and around the vertebral spacers. To assess spine fusion, X-rays (0, 2, and 3 months), CT images (2 and 3 months), and microCTs (explanted 3 month specimens) were obtained. Spinal fusion criteria were either > 50% contiguous bone volume within the spacer or contiguous bone formation within the callus totaling > 1/3 anterior vertebral body circumference. All animals were sacrificed at 3 months after surgery. The results showed significantly increased bone formation in NELL-1 treated animals relative to controls. CT images at three months demonstrated successful spinal fusion in 100% of the 0.6mg/ml NELL-1 dose sites and none of the controls. Representative post-sacrifice microCT images of L5–6 from two controls (right) and two NELL-1 animals (left) also confirmed this finding (Figure 1). MicroCT analysis of central bone within the spacers revealed increased bone density and bone volume in NELL-1 specimens relative to controls. We conclude that all three NELL-1 doses induced significantly more bone formation than controls. 0.6mg/ml NELL-1 appears to induce the most robust bone, and this initial finding will be investigated in a larger follow-on study. Overall, NELL-1/DBX at the 3 month time point may induce comparable sheep interbody fusion to published BMP-2 studies carried out to the 6 month time point. 

Figure  .  

Disclosures: S.S. Lu, None.

This study received funding from: UC-Discovery Grant & Bone Biologics.

M201

TGF-beta Suppresses POEM Expression in Osteoblasts.

A. Miyazono*¹, A. Yamada*¹, N. Morimura*², D. Suzuki*¹, M. Kobayashi*³, M. Takami¹, K. Tezuka⁴, M. Yamamoto³, R. Kamijo¹. ¹Department of Biochemistry, Showa University, Tokyo, Japan, ²Laboratory for Comparative Neurogenesis, RIKEN Brain Science Institute, Saitama, Japan, ³Department of Periodontology, Showa University, Tokyo, Japan, ⁴Graduate School of Medicine, Gifu University, Gifu, Japan.

POEM (preosteobiast epidermal growth factor-like repeat protein with MAM domain), also known as nephronectin, is an extracellular matrix protein that is thought to play a critical role in the development and functions of various tissues, such as those of the kidney, bone, muscle, and endocrine organs, through interaction with other extracellular molecules. In the present study, we found that transforming growth factor-beta (TGF-beta) dose-dependently and strongly inhibited POEM expression in mouse osteoblastic cell lines, MC3T3-E1 and UAMS-32. Inhibition of POEM expression by TGF-beta could be seen at concentrations exceeding 10 pg/ml. The time-dependent effects of TGF-beta on the reduction of POEM expression were further examined using 1ng/ml of TGF-beta. A decrease in POEM mRNA was detected at 3 hours after the addition of TGF-beta and the decrease continued up to 24 hours, demonstrating that the TGF-beta induced decrease of POEM gene expression occurs in both time- and dose-dependent manners. When the cells were pretreated with a selective inhibitor of ALK5 (TGF-beta R1, a TGF-beta type 1 receptor) prior to the addition of 1 ng/ml of TGF-beta, down-regulation of POEM expression was completely blocked. These results suggest that regulation of POEM expression by TGF-beta occurs via TGF-beta R1. Further, bone morphogenetic protein-2 (BMP-2) slightly blocked the effect of TGF-beta toward the reduction of POEM expression. TGF-beta is a key regulator of bone matrix properties and composition, and it inhibits the expression of RUNX2 which is a critical transcriptional regulator of osteoblast differentiation in MC3T3-E1. Therefore to elucidate the relationship between the inhibition of osteoblast differentiation and reduction of POEM expression by TGF-beta, effect of TGF-beta on the expressions of alkaline phosphatase (ALP), osteocalcin, and bone sialoprotein (BSP) were examined. Each of those genes was down-regulated by TGF-beta in a manner consistent with that of POEM mRNA. These results indicate that POEM is a novel marker gene for osteoblast differentiation similar to others, such as ALP, osteocalcin, and BSP. In addition, reduced POEM expression may contribute to the inhibition of osteoblast differentiation by TGF-beta.

Disclosures: A. Miyazono, None.

M202

See Sunday Plenary Number S202

M203

A Novel Activin/Nodal-binding Protein, G11, Inhibits Matrix Mineralization in Osteoblasts.

Y. Mochida, D. Parisuthiman*, M. Katafuchi*, P. Atsawasuwan*, M. Kaku*, M. Yamauchi. Dental Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

Transforming growth factor-beta (TGF-β) superfamily consists of three major subfamilies including TGF-β, bone morphogenetic protein (BMP) and activin/nodal, and they are known to be potent effectors in almost all crucial biological/developmental/regenerative events. Currently, however, the presence of activin/nodal and, if present, their roles in osteoblasts are largely unknown. In an effort to identify TGF-β superfamily binding proteins, we have taken a bioinformatics approach and just identified a novel gene, G11. The gene structure of G11 is related to chordin family. To investigate the potential function of G11 in osteoblasts, first, its expression pattern during cell differentiation and matrix mineralization was analyzed by quantitative real time PCR. Second, MC derived clones overexpressing G11 (S clones) were established and characterized by analyzing cell proliferation and in vitro mineralization. The phenotypes of S clones were evaluated by comparing to those of two control groups, i.e. MC and a clone transfected with an empty vector (EV clone). The data demonstrated that G11 was highly expressed in the early differentiation stage, i.e. at day 7 in mineralization medium, and decreased thereafter. The cell proliferation rate in S clones was not affected. However, in vitro mineralization in S clones was significantly delayed in comparison to those of controls. In order to gain mechanistic insights into an inhibitory function of G11, the interaction between G11 and TGF-β superfamily members (TGF-β1, -β2, -β3, BMP-2, −4, −6, −7, inhibin-ba, -bb and nodal; all were expressed in MC) was examined by immunoprecipitation-Wesrtern blotting. The data demonstrated that G11 was strongly bound to activin/nodal, and furthermore, G11 enhanced activin/nodal-induced Smad2 phosphorylation in MC cells. These results indicate that G11 is a novel chordin-like extracellular protein expressed in osteoblasts, and that it inhibits in vitro matrix mineralization possibly through its early interaction with activin/nodal.

Disclosures: Y. Mochida, None.

This study received finding from: NIH grants, DE10489 and AR052824.

M204

Collagen Binding Characteristics of Collagen Binding Domain from Clostridium Histolyticum Class I Collaganase.

J. Sakon*¹, P. S. T. Leena*¹, O. Matsushita*². ¹Biochemistry, University of Arkansas, Fayetteville, AR, USA, ²Molecular Biology, Kitasato University School of Medicine, Kanagawa, Japan.

Weekly intraperitoneal injection of a fusion protein of PTH(1–33) with CBD for 8 weeks in mice showed 15% BMD increase. Clostridium histolyticum ColG collagenase activated by Ca²⁺ is responsible for extensive tissue destruction, and the CBD is a segment of the multi-domain enzyme. Binding of two Ca²⁺ on CBD is co-operative and is both enthalpically and entropically driven (K_d1 = 2.13microM; K_d2 = 4.63microM). Structures in the presence and absence of Ca²⁺ have been solved at ultrahigh resolution (<1.2Ang). N-terminus 14 residues of CBD adopt an alpha-helical conformation; however, an addition of Ca²⁺ unwinds the linker into a new beta-strand. To rule out the crystal-packing artifact, NMR titration studies were done and they confirm the conformational changes upon addition of Ca²⁺. The changes in Stokes and hydrodynamic radii as measured by size exclusion chromatography and dynamic light scattering experiments showed drastic transition upon Ca²⁺ addition. With Ca²⁺ CBD becomes thermally stable (Tm>90C), less susceptible to proteolysis and stable against chemical denaturants. Collagen binding affinity of CBD was detected only in the presence of Ca²⁺. Mutagenesis experiments identified a cluster of Tyr residues involved in collagen binding. The Tyr-rich surface becomes accessible upon Ca²⁺ binding. Co-crystallization of collagen:CBD have yielded no crystals thus far. Docking experiment of CBD with collagen-like peptide G(POG)₆ resulted in 3 different binding orientations. NMR titration of CBD against G(POG)₁₀ eliminated one of the probable in silico orientations. N¹⁵ enriched CBD and unlabeled (POG)₁₀ were used for the titration experiment. Studies thus far demonstrate the drastic structural changes accompanied upon secretion of the enzyme from a bacteria to infected tissues. They also provide atomic insights into how CBD interacts with extracellular collagen.

Disclosures: J. Sakon, None.

This study received funding from: NIH-COBRE.

M205

Smad4 Has a Direct Apoptogenic Role at the Mitochondria.

H. Yuan*¹, T. Qiu¹, J. Huang*², X. Cao¹, M. Wan¹. ¹Pathology, University of Alabama at Birmingham, Birmingham, AL, USA, ²Shihezi University School of Medicine, Shihezi, China.

Smad4, originally isolated from the human chromosome 18q21, is a key factor in transducing the signals of the TGF-β superfamily of growth hormones, which is an important player in regulating bone homeostasis and cancer cell growth and apoptosis. Smad4 plays a pivotal role in mediating antimitogenic and proapoptotic effects of TGF-β, but the mechanisms by which Smad4 induces apoptosis are elusive. Here we report that Smad4 directly translocates to the mitochondria of apoptotic tumor cells. Smad4 overexpression significantly promotes, whereas Smad4 gene silencing by siRNA inhibits, TGF-β- and UV-induced apoptosis in two pancreatic cancer cell lines. To further clarify the mechanism by which Smad4 induces apoptosis, we found that a fraction of Smad4 translocates to mitochondria upon TGF-β treatment or UV exposure by Western blot analysis of cytosol and mitochondrial Smad4 protein. Smad4 mitochondria translocation was also confirmed by Smad4 colocalization with Mitotracker Red observed by confocal fluorescence microscope.

To examine the functional significance of mitochondrial Smad4 localization in apoptosis, we searched for mitochondria proteins that have physical interactions with Smad4 using yeast two-hybrid screening approach. DNA sequence analysis identified 34 positive clones, five of which encoded subunits in mitochondria complex IV, ie. one clone encoded cytochrome c oxidase (COX)II, three clones encoded COXIII and one clone encoded COXVb. The data indicate that Smad4 might associate with mitochondria proteins and may have mitochondria-related function. Strong interaction between Smad4 with COXII was verified in yeast by β-gal activity assays and in mammalian cells by immunoprecipitation assays. Importantly, we isolated the mitochondrial portion and confirm the interaction between COXII and Smad4 in mitochondria upon TGF-β treatment or UV exposure. COXII is encoded by mitochondrial DNA, synthesized in the mitochondria, and inserted into the inner membrane by mitochondrion-dependent pathways. COXII also has been shown to bind directly to cytochrome c and is speculated to regulate apoptosis through this affinity for cytochrome c. The association of Smad4 with COXII in mitochondria demonstrated in the present study implies that Smad4 may regulate cell apoptosis by directly targeting mitochondria.

Disclosures: H. Yuan, None.

M206

Arkadia, an E3 ubiquitin Ligase, Represses Skeletal Muscle Differentiation Through Enhancement of Myostatin and TGF-beta Signaling.

H. Yuzawa*¹, D. Koinuma*¹, S. Maeda¹, M. Takahata*¹, M. Hayashi*¹, K. Miyazawa*², K. Yamamoto*³, T. Imamura¹. ¹Department of Biochemistry, The Cancer Institute of the Japanese Foundation For Cancer Reserch, Tokyo, Japan, ²Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo, Tokyo, Japan, ³Department of Orthopaedic Surgery, Tokyo Medical University, Tokyo, Japan.

Myostatin belongs to transforming growth factor (TGF)-beta family, and act as a negative regulator of skeletal muscle differentiation. Myostatin knockout mice cause dramatic and wide spread increase in skeletal muscle, and naturally occurring myostatin mutations in cattle breeds lead to a heavy muscle condition due to hyperplasia and hypertrophy. Regulation of Myostatin signaling is thus a matter of physiological and pathological importance. However, how positive and negative regulators affect myostatin signaling is still not fully understood. Here we investigated the function of Arkadia in Myostatin signaling during myoblast differentiation. Arkadia was originally identified as an intracellular protein that is essential for formation of a mammalian node during mouse development through enhancement of Nodal signaling. In addition, we have previously reported that Arkadia is an E3 ubiquitin ligase that degrades an inhibitory Smad, Smad7, to enhance TGF-beta family signaling. In the present study, we first showed that mouse myoblast C2C12 cells express increased level of Myostatin and TGF-beta3 during myoblast differentiation. Adenoviral-mediated expression of Arkadia effectively enhanced suppression of myoblast differentiation by Myostatin and TGF-beta3, as determined by myotube formation and target gene expression, including myosin heavy chain. We further examined the role of endogenous Arkadia by lentiviral shRNA knockdown system, and showed that loss of endogenous Arkadia enhances myotube formation. Moreover, protein level of endogenous Smad7 was elevated by knockdown of Arkadia suggesting that Arkadia represses skeletal muscle differentiation through modulation of negative feedback mechanisms. In conclusion, we propose a physiological role of Arkadia as an important negative regulator of myoblast differentiation through enhancement of TGF-beta family signaling. Investigation of expression and activity of Arkadia is thus required in the future.

Disclosures: H. Yuzawa, None.

M207

The Duodenum Rapidly Senses and Modulates Renal Phosphate Reabsorption Via Novel PTH- and Phosphatonin-Independent Pathways.

T. Berndt*, L. Thomas*, T. Craig*, S. Sommer*, X. Li*, E. Bergstralh*, R. Kumar. Nephrology Research, Mayo Clinic Rochester, Rochester, MN, USA.

The mechanism by which phosphorus homeostasis is preserved during alterations in dietary phosphate intake is not completely understood. Net phosphorus balance in mammals is maintained by the absorption of phosphate in the duodenum and jejunum and by the tubular reabsorption of phosphate by the kidney. We have previously demonstrated that renal phosphate reabsorption is altered by the infusion of phosphate into the duodenum by PTH- and phosphatonin-independent pathways. In order to localize the segments of the gastrointestinal tract responsible for this response, we infused phosphate either into the duodenum or the stomach of anesthetized rats previously fed a normal phosphate diet and measured renal phosphate reabsorption following the infusion. After a control clearance, sodium phosphate was administered into the duodenum (n=7) or the stomach (n=6). Five, ten, twenty, and thirty minutes following the infusion of sodium phosphate, renal phosphate reabsorption was measured. The administration of phosphate into the duodenum resulted in rapid and significant increases in the fractional excretion of phosphate (FEP) at 10, 20, and 30 minutes. Basal FEP (mean±SD) was 28.4±8.6%, 5 min = 28.7±7.9%, 10 min = 31.9±7.6%, 20 min = 41.8±9.9%, 30 min = 44.0±11.4%, resulting in a slope/min of 0.6±0.3, P=0.0037. The administration of sodium chloride did not alter the fraction excretion of phosphate in similarly prepared rats (FEP 23.7±7.2%, 20.2±4.0%, 21.6±6.9%, 24.5±8.5% and 24.6±5.4%, resulting in a slope/min of 0.096±0.09, P=0.066.). This effect is independent of PTH and the phosphatonins, FGF-23 and sFRP-4. When sodium phosphate was infused into the stomach, no change in the fraction excretion of phosphaste was observed (FEP was 26.3±6.6%, 25.9±8.0%, 27.4±7.8%, 27.8±6.6%, 29.0±5.8%, resulting in a slope/min of 0.083±0.28, P=0.46).

Our results show that a phosphate sensing mechanism exists in the duodenum but not in the stomach that rapidly decreases renal phosphate reabsorption following the ingestion of a high phosphate meal.

Disclosures: T. Berndt, None.

M208

See Sunday Plenary Number S208

M209

Transgenic Mice Overexpressing sFRP-4 Have Low Bone Mass but Do Not Exhibit Disturbed Phosphate Homeostasis.

H. Y. Cho*, H. J. Sun*, J. Y. Yang*, H. J. Choi*, J. H. Ahn*, S. W. Cho, S. W. Kim, S. Y. Kim*, C. S. Shin. Internal Medicine, Seoul National University College of Medicine, Seoul, Republic of Korea.

Secreted frizzled-related protein-4 (sFRP-4) is a member of secreted modulators of wnt signaling pathways that regulate biological processes ranging from developmental cell fate, cell polarity and tumorigensis. Moreover, sFRP-4 has recently been recognized to play important roles in the pathogenesis of oncogenic osteomalacia as a potential phosphatonin. To investigate the role of sFRP-4 in bone metabolism in postnatal life, we have generated transgenic mice that overexpress sFRP-4 under the control of the serum amyloid P (SAP) promoter, which drives transgene expression postnatally.

Transgene expression was identified in various tissues including serum, bone, liver and kidney in transgenic mice. At 10 week of age, the serum phosphorus concentration and urinary phosphorus excretion in transgenic mice were not significantly different from those of wild-type mice in both female and male. However, transgenic female mice exhibited decreased bone mineral density (0.045 ± 0.001, P < 0.05) and bone mineral content (0.407 ± 0.026, p < 0.05) compared to wild type female mice. Body fat contents were not different between wild-type and transgenic mice. On histological examination, transgenic mice showed decreased osteoid mass, thickness, decreased growth plate width and prolonged reversal phase. Histomorphometric analysis revealed that transgenic mice had decreased osteoid volume (P < 0.05), osteoid thickeness (P < 0.05), and mineralization lag time (P < 0.05) compared to wild type mice. There was no difference in bone resorption parameters. Whereas difference in the mRNA expression of Na/P cotransporters, Npt2a and Npt2c, were not apparent, the 1-a-hydroxylase expression was increased in transgenic mice. Our data do not support the role of sFRP-4 as a phosphatonin but suggest a direct effects of sFRP-4 on new bone formation without disrupting phosphate homeostasis.

Disclosures: H. Y. Cho. None.

M210

See Sunday Plenary Number S210

M211

Evaluation of FGF 23 (c-terminal and intact) ELISA Kits in End-stage Renal Disease Patients.

W. J. Fassbender¹, M. Hanfland-Schmidt*¹, J. Windolf*², U. C. Stumpf². ¹Department of Internal Medicine, Hospital z. Hl. Geist, Kempen, Germany, ²Department of Traumatology and Handsurgery, University Hospital, Duesseldorf, Germany.

Hyperphosphataemia, calcitriol deficency and secondary hyperparathyreoidism (sHPT) are common complications in end-stage chronic kidney disease (CKD). Fibroblast Growth Factor 23 (FGF-23) is a phosphaturic peptide that also inhibits renal 1alpha-hydroxylase activitiy and tubular phosphate reabsorption by inhibition of sodium-dependant renal phosphate transport. Consequences are the decreaese of serum 1,25 dihydroxyvitamin D3 and phosphaturia. Therefore FGF 23 plays a role in hyperphosphataemia associated with CKD and may be involved in the pathogenesis of sHPT. Increased FGF 23 may contribute to maintaining normal serum phoshpate levels in the face of advancing CKD, but up to a reduction of a creatinine clearance lower than 30 ml/min the capacity of this regulative mechanism ends and hyperphosphataemia results. Within end-stage renal disease we find markedly increased serum FGF 23 associated with hyperphosphataemia, phosphaturia and decreased serum calcitriol and sHPT.

Material and methods: We evaluated the actual in Europe available ELISA kits for FGF 23 (c-terminal and intact, “Human Intact FGF 23 ELISA Kit” and “Human FGF 23 (C-Term) ELISA Kit”, Immutopics) for assessment of serum and EDTA-plasma in patients requiring dialysis, as expected with high serum FGF 23 levels compared with values derived from a normal collective of healthy persons with normal renal function. Furthermore preanalytical testing for stability of FGF 23 was performed by comparing samples which were stored at −20° C with samples stored for 6 days at −4° C.

Dialysis patients: 23 patients, 17 male, 6 female, age: 36–79 years (mean age: 62,13).

Collective of healthy subjects: 17 subjects, 8 male, 9 female, age: 24–56 years, (mean age: 33,41).

Results: Dialysis patients: mean values serum FGF 23 15,32 pg/ml (intact) and 2774 U/ml (c-terminal, arithmetic mean). Mean values in EDTA-plasma FGF 23 (intact) 271 pg/ml; FGF 23 (c-terminal) 2606 U/ml;collective of healthy subjects: mean serum values FGF 23 0,64 pg/ml (intact) and 13,67 U/ml (c-termina), arithmetic mean). Mean values of EDTA-plasma FGF 23 (intact) 13,09 pg/ml; FGF 23 (c-terminal) 19,18 U/ml. Discussion:The parallel investigation of serum and EDTA-plasma FGF 23 certificates the advantage of EDTA-plasma in subjects with intact renal function. The significantly increased levels of FGF 23 in patients requiring dialysis demand dilution of the samples. Further investigations concerning age-related changes of serum FGF 23 should be performed. The feed-back mechanism of FGF 23 and serum 1,25 dihydroxyvitamin D3 implements possible future therapeutical options within the treatment of sHPT.

Disclosures: W.J. Fassbender, None.

M212

See Sunday Plenary Number S212

M213

Venous Sampling for FGF23 as a Tool for Pre-Operative Localization of Tumors Giving Rise to Hypophosphatemic Osteomalacia.

E. Hagström*¹, P. A. Westerberg*², G. Toss*³, T. Lindhe*², Ö. Ljunggren², T. E. Larsson². ¹Dept. of Surgical Sciences, Uppsala University Hospital, Sweden, ²Dept. of Medical Sciences, Uppsala University Hospital, Sweden, ³Dept. of Medicine, Linköping, Sweden.

Tumor-induced hypophosphatemic osteomalacia (TIO) is a rare paraneoplastic syndrome, characterized by hypophosphatemia, low or inappropriately normal 1,25(OH)2D3 and rickets/osteomalacia. Increased serum level of Fibroblast Growth Factor-23 (FGF23) has been identified as the causative factor for TIO. In this study, we report on the diagnostic difficulties and tumor localization in a 55-year old male with presumable TIO.

Primary imaging studies, including CT/MRI scans and octreotide scintigraphy, failed to localize the tumor, however consistently elevated FGF23 levels were found (180–220 pg/mL). Venous sampling, followed by intact FGF23 analysis, revealed locally increased FGF23 level in the left femoral vein (343 pg/mL). Octreotide scintigraphy of the upper left leg revealed a 20 mm large tumor located at the left lateral condyle of the femur. Surgical resection of the tumor normalized serum phosphate and vitamin D within 5–10 days, however serum FGF23 declined from 292 to 12 pg/mL 24 hours after surgery. Preoperative bone mineral density (BMD), as determined by DXA analysis, revealed a T-score of −1.6 in lumbar spine and −1.16 in total hip. Lumbar spine BMD was increased by 25% two months after tumor resection. Notably, our patient post-operatively developed a secondary hyperparathyroidism, which may reflect a transient hypocalcemia related to increased bone mineralization. In conclusion, our study demonstrates the diagnostic complexity and difficulties in localizing small TIO tumors. Venous sampling for FGF23 followed by octreotide scintigraphy may constitute a powerful tool for tumor localization in subjects with hypophosphatemic osteomalacia.

Disclosures: E. Hagström, None.

M214

See Sunday Plenary Number S214

M215

Rise in FGF-23 Precedes 1–84PTH and Linear Decline in Calcitriol Precedes Rise in FGF-23 in Non-diabetic Predialysis Patients.

T. Hamano*, K. Tomida*, S. Mikami*, N. Fujii*, T. Ito*, E. Imai*. Nephrology, Osaka University Hospital, Suita, Japan.

Today fibroblast growth factor-23 (FGF-23) was found to be another phosphaturic hormone derived from osteocyte and osteoblast. The reduction in calcitriol synthesis by phosphate load can be explained by the increase in FGF-23. Although many study measured FGF-23 using a two-site ELISA that detects carboxyl-terminal portion of FGF-23, full-length FGF-23 (intact FGF-23) at different glomerular Filtration rate (GFR) is not well documented in large cohort. Our aim is to know at what level of GFR intact FGF-23 and 1–84PTH increase and serum calcitriol decrease significantly. In this cross-sectional observational study (OVIDS-CKD; Osaka Vitamin D Study in patients with CKD), we enrolled non-diabetic patients in outpatients nephrology clinic at two major hospitals in Osaka. Patients who were or had been receiving glucocorticoid, bisphosphonate, vitamin D, calcium, or hormone replacement therapy were excluded. We measured serum phosphorus, calcium, albumin, intact FGF-23, whole PTH, calcitriol, and 25-hydroxyvitamin D (250HD). We check the relationship of fold increase of two phosphaturic hormones and change in other parameters with estimated GFR (eGFR). Patients were divided into 9 groups by eGFR.

