ASBMR 29th Annual Meeting


T083

Phenotypical Heterogeneity Between Osteoclasts Involved in the Endochondral Ossification and Bone Remodeling.

S. Soeta, Y. Izu, S. Kamiya*, H. Amasaki*. Veterinary Anatomy, Nippon Veterinary Life Science University, Musashino-City, Tokyo, Japan.

Osteoclasts at the chondro-osseous junction of the epiphyseal growth plate play an essential role for replacement of cartilage with bone tissues in the endochondral ossification, and contribute to the longitudinal growth of long bones. Therefore, they are thought to be controlled by a different regulatory mechanism from osteoclasts resorbing bone tissues in the modeling and remodeling process. The purpose of this study is to clarify functional heterogeneity between osteoclasts involved in the endochondral ossification and in the modeling and remodeling process in the long bones. We investigated the expression of RANK, RANKL, Flk-1 (VEGF receptor 1), MMP-9, and MMP-13 immunohistochemically in osteoclasts in the tibiae of new-bome and young male rats. Osteoclasts, detected as TRAP-positive multinucleated giant cells, distributed throughout from epiphysis to diaphysis in the tibiae in rats aged 1 to 42 days. In rats aged 1 to 28 days, osteoclasts at the chondro-osseous junction showed RANK, RANKL and MMP-9-immunoreactivities, but not Flk-1 and MMP-13-immunoreactivities. In rats aged 35 and 42 days, however, osteoclasts at the chondro-osseous junction showed Flk-1 and MMP13-immunoreactivities in addition to RANK, RANKL and MMP-9-immunoreactivities. Osteoclasts on the surface of trabecular bones in the diaphysis showed RANK, RANKL, Flk-1, MMP-9 and MMP-13 immunoreactivities in rats aged 1 to 42 days. From the immunohistochemical observations, osteoclasts may be recruited to the chondro-osseous junction without VEGF-Flk-1 pathway, and absorb the cartilage tissue of the epiphyseal growth plate independent on MMP-13 in the growing period. Therefore, osteoclasts involved in the endochondoral ossification in the epiphyseal growth plate may be regulated in a different manner from osteoclasts resorbing bone tissues in the modeling and remodeling process.

Disclosures: S. Soeta, None.

T084

Nicotine and Lipopolysaccharide Stimulate the Formation of Osteoclast-like Cells by Increasing Macrophage Colony-Stimulating Factor and Prostaglandin E2 Production by Osteoblasts.

H. Tanaka*1, N. Tanabe*1, T. Kawato*1, T. Katono*1, A. Namba*2, N. Suzuki*3, M. Motohashi*1, M. Maeno*1. 1Department of Oral Health Sciences, Nihon University School of Dentistry, Tokyo, Japan, 2Department of Oral and Maxillo facial surgery, Nihon University School of Dentistry, Tokyo, Japan, 3Department of Biochemistry, Nihon University School of Dentistry, Tokyo, Japan.

Several studies have indicated that one of the causes of alveolar bone destruction with periodontitis is lipopolysaccharide (LPS) from the cell wall of Gram-negative bacteria in plaque and that tobacco smoking may be an important risk factor for the development and severity of periodontitis. The present study was undertaken to determine the effect of nicotine and LPS on the expression of macrophage colony-stimulating factor (M-CSF), osteoprotegerin (OPG), and prostaglandin E2 (PGE2) in osteoblasts, and the indirect effect of nicotine and LPS on the formation of osteoclast-like cells. Saos-2 cells were cultured with 10−3 M nicotine, or 1 or 10 μg/ml LPS and 10−3 M nicotine, for up to 14 days. The gene and protein expression of M-CSF and OPG were determined using real-time PCR and ELISA, respectively. PGE2 expression was determined using ELISA. The formation of osteoclast-like cells was estimated using tartrate-resistant acid phosphatase (TRAP) staining of osteoclast precursors in culture with conditioned medium from nicotine and LPS-treated Saos-2 cells and the soluble receptor activator of NF-kB ligand (RANKL). M-CSF and PGE2 expression increased markedly in cells cultured with nicotine and LPS compared with those cultured with nicotine alone. OPG expression increased in the initial stages of culture with nicotine and LPS but decreased in the later stages of culture. The conditioned medium containing M-CSF and PGE2 produced by nicotine and LPS-treated Saos-2 cells with soluble RANKL increased the TRAP staining of osteoclast precursors compared with that produced by nicotine treatment alone. These results suggest that nicotine and LPS stimulate the formation of osteoclast-like cells via an increase in M-CSF and PGE2 production and that the stimulation is greater than with nicotine treatment alone.

Disclosures: H. Tanaka, None.

T085

VGLUT1 KO Mice Develop Osteopenia due to an Increase in Osteoclastic Bone Resorption.

S. Uehara*1, S. Senoh*2, M. Nakamura1, T. Ninomiya*3, T. Mizoguchi*3, Y. Kobayashi3, Z. Hua*4, R. H. Edwards*4, Y. Moriyama*2, N. Takahashi3, N. Udagawa1. 1Department of Biochemistry, Matsumoto Dental University, Shiojiri, Japan, 2Department of Membrane Biochemistry, Okayama University, Okayama, Japan, 3Institute for Oral Science, Matsumoto Dental University, Shiojiri, Japan, 4Departments of Neurology and Physiology, University of California San Francisco School of Medicine, San Francisco, CA, USA.

The vesicular glutamate transporter (VGLUT) is responsible for vesicular storage of L-glutamate, and plays an essential role in L-glutamate-mediated chemical transduction in the central nervous system and some peripheral tissues. We previously reported the role of VGLUT in bone metabolism as follows (EMBO J, 25:4175, 2006): (1) VGLUT1 was expressed in the transcytotic vesicle in osteoclasts, and osteoclasts secreted both L-glutamate and bone degradation products upon depolarization or ATP stimuli in Ca2+-and cAMP-dependent manners. (2) Osteoclasts expressed the metabotropic glutamate receptor (mGluR)8. L-glutamate secreted by osteoclasts inhibited the transcytosis and bone resorbing activity of osteoclasts through the mGluR8 in an autocrine manner. (3) VGLUT1 knock out (KO) mice developed osteopenia. In this study, we investigated the mechanism of L-glutamate secretion from osteoclasts and the cause of osteopenia induced by VGLUT1 deficiency in mice in more detail. The results obtained from this work are as follows: (1) we first examined whether the signaling pathway of cAMP-dependent protein kinase (PKA) is involved in the secretion of L-glutamate from osteoclasts. Osteoclasts obtained from the co-culture of mouse osteoblasts and bone marrow cells were treated with KT-5720, a PKA inhibitor, and stimulated with KCl or ATP to induce L-glutamate secretion. KT-5720 scarcely inhibited the KCl and ATP-induced L-glutamate secretion from osteoclasts. (2) To determine the cause of bone loss in VGLUT1 deficiency in mice, we measured serum levels of TRACP5b (a marker of bone resorption) and alkaline phosphatase (a marker of bone formation) in 8-week-old VGLUT1 KO and wild-type mice. Serum levels of TRACP5b in VGLUT1 KO mice were significantly higher than those in wild-type mice. Serum alkaline phosphatase activities were also increased in VGLUT1 KO mice. These results suggest that osteoclasts secrete L-glutamate through PKA independent pathway, and that bone loss in VGLUT1 KO mice is caused by an increase in bone resorption. The results also suggest that the lack of L-glutamate-mediated signals in bone induces a high turnover bone state. Bone histomorphometry of VGLUT1 KO mice is currently analyzed in our laboratories.

Disclosures: S. Uehara, None.

T086

Tumor Cells Metastatic to Bone Express CSF-1 and Support Osteoclast Survival.

K. Yagiz, S. R. Rittling.. Cytokine, Forsyth, Boston, MA, USA.

Tumors metastatic to the bone produce factors that cause massive bone resorption mediated by osteoclasts in the bone microenvironment. The mechanisms that explain the increase in bone resorption in metastasis are unclear. We have hypothesized that osteoclasts in the vicinity of tumor cells are “superactivated”: superactivated osteoclasts are larger than normal osteoclasts with higher resorptive ability. Colony stimulating factor (CSF-1) is strictly required for the formation and survival of active osteoclasts. Aberrant expression of CSF-1, which can be synthesized as a soluble or a membrane-associated protein, has been found in many cancers correlating with disease progression and malignancy. Here we show that r3T cells, a tumor cell line that forms bone metastasis in mice, express abundant CSF-1 in vitro. FACS analysis with an antibody to CSF-1 as well as RT-PCR confirmed that r3T cells express CSF-1 both a secreted and a membrane-associated protein. Murine osteoclast-like cells co-cultured with either mitomycin-c (MMC) treated or glutaraldhyde fixed r3T cells with cytokine RANKL survived in the absence of added CSF-1. In addition, murine osteoclast-like cells were cultured with r3T conditioned medium (CM). Our results showed that r3T cells supported osteoclast formation by providing adequate amounts of CSF-1 protein. Importantly, direct cell-to-cell contact was required for osteoclast survival. Therefore, drugs that could counteract the effect of CSF-1 might be useful for reducing the osteoclast mediated osteolysis. We suggest that tumor cell production of CSF-1 is one of the mechanism by which tumors cause osteoclast superactivation.

Disclosures: K. Yagiz, None.

T087

Hormonal and Pharmacological Modulation of MT2 Melatonin Receptor Inhibits Osteoclast Maturation and Activity.

G. Maillot*1, C. Desrochers*2, R. Doucet*2, E. Levy*1, A. Moreau3. 1Department of Nutrition, Université de Montréal, Research Center CHU Sainte-Justine, Montreal, PQ, Canada, 2Research Center CHU Sainte-Justine, Montreal, PQ, Canada, 3Department of Stomatology & Biochemistry, Université de Montréal, Research Center CHU Sainte-Justine, Montreal, PQ, Canada.

Melatonin (Mel) is known to induce bone formation through osteoblast activation and indirectly through OPG up regulation leading to osteoclast inactivation. However, a direct effect of Mel on osteoclast has never been shown previously. The aim of this study is to investigate the influence of the Mel signaling pathway on osteoclastogenesis and osteoclast activity using in vitro and in vivo models as experimental paradigm. Gene and protein expression of Mel receptor, MT2, were detected in RAW 264.7 cells cultured in presence or absence of RANKL (50ng/ml) whereas MT1 receptor was not detected. Addition of physiological doses of Mel (10−9M) decreased by 60% the number of resorption pits and by 69% the total resorbed area although no significant change was observed in the number of TRAP+ cells. Addition of 4-P-PDOT, a MT2 receptor antagonist, blocks the inhibitory action of Mel suggesting that such effect was mediated through Mel signaling activity. Surprisingly, RAW 264.7 cells treated with increasing doses of 4-P-PDOT (10−10M to 10−4M) alone demonstrated a significant decrease in both number of TRAP+ multinucleated cells and resorption pits in a dose dependent-manner. These effects were neither caused by an increased apoptosis nor due to reduced proliferation since no changes were observed between untreated and 4-P-PDOT treated cells. To further investigate the effect of 4-P-PDOT on bone metabolism, we injected 4-P-PDOT (10mg/kg of body weight) after weaning to male C57B1/6j mice 3 times per week during 4 weeks and performed densitometric (PixiMus II bone densitometer) and histomorphometric analyzes. Preliminary results revealed an increase in bone mineral accretion at the spine and whole body level in treated mice compared to vehicle-injected animals. Overall, these data highlight the importance of the Mel signaling pathway in modulating osteoclast function and show an unrecognized effect of Mel receptor antagonist 4-P-PDOT as a potential anti-osteoporotic drug.

Disclosures: G. Maillot, None.

This study received funding from: Cotrel Foundation Institut de France.

T088

The Osteopetrotic Incisor Absent Rat Exhibits Reduced Osteoclast Activity, Resulting in Normal Endochondral Fracture Union but Delayed Hard Callus Remodeling.

M. M. McDonald*1, A. Morse*1, K. Mikulec*1, C. Godfrey*1, S. Tamara*2, D. Little*1. 1Orthopaedic Research, The Children's Hospital Westmead, Sydney, Australia, 2Science, The University of Technology, Sydney, Australia.

A spontaneous mutation in the incisor absent (ia/ia) rat results in abnormal osteoclasts which display deficient resorption. We investigated the resulting osteopetrotic phenotype to determine the exact age of recovery. Furthermore, we utilized this model to examine fracture repair in the absence of functional osteoclasts.

Samples were harvested from male rats from 3 weeks of age for phenotype analysis. Once the age of recovery was determined, closed fractures were performed in 5 week old male ia/ia and wt/het controls. Harvests were performed at 1, 2 and 3 weeks post fracture, to assess repair within the period of osteoclast dysfunction.

Serum CTX showed decreases up to 9 weeks of age in ia/ia rats (p<0.05), with normalization to control levels at 12 weeks of age. Further, ia/ia primary osteoclast cultures revealed decreased resorption pit formation at 5 and 9 weeks of age compared to control (p<0.01), again normalizing to control levels at 12 weeks.

DEXA scans of tibial metaphyses revealed increases of 40–114% in BMD up to 20 weeks of age in ia/ia rats compared to controls (p<0.01). Histology at this site revealed a constant incline in BV/TV at the proximal region in ia/ia rats such that a 3-fold increase was achieved by 20 weeks compared to controls (p<0.01). In the distal tibial metaphysis however, the BV/TV in the ia/ia rats began to decrease after 9 weeks of age such that it had reduced by half at 20 weeks, suggesting recovery of resorption.

Femur length showed an 11%-14% decrease in ia/ia compared to control rats at all time points examined from 3 weeks of age (p<0.01). However, growth plate height showed a slight increase in ia/ia rats at only 5 weeks of age compared to controls (p<0.05), all other time points showing no differences.

Importantly, initial endochondral fracture union was achieved by 3 weeks post fracture in both ia/ia and controls. QCT revealed significant increases in BMC and volume in ia/ia calluses at 1, 2 and 3 weeks (p<0.01). Controls showed a reduction in callus volume between 2 and 3 weeks, whereas the ia/ia callus volume increased 41%.

In conclusion, the ia/ia rat exhibits a severe osteopetrotic phenotype resulting from reduced osteoclast activity. Recovery from the dysfunctional resorption occurs between 9 and 12 weeks of age. Initial endochondral fracture union was achieved normally in the absence of functional osteoclasts in ia/ia rats, however hard callus remodeling was hindered resulting in a larger, more mineralised callus. This study reveals that osteoclast function is not essential for initial fracture union but is required for hard tissue remodeling.

Disclosures: M.M. McDonald, None.

T089

Normal Endochondral Bone Healing Follows Continuous Bisphosphonate Pre-treatment in a Rat Closed Fracture Model.

A. Morse*, M. McDonald*, K. Mikulec*, H. Mai*, C. Munns, D. G. Little*. Orthopaedic Research, The Children's Hospital Westmead, Sydney, Australia.

Long-term Pamidronate (PAM) therapy is the mainstay of treatment for moderate to severe osteogenesis imperfecta (OI). However, these patients can develop a reduced bone turnover state that may affect fracture healing. We aimed to investigate the effects of continuous PAM pre-treatment on bone healing in a rat model of endochondral fracture repair.

9 week old Wistar rats received closed femoral fractures. 4 weeks prior to fracture PAM dosing commenced subcutaneously (SC) at 0.15mg/kg, 0.5mg/kg and 5 mg/kg twice weekly. Harvests were at 2 and 6 weeks post-fracture.

Radiographic union was observed in all 6 week samples and QCT showed increases of 55–92% in callus BMC and 44–77% in callus volume with PAM treatment over saline controls (p<0.01). These increases were attributed to retention of hard callus, with increases in primary callus BV/TV of 68%–84% at 6 weeks compared to saline (p<0.01). In addition, remodeling of the callus was delayed with decreases of 32% and 45% in callus remodeled neo-cortical bone in the 0.5mg/kg and 5mg/kg PAM groups respectively compared to saline (p<0.01).

Histology revealed that endochondral fracture healing followed a normal timeline regardless of PAM treatment. No differences were observed between groups in callus cartilage content at both 2 and 6 weeks post fracture. One 5mg/kg PAM sample showed retained cartilage at 6 weeks, but had no effect on the overall result.

Systemically, PAM dosing lead to increases of up to 4-fold in metaphyseal BV/TV over saline controls at 6 weeks (p<0.01). Endosteal mineral apposition rate (MAR) was reduced by 6 weeks with PAM treatment, the 0.5mg/kg and 5mg/kg groups showing reductions of 30% and 84% respectively compared to saline (p<0.01). Periosteal MAR however showed no changes with PAM treatment at 6 weeks. Small growth deficiencies of 2–5% in femur length were noted with 0.5 mg/kg or 5 mg/kg PAM compared to saline (p<0.01). The growth rate between the 2 and 6 week time points however remained constant, regardless of treatment. Growth plate height was also unaffected by PAM dosing.

In conclusion, continuous PAM pre-treatment was associated with a small decrease in bone formation and a large reduction in bone resorption, leading to a net increase in callus size. However, this lowered bone turnover did not hinder fracture repair, with endochondral union being achieved normally regardless of treatment. These data suggest that long-term BPs, whilst reducing bone turnover, may not interfere with normal endochondral fracture union in healthy individuals, but may hinder long bone growth. Further work is planned with a longer interval of pre-dosing with Pamidronate as well as experiments in animal models of Osteogenesis Imperfecta.

Disclosures: A. Morse, None.

T090

Anti-GGT Antibody Attenuates Osteoclastic Bone Erosion in CIA Mice.

Y. Ishizuka*1, S. Moriwaki*2, S. Niida2. 1AC Bio Techinologies, Yokohama, Japan, 2Bone & Joint Disease, National Center for Geriatrics & Gerontology, Obu, Japan.

gamma-Glutamyl transpeptidase (GGT/CD224), known as a key enzyme for the catabolism of glutathione and cysteine metabolism, is a newly identified bone-resorbing factor which stimulates osteoclast formation. We found that GGT expression was up-regulated in the inflamed joints of the patients with rheumatoid arthritis (RA). Therefore, we hypothesized that GGT may be involved in the arthritis-related osteolysis. In this study, we demonstrate that the inhibitory effects of anti-GGT monoclonal Ab (GGT-mAb) on the joint destruction in the collagen-induced arthritis (CIA) mice.

Materials and Methods: GGT localization in the synovium of RA patients and CIA mice was determined by immunohistochemistry. In vivo osteoclastogenic activity of GGT was assessed by histological analysis of the rat alveolar bone after infusing the recombinant human GGT (rhGGT) into the jaws. For preparation of CIA mice, DBA/1J mice were immunized with an emulsion containing bovine type II collagen and Mycobacterium tuberculosis in incomplete Freund's adjuvant. GGT-mAbs were generated against rhGGT using BALB/c mice. The GGT-mAbs were administrated via i.p. to CIA mice. The therapeutic effects of GGT-mAbs were evaluated by incidence score, arthritic score and histopathological analysis. Role of GGT in osteoclast formation was investigated using cell cultures, microarray analysis and quantitative RT-PCR.

Results: Immunohistochemistry of the inflamed synovium revealed that GGT was detectable in lymphocytes, plasma cells, and macrophages, as well as capillary vessels. The rhGGT-treated rats exhibited marked alveolar bone destruction with an increase in TRAP-positive osteoclasts. Treatment with GGT-mAbs significantly reduced the number of osteoclasts and the severity of osteolysis in CIA mice. In vitro examinations using bone marrow cultures containing 1,25(OH)2D3 indicated that the osteoclast development was enhanced through the further up-regulation of RANKL expression mediated by GGT stimulation. Moreover, addition of rhGGT accelerated the osteoclastogenesis in the cultures of bone marrow macrophages maintained with M-CSF and RANKL as compared with those of condition without rhGGT.

Conclusion: This study revealed the involvement of GGT in the bone erosion of CIA mice and contributed to further understanding of the arthritis-related bone loss. Thus, GGT antagonists may be new effective agents for attenuating the joint destruction in RA.

Disclosures: S. Niida, None.

T091

Electroneutral Sodium/Bicarbonate Cotransporter NBCn1 Is Expressed in Rat Osteoclast Ruffled Border Membrane.

R. Riihonen1, I. Song*2, S. Nielsen*3, T. Laitala-Leinonen*1, T. Kwon*2. 1Bone Biology Research Consortium, Department of Biomedicine, University of Turku, Turku, Finland, 2Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Daegu, Republic of Korea, 3The Water and Salt Research Center, Institute of Anatomy, University of Aarhus, Aarhus, Denmark.

The aim of this study is to define the expression of the electroneutral sodium/bicarbonate cotransporter NBCn1 in rat osteoclasts and its role for bone resorption. NBCn1 has previously been shown to significantly enhance the cellular uptake of bicarbonate on the basolateral side of the thick ascending limb of the loop of Henle in the kidneys in response to chronic metabolic acidosis, but its function in osteoclasts is unknown.

First, the expression pattern of NBCn1 in bone tissue was determined by analyzing 3-day old rat pup femur and tibia with immunohistochemistry. Additionally, confocal microscopy was utilized to explore the specific localization of NBCn1 in rat osteoclasts. RT-PCR was performed to study NBCn1 mRNA expression in primary rat osteoclasts cultured on bovine bone slices. Moreover, changes of NBCn1 mRNA levels were monitored after exposing osteoclasts to various stimuli such as anoxic conditions, acidic or alkalic culture media and growth factors known to increase the bone resorption rate.

NBCn1 immunostaining was specifically associated with the osteoclast cell membrane facing the bone trabeculae in tissue specimens, and immunocytochemistry revealed a similar expression profile where NBCn1 was exclusively localized in the ruffled border membrane in rat osteoclasts. Using RT-PCR, NBCn1 mRNA expression was detected from rat osteoclast cultures, and the expression levels varied markedly due to changes in the extracellular culture environment.

In conclusion, NBCn1 is localized in the ruffled border membrane of rat osteoclasts and the mRNA expression levels are dependent on the extracellular conditions and the activation status of the osteoclasts. These results suggest a role for NBCn1 in bone resorption, possibly by maintaining an acidic pH in the resorption lacuna by means of bicarbonate uptake during the active bone resorption phase. Thus, we may have found a novel, important player in the osteoclastic bone resorption process and will continue this study by exploring the effects of NBCn1 inhibition on the bone resorption rate.

Disclosures: R. Riihonen, None.

T092

TSG-6, a New Regulator of Bone Remodelling.

D. Mahoney*,1, K. Mikecz*,2, G. Mabilleau*,1, N. A. Athanasou1, T. Ali*,3, C. M. Milner*,3, A. J. Day*,3, A. Sabokbar1. 1Botnar Research Centre, University of Oxford, Oxford, United Kingdom, 2Rush University Medical School, Chicago, IL, USA, 3Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.

TSG-6 (TNF-stimulated gene-6) is a non-constitutively expressed protein which is upregulated by inflammatory mediators and growth factors. The protein is composed almost entirely of LINK and CUB domains; to date a wide variety of protein and glycosaminoglycan ligands have been identified as associating with the LINK module. Animal models of arthritic disease have indicated TSG-6 has anti-inflammatory properties and may protect against cartilage degradation and bone erosion. To determine the mechanism by which TSG-6 affects bone we have characterised; (i) the in vitro effect of TSG-6 on osteoclast activity and osteoblast differentiation, (ii) its mode of action and (iii) its levels in the synovial fluids of inflammatory bone disorders.

Using ELISA and BIAcore analysis, we have shown that TSG-6 binds to a mediator of osteoclastogenesis, sRANKL, as well as BMPs-2, 4, 5, 6, 7, 13 and 14 which promote osteoblastogenesis. In vitro assays indicate that TSG-6 inhibits RANKL-induced human osteoclast formation and activity in a dose-dependent fashion, similar in manner to that of OPG. Furthermore, bone marrow cells isolated from the long bones of TSG-6 deficient mice, give rise to markedly increased resorption as compared to cells isolated from control animals. We have also shown that TSG-6 inhibits the BMP-2 induced alkaline-phosphatase activity of an osteoblastic cell line. Full-length TSG-6 inhibits osteoclastogenesis and BMP-mediated osteoblast differentiation to a greater extent that the isolated LINK and CUB domains: affinity analysis suggests sRANKL and BMP2 interact at composite binding surfaces between the LINK and CUB modules. Quantification of TSG-6 in the synovial fluid of patients with rheumatoid arthritis, osteoarthritis, pyrophosphate arthropathy and gout showed varying levels (0.1–200 ng/ml).

These findings indicate that TSG-6 is a regulator of bone metabolism which can synchronise osteoblast and osteoclast biology. We hypothesise dual functions for this protein; a homeostatic role in non-inflammatory bone environments through inhibition of osteoblast differentiation, and a role in inflammatory bone disease through prevention of osteoclastogenesis. As such TSG-6 has a potential role for the development of therapeutic strategies in bone diseases.

Disclosures: A. Sabokbar, None.

T093

Diphyllin, a Novel Potent V-ATPase Inhibitor, abrogates Acidification of the Osteoclastic Resorption Lacunae and Bone Resorption.

M. G. Sorensen, K. Henriksen, A. V. Neutzsky-Wulff*, C. Christiansen, M. Karsdal.. Nordic Bioscience A/S, Herlev, Denmark.

Dissolution of the inorganic phase of bone by the osteoclasts is mediated through the V-ATPase and CIC-7. Inhibition of the osteoclastic V-ATPase or CIC-7 is a novel approach for inhibition of osteoclastic bone resorption. By testing natural compounds in acidification assays using acridine orange, diphyllin was identified as a potent inhibitor of lysosomal acidification in osteoclasts. We characterized diphyllin with respect to potency in a human osteoclastic bone resorption assay, an acid influx assay, and in a V-ATPase assay. For comparison, diphyllin was tested in an osteoblastic bone formation assay.

Human osteoclasts were generated from CD14+ monocytes cultured with 25ng/ml M-CSF and RANKL. The effect of diphyllin on lysosomal acidification in human osteoclasts was investigated using the dye acridine orange. The effect of diphyllin on osteoclastic bone resorption was measured as the release of CTX-I and calcium into the supernatant, and by scoring pit area. The number of osteoclasts, the TRACP activity, and the cell viability were measured. Diphyllin was tested in the acid influx assay and the V-ATPase assay using bovine chromaffin granules isolated from the medullae of bovine adrenal glands. The effect of diphyllin on bone formation was investigated using MC3T3 cells cultured in medium supplemented with ascorbic acid and beta-glycerol phosphate.

We found that diphyllin inhibited human osteoclastic bone resorption measured by CTX-I (IC50=14nM, Figure 1a), calcium release and pit area, in face of increased osteoclast numbers (Figure 1B) and increased levels of TRACP activity. Moreover, diphyllin dose dependently inhibited lysosomal acidification in human osteoclasts. In the acid influx assay, diphyllin potently inhibited influx (IC50=0.6nM), which is similar to the effect of bafilomycin. Furthermore, diphyllin inhibited the V-ATPase with an IC50 value of 17nM, compared to 4nM for bafilomycin. Finally, diphyllin showed no effect on bone formation in vitro, whereas bafilomycin was highly toxic.

We have identified a natural compound that potently inhibits the lysosomal acidification and the V-ATPase in osteoclasts and thereby abrogates bone resorption. These findings are in alignment with osteopetrotic patients where acidification and resorption are attenuated that result in increased numbers of osteoclasts.

original image

Disclosures: M. G. Sorensen, None.

T094

Canonical NF-κB Pathway Mediates Osteoclast Bone Resorption Activity.

N. S. Soysa*,1, C. N. R. Alles*,1, K. Aoki2, E. Jimi3, 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, 3Biochemistry, Kyushu Dental College, Kitakyushu, Japan.

NF-κB transcription factor is a key determinant in the orchestration of osteoclast formation, differentiation and survival. Nevertheless the mechanistic basis for NF-κB effect on bone resorbing activity of osteoclast has not fully elucidated yet. Hence the main objective of this study was to find the role of canonical NF-κB pathway in osteoclast resorbing activity using the NBD peptide, an established selective inhibitor of IκB-kinase (IKK). BMM cultured on dentine slices and on cover slips in the presence of RANKL and M-CSF were changed after 3 days with 10μM and 20μM of NBD peptides (Wt). Most of the osteoclasts were fully differentiated when the medium was changed and further cultured for 24 hrs in the presence of the peptides. Osteoclasts were stained for TRAP. Apoptosis was detected by DAPI staining and by TUNEL method and pits were measured by using a confocal microscope system. There was no detectable change in osteoclast numbers after changing the medium. Significant reduction in area of resorption was observed in Wt at 10μM and 20μM concentrations (P<0.01 and P<0.001 respectively). Volume of resorption pit was significantly reduced at 20μM (P<0.001). Volume to area ratio which was independent on osteoclast number was also reduced significantly (P<0.05). No difference in number of apoptotic cells was observed between Wt and control. TRAP was measured in the cell lysate and supernatant to measure the percentage TRAP release. Similarly, Wt significantly reduced TRAP release. Cell migration towards RANKL was reduced in treated samples compared to control assessed by transwell migration assay. Then osteoclasts on cover slips were stained for actin rings. Percentage of osteoclasts with actin rings were markedly reduced in peptide treated group (P<0.001). The levels of TRAP, cathepsin K, MMP-9, NFATcl and αvβ3 mRNAs analyzed by real-time RT-PCR were significantly reduced in treated samples (p<0.0001). Taken together these results suggest that NBD peptide reduces osteoclast bone resorbing activity, migration and actin ring formation by down regulating above mRNAs and the inhibitory effect on bone resorption was not due to apoptosis, suggesting the vital role of canonical NF-κB pathway in osteoclast activity. Cathepsin K and MMP-9 and NF-κB dependent genes. Also NF-κB binding sites are present within the promoter region of the NFATc1 gene and NFAT binding sites in the TRAP, Cathepsin K, and αvβ3 promoters as well. Therefore the interplay among NF-κB and NFATc1 seems to be crucial in establishment of osteoclast activity.

Disclosures: N. S. Soysa, None.

T095

Dentine Organic Matrix Down-Regulate Osteoclastic Activity in Root Resorption.

W. Sriarj*,1, B. J. Varghese*,1, K. Aoki2, K. Ohya2, Y. Takagi*,1, H. Shimokawa2. 1Pediatric Dentistry, Tokyo Medical and Dental University, Tokyo, Japan, 2Hard Tissue Engineering, Tokyo Medical and Dental University, Tokyo, Japan.

It has been reported that deciduous tooth roots were more susceptible in resorption than permanent tooth roots, in which resorption did not occur in physiological condition. However, the mechanisms of root resorption are still unclear. Our previous study has shown that osteoclasts cultured on deciduous dentine exhibited a higher level of cathepsin K and MMP-9 mRNA and a higher degree of resorption than cultured on permanent dentine. These could be due to the difference of organic matrices between deciduous and permanent dentine. The purpose of this study is to investigate the effect of organic matrix extracted from bovine deciduous or permanent dentine on the resorptive activity. We extracted dentine organic matrices from bovine deciduous or permanent root dentine using 4M guanidine hydrochloride, pH7.4 (G-ext) then 4M guanidine hydrochloride plus 0.5M EDTA, pH 7.4 (E-ext) sequentially. Osteoclasts, obtained from mouse bone marrow cells co-cultured with osteoblast-rich fraction in α-MEM containing 10% FBS, 1,25-(OH)2-D3 and PGE2, were cultured on the ivory slices with 500μg/ml of G-ext or E-ext for 48 hours. TRAP positive multinucleated cell number, TRAP activity, resorption pit area and the mRNA level of cathepsin K, TRAP, and MMP-9 were determined. We have found that osteoclasts cultured with both E-ext from deciduous and permanent dentine exhibited lower TRAP positive multinucleated cell number and TRAP activity than G-ext as well as resorption pit area. The mRNA level of cathepsin K, TRAP, and MMP-9 were also down-regulated in osteoclasts cultured with E-ext. Furthermore, permanent dentine E-ext seemed to suppress osteoclastic activity more than deciduous one. These findings suggest that some factors in dentine organic matrices E-ext affect osteoclastic activity in root resorption.

Disclosures: W. Sriarj, None.

T096

Homogenous Fluorescence-based Calcium Assay for the Rapid Determination of In Vitro Bone Resorption.