In this study 574 patients were enrolled. In comparison with patients with eGFR more than 80 mL/min/1.73m², intact FGF-23, 1–84PTH, and serum phosphorus increased significantly in those with eGFR<60, eGFR<50, and eGFR<30 mL/min/1.73m², respectively. In patients with eGFR<50 mL/min/1.73m², steeper rise in 1–84PTH was observed than intact FGF-23. Whereas serum 250HD had no correlation with eGFR at all, significant decline in serum calcitriol was observed even in patients with eGFR<80 compared to those with eGFR>80 mL/min/1.73m². Multiple linear regression analysis revealed that significant positive contributors to serum calcitriol were eGFR, 1–84PTH, and 250HD. Negative contributor was intact FGF-23. The significant decline in calcitriol was found even when significant rise in intact FGF-23 was not observed. Therefore the reduction of calcitriol synthesis in earlier stage of CKD seemed not to be explained by the fact that FGF-23 decrease 1 α-hydroxylase in proximal tubules. There might be another mechanism for calcitriol decline in this early CKD stage. The earliest sign for phosphate overload relative to viable nephron numbers was compensative rise in intact FGF-23, not in serum 1–84PTH. Given the origin of this two phosphaturic hormones, this fact might suggest that the bone is more sensitive to minimal phosphate overload than the parathyroid glands. Significant linear decline in calcitriol with CKD progression precedes significant rise in FGF-23.

Disclosures: T. Hamano, None.

M216

Comparison of Rapid Intraoperative Parathyroid Hormone to IRMA in Patients with Hyperparathyroidism.

M. Al Mukaddam*, C. Albany*, C. Hajj-Shahine*, G. El-Haij Fuleihan. Calcium Metabolism and Osteoporosis Program, American University of Beirut-Medical Center, Internal Medicine, Beirut, Lebanon.

The use of rapid intact parathyroid hormone (iPTH) assay is central to the success of minimally invasive parathyroidectomy. Comparing information obtained with this assay and the classical immunoradiometric PTH assay (IRMA) is limited. 142 blood samples collected from 71 patients referred to a tertiary care center for parathyroidectomy were analyzed. PTH levels on the intraoperative blood samples (pre-and post-gland excision) were concomitantly measured with the rapid and the classical IRMA kits. The classical IRMA kit utilizes monoclonal antibodies specific for the mid and C terminal part 39–84 and polyclonal antibodies specific to the N terminal moiety of PTH (CIS Bio International, Gif sur Yvette, France). The rapid iPTH was performed with the electrochemiluminescence immunoassay “ECLIA” (Roche Elecsys 2010 analyzer, Roche Diagnostics, Indianapolis, USA). It employs two monoclonal antibodies that react with epitopes in the amino acid regions 26–32 and 37–42 of PTH.

ECLIA assay (y) values strongly correlated with IRMA assay (x) values; y=1.63x −63.86 (r = 0.956, p<0.0001, pre-gland excision) and y=1.31x +4.22 (r=0.938, p<0.0001, 10–15 minutes post gland excision). This correlation was also evident with the percentage drop in PTH level using the above assays y=0.96x+0.51(r =0.944, p<0.0001). Both assays yielded similar prediction of cure using the 50% drop as cut-off. However, mean pre- and post-gland excision PTH were significantly higher by ECLIA than IRMA, suggesting a systematic difference between the two assays, that is independent of mean PTH levels. 

Table  .  

* paired t-test on log transformed values of PTH, NS: Not Significant

The manufacturers report a normal range for PTH of 15–65 pg/ml with ECLIA, which is very similar to the normal range for IRMA assay of 8–76 pg/ml. However, this study reveals that there is significant difference in the values obtained with the two assays, and underscores the importance of using the same assay when monitoring patients. Further studies should be implemented to elucidate sources of discrepancies between the two assays.

Disclosures: M. Al Mukaddam, None.

M217

See Sunday Plenary Number S217

M218

Ability of XLαs to Mimic Gsα in Mediating Parathyroid Hormone Signaling In Vivo.

C. Aydin¹, L. F. Fröhlich*², D. Ozturk*¹, M. Bastepe¹. ¹Endocrine Unit, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA, ²Institute of Pathophysiology, University of Veterinary Medicine, Vienna, Austria.

XLαs, an imprinted, paternally expressed variant of the α-subunit of the stimulatory G protein (Gsα), can mediate parathyroid hormone (PTH)-induced adenylyl cyclase activation and, thus, is able to replace Gsα in transfected cells. However, XLαs knockout mice show phenotypes that are significantly different from those observed in Gsα knockout mice, suggesting that XLαs is unable to mimic Gsα in vivo. To address this question, we generated a transgenic mouse strain in which the rat type-I γ-glutamyltranspeptidase promoter was employed to target XLαs expression to the proximal tubule, a major site of PTH action. Southern blots identified five different founders (rptXLαs mice), and Northern blots showed that the F1 generation from two of the founders (G14 and G28) expressed transgenic rat XLαs mRNA at the age of two months. In G14 rptXLαs mice, but not in wild-type littermates, RT-PCR using whole kidney total RNA and primers common to both mouse and rat XLαs mRNA yielded a specific amplicon, whose sequence matched the cDNA sequence of rat XLαs. Western blots using a polyclonal antibody against the C-terminus of XLαs detected the XLαs protein in membrane extracts from the proximal tubules of the G14 rptXLαs mice but not of wild-type littermates. Endogenous Gsα mRNA levels appeared similar in transgenic and wild-type animals, which was determined by real-time RT-PCR using total RNA isolated from the proximal tubule. In contrast, the level of 1α-hydroxylase mRNA, a downstream target of Gsα-mediated PTH signaling, was elevated in the G14 rptXLαs mice compared to wild-type littermates. These findings suggest that proximal tubular PTH actions that typically rely on Gsα signaling are enhanced in the G14 rptXLαs mice without changes in Gsα levels, providing strong evidence for the ability of XLαs to function in a manner similar to Gsα in vivo. Since XLαs mRNA is disrupted by most paternal mutations that disrupt Gsα mRNA and is preserved when those mutations are maternal, the Gsα-like activity of XLαs may be important in the pathogenesis of disorders associated with altered Gsα activity, such as pseudohypoparathyroidism, Albright's Hereditary Osteodystrophy, and fibrous dysplasia of bone.

Disclosures: C. Aydin, None.

This study received funding from: NIH/NIDDK (KO1 DK062973 to MB).

M219

See Sunday Plenary Number S219

M220

Estrogen Deficiency and PTHrP Infusion Do Not Fully Reproduce the Bone Loss of Lactation.

S. Brian*, P. Dann*, J. Wysolmerski. Endocrinology and Metabolism, Yale University, New Haven, CT, USA.

Lactation is associated with increased bone turnover and rapid bone loss. Suckling inhibits GnRH secretion, causing hypogonadotropic hypogonadism and low circulating levels of estradiol. In addition, the lactating mammary gland secretes PTHrP into the circulation. Estrogen replacement in lactating mice reduces bone loss by 60%, while disrupting the PTHrP gene in the lactating mammary gland reduces bone loss by 50%. Therefore, we hypothesized that the combination of estrogen deficiency and PTHrP excess is sufficient to explain bone loss in lactating mothers. To test this hypothesis, we attempted to reproduce lactational bone loss in nulliparous mice by suppressing estrogen and raising PTHrP levels. We administered 100 micrograms of leuprolide acetate twice daily to induce hypogonadotropic hypogonadism and lower estrogen levels. PTHrP(1–36) was infused at a rate of 10 pmoles/hr using Alzot miniosmotic pumps. We studied 5 groups of mice: 1) lactating controls, 2) nulliparous controls, 3) PTHrP-treated, 4) leuprolide-treated, and 5) combined PTHrP and leuprolide. All mice were CD-1's between 11 and 14 weeks of age at the beginning of the experiment. BMD was measured by DXA prior to the start of the treatments and again after 11 days, and in lactating mice on day 1 and day 11 postpartum. After 11 days, mice were sacrificed and blood and urine were collected. As expected, lactating mice had lower estradiol levels (4.2pg/ml) than did randomly cycling virgins (13.7 pg/ml). Importantly, leuprolide-treated mice also had suppressed levels of estradiol (3.5 pg/ml) that were equivalent to those of lactating mice. Infusion of PTHrP led to mild hypercalcemia. Urinary C-telopeptide excretion (corrected for creatinine) (CTx) was elevated for all groups as compared to non-lactating controls. However, treatment with leuprolide, PTHrP or the combination did not raise CTx levels as high as those measured in lactating mice. BMD declined by 22% at the spine, 19% at the femur and 16% for the total body in lactating mice. PTHrP treatment caused loses of 8.0% at the spine, 8.7% at the femur and 8.0% at the total body. Interestingly, leuprolide treatment alone caused no bone loss and bone loss in the combined leuprolide and PTHrP group was no worse than with PTHrP treatment alone (5.0% at the spine, 5.1% at the femur and 7.0% for the total body). In conclusion, infusion of PTHrP combined with hypogonadotropic estrogen deficiency does not reproduce the extremely rapid rate of bone loss observed during lactation. These data suggest that either antecedent pregnancy or some other aspect of lactation magnifies bone loss caused by estrogen deficiency and PTHrP.

Disclosures: S. Brian, None.

M221

Activation of the Calcium Sensing Receptor With Cinacalcet Increases Serum Gastrin Levels in Healthy Older Subjects.

L. Ceglia, S. S. Harris, H. Rasmussen*, B. Dawson-Hughes. Bone Metabolism Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, MA, USA.

Gastric acidity is postulated to enhance calcium absorption since calcium is better dissolved at low pH. Extracellular calcium stimulates gastrin and gastric acid secretion in humans. Ex vivo studies indicate that the calcium sensing receptor (CaR), which is expressed on the surface of human G cells and parietal cells, may be involved in this regulation. We evaluated whether cinacalcet (C), a CaR agonist, compared to placebo (P) would increase serum gastrin level (G) and basal gastric acid output (BAO) in healthy subjects.

17 subjects age 47–70 were placed on a metabolic diet with fixed protein, calcium, phosphorus, sodium, magnesium, potassium and vitamin D for 18 days. We measured G, BAO, intact parathyroid hormone (PTH), and 24-hr urine calcium-to-creatinine excretion ratio (Ca/Cr) at baseline (day 8, following a run-in period) and post-intervention (day 18). C was titrated to 30 mg daily. G was measured by the Diasorin method. Gastric volume, pH and acid mEq were measured to calculate BAO.

Mean G was similar in the 2 groups at baseline (C = 33.1 ± 13.9 (SD) pg/ml (n=9), P = 38.5 ± 13.9 pg/ml (n=8)). However, mean change in G differed (C = 7.0 ± 7.3 pg/ml, P = −4.6 ± 6.8 pg/ml, p = 0.004). Mean BAO at baseline was similar in the 2 groups (C = 2.14 ± 1.77 mEq/hr, P = 2.43 ± 1.56 mEq/hr), and mean change in BAO did not differ significantly (C = 1.32 ± 2.37 mEq/hr, P = −0.24 ± 1.08 mEq/hr, p = 0.109). In the group as a whole, change in G correlated with change in BAO (r = 0.530, p = 0.029 (n=17)). Baseline mean PTH was similar in the 2 groups (C = 43.9 ± 16 pg/ml, P = 47.8 ☆ 18.0 pg/ml), and as expected mean change in PTH differed (C = −26.9 ± 11.7 pg/ml, P= 14.3 ± 17.0 pg/ml, p = <0.0001). Ca/Cr did not differ significantly at baseline in the 2 groups (C = 84.9 ± 59.9 mg/g, P = 114.3 ± 80.1 mg/g, p = 0.401), but mean change in Ca/Cr was significantly different (C= 17.9 ± 24.6 mg/g, −12.2 ± 24.3 mg/g, p = 0.023). These results show that activation of the CaR with cinacalcet stimulates gastrin secretion, and that an increase in gastrin is associated with a rise in basal gastric acid output in healthy older subjects. The impact of these changes on calcium absorption requires further investigation.

Disclosures: L. Ceglia. None.

This study received funding from: Jean Mayer Human Nutrition Research Center on Aging at Tufts University.

M222

The Discriminative Power of the 24h-Calcium/Creatinine Clearance Ratio (CCCR), the 24h-Calcium/Creatinine Ratio (CR) and the 24h-Calcium Excretion (CE) for the Separation Between Familial Hypocalciuric Hypercalcemia (FHH) and Primary Hyperparathyroidism (PHPT).

S. E. Christensen¹, P. H. Nissen*², P. Vestergaard¹, L. Heickendorff*², L. Mosekilde¹. ¹Dept. of Endocrinology, C, Aarhus University Hospital, THG, DK-8000 Aarhus C., Denmark, ²Dept. of Clinical Biochemistry, Aarhus University Hospital, THG, DK-8000 Aarhus C, Denmark.

Introduction: The 24h-urine calcium/creatinine clearance ratio (CCCR) is used clinically to separate familial hypocalciuric hypercalcemia (FHH) from primary hyperparathyroidism (PHPT).

A CCCR < 0.01 is often considered to indicate FHH, whereas a CCCR >0.02 may be diagnostic for PHPT. Values between 0.01 and 0.02 are probably inconclusive. The simpler 24h-urine calcium/creatinine ratio (CR) and the 24h-urine calcium excretion (CE) have also been used. However, the discriminative powers of the different indices have not been sufficiently evaluated in well-defined subsets of patients.

Objective: To evaluate the diagnostic powers of CCCR, CR and CE in separating FHH from PHPT, using ROC curve analyses and overlap performance analyses. Materials: We included 59 hypercalcemic FHH-patients characterized by clinically significant mutations in the CaSR gene and 97 hypercalcemic patients with surgically verified PHPT. All patients had a plasma creatinine (P-Cr) level < 150 pmol/l.

Methods: We calculated the CCCR as (P-Ca / 24h-U-Ca) x (24h-U-Cr / P-Cr), the CR as 24h-U-Ca/ 24h-U-Cr (mmot/mmol) and the CE as 24h-U-Ca (mmol/24h). In FHH, all protein coding exons in the calcium sensing receptor gene (CaSR) were sequenced and aligned to GenBank reference sequence NM000388.

Results: Highly significant differences between the two groups were found for all three indices (p<0.00l). The table reports the ROC curve analyses. CCCR had the largest area under the RUC curve and a slightly higher sensitivity and specificity at the optimal separation point compared to the CR and CE. 

Table  .  

CCCR < 0.01 had a positive predictive value of 0.89 for FHH, whereas CCCR > 0.02 had positive predictive value for PHPT of 0.93. Overlap performance analyses supported a two step diagnostic approach based on CCCR determination in all patients with CaSR gene analyses in those with CCCR < 0.02

Conclusions: The diagnostic differentiation between PHPT and FHH should be based on CCCR determination with CaSR analyses in all patients with CCCR < 0.02.

Disclosures: S.E. Christensen, None.

M223

See Sunday Plenary Number S223

M224

The Calcim-Sensing Receptor (CaSR) Dampens the Calcemic Response to Exogenous 1,25 (OH)₂vitamin D In Vivo Independent of Parathyroid Hormone (PTH).

O. Egbuna¹, L. Kantham², S. Quinn², J. Pang*², R. Butters*², M. Pollak*³, E. Brown². ¹Divisions of Endocrinology and Nephrology, Brigham and Womens's Hospital and Beth Israel Deaconess Med Ctr, Boston, MA, USA, ²Division of Endocrinology, Brigham and Womens's Hospital, Boston, MA, USA, ³Division of Nephrology, Brigham and Womens's Hospital, Boston, MA, USA.

The CaSR has known actions on the parathyroid gland, parafollicular C-cell, and kidney. Our studies of the adaptations of mice with targeted disruption of the PTH gene (CaR^+/+ PTH^−/− aka PTH KO)), both the PTH and CaR genes (CaR^−/−PTH^−/− aka DKO) or wild type mice (CaR^+/+PTH^+/+ aka WT) to exogenous 1,25 (OH)₂ vitamin D3, have revealed striking phenotypes that provide new insights on the roles of the CaR and PTH in calcium homeostasis. The 3 groups mice were given 0.5 ng/g BW of 1,25 (OH)₂vitamin D₃ by intraperitoneal injection, and the calcemic response over 72 hrs analyzed. Injections were given while the mice were given free access to a calcium replete diet [0.6% calcium (Ca) w/w chow and plain water] and repeated after conditioning the mice for 18–72 hrs on a Ca-deficient diet (0.01% Ca in the chow and plain water) prior to injection. Peak calcemic responses were observed in all genotypes on either diet at 24 hrs post injection. On the Ca replete diet (top figure), there was a 125% increase in serum calcium (SCa) in the DKO mice at 24 hrs. The increase in SCa was substantially less marked in the (PTH KO) (+41%), and less in the WT mice (+25%). On the Ca deplete diet (lower figure), the calcemic response at 24 hrs in the DKO mice was much less robust- +37%, and was similar to that in the PTH KO mice (+39%) but again was greater than that in the WT mice (+13%).

Lack of the CaSR renders mice lacking PTH markedly more sensitive to the calcemic action of vitamin D₃ than mice lacking PTH alone or WT mice. This exaggerated response appears selective for vitamin D-mediated intestinal Ca absorption rather than bone resorption, since it was observed on a Ca replete but not a Ca deplete diet. Additional factors that may contribute to the increased vitamin D sensitivity of the DKO mice are impaired secretion of calcitonin and decreased renal Ca excretion, although the latter could not explain the differences in the responses on the Ca replete and deplete diets.

Disclosures: O. Egbuna, None.

This study received funding from: NIH-NIDDK.

M225

Testin, a Novel Binding Partner of the Calcium-Sensing Receptor.

A. L. Magno*¹, B. K. Ward*², E. Ingley*¹, A. D. Conigrave³, T. Ratajczak*². ¹Western Australian Institute for Medical Research, University of Western Australia, Perth, Australia, ²Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Perth, Australia, ³School of Molecular and Microbial Biosciences, University of Sydney, Sydney, Australia.

The calcium-sensing receptor (CaR) is a G protein-coupled receptor that can respond to a diverse range of stimuli. Besides its integral role in calcium homeostasis, the CaR is involved in a wide variety of cellular processes through its mediation of different intracellular signalling pathways. To elucidate the mechanisms that convert the various extracellular stimuli for the CaR to specific intracellular responses we used the CaR intracellular tail as bait in a yeast two-hybrid screen of a mouse haemopoietic cell line library. Identified in this library screen was testin, a protein that has been proposed to act as a tumour suppressor and is involved in cell adherence. Testin is a triple LIM domain protein that localises to focal adhesions and actin stress fibres. LIM domains are composed of two zinc fingers that mediate protein interactions important in coordinating specific signalling pathways. Three distinct clones of testin containing a 61 amino acid overlapping region were identified. This overlapping region incorporates the second zinc finger of the first LIM domain. Yeast two-hybrid based deletion mapping studies revealed that testin binds to the membrane-proximal region of the CaR tail, a domain critical for activation of several intracellular signalling pathways. The interaction between the CaR and testin in a mammalian system was confirmed in coimmunoprecipitation studies using lysates from HEK293 cells transiently expressing FLAG-tagged CaR and EGFP-tagged testin. The focus of our current studies is to investigate the possible colocalization of the CaR and testin at focal adhesions and/or actin stress fibres in conjunction with experiments examining testin's potential modulating role in CaR-mediated activation of the mitogen-activated protein kinase, phosphatitylinositol 3-kinase and Rho pathways. In this regard, we have preliminary evidence that overexpression of testin reduces CaR-mediated extracellular signal-regulated kinase phosphorylation in HEK293 cells stably expressing the CaR. In conclusion, we have identified the LIM domain protein, testin, as a novel interaction partner for the CaR that may regulate CaR-mediated signalling linked to the cytoskeleton.

Disclosures: A.L. Magno, None.

M226

Regulation of Calcium-Sensing Receptor by Glial Cells Missing 2 in Hyperplastic Parathyroid Cells.

M. Mizobuchi*¹, C. S. Ritter*¹, G. Sicard*², E. Slatopolsky¹, A. J. Brown¹. ¹Renal, Washington University School of Medicine, St Louis, MO, USA, ²Surgery, Washington University School of Medicine, St Louis, MO, USA.

Glial cells missing 2 (Gcm2) is the key regulating transcription factor for parathyroid gland development. The continued expression of high levels of Gcm2 in mature parathyroid glands suggests that it is required for maintenance of parathyroid cell differentiation. The role of Gcm2 in parathyroid cell physiology, however, has not been fully investigated. In preliminary studies, we confirmed that Gcm2 mRNA continued to be expressed in hyperplastic parathyroid cells placed in culture. Western blot analysis of the human parathyroid tissue using a polyclonal antibody for Gcm2 revealed a band of approximately 57 kDa, the predicted size of the Gcm2 protein. We then examined the effects of Gcm2 silencing on cultured human parathyroid cells. Collagenase-dispersed human parathyroid cells from patients with chronic kidney disease were placed in monolayer cultures, and treated with lentivirus expressing shRNA for human Gcm2 for 24 hours. Fresh medium was added and the cells were harvested after 48 hours. RNA was analyzed for Gcm2, PTH, vitamin D receptor (VDR), calcium-sensing receptor (CaR) and proliferating cell nuclear antigen (PCNA) mRNAs by qPCR. Gcm2 mRNA was decreased by 75 ± 4 % (p < 0.01; mean of 3 cultures). Immunoblot analysis confirmed the decrease in Gcm2 as well. VDR, PTH and PCNA were not significantly affected by Gcm2 silencing, but CaR mRNA was consistently reduced by 48 ± 9% (p < 0.01). Further analysis of CaR mRNA indicated that transcripts containing Exon 1B, derived by transcription from CaR promoter 2, were down-regulated by the Gcm2 silencing. These results indicate that one function of Gcm2 is to maintain high levels of CaR expression in parathyroid cells. Further studies are underway to identify other targets of Gcm2 action.

Disclosures: M. Mizobuchi, None.

M227

See Sunday Plenary Number S227

M228

Calcilytics Block the Suppressive Effect of Calcimimetics on PTH mRNA Levels in Bovine Parathyroid Cells.

B. T. Brinton*¹, M. Chan*¹, J. Li*¹, T. Le-Capling*¹, R. Fantaske*¹, E. F. Nemeth². ¹NPS Pharmaceuticals, Toronto, ON, Canada, ²Dept. Pharmaceutical Sciences, University of Toronto, Toronto, ON, Canada.

Extracellular calcium is the primary regulator of parathyroid cell function and most, if not all its actions are mediated by the calcium receptor (CaR). Thus, activation of CaR by the calcimimetic compound NPS R-568 decreases PTH secretion, PTH mRNA levels, and parathyroid cell proliferation elicited by hypocalcemic conditions. CaR antagonists (calcilytics) increase PTH secretion but, under normocalcemic conditions, do not appear to increase parathyroid cell proliferation. The effects of the calcilytic compound NPS 89636 on PTH synthesis (mRNA levels) in parathyroid cells is reported here.

Dissociated bovine parathyroid (bPT) cells were cultured in media containing 0.9 mM phosphate and various concentrations of extracellular calcium (0.5 to 3 mM) in the presence or absence of NPS R-568 and/or NPS 89636. PTH mRNA levels were determined by real time quantitative PCR. Levels of PTH mRNA were similar following incubation of bPT cells for 24 hrs in media containing 0.5 or 1.8 mM calcium but were reduced by 70% at an extracellular calcium concentration of 3 mM (n=4; p=0.0001). The addition of NPS R-568 to cells cultured for 24 hrs in 1.8 mM calcium reduced PTH mRNA levels by 80%. These inhibitory effects of NPS R-568 were concentration-dependent (IC₅₀ = 42 nM) and stereoselective (no effect with 1 uM NPS S-568) and were essentially the same as our previous results using Northern blots to quantify PTH mRNA. The stability of PTH mRNA, as assessed by comparison to mRNA half-life at 1.8 mM extracellular calcium, was reduced 2.8-fold by 3 mM extracellular calcium and 4.3-fold by 300 nM NPS R-568. The inhibitory effect of 3 mM extracellular calcium was completely blocked by NPS 89636 in a concentration-dependent manner (EC₅₀ = 21 nM). The calcilytic similarly blocked the inhibitory effect of 300 nM NPS R-568 on PTH mRNA levels and shifted the NPS R-568 concentration-response curve to the right. By itself, NPS 89636 did not significantly affect PTH mRNA levels at extracellular calcium concentrations of 0.5 or 1.8 mM. Secretion of PTH, however, was stimulated by NPS 89636 (30–300 nM) at extracellular calcium concentrations of 1.25 to 2 mM. The ability of a calcilytic compound to completely block the effects of extracellular calcium on PTH secretion and mRNA levels demonstrates that both these effects are mediated solely through the CaR. In contrast to PTH secretion, levels of PTH mRNA appear to be maximally elevated at extracellular calcium levels of 1.8 mM and can lowered but not further increased by compounds that act on the CaR.

Disclosures: E.F. Nemeth, NPS Pharmaceuticals 1, 5.

This study received funding from: NPS Pharmaceuticals.

M229

Role of G-alpha-q in Parathyroid Gland Function Elucidated by Mouse Genetic Approaches.

M. Pi, Q. Luo*, L. D. Quarles. The Kidney Institute, KUMC, Kansas City, KS, USA.

The calcium-sensing receptor (CASR) is a heptahelical receptor coupled to G-alpha-i and G-alpha-q. CASR regulates parathyroid gland (PTG) function, including PTH secretion and production as well as chief cell hypertrophy and hyperplasia. The specific and/or separate functions of G-alpha-q and G-alpha-i in regulating these different aspects of PTG function have not been elucidated. To investigate the role of G-alpha-q in the PTG we initially assessed serum PTH levels in heterozygous G-alpha-q-deficient mice. We found that G-alpha-q-deficient mice had small but significant elevation of serum PTH levels compared to wild-type littermates. Next, we created transgenic (Tg^{PTHp−Gqloop}) mice using the human PTH promoter to drive the overexpression of a dominant negative Gαqloop mini-gene to selectively disrupt G-alpha-q function in the PTG We found that the gross appearance, body weight and survival of the Tg^{PTHp−Gqloop} mice were indistinguishable from that of wild-type littermates. We also found that the Gαqloop messenger RNA was expressed in parathyroid gland but not in other tissues of the Tg^{PTHp−Gqloop} mice. Adult Tg^{PTHp−Gqloop} mice exhibited a ∼ 2-fold increase in basal serum PTH levels compared to wild-type mice (43±5 pg/ml vs 25±2 pg/ml; p=0.001), as well as increased PTH mRNA levels in PTG containing tissue. However, stimulation of PTH secretion by lowering the serum calcium with EGTA (300 microM/kg body weight) resulted in identical maximum levels of serum PTH in Tg^{PTHp−Gqloop} and wild-type mice, but the percentage increment in PTH levels was less in Tg^{PTHp−Gqloop} due to the higher baseline PTH values. Of interest, CASR mRNA expression was increased in the PTG, but decreased in the kidney of Tg^{PTHp−Gqloop} compared to wild-type mice. In conclusion, we have demonstrated that the selective targeting of the PTG to investigate signaling mechanisms downstream of CASR is feasible. Our observations suggest a role for G-alpha-q in the regulation of PTH and CASR expression in the PTG but fail to identify G-alpha-q-dependent regulation of PTH secretion in response to hypocalcemic stimuli. Studies are ongoing to determine if further reductions of G-alpha-q, achieved by crossing Tg^{PTHp−Gqloop} onto heterozygous G-alpha-q-deficient mice, will uncover additional effects of G-alpha-q in CASR-dependent signaling. In addition, future studies will use the PTH promoter to separately target G-alpha-i to investigate its function in mediating CASR effects on PTG function.

Disclosures: M. Pi, None.

This study received funding from: P20-RR017686 and R01-AR37308.

M230

Intact Calcium-Sensing Receptor and PTH Genes Are Required to Regulate the Extracellular Magnesium and Calcium Concentrations Independently.

S. Quinn*, O. Egbuna, L. Kantham, R. Butters*, J. Pang*, M. Pollak*, E. M. Brown. Medicine, Brigham and Women's Hospital, Boston, MA, USA.

Although the systems governing Ca²⁺_o and Mg²⁺_o homeostasis share elements in common, e.g., a common mechanism for paracellular reabsorption in the renal cortical thick ascending limb, the circulating levels of these two divalent cations are generally regulated independently of one another. We have studied the adaptations of mice with targeted disruption of the PTH gene (CaR^+/+PTH^−/−), both the PTH and CaR genes (CaR^−/−PTH^−/−) or wild type mice (CaR^+/+PTH^+/+) to variations in calcium or magnesium intake. The results have revealed striking phenotypes that shed light on the roles of the CaR and PTH is permitting independent regulation of Ca²⁺_o and Mg²⁺_o. When maintained on sufficient calcium intake to sustain normocalcemia, CaR^−/−PTH^−/− mice show a dose-dependent, 55% and 33% increases in both blood Ca²⁺_o and Mg²⁺_o, respectively, when Mg²⁺ in the water is increased to 50 and then 100 mM (Figure 1). CaR^+/+PTH^−/− mice show similar, but less marked increases of 24 and 26%, respectively, while wild type mice show 6% and 9% changes. Increasing Ca²⁺ in the water from 0% to 1% and then 2%, produces analogous changes in both blood Ca and Mg elevating the two divalent cations by 106% and 50%, respectively, in the CaR^−/−PTH^−/− mice, by 53% and 17% in the CaR^+/+PTH^−/− mice and by 6% and 3% in the wild type mice (not shown). Thus loss of both PTH and the CaR results in an apparent “default” mode, in which increased intake of either Ca²⁺_o or Mg²⁺_o elevates the blood levels of both divalent cations. This defect is less severe when only PTH is missing, but the full capacity to maintain constancy of both Ca²⁺_o and Mg²⁺_o in the face of markedly increased oral loads of one or the other requires both PTH and the CaR.

Disclosures: S. Quinn, None.

This study received funding from: NIH.

M231

Deletion of the 25 Hydroxy Vitamin D 1α Hydroxylase from Mice Expressing the Null Mutation for the Calcium Sensing Receptor Converts a Hypercalcemic to a Hypocalcemic Hyperparathyroid Phenotype.

C. Richard*¹, P. Miao², R. Samadfam*¹, Y. Wang*¹, G. N. Hendy¹, D. Goltzman¹. ¹Dept of Medicine, Calcium Laboratory, McGill University, Royal Victoria Hospital, Montreal, PQ, Canada, ²Department of Human Anatomy, Nanjing Medical University, Nanjing, China, China.

The calcium sensing receptor (CaSR) is functional mainly in the parathyroid gland and kidney, but likely as well in other tissues such as the gastrointestinal tract and the skeleton. These tissues are also target sites for the action of 1,25-dihydroxyvitamin D [1,25(OH)₂D], Mice which are heterozygous or homozygous for the calcium sensing receptor null mutation (CaSR^+/− and CaSR^−/− respectively) develop hypercalcemia and hyperparathyroidism to a moderate or severe degree, respectively. Mice which are homozygous for targeted deletion of the 25 hydroxyvitamin-D 1α-hydroxylase [1α(OH)ase^−/−] which synthesizes 1,25(OH)₂D develop hypocalcemia, hyperparathyroidism and rickets but survive long term. To assess the interactions between the important calcium regulating molecules, CaSR and 1,25(OH)₂D, we created compound mutants of the CaSR and 1α (OH)ase mutants and assessed their growth, biochemistry and skeletal morphology. CaSR +/-;1α (OH)ase+/+ mice grew normally and exhibited normal longevity. CaSR +/ +;1α (OH)ase-/- mice developed severe rickets and osteomalacia as assessed by faxitron x-ray analysis and von Kossa staining of the skeleton but survived at least 2 months. CaSR +/ -;1α (OH)ase-/- mice developed even more severe rickets, osteomalacia and growth retardation but also survived long term. BMD, assessed by piximus, was reduced by 68% in the lumbar spine and by 49% in the femur compared to wild type mice, but only by 58% and 38% in the lumbar spine and femur respectively of the CaSR +/+;1α (OH)ase-/- mice. Although CaSR-/-;1α (OH)ase+/+ mice develop severe hypercalcemia and most die before or shortly after weaning, deletion of the 1α (OH)ase in CaSR-/-;1α (OH)ase-/- mice facilitated survival for at least 2 months although these animals exhibited the most severe growth retardation, rickets and osteomalacia of all mutants. All mutant animals showed evidence of hyperparathyroidism with CaSR+/-;1α (OH)ase+/+ and CaSR-/-;1α (OH)ase+/+ mice displaying hypercalcemia. In contrast, CaSR+/-;1α (OH)ase-/- mice and CaSR-/-;1α (OH)ase-/- mice were severely hypocalcemic (serum calcium 1.28 and 1.27mM respectively). The results indicate that the phenotypic manifestations of hyperparathyroidism may be markedly influenced by the vitamin D status of animals with CaSR mutations, confirm that elimination of hypercalcemia sustains longevity in mice homozygous for the CaSR null mutation, and suggest that mutations in the CaSR contribute to worsening of skeletal mineralization.

Disclosures: C. Richard, None.

M232

Periarticular Bone Loss in Arthritis: Role of Synovial Glucocorticoid Generation.

M. S. Cooper¹, R. Hardy*¹, E. H. Rabbitt*¹, N. J. Gittoes¹, M. Hewison², C. D. Buckley*¹, K. Raza*¹, P. M. Stewart*¹. ¹University of Birmingham, Birmingham, United Kingdom, ²Cedars-Sinai Medical Centre, Los Angeles, CA, USA.

Periarticular osteoporosis is a common feature of inflammatory arthritis. This feature is related to disease activity and is associated with uncoupling of bone resorption from formation. We previously hypothesised that local glucocorticoid generation within osteoblasts in response to inflammation may underlie this bone loss. An alternative possibility is that excess glucocorticoids are generated from other joint tissues. Primary synovial fibroblasts express the 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) enzyme that generates the active glucocorticoids Cortisol and prednisolone from their inactive counterparts cortisone and prednisone suggesting that synovium might generate glucocorticoids. We have now explored this hypothesis by characterizing glucocorticoid metabolism in synovial tissue explants from patients with rheumatoid arthritis (RA) and osteoarthritis (OA).

Glucocorticoid metabolism was examined in synovial tissue taken from subjects with OA (n=8) or RA (n=12) during orthopedic surgery using radiolabelled steroids and TLC. Immunohistochemistry was used to identify the cellular distribution of enzymes. The functional consequences of enzyme activity on IL-6 production were examined by ELISA.

All synovial biopsies had substantial capacity to activate glucocorticoids (cortisone to Cortisol and prednisone to prednisolone) which was blocked by a specific 11β-HSD1 inhibitor confirming 11β-HSD1 expression. No difference in steroid metabolism was seen between samples from RA and OA subjects. In patients with RA, synovial Cortisol generation increased with the ESR of the tissue donor (R²=0.4, p<0.05). 11β-HSD1 activity had functional consequences with cortisone able to decrease synovial IL-6 production in tissue from patients with RA or OA (31±15% for RA, 36±9% for OA, both p<0.05). Steroid inactivation was also apparent in synovium and was not blocked by inhibition of 11β-HSD1. Using immunohistochemistry 11β-HSD2 expressing cells were identified in RA synovium and expression colocalized with the monocyte marker CD68. This pattern was distinct from 11β-HSD1 which was expressed in fibroblasts.

Synovial tissue metabolises glucocorticoids with the predominant effect being glucocorticoid activation and this increases with inflammation. Endogenous glucocorticoid production in the joint is likely to regulate tissue inflammation but in excess might detrimentally impact on periarticular bone integrity. Additionally, a subset of immune cells expressing 11β-HSD2 within synovium will be resistant to Cortisol/prednisolone through steroid inactivation and this could perpetuate synovial inflammation.

Disclosures: M.S. Cooper, None.

M233

See Sunday Plenary Number S233

M234

The Effects of Glucocorticoid on BMP-2 and BMP-7 Expressions in Bone and Kidney.

L. Cui¹, L. Zhou*¹, T. Wu*². ¹Department of Pharmacology, Guangdong Medical College, Zhanjiang, Guangdong, China, ²Guangdong Key Laboratory for R & D of Natural Drugs, Guangdong Medical College, Zhanjiang, Guangdong, China.

Aims: To determine the effects of long-term over dose of glucocorticoid (GC) on the genes and proteins expressions of bone morphogenetic proteins (BMP)-2 and BMP-7 in the bone tissue and kidneys of rats, to explore the relationships between BMPs changes in kidney-blood-bone and osteoporotic, hence to evaluate the roles of BMP-2 and BMP-7 in GC-induced osteoporotic. Methods: Three months-old Sprague-Dawley male rats were divided to control, GC-treated, GC-treated plus Danshen and GC-treated plus epimedium groups. Rats were oral gavages prenisone (3.2mg/kg/d) in the morning and the other treatments in the afternoon for three months. At endpoint, all rats were sacrificed for the collections of blood, bone and kidneys. The bone histomorphometry parameters in proximal tibial metaphyseal (PTM) were measured. The contents of BMP-7 and BMP-2 in blood serum were determined. RT-PCR was applied to test gene expressions of BMP-2 and BMP-7 mRNA and Immunohistochemistry was applied to analyze the expression locations and quantities of BMP-2 and BMP-7 in kidney and femoral bone. Results: Glucocorticoid induced bone loss by decreasing 28. 96% (P<0.01) in trabecular area and 14.53% in trabecular thickness and 17.93% in trabecular number in PTM respectively, the trabecular separation increased 27.97% (P<0.05) when compared with control. Simultaneously, glucocorticoid decreased the gene and protein expressions of BMP-2 and BMP-7 in femoral bone (P<.05), however, it decreased BMP-7 expression but increased BMP-2 expression in kidney (P<0.05). Glomcrulus and collecting duct atrophy and protein cast were seen in kidneys of glucocorticoid treated rats. The contents of BMP-7 and BMP-2 in blood serum were decreased in the glucocorticoid treated rats. There was a positively correlation (P<0.05) in expressions of BMP-7 among kidney, blood and bone mass and a negatively correlation (P<.05) in expressions of BMP-2 between kidneys and bone mass. Traditional Chinese medicine Danshen and epimedium can prevented glucocorticoid induced bone loss by regulating BMP-2 and BMP-7 expressions in bone and kidneys. Conclusions: Glucocorticoid induce bone loss in rats and down-regulated the expressions of BMP-2 and BMP-7 in bone tissue, down-regulated BMP-7 but up-regulated BMP-2 expression in kidney. BMP-2 and BMP-7 may play an important role in the mechanism of glucocorticouid induced osteoporosis. Some kidneys action Traditional Chinese medicine can prevented glucocorticouid induced osteoporosis by regulating the BMPs expressions in bone and kidneys.

Disclosures: L. Cui, None.

This study received funding from: Guangdong Science & Technology Project.

M235

See Sunday Plenary Number S235

M236

A Vitamin D Deficiency in an Italian Central Town Normal Population (MIMOSA-G project).

P. De Remigis*¹, P. Ranieri*¹, A. De Remigis*¹, B. De Laurentiis*², M. Lattanzio*², L. Vianale*¹. ¹Endocrine Unit, General Hospital, Chieti, Italy, ²Endocrine Laboratory Unit, General Hospital, Chieti, Italy.

In the context of MIMOSA (Mineral Metabolism Osteoporosis in Abruzzo) project, a study was carried out about the correlation between 25-hydroxyvitamin D and PTH levels, in order to unveil vitamin-D deficiency and to schedule a prophylaxis program. 200 healthy subjects were considered; this population, belonged to a town(Guardiagrele of 9970 inhabitants), located in Abruzzo, a Central Italian Region, was selected from a randomized group of 820 subjects, who had ultrasound BMD screening, on the basis of normal t-score at QUS and ruling out other risk factors for osteoporosis, by questionnaire(this project is called MIMOSA-G, because it is restricted to total people of Guardiagrele). The population was homogeneous for environmental factors and nutritional intake, of both genders, ranged from 20 to 85 yrs. They were subdivided in four groups of 25 members, for each gender: a) ranged from 20–50 yrs b) 50–60 c) 60–70 d) over 70. The blood samples were taken in winter; 25-hydroxyvitamin D and PTH were assayed by chemiluminescent method.

In our population of healthy individuals, calcidiol levels are decreasing very significantly from 12.3 ng/ml (young people group) to 8.31 (the oldest one), There is not differences between male and females at any different age classes we considered. On the other hand, in parallel way, PTH levels are increasing significantly from 39.2 pg/ml in young people to 52.9 pg/ml in older ones, showing that a condition of secondary compensatory hyperparathyroidism is realizing by aging.

25-hydroxyvitamin D and PTH levels are negative correlated (correlation coefficient of 0.18). The threshold of 25-hydroxyvitamin D, above which the correlation becomes no significant, is 30 ng/ml (to be considered our biological reference). 50.2% of subjects show a level of 25-hydroxyvitamin D below 8 ng/ml The profile of 25-hydroxyvitamin D graphic indicates the level of a vitamin D is sharply declining in general population over 60 yrs (more evident in females, while in man the curve is smoother), putting at risk of femoral fractures people for secondary hyperparathyroidism. Vitamin D deficiency is shown in Abruzzo severe over all in aged population probably due to inadequate exposure in winter and also insufficient dietary compensation.

Disclosures: P. De Remigis, None.

M237

The Protective Effects of Mechanical Strain on Osteocyte Viability Is Mediated by the Effects of Prostaglandin on the cAMP/PKA and the β-Catenin Pathways.

Y. Kitase*, M. L. Johnson, L. F. Bonewald. Oral Biology, School of Dentistry, UMKC, Kansas City, MO, USA.

Mechanical loading has been shown to prevent or reduce osteocyte cell death or apoptosis in vitro and in vivo. Fluid flow shear stress (FFSS) has been shown to reduce osteocyte apoptosis in response to glucocorticoid (GC). As GC induced osteoporosis is the second most common form of osteoporosis, we sought to identify the molecular mechanisms responsible for the protective effect of mechanical loading. Application of FFSS (16 dynes/cm²) significantly inhibited apoptosis of osteocyte-like MLO-Y4 cells induced by dexamethasone (Dex, 10⁻⁶ M). We hypothesized that PGE₂ is a protective factor for osteocytes as osteocytes release PGE₂ within minutes in response to FFSS. The protective effect of FFSS on Dex-induced apoptosis was significantly inhibited by indomethacin (10⁻⁶ M) while exogenous PGE₂ (10⁻⁶ M) significantly prevented apoptosis induced by Dex. The use of PGE₂ receptor agonists (butaprost, PGE, alcohol, sulprostone) and antagonist (AH6809) showed that the EP2 receptor was responsible. Dex was found to inhibit EP2 receptor and COX-2 expression and PGE₂ production as determined by gene array, real-time PCR and ELISA. As the EP2 receptor is known to signal through the classical cAMP/PKA pathway, the cAMP analog, 8Br-cAMP (100 μM) was tested and found to protect against Dex-induced cell death. However, the PKA inhibitor H89 (5 μM) only partially abrogated the protective effects of PGE₂. As recent studies suggest that PGE₂ can activate the β-catenin signaling pathway in addition to the classical cAMP/PKA pathway, LiCl (10 mM) was tested and found to protect against Dex induced cell death equivalent to FFSS and PGE₂. FFSS, PGE₂ and LiCl increased phosphorylation of both GSK-3α and β. Wortmannin (1μM), a PI3K inhibitor, but not H89, reduced the phosphorylation of GSK-3. FFSS, PGE₂ and LiCl induced nuclear translocation of β-catenin. These studies provide evidence that osteocyte production of PGE₂ in response to mechanical shear stress is protective against GC induced osteocyte cell death, not only through the classical cAMP/PKA pathway, but also the P13K/GSK-3/β-catenin pathway. It will be important to determine if increased mechanical loading alone or in combination with therapeutics will reduce or prevent the detrimental effects of GC on osteocyte viability and bone integrity.

Disclosures: Y. Kitase, None.

M238

PTHrP Regulates Antimicrobial Peptide Production by Skin Keratinocytes.

L. J. Deftos¹, D. W. Burton¹, J. Schauber*², S. Tu*¹, R. A. Dorschner*², R. L. Gallo*². ¹Medicine, Veterans Administration San Diego Healthcare System and University of California, San Diego, CA, USA, ²Dermatology, Veterans Administration San Diego Healthcare System and University of California, San Diego, CA, USA.

PTHrP is expressed in normal skin keratinocytes at relatively high levels, but its function in this tissue is not well studied. Reports have demonstrated that PTHrP expression is increased in skin inflammation and that PTHrP contributes to the progression of other inflammatory diseases, such as rheumatoid and osteoarthritis. In this study, we investigated the interaction of PTHrP with antimicrobial peptides made by keratinocytes. We altered the amount of PTHrP that was expressed by an immortalized keratinocyte cell line (HaCaT) using transfection to increase expression and siRNA techniques to inhibit it. Real-time PCR was used to measure the levels of cellular antimicrobial peptides, including cathelicidin and α- and β-defensins. PTHrP 1–139, 1–141, and 1–173 transfection into HaCaT cells stimulated cathelicidin mRNA expression. While all three isoforms had a stimulatory effect, it was greatest for PTHrP 1–173 (150%), least for PTHrP 1–139 (50%), and intermediate for PTHrP 1–141 (80%), compared to vector control cells. Deletion of the 1–32 domain in the PTHrP expression plasmids prevented the increase in cathelicidin. No PTHrP effects were observed on α- or β-defensin mRNA expression. Inhibition studies using siRNA duplexes targeted to PTHrP 16–22, included in all three isoforms, demonstrated an 89% and 65% decrease in PTHrP and cathelicidin expression, respectively, compared to control (non-silencing) siRNA duplex targeted to a non-mammalian protein from Thermotoga maritimia. Our results demonstrate that PTHrP regulates the expression of cathelicidin in keratinocytes and that both the 1–32 and 140–173 amino acids of PTHrP are required for the maximal stimulatory effect on cathelicidin expression. These data illustrate the contribution of keratinocyte-derived PTHrP to the immune defense of skin. The known interactions of vitamin D with PTHrP posit a novel regulatory network also involving toll receptors for the innate immune pathway in skin and perhaps other tissues.

Disclosures: L.J. Deftos. None.

This study received funding from: Department of Veterans Affairs and N/H.

M239

See Sunday Plenary Number S239

M240

Sex Effects on PTHR1 in Non-Small Cell Lung Carcinoma.

R. H. Hastings*, A. Ko*, R. Quintana*, Y. Rascon*. VA San Diego Healthcare System, San Diego, CA, USA.

Parathyroid hormone-related protein (PTHrP) is associated with increased survival in women with non-small cell lung carcinoma but not in men (Hastings et al. Clin Cancer Res 2006; 12:499–506). The sex dependence could arise from hormonal mechanisms, since lung carcinomas express receptors for gonadal steroids and estrogen is known to upregulate the type 1 PTH receptor (PTHR1). This project tested the effects of estrogen on PTHR1 expression in orthotopic A549 lung adenocarcinomas model in female athymic mice. A549 cells express PTHrP, PTHR1 and estrogen receptor beta. Experimental animals received a subcutaneous estradiol pellet (0.72 mg/60 d to increase serum estradiol levels from control levels of <40 pmol/L, a known anomaly in athymic mice, to over 300 pmol. Immunoblots demonstrated significantly increased PTHRI protein levels in estradiol-treated lung (Figure 1, P < 0.01) at 4 wks after tumor implantation. Estrogen also increased levels in lung carcinomas compared to controls, but the differences were not significant. At this point, our working hypothesis points to pulmonary endothelial cells as the site of estradiol action in lung. PTHrP 1–34 is known to stimulate endothelial cell apoptosis and to decrease tumor angiogenesis (Bakre, Nature Med 2002; 8:995–1003). Thus, the model in Figure 2 could explain the sex-selective association between lung carcinoma PTHrP expression and prolonged survival in female patients. Tumor-derived PTHrP would act on endothelial cells to inhibit angiogenesis, as a result slowing tumor growth and leading to longer survival. The effect would manifest predominantly in females because of estrogen-dependent increases in endothelial PTHRI. Studies are underway to test these hypotheses.