K. E. Renn*, M. J. Brown*. Lonza Walkersville, Inc., Walkersville, MD, USA.

High-throughput assays for measuring in vitro osteoclast-mediated bone resorption are essential tools in the study of bone related diseases. We have developed a homogenous, fluorescence-based assay for the rapid analysis of in vitro osteoclastic activity. The CalciFluor™ Assay quantitatively measures free calcium released during in vitro bone degradation. Normal human osteoclast precursors (OCP) cultured on a human bone substrate in the presence of macrophage-colony stimulating factor (M-CSF) and soluble RANK ligand (sRANKL) exhibit a linear increase in bone degradation over time when assayed using the CalciFluor Assay. Comparable results are obtained when the bone resorption activity of other primary cell types, such as murine bone marrow mononuclear cells, is measured with the assay. Treatment of human OCP with the bisphosphonate alendronate results in a dose-dependent decrease in the amount of calcium released from human bone coated plates. Likewise, OCP cultured in the presence of osteoprotegerin show decreased calcium release from bone, further illustrating that the free calcium elevations result from osteoclast-mediated bone degradation. Results generated with this fluorescent calcium assay are comparable to those obtained with commercially available enzyme immunoassay (EIA) kits detecting collagen degradation products. When the release of either calcium or collagen fragments is measured, actively resorbing osteoclasts show similar time-dependent trends in the amount of bone degraded as well as comparable inhibition profiles in the presence of pharmacologic agents. The CalciFluor Assay is a more rapid, convenient, and economical assay than the current EIA-based assays for bone resorption, and therefore represents an excellent tool for in vitro analysis of osteoclast activity.

Disclosures: M.J. Brown, Lonza Walkersville. Inc. 3.

T097

Lack of Bruton's Tyrosine Kinase Results in Osteoporosis, Despite Defects in Osteoclast Function.

L. Danks*,1, S. Workman*,2, A. D. Webster*,2, B. M. Foxwell*,1, M. Feldman*,1, N. J. Horwood1. 1The Kennedy Institute of Rheumatology, Imperial College, London, United Kingdom, 2Departmeny of Immunology, Royal Free and University College Medical School, London, United Kingdom.

Bruton's tyrosine kinase (Btk) is a member of the Tec family of kinases and mutations in Btk result in a rare disease known as X-linked agammaglobulinaemia (XLA). These patients have an absence of mature B cells resulting in profound hypogammaglobulinemia and recurrent infections. We have previously shown that Btk is not only involved in B cell responses but also plays a role in inflammatory cytokine production in macrophages, however the role of Btk in osteoclast formation and activation is unknown. As the Tec kinases are regulated by the Src family kinases, which are known to have important actions on osteoclast function, we hypothesised that Btk would be a downstream signaling target of Src kinase. Therefore, we investigated the role of Btk in osteoclast differentiation and activation in vitro and examined the effect of Btk deficiency on the bone phenotype of XLA patients.

Human peripheral blood monocytes were differentiated into osteoclasts in the presence of RANKL and M-CSF. Adenoviral overexpression of Btk/GFP during osteoclast culture on hydroxyapatite slides resulted in an unusual actin ring arrangement, the actin rings were smaller and thicker than those of controls. Lacunar pit formation on dentine slices was dose dependently inhibited, whilst there was no effect on TRAP+ osteoclast formation. Osteoclasts derived from PBMC's or CD14+ precursors of XLA patients, compared to control subjects, exhibited a dose dependent and highly significant decrease in bone resorption activity in response to RANKL despite having a dose dependent increase in TRAP+ osteoclast formation. These results suggest that Btk is important specifically for osteoclast resorption activity. The bone phenotype of the XLA patients was examined using serum markers of bone metabolism and bone density analysis. Surprisingly, XLA patients exhibited an osteoporotic bone phenotype with an average 20% reduction in bone density compared to age matched controls.

Our results provide novel evidence that Btk is an important signaling molecule for normal osteoclast activity in vitro however these patients are osteoporotic in vivo. As B cells play a role in the regulation of basal bone turnover, by providing a reservoir of early osteoclast precursors and by direct expression of osteoclast regulatory cytokines, it is likely that the lack of mature B cells in the XLA patients, and the resulting reduction in OPG levels, are able to override the osteoclast defect in vivo. Thus, our novel finding that XLA patients are osteoporotic suggests that absence of B lymphocytes gives rise to an overall decrease in bone density.

Disclosures: L. Danks, None.

This study received funding from: Arthritis Research Council.

T098

NEMO (IKKγ) Modulates Osteoclastogenesis and Bone Erosion.

L. Darwech*, S. Dai*, Y. Abu-Amer.. Orthopedic Surgery and Cell Biology & Physiology, Washington University School of Medicine, Saint Louis, MO, USA.

The transcription factor NF-κB is essential for osteoclastogenesis and is considered an immune-modulator of rheumatoid arthritis and inflammatory osteolysis. Activation of NF-κB subunits is regulated by the upstream IκB kinase (IKK) complex which contains, among other proteins, IKK1, IKK2, and IKKγ; the latter also known as NF-κB essential modulator (NEMO). The role of IKK1 and IKK2 in the skeletal development and inflammatory osteolysis has been described, whereas little is known about the role of NEMO in this setting. In this regard, several case reports identified point mutations that led to osteopetrosis and hypodontia or anodontia with conical incisors. NEMO facilitates and integrates upstream stimuli and appears to assign, by yet unknown mechanisms, signal specificity. Typically, signals such as RANKL or TNF prompt oligomerization of NEMO monomers through well-defined domains termed the coiled-coil-2 (CC2) and Lucine zipper (LZ) motifs. This step is a prerequisite for binding to IKKs and further relaying signal transduction. In fact, blocking NEMO binding to IKKs perturbs NF-κB activation, abrogates osteoclastogenesis, and diminishes inflammatory bone erosion. In this study, we asked whether NEMO is essential for osteoclastogenesis of murine marrow macrophages and whether interruption of NEMO oligomerization impedes the process of osteoclast differentiation in vitro and in vivo. To this end, we generated short peptides overlapping the CC2 and LZ motifs and fused them with a carrier peptide to enable cell membrane translocation. Our results show that the CC2 and LZ short peptides specifically bind to NEMO monomers, prevent trimer formation, and render NEMO monomers susceptible for ubiquitin-mediated degradation. Further, CC2 and LZ peptides attenuate RANKL and TNF-induced NF-κB signaling in bone marrow-derived osteoclast precursors. More importantly, these peptides potently inhibit osteoclastogenesis, in vitro, and arrest RANKL-induced calvarial osteolysis, in mice. To further ascertain its role in osteoclastogenesis, we were able to block osteoclastogenesis using NEMO siRNA knockdown approach. Collectively, our data establish that NEMO is essential for osteoclastogenesis and provide the novel finding that inhibition of NEMO assembly leads to its degradation. More importantly, inhibition of NEMO expression impedes osteoclast formation and arrests osteolysis. Thus, NEMO present itself as a promising candidate for therapeutic intervention.

Disclosures: I. Darwech, None.

T099

NFATc1 Mediates the Stimulatory Effects of Post-translationally Modified Bone Sialoprotein on the Resorptive Activity of Rabbit Osteoclasts.

H. H. Chen*, J. A. R. Gordon*, A. Pereverzey*, S. M. Sims*, G. K. Hunter*, S. J. Dixon, H. A. Goldberg.. CIHR Group in Skeletal Development and Remodeling, The University of Western Ontario, London, ON, Canada.

Cell-adhesion proteins of bone extracellular matrix such as bone sialoprotein (BSP) promote osteoclastogenesis and resorption; however, their mechanism of action has remained elusive. NFATc1 is a transcription factor that plays a critical role in osteoclast differentiation. Our purpose was to determine whether the effects of BSP on osteoclastic resorption involve NFATc1. Osteoclasts were isolated from the long bones of neonatal rabbits. Native BSP (nBSP) from rat long bones (post-translationally modified) and rat recombinant BSP (rBSP, prokaryotically expressed without post-translational modifications) were purified to homogeneity using FPLC. To quantify resorptive activity, osteoclasts were incubated for 24 hours on dentin slices coated with nBSP or rBSP, or uncoated (control). Samples were stained for TRAP activity to determine osteoclast number and then with toluidine blue to determine total area resorbed. In parallel studies, osteoclast attachment and NFATc1 activation were assessed 3 hours after plating on coverslips coated with nBSP or rBSP, or uncoated (control). Immunofluorescence was used to quantify the percentage of osteoclasts demonstrating nuclear localization of NFATc1, which upon activation translocates from the cytosol to the nuclei. Coating of substrates with nBSP or rBSP did not significantly affect the number of osteoclasts attached at 3 or 24 hours. However, nBSP significantly promoted the resorption of dentin slices (2.26 ± 0.26 fold greater than control, mean ± SEM, n = 7), whereas rBSP had no effect (1.06 ± 0.07 fold). Similarly, nuclear localization of NFATc1 was enhanced in osteoclasts plated on coverslips coated with nBSP (1.68 ± 0.05 fold greater than control, n = 3), but not with rBSP (1.05 ± 0.03 fold), suggesting a role for NFATc1 in the activation of resorption. To assess the involvement of NFAT in mediating BSP-induced resorption, we used 11R-VIVIT (a cell-permeable peptide inhibitor of NFAT activation). As expected, 11R-VIVIT decreased NFATc1 translocation in osteoclasts bound to nBSP-coated coverslips. Notably, 11R-VIVIT, but not the inactive control peptide 11R-VEET, blocked the stimulatory effect of nBSP on resorptive activity. These findings establish that interaction of osteoclasts with post-translationally modified BSP enhances NFATc1 activation, which in turn stimulates resorption. This is the first direct demonstration that NFAT regulates the resorptive activity of authentic osteoclasts.

Disclosures: S.J. Dixon, None.

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

T100

AG490, Jak2 Specific Inhibitor, Regulates Osteoclast Survival via Distinct Signaling Pathways. H. B. Kwak*, H. M. Sun*, D. Yang*, H. Kim*, H. Ha*, J. Lee*, Z. H. Lee. Department of Cell and Developmental Biology, School of Dentistry, Seoul National University, Seoul, Korea, Seoul, Republic of Korea.

Osteoclasts are multinucleated cells with the unique ability to resorb bone. Elevated activity of these cells under pathological conditions leads to the progression of bone erosion, such as osteoporosis, periodontal disease, and rheumatoid arthritis. The regulation of osteoclast apoptosis is important for bone homeostasis. In this study, we examined the effect of AG490 (JAK2 specific inhibitor) on osteoclast apoptosis. We show that AG490 inhibits cytochrome c release into cytosol and in turn suppresses caspase-9 and −3 activation, thereby inhibiting osteoclast apoptosis. Also, AG490 stimulated the phosphorylation of ERK and Akt. Adenovirus-mediated overexpression of dominant negative (DN)-Ras and DN-Akt in osteoclasts inhibited the survival of osteoclast despite the presence of AG490. Thus, osteoclast survival in response to AG490 leads to ERK and Akt pathways. Furthermore, constitutive active (CA)-MEK and Myr-Akt suppress the release of cytochrome c into cytosol and inhibit caspase activity. These results suggest that AG490 inhibits caspase activity by activating Akt and ERK and in turn suppresses the apoptosis of osteoclasts.

Disclosures: H.B. Kwak, None.

T101

Expression and Transcriptional Activity of Microphthalmia-Associated Transcription Factor Isoforms During Osteoclastogenesis. M. Murakami1, Y. Iwata1*, M. Funaba2*, 1Laboratory of Molecular Biology, Azabu University School of Veterinary Medicine, Sagamihara, Japan, 2Laboratory of Nutrition, Azabu University School of Veterinary Medicine, Sagamihara, Japan.

Microphthalmia-associated transcription factor (Mitf), a member of the basic helix-loop-helix leucine zipper (bHLH-ZIP) transcription factors, is required for proper development of several cell lineages including osteoclasts, melanocytes, retinal pigment cells and mast cells. Mitf has been implicated to be a key regulator of the later steps of osteoclastogenesis, but the role of Mitf is not fully elucidated; although nine distinct Mitf isoforms, which contain an isoform-specific first exon and are identical exons 2 to 9, have been identified at the RNA level, it is unclear whether any isoforms are unique to the osteoclast lineage cells. The present study examined expression of Mitf isoforms in sRANKL-induced osteoclast-like cells. Previous studies have revealed that tartrate-resistant acid phosphatase (Trap) is a transcriptional target of Mitf in osteoclasts. Thus, we also examined factors affecting Trap gene transcription in HepG2 cells that are responsive to Mitf overexpression. Treatment of RAW264 macrophage-like cells with sRANKL (50 ng/ml) for 72 h resulted in the increase in the number of Trap-positive multinucleated cells. Mitf-A and -J but not the other Mitf isoforms (-B, -C, -D, -E, -H, -M and -mc) were expressed in RAW264 cells, irrespective of the treatment with sRANKL. These results indicate that expression of Mitf isoforms is cell-type specific, and that differentiation of osteoclast lineage cells hardly affects expression pattern of Mitf. In addition, Tfe3, another member of the bHLH-ZIP family, was also significantly expressed throughout the differentiation of RAW264 cells. Transcriptional activation assays using luciferase-based reporter gene containing Trap promoter (-2049 - +1) revealed that overexpression of Mitf-J/-D/-E but not Mitf-A slightly increased luciferase expression. The overexpression of c-Jun but not c-Fos and JunB increased expression of Trap reporter gene, and synergistic effects of c-Jun and Mitfs (-A and -J/-D/-E) on Trap gene transcription were detected. In contrast, no synergism was observed between Mitf and the other AP-1 component (c-Fos and JunB). Distinct expression of Mitf isoforms and functional interaction between Mitf and the other transcription factor such as c-Jun during the terminal differentiation of osteoclasts suggests discrete regulation of osteoclastogenesis.

Disclosures: M. Murakami, None.

T102

FADD-Caspase-8 aXis Regulates Osteoclast Apoptosis. M. Nakamura, T. Akiyama, K. Nakamura, S. Tanaka.. Department of Orthopaedic Surgery, Faculty of Medicine, The University of Tokyo, Tokyo, Japan.

Osteoclasts are terminally differentiated cells and rapidly die through apoptosis in the absence of tropic factors. Apoptosis is genetically programmed cell death to remove unwanted cells from physical status, and is controlled by two distinct signaling pathways; the death receptor-mediated pathway and the mitochondria-mediated pathway. We previously reported that a proapoptotic BH3-only Bcl-2 family member Bim is a key regulator of osteoclast apoptosis, showing the involvement of mitochondrial pathways in the cell death of osteoclasts. On the other hand, the role of the death receptor pathway in osteoclast apoptosis is not fully clarified yet. The death receptor-mediated pathway is activated upon ligand binding to cell surface receptors such as tumor necrosis factor (TNF) receptor that contain cytoplasmic death domains. Ligand binding induces oligomerization of these death domains through ligand-induced receptor trimerization, generates homophilic interaction surfaces for death domain-containing adaptor molecules. FADD (FAS-associating death domain-containing protein) is a universal adapter protein that mediates signaling of death-domain containing members of the TNF receptor superfamily, and plays a critical role in recruiting death-inducing signaling complex and the subsequent activation of initiator caspase-8 and downstream effector caspases. In an attempt to elucidate the role of FADD in osteoclast apoptosis, we constructed an adenovirus vector carrying a dominant negative mutant of FADD (AxFADD-DN), which lacks the death effector domain and therefore cannot activate Caspase-8. Osteoclasts generated in vitro were infected with control adenovirus or AxFADD-DN, which effectively induced FADD-DN expression in the cells, and were subjected to the survival assay. Osteoclasts overexpressing FADD-DN survived longer than control cells after cytokine depletion, which was associated with the downregulation of Caspase-8 activity. Apoptosis of osteoclasts was also suppressed by a specific inhibitor of Caspase-8, Z-IETD-FMK. These results suggest that death receptor & FADD-mediated pathways are crucial for osteoclast apoptosis. To identify death receptors involving in osteoclast apoptosis, we treated the cells with FasL, TNF-α and TRAIL. However, all of them failed to promote the apoptosis of osteoclasts. Our results suggest that the FADD-Caspase-8 axis critically regulates the apoptosis of osteoclasts, which is activated through previously unknown death receptor ligands.

Disclosures: M. Nakamura, None.

T103

RANKL-induced Expression of TRPV2, Calcium Permeable Channel, Is Involved in Osteoclastogenesis via Calcium Signaling Activation. F. Okamoto*, H. Kajiya, A. Nakao*, K. Okabe. Physiological Science and Molecular Biology, Fukuoka Dental College, Fukuoka, Japan.

Osteoclast differentiation from hematopoietic precursor cells is stimulated by receptor activator of NF-κB ligand (RANKL) via activation of transcription factors. Nuclear factor of activated T cells (NFAT) cl is known to be a crucial transcriptional factor for osteoclastogenesis and is activated by calcineurin, a Ca2+/calmodulin-dependent phosphatase. Calcium signaling is considered to be an important regulator during osteoclastogenesis. Although it has been shown that RANKL stimulates PLCγ and then releases Ca2+ from intracellular Ca2+ stores, little is known which intra- and/or extracellular pathways induce osteoclastogenesis. Using a DNA microarray, we found that transient receptor potential V2 (TRPV2), a calcium permeable cation channel, was highly expressed in RANKL treated RAW264.7 cells compared to untreated cells. Thus, the aim of present study was to investigate the expression and role of TRPV2 on osteoclastogenesis via calcium signaling. TRPV2 channels were expressed in RAW 264.7 cells and bone marrow macrophages. RANKL significantly increased TRPV2 expression in both cell types 24 h after treatment. To elucidate the electrophysiological function of TRPV2, non-selective cation currents were recorded from RAW264.7 cells using the whole-cell voltage clamp technique. The current density was significantly increased in RANKL treated cells 24 h after treatment compared to untreated cells. Ruthenium red (10 μM), an inhibitor of TRPV channels, reduced the current density as well as RANKL-induced osteoclastogenesis. Tetracycline-inducible siRNA targeted TRPV2 completely abolished the RANKL-dependent augmentation of non-selective cation currents. Silencing with TRPV2 siRNA in RAW264.7 cells also suppressed RANKL-stimulated osteoclastogenesis. These results suggest that RANKL up-regulates TRPV2 expression which serves as a calcium influx pathway. TRPV2 is involved in the RANKL-evoked calcium signaling and subsequent activation of calcium-dependent transcriptional factors such as NFATcl and AP-1 promoting osteoclastogenesis.

Disclosures: F. Okamoto, None.

T104

Characterizatioin of the Signaling Complex that Mediates Osteoclastic Activation. J. L. Ross*, T. J. Chambers, K. M. Lawrence*., Department of Cellular Pathology, St George's, University of London, London, United Kingdom.

Although the signals responsible for osteoclast differentiation are well characterized, almost nothing is known of those responsible for the activation and modulation of resorption. Confocal microscopy clearly demonstrates that osteoclasts form actin ring structures on mineralized substrates that differ distinctively from those formed on non-mineralized surfaces. Moreover, osteoclasts secrete enzymes when incubated on bone but not on plastic. These findings show that osteoclasts have the ability to distinguish bone from other surfaces, and that mineralized surfaces are essential for the induction of resorptive behaviour.

There is much evidence that the vitronectin receptor (VNR) and c-src are implicated in bone resorption. Recently, it has become clear that in many cases, best exemplified by the immunological synapse of T cells, cell signaling occurs through the formation of large multi-protein complexes that form dynamic physical associations between surface receptors and intracellular signaling molecules. Identifying the components of the multi-protein complex that controls osteoclast resorption may provide new targets for clinical intervention in the treatment of osteoporosis and related diseases.

We therefore performed co-immunoprecipitation experiments, using antibodies to VNR and c-src, to isolate complexes associated with these molecules in resorbing and non resorbing osteoclasts. Osteoclasts were formed on plastic substrates, lifted into suspension, and sedimented onto plastic or bone surfaces. Protein complexes from osteoclasts incubated on bone versus plastic, and with versus without resorption-inducing cytokines, were then compared.

One and 2D gel electrophoresis showed that several proteins were associated with both VNR and c-src under each of the experimental conditions used.

Interestingly co-immunoprecipitates from resorbing cells yielded proteins which were not seen under conditions of no activation. This suggests that in resorbing osteoclasts, additional proteins are recruited to the signaling complex. Furthermore, other proteins appeared to be complexed with VNR and c-src in non-resorbing osteoclasts but absent in activated cells. This implies that some proteins must be removed from the complex in order for resorption to take place, suggesting a novel resorption block mechanism. In order to identify these proteins we used Western immunoblots to probe for known signaling molecules. Using both co-immunoprecipitation antibodies, IL1 receptor, TRAF6, VNR and c-src were found to be present but equal under all conditions, thus eliminating these as differentially-bound candidates. The unidentified novel proteins are currently being identified by mass spectrometry.

Disclosures: J.L. Ross, None.

T105

Elucidating Mechanisms Leading to Increased Resorptive Activity of Large Osteoclasts in Inflammation. D. P. Trebec1, A. Gramoun2*, J. N. M. Heersche2*, M. F. Manolson2, 1Department of Biochemistry, University of Toronto, Toronto, ON, Canada, 2Faculty of Dentistry, University of Toronto, Toronto, ON, Canada.

Large osteoclasts (OCs) (≥10 nuclei) are prevalent in inflammatory diseases characterized by increased bone resorption (e.g. rheumatoid arthritis and periodontal disease). Previously, we have shown that large OCs express higher levels of the activating receptor interleukin-1 receptor-1 (IL-1R1) while the decoy receptor 1L-1R2 was increased in small OCs (2-5 nuclei). In addition, large OCs were found to be more active compared to small OCs in response to interleukin-1 (IL-1)β (Trebec et al. JCB 101: 205-220, 2007). As a result, our aim was to determine how differential expression of IL-1R1 and IL-1R2 leads to increased resorption in large OCs and if IL-1α and IL-1β result in differential effects on OC formation and activity. For all experiments, we used the RAW 264.7 cells differentiated into populations of small and large OCs. In the presence of 50 ng/mL RANKL, RAW cell proliferation rates were unaffected by the addition of IL-1α/β. IL-1α (1 ng/mL) and IL-1β (10 ng/mL) resulted in an increase in TRAP+ cells when added to cultures at day 0 or day 3 but not at day 5. In contrast, TRAP activity revealed no significant differences between the groups. A 2-fold increase in the number of large OCs adopting a “rounded” morphology (indicative of active OCs) was also seen when exposed to 10 ng/mL IL-1β for 24 hours (less so for IL-lα) and was reversed with the addition of 50 ng/mL IL-1 receptor antagonist (IL-Ira). Furthermore, similar to previous results shown with 10 ng/mL IL-1β, large OCs resorbed 3-fold more in the presence of 1 ng/mL IL-1α. Neither the increase in rounded morphology, inhibition by IL-Ira, nor differences in resorptive activity were evident in small OCs in response to either cytokine. Colocalization of IL-1R1 and the integrin αvβ3 was observed in OCs, most notably in OCs plated on fibronectin where they adopted a round morphology. Gramoun et al have shown that OCs on fibronectin are hyperactive. In conclusion, there is a differential response between large and small OCs to IL-1, possibly resulting from variations in their IL-1 receptor levels in turn leading to activation of different signalling pathways. IL-1α was found to be a ∼ 10X more potent than IL-1β. Colocalization of IL-1R1 and αvβ3 could also contribute to the differential signalling responses. Elucidating mechanistic differences between large and small OCs may result in novel targets for therapeutics designed to inhibit excessive resorption by large OCs while maintaining normal bone remodelling.

Disclosures: D.P. Trebec, None.

T106

NF-κB But Not NFATcl Is Involved in AhR - RANKL Crosstalk in Benzo[a]pyrene-Mediated Inhibition of Osteoclastogenesis. I. Voronov*1, K. Li*2, H. C. Tenenbaum2, J. N. M. Heersche*2, M. F. Manolson2, 1Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada, 2Faculty of Dentistry, University of Toronto, Toronto, ON, Canada.

Cigarette smoking is a risk factor for impaired bone healing, however, the molecular mechanisms leading to its detrimental effects are not known. Benzo[a]pyrene (BaP) is an environmental pollutant present in high concentrations in cigarette smoke. BaP binds to the aryl hydrocarbon receptor (AhR), a cytosolic transcription factor (TF), and induces expression of several drug-metabolizing enzymes including cytochrome P450 1B1 (CYP1B1). We have demonstrated previously that osteoclastogenesis is suppressed by BaP, and that RANKL, when delivered in high concentration (100 ng/ml) reverses this inhibition and suppresses BaP-mediated gene expression, suggesting possible crosstalk between AhR and RANKL signaling pathways (Voronov et. al., Biochem. Pharmacol., 70:300-7, 2005). NF-κB and NFATcl are key TFs activated in RANKL-induced osteoclastogenesis. To evaluate the effect of BaP on these TFs, RAW264.7 cells were exposed to 25 or 200 ng/ml RANKL and 10−5 M BaP and analyzed by TF activation ELISA kits. TF activity assays demonstrated that BaP inhibited RANKL-mediated NF-κB activation at 30 min, however, at 60 min, NF-κB activation levels remained above control levels in BaP-containing groups. In contrast, NFATcl activity was significantly upregulated by BaP. To determine the effect of BaP on RANKL-mediated nuclear translocation, the cells were analyzed by immunofluorescence (IF) using an anti-p65 or anti-NFATcl antibodies. IF showed that BaP inhibited RANKL-induced NF-κB nuclear translocation, but had no effect on NFATcl. To investigate the involvement of NF-κB and NFATcl in BaP-mediated gene expression, the cells were cultured in the presence of NF-κB and NFATcl inhibitors. RT-PCR revealed that NF-κB but not NFATcl inhibitors decreased CYP1B1 expression suggesting that NF-κB is involved in BaP-mediated CYP1B1 expression. To assess if there is a direct interaction between AhR and NF-κB, co-immunoprecipitation experiments were performed and revealed an AhR-NF-κB interaction in the presence of BaP.

Based on the above, we speculate that NF-κB but not NFATcl is involved in Ahr - RANKL signaling crosstalk. We conclude that inhibition of RANKL-induced NF-κB activation is one of the possible mechanisms of BaP-mediated inhibition of osteoclastogenesis.

Disclosures: I. Voronov, None.

T107

Densitometric Derived Structural Indices in HR-pQCT Are Independent of Cortical Geometry. A. J. Burghardt, K. Davis*, S. Majumdar.. Radiology, University of California, San Francisco, San Francisco, CA, USA.

High-resolution peripheral quantitative computed tomography (HR-pQCT) has the potential to be a useful method for the longitudinal evaluation of bone quality in human subjects. Current structure analysis methods for this modality calculate BV/TV based on the apparent mineral density of the trabecular compartment and an assumed compact bone mineral density of 1200 mg HA/cm3. This requires accurate calibration of bone mineral densities and adequate correction for beam hardening effects. Using an equivalent calibration and beam hardening correction in a micro-tomography device, it has been shown that cortical geometry can introduce a non-negligible bias in apparent density measures [1]. In this study we evaluate the error in calculating BV/TV densitometrically as it relates to cortical geometry and corrections for beam hardening for HR-pQCT.

Cadaveric distal radii were acquired from 6 donors and a 1cm length was imaged using HR-pQCT (Scanco XtremeCT, 82 μm isotropic nominal resolution) and μCT (Scanco μCT40, 18μm isotropic nominal resolution). For both devices custom beam hardening correction factors were determined using a 200mg HA/cm3 wedge phantom. The datasets were registered based on matching cross sectional areas prior to analysis. BV/TV and Ct.Th measures were determined for each dataset in 1 mm increments along the long axis of the bone. BV/TV was calculated densitometrically as vBMDtrab/1200 mg HA/cm3 for the HR-pQCT images, while a simple voxel counting method was used for the μCT data. Ct.Th was calculated for the μCT data using direct 3D methods.

As reported elsewhere [2], BV/TV determined densitometrically from calibrated HR-pQCT images was somewhat underestimated compared to the reference μCT values. The absolute error in BV/TV exhibited minimal dependence on cortical geometry.

These results suggest that beam hardening effects related to cortical shell geometry are not a significant source of bias in HR-pQCT based measures of trabecular bone structure.

[1] E. Cory et al, ORS 2007

[2] J.A. MacNeil and S.K. Boyd. Med Eng. Phys. 2007 Epub

original image

Disclosures: A.J. Burghardt, None.

T108

Measurement Protocols for the Non-invasive Assessment of Bone Constituent Properties by Raman Spectroscopy. J. H. Cole1, M. V. Schulmerich*1, K. A. Dooley*1, J. M. Kriegl*2, E. Daley*2, S. A. Goldstein*2, M. D. Morris1, 1Department of Chemistry, University of Michigan, Ann Arbor, MI, USA, 2Department of Orthopaedic Surgery, University of Michigan, Ann Arbor, MI, USA.

Techniques that clinically evaluate bone status may assess the mass and organization of the tissue, but no current method can examine features of bone quality such as mineral and matrix properties in vivo, which contribute vitally to skeletal strength and toughness. Previous studies revealed that bone fragility is associated with changes in composition metrics, such as mineral crystallinity or substitution and collagen cross-linking. We recently developed a non-invasive Raman fiber optic probe that allows us to observe bone spectra through the skin and overlying tissue in animal and human cadaveric limbs. The objective of our study was to improve tissue preparation, optimize probe configuration, and examine our ability to measure bone constituent properties non-invasively in different specimens over a range of overlying tissue depths. Excised limbs from human, mouse, rat, and canine specimens were obtained. For the animal limbs, fur was removed in the region of interest (ROI) using a depilatory agent. Glycerol was applied to the ROI to increase light penetration into overlying tissue, thereby improving the Raman bone spectra. Multiple spectra were acquired on each limb. Following transcutaneous measurements, tissues overlying the bone were removed, and Raman spectra were collected from the exposed bone for validation. The contribution of bone (e.g., mineral and matrix) was separated from that of the overlying tissue (e.g., skin and tendon) using multivariate methods. Standard composition metrics including carbonate/phosphate ratio, mineral/matrix ratio, and collagen cross-link ratio were computed using both band heights and band areas. Band height ratios generally gave better accuracy, because they were less sensitive to errors from baseline correction. Bone composition measurements obtained transcutaneously differed by less than 5% from those obtained on the exposed bone, and the two were not significantly different (p > 0.05). The results demonstrate effective measures of bone beneath more than 5 mm of overlying tissue with the expectation of reaching greater depths with further improvements to our system. Non-invasive Raman spectroscopy can accurately assess the contribution of bone composition to bone quality and may prove useful in the clinical assessment of bone fragility.

Disclosures: J.H. Cole, None.

T109

Within Osteons Infrared Parameters Linked to Specific Bone Properties Vary as a Function of Tissue and Animal Age. S. Gourion-Arsiquaud*1, L. M. Havill2, A. L. Boskey1, 1Research, Hospital for Special Surgery, New York, NY, USA, 2Southwest Foundation for Biomedical Research, San antonio, TX, USA.

Fractures are the major problem associated with osteoporosis and represent a major concern of public health. Although BMD is used as a clinical predictor of fracture risk, some patients with fracture incident present normal BMD showing that this is not the only factor affected in osteoporosis. Moreover BMD does not allow understanding the underlying mechanisms involved in these bone diseases. Therefore it was important to find other parameters linked to bone quality. Previous studies on healthy and diseased bones using FTIR spectroscopy have validated parameters that reflect the mineral and matrix organization at a molecular level.

Due to constant bone remodeling, there are tissue age variances within the same bone specimen. In this work we analyze the molecular variations of bone as a function of both tissue and animal age. To address this issue we describe changes of parameters inside osteons from baboons of different ages by FTIR-Imaging and for representative samples also using Raman spectroscopy. The osteon was chosen for this study as it is an active remodeling unit, presenting different tissue ages going from its center to the periphery.