Disclosures: R.H. Hastings, None.

This study received funding from: Flight Attendants Medical Research Foundation, Dept. of Veterans Affairs.

M241

See Sunday Plenary Number S241

M242

Teriparatide [rhPTH(1–34)) Speeds Fracture Healing and Initiates Healing of Non-unions in Humans.

E. N. Schwartz, D. M. Steinberg*. Northern California Institute for Bone Health, Inc., Oakland, CA, USA.

Teriparatide (TPTD) stimulates osteoblast function. Osteoblast function is a key to fracture healing. Numerous studies have shown increased fracture healing with TPTD in a variety of experimental models. In these studies, intermittent low dose TPTD increased callus formation, increased production of bone matrix proteins and increased other potential mechanisms of fracture healing.

There is anecdotal information that TPTD may enhance fracture healing in humans. Accordingly, a variety of acute traumatic fractures (n = 20) of multiple body sites including a stress fracture of the femoral shaft, a fracture of a metacarpal, a fracture of the ankle and a fracture of the thumb all healed within a window of 3–5 weeks when healing of each fracture was estimated to take 6–8 weeks. Fracture healing was assessed by x-ray and CT scanning.

Additionally, these same mechanisms of enhanced fracture healing might also be useful in cases of fracture non-union. Accordingly, a variety of patients with different sites and lengths of time of non-union (n = 10) were treated by intermittent low dose TPTD. These included sites such as navicular bone in the ankle (2 fractures), the ischium, the distal tibial shaft, and other sites. About 50% of the non-unions had been previously operated on (ORIF). About 50% of the treated TPTD non-unions went on to partial or complete fracture healing based on x-ray, CT scanning or operative findings.

The risk factors for healing and non-healing of non-unions are not completely understood at this time.

The cases discussed indicate a possible role for TPTD in acute and stress fracture healing and a possible role in healing of certain fracture non-unions in humans.

Disclosures: E.N. Schwartz, Merck Pharmaceuticals 2, 5, 8; Proctor & Gamble 2, 5; Novartis 2, 5; Amgen 2, 5.

M243

The Effect of Estrogens on Bone Marrow Adipogenesis: A New Role for Sirtuins in Age-Related Bone Loss.

A. Elbaz¹, P. Rivas¹, G. Duque². ¹Lady Davis Institute for Medical Research, Montreal, PQ, Canada, ²Medicine/Geriatrics, McGill University, Montreal, PQ, Canada.

Estrogens (E₂) are known to increase bone mass through the regulation of osteoclastic activity. In the case of age-related bone loss, the role of E₂ remains unclear since the predominant feature of this type of bone loss is not a higher level of osteoclastic activity but the infiltration of bone marrow by fat. In this study, we investigated whether E₂ have an effect on bone marrow adipogenesis and if this potential effect is exerted through the regulation of Sirtl a recently reported suppressor of PPARγ2 in visceral and subcutaneous fat. We hypothesize that E₂ have an inhibitory effect on bone marrow adipogenesis through the induction of Sirtl expression. To examine this hypothesis, young (5 month) and aged (24 month) female C57BI/6J mice (n=10 per-group) were either gonadally-intact, ovariectomized (OVX) or OVX and E₂ (estratien-3,17-_-diol Sterloids Inc., Witten, NH, USA) treated (OVX/E₂) to produce mean circulating levels of E₂ of 12 to 18 ng/ml. After three weeks of treatment, mice were sacrificed and both side tibia and femur dissected and fixed. Hematoxylin/Eosin (H/E), Toluidine Blue and oil red-O stainings were performed. Additionally, expression of Sirtl was determined by immunofluorescence. Post-mortem uterus weight was taken as an index of the effect of estrogen deprivation (OVX) or supplementation (E₂ treatment) and was significantly reduced (P < 0.05) in OVX group (44 ± 1 mg, mean ± s.e.m) compared to intact (76 ± 2 mg) or OVX/E₂ mice (64 ± 3 mg). We found significant differences in fat volume between old OVX and old OVX/E₂ with a significant reduction seen in the treated mice (p<0.001). In contrast, no differences were found between the young groups. Additionally, we found that the reduction in bone marrow fat volume in OVX/E₂ mice correlated with high levels of Sirt1 expression and with reduced levels of PPARγ2 (p<0.01) within the bone marrow. In summary, we have assessed the effect of estrogen on bone marrow adipogenesis in a model of young and old OVX mice. We have found that estrogens only inhibited bone marrow fat infiltration in old mice and that this effect is associated with higher levels of Sirtl expression. This is an initial step in the understanding of the effect of estrogens on bone marrow adipogenesis through the activation of Sirtuins.

Disclosures: A. Elbaz, None.

This study received funding from: Quebec Network on Aging Research-Nutrition Axis.

M244

See Sunday Plenary Number S244

M245

Genetic Determinants of Serum Levels of Free Estradiol and Free Testosterone in Young Adult Swedish Men — The GOOD Study.

A. Eriksson¹, M. Lorentzon¹, S. Nilsson*², L. Vandenput*¹, F. Labrie*³, C. Ohlsson¹. ¹Center for Bone Research at the Sahlgrenska Academy, Göteborg University, Göteborg, Sweden, ²Division of Mathematical Statistics, Chalmers University of Technology, Göteborg, Sweden, ³Laboratory of Molecular Endocrinology and Oncology, Laval University, Quebec, PQ, Canada.

Sex hormones are important for skeletal growth and maintenance. Genetic factors are important regulators of serum sex hormone levels. However, the magnitude of contribution to this regulation from individual genes is not well known. The aim of this study was to investigate which sex hormone-related genes are the most important ones for regulation of calculated free serum estradiol (E2) and testosterone (T) levels, as a result of genetic variations, in young adult men. We used the Gothenburg Osteoporosis and Obesity Determinants (GOOD) study, a population-based study consisting of 1068 young men, age 18.9±0.6 yrs (mean±SD). E2 and T in serum were analyzed using gas chromatography/mass spectrometry (GC-MS). Serum SHBG levels were measured using IRMA. Free E2 (FE2) and free T (FT) were calculated using a method described by Vermeulen et al (1), taking the concentrations of T, E2 and SHBG into account, and assuming an albumin concentration of 43 g/l. Genomic DNA was extracted from whole blood. We selected 51 candidate genes coding for hypothalamic and pituitary hormones regulating sex steroid secretion, enzymes involved in synthesis, metabolism or conversion of sex steroids, sex steroid receptors and carrier proteins. Single nucleotide polymorphisms (SNPs) with a minor allele frequency ≥5% were selected from HapMapData Rel 21a/phasell using a pairwise correlation method (r² ≥0.80) including 10 kb upstream and 5 kb downstream of each gene. Genotyping was performed on the Illumina Bead Station Platform and was successful in ≥ 95% of the study subjects in 626 of 765 selected SNP-s (=81.8%). For FE2 the most strongly correlated SNP was found in CYP19. For FT the corresponding SNP was found in the FSHR (follicle stimulating hormone receptor) gene. An SNP in LHCGR (luteinizing hormone/choriogonadotropin receptor) was the second most strongly correlated SNP for both FE2 and FT2. For each of these genes linear regression with all genotyped SNP-s included were performed. Backward elimination was used to build the best models. For FE2 the best models of SNP-s in CYP19 and LHCGR explained 2.7% and 2.9% respectively of variation in serum levels (p<0.001). For FT SNP-s in FSHR and LHCGR explained 4.0% and 2.7% respectively (p<0.001). In conclusion, genetic variations in CYP19 and FSHR are important determinants of FE2 and FT respectively, in young Swedish men. Genetic variations in LHCGR are important determinants of both FE2 and FT.

(1) Vermeulen A, et al. 1999. J Clin Endocrinol Metab 84:3666–3672

Disclosures: A. Eriksson. None.

M246

See Sunday Plenary Number S246

M247

Equol Producers After Soy Challenge Is Related to High Bone Turnover in Premenopausal Women.

H. Kwak*¹, M. Kim*², C. Hwang*¹, S. Lee*¹, M. Jung*¹, Y. Kang*¹, C. Yim*¹, S. Park*¹, H. Yoon¹, K. Han*¹. ¹Laboratory of Endocrinology, Cheil Hospital and Women's Healthcare Center, Kwandong University College of Medicine, Seoul, Republic of Korea, ²Department of Internal Medicine, Marynoll General Hospital, Inje University School of Medicine, Busan, Republic of Korea.

Soybean isoflavones, the main phyto-estrogen, have been known to be able to modulate the action of endogenous estrogens and metabolism of phyto-estrogens might be affected by the menstrual cycles in premenopausal women. There have been few studies about metabolism of soy isoflavones and metabolites activity on bone in premenopausal women, therefore, we investigated the effect of isoflavones on bone turnover markers after challenge of soy isoflavones in premenopausal women. Premenopausal women were randomly assigned to receive either a 120mg soy extract-containing capsule (isoflavone group) or a lactose-containing capsule as placebo once a day for 3 menstrual cycles. Food records, blood samples, and 24hr urine collections were obtained from each subject at the beginning and end of the study. The isoflavone group showed increased concentrations of urinary isoflavone metabolites (equol, diadzein, genistein and dihydrodaidzein) after administration of soy extract-containing capsules compared to the placebo group. A bone formation marker, serum osteocalcin (OC) was increased after treatment in the isoflavone group compared to the placebo group, but the change of a bone resorption marker, urine deoxypyridinoline (DPD) after treatment was not different between two groups. As urinary isoflavone metabolites showed large inter-individual variation, we performed subgroup analyses according to previous studies. Equol is one of the isoflavone metabolites known to have high activity as diadzein or genistein, so that we divided the isoflavone group into the non-equol producer group (<1000nmol/l of urinary equol) and the equol producer group (>1000nmol/l of urinary equol). Serum OC was significantly increased after treatment in the equol producer group compared to the placebo and non-equal producer groups. And levels of urine DPD didn't change after soy challenge in the equal producer group, however, decreased in the placebo and non-equol producer groups. In conclusion, these data suggested that some premenopausal women who have ability to produce high amount of equol from soy isoflavones would result in relatively enhanced bone turner rate after soy isoflavone intake and high-dose equol might have anti-estrogenic effects on bone metabolism in premenopausal women.

Disclosures: C. Hwang, None.

M248

See Sunday Plenary Number S248

M249

Activation of Farnesoid × Receptor in Breast Cancer Cell Lines by Bone-Derived Lipid.

F. Journe¹, C. Chaboteaux*¹, V. Durbecq*¹, D. Larsimont*¹, G. Laurent*², J. J. Body¹. ¹Institut Bordet, Brussels, Belgium, ²Université de Mons-Hainaut, Mons, Belgium.

The skeleton is the most common site for metastasis of breast cancer. Cancer cells enhance the destruction of the bone matrix and increase the release of many growth factors and cytokines that stimulate metastatic cells proliferation. This vicious cycle is the key mechanism underlying the development of bone metastases. Recent data indicate that bone tissue also contains bile acids (BAs), which are accumulated from serum and which could stimulate the migration of breast tumor cells towards bone (Silva, J Lipid Res 2006). BAs are physiological ligands for farnesoid × receptor (FXR). FXR is a nuclear receptor involved in the regulation of BA metabolism in liver and intestine. In this context, it has been shown to heterodimerize with the retinoid × receptor (RXR). We have recently demonstrated the presence of FXR in human breast cancer specimens. In postmenopausal breast cancer patients, we found a correlation between FXR expression and that of estrogen receptor (ER) and proliferation markers. In the present study, we determined the effects of FXR activation by chenodeoxycholic acid (CDCA) in breast cancer cells, and examined its expression in bone metastasis specimens. We found that 50 μM CDCA stimulated the proliferation of ER(+) MCF-7 cells cultured in steroid-free medium (EC50=15μM), while it had no effect on ER(-) MDA-MB-231 cells. Antiestrogens completely suppressed the mitogenic effect of CDCA in MCF-7 cells. Moreover, CDCA downregulated ER by 40% and stimulated the transactivation of progesterone receptor (PgR) by 316% in MCF-7 cells. Antiestrogens completely abolished PgR induction, and FXR gene silencing (siRNA) inhibited CDCA-stimulated cell proliferation as well as PgR induction. CDCA did not compete with [3H]-17b-estradiol for binding to human recombinant ER immobilized on hydroxylapatite gel. In addition, FXR immunoprecipitation and subsequent ER demonstration by Western blotting revealed physical interaction between both receptors within 30 min. Finally, bone metastases from 32 breast cancer patients were examined for FXR expression by immunohistochemistry. FXR was expressed in 58% of bone metastasis specimens. In conclusion, our data reveal a positive crosstalk between FXR and ER, which accounts for FXR-mediated ER activation and subsequent mitogenic effects of FXR agonists in MCF-7 cells. Therefore, because of significant FXR and ER expressions in bone metastases of breast cancer, bone-derived lipids might contribute to the vicious cycle of tumor-induced osteolysis, especially in postmenopausal or estrogen-depleted cancer patients.

Disclosures: F. Journe, None.

M250

See Sunday Plenary Number S2S0

M251

Degeneration of Intervertebral Disc in Ovariectomized Rats.

M. Kato¹, N. Fujita*¹, N. Hosogane*¹, H. Takaishi*¹, J. Takito*¹, T. Kikuchi*², M. Matsumoto*³, K. Chiba*¹, Y. Toyama*¹. ¹Orthopaedic Surgery, Keio University School of Medicine, Tokyo, Japan, ²National Hospital Organization Murayama Medical Center, Tokyo, Japan, ³Department of Musculoskeletal Reconstruction and Regeneration Surgery, Keio University School of Medicine, Tokyo, Japan.

The intervertebral discs, which consists of water-rich nucleus pulposus and lamellar annulus fibrosus, degenerate with age and become source of low back pain. Estrogen deficiency in postmenopausal state closely associates with osteoporosis and may induce vertebral fractures. The purpose of this study is to investigate whether and how estrogen is involved in the degenerative process of the intervertebral discs. Eight-week-old female Wistar rats were sham operated (Sham) or ovariectomized (OVX). We collected the nucleus pulposus (NP) and the annulus fibrosus (AF) from the intervertebral discs at 1 week, 2 weeks, 3 months, 7 months, and 1 year after the operation. Histological observations at 7 months after the surgery indicated that the cell number of the NP and AF decreased significantly in OVX rats than that in Sham rats. Lamellar structure of the AF was disorganized in OVX rats. T2 weighted MRI images at 1 year after the surgery exhibited lower signal intensity of the NP of OVX rats than that of Sham rats. RT-PCR detected the expression of estrogen receptors (ER)α and ERβ, in both the NP and AF. Immunofluorescence using confocal microscope revealed the nuclear localization of ERα and ERβ in AF cells. In AF cells, nuclear localization of exogenously transfected DsRed-tagged ERα tended to aggregate under estradiol treatment and disperse under estrogen antagonist, IC1182780, administration. Real-time RT-PCR indicated that the expression level of type II collagen of the AF and NP in OVX rats was approximately 0.4 points lower (P<0.05) than that in Sham rats at 2 weeks and 7 months after the surgery. Decreased type II collagen mRNA expression and histological evidence of degeneration in the intervertebral discs of OVX rats suggests that menopause is one of the factors inducing disc degeneration and that estrogen may be involved in intervertebral disc homeostasis.

Disclosures: M. Kato, None.

M252

Co-Chaperone Potentiation of Vitamin D Receptor-Mediated Transactivation: A Role for Bag-1 as an Intracellular Binding Protein for 1,25-Dihydroxyvitamin D₃.

R. F. Chun, M. Gacad*, L. Nguyen*, M. Hewison, J. S. Adams. Endocrinology, Diabetes and Metabolism, Cedars-Sinai Medical Center, Los Angeles, CA, USA.

In recent studies we have proposed that mitochondrial metabolism and nuclear signaling of vitamin D metabolites are not simply dependent on intercompartmental concentration gradients of free hormone in the target cell, but instead involve protein chaperones that interact with and traffic vitamin D metabolites to specific intracellular destinations. For example, the constitutively-expressed member of the heat shock protein-70 family (hsc70) is able to bind both 25-hydroxyvitamin D, (25D₃) and 1,25-dihydroxyvitamin D₃ (1,25D₃), potentiating 25D₃ metabolism and 1,25D₃-mediated gene transactivation. Hsc70 also recruits and interacts with the co-chaperone protein BAG-1. Competitive ligand binding assays showed that, like hsc70, recombinant BAG-1 is able to bind 25D, (Kd=0.57 nM) and 1,25D₃ (Kd=0.19 nM) with high affinity. To investigate the functional significance of this, we transiently overexpressed the naturally-occurring short (S), medium (M) and long (L) variants of BAG-1 in human, 1α-hydroxylase (CYP27b1)-expressing kidney HKC-8 cells stably transfected with a 1,25D₃-responsive 24-hydroxylase (CYP24) promoter-reporter construct. Physiologically relevant concentrations of 25D₃ (200 nM) and 1,25D₃ (5 nM) were able to induce CYP24 promoter activity in these cells. CYP24 reporter activity was amplified 1.5–2.0-fold following over-expression of BAG-1S, L or M. By contrast, BAG-1 isoforms had no effect on metabolism of 25D₃ to either 1,25D₃ or 24,25D₃ by HKC-8 cells, indicating that the induction of CYP24 promoter activity by 25D₃ and potentiation of this effect by BAG-1 involves intracrine conversion to 1,25D₃. Further studies showed that transfection of HKC-8 cells with cDNA for BAG-IS in which the hsc70-binding domain was mutated resulted in decreased binding of 1,25D₃ leading to a concomitant loss of potentiation of CYP24 promoter transactivation. Similar loss or promoter-reporter activity in the presence of mutant BAG-1S was not observed for 25D₃ which retained binding to this form of BAG-1S. These data 1] highlight a novel role for BAG-1 as an intracellular binding protein for 1,25D₃ and 2] suggest that BAG-1 is able to potentiate vitamin D receptor (VDR)-mediated transactivation by acting as a nuclear chaperone for exogenous or endogenously synthesized 1,25D₃. We postulate that hsc70 and BAG-1 interact to provide a chaperone complex for 1,25D₃, but not 25D₃, thereby providing a novel mechanism for fine tuning the intracellular actions of the hormone.

Disclosures: R.F. Chun, None.

M253

See Sunday Plenary Number S253

M254

Expression of the 1,25-Dihydroxyvitamin D₃ Receptor in Multiple Tissues in Adult and Developing Danio rerio (Zebrafish).

T. Craig*, R. Kumar. Nephrology Research, Mayo Clinic Rochester, Rochester, MN, USA.

The receptor for 1,25-dihydroxyvitamin D₃ (VDR) mediates many of the functions of 1,25-dihydroxyvitamin D₃ in cellular growth and development and mineral homeostasis. In order to better understand the functions of VDR, we utilized zebrafish (Danio rerio) as a model organism in which investigations of both adults and developing embryos are facile compared to larger vertebrates. Immunohistochemistry was carried out in decalcified male and female zebrafish adults (5 months age) and in developing fertilized embryos at 72 hours using antibodies against VDR. Mounted sections from formalin-fixed paraffin-embedded tissue blocks were treated with a polyclonal antibody raised in rabbits against the VDR or pre-immune rabbit control serum. Anti-rabbit HRP secondary antibody/diaminobenzidine (DAB) staining, with hematoxylin, as a counter-stain was used to visualize the primary antibody bound to the receptor. We observed intense staining for the VDR in epithelial cells of the intestine, oropharynx, cells covering gill lamellae, osteoblasts, osteocytes, pharyngeal teeth, kidney tubules (but not glomeruli), rods and cones of the retina, spinal cord, and epithelia of the olfactory organ. Less intense staining was observed in the ovaries and testes, with large areas of testes without immunostaining for the receptor.

We conclude that the VDR is expressed in calcium and phosphate transporting epithelia of the intestine, gills, and kidney and in osteoblasts, teeth, and neural cells. The use of zebrafish as a model may allow performance of experiments specific to the VDR that are difficult to carry out in other vertebrates.

Disclosures: T. Craig, None.

M255

See Sunday Plenary Number S255

M256

A Significant Higher Risk for Fall-Associated Fractures with a Creatinine Clearance Below 65.

L. C. Dukas*¹, E. Schacht², M. Runge*³. ¹Acute Geriatric University Clinic, Kantonsspital Basel, Basel, Switzerland, ²Zurich Osteoporosis Research Group ZORG, Zurich, Switzerland, ³Center for Muscle and Bone Research, Aerpah Klinikum, Esslingen, Germany.

A Creatinine clearance of <65ml/min. is associated with decreasing balance and muscle power and an increased risk for falls. In this study we investigated if a Creatinine clearance of <65ml/min. is also associated with a higher fracture incidence. In a cross-sectional study in 1781 German men and women treated for osteoporosis, we assessed the association between the Creatinine clearance and the risk for fractures within the last 12 months. The Creatinine clearance was calculated using the established Cockcroft-Gault formula. Results are from spearman correlation analyses and logistic regression analyses. The p-values are two-sided. 1410 women and 371 men aged 60 years and older with a mean BMI of 26kg/m2 and a mean Creatinine clearance of 60.2ml/min. participated in this study. A Creatinine clearance of <65ml/min. was significantly associated with a 44% higher fall associated fracture incidence compared to a Creatinine clearance of >65ml/min. Participants with a Creatinine clearance of <65ml/min. had significantly more vertebral fractures (P=0.008), hip fractures (p=.002) and fractures of the radius (p=.002) but not significantly more fractures in other localization (p= 0.08), compared to participants with a Creatinine clearance of >65ml/min. We found no association between the Creatinine clearance and non-fall associated fractures (p=.49). We are the first to describe, that patients with a low Creatinine clearance of <65ml/min have a significant higher increased risk for falls and a higher increased risk for fall associated fractures, compared to patients with a Creatinine clearance of >65ml/min. Since the higher risk for falls, associated with a Creatinine clearance of <65ml/min., is due to decreasing calcitriol serum levels, we hypothesize that also the higher fall-associated fracture incidence observed with a Creatinine Clearance of <65ml/min., is due to decreasing calcitriol serum levels.

Disclosures: L.C. Dukas, Teva Pharmaceutical Industries 2.

This study received funding from: TEVA Pharmaceutical Industries.

M257

Examination of Human Retinoid × Receptor alpha Phosphorylation at Serine 260 Using Specific Antibodies in Cancer Cell Lines and Tumor Tissues.

D. C. Huang, X. F. Yang*, L. Nguyen-Yamamoto, R. Kremer. Department of Medicine, Royal Victoria Hospital and McGill University, Montreal, PQ, Canada.

The retinoid × receptor alpha (RXRα) is a member of the nuclear receptor superfamily which regulates transcription of target genes through its heterodimerization with several partners including the vitamin D receptor. In previous studies, we have shown that phosphorylation of serine 260 of the human RXRα disrupts signaling and induces resistance to ligand activation of several partners of RXRα. Here we report the development of novel polyclonal antibodies which specifically react with phosphoserine residue 260 of human RXRα (PS260). Antiserum raised against PS260 or non-phosphorylated human RXRα S260 were purified with PS260 or S260 affinity columns. Specificity of antibodies against PS260 was examined by ELISA and demonstrated selective reactivity with PS260 peptide (Figure). We next examined hRXRα phosphorylation at serine 260 in several cell lines in which MAPKinase/Erk is activated prior to and following treatment with the MAPKinase inhibitorU0126 and showed a strong decrease in hRXRα serine 260 phosphorylation. We next examined human tissues from breast, colon and lung cancers by Western blot analysis using PS260, S260 and ERK antibodies. A strong correlation was observed between ERK expression and hRXRα phosphorylation at serine 260. Furthermore, a positive correlation was observed between tumor staging and hRXRα phosphorylation. Overall, our data indicate that antibodies specifically raised against phosphorylated hRXRα at serine 260 may be useful indicators of tumor progression of several types of cancers.

Disclosures: D.C. Huang, None.

M258

Role of TRPV 6 and β-Glucuronidase in 1,25(OH)2D3- and PTH-Stimulated Calcium Uptake in Intestinal Epithelial Cells.

R. C. Khanal*, N. M. Smith*, T. M. Sterling*, S. Tunsophon*, I. Nemere. Nutrition and Food Sciences, Utah State University, Logan, UT, USA.

We have found that 1,25(OH)₂D₃-stimulated ⁴⁵Ca uptake can be demonstrated in isolated intestinal cells of adult chickens, and that the response is mediated by the 1,25D₃-MARRS receptor, but not the VDR, as judged by preincubation with the appropriate antibodies. Such cells retain hormone-stimulated PKA, believed to mediate ⁴⁵Ca uptake, but lack steroid-stimulated PKC, believed to mediate efflux. PKC mediated efflux is supported by the observation that in freshly isolated cells from young chicks, 1,25(OH)₂D₃-stimulated ⁴⁵Ca uptake can only be demonstrated in cells pretreated with calphostin C, since without the PKC inhibitor, steroid-stimulated uptake is matched by efflux. Moreover, treatment of such cells with phorbol ester rapidly stimulated ⁴⁵Ca efflux. We have also discovered that cultured intestinal cells exhibited hormone stimulated PKC activity after 50 h, but it was diminished by 64 h, and lost by 72 h. Using the 72 h cultured cell model system, we have verified that 300 pM 1,25(OH)₂D₃ stimulated ⁴⁵Ca uptake, and that this response is inhibited by pretreatment with Ab099 against the receptor. To further study the mechanism of calcium uptake, we found that 1,25(OH)₂D₃ also stimulated the release of β-glucuronidase, a lysosomal marker enzyme and activator of TRPV calcium transporters. Moreover, treatment of isolated enterocytes with β-glucuronidase stimulated ⁴⁵Ca uptake in the absence of hormone. To determine whether the PKA signal transduction pathway is responsible for exocytosis of β-glucuronidase, we treated cells with PTH-known to activate PKA but not PKC in the chick model system-forskolin, or phorbol ester. PTH and forskolin-both of which stimulate ⁴⁵Ca uptake-were each found to stimulate β-glucuronidase release, while phorbol ester had no effect. Finally, using siRNA to either TRPV 6 or β-glucuronidase abolished 1,25(OH)₂D₃-stimulated ⁴⁵Ca uptake, relative to controls transfected with scrambled siRNA.

Disclosures: R.C. Khanal, None.

M259

See Sunday Plenary Number S259

M260

Derepression of CYP27B1 Gene Expression Mediates DNA-Demethylation.

M. Kim*, I. Takada*, K. Takeyama*, S. Kato. Institute of Molecular and Cellular Biosciences, Tokyo, Japan.

25(OH)D3 1α-hydroxylase (CYP27B1) gene is a key enzyme in vitaminD synthesis, hydoxylating 25(OH)₂D₃ to the active form of vitaminD, 1α,25(OH)₂D₃. 1α,25(OH)₂D₃ negatively regulates gene expression through Vitamin D receptor(VDR) binding to promoter, while PKA signaling activated by parathyroid hormone induced gene expression. The transactivation function by VDIR, a bHLH transcriptional factor recognizing negative VDRE in CYP27B1 promoter(1αnVDRE), was found to be suppressed by liganded VDR/HDAC co-repressor complex(EMBO J. 23, 1598, 2004). We further reported that Dnmts interacts with VDIR /VDR/HDAC co-repressor complex, 1α,25(OH)₂D₃-dependent transrepression of CYP27B1 gene links repressive histone modification with epigenetic DNA methylation.

Now we reports that DNA methylation and demethylation have a critical role of CYP27B1 gene regulation. ChIP assay showed that Dnmt and HDAC sequentially interacts with 1αnVDRE, and then sequential changes in DNA methylation pattern were occurred. However, DNA methylation status couldn't induced of heterochromatin formation of CYP27B1 gene, instead that, DNA demethylation occurred on CYP27B1 promoter by depleting of 1α,25(OH)₂D₃. Furthermore, demethylation was increased when activates PKA signaling under cAMP pathway. Demethylation was observed later for one hour since 1α,25(OH)₂D₃ is depleted. These changes occured in the absence of cell growth or cell division. Furthermore, demethylation was inhibited by a treatment with actinomycin, a transcription inhibitor. These results suggest that this demethylation links to transcriptional mechanism. Thus, we propose the presence of demethylase enzyme that removes 1α,25(OH)₂D₃-induced DNA methylation.

Disclosures: M. Kim, None.

This study received funding from: ERATO, Japan Science and Technology Agency.

M261

Personal Ultraviolet Exposure and Vitamin D Synthesis.

M. G. Kimlin, A. Brodie, C. A. Lang. Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Australia.

The association between exposure to ultraviolet radiation and the synthesis of Vitamin D [25(OH)D] is an area that is under increasing scrutiny. It is currently assumed that incidental UV exposure, particularly in a sunny climate should provide adequate vitamin D status for the population.

This research was undertaken to test this assumption among healthy free-living adults aged 19 to 82 years, in southeast Queensland, Australia (27°S), during late February/early March 2007 (Australian summer). 42 adults (17 males, median age 43 years) participated in this project, by having a blood sample taken to assess baseline serum 25(OH)D status and answering a self-reported questionnaire which sought demographic data and information about sun exposure. Participants were distributed with UV dosimeters to assess personal sun exposure. A second blood sample at 48-hour post-baseline blood, was collected along with the used UV dosimeters. This research was approved by Queensland University of Technology Human Research Ethics Committee and conducted under the guidelines of the Declaration of Helsinki.

The mean blood serum 25(OH)D at baseline was 75nm/L (min., 16, max., 160 nm/L) and 48 hours post baseline was 79nm/L (min., 21, max., 183 nm/L). The median personal UV exposure in the first 24 hours (as measured with the UV dosimeters) was 1.05 MED (minimal erythemal dose) with a range of 0 MED to 15.8 MED and in the final 24 hours the median personal UV exposure was 1.92 MED with a range of 0.2 MED to 7.4 MED. No significant correlation was found between personal UV exposure over the 48 hour period and change in 25(OH)D status between baseline and final collection. Additionally, self reported time in the sun (during this 48 hour period) was not related to change in 25(OH)D status. No relationships were also found between sunscreen use, age or gender. These results suggest that short term sun exposure does not impact on the 25(OH)D status, even though high exposures were of such intensity to promote the production of 25(OH)D.

Disclosures: M.G. Kimlin, None.

This study received funding from: Institute of Health and Biomedical Innovation.

M262

See Sunday Plenary Number S265

M263

Seasonal Skin Colour Variations and the Impact on Vitamin D Status.

C. A. Lang¹, A. Brodie¹, S. Harrison*², M. G. Kimlin¹. ¹Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Australia, ²Skin Cancer Research Group, James Cook University, Townsville, Australia.

The influence of sunlight exposure on Vitamin D [25(OH)D] status is an intriguing and complex question. The current theory is that incidental UV exposure, particularly in a sunny climate should provide adequate 25(OH)D, however, an individual's characteristics, such as skin colour also contributes to an individual's potential to synthesize 25(OH)D. The aim of this research was to investigate the relationship between skin colour and 25(OH)D status of healthy adults in the 2007 summer and the 2006 winter in Queensland, Australia (27°S). A sample of blood was collected to assess 25(OH)D status at each time point. Skin colour was determined using a Minolta 2500D spectrophotometer recording spectral skin reflectance on the dorsum of the hand, forehead and upper inner arm. Melanin Density (MD) was calculated using the formula described by Dwyer et al. This research was approved by Queensland University of Technology Human Research Ethics Committee and conducted under the guidelines of the Declaration of Helsinki. Sixty participants (18m / 420 with a median age 53 years (18 to 87 years) completed both summer and winter cross-sectional surveys. 53% described themselves as having a fair complexion, 25% medium and 18% an olive/dark complexion. There were 60% of the sample with an adequate (>50nm/l) 25(OH)D level in winter compared with 86.7% in summer (p=0.005). Vitamin D levels in winter (median 53.5 nm/l, interquartile range (IQR) 40.3nm/l) were significantly lower (z = −4.28, p<0.001) than summer (median 72.0 nm/l, IQR 38.8 nm/l). There was a median seasonal difference in 25(OH)D of 16.0 nm/l, with a wide range (-43.0 to 69 nm/l) that was not associated with age, gender, occupation or self reported skin colour. Median MD levels of a high UV exposure site (forehead) were significantly lower (z = −3.59, p<0.001) in winter (median 2.55 (IQR 1.74) than summer (median 3.02, IQR 1.61). There was a significant inverse correlation between the winter levels of forehead MD and 25(OH)D status (r=-0.353, p=0.006) and no correlation between the summer forehead MD and 25(OH)D (r = 0.012, p=0.927).

These results indicate that seasonal variations in skin colour, due to seasonal exposure, cannot be used as a crude indicator for 25(OH)D status. Why the forehead MD was correlated in winter but not summer remains unanswered. Further research is required into the interactions between the solar UV environment and Vitamin D synthesis, particular with respect to seasonal variability of skin colour.

Disclosures: C.A. Lang. None.

This study received funding from: Institute of Health and Biomedical Innovation.

M264

Vitamin D₂ Supplementation Suppresses Endogenous Levels of 25-Hydroxyvitamin D_3; Clinical Application of A New, Direct Immunoassay for the Specific Detection of 25-Hydroxyvitamin D₃.

B. Li*¹, I. Byrijalsen¹, C. Christiansen¹, P. Glendenning*², D. Henriksen*³, S. Vasikaran*², P. Qvist¹. ¹Nordic Bioscience A/S, Herlev, Denmark, ²Department of Core Clinical Pathology & Biochemistry, Royal Perth Hospital, Perth, Australia, ³Sanos Bioscience A/S, Roedovre, Denmark.

The nutritional vitamin D status is assessed on the basis of 25-hydroxyvitamin D (25(OH)D) levels in circulation, however there is no consensus on the relative potency of the two main vitamin D forms, D₃ and D₂. Therefore it is important to have available specific data for the endogenous form 25(OH)D₃ as well as for the corresponding D₂ forms. The present study was performed to determine the circulating levels of 25(OH)D₃ during vitamin D₂ and D₃ supplementation, respectively, using a new, direct immunoassay specific for 25-hydroxyvitamin D₃.

Serum samples were obtained at baseline and three months after supplementation with vitamin D₃ (39 participants, 400–800 IU/day) and vitamin D₂ (45 participants, 1000 IU/day), respectively. 25(OH)D₃ levels were determined by a new direct, vitamin D₃ specific ELISA based on polyclonal antibodies with <1% cross-reactivity to 25(OH)D₂ (Nordic Bioscience Diagnostics A/S, Herlev, Denmark). The direct 25(OH)D₃ ELISA demonstrated excellent correlation (r=0.95) to 25(OH)D₃ levels determined by HPLC through the Vitamin D External Quality Assessment Scheme (DEQAS, UK).

In the vitamin D₃ treated group, the relative levels of 25(OH)D₃ increase 21% (post vs pre treatment in individuals) after 3 months treatment (P<0.01). In contrast, vitamin D₂ supplementation induced 15% reduction (post vs pre treatment in individuals) compared to pre-treatment (P<0.01). The data suggest, that the decrease of 25(OH)D₃ level in vitamin D₂ group could be caused by the vitamin D₂ supplementation, which could give rise to competition between vitamin D₂ and vitamin D₃ for the substrate of vitamin D 25-hydroxylase.

Further studies are needed to confirm the suppression of endogenous vitamin D₃ levels during vitamin D₂ supplementation and its clinical relevance.

Disclosures: B. Li, Nordic Bioscience A/S 3.

M265

See Sunday Plenary Number S265

M266

Fetal Hypervitaminosis D Results in Neonatal Lethality.

L. Lieben*, R. Van Looveren*, R. Bouillon, G. Carmeliet. Laboratory of Experimental Medicine & Endocrinology, K.U. Leuven, Leuven, Belgium.

1,25(OH)₂vitaminD₃ [1,25(OH)₂D₃] is the major regulator of calcium homeostasis but is dispensable for fetal bone development. Hypervitaminosis D is deleterious during adult life but its effect on embryonic development is still enigmatic. To this end, vitamin D receptor deficient (VDR^−/−) females, showing highly elevated serum 1,25(OH)₂D₃ levels (p<0.001 vs. VDR^+/+ mothers) were mated with VDR^+/− males generating offspring that either can (VDR^+/−) or cannot respond (VDR^−/−) to possible high 1,25(OH)₂D₃ levels. VDR^−/− mice conceived less frequently, but both genotypes were bom in the anticipated Mendelian ratio (47% VDR^+/− and 53% VDR^−/− pups). Although genotypes were indistinguishable in appearance and weight at birth, 97% of VDR^+/− pups died immediately thereafter. Further analyses were therefore performed at embryonic day 18.

No manifest difference in serum calcium levels was observed between genotypes. Surprisingly, bone mass was decreased in VDR^+/− embryos, whereas it was normal in VDR^−/− embryos. More specifically, femoral calcium content was decreased by 30% in VDR^+/− fetuses as compared to VDR^−/− littermates and to VDR^+/+ embryos of VDR^+/+ mother (p<0.001). This is primarily due to reduced cortical bone volume as shown by histomorphometry (-50% vs. VDR^+/+ fetuses of VDR^+/+ mother; p<0.001). Transplacental calcium transport was significantly decreased in VDR^+/− fetuses (-20%; p<0.05), although no difference was found in the placental TRPV6 and calbindin-D_pk mRNA level between genotypes. In addition, amniotic fluid calcium levels were increased in VDR^+/− embryos (1.2x; p<0.001) despite increased TRPV5 mRNA expression (5x; p<0.001) compared to VDR^−/− littermates. These data indicate that bone mineralization is impaired in VDR^+/− fetuses of VDR^−/− mothers, associated with normocalcemia and increased calcium excretion.

A likely explanation are the increased 1,25(OH)₂D₃ levels detected both in VDR^−/− (14x) and VDR^+/− fetuses (9x) of VDR^−/− mothers compared to VDR^+/+ embryos of VDR^+/+ mothers (p<0.001), suggesting diffusion of 1,25(OH)₂D₃ through the maternal-fetal barrier. VDR^+/− fetuses responded to this elevated 1,25(OH)₂D₃ level with increased mRNA level of fibroblast growth factor 23 in the femur (5x; p<0.001) and 24-hydroxylase in the kidney (1.7x; p<0.05) and decreased renal expression of 1α-hydroxylase (-80%) as compared to VDR^+/+ embryos of VDR^+/+ mothers.

In conclusion, our data indicate that 1,25(OH)₂D₃ diffuses through the maternal-fetal barrier and that fetuses control their serum 1,25(OH)₂D₃ levels by activating feedback loops. Moreover, excessive 1,25(OH)₂D₃ is toxic for the fetus resulting in decreased bone mineralization and neonatal lethality.

Disclosures: L. Lieben. None.

M267

Effect of Hypercalciuria on Bone Density and Strength in Genetic Hypercalciuria Stone-Forming Rats.

M. D. Grynpas¹, A. Ng*¹, S. D. Waldman*², D. A. Bushinsky³. ¹Pathology, Mount Sinai Hospital Research Institute, Toronto, ON, Canada, ²Chemical Engineering, Queens University, Kingston, ON, Canada, ³Nephrology/Medicine, University of Rochester, Rochester, NY, USA.

Hypercalciuria is the most common metabolic abnormality in patients with calcium (Ca) containing kidney stones. Patients with hypercalciuria often excrete more Ca than they absorb, indicating a net loss of total body Ca, almost certainly derived from bone. A number of studies have shown a decrease in bone mineral density (BMD) and an increase in fracture rate in hypercalciuric stone formers compared to controls. However, in human stone formers it is difficult to determine if the reduction in BMD is due to a primary bone disorder or to any number of dietary factors that may be altered in stone formers over their lifetime. To study the independent effect of hypercalciuria on bone, we utilized the genetic hypercalciuric stone-forming (GHS) rats which were established by successively inbreeding the most hypercalciuric progeny of hypercalciuric Sprague-Dawley rats. Sixteen 46th generation female GHS and control (Ct1) rats were placed in metabolic cages and fed a 1.2% Ca diet for 6 weeks, and then their femurs, tibiae, humeri and lumbar spines were studied for BMD, histomorphometry, image analysis and mechanical tests. All comparisons are to Ctl rats. Urine Ca excretion was significantly greater in the GHS rats. BMD in both the femur and vertebrae (L1-L5) was significantly lower in the GHS rats. In the femur, trabecular bone volume and thickness were significantly lower in the GHS rats; however, trabecular number was not altered. Neither osteoid volume nor osteoid surface was altered in GHS rats. In the femur, the number of end points, multiple points, the length of node-to-free struts and the length of the node-to-node struts were each significantly higher in the GHS rats. In the humeri, failure stress was not different; however, failure strain and toughness were lower and modulus of elasticity higher in the GHS rats. Cortical and trabecular hardness was lower in the GHS compared to Ctl. Backscattered imaging (BSE) revealed that the GHS rat cortical bone is more mineralized than that of Ctl. Thus, the GHS rats, fed an ample Ca diet, have reduced bone mineral density with reduced trabecular volume, mineralized volume and thickness and their bones are more brittle and fracture prone, indicating that hypercalciuric stone-forming rats have an intrinsic bone disorder that is not secondary to diet.

Disclosures: D.A. Bushinsky, None.

This study received funding from: NIH.

M268

See Sunday Plenary Number S268

M269

Seasonal Changes in Bone and Body Mass in C57BL/6J Inbred Mice Are Gender and Compartment Specific.

K. M. Delahunty*¹, L. G. Horton¹, H. F. Coombs*¹, W. G. Beamer¹, M. L. Bouxsein², C. J. Rosen¹. ¹The Jackson Laboratory, Bar Harbor, ME, USA, ²Beth Israel Deconess Medical Center, Boston, MA, USA.

Seasonal bone loss in humans has been thought due to changes in vitamin D. We previously reported that C57BL/6J (B6) mice at The Jackson Laboratory had lower areal (a)BMD, measured by DXA, in Winter versus Summer despite no changes in 250H vitamin D. However, these data were obtained retrospectively. To test the hypothesis that seasonal bone loss occurs in B6 and to characterize physiological changes, we performed a cross sectional study in July 2006 (Summer), and February 2007 (Winter) using 16 week B6 virgin female (n=20–24) and male (n=18–21) mice from the same colony and animal room. We measured morphometric phenotypes by DXA, total femoral volumetric (v)BMD by pQCT, trabecular bone by Micro CT40, and serum IGF-I by RIA. The mice were fed the defined NIH31 diet, 16% fat by kcal, and unrefined for nutrient sources.

Female body weight, percent body fat and femur length did not show seasonal changes, but serum IGF-I was 25% lower in Winter than in Summer (p<0.006). In Winter, males had higher body weight (p<0.013), but no increase in percent body fat or femoral length, while serum IGF-I was reduced (∼25%, p<0.001). Total femoral vBMD was also lower in Winter than Summer for both females (p=0.017) and males (p<0.01). But in the mid shaft of the femur, during Winter males had markedly reduced percent bone area (p<0.003) accompanied by expansion of periosteal (p=0.005) and endosteal (p<0.002) circumferences. Females in Winter had no changes at the mid shaft, but a decrease in total femoral mineral (p=0.042). By contrast for the distal femur, females had significantly lower BV/TV in Winter than in Summer (p=0.02), with fewer trabeculae (p=0.02), more spacing (p=0.03), and lower connectivity density (p<0.05); males had no seasonal changes in trabecular bone. Summary: Seasonal changes in bone, body mass and IGF-I occur in B6 mice despite controlled conditions that include: a) constant diet, b) 14:10hr light:dark cycle, c) minimal changes in humidity, and d) constant room temperatures. Seasonal changes in males resulted in compensation for Winter bone loss by expansion of periosteum and preservation of trabecular bone. In females, both BMC and trabecular bone were decreased in the Winter. Mechanistically, the cues — environmental or endogenous — driving these skeletal changes are unknown. Further studies that control for factors such as nutrient composition, are necessary to gauge the true magnitude of seasonal change, the mechanisms involved, and the relevance for future mouse and human studies.

Disclosures: L.G. Horton, None.

This study received funding from: NIAMS 45433.

M270

See Sunday Plenary Number S270

M271

Genetic Interaction Between pS3 and Atm in Bone Development.

Y. Hu*, W. Leong*, B. Au*, B. Li. Institute of Molecular and Cell Biology, Singapore, Singapore.

p53 and Atm play critical roles in DNA damage response with p53 being a target of the Atm kinase. Recent studies show that p53 is a negative regulator of osteoblast differentiation, osteoblast-dependent osteoclastogenesis and bone remodeling (Wang et al. J Cell Biol 172:115, 2006; Zambetti et al. J Cell Biol 172:795, 2006; Lengner et al. J Cell Biol 172:909, 2006). p53(-/-) mice display a high bone mass phenotype; p53(-/-) osteoblasts show accelerated differentiation and increased expression of osteoblast-specific transcription factor, osterix. On the other hand, Atm plays a positive role in regulating expression of osterix. Atm(-/-) mice show reduced bone mass, decreased bone formation rate and defective differentiation of osteoblasts (Rasheed et al. Hum. Mol Genet. 15:1938, 2006; Hishiya et al. Bone 37:497, 2005.) To study the relationship of these two genes in bone development, we generate p53 and Atm double knockout mice. By using calcin double labeling method we found P53(-/-)Atm(-/-) mice have a high bone mass, increased bone formation rate. The p53(-/-) Atm(-/-) osteoblasts show accelerated differentiation justified by ALP and Von Kossa staining. The cells also show increased expression of osterix but not Runx2. Overall, these results indicate that p53 may function downstream of Atm in regulating bone development.

Disclosures: Y. Hu, None.

This study received funding from: A-Star.

M272

See Sunday Plenary Number S272

M273

A Functional RNAi Screening for Runx2-regulated Genes Corresponding to Ectopic Bone Formation in Human Spinal Ligaments.

M. Kishiya*¹, K. Furukawa*¹, K. Kanemaru*¹, H. Kudo*², T. Numasawa*², T. Yokoyama*², S. Toh*², S. Motomura*¹. ¹Pharmachology, Hirosaki University School of Medicine, Hirosaki, Japan, ²Orthopaedic Surgery, Hirosaki University School of Medicine, Hirosaki, Japan.

Ossification of the posterior longitudinal ligament of the spine (OPLL) is characterized by ectopic bone formation in the spinal ligaments which enlarges with time and compresses spinal cord, resulting a serious neurological symptoms. We have previously reported that osteoblast differentiation keyfactor (Runx2) were enhanced in OPLL cells. To clarify a role of Runx2 in the ectopic ossification, effects of osteogenic medium (OS:48h) and RNA interference (using siRNAs targeted to Runx2) on gene expressions were analyzed by genome-wide linkage analysis (microarray) and the results were confirmed by real-time PCR in the ligament cells derived from tissues of OPLL and non-OPLL (CSM; Cervical spondylotic myelopathy) patients obtained during surgery to decompress the spinal cord for myelopathy.