The following parameters were examined across individual osteons in femoral samples from 27 baboons aged 0-30 years: Mineral/matrix ratio, a quantitative assessment of the mineral content; Crystallinity ratio linked to the hydroxyapatite (HA) crystal size; Carbonate to phosphate ratio, and Collagen cross-link ratio which represent, respectively, the incorporation of carbonate into the HA lattice and the maturity of collagen. We found that mineral/matrix ratio and crystallinity ratio increase with tissue age whereas carbonate to phosphate ratio decreases. These data improve our knowledge on the mechanisms involved in bone tissue aging. The Raman data validate and complete these IR results. The parameters show a similar trend within osteons regardless the age of the animal. Only the absolute values of these ratios differed. This work demonstrates not only that FTIR imaging allows the co-localization of important mineral and matrix properties of bone at the microscopic level but also that osteon analysis can provide reproducible characterization of these properties. Indeed, this work set up a basis which will permit using normal osteonal data as a baseline against which those of samples from bone disease subjects can be compared. This should allow characterization of which bone properties are associated with the metabolic bone diseases. On the same way osteonal studies should provide information on the efficacy of therapies used in the treatment of bone diseases.

Disclosures: S. Gourion-Arsiquaud. None.

T110

Assessment of Bone Tissue Mineralization: Evaluation of Polychromatic μCT Mineralization Measurement by Comparison to Synchrotron Radiation μCT. G. J. Kazakia, A. Burghardt, S. Majumdar.. Radiology, UC San Francisco, San Francisco, CA, USA.

The goal of this study was the quantitative evaluation of micro-computed tomography (μCT)-based measurement of degree of mineralization of bone (DMB). While μCT systems are used widely to assess bone structure, beam-hardening effects due to the polychromatic source complicate assessment of DMB. Beam-hardening correction algorithms have recently been introduced to overcome this limitation. Synchrotron radiation μCT (SRμCT) is an appropriate standard for spatially-resolved DMB evaluation due to the monoenergetic, high flux, parallel beam that provides high spatial resolution and accurate attenuation measurement. Therefore we proposed to compare μCT data to those obtained by SRμCT to evaluate the accuracy of μCT mineralization measurement.

Cylinders of trabecular bone (8 × 4 mm, n = 14) were harvested from proximal femurs, proximal tibiae, and vertebrae of human cadavers in an effort to include a range of structure and mineralization. Specimens were imaged using a desktop μCT system (μCT-40, Scanco Medical AG) at a resolution of 8 microns and energy settings optimized for mineralized human trabecular bone (70 kV, 114 mA). Density-calibrated μCT reconstructions were created using a hydroxyapatite (HA) phantom.

The specimens were then scanned at a synchrotron beamline equipped with a μCT stage (National Synchrotron Light Source, BNL) at low energy (26keV) with a voxel size of 7.5 microns. Density-calibrated reconstructions were created using the μCT HA phantom. SRμCT data were segmented using a global threshold criterion. Segmentation of the μCT data was performed by matching the volume fraction (BV/TV) determined by SRμCT analysis. Mean DMB (g/cc) and BMC (g) were calculated for each specimen from the μCT and SRμCT reconstructions. Following imaging, ash weight (g) and density (g/cc) were measured using established gravimetric protocols.

Desktop μCT DMB measurements were well-correlated to SRμCT DMB values when anatomic sites were considered individually (R2 = 0.80 tibia & vertebra; 0.99 femur) but not when pooled (R2 = 0.21). At all sites evaluated, μCT underestimated DMB as compared to SRμCT (12 to 18%, p < 0.001). At all sites, BMC derived from both μCT and SRμCT were well-correlated with ash weight (R2 = 0.98). DMB was correlated to ash density only for proximal femur cores (R2 = 0.55 for both μCT & SRμCT). These results suggest that trabecular bone structure is critical in tomography-based determination of mineral density. We found that bone samples from sites with high BV/TV (proximal femur) produce more accurate DMB measures than low BV/TV samples. Partial volume and segmentation artifacts, which are more prevalent in low BV/TV imaging, likely contribute to errors in DMB.

Disclosures: G.J. Kazakia, None. This study received funding from: NIH.

T111

Analysis of Differing Approaches for the Assessment of Large Scale Transcriptional Profiling of Fracture Healing. J. E. McLean1, L. J. Silkman*1, T. A. Einhorn*1, T. F. Smith*2, L. C. Gerstenfeld1, 1Orthopeadic Research, Boston University, Boston, MA, USA, 2Biomedical Engineering, Boston University, Boston, MA, USA.

Fracture healing is a synchronous temporal process of one round of endochondral bone formation followed by an extended period of remodeling. The aim of this study was to compare three approaches to the normalization of large scale transcriptional profiling. The methods used were: the profile of unfractured bone as the reference; an exogenously added artificial gene sequence with no known correlate in the mouse genome as reference “alien gene”; and a set of standard genes “housekeeping genes”. Standard femur fractures were generated in C57/B6 mice and assessed at days 3, 7, 10, 14, and 21. ∼21,000 genes were examined. The hybridization of each spot was assessed using three separate detection channels and spots were measured as a ratio intensities. Separate ratios were determined using the biological reference and the exogenously added Alien gene, which were both co-printed with each gene spot on the chip included with the hybridization cDNAs. Sixty standard genes were empirically compiled based on both their invariant expression from the biological reference and low standard deviation. Sixty genes with the largest ratio differences from their reference for four basic expression patterns increasing, decreasing, and two types of complex behavior over time were used to assess statistical reproducibility of the data. The Alien and biological reference had statistically significant correlation for all sets at (p<0.002). The correlation between the standard genes and the biological reference showed statistical significance (all p<0.05) and was only significant at p<0.05 for two out of four sets of examined mRNAs when compared to Alien. The use of the biological reference excludes ratio intensity measurements from being made for all genes that are not actively transcribed in the unfractured bone sample. This analysis method excluded ∼2000 mRNAs that are newly transcribed following fracture, as determined via ratio to Alien reference. Using fixed in-spike concentration of Alien also enables absolute ratio intensity measurements of the expressed levels for individual mRNAs to be made. Validation of expressed levels between gene transcripts can be assessed, by examining the ratios of Colla1 and Colla2, splice variants of Col2a1 and a selected group of ribosomal subunit proteins. The inherent advantages of using an exogenous reference enables absolute determination of individually expressed mRNAs species to be made and provides the greatest degree of accuracy of both inter-chip and inter-experimental comparisons.

Disclosures: J.E. McLean, None. This study received funding from: NIH.

T112

Microscopic Imaging of Bone Composition En Block with Synchrotron Infrared Microspectroscopy. L. M. Miller1, T. C. Feldman*2, A. Schirmer*3, R. J. Smith*1, S. Judex3, 1National Synchrotron Light Source, Brookhaven National Laboratory, Upton, NY, USA, 2Materials Science and Engineering, Stony Brook University, Stony Brook, NY, USA, 3Biomedical Engineering, Stony Brook University, Stony Brook, NY, USA.

Understanding changes in the chemical makeup of bone is important for diagnosing and treating bone disease. Fourier transform infrared microspectroscopy (FTIRM) is a well-established imaging technique that provides information on both the mineral and matrix composition of bone. One drawback of the technique is that FTIRM data are typically collected in a transmission geometry, thus requiring plastic-embedding and microtoming of very thin (3 – 5 micrometer) sections of bone. In this work, we have established a new method for collecting FTIRM data in a reflection geometry from the surface of a bone block using synchrotron light. This study will demonstrate the accuracy and quality of reflection FTIRM, while investigating spatially-resolved chemical changes in developing mouse bone. Tibiae of female BALB mice were harvested at 8 time points distributed between 1d and 40d of age and embedded in poly methyl methacrylate (PMMA). Thin sagittal sections (3 micrometers) were cut from the surface of each embedded bone. FTIRM was performed on both the thin section and the surface of the sample block in transmission and reflection geometry, respectively. The results from the two imaging methods were compared using visible landmarks present in both the thin section and the embedded bone. Specifically, the mineralization (phosphate/protein ratio), carbonate accumulation (carbonate/phosphate ratio), crystallinity, and collagen cross-linking were assessed. The results of this work will be presented as a validation of this new methodology, which will increase the applicability of FTIRM by expanding its use to those samples that cannot be processed into thin sections for analysis.

Disclosures: L.M. Miller, None. This study received funding from: U.S. Department of Energy.

T113

Back-Scattered Electron Imaging of Bone Mineralization in Osteoarthritis. P. Sutton-Smith*, N. Fazzalari, J. Kuliwaba, H. Beard*, B. Ma*., Tissue Pathology, Institute of Medical and Veterinary Science, Adelaide, Australia.

Osteoarthritis affects a large proportion of Western society. There is controversy over the degree and nature of bone changes in this disease with presentation of heterogeneous tissue-level morphology. Studies of sub-chondral bone have shown increased bone volume and decreased bone mineralization, but more distal sites have not been systematically investigated. We have used a quantitative back-scattered electron imaging technique to analyze inter-trochanteric trabecular bone samples from 22 patients (11 males aged 70±12 years, 11 females aged 68±13 years) undergoing total hip replacement for oseoarthritis. These are compared with a group of 20 skeletally normal post mortem controls (12 males aged 60±14 years, 9 females aged 63±14 years) with both groups including only individuals aged greater than 40 years. Bone samples were fixed and embedded in methylmethacrylate resin for histomorphometry. These blocks were then polished, carbon coated and examined in a Philips XL20 scanning electron microscope which was calibrated with carbon/aluminium standards. Data were pooled to create a mineralization distribution for each of the groups, as well as analyzing the weight percent calcium (wt%Ca) and trabecular bone volume fraction (BV/TV[%]) data for individuals. The mineralization of the osteoarthritis group was less than that of the control group (24.2 wt%Ca versus 25.3 wt%Ca, respectively). The osteoarthritis mineralization distribution was a lower, broader curve consistent with the heterogeneous tissue-level morphology characteristic of osteoarthritis. Statistical analysis of individual's values revealed a significant lowering of the weight percent calcium in osteoarthritis compared to controls (24.4 wt%Ca versus 25.1 wt%Ca, respectively, p<0.001) concomitant with a significant increase in BV/TV(%) (10.4% versus 7.6%, respectively, p<0.02). These results are similar to those reported for sub-chondral bone in osteoarthritis and imply an increased rate of bone turnover associated with a net positive gain in bone volume. These findings at a site distal to the primary disease process have implications for systemic changes of bone metabolism in osteoarthritis.

Disclosures: P. Sutton-Smith, None.

T114

Osthol Stimulates Pre-osteoblast Proliferation and Differentiation Via BMP-2/RUNX2/SMAD1 Pathway. D. Tang*1, S. Cheng*1, O. Shi*1, D. Chen2, Y. Wang*1, 1Orthopaedics, Institute of Spine, Shanghai University of Traditional Chinese Medicin, China, 2Orthopaedics, Center for Musculoskeletal Research, University of Rochester, NY, USA.

Osthole, also named 7-methoxy-8-isopentenoxycoumarin, is a coumarin extracted from cnidii fructus. In the present researches, it has various of pharmacological effects, such as anti-arhythmia, anti-immunity, anti-apoptosis, anti-tumor, anti-inflammation, etc. It was also found that osthole has effects on osteoporosis using ovariectomized (OVX) rats and human osteoblast-like cell lines. The purpose of this study was to examine effects of osthole on the pre-osteoblast related with the dose and find its possible mechanisms for anti-osteoporosis. OCT-1 cells (a pre-osteoblast cell line) were used, cultured with 3 dose of osthole for 48h, or with purified BMP-2 protein. We first determined the effect of osthole on cell proliferation by MTT assay, alkaline phosphatase (ALP) activity in the cells was also assayed after appropriate treatment periods. The real time RT-PCR was employed to quantify the changes in mRNA levels for bone morphogenetic protein-2 (BMP-2) and RUNX2 of cells. At last we used the Western Blotting analysis to evaluated the expression of Smadl protein.

As a result, osthole (10ug/ml) can promote the OCT-1 cells proliferation, but osthole (30ug/ml) and osthole (60ug/ml) had no effect on the proliferation of these cells. Various dose of osthole can increase the ALP activity of OCT-1 cells, the effect of 30ug/ml was the best, and 10ug/ml followed. From the real time RT-PCR analysis. Various dose of osthole can enhance the BMP-2 mRNA levels, the effect of 10ug/ml was the best, and 30ug/ml followed. In addition, we found that osthole (10ug/ml) can enhance the BMP-2 mRNA levels significantly, but osthole (60ug/ml) decreased the BMP-2 mRNA levels significantly. From the Western Blotting analysis, osthole (10ug/ml) and osthole (30ug/ml) can stimulate the expression of Smadl protein, but osthole (60ug/ml) had the opposite effect. In this study, we demonstrate that osthole is a promising agent for anti-osteoporosis, but the effect of osthole is related with the dose. Only suitable dose of osthole can stimulate bone formation, while exorbitant dose of osthole may inhibit bone formation.

Disclosures: D. Tang, None.

T115

A New Approach to Assess Bone Formation Rates and Immunohistochemistry / in situ Hybridization in the Same Bone Specimen. J. Zhang, G. E. Gutierrez, M. Zhao, S. A. Munoz*, J. R. Edwards, G. R. Mundy.. Center for Bone Biology, Vanderbilt University, Nashville, TN, USA.

Bone formation rate is the key parameter in the in vivo characterization of osteoblast activity and bone growth/remodeling. The standard procedure to assess new bone formation is to use calcium-seeking fluorochromes which are visualized in undecalcified, plastic embedded sections of bone using fluorescence microscopy. Not only is this method time consuming and laborious, more importantly, it limits the application of immunohistochemistry (IHC)/in situ hybridization (ISH) on the same sections/specimens. Consequently, two separate bones (left and right) of an animal must be used for assessing bone formation or IHC/ISH experiments. This is problematic because we have observed that BMD and structural property differ between left-sided and right-sided bones. This suggests that experiments performed on different bones from the same animal may generate different biological information and lead to inaccurate conclusions. Our goal is to develop methods that enable the evaluation of all bone parameters including bone formation rate, bone /osteoid volume, osteoblast and osteoclast numbers, protein and gene expression profiles (IHC, ISH) using one bone specimen. To achieve this goal, the most challenging problem is to assess bone formation rate in decalcified specimens. We have tested three fluorochromes, two fixation buffers and two decalcification solutions. We found that the visualization of calcein green is superior to calcein blue and alizarin red in formalin fixed, EDTA-decalcified paraffin embedded bone sections; the fixation and decalcification times are also critical for preserving calcein green labeling. All three fluorochromes were not preserved in ethanol or formalin fixation following acid decalcification. Optimizing the time for different specimens, we have effectivelly assessed mineral apposition rates and other parameters of bone remodeling, as well as IHC and ISH in the same slides/specimens in different developmental stages of tooth and bone. In conclusion, we have successfully developed a simple method to assess new bone formation rates and protein/gene expression profiles in the same bone specimen. This procedure greatly increases the efficiency and reliability of the results.

Disclosures: J. Zhang, None.

T116

IGF-1 Regulation of Collagen II and Matrix Metalloproteinase-13 Gene Expression in Rat Endplate Chondrocytes via Distinct Signaling Pathways. M. Zhang*1, O. Zhou*1, Y. Dong2, T. Li2, O. Sh*1, Y. Wang*1, 1Orthopaedics, Institute of Spine, Shanghai University of Traditional Chinese Medicin, China, 2Orthopaedics, Center for Musculoskeletal Research, University of Rochester, NY, USA.

It is well known that IGF-1 exerts positive anabolic effects on chondrocytes in vivo and in vitro. However, little is known on IGF-mediated regulation of collagen II, matrix metalloproteinase-13(MMP13) gene expression and the involved intracellular signaling pathway in endplate chondrocytes. In the present study, chondrocytes isolated from normal rat endplate cartilage were stimulated with IGF-1 in monolayer culture. The technique of real-time PCR was employed to quantify the changes in mRNA levels for collagen IIα and mmp13 in rat endplate chondrocytes in response to IGF. The data were normalized to mRNA levels of β-actin, a constitutively expressed gene. Expression of signaling proteins was evaluated by western blot analysis. Cells were also treated with pharmacologic agents that block PI3K and MAPK signaling pathways. IGF-1 increased the collagen IIα mRNA expression in a time- and dose-dependent manner. With the treatment of IGF-1 (100 ng/ml), the expression of collagen IIα mRNA in rat endplate chondrocytes reached the highest value at 24 h and then decreased gradually along with time. IGF-1 (100 ng/ml) increased collagen IIα mRNA levels in rat endplate chondrocytes by 3.89 fold after treatment 24 h. Additionally, IGF-1 decreased MMP13 mRNA expression in rat endplate chondrocytes. IGF-1 (100 ng/ml) decreased MMP13 mRNA level by 0.55 fold after treatment 24 h. Furthermore, IGF-1 activated (phosphorylation) members of both the P13K pathway and the ERK/MAPK pathway, Akt and ERK1/2. Coincubation of IGF-1 with PI3K inhibitor wartmannin significantly blocked the stimulatory effect of IGF-1 on collagen IIa mRNA expression, but did not significantly inhibit IGF-induced repression of MMP13 mRNA expression. In contrast, the ERK/MAPK inhibitors PD98059 was able to block partially IGF-stimulated collagen IIα mRNA expression, but significantly blocked IGF-induced MMP13 mRNA repression. These data suggested that IGF-1 stimulation of PI3K signaling pathway is responsible for the ability of IGF-1 to increase collagen IIα mRNA expression, and IGF-1 stimulation of ERK/MAPK signaling pathway is responsible for the ability of IGF-1 to inhibit MMPI3 mRNA expression.

Disclosures: M. Zhang, None.

T117

Activation ERK1/2 Prevents the FasL-induced Apoptosis Anulus Fibrosus Cells in Insulin-like Growth Factor-1-Treated. Q. Zhou*1, M. Zhang*1, Y, Dong2, T. Li2, O. Shi*1, Y. Wang*1, 1Orthopaedics, Institute of Spine, Shanghai University of Traditional Chinese Medicine, China, 2Orthopaedics, Center for Musculoskeletal Research, University of Rochester, NY, USA.

FasL binds to Fas to induce cell apoptosis. Insulin-like growth factor-1(IGF-1) decreases apoptosis in several cell types. There have been no descriptions showing that through activation Erk1/2, increasing focal adhesion kinase (FAK) expression on anulus fibrosus cells with IGF-1.In this present study, rat anulus fibrosus cell were cultured and treated with antibody FasL, with or without human recombinant IGF-1. Cellular morphology was examined by light microscopy and reverse transcription polymerase chain reaction (RT-PCR). Apoptotic changes were evaluated by transmission electron microscopy, TUNEL staining, and immunostaining of Bax and bcl-2. Real time RT-PCR analysis showed mRNA levels of FAK, collagen II and aggrecan. Values are normalized to β-actin from three independent experiments. Western blot was performed to assay signaling proteins FAK and Erk1/2 phosphorylation and assess its relation to IGF-1. Anulus fibrosus cells expressed collegenll and aggrecan in vitro that maintenanced cell characteristic in vivo. FasL can induce anulus fibrosus cells apoptosis. TUNEL staining confirmed increased apoptosis ratio in antibody treated cells. Expression of bcl-2 was decreased by FasL, while expression of Bax was increased. FasL treatment caused FAK, collagen II and aggrecan mRNA expression approximately 2.84, 3.43, 3.91-fold down-regulated when compared to untreated controls. Simultaneous treatment with IGF-1 inhibited the effect of FasL on FAK, collagen II and aggrecan mRNA expression, increased 3.63,11.32,2.16-fold after treatment 24 hours. FasL treatment inhibited members of ERK/MAPK pathway, Erkl/2 phosphorylation. But not significant affected FAK phosphorylation. Co-treatment of IGF-1 can activate Erk1/2 (phosphorylation) but not significant activate FAK in short time. These data suggested that IGF-1 through activation Erk1/2 and up-regulated the FAK expression protects anulus fibrosus cells from FasL-induced apoptosis.

Disclosures: Q. Zhou, None.

T118

Dominant X-linked Hyposphatemic Rickets: A New Mutation of PHEX Gene. L. Masi1, S. Carbonell Sala*1, A. Gozzini*1, I. Pela*2, A. Amedei*1, A. Falchetti1, E. Luzi*1, S. Ottanelli*1, M. L. Brandi1, 1Internal Medicine, University of Florence, Florence, Italy, 2Pediatric, University of Florence, Florence, Italy.

Hypophosphatemic rickets is a group of disorders with hyphophsphatemia, hyperphosphaturia, normal levels of vitamin D and parathyroid hormone (PTH), bone deformities, osteomalacia/rickets. X-linked hypophosphatemic rickets (XLH) is one of the most common form of the familial hypophosphatemic rickets and in 60-80% of cases bear of mutation in PHEX gene (Xp22.2-p22.1). PHEX encodes for an endopeptidase membre M13Zn-metalloproteinase family, involved in the regulation of phosphate homeostatis. PHEX inactivating mutations causes XLH. These mutation enable the accumulation of phosphaturic factors and/or mineralizzation inhibitors. In the present study we descrive a 3 years old female referred to our Center, exhibiting clinical features of a clear hyperphosphaturia, hypophosphatemia, normocalcemia, PTH circulating levels at the upper values of the normal range and normal values fo vitamin D. She showed deep astenia, muscle pain and spasms, bowed legs and cranial deformities. The parents feel well and were not consanguineous. The patient and her parents underwent PHEX mutational analysis upon administration of an informed consent from (in case of minor patient signed by legal tutor). Genomic DNA has been extracted by peripheral blood leukocytes. The 22 exons and the intron-exon boundaries of PHEX have been investigated by PCR and direct - sequencing (ABI-Prism 3100) protocol. It has been identified a hemizigous mutation of PHEX IVS1078+1 position, causino nucleotide ch'ange in a splice donor site that may cause an abnormal splicing phenomenon. This nucleotide substitution has been never described on PHEX database (http://www.phexdb.mcgill.ca/). Up today, no functinal analysis for this mutation has been performed. In the present study a mini-gene analysis is undergoing to evaluate the alternative splicing pattern. Finally, we are planning to use cellular models, either obtained from patients and engineered by transfection methods to evaluate the functionality of the mutated gene. These approaches will be helpful to better understand the molecular mechanism of PHEX action and could provide highlight for for future targeted therapies.

Disclosures: L. Masi, None.

T119

Analysis of the Function of the CTCF-binding Site Between the Rxrb and the Col11a2 Genes. J. Murai*1, M. Okamoto*1, H. Yoshikawa1, N. Tsumaki2., 1Orthopaedic Surgery, Osaka University Graduate School of Medicine, Suita, Japan, 2Bone and Cartilage Biology, Osaka University Graduate School of Medicine, Suita, Japan.

The α2(XI) collagen chain gene (Col11a2) is specifically expressed in cartilage. The 1st intron sequence of the Col11a2 gene acts as the enhancer that activates expression in cartilage and silencer that inactivates expression in non-cartilaginous tissues. The 5′-end of the Col11a2 gene resides very closely to the 3′-end of the retinoid-X-receptor β gene (Rxrb) which is ubiquitously expressed. We hypothesized the enhancer/silencer blocking elements between these genes. So far, almost all of the characterized enhancer blocking elements identified in vertebrates are bound by the CTCF protein. At the last this annual meeting, we reported the CTCF binding site between the Col11a2 and the Rxrb genes. The purpose of this study is to investigate the function of the CTCF binding site. We prepared three transgene constructs by using the human bacterial artificial chromosome (BAC) DNA clone which is 160 kb in length covering entire COL11A2 and RXRB genes. The wild type (WT) construct was generated by placing the LacZ sequence at the downstream of the RXRB gene promoter in the BAC clone. The mutantion construct (Mut) was made by introducing 14 bp-substitution mutation in the CTCF binding site in the WT. The deletion construct (Del) was produced by deleting the 507bp sequence around the CTCF binding site in the WT. We generated transgenic mice bearing these constructs and analyzed LacZ expression patterns by staining the 13.5 d.p.c. founder embryos with X-gal. All of the 4 transgenic mice bearing the WT transgene expressed the LacZ gene in all tissues. This ubiquitous expression pattern of the LacZ was consistent with the expression pattern of the endogenous Rxrb gene. On the other hand, 2 out of 3 transgenic mice bearing the Mut and 2 of 4 mice bearing the Del expressed the LacZ genes specifically in cartilage. Abolition of the CTCF-binding site specifically inhibited expression of the LacZ in non-cartilaginous tissues, suggesting that the CTCF-binding site blocks silencer activities of the Col11a2 regulatory sequences. Next, we established stable transformant clones of rat chondrosarcoma cells bearing the WT, Mut and Del BAC DNA respectively. By the real time RT-PCR, we specifically amplified the human RXRB and COL11A2 mRNA sequences that derived from the transgene. The expression levels of the puromycin resistance gene were used as the internal control. The mean expression levels of the RXRB genes of the Mut and Del clones were significantly higher than that of the WT clones. The abolition of the CTCF-binding site increased expression of the RXRB in chondrosarcoma cells, suggesting that the CTCF-binding site blocks enhancer activities of the Col11a2 in cartilage.

Disclosures: J. Murai, None.

T120

Identification of Hs.43125 as an Articular Chondrocyte-Specific Gene. S. Shin*, J. Chun*, Department of Life Science, Gwangju Institute of Science and Technology, Gwangju, Republic of Korea.

Developmental process of endochondral bones is initiated by chondrogenesis of mesenchymal cells and subsequent hypertrophic maturation. During this developmental process, chondrofying mesenchymal cells produce a variety of signaling molecules to coordinately regulate cartilage and bone development. We searched expressed sequence tags (ESTs) cluster library of human normal cartilage in Unigene transcriptome database to identify unrevealed signaling molecules which are produced during limb development. Hs.43125 was identified as a novel cartilage-specific gene and seleted for functional characterization. Hs.43125 is composed of four different exons and signal peptide sequence for secretion. When myc-tagged Hs.43125 cDNA was ectopically expressed in primary articular chondrocytes, Hs.43125 encodes a 17 kDa secretory. The molecular weight of secreted Hs.43125 was smaller than cellular protein indicating post-translational modification during secretion. In attempt to examine the role of Hs.43125, expression pattern of Hs.43125 was examined during chondrogenesis of mesenchymal cells and hypertrophic maturing of chondrocytes. Hs.43125 expression was low in mesenchymal cells, increased during chondrogenesis and decreased during hypertrophic maturation of chondrocytes in vitro. In situ hybridization analysis also indicated that Hs.43125 is expressed in proliferating chondrocytes of developing cartilage of mouse embryo at 14.5 day. Our results suggest that Hs.43125 may have a role in chondrogenesis, maintanance of differentiatied chondrocyte phenotypes, and/or transition of prehypertrophic chondrocytes to hypertrophic chondrocytes.

Disclosures: S. Shin, None.

T121

Carbonic Anhydrase II Regulates Differentiation and Proliferation of Ameloblast via Intracellular pH-dependent Mechanism. X. Wang*1, T. Suzawa1, M. Nakamura*2, B. Zhao*1., R. Yasuhara*1, T. Inoue*3, Y. M. Masuda*4, K. Matsumoto*4, R. Kamijo*1, 1Department of Biochemistry, Showa University School of Dentistry, Tokyo, Japan, 2Department of Oral Anatomy, Showa University School of Dentistry, Tokyo, Japan, 3Department of Oral Physiology, Showa University School of Dentistry, Tokyo, Japan, 4Department of Clinical Cariology & Endodontology, Showa University School of Dentistry, Tokyo, Japan.

This study aimed to clarify the involvement of carbonic anhydrase II (CAII), a zinc metalloenzyme, on ameloblast differentiation and enamel biomineralization. We identified the expression level of CAII mRNA is strongly up-regulated in differentiated enamel epithelial tissues by means of microarray. Immunohisotochemical analysis revealed that CAII was expressed intensively in secretory stage ameloblast in enamel epithelium. In an ameloblast primary culture, the CA enzyme activity measurement showed that activity of CA was increased along with differentiation of ameloblast. RT-PCR indicated that the expression level of amelogenin, a marker for secretary ameloblasts, was enhanced by ethoxizolamide (EZA), a CA inhibitor, as well as by CAII antisense oligonucleotide (CAIIASO). In contrast, expression of EMSP-1, a marker for mature ameloblast, was inhibited by them, suggesting that inhibition of CAII activity could inhibit differentiation of ameloblast. MTS assay also showed they promoted proliferation of ameloblast. Inhibition of ameloblast differentiation by EZA and CAIIASO was confirmed by tooth germ organ culture, since they induced disorderly arrayed ameloblasts with poor polarity. Intracellular pH of ameloblast was labeled with BCECF-AM, and was controlled by the K+-nigericin method. EZA and CAIIASO elevated intracellular pH in amoeloblast, and an artificial decrease of intracellular pH abolished the effects of CAIIASO on ameloblast. These results suggest the novel role of CAII during amelogenesis, that is, controlling differentiation and proliferation of ameloblast. The results also suggest controlling intracellular pH might be primary mechanism of CAII in ameloblast.

Disclosures: X. Wang, None.

T122

Regulation of Matrix Metalloproteinases Expression by EPASI in Articular Chondrocytes. S. Yang*, J. Cho*, J. Chun*, Department of Life Science, Gwangju Institute of Science and Technology, Gwangju, Republic of Korea.

We identified and selected for functional characterization of endothelial PAS domain protein 1 (EPASI), also referred as a hypoxia-inducible factor 2α, by screening transcriptome database for expressed sequence tags (ESTs) using osteoarthritic cartilage library in unigene. Because EPASI plays essential roles in hypoxia and many pathogenic conditions, this study examined a role of EPASI in articular chondrocytes. EPASI expression was significantly increased in human osteoarthritic cartilage compared with normal cartilage. In primary culture articular chondrocytes, EPASI expression was significantly increased during dedifferentiation of chondrocytes caused by interleukin-1beta (IL-1β), epidermal growth factor (EGF), retinoic acid (RA), or serial subculture as monolayer. Dedifferentiation of chondrocyte accompanies induction of several matrix metalloproteinases (MMPs) such as MMP-1, −3, −9, −12, and −13, without effects on MMP-2, −14, and −15 expressions. Upregulation of EPASI during dedifferentiation of chondrocyte occurs prior to MMP expression. We, therefore, examined whether EPASI mediates upregulation of MMPs. Overexpression of EPASI by adenovires-EPASI (Ad-EPASI) induced MMP-1, −3, −9, and −13 expressions, whereas expression patterns of MMP2-, −14, and −15 were not changed. Knockdown of EPASI by siRNA blocked IL-1β-induced expression of MMP-3, −9, and −13. Our results suggest that EPASI may play a role in cartilage destruction by modulating MMP expression.

Disclosures: S. Yang, None.

T123

Identification and Characterization of MicroRNAs Expressed During Chondrogenesis of Mesenchymal Cells. S. Yu. J. Chun*, Life Science, GIST, Gwangju, Republic of Korea.

microRNAs (miRNAs) play important roles in regulating the expression of specific mRNAs at both transcriptional and posttranscriptional levels. In attempt to understand more information in on-off of mRNA expression during chondrogenesis, we investigated miRNAs expressed in chondrifying mesenchymal cells derived from mouse embryo limb buds. We identified three new miRNAs from chondrifying mesenchymal cell. Among the identified miRNAs, expression of miRNA 542-3t was decreased during chondrogenesis of mesenchymal cells, and its expression was specifically detected in developing cartilage tissue. By computational analyses, 6130401L20Rik was identified as a potential target gene of miRNA 542-3t. Consistent with the expression pattern of miRNA 542-3t, a target gene expression was increased during chondrogenesis, and its expression was detected in developing cartilage. Our results suggest specific that miRNA 542-3t may regulate transcription of mRNAs such as 6130401L20Rik during mammalian limb development.

Disclosures: S. Yu, None.

T124

Effect of Estrogen Deficiency on Gene Expression Pattern in the Bone Tissue of Postmenopausal Versus Premenopausal Healthy Women. B. Balla*1, J. Kósa*1, J. Kiss*2, A. Borsy*3, J. Podani*4, I. Takács1, Á. Lazáry1, Z. Nagy1, K. Bácsi*1, G. Speer*1, L. Orosz*3, P. Lakatos1, 11st Department of Internal Medicine, Semmelweis University, Budapest, Hungary, 2Department of Orthopedics, Semmelweis University, Budapest, Hungary, 3Institute of Genetics, Agricultural Biotechnology Center, Gödöll, Hungary, 4Department of Plant Taxonomy and Ecology, Eötvös Loránd University, Budapest, Hungary.