Microarray demonstrated 22 candidate genes regulated by Runx2 in OPLL cells. In addition to osteogenic markers, any chondrogenic markers and angiogenic markers were influenced by OS induction and RNAi for Runx2. Particulary, Aggrecan-1 and angiopoietin-1 were significantly increased by OS induction in OPLL cells but not in non-OPLL cells and decreased by siRNAs for Runx2, but Runx2 were not decreased by siRNAs for angiopoietin-1. Furthermore, VEGF-A down-regulated by OS induction was up-regulated by siRNA for Runx2 or angiopoietin-1. In our study, we suggested that OPLL development result in forming the metaplasia of spinal ligament cells and was derived through a mechanism of endochondral ossification. Furthermore, angiopoietin-1 and VEGF-A is downstream of Runx2 both in OPLL cells and osteoblasts. In our present study, we investigated any circulation factors in OPLL patients. OPLL patients was bleeding tendency in the perioperative period. Particularly, blood loss after surgery was significantly increased than non-OPLL patients. Other factors (function of platelets, coagulation abnormality. Hypertension, operation time … et al) was not significant between OPLL patients and non-OPLL patients. These data supported that OPLL patients find abnormality of microangigenic function.

At least we can say that there is some remarkable relation in OPLL development. Signaling pathway between skeletogenesis and vasculogenesis or angiogenesis could have been on contact with Runx2, events that may regulate angiogenesis, and OPLL growth. These data may become a key to elucidate the disease state of OPLL between ectopic bone formation and angiogenesis. Angiopoietin-1 may play an important role in the disease state of OPLL.

Disclosures: M. Kishiya, None.

M274

Dwarfism: A Comparative Analysis of Bone Mineral Density. D. C. Andrade*¹

M. G. Pippa*², S. R. Eis³, C. A. Brandao⁴, C. A. Zerbini². ¹Clinical Research, Centro Paulista de Investigação Clínica, Sao Paulo, Brazil, ²Rheumatology, Hospital Heliopolis, Sao Paulo, Brazil, ³Clinical Research, Centro de Diagnóstico e Pesquisa da Osteoporose do Espírito Santo, Vitoria, Brazil, ⁴Bone Densitometry, Fleury Medicina e Saúde, Sao Paulo, Brazil.

Dwarfism is a medical condition which result in short stature. It refers to an adult height of 4 feet 10 inches or under and can be caused by a wide variety of conditions, most of which are genetic. The most common type, accounting for 70% of all cases of short stature, is called achondroplasia. This one results from a spontaneous mutation in one gene or a child can inherit the gene from a parent who has achondroplasia. Characteristically, the arms are shorter than the forearms and the thighs are shorter than the legs, although trunk growth is nearly normal. Little is known about bone mass adjusted for body size and body composition compartments in children with dwarfism. Our objective was to evaluate the influence of short high in bone mineral density (BMD). We compared BMD results of dwarfs with a normal population. Twenty females and males dwarfs and were studied. All of them were submitted to dual-energy X-ray absorptiometry (DXA) in order to analyze BMD of lumbar spine. Control group was formed by normal females and males, that were matched by sex and age. Areal BMD was measured and reported in g/cm². Adjusted BMD was calculated by the relation between bone mineral content (BMC)/ high and results were given in g/m². Apparent BMD (App BMD) was calculated according to Bachrach. Variables were evaluated as descriptive patterns. The adherence to normal curve was evaluated by Kolmogorov Smimov test. To compare the difference of quantitatives variables between dwarfs and control group the Student t test was used. The effective P value to be considered significant was < 0.05. Data were analysed using the statistical computer program Minitab vertion 15.0%. Dwarf population showed lower means for all anthropometric values, but BMI [(weight = 52.8 Kg); (high = 1.23 m);(BMI = 34.4 Kg / m2)] when compared to control group [(weight = 65.3 Kg); (height = 1.62 m);[(BMI = 34.4 Kg / m2)]. Analysing bone meass we observed that there were no significant differences between Dwarfs and control group concerning to areal L1-L4 BMD(1.035 g/cm²; 1.122 g/cm²) p = 0.158, L1-L4 Z-score (-0.71 SD; −0.44 SD) p = 0.532, and L1-L4 App BMD(0.308 g/cm³; 0.313 g/cm3¹ p = 0.735. Dwarfs presented lower values of L1-L4 BMC (48.40g) and L1-L4, area BMD (45.65) when compared to control group L1-L4 BMC (58.50g) p = 0.040; L1-L4 area BMD (51.67g) p = 0.026. Interestingly Adjusted L1-L4 was higher in Dwarfs when compared to controls (0.837 g/cm² / 0.697 g/cm² respectively), p = 0.001. Our results suggest BMD in Dwarfs should be based on analysis of adjusted L1-L4 BMD instead of App BMD.

Disclosures: D.C. Andrade, None.

M275

See Sunday Plenary Number S275

M276

Analysis of Cranial Defect Healing in CXC Receptor2 Knock-out (CXCR2^−/−)

Mice. D. S. Bischoff*¹, T. Sakamoto*¹, N. S. Makhijani*¹, H. E. Gruber², D. T. Yamaguchi¹. ¹Research Service, VA Greater Los Angeles Healthcare System, Los Angeles, CA, USA, ²Orthopaedic Surgery, Carolinas Medical Center, Charlotte, NC, USA.

The potential role of CXC chemokines bearing the glu-leu-arg (ELR⁺) motif in bone repair was studied using a mouse cranial defect (CD) model in CXCR2^−/− mice. Inflammation is the initial step in bone repair. ELR⁺ CXC chemokines are released by inflammatory cells and serve as angiogenic factors. Previously we have shown that osteogenic differentiation of hMSCs increases expression of the ELR⁺ CXC chemokine, IL-8 (CXCL8). CXCL8 signals through the CXCR2 receptor in mice. We created 1.8 mm CDs in 6 week old male CXCR2^−/− and wild-type (WT) mice and allowed the CDs to repair over a 12-week period. CXCR2^−/− mice were significantly reduced in size and weight compared to their WT littermates. Weight, length, and DEXA measurements were taken (total, femur, tibia, radius, humerus, and vertebrate) before surgery and 6 and 12 weeks after surgery. CXCR2^−/− mice were significantly smaller in weight (54% of WT at 6 weeks, 75% at 12 and 18 weeks, p<0.0001) and length from base of tail to nose tip (82% at 6 weeks, 90% at 12 and 18 weeks, p<0.0004). DEXA analysis indicated that bone mineral density (BMD) and bone mineral content (BMC) were significantly decreased in CXCR2^−/− mice in all bones, and total area (TA), bone area (BA), total tissue mass (TTM), and lean body mass (LBM) significantly decreased in all with the exception of the vertebrae. There was no difference in % fat between CXCR2^−/− and WT mice. Normalizing for the size differences with weight or LBM, BMD is significantly decreased in CXCR2^−/− mice only at 6 weeks whereas BMC is slightly increased in the CXCR2^−/− mice at 18 weeks of age. CDs were analyzed using the BIOQUANT system. Although the CDs were not completely healed even after 12 weeks, preliminary analysis indicates that there was significant bone in-growth in both the CXCR2^−/− and WT CDs. Old and new bone were identified using polarized light and quantitated for numbers of osteocytes (OCy) and blood vessels (BV) around the original CD. New bone in CXCR2^−/− mice had more OCy than WT (2618 vs 2049 OCy/μm²). CXCR2^−/− old bone had about half as many OCy as new bone but still more than WT old bone (1282 vs 1043 OCy/μm²). In new bone, the number of BV in WT was ∼2X more than seen in CXCR2^−/− (43 vs 20 BV/μm²). The number of BV in the old bone was more closely matched (9 vs 11 BV/μm² for WT and CXCR2^−/− respectively). Conclusion: CXCR2^−/− mice have 1) reduced weight and size; 2) reduced BMD at 6 weeks of age but not at later time points; 3) increased BMC at 18 weeks of age; 4) increased number of OCy in new bone; and 5) decreased number of BV in new bone.

Disclosures: D.S. Bischoff, None.

This study received funding from: VA Merit Review.

M277

See Sunday Plenary Number S277

M278

Significant Lower Balance and Muscle Power Performance and Higher Risk for Falls in Elderly People with a Decreasing Creatinine Clearance.

L. C. Dukas*¹, E. Schacht², M. Runge*³. ¹Acute Geriatric University Clinic, Kantonsspital Basel, Basel, Switzerland, ²Zurich Osteoporosis Research Group ZORG, Zurich, Switzerland, ³Center for Muscle and Bone Research, Aerpah Klinikum, Esslingen, Germany.

A Creatinine clearance (CrCl) of <65ml/min. is associated with a significant increased risk for falls. In a German study a decreasing creatinine clearance was associated with significantly decreasing muscle power and balance. We assessed this association in a multinational study. For this cross-sectional study data were collected from 7 European countries including Russia. 1190 women and 127 men aged 60 years and older with a mean BMI of 27.2kg/m2 and a mean CrCl of 58.6ml/min., freshly diagnosed and therefore untreated for osteoporosis, participated in this study. The CrCl was calculated using the established Cockcroft-Gault formula. Results are from spearman correlation analyses and from logistic regression analyses. The p-values are two-sided.

A decreasing CrCl was multivariate controlled significantly associated with lower performance in the Timed-up-and-go test (TUG) (corr-0.1341, p<.0001) and in the Chair Rising test (corr — 0.1266, p<.0001). Performance in the Tandem Stand was not associated with the CrCl. A CrCl of <65ml/min. was multivariate controlled a significant risk factor for falls.

We found a significant correlation between decreasing CrCl and lower performance in balance and muscle power tests. However, contrary to the findings in another study the performance in the Tandem Stand test was not significantly associated with a decreasing CrCl. Furthermore we found that a CrCl<65ml/min is, independent from the performance in muscle and balance tests, a significant risk factor for falls. Our results together with the results from already published studies allows us to suggest a low CrCl of <65ml/min. to be a diagnostic tool to identify elderly frail patients.

Disclosures: L.C. Dukas, Teva Pharmaceutical Industries 2.

This study received funding from: TEVA Pharmaceutical Industries.

M279

See Sunday Plenary Number S279

M280

Candidate Genes of Every Known QTL of BMD in Whole Mouse Genome.

O. Xiong*, Y. Jiao, W. Gu. Departments of Orthopaedic Surgery — Campbell Clinic, University of Tennessee Health Science Center, Memphis, TN, USA.

One difficulty in the identification of candidate genes for QTL is that the genomic region of a QTL is generally considered as too large to search the candidate genes. In this study, we systematically examined all the genes in each of QTL of BMD identified from mouse study.

Methods: To identify every QTL, literature search was conducted with key words “Bone” and “QTL” in PubMed for every publication up to January 2007. Genes within a QTL region are obtained from Ensembl database (http://www.ensembl.org/index.html). For every known gene in QTL, their potential connection with BMD was evaluated by searching information from Online Mendelian Inheritance in Man (OMIM) at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM and PubMed at http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed. Searching terms are the combination of the symbol of the gene with any of those eight key words: bone mineral density, BMD, bone strength, bone size, osteoporosis, osteoblast, osteoclast, and fracture.

Results: We obtained candidate genes for every known BMD QTL. A total of 74 BMD QTL cover 1,210,895,314 bp genomic sequence which is roughly 46% of the total mouse genome sequences. Every autosomal chromosome except chromosome 8, contains at least one BMD QTL. Within the 1,210,895,314 genomic sequences of total of 74 BMD QTL, a total of 15,075 genes have been located. Among all those genes, 290 have been nominated as candidate genes according our bioinformatics search. For any potential candidate, at least the abstract of one reference was read to confirm the link between the gene and BMD. For a gene with more than one reference that indicates its candidacy, at least two references were read and citied in this study. Many BMD QTL have been located on the gene rich regions of the genome. The average gene density through whole mouse genome is about one gene per 95,977 bp. Within the total of 1,210,895,314 BMD QTL genome sequences, there is about one gene per 80,324 bp, while in the genome region outside of BMD QTL regions, there is about one gene per 114,894 bp.

Discussion and Conclusion: Our search reveals that a very large number of candidate genes exists within QTL regions. For most of them, we do not aware any report on the investigation or even being listed as candidates. The other important finding is that the association between polymphism of many candidate genes and BMD has been reported in human studies. As the rapid development of technologies and genome projects such as knockout of genes in whole mouse genome, information of gene function accumulates in an unexpected speed. Those information should be useful for our gene identification of QTL. A comprehensive search of candidate genes for known QTL may provide unexpected benefit for QTL studies.

Disclosures: W. Gu, None.

M281

See Sunday Plenary Number S281

M282

Identification of the CACNA2D2 Gene on Chromosome 3p21 as a Novel Susceptibility Gene for Osteoporosis.

O. Huang*¹, C. Cheung*¹, P. Fong*², M. Ng*¹, W. Mak*², V. Chan*¹, P. Sham*², A. Kung¹. ¹Medicine, The University of Hong Kong, Hong Kong, China, ²Genome Research Center, The University of Hong Kong, Hong Kong, China.

Evidence of linkage of chromosome 3p to bone mineral density (BMD) has been previously reported in individual as well as meta-analysis of genome wide linkage scans. To identify the quantitative trait loci gene underlying BMD variation at chromosome 3p, we performed a region-wide association study with 200 single-nucleotide polymorphisms (SNPs) across 6Mb of chromosome 3p21 in 1, 243 case-control Chinese subjects. The cases were subjects with low BMD (Z-scores ≤ −1.28, equivalent to the lowest 10 percent of the population) at either the L1–4 lumbar spine or femoral neck. Control subjects had high BMD (Z-score ≥ +1.0) at the corresponding sites. Two hundred tag SNPs were selected and genotyped using the high-throughput Sequenom genotyping platform. One hundred thirty-five SNPs met quality control criteria (genotyping call rate >0.9, minor allele frequency > 0.01, duplicate error rate <0.02 and Hardy-Weinberg equilibrium p values >0.01) and were analyzed for their association with BMD measured using dual-energy x-ray absorptiometry. Binary logistic regression analyses were performed to test for associations between each SNP genotype and BMD. Haplotype association analyses were performed by WHAP. Five SNPs (rs11918619, rs1471217, rs2336664, rs7626551, and rs2257216) were associated with spine BMD and two SNPs (rs2236953, rs2624839) with femoral neck BMD (p<0.01) in single marker associations. The region that contained 6 SNPs, located within the CACNA2D2 gene, showed the strongest association with femoral neck BMD in the haplotype analyses (permutation p<0.002). In addition, we consistently detected significant associations for the haplotype CCGATGCCAC of the CACNA2D2 gene with BMD at the spine, trochanter and total hip region. Our results suggest that CACNA2D2, which encodes a voltage-dependent calcium channel subunit. is a novel susceptibility gene for BMD variation in Chinese and is likely to be involved in the pathogenesis of osteoporosis. Replication study is currently being conducted in Japanese population.

Disclosures: Q. Huang, None.

M283

Lack of Major Survival Benefit of Vitamin D Receptor Gene Polymorphisms.

A. Arabi¹, L. Zahed*², Z. Mahfoud*³, J. Maalouf¹, M. Choucair*¹, M. Nabulsi*⁴, G. El-Hajj Fuleihan¹. ¹Calcium Metabolism and Osteoporosis Program, American University of Beirut, Beirut, Lebanon, ²Pathology and Laboratory Medicine, American University of Beirut, Beirut, Lebanon, ³Epidemiology and Population Health, American University of Beirut, Beirut, Lebanon, ⁴Pediatrics, American University of Beirut, Beirut, Lebanon.

Vitamin D has recently emerged as an important risk factor for several health problems associated with increased mortality. Vitamin D receptor (VDR) is widely expressed in most immune cell types and other tissues, and prostate, breast and colon cancer cells exhibit increased levels of VDR protein when compared to their normal counterparts. This raises the possibility that some VDR genotypes maybe associated with higher mortality rate and therefore their frequency distribution may decrease in the surviving elderly.

The aim of this study was to look for a survival benefit of VDR gene polymorphisms by comparing the genotype frequencies in children to those in the elderly in a population of the same ethnic background.

Data from 203 elderly subjects aged 65–85 years who participated in a population-based study assessing the prevalence of osteoporosis, and data from 336 children aged 10–17 years who participated in a vitamin D trial were used. In both groups, polymorphisms in the VDR gene were assessed with the restriction enzymes BsmI, TaqI and ApaI.

The frequency distributions of VDR genotypes were similar to those reported in white populations. The heterozygote genotype for all enzymes was the most frequent in both age groups (Table 1). The frequencies of genotypes in both age groups were in Hardy-Weinberg equilibrium. There was no difference in the frequency distribution of VDR genotypes between the young and the elderly, both by gender and in the overall group. The lack of difference in the frequency distribution of VDR genotypes between the children and the elderly within the same population renders the role of VDR as a major modulator of the relationship between vitamin D and conditions associated with high mortality unlikely.

Table Table 1.. Frequency distribution of VDR genotypes according to the restriction enzyme in children and in the elderly*

* No significant difference between the children and the elderly within gender or in the overall group.

Disclosures: A. Arabi, None.

M284

The Decreased Activity of Lactase Phlorizin Hydrolase and Bone Mineral Density in Postmenopausal Women.

K. Bácsi*, J. P. Kósa*, B. Balla*, Á. Lazáry, Z. Nagy, I. Takács, G. Speer*, P. Lakatos. First Department of Internal Medicine, Semmelweis University, Budapest, Hungary.

The CC genotype of the −13910 T/C dimorphism (LCT) related to lactose intolerance and decreased calcium absorption from gut is accompanied with reduced activity of lactase phlorizin hydrolase. We hypothesized that the altered calcium absorption throughout life has an impact on bone mineral density (BMD) of postmenopausal women. We studied 200 osteoporotic, 235 osteopenic and 160 healthy women. Genotyping, osteodensitometry at the lumbar spine, total hip, femur head, Ward's triangle and radius as well as serum calcium measurements were carried out in all subjects. The −13910 T/C polymorphism was significantly associated with the BMD at the Ward's triangle (Z-score (TT) = 0.082±0.787, Z-score (TC) = −0.I79±0.829, Z-score (CC) = −0.334±0.871; p=0.028) and in a recessive model at the radius (Z-score (TT+TC) = 0.406±1.322, Z-score (CC) = 0.105±1.415; p=0.038) in the whole study population. Similar relationship was seen at the femoral head (Z-score (TT+TC) =0.595±1.02, Z-score (CC) = 0.148±1.063; p=0.013) but only in the unified groups of osteoporotic and healthy women. Also, we found significant association between genotypes and both the total hip density (Z-score (TT) = −0.717±0.841, Z-score (TC) = −1.106±0.877, Z-score (CC) = −1.003±0.826; p=0.043) and the Ward's triangle density (Z-score (TT) = −0.375±0.606, Z-score (TC) = −0.819±0.485, Z-score (CC) = −0.88±0.541; p=0.015) in osteoporotic and osteopenic patients together. All results remained significant after adjustment for menopausal age and body mass index. We could not detect any associations between genotypes and BMD at the lumbar spine in the study groups. Our data suggest that the −13910 T/C dimorphism might have an influence on bone mass at cortical but not trabecular bone.

Disclosures: K. Bácsi, None.

This study received funding from: ETT 022/2006, NKFP-1A/007/2004, NKFP-1A/-002/2004.

M285

An Interaction Between Dietary Fat and Polymorphisms in a Previously Undetected, Alternate 3' UTR of the PPARG Gene Influences Bone Mass in Both Mice and Humans.

C. L. Ackert-Bicknell¹, J. Graber*¹, K. Cho², S. Demissie*², J. Ordovas*³, W. G. Beamer¹, D. P. Kiel⁴, C. J. Rosen¹. ¹The Jackson Laboratory, Bar Harbor, ME, USA, ²Boston University School of Public Health, Boston, MA, USA, ³Tufts University, Boston, MA, USA, ⁴Hebrew SeniorLife, Boston, MA, USA.

We previously identified a Quantitative Trait Locus (QTL) for volumetric BMD on mouse Chromosome (Chr) 6 and now name Peroxisome Proliferator-Activated Receptor-gamma (Pparg) as a likely candidate gene. The B6.C3H-6T (6T) congenic mouse was created for the purpose of understanding the biology underlying this QTL and was generated by introgressing a 30 cM region of Chr 6 from C3H/HeJ (C3H) onto C57BL/6J (B6). Female 6T mice have low BMD and increased adipocyte infiltration in bone marrow. PPARG is a nuclear receptor known to be key for maturation of adipocytes. Existing annotation of PPARG indicated a 3' Untranslated Region (3' UTR) with an approximate length of 210 nucleotides (nt). Examination of the pattern of evolutionary conservation downstream of this site led us to predict a previously undetected alternative 3' processing site that would result in a longer (∼3450 nt) 3' UTR. We have sequenced the genomic region between the two 3' processing sites and found 25 polymorphisms in C3H, when compared to B6. Dietary fat is able to activate PPARG and female 6T mice fed a high fat diet (60% fat) have lower aBMD than low fat (11% fat) fed 6T mice. There is no difference in aBMD in B6 mice fed the two diets. Using Real Time RT-PCR, we examined expression of Pparg transcripts containing this alternate 3' UTR in bone. There was no difference between B6 mice fed the low and high fat diets, (fold = 1.52±0.4, p = 0.318) and between B6 and 6T mice fed a low fat diet (fold = 1.8±0.6, p = 0.18). 6T mice fed high fat had a 4.3±1.4 fold increase compared to B6 mice fed low fat (p =0.036) and a 2.25±0.6 fold increase compared to 6T mice fed low fat (p=0.016). We examined 13 SNPs in the PPARG gene in the Framingham Offspring Cohort. For three of these SNPs, rs1152004, rs1175381 and rs1186464, we found a significant interaction with dietary fat intake for the phenotype of DXA BMD of the trochanter region in women. SNPs rs1152004 and rs1175381 are located in a region of high evolutionary conservation very near to the alternative 3'-processing site in PPARG The usage of alternative 3' UTR can affect transcript stability and translation efficiency. These findings suggest that dietary fat has a significant influence on BMD but is dependent on alleles in the rare, extended 3' UTR of PPARG.

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

This study received funding from: NIAMS 45433.

M286

Association of Luteinizing Hormone Receptor Polymorphism with Bone Mineral Density in Young Men — Results from Odense Androgen Study.

C. L. Brasen*¹, T. L. Nielsen*², B. Abrahamsen³, L. Christiansen*⁴, C. Hagen*², M. Andersen*², K. Brixen², L. Bathum*¹. ¹Dept. of Biochemistry, Pharmacology and Genetics, Odense Universitetshospital, Odense C, Denmark, ²Department of Endocrinology, Odense Universitetshospital, Odense C, Denmark, ³Department of Medicine & Endocrinology, Copenhagen University Hospital Gentofte, Hellerup, Denmark, ⁴Department of Epidemiology, Institute of Public Health, University of Southern Denmark, Odense C, Denmark.

The N312S polymorphism in the luteinizing hormone receptor (LHR) has previously been shown to be associated with genital undermasculinization. We investigated the hypothesis, that the N312S polymorphism in LHR is associated with bone mineral density (BMD).

We genotyped 783 healthy men aged 20–29 years from the population-based Odense Androgen Study for the N312S polymorphism in LHR and investigated association with BMD in the spine, hip, truncus and extremities as well as whole body BMD. 406 of the participants underwent MRI scans to determine visceral and subcutaneous adipose tissue(VAT and SAT), muscle tissue and femoral compacta area. We furthermore tested for association with testicular volume, height, lean body mass, carboxy-terminal telopeptide of type I collagen(ICTP), bone-specific alkaline phosphatase(bone ALP), luteinising hormone, testosterone, oestrogen, dihydrotestosterone(dht) and androstenedione. Genotype distribution (AA, 0.20; AG 0.48; GG, 0.32) was consistent with the Hardy-Weinberg equilibrium. Using the entire study group, we found significant association between LHR alleles and testicular-volume (p=0.03) as well as with BMD in spine (p=0.04) and trend association between LHR alleles and BMD in truncus and whole body BMD.

Stratifying participants into “sedentary” and “non-sedentary”, we found that in the sedentary participants (n=615), LHR N312S was significantly associated with testicular volume, BMD in spine, truncus, and whole body. The results also indicated a trend association with BMD in hip and extremities, as well as the level of ICTP.