Estrogen deficiency at the time of menopause results in marked increase in bone resorption and formation leading to rapid bone loss. The aim of our investigation was to determine genes characterized by significantly changed mRNA expression rates in postmenopausal vs. premanopausal healthy bone tissue and describe the interrelationship among these genes using multidimensional data analysis. Ten bone tissue samples from postmenopausal non-osteoporotic female patients (mean age: 53.50 ± 4.12 years, T-score > -I SD) and seven bone tissue samples from premenopausal healthy women (mean age: 52.14 ± 2.34 years, T-score > −1 SD) were examined in our study. Messenger RNA was prepared from each sample and reverse transcribed to cDNA. The expression differences of selected 118 genes were analyzed by TaqMan Gene Expression Assay in Real-Time PCR system. Statistical methods were performed using Mann-Whitney U test, canonical variates analysis and principal components analysis (PCA). Mann-Whitney U test indicated significant differences in the expression of 29 genes between post- and premenopausal healthy individuals (p ≥ 0.05). Twenty eight genes, including extracellular matrix molecules and digesting enzymes (COL2A1, COL3A1, COL5A1, COL5A2, COL9A1, COL12A1, COLI5A1, MGP, BGLAP, FN1, MMP13, BMP1), TGF-beta/BMP pathway (BMPR1A, TGFB2, TGFB3, TGFBR2, SMAD4), transcription factors (RUNX2, SP7, TCF7L2, SOX9), growth factors (PDGFA, FGFR1) and other candidate genes (TNFSF11, IL6, ALPL, IGSF4, TRIB2) were significantly upregulated in postmenopausal women compared to premenopausal ones. Only one gene (ENOI) showed downregulation in the bone tissue after menopause. Appling canonical variates analysis the groups of post- and premenopausal patients are separable by 12 genes coding extracellular matrix molecules which have the best discriminatory power. Based on the multiple mRNA expression profiles of 118 genes, post- and premenopausal states could be differentiated by enhanced postmenopausal gene expression rate using PCA. Significant differences observed in gene expression profiles of estrogen deficient human bone tissue provide further insight into the process of postmenopausal changes of bone metabolism. The menopausal states of bone tissue have been unambiguously defined by their complex gene transcription patterns.

Disclosures: B. Balla, None. This study received funding from: NKFP-1A/007/2004, NKFP-1A/002/2004. ETT022/2006.

T125

Novel Transcription Factor-like Function of MMP-3/stromelysin-1 that Regulates Connective Tissue Growth Factor (CTGF/CCN2) Gene Transcription. T. Eguchi1, S. Kubota*1, K. Kawata*1, Y. Mukudai*1, L. Yanagita*1, T. Ohgawara*1, J. Uehara*2, S. Ibaragi*3, M. Takigawa1, 1Dept. of Biochemistry and Molecular Dentistry, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan, 2Dept. of Oral & Maxillofacial Rehabilitation, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan, 3Dept. of Oral & Maxillofacial Surgery, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama, Japan.

Connective tissue growth factor (CTGF/CCN2) is the second member of CCN protein family and a multifunctional growth factor involved in the development and remodeling of matrix-rich tissues such as cartilage and bone. For example, we have shown that CCN2 promotes endochondral ossification by acting on chondrocytes, osteoblasts and endothelial cells. Its expression is extremely high in chondrocytes especially in hypertrophic chondrocytes. Enhanced production of CCN2 in HCS-2/8 human-derived chondrocytic cells is found to be mediated by the TRENDIC (transcription enhancer dominant in chondrocytes). Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidase also involved in the development and remodeling of cartilage and bone. Here, we report that human MMP3/stromelysin-1 in the nucleus binds to the TRENDIC, and displays a transcription factor-like function. The MMP3 was detected in the nuclear extract of the HCS-2/8 cells in vitro, and also in the nucleus of the normal and osteoarthritic chondrocytes in vivo. Externally added recombinant human MMP3 was internalized into the HCS-2/8 cells and translocated to the nucleus. Also, six putative nuclear localization signals were found in MMP3 and were capable of trans-locating the fused green fluorescent protein to the nucleus. MMP3 interacted with enhancer sequences including TRENDIC in the CCN2 promoter. MMP3 overexpression resulted in activating the CCN2 promoter; whereas, a TRENDIC-mutant of the promoter lost the response. Knocking-down of MMP3 also suppressed CCN2 expression. Notably, an MMP3 inhibitor specifically suppressed the CCN2 promoter activity in the cells. Furthermore, we identified several nuclear MMP3 associated proteins (NuMAPs) in the cells. These finding indicates that MMP3 has dual functions. One is as an extracellular matrix-degrading activity. The other is novel transcription factor-like function for CCN2/CTGF, which results in production of ECM. This extra- and intra-cellular, dual function of MMP3 may play an important role in development and matrix remodeling of cartilage and bone.

Disclosures: T. Eguchi, None. This study received funding from: MEXT, JSPS.

T126

Epigenetic Status Monitored by DNA Methylation in the 5′-flanking Regions of CpG-rich Promoters Are Stable During Chondrogenesis in Pellet Cultures of Pluripotent Human Mesenchymal Progenitor Cells. Y. Ezura1, I. Sekiya*2, T. Muneta*3, M. Noda1, 1Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan, 2Section of Cartilage Regeneration, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan, 3Section of Orthopedic Surgery, Graduate School, Tokyo Medical and Dental University, Tokyo, Japan.

Developmental skeletogenesis requires multistep cellular events, including progenitor cell recruitment, proliferation, differentiation and multiple interactions of different types of cells. The entire actions of the processes are believed to be programmed in the genomic sequence of the cells; however investigations indicating the additional involvement of “epigenetic” regulation also exist. Here, to investigate the possible involvement of “epigenetic” controls of gene expression during skeletogenesis, we analyzed DNA methylation of CpG-rich promoters for several genes, using genomic DNA samples obtained from the experimental chondrogenesis assay of mesenchymal progenitor cells derived from human synovial tissues. Chondrogenic pellet-cultures were performed according to a standard protocol using rhBMP2 and TGF beta in the medium, and genomic DNA was isolated before and after 3 weeks of pellet-culture. This resulted in highly differentiated chondrocytes producing large amount of cartilage matrix. Candidate genes for the epigenetic control were selected by analysis of expression profiling data accomplished by Affymetrix DNA microarray. By searching dramatically upregulated or downregulated genes during the chondrogenesis, 40 genes were chosen. Among them, genes having extreme CpG-rich promoter were examined at first, for the extent of DNA methylation using bisulfite DNA sequencing method. Consistent with previous studies, most of the CpG-rich promoters were hypomethylated in the progenitor cells before pellet-culture, even for the genes completely silenced before differentiation. We observed stable, similar level of methylation after 3 weeks of pellet-culture in most case. In contrast, we detected highly methylated CpG sites in the promoter region of moderate CpG content, like matrillin-4 gene promoter that is transcriptionally repressed before differentiation; however, the methylation levels were similar even after the induction of chondrogenesis despite apparently detectable levels of the expression. We concluded regarding the genes examined in this study that epigenetic status such as DNA methylation would stably exist during chondrogenic differentiation at least in the context of pellet cultures of the human mesenchymal progenitor cells.

Disclosures: Y. Ezura, None.

T127

Bone Remodeling after Achievement of Osseointegration by Titanium Implantation in Rat Maxillae. M. Haga*1, N. Fujii*2, K. Nozawa-lnoue*3, K. Uoshima*2, S. Nomura*1, T. Maeda*3. 1Div. of Oral Health in Aging and Fixed Prosthodont., Niigata Univ. Grad. Sch. of Med. & Dent. Sci., Niigata, Japan, 2General Dentistry and Clinical Education Unit, Niigata Univ., Niigata, Japan, 3Div. of Oral Anat., Niigata Univ. Grad. Sch. of Med. & Dent. Sci., Niigata, Japan.

Osseointegration is regarded as the most appropriate bone-implant interface as it affords a favorable prognosis over an extended period. Previous reports have indicated the existence of pre-existing bone with empty osteocytic lacunae even after achievement of osseointegration. However, little information is available regarding the fate of this injured bone. It is also unclear whether bone remodeling takes place around the implant. The present study was therefore undertaken to examine the response of the surrounding bone around a titanium implant on an animal model using rat maxillae.

Following extraction of the upper first molars of male Wister rats (4-week-old; n-80), pure titanium implants were installed in the prepared bone cavities by drilling at 1 month after tooth extraction. Under deep anesthesia, the animals were sacrificed at 1 to 12 months after implantation. Decalcified paraffin sections were processed for double staining with alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP). For histologic observations, some sections were stained with hematoxylin and eosin (H-E), or Azan. An additional 14 rats were injected with calcein for dynamic labeling of bone formation at 5 and 20 days before sacrifice. Undecalcified sections were prepared in a cryostat with a tungsten knife.

The present animal model achieved osseointegration between the implants and alveolar bone by 1 month post-implantation, but the injured bone with empty osteocytic lacunae remained in the pre-existing bone. Surface analyses showed that the pre-existing bone with empty osteocytic lacunae decreased gradually to disappear completely at 3 months after implantation. Many ALP-positive osteoblasts and TRAP-reactive osteoclasts localized on the surface of the bone along the implant at 1 month post-implantation and remained until 6 months after implantation. The newly-formed bone around the implants thickened and exhibited the same histological features as compact bone at 3 months. Two calcein-labeled lines were recognizable in the newly-formed bone around implants throughout this observation period.

These morphological data suggest the involvement of bone remodeling in replacing newly-formed immature woven bone and injured pre-existing bone with compact bone around titanium implants.

Disclosures: M. Haga, None.

T128

The Increase of the Mechanical Strength of Novel Unidirectional Porous Hydroxyapatite Ceramics In Vivo. M. Iwasashi1, M. Sakane1, Y. Shirai*2, Y. Suetsugu*2, T. Tateishi*2, N. Ochiai*1, 1Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan, 2Biomaterials Center, National Institute for Materials Science, Tsukuba, Japan.

Background While the porous Hydroxyapatite (HAp) ceramic has interconnection between the pores, a few ostegenesis occur at the deep area of the HAp material. We have recently developed a unidirectional pores HAp ceramic, which have continuous interconnection through the material.

Purposes The purposes of this study were to observe osteoconductivity of the novel HAp ceramic, and to evaluate the increase of mechanical strength after implantation in the femur of rabbit.

Materials and Methods: Materials for this study were provided by KURARAY CO., LTD. (Kurashiki, Japan).

Animal experiments were carried out in our university and governmental guidelines for proper conduct of animal experiment. Japanese white rabbits were used in this study. Under intravenous injection of thiopental, the knee joint was exposed. Intramedullary, a tunnel was made with drilling of the femur, the cylindrical unidirectional porous HAp (6mm in diameter, 7mm in height) were implanted in the femur. The direction of the interconnected pore is parallel to the axis of the femur. Two, six and twelve weeks after implantation, the femurs were harvested and the 4×4×5mm HAp samples were cut down. The compression strength was measured using a uniaxial mechanical testing apparatus with speed of 0.5mm/s. The same size samples before implantation were measured as a control. For statistical analysis, Tukey-Kramer test were used and P < 0.05 was considered statistically significant. Some of the femurs were used for histological evaluation.

Results: The compression strength of samples before implantation was 13.8±1.4MPa, and that of samples at two, six, and twelve weeks was 10.9±3.9,21.6±9.2,47.0±12.7MPa. The compression strength at six and twelve weeks increased significantly. Histologically, new bone and osteogenetic cells were observed inside of the HAp after two weeks.

Discussion: The microstructure of unidirectional porous HAp is oval pores 100 ∼ 300μm in diameter penetrate through the material. The structure is advantageous not only about compression strength but also migration for osteogenic and angiogenic cells inside of the HAp, and contributed to excellent new bone formation at two weeks. The shape of new bone is columns because osteogenesis occurred along the inner wall of the HAp. Beside, early angiogenesis promote bone remodeling. Hence, the mechanical strength at twelve weeks gained 3.4 times as much as before implantation.

Conclusion: Unidirectional porous HAp has excellent osteoconductivity and increase of mechanical strength with time in vivo.

Disclosures: M. Iwasashi, Materials were provided by Kuraray Co., Ltd. (Kurashiki, Japan) 9.

T129

Identification of Calcyclin ExpresSion In Rat Amelogenesis Using Acp Ddrt-per Method. Y. Kawano*, S. Kinosita-kawano*, K. Nozawa-inoue*, A. Suzuki*, T. Maeda*, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan.

Ameloblasts are involved in enamel secretion and mineralization. Unmineralized enamel is secreted by secretory ameloblasts and subsequently mineralized by maturation stage ameloblasts. The transformation from secretory into maturation stage ameloblasts during amelogenesis is presumed to be accompanied by changes in gene expression in each cell type. The goal of this study was to identify the genes expressed specifically in ameloblasts that might be involved in ameloblast differentiation, enamel formation or maturation.

Annealing controlled primers (ACP) differential display PCR screening was performed to compare RNA isolated from secretory ameloblasts with those from maturation stage ameloblasts. Using 120 different combinations of primers, we identified and cloned 36 distinct differential display fragments with sizes ranging from 250 to 1000 bp. Seventeen of 39 differentially expressed genes (DEGs) were sequenced and identified. Seven DEGs have significant sequence similarities to already published genes whose functions in the rat incisor are known. The rest is homologous to genes expressed in other tissues, but is not known to be expressed in the incisor. One of the DEGs showed 100% homology to calcyclin (S100A6).

Calcyclin belongs to a family of S100A calcium binding proteins that bind calcium molecules through conserved EF-hand domains. Recently a wide variety of cellular functions has been attributed to the products of this gene family. In particular, calcyclin has been implicated in intracellular calcium homeostasis and signaling, ion transport, and cell proliferation. Quantitative RT-PCR confirmed that calcyclin has specific expression patterns in differentiating ameloblasts during odontogenesis. Calcyclin mRNA was less expressed in secretory stage, but highly in both early and late maturation stages in rat incisor. Immunohistochemistry also showed that calcyclin proteins are expressed in maturation stage of ameloblasts, but not in papillary layer cells. Furthermore, odontoblasts and cementoblasts showed immunopositive reaction for calcyclin, while osteoblasts on alveolar bone were devoid of immunoreaction.

Using ACP, we found 39 DEGs in secretory and maturation stage ameloblasts. Calcyclin was identified to be 100% homologous to one of the DEGs. We confirmed the existence of calcyclin in the differentiating ameloblasts using qRT-PCR and demonstrated its expression in ameloblasts, odontoblasts and cementoblasts by immunohistochemistry. Our findings suggest a role for calcyclin in amelogenesis, especially in enamel maturation, dentinogenesis, and cementogenesis.

Disclosures: Y. Kawano, None.

T130

The Effects of Heme Oxygenase-1 on Collagen Induced Arthitis Model. H. Kim*1, M. Lee*2, J. Oh*3, J. Kim*4, K. Yun*1, 1Pathology, Wonkwang University Hospital, Iksan Cholla-bukdo, Republic of Korea, 2Rheumatology, Wonkwang University Hospital, Iksan Cholla-bukdo, Republic of Korea, 3Anatomy, Wonkwang University School of Medicine, Iksan Cholla-bukdo, Republic of Korea, 4Orthopaedic Surgery, Wonkwang University Hospital, Iksan Cholla-bukdo, Republic of Korea.

Introduction: Heme oxygenase-1 (HO-1), an inducible heme-degrading enzyme, is expressed by macrophages and endothelial cells in response to various stress. It is also an important mediator of inflammation.

Purpose: To determine the effects of HO-1 modulation on the collagen-induced arthritis (CIA) model and observe the VEGF expression in human synovial fibroblast

Method: DBA/1J mice were treated with an inhibitor of HO-1, tin protoporphyrin IX (SnPP), or with an inducer of HO-1, cobalt protoporphyrin IX (CoPP), from day 1 to day 35 after CIA induction. The clinical evolution of disease was visually monitored. At the end of the experiment, joints were examined for histopathologic changes. VEGF expression in paws were measured by immunohistochemical stain. mRNA expression of VEGF stimulated with TNF-alfa, COPP and SnPP, accessed in human synovial fibroblast by RT-PCR

Results: Administration of cobalt protoporphyrin IX significantly induced the inflammatory response, with increased arthritis index and expression of VEGF in paw of arthritis models. Treatment with SnPP significantly reduced the severity of CIA, with inhibition of joint inflammation and cartilage destruction. The expression of VEGF were also significantly reduced by SnPP treatment in paw. CoPPIX as inducer of HO-1, increased VEGF expression dose dependently in synovial fibroblast. In contrast, inhibition of HO-1 activity by SnPPIX reversed CoPPIX-induced VEGF production.

Conclusion: The effects of HO-1 induction in rheumatoid arthritis result in aggravation of arthritis via up-regulation of VEGF. We concluded that inhibition of HO-1 could be a therapeutic target of rheumatoid arthritis

Disclosures: H. Kim, None. This study received funding from: Wonkwang University grant 2004.

T131

Loss of Annexin VI Affects Endochondral Bone Formation. H. Kim1, S. E. Moss*2, T. Kirsch1, 1Orthopaedics, University of Maryland School of Medicine, Baltimore, MD, USA, 2Division of Cell Biology, University College London, London, United Kingdom.

Annexin VI is a Ca2+ and membrane-binding protein, which is involved in a various cell functions. We have shown that annexin VI is expressed by hypertrophic growth plate chondrocytes and osteoblasts together with other annexins, including annexin II and V. These annexins control Ca2+ homeostasis in growth plate chondrocytes and matrix vesicles thereby regulating terminal differentiation and mineralization events (Kirsch 2005 Front Biosci 10:576-581). To determine the exact function of annexin VI in skeletal tissues, we analyzed long bones of annexin VI-/- mice histomorphometrically and analyzed the function of matrix vesicles isolated from long bones of 5 months old mice. Matrix vesicles are released by mineralization-competent growth plate chondrocytes and osteoblasts and these particles initiate the mineralization process. We have shown that annexins II, V and VI have the critical role of mediating influx of Ca2+ into the vesicles enabling the formation of the first mineral phase inside the vesicles. Newborn annexin VI-/- mice were ∼20% shorter and lighter than wild type littermates. Histomorphometric analysis of the femoral and tibial growth plates of these animals revealed that the lengths of the growth plates (proliferative and hypertrophic zones) were reduced by ∼40% in the annexin VI-/- mice compared to the lengths of the growth plates of wild type littermates. The length of the proliferative zone of the annexin VI-/- mice is similar to the length of the proliferative zone of the wild type littermates. However, the length of the hypertrophic zone is markedly reduced in the growth plates of the annexin VI-/- mice. At 4 weeks of age the length of the growth plate in the annexin VI-/- mice was enlarged compared to the length of the growth plates of the wild type littermates, suggesting that endochondral bone formation is delayed in annexin VI-/- mice. At 10 weeks of age the annexin VI-/- mice showed reduced bone volume compared to the bone volume of the wild type littermates. Matrix vesicles isolated from long bones of annexin VI-/- mice contained more annexin II and annexin V than the amount of these annexins in matrix vesicles isolated from wild type littermates. However, vesicles isolated from annexin VI-/- mice contained less Ca2+ and Pi than the amount of these mineral ions in vesicles isolated from wild type littermates. In addition, matrix vesicles isolated from annexin VI-/- mice were not able to mineralize in vitro. These findings reveal that annexin VI plays an important regulatory role in terminal differentiation and mineralization events of growth plate chondrocytes and osteoblasts and a loss of annexin VI results in delayed endochondral bone formation and bone loss in older annexin VI-/- mice.

Disclosures: H. Kim, None. This study received funding from: National Institutes of Health.

T132

PHD1 and PHD3 Are Expressed in Bone Cells but Are Not Essential for Bone Homeostasis. K. Laperre*1, N. Smets*1, R. Bouillon1, P. Carmeliet*2, G. Carmeliet1, 1Laboratory of Experimental Medicine and Endocrinology, Katholieke Universiteit Leuven, Leuven, Belgium, 2Center for Transgene Technology and Gene Therapy, VIB, Katholieke Universiteit Leuven, Leuven, Belgium.

Hypoxia induces vascular endothelial growth factor (VEGF) expression, an essential factor for angiogenesis. Signalling occurs via stabilization of hypoxia inducible factor (HIFα) that binds to its response element in the VEGF promoter. During normoxia HIFα activity is inhibited by prolyl hydroxylase domain containing proteins (PHD1, PHD2 and PHD3) targeting HIFα for proteasome mediated degradation. Both the 3 VEGF isoforms and HIFα are essential for bone development by affecting angiogenesis and/or bone cell function. The role of PHDs in bone biology is however not yet elucidated.

In this study, we report that PHDs are expressed in primary murine osteoblast, osteoclast and chondrocyte cell cultures as analysed by quantitative PCR. Abundant mRNA levels were observed for PHD1, less expression was detected for PHD2 whereas PHD3 mRNA levels were very low. Exposure of these cultures to hypoxia for 24 hours resulted in a 3 to 4 fold induction of VEGF mRNA expression in all cell types (p<0.001). In addition, PHD2 and mainly PHD3 mRNA levels were highly induced in the 3 cell types (p<0.0001) whereas PHD1 mRNA expression was not affected by hypoxia.

The role of PHD1 and PHD3 in bone homeostasis was investigated by analyzing the bone phenotype of PHD1 and PHD3 knock out (KO) mice. PHD2 KO mice are early embryonic lethal precluding investigation. PHD1 and PHD3 KO mice are viable and show a normal growth curve. Bone modeling and remodeling were investigated in respectively 4 and 14 week old mice. Femur length was not different between PHD1 and PHD3 KO and their respective wild type (WT) littermates. Trabecular parameters of isolated femurs were analysed by peripheral quantitative computed tomography (pQCT). Trabecular density and content were not different between the genotypes. Histomorphometry on Von Kossa stained sections of PHD3 KO and WT mice confirmed these data. In addition, serum osteocalcin levels also revealed no difference between PHD1 and PHD3 KO mice and their respective WT littermates. A possible explanation for the normal bone phenotype is compensation by the other PHDs. Osteoclasts and chondrocytes lacking PHD1 showed an increased expression of PHD2 and PHD3. This compensatory increase was also observed in PHD3 deficient osteoclasts.

In conclusion, the 3 PHDs are expressed in the major bone cell types but lack of PHD1 or PHD3 has no major impact on bone modeling and remodelling.

Disclosures: K. Laperre, None.

T133

The New Surface Modificated Demineralized Bone Matrix by rhBMP-2-collagen Hybrid Coating. K. Lee*1, Y. Shim*2, J. Jang*2, H. Kim*3, Y. Choi*2, K. Lee*2, B. Ko*2, M. Ryu*2, I. Kim*2, S. Moon*3, H. Lee*3, 1Medical school, Yonsei University, Seoul, Republic of Korea, 2Korea Bone Bank Co., Ltd., Seoul, Republic of Korea, 3Medical School, Yonsei University, Seoul, Republic of Korea.

The use of bone grafts is increasing considerably in orthopaedics and dental practice. Also demineralized bone matrix (DBM) is inherently osteoinductive and it has potential appeal as a bone graft substitute. DBM directly induces new bone formation when implanted subcutaneously or intramuscularly. Allogeneic DBM has intrinsic shortcomings related to procuring, processing and characterizing bone from a human donor pool. Xenogeneic bone represents an unlimited supply of available material if it can be processed to render it safe for transplantation to the human host. Processed xenogeneic DBM still appear a promising alternative to autografts and allografts although several such graft materials were found to be unsatisfactory because of poor histocompatibility which apparently stemmed from the difficulties in manufacturing such a complex material. However, there is lack of consensus about whether DBM from one species can facilitate osteoinduction in a different species, even among lower-order animals. To improve the property of xenogeneic DBM, we modified the surface of DBM by rhBMP-2-collagen hybrid coating. In vitro test, surface modified xenogeneic DBM and human mesenchymal stem cell were cocultured by alginate bead culture. And In vivo test, DBM was implanted in fibular fractured Newzealand white rabbit

Disclosures: K. Lee, Korea Bone Bank Co., Ltd. 3. This study received funding from: Korea Bone Bank Co., Ltd.

T134

Validation of a Simple Isotope Method for Estimating True Calcium Fractional Absorption in Adolescent Girls. W. Lee*1, G. P. McCabe*2, M. E. Wastney3, B. R. Martin4, C. M. Weaver4, 1Agricultural and Biological Engineering, Purdue University, West Lafayette, IN, USA, 2Statistics, Purdue University, West Lafayette, IN, USA, 3Metabolic Modeling Services, Blenheim, New Zealand, 4Foods and Nutrition, Purdue University, West Lafayette, IN, USA.

A 5-hr serum oral isotope method has been validated against a classic double isotope or ratio method for assessing calcium absorption in adults (Heaney and Recker Ann Intern Med 103:516, 1985). A simple approach for calculating absorption has not been developed in children. Our goal was to develop an equation to predict absorption using a single serum measurement of specific activity (SA, expressed as the fraction of dose of single oral tracer per gram calcium) and physiological factors and to also identify the best time points to measure SA. Subjects were 45 adolescent girls aged from 10 to 15 yr participating in metabolic studies. Tracer data following oral (44Ca) and intravenous (42Ca) administration of calcium stable isotopes and SA in serum and urine from various time points up to 4 d were used. Absorption was calculated as the ratio of oral to IV tracer in 24-hr urine. In addition to SA, physiological factors (postmenarcheal age and height) were used as predictors in multiple regression analysis. The 4-hr serum SA showed the highest R2 value with the 24-hr urine ratio. Measured serum SA values between 2.5 and 12 hr can be used with the same equation to predict the 24-hr urine ratio but with lower precision. A similar approach was used to develop equations for urine sampling following oral stable calcium isotope administration. We conclude that the developed equations can be used with a single blood draw or urine collection to evaluate calcium absorption in adolescents.

Disclosures: W. Lee, None.

T135

Does Increased Local Bone Resorption Result in Increased Cartilage Degradation?D. J. Leeming1, I. Byrjalsen1, M. G. Sørensen1, M. Koizumi*2, P. Ovist1, M. Fregerslev*3, N. Lynnerup*1, C. Christiansen*1, M. A. Karsdal1, 1Nordic Bioscience, Herlev, Denmark, 2Cancer Institute Hospital, Tokyo, Japan, 3Institute of Forensic Science, University of Copenhagen, Copenhagen, Denmark.

An association between bone and cartilage degradation in osteoporotic and arthritic patients has previously been reported. Breast and prostate cancer patients often develop lesions of locally high bone turnover, when the primary tumor metastasizes to the bone. The objective of the present study was to determine whether increased bone turnover is associated with an increase in cartilage degradation in an aggressive bone disease such as metastatic cancer.

The study population included 132 cancer patients; 90 breast cancer (45 with bone metastasis (BM)) and 42 prostate cancer (17 with BM). Presence of BM was determined by Tc99 scintigraphy and the skeletal involvement was graded according to the Soloway score 1-4. Urine samples were obtained and bone resorption assessed by CrossLaps ELISA (CTX-I), and cartilage degradation by CartiLaps ELISA (CTX-II). Human cortical bones were collected during autopsies and bone slices were prepared. Human osteoclasts were made by isolated of CD14+ monocytes from human peripheral blood, followed by culturing in the presence of 25ng/ml of M-CSF and RANKL in 2% serum for 28 days. Osteoclastic resorption was investigated by CTXI and CTXII in the conditioned medium. Additionally, bone slices were decalcified using EDTA and treated by cathepsin K in-vitro and markers assessed.

Patients with bone metastases revealed significant increased levels of CTXI at all Soloway scores (p<0.001). In contrast CTXII was not elevated until at Soloway score 3 (p<0.01). The ratio CTXII/CTXI decreased at all Soloway scores (score 1-2: p<0.01; score 3-4: p<0.001). The percentage increase in CTXI was 70 and 903% at score 1 and 4, respectively. CTXII was elevated by 99% at score 3; by 128% at score 4. Human osteoclastic bone resorption on human bone resulted in release of CTXI fragments but not CTXII fragments. In alignment, cathepsin K treatment of human bone released CTXI, but not CTXII fragments. Treatment of cartilage did not result in CTXI or CTXII release.

Human osteoclasts and cathepsin K were able to release CTXII fragments from human bone. Cartilage degradation was not elevated at high bone resorption levels until the late stage in the pathology of the cancer invasion involving more than 6 skeletal lesions (score 3). This disassociation of localised bone resorption and cartilage degradation in early stages of cancer patients with bone metastases suggests that other metabolic pathways are responsible for the association of bone resorption and cartilage degradation as is seen in osteoporosis and osteoarthritis

Disclosures: D.J. Leeming, Nordic Bioscience 3.

T136

Biosynthesis of Menaquinone-4 (Vitamin K2) from Phylloquinone (Vitamin K1) and Menadione (Vitamin K3) in Bone. K. Nakagawa*, Y. Shimomura*, Y. Suhara*, T. Okano.. Department of Hygienic Sciences, Kobe Pharmaceutical University, Kobe, Japan.

Vitamin K is essential for the modification of glutamic acid residues of specific substrate proteins into γ-carboxyglutamic acid (Gla) residues. In bone, osteocalcin, which has three Gla residues, is thought to be involved in the regulation of mineralization. Vitamin K compounds share a common chemical structure consisting of a naphthoquinone nucleus capable of redox cycling. Vitamin K1 (phylloquinone: PK) has a long phytol side-chain, whereas vitamin K2 has an unsaturated side-chain containing 1-13 isoprene unit. Menaquinone-4 (MK-4), vitamin K2, possesses a significant pharmacological activity on bone formation and is used as a medication for osteoporosis in Japan. The daily dietary intake of vitamin K is mainly (>90%) in the form of PK. MK-4 may be present in low levels in food products. However, MK-4 is found in most tissues. In general, tissue concentrations of MK-4 exceed those of PK except for liver, where relatively low MK-4 levels are found. Therefore, it has been recognized that PK is converted into MK-4 and accumulates in extrahepatic tissues including bone. Menadione (K3), commonly added to animal diets, is converted into MK-4 when administered to animals. However, the conclusive scientific evidence for the conversion of PK into MK-4 is exiguous. To elucidate the conversion of PK and K3 into MK-4, we synthesized deuterium-labeled PK, MK-4 and K3. PK or K3 with a deuterium-labeled 2-methyl-1,4-naphthoquinone ring was given orally to mice and bone were collected for LC-APCI-MS/MS analyses. Deuterium labeled-PK (PK-d7) and deuterium labeled-K3 (K3-d8) administration resulted in the accumulation of MK-4-d7 in tissues, particularly in liver, brain and bone. This is the fust direct evidence using deuterium labeled compounds and LC-APCI-MS/MS analysis that PK and K3 are converted into MK-4 in bone. Moreover, administration of the anticoagulant warfarin, an inhibitor of vitamin K reductase in vitamin K cycle, significantly blocked the conversion of PK-d7 and K3-d8 to MK-4-d7 in bone. Interestingly, MK-4 epoxide, an oxidized form of hydroxymenaquinone-4 (a reduced form of MK-4) generated by γ-glutamyl carboxylase during the turn-over of the vitamin K cycle, was found at higher concentrations than PK epoxide. This result suggests that in bone, MK-4 is rapidly and remarkably metabolized to the epoxide form via a hydroquinone form. Our results indicate that MK-4 is the true physiologically active form for bone formation and that it is derived from dietary vitamin K.

Disclosures: K. Nakagawa, None.

T137

Plasminogen Activator Inhibitor-1 Increases Femoral Mineralization Independent of Estrogen Status Through Modulation of Fibronectin Matrix. S. M. Nordstrom, J. W. Covington*, D. E. Vaughan*, Pharmacology, Vanderbilt University, Nashville, TN, USA.

Plasminogen activator inhibitor-1 (PAI-1) is a recognized regulator of matrix remodeling in cardiovascular, renal, and tumor biology, while its role in bone matrix is less well characterized. Previously, we reported a gender-specific and age-dependent increase in femoral strength and mineralization in mice that over-express human PAI-1 (PAI-1 .stab). In this study, we investigated the female-specificity of this phenotype and the mechanism by which PAI-1 influences bone remodeling. PAI-1.stab and WT females were ovariectomized (OVX) or SHAM-operated (SHAM) at 16 weeks (n = 11-12). Total skeletal (tBMD) and vertebral bone mineral density (vBMD) were measured by DEXA at baseline and at 2-week intervals for 12 weeks. Serum osteocalcin and TRACP5b were determined by EIA. Femora were analyzed for trabecular bone histomorphometry. Primary adherent bone marrow cells were evaluated for fibronectin (Fn) matrix deposition by immunocytochemistry while Fn transcription was determined by real-time RTPCR in primary calvarial osteoblasts. PAI-1 .stab mice had increased tBMD (p = 0.0014) and a differential pattern of remodeling (p = 0.0166) from 16-28 weeks of age under basal conditions (SHAM). This phenotype was maintained in the setting of estrogen-deficiency. PAI-1.stab mice exhibited a trend towards increased vBMD with a significant difference in the remodeling pattern (p = 0.0022). Genotype did not influence trabecular architecture of SHAM or OVX femora. However, PAI-1.stab mice had significantly reduced osteoid surface and a 1.7-fold greater mineral apposition rate under both SHAM and OVX conditions (p<0.001). Serum osteocalcin was comparable between genotypes in SHAM and OVX, while TRACP5b levels were decreased in PAI-1.stab OVX mice (p<0.001). PAI-1.stab primary adherent bone marrow cells exhibited increased Fn matrix formation from 2-14 days in culture (p<0.0001) and a greater rate of Fn deposition (p = 0.0014) compared to WT cells. Fn mRNA was increased 2-fold in PAI-1.stab osteoblasts compared to WT osteoblasts. Taken together, these studies indicate that PAI-1 over-expression results in increased bone mineralization without alteration of bone architecture. This effect is independent of estrogen and occurs in conditions of basal bone remodeling as well as in accelerated remodeling following OVX. Increased mineralization results, in part, from alterations in the Fn component of bone matrix leading to aberrant osteoblast/osteoclast mineralizing activity. This newly recognized role of PAI-1 in matrix synthesis and mineralization suggests novel targets for the prevention and treatment of bone disorders in humans.

Disclosures: S.M. Nordstrom, None.

T138

The Overexpression of Type III Na-dependent Phosphate Transporter Pit-1 in Rats Develops the Progressive Nephrotic Syndrome. S. Sekiguchi, S. Asano*, K. Nishiwaki-Yasuda*, A. Yokovama*, K. Inagaki*, H. Kakizawa*, N. Hayakawa*, N. Oda*, A. Suzuki, M. Itoh*., Department of Internal Medicine, Fujita Health University, Aichi, Japan.

Phosphate uptake at the cellular membrane is essential to maintain the cell activity because phosphate has to be supplied for ATP synthesis. Generally, the extracellular signal to stimulate cellular proliferation also induces the enhancement of phosphate uptake, that is. Pi transport activity. On the contrary, accumulating evidence suggests that phosphate overload from the extracellular milieu would be stressful for the cells, because phosphate uptake itself might induce the formation of apoptosome, resulting in the apoptosis of the cells. In the present study, we investigate the effect of overexpression of Pit-1, a type III Pi transporter, on the kidney which is a major phosphate-handling organ in Pit-1 transgenic rats (Tg). Survival was much shorter in Tg rats with death already occuring between the 7 and 9 month of age. The decrease of serum albumin started at the age of 2 months old, and progressively deteriorated. Tg rats developed massive proteinuria at 3 months of age, associated with hyperlipidemia, suggesting the progress of nephrotic syndrome in Tg rats. On the contrary, serum creatinine levels in Tg were not different from those in Wt. At their birth, the difference of electromicroscopic (EM) finding of the glomerulus between Tg and wild type rats (WT) was not apparent, but at 8 weeks after birth, the glomerulus of Tg has started to shrink compared to that of WT. Their glomerular abnormality assessed by EM images were also observed in 8 weeks old Tg rats, that is, glomerulus exihibits diffuse loss of foot processes of visceral epithelial cells (podocytes), thickenened basement membrane and the massive deposition of abnormal protein in the glomerulus. These findings suggest that early onset of the development of the damage of glomerulus due to overexpression of Pit-1 phosphate transporter.

In conclusion. Pit-1 overexpression in rats induces the phosphate-dependent cellular stress, which results in the damage of the filtration at gromerulus without affecting renal function itself. This might be a suitable animal model to analyze the pathogenesis of nephrotic syndrome.

Disclosures: S. Sekiguchi, None.

T139

Cementoblast Cultured on Fibrin Induces Fibrinolysis and Its Apoptosis. H. Jung*, Y. Choi*, J. Baek, H. Ryoo, K. Woo. Dept. of Cell and Developmental Biology, Seoul National University, School of Dentistry, Seoul, Republic of Korea.

Cementum is a mineralized tissue covering roots of teeth that provide for the attachment of periodontal ligament to roots and surrounding alveolar bone. During periodontal regeneration after disease or surgery, cementum is thought to play a critical role in the reparative process. However, it has remained a poorly defined tissue at the cellular and molecular level. We intended to examine effect of fibrin, the natural provisional matrix during wound healing, on cementoblast differentiation. A cementoblast cell line OCCM30 was grown on fibrin matrix and the phenotypes were examined. Unexpectedly, it was observed that the fibrin matrix, on which cementoblasts were cultured, was degraded and the cementoblasts on fibrin underwent apoptosis. In contrast, fibrin matrix promoted expressions of osteoblastic phenotypes in MC4 cells without any observable degradation. The degradation of fibrin was quantified through measuring fluorescence-labeled fibrin released to culture medium. The apoptosis was confirmed by TUNEL staining and activation of caspase 3. Next, we surveyed expression of enzymes responsible for fibrin degradation. Tissue-type plasminogen activator was expressed in cementoblasts grown on fibrin. Aminocaporic acid, an anti-fibrinolytic agent, reduced the fibrin degradation and apoptosis of cementoblast in a dose-dependent manner. Taken together, these results imply that fibrin matrix in periodontal wounds may impair regeneration of cementum.

Disclosures: K. Woo, None.

T140

Analysis of In Vivo Responses to Hydrogen Peroxide Purified PHBV Biomaterial Implanted in a Murine Tibial Defect. A. C. K. Wu*1, S. Toulson*2, A. Pettit*3, L. Grøndahl*4, E. J. Mackie*2, A. I. Cassady*3, 1School of Biomedical Sciences, University of Queensland, Brisbane, Australia, 2Faculty of Veterinary Science, University of Melbourne, Melbourne, Australia, 3Institute for Molecular Bioscience, University of Queensland, Brisbane, Australia, 4School of Molecular and Microbial Sciences, University of Queensland, Brisbane, Australia.

The utilization of biomaterials for the repair of critical bone defects has become an emerging field due to the limitations of current therapeutic options. Optimal bone biomaterial should be able to provide structural support for bone regeneration and elicit minimal inflammatory response and toxic effects when implanted. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) is a naturally occurring biodegradable polymer that possess properties include slow rate of degradation in biological environments and can be tailored into desired building blocks due to its thermoplastic nature. We have previously reported that PHBV is contaminated with bacterially derived impurities and have developed protocol that can eliminate impurities from the material. The purified PHBV have a much improved biocompatibility as tested by the in vitro tissue culture assays. The aim of the current study was to further validate the biocompatibility of purified PHBV under in vivo conditions. Cylindrical blocks of the materials were implanted into a murine tibial cortical defect, consisting of a hole drilled through the diameter of the tibial diaphysis. The defect was either filled with a material plug or left unfilled as a control. The animals were sacrificed at one week and four weeks after surgery and the extracted tibiae were decalcified and paraffin embedded. Bone sections were examined using standard histological staining as well as immunohistochemical staining to determine the extent of the cellular response surrounding the implant. The PHBV implant induced a mild inflammatory response one week after injury, which persisted at four weeks. Graunloma type tissues were formed at the implant tissue interface at the periosteum encapsulating the implants four weeks post surgery. Our data indicate that solid PHBV biomaterials induce a mild tissue reaction with no evidence of osteointegration into the implant at the tissue interface during the study time frame.

Disclosures: A.C.K. Wu, None.

T141

Using Replication Competent Avian Virus System to Delivery Short Hairpin SiRNA to Silence Gene Expression of Wdr5 in Chicken Limb Bud During Embryogenesis. S. Zhu, F. Gori.. Endocrine Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA.

Wdr5, a WD40 protein, is expressed in chondrocytes and osteoblasts in vitro and in vivo. Targeted expression of Wdr5 to osteoblasts, using the 2.3-kb fragment of the mouse type 1 collagen promoter results in accelerated osteoblast differentiation during skeletal development. A novel finding in these studies was expansion of the hypertrophic chondrocyte layer, suggesting that Wdr5 expression in the bone collar acts in a paracrine fashion to regulate chondrocyte differentiation. In addition, reduction of endogenous Wdr5 protein levels using plasmid-based small interfering RNAs (siRNAs) markedly inhibits osteoblast differentiation of MC3T3-E1 cells, suggesting that Wdr5 is required for osteoblast differentiation in vitro. To address whether Wdr5 is essential for osteoblast and chondrocyte differentiation in vivo during endochondral bone formation, a replication competent avian virus system (RCAN) is being generated to express short hairpin RNA (shRNA) under the control of the U6 RNA polymerase III (U6 pol III) promoter to specifically silence the expression of Wdr5 throughout the developing chicken limb. Four sites targeting the coding sequence of the chicken Wdr5 were chosen using the Ambion web site algorithm. Four RCAN vectors containing the selected Wdr5-shRNA sequences and one RCAN vector containing a scrambled-shRNA (RCAN-control) to use as a control have been generated. To verify the infection efficiency of RCANs, Green Fluorescence Protein (GFP), driven by cytomegalovirus (CMV) promoter, was cloned into the RCAN constructs upstream the U6 Pol III-shRNA cassette. To examine the role of Wdr5 on limb development in vivo, high-titer RCAN-shWdr5 and RCAN-control retroviral inoculates are being injected into the right wing of E3.5 chick embryos, a stage analogous to mouse embryo E12.5. The contralatered wings will serve as control. The effects of the differing degrees of Wdr5 silencing will be investigated histologically and by in situ hybridization analysis. These studies should offer a rapid and efficient method of analysis the function of Wdr5 during embryonic skeletal development. Furthermore, they will allow us to dissect the roles of other genes that interact with Wdr5.

Disclosures: S. Zhu, None. This study received funding from: NIH.

T142

Podocan Affects Cell Proliferation and In Vitro Mineralization Possibly by Inducing Cellular Senescence in MC3T3-E1 Cell. M. Katafuchi1, Y. Mochida2, P. Atsawasuwan*2, M. Sricholpech*2, M. Bahadoran*3, M. Kaku*2, T. Matsuura1, M. Yamauchi2, 1Department of Oral Rehabilitation, Fukuoka Dental College, Fukuoka, Japan, 2Dental Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA, 3School of Dentistry, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

Recently we have reported that podocan (Podn), a recently identified small leucine-rich proteoglycan/glycoprotein (SLRP), is expressed in preosteoblastic cell line, MC3T3-E1 cells (MC). In this study, to explore the potential function of Podn in bone biology, MC derived clones stably overexpressing Podn (S clone) were generated and partially characterized by comparing with two control groups, MC and a clone transfected with an empty vector (Ev clone). To establish the S clones, MC was transfected with pcDNA3 Podn HA-tag construct using FuGENE6 transfetion reagent. After transfection, cells were cultured and exposed to 400 ug/ml of G418 antibiotics for 4 weeks to select stably transfected clones. The overexpression was confirmed by immunoprecipitaion followed by western blot analysis with anti-HA antibody. Seven S clones were selected and analyzed for cell proliferation, in vitro mineralization and cellular senescence. The cell proliferation rate evaluated by cell counting at day 5 of culture was significantly decreased in all S clones in comparison to that of controls (25-58% of the controls). When cells were cultured in α-MEM in the presence of 10% FBS, 2 mM β-glycerophosphate and 50 μg/ml ascorbic acid for up to 4 weeks, the onset of mineralization evaluated by Alizarin red staining was significantly delayed in 60-70% of S clones compared to controls. To investigate the potential cause of these phenotypes, cells (10,000 cells/mL) were cultured for 24 hours and evaluated for cellular senescence by staining for senescence-associated-β-galactosidase (SA-β-gal) activity. In S clones, the number of stained cells was significantly increased when compared to controls (by ∼6 fold). These results indicate that Podn may induce cellular senescence in MC resulting in decreased cell proliferation and delayed mineralization in vitro.

Disclosures: M. Katafuchi, None. This study received funding from: NIH grants DE10489 and AR052824.

T143

Abnormal Collagen Type 1 Production by Human Osteoarthritic Osteoblasts. D. Couchourel*, I. Aubry*, A. Delalandre*, D. Lajeunesse., Rhumatology, Centre Hospitalier de l'Université de Montréal (CHUM), Montréal, PQ, Canada.

Osteoarthritis (OA) is characterized by articular cartilage loss, bone sclerosis and synovial inflammation. Bone sclerosis is due to an abundant osteoid matrix that does not mineralize normally. Here, we prepared primary normal and OA osteoblasts (Ob) from subchondral bone of tibial plateaus to study in vitro the mechanisms responsible for this abnormal sclerosis. The expression of collagen type 1 al chain (COLL1A1) and a2 chains (COLL1A2) was determined by real-time PCR in parallel with in vitro mineralization evaluated by alizarin red staining. The SaOS-2 cell model, which shows high rates of mineralization, and OA Ob were used to determine the impact of either COLL1A1 or COLL1A2 overexpression or siRNA inhibition on the mechanism of mineralization. Last, we determined if a putative factor released by OA Ob could inhibit mineralization using conditioned-media (CM) from OA Ob on SaOS-2 cells while SaOS-2 CM was used to determine if this could correct OA Ob mineralization. In vitro alizarin red staining was significantly reduced in OA Ob compared to normal under basal condition and following BMP-2 treatment indicating a reduced mineralization capacity. Mineralization was reduced in OA Ob due to an increase of COLL1A1 expression compared to normal with no significant changes in COLL1A2. The COLL1A1 to COLL1A2 ratio was 7.2 ± 0.2 in OA Ob whereas it was 2.5 ± 0.6 in normal Ob at day 30 post-confluence. The COLL1A1 to COLL1A2 ratio in SaOS-2 cells varied from 7.5 ± 1.5 at day 0 to 1.5 ± 0.2 at day 14 post-confluence whereas the mineralization progressively increased. Overexpressing COLL1A1 in SaOS-2 cells reduced whereas COLL1A1 siRNA transiently increased mineralization. In OA Ob, overexpressing COLL1A2 increased mineralization by 26% whereas the COLL1A1 to COLL1A2 ratio decreased by 28.8%. Furthermore, siRNA inhibition of COLL1A1 expression in OA Ob increased mineralization by 43.62%. Last, SaOS-2 CM increased the mineralization of OA Ob while OA Ob-CM reduced mineralization of SaOS-2 cells but without any significant changes in the COLL1A1 to COLL1A2 ratio in either cells. This study suggests that the abnormal mineralization of OA bone tissue observed in vivo may be linked, in part, with the abnormal expression of COLL1A1 by OA Ob and their incapacity to down-regulate their COLL1A1 to COLL1A2 ratio. This assessment is illustrated by the fact that when this ratio is altered in primary OA Ob and in SaOS-2 cells mineralization is not optimal. In addition, this could also be linked with the expression of a putative soluble factor that alters mineralization without altering the COLL1A1 to COLL1A2 ratio.

Disclosures: D. Lajeunesse, None.

T144

An Active Fragment of Secreted Phosphoprotein-24 Enhances but Is Not Indispensable for BMP-Mediated Adult Human Bone Marrow Derived Mesenchymal Stem Cell Differentiation to Osteoblasts. A. Maiti1, O. Korchynskyi1, S. C. Ramage2, A. P. Pacitti*1, M. J. Beckman2, 1Orthopaedic Surgery, Virginia Commonwealth University, Richmond, VA, USA, 2Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA, USA.

Mesenchymal stem cells (MSCs) are multipotent cells that can proliferate and differentiate into multiple tissues. The recruitment and differentiation of MSCs into distinct tissues is a precondition for in situ tissue engineering. MSCs play a major role in osteogenesis and their osteogenic potential depends on several factors including BMP/Smad induced transcription and canonical Wnt/β-catenin signaling. Glucocorticoid signaling is required for normal bone formation, and synthetic glucocorticoids such as dexamethasone (Dex) promote osteoblastic differentiation of MSCs. We examined the osteogenic regulation of secreted phosphoprtotein-24 (Spp24), a 24 kDa bone matrix protein hypothesized to possess osteoinductive properties. Adult human bone marrow derived MSCs were isolated by plastic adherence. Both Dex and BMP-2/7 heterodimer accelerated MSC differentiation to osteoblasts (OB) in a 14 day period in the presence of ascorbic acid and β-glycerolphosphate. We used a polyclonal rabbit anti-bovine Spp24 antibody to detect human endogenous Spp24. Gene expression of OB lineage markers were measured by Real-Time RT-PCR and alkaline phosphatase and Alizarin Red staining were performed as measures of an OB-specific marker and OB matrix mineralization, respectively. Western Blot of differentiated MSCs revealed a predominant 18 KDa fragment absent in undifferentiated MSCs. The formation of the 18 kDa fragment of Spp24 in Dex-induced differentiation corresponded with increased gene expression of OB-specific markers (Runx2, Osterix, osteocalcin and collagen I) and alkaline phosphatase staining. Temporal regulation of this putative active form of endogenous human Spp24 was seen at days 7-10 of Dex-induced OB differentiation. To examine the effect of Spp24 on BMP-mediated osteoblastogenesis we replaced Dex with BMP-2/7 as the differentiation mediator and performed adenoviral transduction of Adv5CMV-LacZ control or recombinant Adv5CMV-Spp24 over 12 and 14 day time frames. Expression of full-length mouse Spp24 (mSpp24) enhanced of BMP-mediated matrix mineralization, but without BMP-2/7, Spp24 had only a minor effect at day 12. At day 14 mSpp24 expression induced significant osteoblastogenesis assessed by gene, protein markers and Alizarin Red staining. These results highlight a critical processing step during osteoblastogenesis that produces an activated 18 kDa fragment of Spp24 involved in the enhancement of bone matrix mineralization by its cooperation with BMP.

Disclosures: A. Maiti, None.

T145

Cleavage Products of Amyloid Precursor Protein Regulate Osteoblast FunctionJ. McLeod*1, N. Curtis*2, M. Fagan*2, P. Genever*1, 1Dept. of Biology, University of York, York, United Kingdom, 2Dept. of Engineering, University of Hull, Hull, United Kingdom.

γ-secretase is an intramembrane protease responsible for the cleavage of numerous molecules which regulate osteoblast activity, such as Notch and EphrinB2. Cleavage of its substrates results in release of a fragment into the extracellular space, and a C-terminal peptide into the cytoplasm. Amyloid precursor protein (APP) is also processed by γ-secretase, though its role in bone is unknown. Amyloid-β (Aβ) peptides are secreted into the extracellular space as a result of γ-secretase activity on APP, and in the brain can aggregate to form the plaques characteristic of Alzheimer's disease (AD). The amyloid intracellular domain (AICD) binds to the nuclear adaptor protein Fe65 in the cytoplasm, and translocates to the nucleus to form a transcriptionally active AFT complex with Tip60. In this study the expression, cleavage and potential function of APP were investigated during osteogenesis. By qRT-PCR and western blot analysis, expression of all γ-secretase subunits (PS1, PS2, APH-1α, APH-1β, Net, PEN-2) was confirmed. A specific fluorimetric assay showed significant increase in enzyme activity during osteogenic differentiation of human bone marrow stromal cells (MSCs). Using RT-PCR we identified expression of a longer isoform of APP in osteoblasts compared to neuronal APP, which contained an additional Kunitz protease inhibitory domain that promotes aggregation of Aβ peptides. Western blot analysis confirmed expression and processing of APP throughout osteogenesis, and treatment of samples with γ-secretase inhibitors confirmed that this enzyme specifically cleaves APP within differentiating MSCs. Expression of Fe65 and Tip60 throughout in vitro osteogenesis was confirmed by RT-PCR and western blot analysis. Using confocal microscopy we showed that over-expression of both AICD and Fe65 in C3H10T1/2 stromal cells resulted in colocalisation within discrete domains of the nucleus. A specific chemiluminescent immunoassay demonstrated secretion of Aβ during osteogenesis. MSCs showed a significant increase in adhesion to extracellular matrices containing aged Aβ plaques compared to non-aged Aβ peptide controls and Aβ plaques were localized to the endosteal and periosteal surfaces in sections of adult rat ulna. By μCT analysis (15 μm resolution) of vertebrae from a 12 month old AD mouse model, Tg2576, which over-expresses a mutant APP resulting in increased γ-secretase cleavage, we showed a 20% decrease in bone volume compared to wild type controls (10.60mm3 vs. 8.47mm3; wild type vs. transgenic). These findings indicate that APP may function as a novel regulator of osteoblastic activity both in vitro and in vivo, with functions ranging from adhesion to regulation of gene transcription.

Disclosures: J. McLeod, None.

T146

The Effect of IL-6 and sIL-6R on the Expression of Cartilage Matrix Proteins. A. Namba*1, Y. Aida*2, Y. Watanabe*2, H. Tanaka*3, O. Shimizu*1, N. Suzuki*4, M. Maeno*3, 1Departments of Oral and Maxillofacial Surgery, Nihon University School of Dentistry, Tokyo, Japan, 2Department of Fixed Prosthodontics, Nihon University School of Dentistry, Tokyo, Japan, 3Department of Oral Health Sciences, Nihon University School of Dentistry, Tokyo, Japan, 4Department of Biochemistry, Nihon University School of Dentistry, Tokyo, Japan.

Elevated concentrations of interleukin-6 (IL-6) and soluble IL-6 receptor (sIL-6Rα) in the synovial fluid of the temporomandibular joint (TMJ) have been implicated in the joint cartilage destruction associated with TMJ disorders. Recently, we demonstrated that IL-1β promotes the resolution system of cartilage matrix turnover through an increase in inflammatory cytokine production by chondrocytes and that it also may promote the autocrine action of IL-6 through an increase in IL-6Rα expression in human chondrocytes (Life Sci 79, 764-771, 2006). In the present study, we examined the effect of IL-6 and sIL-6Rα on cell growth and alkaline phosphatase (ALPase) activity. We also examined the expression of Sox-9, cartilage matrix proteins (including type II collagen, aggrecan core, and link protein), bone morphogenetic protein (BMP)-7, and BMP receptors (BMPR) in human chondrocytes. The cells were cultured with or without 50 ng/mL IL-6 and/or 30 ng/mL sIL-6Rα for up to 28 days. The levels of mRNA expression for Sox-9, the cartilage matrix proteins, BMP-7, and BMPR were determined using real-time PCR; protein levels were determined using ELISA. The proteoglycans of the extracellular matrix were stained with Alcian blue. Cell proliferation increased slightly in the presence of both IL-6 and sIL-6Rα after 7 days of culture. ALPase activity, a marker enzyme used to differentiate osteoblasts and chondrocytes, decreased markedly in the presence of both IL-6 and sIL-6Rα after 10 days of culture. The expression of Sox-9, aggrecan core, and Alcian blue-positive proteoglycans in the extracellular matrix did not change in the presence or absence of IL-6 and sIL-6Rα, whereas the expression of type II collagen, link protein, BMP-7, and BMPR increased in the presence of both IL-6 and sIL-6Rα after 7 days of culture. These results suggest that IL-6 in the presence of sIL-6Rα suppresses the differentiation of chondrocytes and induces the repair of arthrodial cartilage through the increased expression of cartilage matrix proteins, BMP-7, and BMPR.

Disclosures: A. Namba, None.

T147

Histological and Biochemical Characterization of Mandibular Bones in Senile Osteoporotic Mice. K. Tokutomi1, T. Matsuura1, Y. Daigo*1, M. Katafuchi*1, K. Toh*1, H. Sato1, M. Yamauchi2, 1Oral Rehabilitation, Fukuoka Dental College, Fukuoka, Japan, 2Dental Research Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.

The quantity and quality of jaw bone are an important determinant for the outcome of many aspects of dental treatment. However, relatively little is currently known about the age-related changes of jaw bone. In this study, the mandibular bones of SAMP6 mice, a senile osteoporosis mouse model, were partially characterized and compared to those of SAMR1 mice as control. A total of twelve 6 month-old male mice of each strain were used. Static bone morphometric analysis was performed using 6 mice from each strain to assess cortical thickness index (CTI) at the mandibular ramus, bone area (B. Ar / T. Ar), trabecular width (Tb. Wi), trabecular number (Tb. N) and trabecular separation (Tb. Sp) at the interraducular septum between the distal root of the first molar and the mesial root of the second molar. Using other 6 mice from each strain, bilateral condylar necks and mandibular rami were dissected, pulverized, weighed, hydrolyzed with 6N HCl and subjected to amino acid analysis to determine collagen content (μg/mg bone powder) and the extent of lysine hydroxylation (moles of hydroxylysine/mole of collagen). Statistical analysis was performed by Student t-test. Both CTI and B.Ar/T.Ar values of SAMP6 (20.1±1.3% and 62.9±4.6%) were significantly lower than those of SAMR1 (21.9±1.4% and 77.0±4.8%) (p = 0.046 and p<0.001). The values of Tb.Wi (75.9±16.1 μm) and Tb.N (8.62±1.79/mm) of SAMP6 showed a trend of decrease but not statistically significant comparing to those of SAMR1 (81.6±15.1 μm and 9.72±1.28/mm, respectively) (p = 0.542 and p = 0.248). The Tb.Sp value of SAMP6 (45.6±14.2 μm) was significantly higher than that of SAMR1 (23.5 ±2.8 μm) (p = 0.004). Biochemical analysis revealed that collagen content of SAMP6 (126.4±9.7 μg/mg) was significantly lower than that of SAMRI (149.6±10.3 μg/mg) (p<0.001) and that the extent of lysine hydroxylation of SAMP6 (51.10±0.39) was higher than that of SAMRI (48.54±1.42) (p = 0.040). These results indicate that the mandible of SAMP6 exhibits not only reduced bone mass but also altered quantity and quality of collagen matrix. The latter may contribute to a mineralization defect seen in osteoporotic patients.

Disclosures: K. Tokutomi, None.

T148

Enamel Phenotype and Sexual Dimorphism in Dentin of dspp-null Mouse Molars. K. Verdelis1, J. T. Wright*2, N. Haruyama*3, A. Kulkarni*3, L. Lukashova*1, A. L. Boskey1, 1Research, Mineralized Tissues Laboratory, Hospital for Special Surgery, New York, NY, USA, 2School of Dentistry, University of North Carolina, Chapel Hill, NC, USA, 3NIDCR, NIH, Bethesda, MD, USA.

The dentin phenotype in dentin sialophosphoprotein (dspp) depleted 70 day-old mice resembles that in humans with dentinogenesis imperfecta type III. Our purpose was to quantitatively compare the dentin and enamel mineral density and the amount of dentinal and enamel tissue formed in normal(WT) and dspp-/- (KO) male and female mature molars.

Extracted 1st (m1) and 2nd (m2) lower molars from 8 WT and 9 KO 7-8 mo-old male and female mice (n total = 33) were analyzed on a 1172 (Skyscan, Be) μCT system at 8 μm isotropic voxel resolution. Parameters calculated from the resulting mineral distributions (1st molars presented in Fig. 1) were: average mineral density (dentin-enamel), mineralized tissue volume (dentin), heterogeneity index (calculated by width at half height of mineral density distribution peaks- dentin).

WT molar crowns had a lower mineral density in both male and female animals (male m1 and m2 = −11 to −16%, p<0.01, female m1 and m2 = −6 to −7%, p<0.01). The total volume of dentin was, as initially described, much lower in male KO (-16 to −36%, p<0.05), while in female KO this volume was the same (m2) or there was a trend for increased volume ( = +8%, p = 0.06- m1), probably due to enlarged pulp space. Dentin heterogeneity was also sex-dependent, agreeing with the earlier results in KO male (+20 to 28%, p<0.05), but lower in female KO animals (-18 to −22%, p<0.05). Root dentin showed similar results. Enamel presented phenotypic differences between KO and WTmice that were not sex-dependent, both in mineral density (male = −5%, p<0.05, female = −2 to −4%, p<0.05) and volume of tissue (male = +21 to 29%, p<0.05, female = +22%, p<0.05). In KO males only (5 of 8 animals) dystrophic calcification of the pulp and root canals was present.

The results showed that the described dentinogenesis imperfecta III dentin phenotype of the dspp-/- mice is sex-dependent, implying possible X-chromosome located protein partners for dspp-/- in its signaling function in dentin and possibly in the pulp. They also showed that the final mineral density difference of KO dentin is moderate and that there are some enamel changes as well in the dspp-/- mice.

Supported by NIH grants DE 04141 and AR-46121.

Figure Fig. 1:.

Distribution of mineral densities in 1st molars

Disclosures: K. Verdelis, None.

This study received funding from: NIDCR. NIH.

T149

Bone Sialoprotein-Mediated Osteoblast Differentiation and Mineralization Is Principally Dependent on the Intrinsic Characteristics of Responding Cells. J. G. Yost*, Q. Lu*, M. Rodova*, J. Wang. Orthopedic Surgery, The University of Kansas Medical Center, Kansas City, KS, USA.

Bone sialoprotein (BSP) is one of the major non-collagenous phosphoproteins in bone and tooth, which stimulates matrix mineralization and osteogenesis in surgically created rat calvarial defects, but not in rat thoracic subcutaneous tissue (Calcif Tissue Int 2006; 79:179). This has raised an important question of whether BSP-mediated osteogenesis is dependent on local environment and/or cellular conditions. This study tests our hypothesis that the effect of BSP on osteoblast differentiation and function is principally dependent on the characteristics of responding cells. We first examined the origins of BSP-responsive cells at the cranial defect site by using a nitrocellulose membrane to separate BSP-collagen implants from the host bone/periosteum, the dura that is located at the bottom of the defect, and the skin flap covering the defect. The results demonstrated that bone-forming cells observed in the mid-portion of BSP-treated defects are primarily derived from the dura and to a much lesser extent from the host bone and periosteum. Cells derived from the skin flap do not differentiate into osteoblasts or mineralize. To eliminate the effects of local environment on BSP function, primary cells isolated separately from the dura, cranial subcutaneous or thoracic subcutaneous tissues were cultivated in the same conditions of α-MEM media. When the cells reached 75-80% confluence, they were transfected with a BSP expression lentiviral vector with the addition of 10% FBS, 50 μg/ml ascorbic acid and 5 mM β-glycerophosphate. Transfection of rat dural cells with a lentiviral BSP expression vector, but not an empty vector, led to the expression of osteocalcin and matrix mineralization at 14 days after transfection; while transfection of rat cranial and thoracic subcutaneous cells with a lentiviral BSP expression vector did not result in osteocalcin expression or mineralization even by 21 days. Quantitative real-time PCR analyses demonstrated that the expression levels of Runx2 and Osterix were significantly higher in normal dura than in normal cranial and thoracic subcutaneous tissues, while there were no significant differences in the expression levels of osteocalcin, BSP and osteopontin between these tissues. These results suggest that the dura-derived cells, at least some of them, are Runx2/Osterix expressing osteoprogenitors or preosteoblasts, which can be further differentiated into functional osteoblasts upon BSP treatment, and that BSP-mediated osteoblast differentiation and mineralization is principally dependent on the intrinsic molecular and cellular characteristics of responding cells.

Disclosures: J. Wang, None.

T150

Focal Bone Loss in Mice with Transient Muscle Paralysis. B. J. Ausk*, P. Huber*, S. Srinivasan, T. S. Gross.. Orthopaedics and Sports Medicine, University of Washington, Seattle, WA, USA.