Additional stratification by smoking showed that in the group of sedentary ever-smokers (n=199) LHR alleles were significantly associated with BMD in spine, truncus, extremities and whole body, (p<0.01 for all), ICTP (p=0.04) and bone ALP (p=0.002). The difference in mean BMD in the spine equals 5.9%. Furthermore there was a trend association with BMD in hip and lean body mass. Finally we found a trend association with VAT, SAT and femoral compacta area.

These findings strengthen the hypothesis that the LHR N312S polymorphism is associated to genital undermasculinization and hereby low BMD. More studies are needed to verify these results. Interaction with other polymorphisms will be discussed.

Disclosures: C.L. Brasen, None.

M287

Study of 7 Polymorphisms Genes (LCT, MTHFR, COLIA-1 and Promoters of RANKL, OPG, IL-6 and TCIRG1) Previously Described as Linked to Osteoporotic Fractures: Lack of Association in French Women.

V. Breuil¹, D. Quincey*², J. Testa*³, C. Albert*¹, C. H. Roux*¹, Z. Mroueh*², H. Chami*¹, O. Brocq*¹, C. Grisot*¹, L. Euller-Ziegler¹, G. Carle². ¹Rheumatology, CHU de Nice — Medical School, Nice, France, ²CNRS — Faculté de Médecine, GEPITOS-Université de Nice Sophia Antipolis, Nice, France, ³Informatique Médicale, CHU de Nice — Medical School, Nice, France.

Osteoporosis (OP) is have a strong genetic determination and numerous polymorphisms ar reported to be associated with it. However, theses associations depend on the population studied. Thus, we tested in a French population 7 genes polymorphisms [Lactase Transferase (LCT), RANKL promoter, Collagen lot (COLIA-1), OPG promoter, IL-6 promoter, methylenetetrahydrofolate reductase (MTHFR), and TCIRG1 promoter] previously reported to be associated with OP fractures.

Two groups of postmenopausal women were selected, 50 to 80 years old: one control group free of OP fracture and with normal BMD (T-score >-ISD at hip and spine) in without any anti-OP treatment, and one group of OP women with at least one fracture and a low BMD (T-score <−2.5 SD at hip and/or spine); secondary OP were excluded. For each patient, genomic DNA was isolated from peripheral blood and polymorphisms genotyped by PCR and restriction analysis. The univariate statistical analysis used Chi² or Fisher test (qualitative variables) and Kruskall-Wallis (quantitative variables); the multivariate analysis used logistic regression.

159 women (89 OP and 70 controls) were enrolled. The 2 groups were similar regarding age of menopause, statine, betablockers, neuroleptics or antidepressants use. In the OP group, there was significant higher rate of family history of OP, higher age, age of puberty and loss of height, as well as significant lower weight, height, body mass index, high blood pressure. In the OP group, 47 vertebral fractures and 62 non vertebral fractures were recorded. The analysis of each polymorphism didn't show any significant differences between the 2 groups. To take in account the polygenic pattern of the disease, we tested wether the OP group presented significantly more polymorphisms on the 7 genes than the control group and failed to detect any difference. However, we found a significant association between the polymorphism of IL-6 or TCIRG1 promoter and family history of OP (p= 0.01), as well as other polymorphisms associated with hyperlipidaemia, height, loss of height, BMI, and diabetes.

In our French population, we did not found the polymorphisms associations with OP fractures previously described. This could be related to the sample size, even if some of the previous studies enrolled a similar number of subjects, or, as previously described for numerous polymorphisms, to a geographical factor.

Disclosures: V. Breuil, None.

This study received funding from: PHRC régional du CHU de Nice, Lilly.

M288

See Sunday Plenary Number S288

M289

A Haplotype-Based Analysis of the LRP5 Gene in Relation to Osteoporosis Phenotypes.

L. Agueda*¹, X. Nogues², M. Bustamante*¹, S. Jurado*², N. Garcia-Giralt*², J. Garces*², L. Mellibovsky*², S. Balcells*¹, A. Diez-Perez², P. Grinberg*¹. ¹Genetics, University of Barcelona, CIBERER, IBUB, Barcelona, Spain, ²URFOA, IMIM, Hospital del Mar, Autonomous University of Barcelona, Barcelona, Spain.

LRP5 encodes the low-density lipoprotein receptor-related protein 5, a transmembrane protein involved in Wnt signaling. In bone, LRP5 is an important regulator of osteoblast growth and differentiation, affecting peak bone mass in vertebrates. Rare mutations in LRP5 result in severe phenotypes associated with bone. Whether common variants in LRP5 are associated with normal BMD variation or osteoporosis phenotypes remains controversial. In this study, we employed a haplotype-based approach to comprehensively examine the contribution of common variation at the LRP5 genomic region to osteoporosis susceptibility.

A total of 24 SNPs were selected to cover a 150 kb genomic region including the LRP5 gene and flanking regions, about 7 kb at each side. Most of these SNPs were tag-SNPs selected based on HapMap-CEU data. The remaining were either missense changes or SNPs previously studied by others. Genotyping was performed using SNPlex or TaqMan methodologies. Association, both at single SNP or block-haplotype level, was tested against LS-BMD, FN-BMD and osteoporotic fracture in a cohort of 964 Spanish postmenopausal women (BARCOS cohort). Statistical methods included ANCOVA and logistic regression.

At the single SNP level, a significant association with LS BMD was found for SNP#1, located in the promoter region (p=0.011, recessive model). The SNP#6 (intron 1) was associated with both LS BMD (p=0.025, additive model) and FN BMD (p=0.031, recessive model). Three SNPs, SNP#11 (intron 1; p=0.007, additive model), SNP#13 (intron 5; p=0.041, recessive model) and SNP#15 (intron 5; p=0.019, recessive model) were significantly associated with the risk of osteoporotic fracture, with ORs of 1.70, 1.66 and 1.79, respectively. In general, haplotype analyses did not provide additional information to that obtained from SNP analyses. The only exception was one block-haplotype (tagged by SNPs 10, 11 and 12), which showed a significant association with LS BMS (p=0.043), while none of the tag-SNPs gave a significant result on their own.

In conclusion, we found that genetic variation in the first half of the LRP5 genomic region was associated with osteoporotic phenotypes in the BARCOS cohort.

Disclosures: X. Nogues, None.

M290

An α_vβ₃ Integrin Antagonist (MK-0429) Inhibits Osteolytic Lesions in Breast Cancer and Melanoma Lung Metastases.

M. Pickarski, G. Wesolowski, G. Neusch*, T. Prueksaritanont*, L. T. Duong. Merck Res. Labs., West Point, PA, USA.

There is significant evidence to support multiple roles of α_vβ₃ integrin in mediating osteoclastic bone resorption, tumor progression and metastasis. Increased expression of α_vβ₃ in several tumors, including breast cancer and melanoma, is correlated with a greater metastatic potential and the development of bone metastases. Previously, we demonstrated the efficacy of MK-0429 as a safe, well tolerated, orally active antiresorptive in reversing estrogen-deficiency induced bone loss in preclinical animal models and in postmenopausal women. Here, we evaluated the efficacy of MK-0429 in inhibiting the proliferation of breast cancer (BrCa), prostate cancer (PCa) and melanoma cells. 

Table  .  

MK-0429 also inhibited MDA-MB-231 breast cancer (IC₅₀ = 2.1 nM) and PC3-M prostate cancer (IC₅₀ = 4.1 nM) cell migration to vitronectin; and blocked MDA-231 matrigel invasion (IC₅₀ = 7.0 nM), comparable to its potency to inhibition of osteoclastic bone resorption in vitro (IC₅₀ = 12 nM). In this study, MK-0429 was evaluated in the prevention of bone metastasis growth using the intra-tibial engraftment model of MDA-MB-231 breast carcinoma in nude rats. Bone loss and skeletal tumor growth were determined by μCT and histology over a 6-week period. Compared to vehicle, treatment with oral MK-0429 at 100, and 300 mg/kg, bid, significantly prevented osteolytic lesions by 22 and 42%, respectively, compared to 45% with zoledronic acid (ZOL, 7.5 μg/kg, sc, wkly). Compared to vehicle, MK-0429 also reduced tumor volume by 40 and 66%, vs. a 56% reduction with ZOL. MK-0429 significantly inhibited cortical disruption, tumor necrosis, tumor infiltration between the trabecular bone plates, and invasion of the bone marrow. While having no effect on melanoma growth in an s.c. xenograft model, MK-0429 reduced the incidence of B16-F10 pulmonary metastases by 60% vs. vehicle. Taken together, these data show that MK-0429 had both anti-resorptive and anti-metastatic activities in preclinical models of breast cancer bone metastasis, as well as extraskeletal activity in blocking melanoma lung metastasis. Compared to the i.v. bisphosphonates, MK-0429 may represent a novel oral therapy for the treatment of patients with bone metastasis.

Disclosures: L.T. Duong, Merck & Co. 3.

M291

See Sunday Plenary Number S291

M292

Prevalence of Vitamin D Insufficiency in Patients with Breast Cancer.

N. Napoli, C. Ma, S. Vattikuti*, M. R. Azadfard*, A. Rastelli, M. Ellis*, R. C. Armamento-Villareal. Medicine, Washington University School of Medicine, St. Louis, MO, USA.

Previous studies have shown that aromatase inhibitor therapy in patients with ER+/PR+ breast cancer is associated with bone loss and increased incidence of fractures. As these studies were not designed to investigate skeletal outcomes as the primary endpoint, the vitamin D status of these patients remains undetermined. The objective of our study is to determine the incidence of vitamin D deficiency in patients with newly-diagnosed breast cancer who are about to initiate aromatase inhibitor therapy. We have recruited 80 postmenopausal patients (69 Caucasians, 11 African-Americans) between 40 to 80 years old from March 2006 to March 2007, with newly-diagnosed Stage 1 to 111a, ER+/PR+ breast cancer who will be initiated on aromatase inhibitors by their oncologists. Table 1 shows the clinical data of the whole study population. Baseline vitamin D levels were available in 70 patients. Analysis of serum vitamin D levels showed that 60 out of 70 patients (85.7%) of which 51 were Caucasians and 9 were African-Americans, have insufficient levels, i.e. < 30 ng/ml (31 patients between 20–30 ng/ml, 25 between 10–19 ng/ml and 4 have < 10 ng/ml). The vitamin D levels were significantly lower in African-Americans compared to Caucasians (16.3 ± 10.8 ng/ml vs 22.3 ± 8.3 ng/ml, p=0.04). Analysis according to the time of presentation showed no seasonal variation in vitamin D levels (April to September =23.2±9.1 vs October to March = 20.5±8.8 ng/ml, p=0.24). In summary, insufficient vitamin D levels is common among patients with breast cancer perhaps from a combination of poor sunlight exposure (from lack of physical activity) and poor intake (from lack of appetite). The prevelance of vitamin D insufficiency appears to be higher in our cohort than what has been reported in the literature for otherwise healthy postmenopausal women without breast cancer (Holick MF, et al. J Clin Endocrinol Metab. 2005 Jun;90(6):3215–24. Epub 2005 Mar 29). Low vitamin D levels may aggravate bone loss from aromatase inhibitors, thus, routine vitamin D determination followed by appropriate supplementation may be necessary in breast cancer patients, most especially in those who will be starting aromatase inhibitor therapy.

Clinical data of the whole study population (N=80) 

Table  .  

Disclosures: N. Napoli, None.

M293

See Sunday Plenary Number S293

M294

6-Thioguanine Acts on Gli2 to Decrease PTHrP Expression and Osteolysis.

M. P. Nguven¹, J. A. Sterling², A. J. Roberts*², G. R. Mundy². ¹Department of Medicine, Vanderbilt University, Nashville, TN, USA, ²Center for Bone Biology, Department of Clinical Pharmacology, Vanderbilt University, Nashville, TN, USA.

Breast and lung tumor cells frequently metastasize to bone, where they cause osteolytic bone destruction. Parathyroid hormone-related peptide (PTHrP) has been identified as one of the major mediators of osteolysis and hypercalcemia in tumors that metastasize to bone. Thus, compounds that inhibit tumor cell production of PTHrP can potentially be clinical treatments for osteolysic metastases. Our laboratory has previously demonstrated that guanosine nucleotides, represented by 6-thioguanine (6-TG), reduces PTHrP production, tumor-induced osteolysis, and tumor burden in bone. 6-TG was identified from screening a diverse library of small molecular weight chemicals that inhibit PTHrP promoter activity, using a cell-based screen with PTHrP promoter as the molecular target (Gallwitz et. al, JCI 2002). This observation raises the possibility that 6-TG or a related compound may be effective in reducing PTHrP production by cancer cells and thus provides a useful therapeutic approach to hypercalcemia and osteolysis caused by PTHrP excess. In this study, we have attempted to determine the molecular mechanism by which 6-TG inhibits the PTHrP promoter. Recently, we have found that PTHrP production in cancer cells is regulated by activation of the developmental Hedgehog (Hg) pathway, specifically by the Hh pathway transcriptional activator, Gli2. We hypothesized that 6-TG inhibits PTHrP through regulating Gli2 expression. To test this hypothesis we utilized the osteolytic and PTHrP producing tumor cells, RWGT2, a human squamous cell carcinoma, and MDA-231, breast carcinoma cells. We treated these cells with either 6-TG or purine as a negative control. We found that 6-TG treatment of RWGT2 and MDA-231 human cancer cells decreases Gli2 expression by Western blot and real-time PCR by approximately 10-fold. These effects correlated with decreases in PTHrP expression by realtime PCR and PTHrP promoter-luciferase activity. Since 6-TG can decrease Gli2 mRNA expression, it is also possible that 6-TG directly affects Gli2 promoter activity. We have therefore cloned the Gli2 promoter to determine if 6-TG can repress Gli2 promoter activity in osteolytic tumor cells. Currently, our data demonstrates that 6-TG inhibits PTHrP through regulating Gli2, suggesting that specific inhibitors of Gli2 expression may be a potential approach for treating osteolytic metastases.

Disclosures: M.P. Nguyen, None.

M295

See Sunday Plenary Number S295

M296

Non-Canonical Hedgehog Signaling Regulates Gli2 and PTHrP Expression in Osteolytic Cancer Cells.

J. A. Sterling¹, S. S. Padalecki², B. G. Grubbs², M. P. Nguyen¹, A. J. Roberts*¹, B. O. Oyajobi², G. R. Mundy¹. ¹Center for Bone Biology, Vanderbilt University, Nashville, TN, USA, ²University of Texas Health Science Center, San Antonio, TX, USA.

Cancer cells cause osteolysis and hypercalcemia by over-production of PTHrP. We have shown previously that PTHrP expression and osteolysis by cancer cells are regulated by Gli2, a Hedgehog (Hh) signaling molecule. Gli2 is a developmental protein mis-expressed in some tumors. Understanding whether Gli2 expression in cancer cells is regulated through Hh receptor signaling or other mechanisms is important for target-based drug approaches for inhibiting Hh signaling and PTHrP in osteolytic breast cancer. To clarify this, we have used several approaches. We examined cancer cells associated with increased PTHrP expression for expression of Hh signaling receptors and determined the effects of specific receptor antagonists. The absence of these receptors would indicate the Hh pathway is stimulated independently of Hh ligand. We also determined the effects of antagonists specific for Gli2. Hh molecules utilize two receptors Smoothened (Smo), the signaling receptor, and Patched (Ptc). We found that osteolytic MDA-231 human breast cancer cells express Ptc but do not express Smo. Without the presence of the signaling receptor, the observed increase in Gli2 is unlikely due to canonical signaling. To verify that Gli2 is not regulated by canonical Hh signaling, we utilized the Hh antagonist cyclopamine. Cyclopamine had no effect on growth of MDA-231 cells in vitro and did not decrease Gli2 or PTHrP expression by real-time PCR. When athymic nude mice were inoculated with MDA-MB-231 cells via the intracardiac route, and treated with 10mg/kg/day of cyclopamine, there was no inhibitory effect on tumor-induced osteolysis. However, overexpression of intracellular molecules that modulate Hh signaling by down-regulating Gli2 protein in MDA-231 cells decreased PTHrP expression. Specifically, Suppressor of Fused (SuFu), a protein that binds to Gli2 and prevents it from translocating to the nucleus, blocked Gli2 stimulation of PTHrP, while a dominant-negative SuFu increased PTHrP promoter activity. Furthermore, overexpression of a dominant-negative β-TrCP, the E3 ligase which targets Gli2 for ubiquitination and proteasomal degradation, increased PTHrP promoter activity by inhibiting the degradation of Gli2. Together, our data confirm that Gli2 mediates PTHrP expression, and that Gli2 expression is not driven by signals initiated by Hh in breast cancer. Although these data suggest Hh antagonists such as cyclopamine will not be effective treatments for osteolytic bone metastases, they do indicate the potential utility of targeting Gli2 degradation.

Disclosures: J.A. Sterling, None.

M297

TGFβ Drives Expression of Osteogenic Molecules in Bone Metastatic Breast Cancer Cells: A Potential Mechanism for Breast Cancer Osteomimicry.

M. Ni*¹, J. A. Sterling², A. Roberts*², L. Lin*³, G. R. Mundy², R. L. Caldwell⁴. ¹Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, TN, USA, ²Department of Medicine/Clinical Pharmacology, Vanderbilt University School of Medicine, Nashville, TN, USA, ³Department of Phamacology, Vanderbilt University School of Medicine, Nashville, TN, USA, ⁴Orthopaedics and Rehabilitation, Vanderbilt University School of Medicine, Nashville, TN, USA.

As tumor cells leave the primary environment, expression of molecules important for homing to the targeted organ ensues. For bone metastatic cancer cells, these molecules are similar to those expressed by bone cells, a process called osteomimicry. They include Runx2 and other molecules in the Wnt/B-catenin pathway which are important in osteoblast differentiation. Several groups have reported similar findings, yet the significance of these molecules is unclear. We have previously demonstrated that osteolytic breast cancer cells express the Hedgehog (Hh) signaling molecule Gli2, which drives BMP2 expression and the Wnt/B-catenin pathway in osteoblasts. We hypothesized that expression of BMP2 and Wnt/B-catenin pathway components are dependent on activation of the Hh pathway in breast cancer cells. We found that wild type (WT) MDA-MB-231 human breast cancer cells express Gli2 and BMP2. Since breast cancer cells in bone are exposed to high concentrations of TGF-b, we examined these cells for Gli2 and BMP2 regulation by TGF-b. We also examined MDA-MB-231 cells deficient in TGF-b signaling (DN-TGF-betaRII). In WT MDA-MB-231 cells, we found high levels of BMP2 expression that were increased after TGF-beta treatment. BMP2 expression was absent in DN-TGF-betaRII cells. We found TGF-beta treatment increases BMP receptor type II (BMPRII) expression in WT cells, suggesting that bone metastatic breast cancer cells are able to respond to the observed increase in BMP2. To determine if TGF-beta could increase BMP2 signaling, we tested the effect of TGF-beta on a SMAD6 promoter-reporter construct, revealing that TGF-beta increases activity from the SMAD6 promoter. Contrary to other tumor cell types that exhibit decreased proliferation after BMP2 treatment, cell proliferation assays did not indicate a BMP2-mediated growth inhibition in WT breast cancer cells. We then examined an MDA-MB-231 clone over-expressing a dominant-negative, kinase-deficient BMPRII and found decreased tumor cell proliferation compared to WT cancer cells. Our data suggest that the Hh pathway, and specifically Gli2, may drive expression of downstream molecules implicated in osteomimicry, and that the process is enhanced by TGF-beta expression in the bone metastatic microenvironment.

Disclosures: J.A. Sterling, None.

This study received funding from: Vanderbilt University Tumor Microenvironment Network and Developmental Funds from the Department of Orthopaedics and Rehabilitation.

M298

See Sunday Plenary Number S298

M299

Fracture Risk Factors in Breast Cancer Survivors with Chemotherapy-induced Amenorrhea.

K. M. Winters-Stone*¹, L. Nail*¹, A. Schwartz*², K. Witzke*³. ¹School of Nursing, Oregon Health & Science University, Portland, OR, USA, ²School of Nursing, Arizona State University, Tempe, AZ, USA, ³Kinesiology, California State Univ., San Marcos, CA, USA.

Premenopausal breast cancer survivors (BCS) who experience chemotherapy-induced amenorrhea may have elevated fracture risk due to both chemotherapy and hypoestrogenism. Low estrogen is associated with bone loss and possible neuromuscular declines, whereas chemotherapy for breast cancer is also associated with bone loss, and with weight gain and neurological symptoms. Our pilot study aimed to describe the baseline and one-year change in fractures, falls and bone health in breast cancer survivors with premature menopause within one year following treatment completion. We collected baseline and one year follow-up data to describe changes in fractures and risk factors over time in premenopausal women with early stage breast cancer who experienced chemotherapy-related loss of menstrual cycles (N=47; mean age: 46 yrs; 12.2±4.5 mos. post-chemotherapy). Data on premenopausal, cancer-free controls (N=30; mean age: 41 yrs) were used as a reference group. Bone health was assessed by measuring bone mineral density (BMD) of the proximal femur (hip) and lumbar spine via DXA (Hologic Discovery Wi) and by measuring bone turnover via levels of serum osteocalcin (OC) and urinary deoxypyridinoline cross-links (Dpd). DXA data were also described as T-scores derived from spine and hip BMD values categorized as either normal (≥-1) or low (<−1). Baseline fall and fracture history were assessed retrospectively by questionnaire and prospectively over 12-months by monthly postcards. Comparisons between groups were determined by ANCOVA on baseline and 12 month values, adjusting for group differences in age and by chi-square for frequency data. There were no significant differences between groups for spine or hip BMD at either time point. However, more BCS were classified as having low spine BMD compared to controls both at baseline 39% vs. 17% for BCS and controls, respectively, p=.06) and at follow-up (44% vs. 20% for BCS and controls, respectively, p<.05). Levels of both OC and DpD were significantly greater in BCS versus controls both at baseline and at 12-mos. follow-up (p<.01). In BCS, 11% had a history of fracture at baseline compared with 3% of controls (n.s.) and fracture rates over 1 yr were similar between groups. At baseline 42% of BCS had fallen in the last year compared to 50% of controls (n.s.), but over 1 year 70% of BCS experienced 1+ falls compared to 43% of controls (p=.05). Prematurely menopausal breast cancer survivors should be screened for bone health and fall history and if positive should be considered for therapeutic intervention to decrease fracture risk, such as exercise and/or medication.

Disclosures: K.M. Winters-Stone, None.

This study received funding from: PHS Grant 5 M01 RR00334 and the OHSU Medical Research Foundation.

M300

See Sunday Plenary Number S300

M301

Bone Metastases Select for Integrin α2β1 Negative Cells in Prostate Cancer Cell Line DU145.

J. Yin, K. Tracy*, Y. Ward*, K. Kelly*. National Cancer Institute, Bethesda, MD, USA.

The metastatic process involves making and breaking contacts with different extracellular matrix (ECM) components in the primary tumor and metastatic sites. ECM binding is mediated by cell surface integrins. The integrin expression patterns in prostate cancer bone metastasis have not been well studied. To determine whether changes in integrin expression affect prostate cancer metastasis to bone, we studied the integrin expression profile of bone tropic cells in a xenograft model of prostate cancer metastasis. DU145 cells expressing the Ras effector mutant, Ras^V12G37, gain the ability to colonize bone, in part as a result of activating the Ral Pathway. Ras signaling through growth factor receptors is commonly activated in prostate cancer progression, and Ral is an important downstream pathway of Ras. All clones isolated from bone metastasis had increased bone metastatic activity compared with the parental DU145/Ras^V12G37 cells as evidenced by development of more and larger bone metastases at earlier times. The DU145 cell line expresses low levels of integrin α1, and high level of integrin α2, αv, β1, and β3. Although over-expression of Ras^V12G37 in a polyclonal population of DU145 cells did not significantly alter the expression of integrins α1, αv, β1 and β3, about 10% of cells demonstrated loss of integrin α2 expression. Interestingly, integrin α2 expression was lost in 4 out of 5 cell lines isolated from bone metastases, indicating selection within the population for rare α2β1 negative cells. In addition, loss of integrin α2 expression was correlated with high Ras expression and high RalA activity. We then performed cell adhesion assays on several ECM components including collagens 1 and IV, laminin and fibronectin. All isolated bone metastatic cell lines with low integrin α2 expression lost adhesiveness to collagen I and IV, while adhesion to laminin or fibronectin remained unchanged. These results suggest that cells with higher RalA activity and loss of integrin α2 are selected in the development of bone metastasis. Downregulation of integrin α2 has been observed in prostate cancer patients, and v-Ras has been shown to down-regulate integrin α2 expression. Our results indicate that high Ras activity and/or loss of integrin α2 may be a marker of prostate cancer bone metastasis.