The mechanisms by which muscle function locally mediates bone homeostasis are unclear. We have previously reported that transient muscle paralysis rapidly induces osteoclast mediated trabecular and cortical bone loss in bones nearest the paralyzed muscles. Given this spatial relation, we hypothesized that resorption within a given bone would be focused on the cortex adjacent to the paralyzed muscle. Ten female C57B6 mice (16 wk) received 1M injections of Botox (2.0 units/100 g) in the right calf immediately following a μCT scan of a 2 mm mid-diaphyseal region of the tibia. At 21 d, a second μCT scan of the same region was obtained. Pre- and post-scans of individual mice were aligned and changes in cross-sectional area determined at three 2-D cross-sections located at the distal (A, 1.4 mm proximal to the tib-fib junction), middle (B, 2.4 mm proximal) and proximal end (C, 3.4 mm proximal) of the scanned volume. Composite 2-D images at each location were partitioned to identify focal bone loss in the posterior third of bone (i.e., adjacent to paralyzed calf muscle; Calf) compared to the remainder of the cross-section (Non-Calf). Mean (± s.e.) whole cross-sectional bone area was significantly reduced throughout the scanned volume (A: −0.073 ± 0.009 mm2; B: −0.071 ± 0.012 mm2; C: −0.051 ± 0.009 mm2, all p < 0.001). Bone loss arose entirely via endocortical expansion and the magnitude of the loss did not differ statistically across the scanned volume (p > 0.3). While bone loss at the most distal location (A) was focused in the Calf Sector (18.8 ± 2.2% vs 8.3 ± 1.3%, p = 0.001), bone loss at the most proximal location (C) was primarily associated with the Non-Calf Sector (9.4 ± 1.6% vs 3.1 ± 0.9%, Φ = 0.004). The center of the scanned region (B) demonstrated bone erosion in both cortical sectors (13.7 ± 2.4% vs 9.2 ± 1.9%, Calf vs Non-Calf, respectively, p > 0.15). These data clearly indicate that the endocortical resorption induced within the bone by transient muscle paralysis, though focal and highly reproducible across mice, was not exclusive to cortical areas adjacent to the paralyzed muscle. As the paralyzed calf spanned the entire scanned bone volume (no muscle insertions within 6 mm of scanned volume), the shift of resorption from anterior predominant to posterior predominant within this region suggests that the dynamics governing focal bone loss have complexities beyond that originally hypothesized. Instead, the data suggest that osteoclast recruitment to bone surfaces is not a random process but is either the result of active osteoclast chemotaxis or focal inhibition of osteoclast attachment.

Disclosures: B.J. Ausk, None.

T151

The Increased vBMD and Bone Area due to Mechanical Loading Gradually Decreased Following Cessation of Loading. C. Kesavan, D. J. Baylink*, P. Gifford*, S. Mohan. MDC, J.L. Pettis VAMC, Loma Linda, CA, USA.

Dynamic loads lead to increases in volumetric (v) BMD and cross sectional area. However, the issue of how long these gains are maintained after cessation of loading has not been fully understood. To address this question, we performed a long term study in which skeletal changes were monitored every 2-4 weeks for a 12 week period following cessation of external loading. A four-point loading device was used to apply external loading (9N at a frequency of 2Hz for 36 cycles once per day for 12 days) to the right tibia of 10-week old female mice. Skeletal changes were monitored by in vivo pQCT. The value of the left non-loaded tibia was subtracted from the value of the right loaded tibia to determine the changes associated with loading at each time point. Two-weeks of four-point loading caused a drastic 40% increase in total area (TA) and 15% increase in total vBMD. However, the increase in size due to external loading did not continue once the external loading was stopped. Furthermore, cessation of loading resulted in a continuous loss of both bone size (TA) and vBMD. The vBMD returned to normal at 12 weeks with a half life of 6 weeks. The TA declined with a half life of 8.5 weeks and was still significantly elevated at 12 weeks. Thus, the decline in elevated TA proceeded at a much slower pace than the loading induced increases in vBMD. Conclusions: 1) External loading-induced increases in bone are lost over a period of time but at a much slower pace than the induction of the increases; 2) While a single burst of external loading provides increased bone mass temporarily, periodic loading may be necessary to maintain long term bone strength.

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Disclosures: C. Kesavan, None.

T152

Identifying Mechanical Loading QTL by Gene Expression Changes for Alkaline Phosphatase and Bone Sialoprotein in C57BL/6J (B6) × C3H/HeJ (C3H) Intercross. C. Kesavan, D. Baylink*, S. Kapoor*, S. Mohan. MDC, J.L. Pettis VAMC, Loma Linda, CA, USA.

Previous studies have shown that mechanical loading (ML) 1) produces a more robust skeletal anabolic response in B6 mice than C3H mice, which is largely mediated by genetic differences and 2) induces greater expression of bone formation (BF) marker genes in the bones of B6 mice compared with C3H mice. We, therefore, tested the hypothesis that the variation in skeletal anabolic response to ML in the F2 mice of B6 and C3H intercross is largely due to changes in BF response. To examine this, we measured expression changes in two BF markers, namely bone sialoprotein (BSP) and alkaline phosphatase (ALP) in the loaded and non-loaded bones of 10-week F2 female mice (n = 241). Loading was performed daily on the right tibia by four-point bending using a 9 Newton at 2Hz, 36 cycles, for 12 days. The left tibiae were used as internal controls. The expression levels of ALP and BSP were quantitated after normalization with two house keeping genes, actin and PPIA. The mean increase in gene expression in the F2 mice, expressed as fold change, ranged from 3.0 ± 2.8 for BSP and 2.7 ± 2.8 for ALP, and showed significant correlation (r = 0.25 to 0.36, p<0.01) with changes in BMD and bone size. A genome-wide search using 111 microsatellite markers with 15 cM intervals in the 

Table  .  
  1. *determined using MapQTL program; ap<0.01, bp<0.05, cp<0.1

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F2 mice revealed QTLs on Chrs 8, 9, 17 and 18, which corresponded to ML QTLs we previously identified using changes in BMD and bone size as end points. We identified two new ML QTLs on Chrs 16 and 19 using gene expression data. In conclusion: 1) Identification of several common QTL for BMD, BSP and ALP phenotypes suggests that the skeletal response to ML is largely mediated by increased BF; 2) Identification of genes that are involved in regulating BSP and ALP expression in response to ML will lead to improved understanding of the molecular pathways regulating the bone response to ML.

Disclosures: C. Kesavan, None.

T153

Comparative Analysis of Expression Profile in the Anatomically Separated Parts of Femurs Prepared from Unloading Induced Bone Loss Model of Tail-suspended Mice. J. Nagata, Y. Ezura, M. Noda. Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan.

In vivo experimental analysis for disuse osteoporosis has been effectively carried out by rodent experiment of hindlimb unloading obtained by “tail-suspension”. Applications of knock-out mice for this experiment unveiled molecular involvement of several genes such as osteopontin, for the unloading induced bone-loss. However, the entire process of the molecular events involved in this phenomenon is largely unknown. To obtain insights into the integrative understandings of the molecular events, we performed DNA microarray analysis on tissues prepared from unloaded femurs of the tail-suspended mice. Tail suspension was carried out using 8-week old C57B16 mice (n = 5) for 2-weeks, and we compared them with normally loaded mice (n = 5). Whole femora were dissected out from left hindlimbs, and separated into three parts; (A) bone marrow flushed out from mid shaft 1/3, (B) mid shaft cortex, and (C) distal and proximal epiphyses and metaphyses. Total RNA was isolated from these samples, and only the quality checked samples were used for expression analyses. Micro-CT analysis on contralateral femora confirmed significant decrease of bone volume in the distal end. Expression profile for up to 39,000 transcripts was analyzed by Affymetrix DNA microarray (Mouse Expression 430_2.0_Array) for three different parts of femurs (A to C). The analyses displayed significantly different patterns. Partial resemblance and reasonable shift observed in the expression pattern of some genes indicated a possibility for analytical reconstruction of changes in proportion of different types of cells and/or changes in expression levels of the gene in specific type of cells in the tissue. For this sake, we first specified significantly up-regulated and down-regulated genes in each type of anatomical site origins (A to C), considering data conceivability regarding flag indication for the spot signal and consistency between the triplicated samples, as well as the statistical significance of the differential expression between loaded and unloaded samples indicated by p-values for Student's t-test. Upregulated 102 and down-regulated 83 were selected from the data for tissue preparation (A). Similarly for preparation (B) and (C), 91 and 46 upregulated and 121 and 50 down-regulated genes were selected. Comparative analysis of these genes among the data obtained from different tissue preparation should clarify the specific cellular events occurring in the unloaded bones in vivo.

Disclosures: J. Nagata. None.

T154

Collagen and Osteopontin Upregulation by Mechanical Loading of Bone Cells in Porous 3D Scaffolds. A. Sittichockechaiwut, L. E. Brown*, G. C. Reilly. Engineering Materials, Univeristy of Sheffield, Sheffield, United Kingdom.

Previously, we showed that polyurethane foam scaffolds seeded with osteoblastic cells and loaded in a biodynamic chamber attached to a mechanical testing machine (Bose ElectroForce 3200) provide a good model for analysis of mechanotransduction in 3 dimensions. Under static culture conditions MC3T3-E1 and MLO-A5 cells settled throughout the scaffolds and were shown to be viable up to 15 days of culture. Since MLO-A5s have been demonstrated to be rapid matrix producers (Kato et al. JBMR 2001) we used these cells to investigate the effects of compressive loading in the 3D system. Cell-seeded scaffolds were loaded in compression with a sine wave at 1Hz, 5% strain for 2 hours per day at days 5, 7 and 9 of culture. Collagen content, as assayed by colorimetric analysis of sirius red staining, was significantly (p<0.05) higher in loaded scaffolds even if the loading was reduced to days 5 and 7 only. There was no effect of 2 hrs of loading on cell number. However, reducing the length of the loading period to 0.5 or 1 hr eliminated the difference in collagen content but caused an increase in the final number of viable cells in loaded scaffolds. To asses loading induced changes in matrix gene expression 2 types of culture were set up. 1) MLO-A5 cells in polyurethane foams (BritishVita). 2) MC3T3-E1 cells in collagen sponges (BD biosciences). In both experiments cells were cultured until day 5 then subjected to a single bout of 2 hours of loading at 5% strain, 1 Hz. In both experiments an increase in osteopontin mRNA was detectable in loaded samples by PCR. Positive effects of mechanical loading on bone matrix production can clearly be demonstrated in 3D porous scaffolds.

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Disclosures: G.C. Reilly, None

This study received funding from: The Royal Society of London.

T155

Continuous Local Infusion of Basic Fibroblast Growth Factor Enhances Consolidation of the Bone Segment Lengthened by Distraction Osteogenesis in Rabbit Experiment. A. Abbaspour, S. Takata, K. Sairyo*, S. Katoh*, K. Yukata*, N. Yasui*, The University of Tokushima, Tokushima, Japan.

Experimental tibial lengthening was achieved in 52 rabbits to examine the effect of continuous local infusion of recombinant human fibroblast growth factor-2 (rhFGF-2) on bone regeneration during distraction osteogenesis. The tibia was separated by osteotomy and was subjected to slow progressive distraction (rate:0.35 mm/12h) using a monolateral external fixator. The lengthening process consisted of a lag phase for one week after osteotomy, a distraction phase for two weeks, and a consolidation phase for five weeks in this experiment. At various stage of the distraction, rhFGF-2 was infused continuously for two weeks into the lengthened segment [rate:14.28 μg/day(60 μl/day)] using an osmotic pump implanted under the skin. Bone healing was significantly accelerated when rhFGF-2 was infused in the beginning of consolidation phase, but not in the distraction phase nor in the lag phase. Infusion of normal saline (N/S) using the same osmotic pump had no effect. Dual energy X-ray absorptiometry (DXA) and peripheral quantitative computerized tomography (pQCT) studies demonstrated that rhFGF-2-treated tibia had higher bone mineral density (BMD), higher bone mineral content (BMC) and increased cortical bone thickness (CBT) than N/S-treated tibia. In addition, three-point bending test demonstrated that rhFGF-2-treated bone had significantly stronger mechanical properties than N/S-treated bone. Finally, distribution of the infused materials was checked by using Indian ink or Urographin. The dyes distributed widely but exclusively in the lengthened segment. Based on these results we conclude that direct delivery of rhFGF-2 into the lengthened segment can shorten the consolidation phase of limb lengthening and the method is applicable to the clinical treatment.

Disclosures: A. Abbaspour, None.

T156

Limited Collagenase Digestion Increases Solubility and Induction Potential of Human Demineralized Bone Matrix (DBM) In Vitro. H. Tang, I. Shanahan*, M. Diegmann*, N. Forsyth*, D. Knaack*, K. Behnam. R&D, Osteotech, Inc., Eatontown, NJ, USA.

When processed in a manner consistent with the preservation of native growth and differentiation factor activity, human demineralized bone matrix has the ability to induce the formation of endochondal bone heterotopically in athymic rodents. Histological demonstration of the formation of ectopic mineralized bone, bone marrow, osteoid, and cartilage is generally considered to be the gold standard for the measurement of osteoinductive activity. While quantitative and semiquantitative histomorphometric methods for defining the osteoinductive potential of DBM have been previously proposed, for the purpose of testing lots of DBM produced from individual donors a predictive in vitro alternative would be desirable. Previously, quantitative in vitro methods for the measurement of the induction of osteoblastic markers in clonal cells have been proposed. We find that a significant limitation of many of these assays is the relative insolubility of the collagenous matrix in the cell culture media. Limited digestion of human demineralized bone matrix with purified bacterial collagenase increases both the solubility of DBM in cell culture media and the ability of the residual matrix to upregulate alkaline phosphatase activity in clonal C2C12 myoblasts. Partial digestion of DBM for 60 minutes increases the ability of the material to induce AP activity in C2C12 cells more than 10 fold 4 days post-treatment but the same treatment has no effect on the activity of guanidine hydrochloride inactivated DBM. The partially digested DBM begins to dissolve into the cell culture medium within 4 to 5 hours, with scant amounts of residual DBM remaining after 48 hours. Complete solubilization of the matrix is dependent on the presence of cells but direct cell contact with the matrix is not required. The capability of DBM to induce alkaline phosphatase activity is dependent both on the dose of the DBM added and the duration collagenase digestion [data not shown]. These results indicate that the solubility of DBM in cell culture media influences the release kinetics of native growth factors which reside within the collagenous matrix.

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Disclosures: K. Behnam, Osteotech, Inc. 1, 3.

This study received funding from: Osteotech, Inc.

T157

Continued Enhanced Fgfr2 Function Delayed Fracture Healing in Mice. L. Yin, X. Du, Z. Liu, N. Su, H. Qi, J. Yang, L. Zhao, L. Chen. Trauma Center, Research Institute of Surgery, Dapin Hospital, Third Military Medical University, chongqing, China.

To evaluate the role of FGFR2 in fracture healing, this study was conducted using twelve-week-old female mice carrying gain-of-function substitution (Ser252Trp) in Fgfr2 or their wild-type littermates. After been anesthetized, mice were subjected to an open mid-diaphyseal fracture of its right femur, a 0.5-mm-diameter metal pin was inserted through the patellar tendon into the medullary canal of the tibia to fix the fracture. Mice were sacrificed at day 5, 7, 10, 14 and 21 after fracture. Digital radiographs of right hind limbs were obtained. After been demineralized, embedded and sectioned, H.E. staining and safranin O/fast green staining were undertaken to show the histological structure of the callus. In situ hybridization using digoxigenin-labeled probes were performed to detect the expression of Fgfr2, Col2(Collagen 2), Ihh(Indian hedgehog) and BMP4(bone morphogenetic protein 4) in callus. The results showed that all the wild-type mice had radiographic evidence of bone union compared with delayed fracture union in mutant-type mice. At 7 days post-fracture, marked chondrogenesis appeared in callus of wild-type mice but not in mutant-type mice. By 21 days, fracture healing was completed in wild-type mice. In contrast, at this time, the fracture healing was not finished in mutant-type mice, with persistence of fibrous scar and cartilage in the fracture gap and bone marrow cavity. The expression of Fgfr2 was present in both wild-type and mutant-type calluses. The initiation of Ihh and Col2 expression in callus was delayed in mutant-type mice than that in wild-type mice. No significant difference in the expression of BMP4 between two groups was observed. Therefore, this study indicated that constitutively activated Fgfr2 function will suppress the endochondral ossification in fracture callus and delay the healing of fracture through the inhibition of the Ihh expression in fracture site.

Disclosures: L. Chen, None.

This study received funding from: 973 Research Projects, No 2005CB522604; National Natural Science Foundation of China, No. 30672121: Opening Project of State Key Laboratory of Trauma, Burn and Combined Injury, No 2006A-4.

T158

FGF-23 Controls NaPiIIa Trafficking via Crosstalk Between the PI-3 kinase and MAP kinase Pathways. M. Khan*, S. M. Jan de Beur.. Medicine, Johns Hopkins Medical Institute, Baltimore, MD, USA.

Fibroblast Growth Factor 23 (FGF-23) is an important physiological regulator of phosphate homeostasis and its excess or deficiency has been implicated in several human disorders of phosphate homeostasis. FGF-23 exerts its phosphaturic action by promoting internalization of the type IIa and type IIc sodium-phosphate co-transporters (NaPiIIa, NaPiIIc) from the renal brush border membrane and preventing the compensatory increase in 1,25(OH)2D3 by reducing renal 1α hydroxylase expression. FGF-23 can bind several known FGF receptors (FGFR) and activate MAP kinase signaling. However, it is not known which of FGFR signaling pathways mediate the diverse actions of FGF-23. We sought to distinguish which FGFR signaling pathway regulates FGF-23-mediated NaPiIIa internalization. Previously, we reported that FGF-23 stimulates ERK1/2 (ERK) phosphorylation and that inhibiting ERK signaling with U0126 abolished FGF-23-mediated NaPiIIa internalization in opossum kidney cells (OK/e). To interrogate other FGFR signaling pathways such as the phosphatidylinositol-3 kinase (PI-3 kinase) pathway, we measured phosphorlation of Akt by immunoblot but observed no increase in pAkt upon stimulation with FGF-23 (0.1 ng/ml-100ng/ml). Yet, surprisingly, inhibition of PI-3 kinase with LY294002 partially blocked FGF-23-mediated NaPiIIa internalization. In tyrosine kinase receptor signaling in general and FGFR signaling specifically, the MAP kinase pathway may be activated by a number of upstream molecules including Ras, phospholipase Cγ (PLCγ) via protein kinase C (PKC), and PI-3 kinase via PDK-1. In order, to further dissect the inputs to the MAP kinase pathway, we measured pERK by immunoblot in FGF-23 stimulated OK/e cells treated with the farnesyl transferase inhibitor (FTI-277) that induces accumulation of non-famesylated cytoplasmic Ras to form inactive Ras/Raf complexes. OK/e cells were preincubated with 2 μm FTI-277 for 1-24 hrs and then stimulated with FGF-23 (10 ng/ml) or PTH (1-84, 10−7M). MAP kinase activation was measured by immunoblot of whole cell lysate with a pERK antibody. In OK/e cells treated with FGF-23, FTI-277 did not appreciably reduce pERK compared to untreated FGF-23-stimulated cells. In contrast, PTH-stimulated ERK phosphorylation was completely abolished with FTI-277 treatment. U0126 blocked both FGF-23 and PTH-stimulated ERK phosphorylation. Taken together, these data suggest that the PI3 kinase pathway regulates FGF-23-mediated NaPiIIa internalization through crosstalk with the MAP kinase pathway and that this is a distinct signaling pathway from PTH-mediated NaPiIIa trafficking.

Disclosures: S.M. Jan de Beur, None.

This study received funding from: Naitonal Institutes of Health.

T159

Age-Dependnet Regulation of FGF23 Production in Murine Bone. T. Masanori1, B. Yuan2, Y. Xing*2, K. Ava*1, T. Morishima*1, M. K. Drezner2, H. Tanaka1, 1Department of Pediatrics, Okayama Univ., Okayama City, Japan, 2Department of Medicine, Univ of Wisconsin and GRECC, William F. Middleton VAMC, Madison, WI, USA.

The detailed physiological regulation of FGF23 production remains unknown. However, the principal location of the hormone in osteoblasts and variation in the amount present during matrix mineralization suggests FGF23 production may be age-dependent (Bone. 2007 Feb 8 [epub ahead of print]). Thus, in the present study, we examined the influence of age on FGF23 production in mice. Initially, we measured serum FGF23 (s-FGF23) in wild type (WT) C57BL/6J mice at 2, 4 and 12 weeks of age. s-FGF23 was highest at 2 weeks and declined thereafter (516.5±48.3, 373.3±56.4 and 106.8±8.4 pg/ml, respectively; p<0.05). In accord, FGF23 mRNA expression in WT-mice significantly (p<0.05) decreased at 4 (41.5±9.1%) and 12 (77.9±8.4%) weeks of age. Interestingly, hyp-mice with a pathological abnormality of FGF23 metabolism, had superimposed on this defect a progressive age-dependent decrease in FGF23 mRNA production, significantly (p<0.05) declining by 42.2±9.6% at 4 weeks and 89.9±5.3% at 12 weeks. Accordingly s-FGF23 similarly decreased (4811.8±184.6, 3391.2±158.1 and 1367.4±59.2 pg/ml; p<0.05). To examine if the age-dependent modulation of FGF23 expression is PHEX independent, we established an in vitro system using osteoblasts derived from SV40 Tg mice (SV-Ob). Cells were maintained in αMEM containing 10% FBS, ascorbate and β-glycerophosphate (VC-GP). The SV-Ob cells proliferated and reached sub- confluence at day 6 of incubation and after 12 days, mineralization of extra-cellular matrix was evident. After 3 days of incubation proliferating cells displayed abundant FGF23 mRNA expression, which decreased with maturity at 6 days (97.4±1.2%; p<0.05) and 12 days (99.1±0.2%; p<0.05) of incubation. Incubation of SV-Ob cells in conditioned medium from SV-Ob cells at day 6, but not day 12, of culture significantly elevated FGF23 mRNA expression compared to that in cells incubated with fresh VC-GP medium (4.86±0.90 fold; p<0.05), indicating that a factor(s) produced by immature osteoblasts plays an important role in regulation of FGF23 production. Interestingly, exposure of SV-Ob cells to d6 medium, containing the phex inhibitor, MH2-64-C, did not modulate the effect on FGF23 expression. Moreover, SV-Ob cells, transfected with pcDNA/phex-WT vector encoding the mouse phex protein, displayed FGF23 mRNA expression no different than that in SV-Ob cells transfected with an empty vector. Our observations clearly indicate that FGF23 production in murine osteoblasts is regulated by an age-dependent mechanism, which results in a PHEX independent decline of FGF23 mRNA with maturity.

Disclosures: T. Masanori, None.

T160

Overexpression of Low Molecular Weight Isoform of FGF2 partly prevents Bone Loss in Ovariectomized Mice. L. Xiao1, J. D. Coffin2, M. M. Hurley1, 1University of Connecticut Health Center, Farmington, CT, USA, 2The University of Montana, Missoula, MT, USA.

In humans, there are multiple high molecular weight (HMW, 22, 23, 24kDa) nuclear isoforms of basic fibroblast growth factor (FGF2) and a low molecular weight (LMW, 18kDa) isoform that is exported from cells. We previously reported that targeted over-expression of the LMW FGF2 isoform in preosteoblasts and mature osteoblasts increased bone mass in vivo and in vitro. To determine whether LMWFGF2 protects against ovariectomy induced bone loss, female Col3.6/GFP transgenic mice (VectorTg) and Col3.6/LMWFGF2/GFP transgenic mice (LMWTgFGF2) were ovariectomized (OVX) or sham-operated (sham) at 14 weeks of age and sacrificed 6 weeks post surgery. Six weeks after OVX, there were significant reductions in uterine weight in both VectorTg-OVX and LMWTgFGF2-OVX groups compared to the sham group. Body weight was significantly increased only in the VectorTg-OVX group compared with VectorTg-sham group. DEXA analysis showed a 4.6% and 9% significant reductions in femoral bone mineral density (BMD) and bone mineral content (BMC) in VectorTg-OVX group compared with VectorTg-sham group (p<0.05). There was no significant reduction in BMD and BMC in LMWTgFGF2-OVX group compared with LMWTgFGF2-sham group. Micro-CT analysis revealed that femoral trabecular bone volume/total volume (BV/TV), connective density (Conn-Den) and trabecular number (Tb.N) were significantly decreased by 32%, 38% and 18% respectively in VectorTg-OVX compared with the VectorTg-sham group. However, there were only 24%, 22% and 13% decrease in femoral BV/TV, Conn-Den and Tb.N respectively in LMWTgFGF2-OVX group compared with LMWTgFGF2-sham group. At 2 and 3 weeks mineralized bone nodules were increased in ex vivo bone marrow stromal cell cultures from LMWTgFGF2-sham mice compared with VectorTg-sham and was further increased in cultures from LMWTgFGF2-OVX mice compared with VectorTg-OVX. Northern analysis showed that mRNA for Osteocalcin (OC); a late marker of differentiated osteoblasts; was similar between VectorTg-sham and VectorTg-OVX in 3 week cultures. However there was a 1.75 fold increase in LMWTgFGF2-sham compared with Vector-sham and a 1.91 fold increase in LMWTgFGF2-OVX compared with Vector-OVX group. Similar data was observed in two independent lines of LMWTgFGF2 mice. These data indicate that targeted over-expression of LMW isoform of FGF2 protein in preosteoblasts and osteoblasts partially protected against OVX-induced bone loss in vivo and in vitro.

Disclosures: L. Xiao, None.

T161

Bone Regeneration with Local Controlled Application of Granulocyte Colony-Stimulating Factor (G-CSF) in a Bone Defect of Rabbit Ulna. K. Ishida*1, R. Kuroda*1, K. Sasaki*1, Y. Mifune*1, K. Tei*1, M. Miwa*1, T. Matsumoto*1, Y. Tabata*2, M. Kurosaka*1, 1Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe, Japan, 2Department of Biomaterials, Field of Tissue Engineering, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.

It is recognized that re-establishment of vascularity is an early event in fracture healing and the appropriate vascularization is emerging as a prerequisite for bone regeneration. G-CSF is a cytokine known to have an angiognenesis-promoting effect by recruitment of bone marrow cells. In this study, we investigated that the local applied G-CSF enhances bone regeneration via revascularization and osteoblast mobilization by controlled release from gelatin hydrogel. Methods: We have designed a biodegradable catonized gelatin (CG) hydrogel as the drug delivery system for G-CSF. All animal procedures were approved by the Animal Research Committee of Kobe University Graduate school of Medicine. 24 Japanese white rabbits were used in this study. A segmental bone defect (20 mm) was created at the diaphysis of rabbit ulna. The defects were treated in two groups as follows: Group G0: defects were filled with CG hydrogel only, Group G5: defects were filled with CG hydrogel with G-CSF (5μg). Every defect of the radiographs was obtained at 2, 4, and 8 weeks after surgery. Semiquantitative radiographic evaluations were performed using Image J and the radiographic healing was graded according to modified Cooks' grading scale. Toluidine blue staining was performed to see endochondral ossification. Histochemical staining for isolectin B4 was performed as an assessment of capillary invasion and immunofluorescent staining for osteocalcin were performed as an osteoblast marker. Results: Radiographic semiquantitative assessment revealed that there were more bone formation in group G5 than in group G0 at every weeks. Radiographic grading also revealed that bone formation was significantly promoted in group G5 at 4 and 8 weeks. Toluidine blue staining revealed that more cartilaginous matrix was seen in group G5 at as early as 2 weeks. Histochemical staining for isolectin B4 showed that capillary density was significantly increased in group G5 at 2 weeks. Osteoblast density was also significantly increased in group G5 at 2 weeks and these results revealed that revascularization and osteoblast mobilization into the bone defects were promoted in group G5. Conclusions: The present study demonstrates that local controlled application of G-CSF promotes rabbit bone regeneration. The reasons seemed that the G-CSF promoted revascularization and osteoblast mobilization. G-CSF had further possibility to apply other situations that needs both bone repair and neovascularization.

Disclosures: K. Ishida, None.

T162

Immunoregulatory Role of RANKL-stimulated Dendritic Cells on Autoimmune Arthritis in MRL/1pr Mice. T. Izawa*1, N. Ishimaru*2, K. Moriyama3, Y. Hayashi*2, 1Department of Orthodontics and Dentofacial Orthopedics, Institute of Health Biosciences, Tokushima University, Graduate School, Tokushima, Japan, 2Department of Oral Molecular Pathology, Institute of Health Biosciences, Tokushima University, Graduate School, Tokushima, Japan, 3Department of Maxillofacial Orthognathics, Tokyo Medical and Dental University, Graduate School, Tokyo, Japan.

Recent studies have established that receptor activator of NF-kB(RANK) /RANKL signaling plays a key role for maintenance of dendritic cells (DCs) in the periphery. However, the association of the RANKL signaling for DCs with autoimmunity has been obscure. In this study, we investigated the immunoregulatory role of DCs through RANKL signaling on autoimmune arthritis using MRL/1pr mice.

The phenotypes and functions were analyzed using DCs of spleen and lymph nodes, or bone marrow-derived DCs (BMDCs) from MRL/1pr and MRL+/+ mice. The surface markers on the peripheral DCs and RANKL-stimulated BMDCs were analyzed by flow cytometer. Apoptosis- and cell cycle-related molecules of the DCs were detected by Western blot analysis. In addition, the adaptive transfer of RANKL-stimulated BMDCs was performed into MRL/1pr mice as recipients to modulate the arthritis lesions.

We found that the number of CD11c'CD11b'CD8a' myeloid DCs in the periphery from MRL/1pr mice was significantly increased comparing with that from control mice, and that the MRL/1pr DCs could survive much longer than control DCs. In addition, the expressions of Bcl-x and Bcl-2 and the nuclear transport of NF-κB of RANKL-stimulated BMDCs from MRL/1pr mice were considerably upregulated compared with those from control mice. On the other hands, Fas expression of normal BMDCs was significantly increased by RANKL stimulation. Moreover, the adoptive transfer of RANKL-stimulated BMDCs resulted in accelerated and severe autoimmune arthritis of MRL/1pr recipients. These findings suggest that the crosstalk between RANKL and Fas signaling in DCs might influence the maintenance of DCs in the periphery and the development of autoimmunity.

Disclosures: T. Izawa, None.

T163

SDF-1/CXCR4 Is Essential for In Vivo Endochondral Bone Repair. I. Kitaori*1, H. Ito1, E. M. Schwarz2, T. Nakamura1, 1Orthopaedic Surgery, Graduate School of Medicine, Kyoto University, Kyoto City, Japan, 2The Center for Musculoskeletal Research, University of Rochester, Rochester, NY, USA.

INTRODUCTION A chemokine, SDF-1, and its receptor, CXCR4, have been well known to play a crucial role in homing of hematopoietic stem cells (HSCs) to bone marrow niche. Additionally accumulating data have shown that SDF-1 is highly expressed in some damaged organs, such as myocardial infarction and brain injury, and recruits circulating mesenchymal stem cells (MSCs) to the damaged lesion, allowing them to start normal organ repair. But it is still unknown whether SDF-1 is involved in bone repair. Using murine autograft and allograft models, we tested the hypothesis that SDF-1 is essential in endochondral bone repair. METHODS We used in-vivo 4-mm segmental femoral autograft and allograft model of C57B1/6 mice. For autograft model, the freshly removed bone was transplanted back to the same mouse, and secured by a metal pin. For allograft model, graft bones, which had been harvested from ICR mice and kept frozen for at least 24 hours, were applied to the defects. For a loss of function of study in autograft model, mice received anti-SDF-1 neutralizing antibody or control IgG injection peritoneally after the implantation. For a gain of function study in allograft model, rmSDF-1 protein or adeno-SDF-1 was loaded locally around the graft bones. For an in-vivo chemotaxis assay, pre-cultured bone marrow stromal cells (BMSCs) were labeled with BrdU and transplanted through tail vein at the end of the surgery. Bone grafts and surrounding tissue were harvested at days 0.5, 1, 2, and 3 after transplantation for RNA extraction and at days 7, 14, and 28 for histological analysis. The expression of SDF-1 mRNA was measured by RT-PCR. To detect the labeled-BMSCs immunohistostaining using anti-BrdU antibody was examined. RESULTS & DISCUSSION In this study we demonstrated the involvement of SDF-1 in endochondral bone repair. The mRNA expression of SDF-1 was increased in autograft at days 2 after transplantation, while no increase was detected in allograft. The transplanted BMSCs were detected around the grafted bones at days 7. Moreover the new bone formation around the autograft was significantly inhibited by the injection of anti-SDF-1 antibody. These data indicated that SDF-1 had an essential role in the acute phase of normal bone repair. The gain of function study, however, showed no remarkable increase in the bone formation, suggesting that SDF-1 may need to collaborate with other molecules to have a successful effect.

Disclosures: T. Kitaori, None.

T164

Intracellular Cytokine Profile of RANKL Expressing Lymphocytes. D. Krenbeck, U. Semrad*, M. Rauner*, D. Stupphann*, J. Patsch, M. Willheim*, P. Pietschmann. Department of Pathophysiology, Medical University of Vienna, Vienna, Austria.

Cytokines play an important role in regulation of bone mass by influencing Receptor activator of NFκb (RANKL). For example, TNFα increases bone resorption by elevating RANKL expression while IFNγ suppresses osteoclastogenesis by inhibiting RANK/RANKL signalling. Lymphocytes are major producers of cytokines and as they express RANKL, they are thought to be involved in bone destruction in a number of diseases including rheumatoid arthritis, parodontitis and possibly osteoporosis. Yet their function in bone metabolism is still poorly understood. Therefore we studied expression of surface and intracellular RANKL in lymphocytes as well as cytokine production by flow cytometry.

Peripheral blood mononuclear cells were isolated from young healthy subjects and stimulated for 4 hours with ionomycin (1.25 μM), phorbol 12-myristate 13-acetate (10 ng/ml) and brefeldin A (10 μg/ml). Harvested cells were incubated with an anti human RANKL specific monoclonal antibody followed by saponin treatment and further incubation with antibodies against IFNγ, TNF-α and CD4. To detect intracellular RANKL cells were permeabilised prior to staining.

Data was acquired on a FACSCalibur flow cytometer (Becton Dickinson) and analysed with the CellQuest Pro Software. RANKL positive cells were gated to illustrate their intracellular cytokine pattern.

In most of the cells intracellular RANKL was detected whereas only few cells were positive for surface RANKL. The latter were predominantly negative for IFNγ (80±7%) but expressed TNFα (47±26%). By gating on CD4 positive lymphocytes a similar pattern in RANKL and cytokine expression was observed.

Our data suggest that lymphocytes are involved in regulation of bone mass predominantly by the secretion of RANKL, rather than regulatory interaction of surface-expressed RANKL with target cells. Yet a small sub-population of lymphocytes expressing surface RANKL but not IFN γ may act on osteoclasts and their precursors directly via cell-cell contact.

Disclosures: D. Krenbek, None.

T165

TNF-alpha Dependent Redox Signals Upregulate Msx2 Gene Transcription via Stress Activated Protein Kinases in Arterial Myofibroblasts. C. F. Lai-Huang, J. S. Shao, E. Huang*, Z. Al-Aly*, S. L. Cheng, D. A. Towler. Internal Medicine - Bone and Mineral Diseases, Washington University School of Medicine, St. Louis, MO, USA.

Aortofemoral calcification is prevalent in type II diabetes (T2DM), tracking metabolic syndrome parameters and increasing the risk for lower extremity amputation. LDLR-/- mice fed high fat diets (HFD) develop T2DM, and accumulate aortic calcium, mediated via BMP2 and Msx2-Wnt osteogenic mechanisms that resemble craniofacial mineralization. Low-grade inflammation - including elevated serum TNF-alpha – is characteristic of T2DM. TNF-alpha upregulates myofibroblast BMP2, a potent stimulus for aortic Msx2 expression. We wished to determine if TNF-alpha augmented Msx2 expression in arterial myofibroblasts, potentially via autocrine BMP2 signals. TNF-alpha (10 ng/ml) upregulated both BMP2 and Msx2 mRNA accumulation 4-fold in aortic myofibroblasts following 6 hours of treatment. However, detailed time course reveals that Msx2 induction actually precedes BMP2 expression, with 2-fold Msx2 induction observed within 1 hour of treatment. Moreover, addition of 250 ng/ml recombinant noggin, an inhibitor of BMP2 and BMP4, did not inhibit Msx2 induction, providing further evidence of regulation independent of autocrine BMP2 signals. Msx2 induction was maximal by 3 hours, and dose-response studies revealed an ED50 for TNF-alpha of 2 ng/ml. Induction was completely abrogated by actinomycin treatment, and dose-dependently prevented by DRB, indicating Msx2 induction was transcriptionally regulated by TNF-alpha. Inhibition of the IKK-NF-kappaB pathway with BAY 11-7082 did not inhibit (actually enhanced) Msx2 induction. However, the JNK/SAPK inhibitor SP600125 significantly reduced TNF-alpha actions. JNK/SAPK is a critical component of the oxidative stress signaling cascade downstream of NADPH oxidase. Therefore we examined the effects of apocynin (NADPH oxidase inhibitor), SIN-1 (superoxide and nitric oxide precursor) and H202 on Msx2 expression. Apocynin reduced both basal and TNF-alpha stimulated Msx2 expression, indicating a role for NADPH oxidase redox signaling in aortic myofibroblasts. SIN-1 treatment had no effect. However, H202 dose-dependently upregulated Msx2 mRNA accumulation, phenocopying the response to TNF-alpha. Finally, mice with vascular TNF-alpha augmented by a SM22- TNFalpha transgene exhibit higher levels of aortic Msx2 gene expression vs. non-transgenic sibling cohorts, providing further evidence for a vascular TNFalpha - Msx2 regulatory axis in vivo. Thus, arterial myofibroblast Msx2 gene transcription is directly stimulated by TNF-alpha, entraining vascular Msx2 expression to inflammation via redox-regulated JNK/SAPK signal cascades.

Disclosures: C.F. Lai-Huang, None.

This study received funding from: National Institutes of Health.

T166

The Role of Wnt3A Signaling in Maturation of Rat Osteoblasts. L. Ling*, C. Dombrowski*, L. M. Haupt*, V. Nurcombe*, S. M. Cool. Stem Cells and Tissue Repair, Institute of Molecular and Cell Biology, Singapore, Singapore.

Recent studies have indicated that heparan sulfate (HS) sugar-dependent Wnt signaling is crucial to bone formation. These sugars are manufactured in the Golgi, and are heavily reliant on the HS-synthesizing enzymes glucosaminyl N-deacetylase/N-sulfotransferase-1 (NDST-1) and HS 2-O-sulfotransferase (HS-2-OST). NDSTs and HS-2-OST are responsible for HS N-sulfation and O-sulfation, respectively; NDST1 is also a key enzyme directing further HS modifications including O-sulfation. Recent studies have indicated that HS regulates proliferation and differentiation of osteoblasts and improves bone healing. The work here was designed to investigate the function of Wnt3A during the maturation of primary osteoblasts derived from new-born rat calvaria. Sequential digests of rat calvaria were able to release cells characteristic of the various stages of osteogenic development. Preosteoblasts (fraction 3) were most sensitive to 1,25α-vitaminD3 and parathyroid hormone, whilst more mature osteoblasts (fraction 5) exhibited the greatest alkaline phosphatase (ALP) activity as well as collagen Iα1 and osteocalcin (OC) expression. The osteogenic potential of F3 and F5 were further demonstrated by Alizarin Red and von Kossa staining. The mRNA level of target genes and the expression of target proteins were determined by RQ-PCR and Western Blot analysis, respectively, and cell proliferation determined by the GUAVA PCA-96 Viacount system. We found that the levels of NDST1 and HS-2-OST were at least 5-fold higher in F3 than F5, suggesting that HS sugars are intimately involved in the maturation of pre-osteoblasts to osteoblasts. We further demonstrated that Wnt3A increased NDST1 expression in F5 (2.7-fold) and F3 (1.4-fold). Wnt3A and LiCl (an activator of Wnt signaling) both inhibited cell proliferation while increasing ALP activity and OC expression in both fractions, inducing a higher ALP activity in F3 (2.1-fold) than F5 (1.5-fold) but a higher OC expression in F5 (5.0-fold) than F3 (1.7-fold). Heparin dose-dependently increased Wnt3A-stimulated ALP activity in F3, but decreased it in F5. We also observed that activation of Wnt signaling dramatically increased noggin expression, a BMP inhibitor, while decreasing RUNX2 expression. In conclusion, Wnt3A signaling regulates the maturation of calvaria osteoblasts by switching the cells from proliferation to differentiation. As Wnt3A is known to interact with BMP2 signaling which is also mediated by HS, the sugar may be orchestrating the availability of these factors to precursor cells.

Disclosures: L. Ling, None.

This study received funding from: Institute of Molecular and Cell Biology, A *STAR.

T167

Osteprotegerin as Cardiovascular Risk Marker in Patients with Type 2 Diabetes Mellitus. P. Rozas-Moreno*, M. Muñoz-Torres, G. Alonso*, D. Fernández-García*, I. Luque-Fernández*, A. Sebastian-Ochoa*, F. Escobar-Jiménez*, Bone Metabolic Unit. Department of Endocrinology, University Hospital San Cecilio, Granada, Spain.

Background: Type 2 diabetes mellitus patients have a high prevalence of cardiovascular disease. The classic cardiovascular risk factors seem not to completely explain such increase in vascular disease in type 2 diabetes mellitus. Hyperhomocysteinemia and enhanced carotid intima-media thickness (CIMT) are both predictors of cardiovascular disease in diabetic patients. It have been postulated that osteoprotegerin (OPG) could be the physiopathologic link between osteoporosis and vascular disease. On the other hand, elevated serum levels of OPG, a key cytokine in the bone remodelling process, have been related to an increased mortality due to cardiovascular events in type 2 diabetes mellitus patients.

Objetive: To analyze the association between serum OPG and some of the cardiovascular risk factors observed in type 2 diabetes mellitus (Arterial hypertension [HT], hyperlipidemia, obesity, smoking, hyperhomocysteinemia, CIMT).

Patients and methods: 72 patients with type 2 diabetes mellitus were studied. All of them attended to the Endocrinology Service of University Hospital “San Cecilio” of Granada, Spain. Arterial hypertension was defined as blood pressure = 140/ 90 mmHg and/or actual anti-hypertensive treatment. Serum levels of homocystein, LDLc, HDLc and OPG were measured (OPG ELISA BI-20402, BIO-MEDICA-GRUPPE WIEN, Austria). Hyperlipidemia was defined according to the ATPIII criteria (statins treatment, LDLc = 130 mg/dl, HDLc < 40/50 mg/dl in males and females respectively). Obesity was graded using the Body Mass Index (BMI: Kg/m2). The CIMT was detemined by means of vascular Eco-doppler (TOSHIBA Vision 6000) of both carotid arteries at the level of 10 mm proximal from the carotid branch off using a mode B, 7.5 MHz probe. A value = 0.9 mm was considered as pathologic.

Results: The study population included 41.7% females and 58.3% males, mean age 57.9 ± 6.3 years-old. Males showed smaller serum levels of OPG than females (4.70±1.8 vs 5.98 ± 2.6 pmol/L respectively, p = 0.02). Serum levels of OPG were positively related to age in both genders (r = 0.31, p = 0.01). In female patients OPG levels were not significatively related to none of the studied variables. In males the serum levels of OPG were significatively related to presence of HT (Yes/No: 5.59±2.3 vs 4.06±1.5 pmo/L, p = 0.01), CIMT = 0.9 mm (Yes/No 5.09±1.6 vs 3.9±1.9 pmol/L, p = 0.03), presence of carotid plaque (Yes/No: 5.46±1.6 vs 4.22±1.8 pmol/L, p = 0.047) and BMI (r = 0.3, p = 0.05).

Conclusion: Our results suggest that the serum levels of OPG could contribute additional information about the cardiovascular risk in males with type 2 diabetes mellitus.

Disclosures: M. Muñoz-Torres, None.

T168

Local CNP Clearance System as a Regulator of CNP-GC-B System for Bone Growth. Y. Nakatsuru*, Endocrinology and Metabolism, Kyoto University, Kyoto, Japan.

Natriuretic peptide family consists of three endogenous ligands, ANP, BNP and CNP. They exert their biological actions by producing intracellular cyclic GMP through two subtypes of membranous guanyulyl cyclase (GC), GC-A and GC-B. The third natriuretic peptide receptor is called clearance receptor (C-receptor), which has no GC activity and is involved in the clearance of ligands. ANP and BNP are widely known as regulators of cardiovascular homeostasis through GC-A, whereas recent studies elucidated that CNP plays pivotal roles in the regulation of endochondral bone growth via GC-B. A previous study demonstrated that C-receptor deficient mice exhibit skeletal overgrowth like transgenic mice with targeted overexpression of CNP in cartilage. Here we examined the significance of natriuretic peptide clearance system in the growth plate using fetal mouse tibial organ culture. A specific agonist for C-receptor, C-ANF, increased the production of intracellular cGMP dose-dependently, and accordingly, elongated the tibial explants and their cartilaginous primordium significantly. Histological examination reveled that the width of the growth plate is widened in the C-ANF treated group. On the other hand, C-ANF did not increase the length of tibial explants from CNP depleted mice. These results suggest that C-ANF raised local CNP concentration in the growth plate cartilage. We conclude that local CNP clearance system is essential for the regulation of growth promoting effect of CNP-GC-B system in the growth plate.

Disclosures: Y. Nakatsuru, None.

T169

Involvement of Connexin43 in Interleukin-1β-induced Osteoarthritis-Associated Changes in Synovial Fibroblasts. C. Niger, F. D. Howell*, J. P. Stains. Orthopaedics, University of Maryland, Baltimore, MD, USA.

Interleukin-1 beta (IL-1β), produced by activated synoviocytes, has been reported to play a prominent role in the pathology of osteoarthritis (OA). In vivo studies have shown an enhancement in connexin43 (Cx43)-mediated gap junctional intercellular communication between synovial lining cells as one of the changes observed in OA. The purpose of this study is to examine the role that Cx43 plays in the IL-1β-induced changes in OA-associated gene expression observed in synovial fibroblasts.

We show that treatment of a rabbit synovial fibroblast cell line (HIG-82) with IL-1β stimulates the synthesis of Cx43 protein in a dose- and time-dependant manner. Additionally, by immunocytochemistry, we observe a large scale redistribution of Cx43 from the cytosol to the plasma membrane, following treatment with 1 ng/ml IL-1β for 16h. By real time PCR, we detect that IL-1β treatment or transient transfection of Cx43 in synovial fibroblasts were both effective at increasing gene expression of several OA-associated genes, including matrix metalloproteinases (MMP-1 and −13) and cyclooxygenase-2. Similar results for secreted MMP activity in conditioned media were also observed. Surprisingly, overexpression of Cx43 is as effective, if not more effective, than IL-1β treatment at inducing the expression and/or activity of OA-associated genes. Thus, it appears that IL-1β increases Cx43 levels and alters localization, and that this change in Cx43 is sufficient to induce OA-associated gene expression and MMP activity. In order to elaborate on these findings, we examined the signal transduction cascades downstream of IL-1β treatment. We show that IL-1β potently activates the extracellular signal regulated kinase (ERK) cascade. Furthermore, inhibition of the ERK cascade with U0126 abrogates the upregulation of Cx43 expression, the cellular redistribution of Cx43 to the plasma membrane, and the associated changes in gene expression and MMP activity. Importantly, Cx43 overexpression in conjuction with IL-1β does not synergize to increase transcription of OA-associated genes, nor does Cx43 overexpression potentiate ERK activation by IL-1β.

Together, our data show that Cx43 is a major target of IL-1β action on synovial fibroblasts. The data suggest that IL-1β acts on Cx43 expression and cellular distribution in an ERK-dependent manner, and the subsequent effects of these changes on Cx43 are sufficient to induce OA-associated changes in the phenotype of these cells.

Disclosures: C. Niger, None.

T170

Role of Interleukin-6 Signalling in c-Src-mediated Osteoblast Differentiation. B. Peruzzi*1, M. Longo*1, N. Rucci1, F. De Benedetti*2, A. Teti1, 1Experimental Medicine, University of L'Aquila, L'Aquila, Italy, 2Ospedale Pediatrico Bambino Gesù, Rome, Italy.

The non-receptor tyrosine kinase c-Src plays unique roles in the regulation of bone cell activity. In osteoblasts (OBs) c-Src maintains an “immature” state in which cell proliferation is very active and further differentiation is inhibited. In a large-scale transcriptome study of mouse primary OBs treated with the c-Src inhibitor PP1, we noted a ∼60% expression decrease of the pro-inflammatory cytokine interleukin-6 (IL-6), confirmed by RT-PCR and ELISA assay. Our previous work had shown that NSE/hIL-6 transgenic mice, which have high levels of circulating IL-6, display reduced OB activity, therefore we hypothesised that c-Src and IL-6 could be functionally associated in regulating OB function and that they could contribute to imbalanced bone remodelling in pathologic situations. In a time-course experiment in which we treated mouse primary OBs with PP1, we investigated IL-6 expression and found a time-dependent reduction of IL-6 mRNA in PP1- vs. vehicle-treated OBs, also confirmed at level of secreted protein by ELISA assay. The same result was also obtained in OBs in which c-Src was inhibited by siRNA treatment for 48 hours and by retroviral infection with a dominant-negative-Src isoform. In order to explain how c-Src inhibition influenced IL-6 expression, we tested the involvement of “Signal Transducer and Activator of Transcription” (STAT) proteins, among which STAT3 is described to drive the trascription of the IL-6 gene and to be a c-Src substrate. In the PP1 time-course experiment we showed that STAT3-phosphorylation at the tyrosine 705 (a c-Src substrate) was decreased following c-Src inhibition, suggesting involvement of this transcription factor in the c-Src-dependent regulation of IL-6 expression. We then tested a possible role for STAT3 on c-Src signalling. We inhibited STAT3 by siRNA and found a reduced c-Src-activating phosphorylation and increased osteoblast differentiation markers. Consistent with our hypothesised IL-6 role, we observed a time-dependent increase of c-Src activating phosphorylation in primary OBs treated with recombinant human IL-6 (rhIL-6) for 1 week. In conclusion, we have obtained evidence of a relevant interplay between c-Src and IL-6 in OB differentiation, partly based on STAT3-dependent transcription.

Disclosures: B. Peruzzi, None.

T171

Characterization of a New Traumatic Mouse Model of Heterotopic Ossification. B. E. Rapuano*1, A. Grose*2, E. Tomin*1, J. M. Lane*3, D. Helfet*3, 1Research, Hospital for Special Surgery, N.Y., NY, USA, 2Orthopedics, Upstate Medical Center, Syracuse, NY, USA, 3Orthopedics, Hospital for Special Surgery, N.Y., NY, USA.

Purpose: To develop a physiologically relevant traumatic model of heterotopic ossification (HO) in the mouse, quantify the amount of ectopic bone which forms in the model, and begin to characterize the molecular regulators that mediate the formation of HO.

Methods: Mice from the C57BL/6J strain underwent a surgical procedure in which the quadriceps in recipient mice were subjected to surgical trauma by clamping and bone marrow cells from donor mice were transplanted into a pouch created surgically in the clamped area in recipient mice. Some recipient mice were sacrificed 42 days following surgery, the femurs with attached quadriceps were removed from the operated hindlimbs, and HO was quantified by microCT analysis. Other recipient mice were sacrificed at 2, 7 and 14 days following surgical induction of HO and femur / muscle samples were removed, fixed, embedded in paraffin and sectioned for histology. The expression of bone morphogenetic protein (BMP) 2, 4 and 7 was analyzed immunohistochemically in deparaffinized sections.

Results: HO (1.47 +/- 0.3 mm3; mean +/- S.E.) formed in the quadriceps of all of the operated limbs (12 of 12). Bone mineral density, tissue mineral density and bone volume fraction were 273 +/ 55 mg/cc, 504 +/ 75 mg/cc and 0.42 +/- 0.02, respectively (N = 12 mean +/- S.E.). BMP positive cells were found at the site of tissue injury several weeks before the development of HO.

Conclusions: By transplanting bone marrow containing stromal cells into a site of muscle injury, traumatic HO can be formed reproducibly in an animal model in which putative molecular regulators of the human disorder are expressed prior to the formation of ectopic bone. Therefore, we have developed a new mouse model of traumatic HO that may be more physiologically relevant than previous models that rely on exogenous BMP's or demineralized bone matrix.

Disclosures: B.E. Rapuano, None.

This study received funding from: AO Foundation.

T172

Connective Tissue Growth Factor Induces NFAT and Osteoblastogenesis In Vitro. A. Smerdel-Ramoya, S. Zanotti, E. Canalis. Research, Saint Francis Hospital and Medical Center, Hartford, CT, USA.

Connective Tissue Growth Factor (CTGF) is a member of the CCN family of secreted proteins that regulates multiple cellular functions. Previously, we demonstrated that osteoblasts express CTGF, but its role in osteoblastic differentiation and function remains unclear. To define the function of CTGF, ST2 stromal cells were transduced with a retroviral vector expressing CTGF under the control of the cytomegalovirus promoter. Overexpression of CTGF induced osteoblastogenesis, increased mineralized nodule formation, alkaline phosphatase activity and osteocalcin mRNA levels. Although, CTGF can interact with BMPs, it did not enhance the effect of BMP-2 on Smad signaling or the effect of Wnt3a on Wnt/β-catenin signaling. To investigate alternate mechanisms involved in the osteoblastic differentiation induced by CTGF, we examined CTGF activity on the nuclear factor of activated T cells (NFAT), a transcription factor that cooperates with osterix in the formation of bone. CTGF increased the transactivation of a transiently transfected IL4-9xNFAT-Luc construct, where 9 repeats of NFAT sites direct luciferase expression. The effect was reduced by FK506, an inhibitor of calcineurin, suggesting that CTGF acts on the calcineurin dependent pathway to activate NFAT. In addition, CTGF increased the transcription of Hes-1, a transcription factor known to regulate osteoblastogenesis and NFAT expression. Down regulation of Hes-1 using RNA interference (RNAi) resulted in a decrease of NFAT activation by CTGF, indicating that the induction of Hes-1 by CTGF explains in part its effect on NFAT transactivation. Although, hes-1 is a Notch target gene, its induction by CTGF cannot be explained by activation of the canonical Notch RBPJk/CSL signaling pathway, since CTGF opposed Notch signaling. Down-regulation of CTGF expression by RNAi suppressed NFAT and Hes-1 transactivation confirming that both, NFAT and Hes-1, are dependent on CTGF expression. In conclusion, CTGF overexpression induces osteoblastic differentiation possibly by enhancing NFAT and Hes-1 signaling.

Disclosures: A. Smerdel-Ramoya, None.

T173

NOX Expression in Odontoblasts and Cementoblasts of NOS Knock-out Mice. Y. Sugawara, H. Watanabe, T. Yanagisawa*, Ultrastructural Science, Tokyo Dental College, Chiba, Japan.

Reaction systems of nitric oxide (NO) and reactive oxygen species (ROS) are intricately combined and are involved in the control of many cell functions such as tissue homeostasis, as well as tissue damage and diseases. Oral tissues are sensitive to the effects of their gas environment, but there are a few reports on these effects. In this study, we performed the following experiment to evaluate the protein levels of ROS synthetase, superoxide dismutase(SOD), and nitric oxigen synthetase(NOS) in the dental pulp, odontoblasts, periodontal ligament and cementoblasts of normal and NOS1 (n-NOS), NOS2 (i-NOS), and NOS3 (e-NOS) knock-out (KO) mice and to clarify the interrelations of the kinetics of various enzymes in the absence of n-,i-,e-NOS.

After anesthetizing 5-week-old normal and NOS1, 2, and 3 KO mice, immunohistochemical evaluation were performed using NOXO, NOXA, NQO, and Mn-SOD antibodies. The expression of NOXA, NOXO, and NQO in odontoblasts and cementoblasts were weakly positive in the control mice and strongly positive in the NOS2KO and NOS3KO mice. The Mn-SOD expression in odontoblasts and cementoblasts were weakly positive in the control mice and strongly positive in the NOS2KO and NOS3KO mice. From these results, ROS were considered to be generated by NOX1 in odontoblasts and cementoblasts, and this ROS generation to be affected by i-NOS and e-NOS. futhermore, as Mn-SOD and NOX were strongly positive in the same areas, increases in ROS are considered to be accompanied by increases in Mn-SOD.

Disclosures: Y. Sugawara, None.

T174

Adiponectin, Anabolic to Bone In Vitro but Negative Effects In Vivo. G. A. Williams*1, K. E. Callon*1, J. Lin*1, M. Watson*1, D. Naot1, Y. Wang*2, A. Xu*1, J. Cornish1, I. R. Reid1, 1Medicine, University of Auckland, Auckland, New Zealand, 2Genome Research Center, University of Hong Kong, Hong Kong, Hong Kong, 3Medicine, University of Hong Kong, Hong Kong, Hong Kong.

Adiponectin, the most abundant adipose-specific protein, regulates energy homeostasis as well as glucose and lipid metabolism. Adiponectin is reduced in obesity, insulin resistance and type 2 diabetes, and plasma concentrations are inversely related to body weight.

Body weight is an important risk factor for vertebral and hip fractures, ranking in importance alongside that of age. There is now an accepted positive association of fat mass with the secretion of bone active hormones from the pancreatic beta cell and from the adipocyte.

We have investigated the effect of adiponectin on bone cells in vitro as well as to determine the bone phenotype of adiponectin knockout mice. Adiponectin significantly stimulated osteoclastogenesis by 21% at 0.1 μg/mL, but markedly inhibited this process by 26% and 54% at 1 and 5 μg/mL, respectively. It had no effect on bone resorption in the isolated mature osteoclast assays. Adiponectin was dose-dependently mitogenic to primary osteoblasts (24% increase at 5 μg/mL).

Male adiponectin-deficient (Ad-KO) and wild-type (WT) C57BL/6J mice were examined at 8 and 14-weeks of age. We scanned the left proximal tibia using micro-CT at 5μm resolution and analysed bone micro-architecture by 3D analysis. We found significant increases in trabecular bone volume (BV/TV) (15.92±1.63% in Ad-KO vs 12.19±0.72% in WT, p = 0.02) and trabecular number (3.20±0.18mm−1 in Ad-KO vs 2.32±0.12mm−1 in WT, p = 0.0009) in 14-week old Ad-KO mice compared to controls. Similar differences between WT and Ad-KO were present in 8-week old animals, but these did not reach statistical significance.

Our results demonstrate that although adiponectin can be anabolic in vitro, adiponectin-deficient animals have increased number and volume of trabeculae, indicating that adiponectin is inhibitory to bone accrual in vivo. The latter data concur with the observations from epidemiological studies in humans that adiponectin negatively correlates with both fat mass and bone mass. Therefore, adiponectin may be a contributor to the link between fat and bone mass.

The actions on bone of novel hormones related to nutrition are an important area of further research. An understanding of this aspect of bone biology may open the way for new treatments of osteoporosis. The role of weight maintenance in the prevention of osteoporosis is an important public health message that needs to be more widely appreciated.

Disclosures: G.A. Williams, None.

This study received funding from: HRC.

T175

EGF-like Ligands Stimulate Osteoclastogenesis by Regulating Expression of Osteoclast Regulatory Factors by Osteoblasts: Implications for Osteolytic Bone Metastases. J. Zhu1, X. Jia1, G. Xiao2, Y. Kang3, N. C. Partridge1, L. Oin1, 1Physiology and Biophysics, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ, USA, 2Medicine, University of Pittsburgh, Pittsburgh, PA, USA, 3Molecular Biology, Princeton University, Princeton, NJ, USA.

Epidermal growth factor (EGF)-like ligands and their receptors constitute one of the most important signaling networks functioning in normal tissue development and cancer biology. In vivo mouse models suggest this signaling network plays an important role in bone metabolism. Previous studies found that EGF stimulates bone resorption in organ cultures but the mechanism was unknown. Here we report that EGF-like ligands stimulate formation of TRAP-positive osteoclastic cells in a coculture system containing bone marrow macrophage and osteoblastic MC3T3-E1 cells. Western blots and [1251]EGF ligand binding experiments revealed that MC3T3-E1 cells express EGF receptor (EGFR) while osteoclasts do not, indicating EGF-like ligands do not function directly on osteoclasts. Conditioned medium from EGF-treated MC3T3-E1 cells generated more multinucleated TRAP-positive osteoclasts in the primary osteoclastic culture than conditioned medium from control-treated MC3T3-E1 cells, suggesting that EGF-like ligands regulate the production of secretory factors from osteoblasts. Using quantitative RT-PCR and ELISA, we identified that EGF-like ligands have no effect on RANKL expression but decrease its decoy receptor osteoprotegerin (OPG) expression and increase monocyte chemoattractant protein 1 (MCP1) expression in both MC3T3-E1 and mouse bone marrow primary osteoblastic cells. MCP1 is a chemokine that recruits osteoclasts and stimulates their fusion. Addition of exogenous OPG completely inhibited osteoclast formation stimulated by EGF-like ligands in the coculture system, while addition of a neutralizing antibody against MCP1 exhibited partial inhibition. Coculture with bone metastatic breast cancer cells, MDA-MB-231, which express significant levels of EGF-like ligands, had similar effects on the expression of OPG and MCP1 in the osteoblastic cells and these effects could be partially abolished by EGFR inhibitor PD153035. In summary, we demonstrate that EGFR signaling stimulates osteoclastogenesis indirectly by regulating OPG and MCP1 expression in osteoblasts. Since a high percentage of human carcinomas express EGF-like ligands, our findings suggest a novel mechanism for osteolytic lesions caused by cancer cells metastasizing to bone.

Disclosures: J. Zhu, None.

This study received funding from: NIDDK.

T176

Glucocorticoid Enhances the Expression of a Wnt Antagonist, Secreted Frizzled-Related Protein 3, in Cultured Human Osteoblasts. K. Ohnaka*1, Y. Matsuzaki*1, M. Tanabe*1, M. Adachi*1, H. Kawate*1, R. Takayanagi2, 1Department of Geriatric Medicine, Kyushu University, Fukuoka, Japan, 2Department of Medicine and Bioregulatory Science, Kyushu University, Fukuoka, Japan.

Glucocorticoid-induced osteoporosis (GIO) is one of the most frequent and serious problems of long-term glucocorticoid therapy. Although the major cause of GIO is considered to be impairment of bone formation, detailed mechanism underlying GIO remains to be fully elucidated. Recently, the Wnt signal emerged as a novel key pathway for promoting bone formation. In this study, we investigated the effect of glucocorticoid on a Wnt antagonist, secreted frizzled-related protein (sFRP), in cultured human osteoblasts and an osteosarcoma-derived cell line, MG-63 cells. The expression of mRNA for secreted frizzled-related protein 3 (sFRP3) was markedly induced by dexamethasone among sFRP families. The expression of sFRP3 was also enhanced by hydrocortisone and prednisolone, but not by 17-β estradiol, dehydrotestosterone or 1,25-dihydroxyvitamin D3. Similar effect of dexamethasone on sFRP3 expression was observed in MG-63 cells.

To examine the effect of sFRP3 on osteoblast function, we then generated an expression vector containing entire coding region of human sFRP3 cDNA (pcDNA-sFRP3-HA). Transient transfection of pcDNA-sFRP3-HA suppressed the Wnt3a-induced the accumulation of cytosolic β-catenin in MG-63 cells. Transfection of sFRP3 also decreased the Tcf/Lef-dependent transcriptional activity in MG-63 cells. Furthermore, sFRP3 suppressed the proliferation of MG-63 cells.

These data suggest that glucocorticoid may impair osteoblast function and bone formation by enhancement of sFRP3 in human osteoblastic cells, which may be involved in the pathogenesis of GIO.

Disclosures: K. Ohnaka, None.