Disclosures: J. Yin, None.

This study received funding from: NCI.

M302

Up-regulation of TACE in Monocytes Ameliorates their Deflected Differentiation into Osteoclasts and Dendritic Cells in Myeloma.

M. Abe¹, M. Hiasa*², S. Kido¹, K. Takeuchi*¹, K. Kitazoe*¹, A. Oda*¹, H. Amou*¹, K. Kagawa*¹, T. Hashimoto*¹, S. Ozaki*¹, T. Matsumoto¹. ¹Department of Medicine and Bioregulatory Sciences, University of Tokushima Graduate School of Medicine, Tokushima, Japan, ²Orthodontics and Dentofacial Orthopedics, University of Tokushima Graduate School of Medicine, Tokushima, Japan.

Monocytes are a common precursor for both osteoclasts (OC) and dendritic cells (DC). M-CSF and RANK ligand induce osteoclastogenesis from monocytes, while GM-CSF and IL-4 in combination trigger the differentiation of DC. We demonstrated that GM-CSF and IL-4 up-regulate TNF-alpha converting enzyme (TACE) expression and activity in monocytes, which cleaves membrane-bound M-CSFR to disrupt M-CSF signaling and inhibit induction of osteoclastogenesis. Multiple myeloma (MM) cells affect the reciprocal regulation of monocytic differentiation into OC and DC lineages towards OC lineage to cause devastating bone destruction and immune suppression in MM. In the present study, we therefore explored a role of up-regulation of TACE in monocytes in the skewed induction of OC and DC differentiation by MM cells. GM-CSF and IL-4 up-regulated the levels of soluble M-CSFR in the co-cultures of monocytes with U266 MM cells. Addition of TAPI-0, a TACE inhibitor, substantially reduced the soluble M-CSFR levels enhanced by GM-CSF and IL-4, suggesting that up-regulation of TACE by GM-CSF and IL-4 mediates cleavage of cell-surface M-CSFR on monocytes in the presence of MM cells. Importantly, the GM-CSF and IL-4 treatment also disrupted osteoclastogenesis in rabbit bone cell cultures enhanced by U266 MM cells as well as by M-CSF and RANK ligand. Addition of TAPI-0 restored the MM cell-mediated osteoclastogenesis suppressed by GM-CSF and IL-4. In contrast, TAPI-0 potently suppressed CD1a+ DC differentiation induced by GM-CSF and IL-4 in the presence of MM cells. These results suggest that up-regulation of TACE by GM-CSF and IL-4 is able to disrupt osteoclastogenesis enhanced by MM cells, while inducing DC formation. Therefore, GM-CSF and IL-4 in combination or other alternatives to up-regulate endogenous TACE activity in monocytes may have a potential as a novel therapeutic maneuver to drive into DC lineage the monocytic differentiation deflected to OC lineage in MM.

Disclosures: M. Abe, None.

M303

See Sunday Plenary Number S303

M304

In Vitro Effects of Zoledronic Acid Alone or in Combination with Radiation Therapy on Cells of the Metastatic Bone Environment.

S. A. Arrington, B. S. Margulies*, E. R. Fisher*, M. J. Allen. Orthopedic Surgery, SUNY Upstate Medical University, Syracuse, NY, USA.

The specific aim of this current study was to determine the effect of zoledronic acid (ZA) on individual cell types involved in the metastatic bone environment. In addition we wanted to analyze whether ZA increased breast cancer cell sensitivity to radiation therapy. Mouse marrow stromal cells (ST2), mouse osteoblast-like cells (MC3T3), and human breast cancer cells (F10) were cultured with up to 100μM of ZA for two and seven days. Additionally, F10 cells were treated 24 hours after seeding with radiation (0 Gy to 20 Gy) alone or in combination with 5 μM ZA. Cells were assayed 7 days post-treatment. Cell viability was measured using an MTT assay. Treatment affects on cell cycle were analyzed using flow cytometry. ZA caused a dose-dependent decrease in cell viability with IC₅₀ ranging from 55 μM (F10 cells) to 7 μM (ST2 cells) and 5 μM (MC3T3 cells) 7 days post-treatment. This indicates that F10 breast cancer cells are less sensitive to ZA. F10 cells treated with 5 Gy radiation in combination with ZA showed significant decreases in cell viability compared to cells treated with 5 Gy alone (p=0.0406). Cell cycle analysis revealed G2/M arrest in ST2 cells treated with 5 μM ZA, where as F10 cells displayed an increase in G0/G1. F10 cells treated with radiation showed a dose dependent increase in G2/M arrest, which was further increased by the addition of 5 μM ZA (Figure 1). These results indicate that when F10 cells are exposed to ZA, they may be blocked from entering S-phase. There was a dramatic increase of cells in G2/M after treatment with 10 Gy and 5 μM ZA, compared to treatment with 10 Gy alone. Taken together these results indicate that even though breast cancer cells are less sensitive to the effects of ZA, compared to marrow stromal cells and osteoblasts, ZA is still effective in causing disruptions to the cell cycle and decreasing cell viability. These effects are further enhanced when ZA is combined with radiation therapy. These findings correlate well with previous work in our mouse model, which also demonstrated decreased tumor damage in tumor-burdened bones treated with radiation and ZA compared to radiation alone.

Disclosures: S.A. Arrington, None.

This study received funding from: CDMRP Department of Defense.

M305

See Sunday Plenary Number S305

M306

Anti-tumor Actions of 2-Methoxyestradiol Is Accompanied by an Increase in Osteoprotegrin Expression in Osteosarcoma Cells.

M. Benedikt*, J. P. Szatkowski*, K. L. Shogren*, G. Sarkar*, M. J. Yaszemski, A. Maran. Orthopedic Research, Mayo Clinic, Rochester, MN, USA.

Osteosarcoma is the most common bone malignancy that primarily affects children and young adults. About 30% of patients diagnosed with osteosarcoma will develop metastatic disease. Although a combination of surgery and adjunctive chemotherapy has resulted in improved survival rates, the mortality rate remains very high. 2-Methoxyestradiol (2-ME), a naturally occurring metabolite of 17β-estradiol has been implicated in the inhibition of tumor cell proliferation. Previous results show that 2-ME did not affect the growth of normal osteoblasts in vivo and in vitro, but inhibited the proliferation and induced apoptosis in osteosarcoma cells. Several recent studies have demonstrated that the expression of bone remodeling proteins osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL) are altered in different human cancers. To investigate whether the OPG/RANKL system is involved in the anti-tumor actions of 2-ME, we studied the expression of OPG protein in 2-ME-treated human osteosarcoma cells. Western blot analysis showed that OPG protein levels increased by 2-fold in 2-ME (10μM)-treated MG63 osteosarcoma cells. Furthermore a tumorigenic estrogen metabolite, 16α-hydroxyestradiol which does not induce cell death, had no effect on OPG protein levels. Our results also show that 2-ME treatment increased OPG protein levels in TE85, KHOS, 143B, ROS, SAOS, LM8 human osteosarcoma cells. Thus, these findings suggest that there may be a close association between OPG regulation and 2-ME-mediated cell death in osteosarcoma cells.

Disclosures: M. Benedikt, None.

This study received funding from: National Institute of Health and Mayo Clinic.

M307

See Sunday Plenary Number S307

M308

C-terminal PTHrP(109–119) in Human Serum and Urine Is a Useful Biomarker for Malignancies.

D. Burton*¹, C. Chalberg*¹, K. C. Smith*¹, R. L. Fitzgerald², D. A. Herold*², L. J. Deftos¹. ¹Medicine, Veterans Administration San Diego Healthcare System and University of California, San Diego, CA, USA, ²Pathology, Veterans Administration San Diego Healthcare System and University of California, San Diego, CA, USA.

Since PTHrP is the most common oncoprotein secreted by human cancers it is a logical biomarker for many malignancies. However, most serum immunoassays for PTHrP can only detect this oncoprotein when it is markedly elevated, usually late in the course of the cancer. This lack of sensitivity limits the clinical value of contemporary PTHrP assays, both research and commercial. In order to address this limitation, we applied newly-developed PTHrP immunoassays to the measurement of PTHrP in the serum and urine of human subjects. We utilized PTHrP immunoassays with differing immunochemical formats to evaluate the PTHrP signature in the serum and urine of patients with calcium and skeletal disorders. In the present study, we collected serum or urine samples from over 200 patients that were either normo- or hypercalcemic (Ca²⁺ > 10.6 mg/dL). We employed three human PTHrP epitope-specific immunoassays directed against human PTHrP amino acids 1–34, 38–64 and 109–119 with assay limits of detection of 2.0, 0.6 and 0.8 fmol/tube, respectively. The PTHrP (1–34) (N-PTHrP) species was undetectable in all urine specimens tested, while the PTHrP(38–64) form averaged 0.025 pmol/L. The PTHP(109–119) (C-PTHrP) fragment was the most readily detectable form of PTHrP present in urine, averaging 0.52 pmol/L in our patient survey. Similar PTHrP fragment ratios were observed in the sera. Serum C-PTHrP levels ranged from undetectable to 3.1 nmol/L. Cancer patients had higher serum C-PTHrP levels than normal, non-tumor bearing subjects (p < 0.05). There was not a significant correlation between serum Ca2+ and serum C-PTHrP, which likely indicates inactivation by proteolysis of the hypercalcemia inducing N-PTHrP fragment. We next analyzed the serum and urine PTHrP from cancer patients using size-exclusion chromatography (Sephacryl S100 beads). Immunoreactive C-PTHrP peaks were observed in both fluids, but the N-PTHrP assay did not detect any PTHrP in the fractions. These results demonstrate the stability of the C-PTHrP species in biological fluids and the processing of the N-PTHrP species. Extension of our studies should lead to the development of testing procedures that may be useful in the early diagnosis and clinical management of patients with PTHrP-expressing cancers.

Disclosures: D. Burton, None.

This study received funding from: Department of Veterans Affairs.

M309

See Sunday Plenary Number S309

M310

The Ubiquitin-Proteasome Pathway Is Dysregulated in Myeloma Cells in the Bone Microenvironment In Vivo.

C. M. Edwards, J. A. Fowler, R. L. Caldwell, A. L. Bates*, J. R. Edwards, J. Zhang*, P. E. Foehr*, R. Parker*, A. Roberts*, G. R. Mundy. Vanderbilt Center for Bone Biology, Vanderbilt University, Nashville, TN, USA.

There is abundant evidence that myeloma cells are dependent on host cells for their aggressive behavior in vivo. Recent basic and clinical data shows that myeloma cells are exquisitely sensitive to proteasome inhibition, suggesting that the ubiquitin-proteasome pathway, which is present in all cells, is critical for myeloma cell growth and survival. We hypothesized that activity of the ubiquitin-proteasome pathway in myeloma cells may be increased by and dependent on host cells. We have examined this new concept using the well-characterized 5TGMI murine model of myeloma. 5TGM1-GFP cells were inoculated into C57BlKaLwRij mice, resulting in tumor growth within the bone marrow and development of the characteristic osteolytic bone disease of myeloma. 5TGM1-GFP cells were then isolated from the bone marrow of myeloma-bearing mice by FACS and compared with pre-inoculation cells. A significant increase in chymotrypsin-like proteasome activity was found in 5TGM1 myeloma cells following in vivo growth in bone, as compared to the pre-inoculation cells. Protein profiling by MALDI mass spectrometry revealed a number of regulated proteins, including a reduction in free ubiquitin in myeloma cells following in vivo growth in bone. This decrease in free ubiquitin expression was confirmed by western blotting. Real time PCR demonstrated no significant difference in expression of ubiquitin mRNA in myeloma cells following in vivo growth in bone, suggesting post-translational regulation of ubiquitin in the bone microenvironment. Consistent with an increase in proteasome activity, a decrease in expression of β-catenin, which is degraded by the proteasome, was also demonstrated in myeloma cells isolated from bone when compared to the pre-inoculation cells. Treatment of pre-inoculation 5TGM1 myeloma cells with conditioned media from 14M1 stromal cells, which were isolated from myeloma-bearing mice, demonstrated a significant increase in proteasome activity suggesting the involvement of a soluble factor released from the host microenvironment in mediating the increase in proteasome activity. Our data demonstrate that the ubiquitin-proteasome pathway is dysregulated in myeloma cells within the bone microenvironment in vivo, associated with a reduction in free ubiquitin and an increase in proteasome activity. This suggests that the ubiquitin-proteasome pathway in myeloma cells may be controlled by host factors, and that these host-tumor interactions may contribute to the survival of myeloma cells within the bone microenvironment in vivo.

Disclosures: C.M. Edwards, None.

M311

See Sunday Plenary Number S311

M312

Prevalence of Secondary Causes of Osteoporosis Among Breast Cancer Patients with Osteoporosis and Osteopenia.

P. M. Camacho¹, A. Dayal*¹, J. Diaz*¹, F. Nabhan*¹, M. Agarwal*², J. Norton*³, P. Robinson*⁴, K. S. Albain*⁴. ¹Endocrinology and Metabolism, Loyola University Chicago, Maywood, IL, USA, ²Medicine, Christ Hospital, Oak Lawn, IL, USA, ³Cancer Center, Loyola University Chicago, Maywood, IL, USA, ⁴Hematology/Oncology, Loyola University Chicago, Maywood, IL, USA.

Purpose: The main objective of this study was to determine the prevalence of secondary causes of osteoporosis among breast cancer patients being evaluated for osteopenia (low bone mass) and osteoporosis.

Methods: We conducted a retrospective chart review of 238 postmenopausal women consecutively referred to Loyola University Medical Center Endocrinology clinics from 2000–2005, for the management of osteoporosis or osteopenia. Patients were divided into two groups: those without a history of breast cancer or NBC group (N=174), and those with breast cancer or BC group (N=64). The BC group was comprised of patients with early stage breast cancer in the midst of or considering adjuvant hormonal therapy with aromatase inhibitors. Histories and biochemical data from their initial consultation were analyzed. Statistical analysis of each patient population was performed to elucidate the prevalence of secondary causes of osteoporosis in patients with breast cancer relative to the group of patients without breast cancer.

Results: The demographics of the two groups differed in age (64.2 ± 14.2 in NBC versus 59.5 ± 10.6 in BC group, p=0.015), mean weight (63.5 ± 13.7 in NBC versus 73.62 ± 20.95 kg in BC, p <0.001), 25 OH Vitamin D levels (28.7 ± 13.1 in NBC versus 34.03 ± 15.1 ng/ml in BC, p = 0.019) and degree of bone loss (spine T score of −1.966± 1.34 in NBC versus-0.918±1.41 in BC, p < 0.001). The presence of at least one secondary cause of osteoporosis was seen in 78.1% of the breast cancer patient group (excluding cancer-related therapies), and in 77% of the non-breast cancer group. Newly diagnosed metabolic bone disorders were seen in 57.8% of the breast cancer population. The most common secondary cause in both groups was vitamin D deficiency, which was seen in 37.5% of the breast cancer group and 51% of the non-breast cancer group. In the BC group, this was followed by idiopathic hypercalciuria (15.6% versus 8% in NBC, trend towards higher prevalence in BC than the NBC group p=0.085), normocalcemic hyperparathyroidism (3.1%) and primary hyperparathyroidism (1.6%).

Conclusion: We found a high prevalence of secondary causes of osteoporosis among breast cancer patients undergoing or considering adjuvant hormonal therapy with aromatase inhibitors. Previously published reports of bone loss and fractures seen in patients on such agents may have been partly due to the presence of these disorders. It is prudent to obtain a baseline DXA and to screen patients with breast cancer for secondary causes of bone loss.

Disclosures: P.M. Camacho, None.

This study received funding from: Proctor and Gamble.

M313

Environmental Influences on Variability in Levels of Bone Turnover Markers.

O. S. Donescu¹, M. C. Battie*², T. Videman*³. ¹Research, Toronto Rehabilitation Institute at University of Toronto, Toronto, ON, Canada, ²Physical Therapy, University of Alberta, Edmonton, AB, Canada, ³Rehabilitation Science, University of Alberta, Edmonton, AB, Canada.

Biochemical markers may capture the imbalance between bone formation and resorption and may also allow quantitative evaluation of rates of bone loss. Therefore, determinants of marker levels may also provide insights into factors influencing bone turnover and osteoporosis. The aim of the study was to estimate the influence of environmental factors (e.g. lifetime physical activities at work and leisure time, calcium intake, smoking, alcohol and coffee consumption) in determining procollagen type I amino-terminal propeptide (PINP), type I collagen carboxy-terminal telopeptide (ICTP) and urinary amino-terminal type I collagen telopeptide (NTx) marker levels in 147 monozygotic (MZ) and 153 dizygotic (DZ) male twin pairs.

Biochemical markers of bone turnover originating from type I procollagen synthesis or type I collagen breakdown were examined in men using a classic twin study design based on MZ and DZ twins. An extensive, structured interview was conducted to obtain data on exposure to suspected environmental and behavioral risk factors, such as recent and lifetime experiences of smoking, alcohol, coffee consumption, dietary calcium intake, lifetime leisure time activities and sport participation, sitting at work, job heaviness, as well as medical history and medication use. Clinical examinations of each subject were also conducted, including dual magnetic resonance imaging (MRI) of the lumbar spine, serum and urine samples (obtained over a one and a half-day period) and lifting tests. After excluding subjects with medical conditions or taking medications thought to influence biological variation of markers, 98 MZ and 108 DZ male twin pairs were left. Subjects age ranged from 40–56 years (48 +/- 7.9) for MZ and 42–56 (49+/-6.7) for DZ twins.

The effect of dietary calcium, smoking, coffee and alcohol, as well as lifetime physical activity (e.g. leisure time activities, sport participation, occupational loading) were not significant determinants of biochemical marker levels in this group of adult men. The genetic and environmental variance in bone markers PINP, NTx and ICTP was largely independent from the environmental or behavioral factors studied, suggesting that these factors have a minor influence in bone formation and degradation in men. In conclusion, possible interventions focusing on these behavioral factors in adulthood might be of limited value in targeting bone resorption or formation.

Disclosures: O.S. Donescu. None.

M314

Monitoring Therapy: Are Bone Resorption Markers (BRM) During Osteoporosis Treatment with Bisphosphonates Within the Lower Half of the Premenopausal Range?

D. A. Eekman*¹, H. J. G. M. Derikx*², I. E. M. Bultink*¹, A. P. van Zanten*³, W. F. Lems¹. ¹Rheumatology, VU University Medical Center, Amsterdam, The Netherlands, ²Rheumatology, Slotervaart Hospital, Amsterdam, The Netherlands, ³Clinical Chemistry, Slotervaart Hospital, Amsterdam, The Netherlands.

Background and objectives: Although oral bisphosphonates (OB) are efficacious, adherence to anti-osteoporotic treatment is generally poor. It has been been suggested that adequate monitoring of the effects of therapy with BRM has a positive effect on adherence (1). We observed in which proportion of patients, diagnosed with osteoporosis and treated with an OB, the urinary excretion of the total fraction of deoxypyridinoline (DPD) is in the lower half of the premenopausal range. (2)

Patients and methods: all consecutive patients visiting the osteoporosis outpatient clinic of the Slotervaart hospital using an OB for at least three months were enrolled. Urine samples were collected by asking the patients to collect their fasting second morning-void. Total DPD was determined by high performance liquid chromatography using commercial reagents (Chromsystems, München, Germany). Day to day variation of this technique is lower than 8%. The mean (SD) urinary excretion of DPD is 9.3 (2.2) μmol/mmol creatinine, based on fasting second morning-void samples of 35 healthy premenopausal women at 3 consecutive moments.

Results: 38 patients were enrolled, 31 women and 7 men, mean age (SD) 69 (11) years. The mean BMD (SD) of the spine and hip was 0.800 (0.147) g/cm² and 0.704 (0.086) g/cm², respectively. All patients were treated with bisphosphonates: alendronate (22), risedronate (11), ibandronate (5).

The mean (SD) urinary excretion of DPD was 8.86 (6.44) μmol/mmol creatinine. In 71% of the patients the DPD excretion was below the premenopausal mean (<9.3 μmol/mmol creatinine).

Conclusion: In our ongoing study in patients with osteoporosis chronically treated with bisphosphonates, bone resorption was within the premenopausal range in more than 70% of the patients: the (biological) target of therapy.

Our data illustrate that measuring BRM is feasible in a non-academic outpatient clinic, and suggest that measurement of urinary excretion of DPD might be a valuable tool to improve adherence to therapy.

Reference:

1) Delmas E.P. et al J Clin Endocrinol Metab. 2007 Apr;92(4): 1296-304

Disclosures: D.A. Eekman, None.

M315

Changes in Bone Resorption Markers During Osteoporosis Treatment with Oral Bisphosphonates.

D. A. Eekman*¹, H. J. G. M. Derikx*², I. E. M. Bultink*¹, A. P. van Zanten*³, W. F. Lems¹. ¹Rheumatology, VU University Medical Center, Amsterdam, The Netherlands, ²Rheumatology, Slotervaart Hospital, Amsterdam, The Netherlands, ³Clinical Chemistry, Slotervaart Hospital, Amsterdam, The Netherlands.

Background: Adherence to anti-osteoporotic treatment is generally poor; it has been suggested that monitoring therapy with measurement of bone resorption markers (BRM) might improve it. (1) In large observational studies it has been shown that already after 3 months of therapy, BRM can be decreased by more than 30%. (1) Several BRM are available; we investigated the changes in the total fraction of deoxypiridinoline (DPD) during treatment with bisphosphonates.

Objectives: to observe in daily practice in which proportion of patients, diagnosed with osteoporosis and subsequently treated with bisphosphonates, the urinary excretion of DPD has decreased by 30% or more, at least 3 months after starting with therapy.

Patients and methods: in this ongoing project, all consecutive patients who come to the osteoporosis outpatient clinic of the Slotervaart Hospital and start with bisphosphonates are enrolled. Urine samples are collected by asking the patients to collect their second fasting urine. DPD is determined by high performance liquid chromatography using commercial reagents (Chromsystems, München, Germany). Day to day variation of this technique is lower than 8 %. Reference values were determined in a group of 35 healthy premenopausal women as 6.6–15.4 μmol DPD / mmol creatinine.

Results: so far 19 patients are enrolled, 18 women and 1 man, mean (SD)age 67 (15) years. The mean BMD of the spine was 0.806 g/cm² and of the hip 0.699 g/cm². All patients started with bisphosphonates: alendronate (7), risedronate (5), ibandronate (6) and pamidronate (1). Seventy-nine percent of the patients were treated with calcium/vitamin D.

During anti-osteoporotic treatment the mean (SD) decrease in urinary excretion of DPD was 36% (18). In 12 of the 19 patients (63%), the urinary excretion decreased by more than 30%. In 6 of the 7 patients we found an explanation for the non-occurrence of a decrease in bone resorption: secondary osteoporosis in 3 and in 3 patients it appeared that they had not taken their medication adequately.

Conclusion: In 63 % of the patients we found a favourable bone marker response. In the others we found either a cause for secondary osteoporosis, or a failure in adherence to therapy. We think that this ongoing project (so far in a limited number of patients) emphasizes that it might be useful to use the markers of bone resorption during osteoporosis treatment with bisphosphonates in daily practice.

Reference:

1) Delmas E.P. et al J Clin Endocrinol Metab. 2007 Apr;92(4):1296-304

Disclosures: D.A. Eekman, None.

M316

See Sunday Plenary Number S316

M317

Markers of Bone Turnover in Peripubertal Girls.

K. M. Fagerlund*¹, M. Lehtonen-Veromaa*