T177

Identification and Functional Involvement of Lipocalin 2 in Glucocorticoid-induced Growth Retardation. H. C. Owen*1, S. F. Ahmed2, C. Farquharson1, 1Bone Biology Group, Roslin Institute, Edinburgh, United Kingdom, 2Bone and Endocrine Research Group, Royal Hospital for Sick Children, Glasgow, United Kingdom.

Glucocorticoids (GC) are commonly used for immunomodulation and steroid replacement. Long-term use can, however, result in side effects including growth retardation in children. This is thought to be due to their actions on growth plate chondrocytes. To gain an insight into the mechanisms involved in GC-induced growth retardation, we performed Affymetrix microarray analysis of the murine chondrogenic cell line ATDC5, incubated with 10−6M dexamethasone (Dex) for 24h. Differential expression of selected genes was confirmed by qRT-PCR. Genes confirmed as down-regulated included secreted frizzled-related protein and IGF-1, whilst upregulated genes included serum/GC-regulated kinase, connective tissue growth factor, and lipocalin 2. Lipocalin 2 is an acute phase transport protein which we have shown is expressed in proliferative chondrocytes within the growth plate. In this study, qRT-PCR analysis confirmed that lipocalin 2 gene expression increased in ATDC5 cells by 40-fold after 24h Dex. Expression further increased after 48h (75-fold) and 96h (84-fold) Dex and this response was Dex concentration-dependent. Western blotting confirmed that Dex also increased lipocalin 2 protein expression. The lipocalin 2 response was not blocked by cycloheximide or the p38 inhibitor SB203580, but was blocked by the GC-receptor antagonist RU-486 and the Nuclear-Factor kappa B (NFκB) inhibitor TLCK. The lack of a cycloheximide effect implies a direct action of Dex on lipocalin 2 expression, which is consistent with the presence of a glucocorticoid response-element on the lipocalin 2 promoter. Dex also caused an increase in lipocalin 2 expression in primary murine chondrocytes at 6h (2.5-fold, p<0.05), and this was maintained for up to 72h. Proliferation in ATDC5 cells stably transfected to overexpress lipocalin 2 was slower than control cells (49%, p<0.05) and lipocalin 2 overexpression caused an increase in proteoglycans (66%, p<0.05) and collagen type-X expression (4-fold, p<0.05). Alkaline phosphatase activity and expression was unaffected. The effects of lipocalin 2 overexpression on chondrocyte proliferation (64%, p<0.05) and collagen type-X expression (8-fold, p<0.05) were further exacerbated with the addition of 10−6M Dex. This synergistic effect may be explained by a further increase in lipocalin 2 expression with 10−6M Dex in transfected cells (45%, p<0.05). These results suggest that lipocalin 2 may mediate Dex effects on chondrocytes through an NFκB-dependent pathway, and therefore provides a potential novel mechanism for GC-induced growth retardation.

Disclosures: H.C. Owen, None.

This study received funding from: BBSRC.

T178

The Temporal Feature of Bone Marrow Fat Cells in the Process of Steroid-Associated Osteonecrosis Development. H. Sheng*1, G. Zhang*1, L. Qin*1, W. Cheung*1, C. Chan*1, Y. Wang*2, H. Wang*3, K. Lee*4, K. Leung*1, 1Orthopaedics & Traumatology, the Chinese University of Hong Kong, Hong Kong, Hong Kong, 2Diagnostic Radiology and Organ Imaging, the Chinese University of Hong Kong, Hong Kong, Hong Kong, 3Fudan University, Shang Hai, China, 4Lee Hysan Clinical Research Laboratory, the Chinese University of Hong Kong, Hong Kong, Hong Kong.

The fat volume of bone marrow in steroid-associated osteonecrosis (ON) increases greatly, but the dynamic change of fat cells in the process of ON development is still unknown. This study aimed to explore the time course change of bone marrow fat cells using our established ON model. Thirty-two rabbits were divided into control (n = 16) and steroid-treatment group (n = 16). Each of group was further divided into the early (n = 8) and late group(n = 8) in terms of sacrifice time. The rabbits in steroid-treatment groups were injected with once lipopolysaccharide and three times of methylprednisolone to induce ON. The rabbits in control groups were injected with normal saline accordingly. The blood perfusion function of the femoral heads were measured by dynamic contrast magnetic resonance imaging (MRI) at week 0, week 2 and week 4. The rabbits were sacrificed at week 2 and week 4, the bilateral femurs were harvested and processed for bone marrow fat cell density, fat cell diameter and fat cells area percentage analysis histomorphometrically and ON evaluation histopathologically. The dynamic MRI showed the blood perfusion function decreased continuously in steroid-treatment group, the maximum enhancement decreased from 55% in week 2 to 30% in week 4. The ON incidence increased from 25%(2/8) in week 2 to 87.5%(7/8) in week 4. The fat cells area percentage in steroid-treatment early and late group increased by 44% and 83.4% compared with control group. The fat cells density in early and late group increased by 67.1% and 54.4% than control group, but there is no significant difference between the treatment groups; The fat cells diameter in late group was much larger than control group, but the fat cell diameter in early group was smaller than control group; The fat cells size profile showed most of the increased fat cells were small size cells ranging from 30 to 40μm in diameter in early group, but large size cells ranging from 50 to 60μm in the late group. The marrow fat cells featured increase in number of small size fat cells in the early stage and enlargement in size of fat cells in the late stage in ON development. These imply the active adipogenesis in the early stage and excessive lipogenesis in the late stage in the process of steroid-associated ON development. The forming new hypothesis is inhibiting adipogenesis in early stage and decreasing fat cells lipogenesis in late stage would prevent steroid-associated ON development.

Disclosures: H. Sheng, None.

This study received funding from: RGC(CUHK4503/06M) and IFTF(ITS/012/06).

T179

Transcription Factor E4bp4 Is Induced by Glucocorticoids and Represses Promoter Activity in Osteoblasts. S. M. Woo*1, I. C. Ozkurt*2, S. Tetradis1, 1UCLA School of Dentistry, Los Angeles, CA, USA, 2Ankara University, Ankara, Turkey.

Chronic, systemic use of glucocorticoids (GCs) dramatically inhibits bone formation. GCs mediate their effects by binding to and activating the glucocorticoid receptor to regulate downstream gene expression. GCs profoundly repress osteoblastic genes. However, the molecular mechanisms mediating GC repression of osteoblastic gene expression remain largely unknown. E4bp4, a transcription factor acting as a transcriptional repressor in osteoblasts, is induced by GCs in fibroblasts. Thus, we hypothesize that E4bp4 participates in the GC repression of osteoblastic genes. Here, we study GC-induced E4bp4 gene expression and explore E4bp4 involvement in GC regulated promoter activity in osteoblasts. Calvariae-derived murine osteoblasts (MOBs), calvarial cultures, and 6-8 day old CD-1 mice were treated with Dexamethasone (Dex), and E4bp4 expression was measured by Northern and Western blot assays and quantitative real-time PCR. Protein-DNA binding was evaluated by electrophoretic mobility shift assays and promoter activity by luciferase assays. MOBs were infected in vitro with an adenovirus overexpressing E4bp4, and transcriptome profiling was completed using the Affymetrix GeneChip 430A 2.0 Mouse Array and GeneSifter microarray analysis software. Dex increased E4bp4 mRNA and protein expression that peaked at 4-6 h and 1μM Dex, with elevated levels even at 24 h. Pre-treatment with the protein synthesis inhibitor cycloheximide did not prevent E4bp4 induction, showing that E4bp4 is a Dex-induced primary response gene. Nuclear proteins from MOBs treated with Dex specifically bound the E4bp4 response element (EBPRE), but not mutated EBPREs. Co-incubation with E4bp4-specific antibodies showed that the Dex-induced EBPRE-bound nuclear proteins contained E4bp4 protein. Furthermore, Dex treatment attenuated activity of a chimeric promoter containing three tandem repeats of wild-type but not mutant EBPRE, suggesting that E4bp4 protein mediates promoter repression by Dex. Adenoviral-mediated E4bp4 overexpression upregulated 415 and downregulated 337 genes more than 1.8 fold. Gene ontology (GO) grouping summarized the molecular function, biological process, and cellular component of regulated genes. Interesting GO groups with z-score >2 included multicellular organismal development, cell surface receptor linked signal transduction, extracellular space, extracellular matrix, receptor binding, and receptor activity. We are currently investigating direct regulation of individual genes by both GCs and E4bp4. These results support a role of E4bp4 in mediating glucocorticoid-induced gene repression in osteoblasts.

Disclosures: S.M. Woo, None.

This study received funding from: NIDCR R01 DE013316. NIDCR T32 DE007296.

T180

A Novel Mechanism of Glucocorticoid Action-Inhibition of Cox-2 Transcription by Glucocorticoid Induced Leucine Zipper (GILZ). N. Yang*, X. Shi. Institute of Molecular Medicine & Genetics, Medical College of Georgia, Augusta, GA, USA.

Cyclooxygenase-2 (COX-2) plays an important role in rheumatoid arthritis (RA) and therefore, it has been a major target for anti-arthritis therapies. The expression of COX-2 is induced by inflammatory cytokines such as TNF-α and IL-1β, and inhibited by glucocorticoids (GCs). However, the molecular mechanisms underlying the anti-inflammatory and immune suppressive actions of GCs are not well defined. Importantly, long-term GC therapy causes, among other adverse effects, rapid bone loss resulting in osteoporosis, therefore, their use is limited. Here we report that a recently identified GC-inducible protein, GILZ, mimics GC action and inhibits inflammatory cytokine-induced COX-2 expression in bone marrow mesenchymal stem cells (MSCs), the cells that have been recently implicated in the pathogenesis and progression of RA. Using a retrovirus-mediated gene expression approach we demonstrate that overexpression of GILZ inhibits TNF-α and IL-1β-induced COX-2 mRNA and protein expression and knockdown of GILZ by shRNAi abolishes GC inhibition of cytokine-induced COX-2 expression. Consistent to these results, overexpression of GILZ also inhibits NF-κB- and AP-1-mediated COX-2 promoter activity. Finally, we show that GILZ inhibits COX-2 expression by blocking NF-κB nuclear translocation and by inhibiting AP-1 binding to the COX-2 promoter region. Our results suggest that GILZ is a key GC effect mediator and that GILZ may have pharmacotherapeutic value for novel anti-inflammation therapies.

Disclosures: N. Yang, None.

This study received funding from: Arthritis Foundation.

T181

Glucocorticoid Excess in Mice Leads to Early Activation of Osteoclastogenesis and Prolonged Suppression of Osteogenesis Through Gene Expression Profiling. W. Yao, Z. Cheng, C. Busse*, S. Rao*, N. Lane. Center for Healthy Aging, UC Davis Medical Center, Sacramento, CA, USA.

Glucocorticoid (GC) excess induces alterations in bone metabolism that weakens bone structure. We performed genomewide cDNA microarray on whole bone RNA (Affymetrix Mouse Genome 430 2.0 array) to identify genes associated with bone metabolism in GC-treated mice. Long bones from mice exposed to GC excess were collected at 0, 3, 7, 28 and 56 days (5mg prednisolone 60-day slow release pellet, n = 8-12/group). The success of this animal model was confirmed by changes in bone turnover markers as well as bone microarchitecture measured by microCT. Differences in gene expression with time were calculated by unequal-variance ANOVA with a threshold of > 2fold and p < 0.01. The significantly expressed1179 transcripts were then further grouped by Gene Functions using GeneShifer Software. Quantitative real-time PCR was performed on selected genes to confirm microarray results.

Results: Compared to placebo treated mice, GC-treated mice lost trabecular bone volume at the distal femur (16% by d28 and was unchanged at d56). Serum measurements of osteoclast maturation, TRAP5b, increased by 18% at d7 and d28, and increased by 15% at d56; and osteoblast maturation, osteocalcin decreased by 50% from d28-d56. Compared to placebo treated mice, GC excess was associated with increased expression of genes involved with osteoclast activation (csfl, c-fms, c-fos, adam8, Trem2, Oscar, Nfactc1) that peaked at d7; osteoclast cytoskeleton reorganization (β3, Ibsp, c-src, vav3, ATPase) and genes associated with matrix degradation peaked at d 28 (cathepsins, mmps and proteases). At d28 and d56, genes associated with osteoblast activation and function had decreased expression (BMPs 2, 5, 8, 12, 13, TGFβ1) and Wnt inhibitors such as Wifl, Dkkl and Sost had increased expression compared to placebo treated animals. In addition, with GC excess, genes expressed by osteocytes and associated with bone mineralization, DMP1 and Phex, had increased expression at d28 and d56. RT-PCR confirmed our microarray findings in selected genes.

In conclusion, GC excess is associated with early activation of osteoclastogenesis and delayed but prolonged suppression of osteogenesis and matrix mineralization. These rapid and sustained alterations in bone cells maturation and activity help to explain the significant deterioration in bone strength in the presence of GCs.

original image

Disclosures: W. Yao, None.

This study received funding from: R01 AR043052-07, 1K12HD05195801.

T182

Osteoblast-Targeted Disruption of Glucocorticoid Signalling Delays Intramembranous Bone Development In Vivo. H. Zhou, W. Mak, Y. Zheng, C. R. Dunstan, M. J. Seibel. Bone Research Program, ANZAC Research Institue, University of Sydney, Sydney, Australia.

Transgenic (tg) expression of 11beta-hydroxysteroid dehydrogenase type 2 (HSD2), a glucocorticoid (GC) inactivating enzyme, under the control of a 2.3kb collagen type I promoter (Col2.3-HSD2) abrogates intracellular GC signalling in mature osteoblasts. Adult Col2.3-HSD2 tg mice exhibit a mild osteoporotic phenotype but the skeleton otherwise appears normal (1). To investigate the effects of osteoblast-targeted disruption of GC signalling on early skeletal development, calvaria and tibiae of wild-type (WT) and Col2.3-HSD2 tg mice aged 0, 1, 7, 10 and 14 days were analysed.

HSD2 mRNA and protein expression was present in the calvaria and long bones of tg mice but absent in the bones of WT mice. Calvaria from tg mice demonstrated a markedly altered phenotype: compared to their WT littermates, tg mice had poorly formed parietal bones with large amounts of cranial cartilage still present at day 0. A similar picture was seen at day 1, with the extent of parietal bones being reduced by 33% relative to WT mice. The cranial cartilage plate was present in all tg mice but absent in WT mice. In 7-day old mice, the cranial cartilage plate was reduced in size but still present in tg animals, but formation of parietal bones was complete and bone area was similar to WT mice. Calvaria appeared normal at 2 weeks of age. Immunohistochemical analyses showed reduced protein expression of MT1-MMP, an enzyme essential for calvarial cartilage removal in the cranial cartilage of tg mice. TUNEL staining indicated, reduced chondrocyte apoptosis. In contrast, no phenotype was observed in the long bones (tibiae) of tg mice.

Our results indicate that osteoblast-targeted disruption of GC signalling results in delayed calvarial (intramembranous) development, without affecting the (endosteal) formation of long bones. These findings further suggest that in neonatal mice, osteoblasts regulate cranial cartilage removal, a function that appears to be GC-dependent and mediated through activation of chondrocytic MT1-MMP expression. Our studies point to a novel role for both GC and osteoblasts in intramembranous bone development. (1) Sher et al Endocrinology 145:922-9, 2004.

Disclosures: H. Zhou, None.

This study received funding from: NHMRC #402462.

T183

Transgenic Disruption of Glucocorticoid Signalling in Mature Osteoblasts Attenuates KRN Serum-induced Arthritis In Vivo. H. Zhou1, E. Buttgereit*2, T. Gaber*2, R. Kalak*1, R. Dragovic*1, D. Huscher*2, R. H. Straub*3, J. Modzelewski*1, C. R. Dunstan1, M. J. Seibel4, 1Bone Research Program, ANZAC Research Institue, Sydney, Australia, 2Dept. of Rheumatology & Clinical Immunology, Charité (CCM) and DRFZ, Berlin, Germany, 3Dept. of Internal Medicine I, University Hospital, Regensburg, Germany, 4Dept. of Endocrinology & Metabolism, Concord Hospital, Sydney, Australia.

Transgenic (tg) overexpression of 11beta-hydroxysteroid dehydrogenase type 2 (HSD2), a glucocorticoid (GC) inactivating enzyme under the control of a 2.3Kb collagen type I promoter (Col2.3-HSD2), abrogates intracellular GC signalling exclusively in mature osteoblasts. Since GC are important immune modulators, we investigated the impact of osteoblast-targeted disruption of GC signalling on joint inflammation and bone catabolism using the KRN serum transfer model of autoimmune arthritis.

KRN arthritis was induced in 5-week-old male Col2.3-HSD2-tg mice (tg-KRN, n = 28) and their wild-type (WT) littermates (WT-KRN, n = 27). Twelve tg and 13 WT mice served as controls (CTR) receiving either normal serum or PBS. Body weight and paw swelling (ankle size, clinical arthritis, scored 0-6) were assessed daily from induction (day 0) to day 14. Serum levels of IL-1beta, IL-2, IL-4, IL-6, IL-10, IL-12, TNF-alpha, IFN-gamma, G-CSF, M-CSF and corticosterone (CS) were determined in CTR, and in KRN animals on days 7 and 14. Micro-CT of the tibia and the paw skeleton was employed to assess for morphological changes.

Both tg-KRN and WT-KRN developed acute arthritis. However, the inflammatory response was significantly blunted in tg mice from day 7 onwards (Fig. 1). Compared to CTR, tibia bone volume (BV/TV) was significantly reduced in WT-KRN but not in tg-KRN on day 14. As compared to day 7, mean serum M-CSF levels were significantly lower on day 14 in tg-KRN but not in WT-KRN. Compared to CTR, serum TNF-alpha, IL-6 and IL-12 levels were significantly lower in tg-KRN on day 14. However, there were no differences in cytokine or CS levels between tg-KRN and WT-KRN at any time point, suggesting that in HSD2-tg mice the inflammatory process was attenuated through local mediators.

We conclude that osteoblasts are able to significantly modulate local inflammatory responses via a glucocorticoid-dependent pathway.

original image

Disclosures: H. Zhou, None.

This study received funding from: NHMRC #402462.

T184

Regulation of Oxidative Stress and Osteoblast Apoptosis by Estrogens Is Preserved in Mice in which the ER Cannot Directly Interact with DNA. M. Almeida, M. Martin-Millan*, L. Han, A. Warren*, V. Lowe*, R. S. Shelton*, A. DeLoose*, R. S. Weinstein, T. Bellido, C. A. O'Brien, 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.

To investigate the role of the ERα, and in particular its non-ERE-mediated signaling, in skeletal homeostasis we have crossed knock-in mice in which classical ERα signaling through ERE sites has been selectively eliminated while preserving non-classical signaling (ERαNERK1/+), with heterozygous mice of the ERα gene, to generate ERαNERK1/− mice. We report that 17β-estradiol (E2) at 10−8 M stimulated apoptosis of mature osteoclasts in bone marrow cultures from ERαNERK1/− mice as effectively as in cultures from wild-type control mice. Likewise, E2 prevented etoposide-induced apoptosis in osteoblastic cultures derived from calvaria of either type of mouse. In agreement with the in vitro data, ovariectomy (ovx) upregulated osteoclastogenesis in wild type as well as in ERαNERK1/− mice as measured in ex vivo bone marrow cultures. Ovx also caused an increase of osteoblast apoptosis in ERαNERK1/− and wild-type mice; and E2 replacement prevented the phenomenon in both. Further, ovx led to an increase in reactive oxygen species (ROS) levels and a decrease in glutathione reductase activity in the bone marrow; as well as an increase in the phosphorylation of p66shc (a key component of a signaling cascade that is activated by ROS and stimulates apoptosis) in vertebral lysates from both wild-type controls and ERαNERK1/− mice. E2 replacement prevented all these changes in both type of mice. Lastly, there was a progressive decrease in baseline BMD in the femur and spine from ERα+/+, to ERα+/−, to ERα1/+, to ERα1/− mice. Moreover, ERαNERK1−/− mice exhibited a decrease in bone size and osteoblastogenesis as measured by the number of colony forming units-osteoblasts in ex vivo bone marrow cultures; as well as a decrease in osteocalcin levels in the serum. These results strongly suggest that the antioxidant properties of estrogens, as well as their ability to control osteoblast and osteoclast apoptosis and osteoclastogenesis do not require classical DNA binding of the ERα; and are consistent with evidence that trabecular (but not cortical) bone mass remains estrogen responsive in the ERαNERK1/− mice. The decrease in bone size, BMD and osteoblastogenesis in the ERαNERK1/− mice may be explained by evidence presented elsewhere in this meeting that the unliganded wild type ERα (presumably through its ability to bind directly to DNA) is a co-activator of BMP-induced osteoblastogenesis and thereby bone formation and growth during development.

Disclosures: M. Almeida. None.

T185

Estrogens or Androgens Attenuate p66shc Phosphorylation via an ERK and PKCβ Signaling Cascade: a Critical Mechanism of their Protective Effects Against Oxidative Stress and Bone Loss. M. Almeida, M. Martin-Millan, L. Han, A. Warren*, V. Lowe*, T. Bellido, R. L. Jilka, C. A. O'Brien, 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.

Phosphorylation of the adapter protein p66shc is not only required for transduction of oxidative stress signals leading to apoptosis but also amplifies such stress by generating reactive oxygen species (ROS) in the mitochondria. Based on this and evidence that ovariectomy (ovx) or orchidectomy of 5 month-old C57BL/6 mice increases the phosphorylation of p66shc in vertebral lysates, while replacement with 17β-estradiol (E2) or DHT prevent this phenomenon as effectively as the antioxidant N-acetyl-L-cysteine (NAC), we have investigated the molecular basis of the effects of sex steroids on p66shc. We report that exposure of several osteoblastic cell lines as well as primary cultures of calvaria osteoblasts to H2O2 stimulated the phosphorylation of p66shc within 5 min, and that pre-treatment of all these cells for 1 h with either E2 or DHT prevented this effect. Further, overexpression of p66shc in C2C12 cells induced apoptosis both under basal condition and in the presence of H2O2. The suppressive effect of sex steroids on H2O2-induced phosphorylation of p66shc was inhibited by the ERK-specific inhibitor PD98059 indicating that this is a kinase-mediated action of the ER. Consistent with this finding E2 inhibited the pro-apoptotic effect of H2O2 as well as p66shc phosphorylation in calvaria derived osteoblast cultures from ERαNERK1/− mice, a knock-in mouse mutant of the ERα that lacks classical DNA binding on ERE elements. Moreover, the increase in osteoblast apoptosis and p66 phosphorylation in the vertebra following ovx was suppressed by E2 replacement in both wild-type and ERαNERK1/− mice. To further dissect the mechanism by which sex steroids antagonize p66shc phosphorylation induced by oxidative stress we searched for and found that PKCβ activity was required for H2O2-induced p66shc phosphorylation, as it was blocked by the specific PKCβ inhibitor hispidin. Furthermore, activation of PKC by PMA induced p66shc phosphorylation and stimulated apoptosis in C2C12 cells; and both effects were prevented by hispidin. Finally, both phosphorylation of p66shc as well as apoptosis induced by PMA were prevented by E2 suggesting that modulation of PKCβ activity might mediate the anti-apoptotic effects of sex steroids in osteoblastic cells. These results strongly suggest that sex steroids attenuate osteoblast apoptosis induced by oxidative stress by modulating a cascade of cytoplasmic kinases culminating with inhibition of p66shc phosphorylation.

Disclosures: M. Almeida, None.

T186

Estrogens Attenuate IL-6, and TNFα Production in Osteoblastic Cells by Decreasing Oxidative Stress and its Effects on NFκB Activation. M. Almeida, 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 Central Arkansas Veterans Healthcare System, Little Rock, AR, USA.

Reactive oxygen species (ROS) may play a critical pathogenetic role in osteoporosis, and the anti-osteoporotic effect of estrogens may result from the ability of these hormones to protect against oxidative stress. Indeed both aging and estrogen deficiency increased oxidative stress in the bone of both male and female C57BL/6 mice as well as the number of osteoclast progenitors in the bone marrow. Based on this and the well known relationship among IL-6, TNFα, osteoclastogenesis, and estrogens we tested the hypothesis that estrogens exert their anti-osteoclastogenic effects by enhancing defense against ROS. We report that increasing levels of ROS, decreasing GSR activity, and increasing phosphorylation of p66shc between 8 and 31 month of age in C57BL/6 female mice were temporally associated with a progressive increase in the expression of the osteoclastogenic cytokines TNFα and IL-6, as measured by RT-PCR in calvaria. Consistent with the notion that the in vivo changes in oxidative stress and cytokine production were causally related, H2O2 up-regulated the expression of both TNFα and IL-6 in cultures of the UAMS32 osteoclast supporting stromal cell line. And, 17β-estradiol (E2) at 10−8 M potently attenuated the effects of H2O2 on the production of both cytokines. In line with this finding, H2O2 strongly activated NFκB within 1 h, as determined by the phosphorylation of the NFκB inhibitor kinase Iκβ, by Western blot. Consistent with the inhibitory effect of E2 on the expression of IL-6 and TNFα upon H2O2 stimulation, pre-treatment with E2 for one hour abrogated the effect of H2O2 on NFκB activation. Finally, to determine whether the increase in osteoclastogenesis that follows estrogen deficiency is mediated by the anti-oxidant actions of estrogen, we treated ovx mice with E2 or the antioxidant N-acetyl-L-cysteine (NAC). NAC was as effective as E2 in preventing the increase in the number of osteoclast progenitors as determined by ex vivo bone marrow cultures. Consistent with this, administration of L-buthionine-(S,R)-sulphoximine (BSO), a specific inhibitor of glutathione synthesis, for 6 weeks, or the oxidant-producing herbicide paraquat for 4 weeks, to 5 month-old C57BL/6 mice increased both ROS levels as well as the number of osteoclast progenitors in the bone marrow. Taken together with evidence that estrogens directly suppress p66shc phosphorylation, presented elsewhere in this meeting, these results suggest that estrogens attenuate IL-6 and TNFα production in stromal/osteoblastic cells by decreasing oxidative stress and its effects on NFκB activation.

Disclosures: M. Almeida, None.

T187

Premenopausal Women with Short Luteal Phase Length Have Reduced vBMD, but Normal Bone Bending Strength at the Tibial Midshaft. B. C. Kaufman*1, R. J. Wetzsteon1, M. S. Kurzer*2, J. C. Prior*3, M. A. Petit1, 1Kinesiology, University of Minnesota, Minneapolis, MN, USA, 2Food Science and Nutrition, University of Minnesota, Minneapolis, MN, USA, 3Vancouver General Hospital, Vancouver, BC, Canada.

Progesterone is hypothesized to have an important influence on bone metabolism, which could influence bone health in women with a shortened luteal phase. The purpose of this cross-sectional study was to explore the relationship between luteal phase length (LPL) and bone parameters in healthy, sedentary, pre-menopausal women. We used baseline data from a subset of women (n = 32, aged 18-30yr; mean BMI = 22.9±3.1) enrolled in a study of physical activity and ovulation (WISER). Menstrual cycle characteristics were assessed by questionnaire and confirmed with ovulation kits over 5 menstrual cycles. Volumetric bone mineral density (vBMD, mg/mm3), bone geometry (Total Area), and estimated bone strength (polar strength strain index, SSI) were assessed by pQCT (XCT 3000) at a distal (8%) and midshaft (50%) site of the left tibia. Participants were split into two groups based on average luteal phase length of < 10.9d (short LPL, n = 12) or > 11.0d (normal LPL, n = 20). Groups were similar in age, height, weight, %fat and %lean mass. There were also no differences between groups in physical activity (assessed by questionnaire), total calorie intake (from 3d food records), or average menstrual cycle length (29 and 30d respectively). At the tibial midshaft, total vBMD was significantly lower (-5.9%, p = 0.005) in the women with short LPL, while total bone area was significantly higher (+13%, p = 0.013) resulting in a non-significant difference in SSI between groups (p > 0.05). Similar trends were seen at the distal site, but differences in bone parameters were not significant. Results remained significant after adjusting for tibial length and body weight. These data suggest that sedentary women with shortened luteal phase length may have reduced vBMD that seems to be compensated for by increased bone area, resulting in bone bending strengths similar to sedentary women with normal luteal phase length at the tibial midshaft.

Disclosures: B.C. Kaufman, None.

T188

Identification of Primary Target Cells for the Classical Genomic Effects of Estrogen and Raloxifene in Bone. C. Håkansson*1, P. T. van der Saag*2, J. Å. Gustafsson*3, H. Carlsten*1, C. Ohlsson4, M. K. Lagerquist4, 1Dept. of Rheumatology and Inflammation Research, Inst. of Medicine, Gothenburg, Sweden, 2Hubrecht Lab., Netherlands Inst. for Developmental Biology, Utrecht, The Netherlands, 3Dept. of Bioscience at NOVUM, Karolinska Institute, Huddinge, Sweden, 4Dept. of Internal Medicine, Inst. of Medicine, Gothenburg, Sweden.

Estrogen, as well as the selective estrogen receptor modulator raloxifene, has bone protective effects, but the mechanism behind these effects is still not completely characterized. Recently, immune cells in the bone marrow have been implicated to be involved in estrogenic effects on bone. The aim of this study was to identify primary target cells in bone marrow for the classical genomic effects of estrogen and raloxifene in an attempt to clarify the mechanism behind the bone protective effects of these compounds. For this purpose we have used a reporter mouse with a luciferase reporter gene under the control of three estrogen-responsive elements (EREs). These mice enable detection of in vivo activation of gene transcription via binding of estrogen receptors (ERs) to ERE-elements. Three-month-old ovariectomized (ovx) mice were given a single dose of 17β-estradiol (E2) (50μg/kg, s.c.) or the raloxifene-analog LYI17018 (3mg/kg, s.c.) and the experiment was ended after 10h. The doses were chosen because of their equipotent effects on bone mass.

Bone marrow was flushed from femur and sorting of various cell populations was performed using FACSAria, resulting in cell populations with >95% purity.

In total bone marrow, E2 increased luciferase activity to 12000 RLU/106 cells and raloxifene to 700 RLU/106 cells (p<0.01). In the B cell fraction (CD19° cells), E2 increased luciferase activity slightly (1600 RLU/106 cells), while raloxifene had no effect. No effect of either E2 or raloxifene was found in the T lymphocyte or the granulocyte population, while the cell fraction lacking both lymphocytes and granulocytes exhibited the greatest luciferase activity/106 cells (40000 RLU for E2 and 6000 RLU for raloxifene).

In conclusion, neither lymphocytes nor granulocytes are primary targets for the classical genomic effects of E2 or raloxifene in bone marrow. Furthermore, raloxifene can activate gene transcription via ER binding to EREs in the bone marrow, but not to the same extent as E2 at doses that give equipotent effects on bone mass.

Disclosures: M.K. Lagerquist, None.

T189

Estrogen Receptors in Adolescent Idiopathic Scoliosis (AIS). D. Leboeuf*1, K. Letellier*2, B. Azeddine*2, G. Grimard*3, S. Parent*3, H. Labelle*3, A. Moreau4, F. Moldovan*4, 1Sciences Biomédicales, Université de Montréal, Montréal, PQ, Canada, 2Biologie Moléculaire, Université de Montréal, Montréal, PQ, Canada, 3Hǒpital Sainte Justine, Montréal, PQ, Canada, 4Centre de Recherche de l'Hǒpital Sainte Justine, Montréal, PQ, Canada.

AIS occurs and progresses during puberty, a period greatly influenced by hormones. This, and the fact that most severe cases of scoliosis affect girls, points to a possible role for estrogens in the onset and the progression of AIS. Estrogen signaling occurs mainly via two nuclear receptors, ERα and ERβ. Here we investigate the expression of ERα and ERβ in AIS and control (trauma) patients. RNA extracted from osteoblasts of AIS and control patients was investigated by RT-PCR using primers covering all 4 domains of ERα and ERβ (protein-protein interaction, hinge, ligand-binding and DNA binding domains). In addition, ERα and ERβ were detected by immunohistochemistry on spinal biopsy tissues and on osteoblasts exposed in vitro to 17-β-Estradiol.

All AIS patients (n = 10, age = 14±2.02, Cobb = 59.37±20.9) and controls (n = 8, age = 14.7±3.0,) show transcripts of many isoforms of ERα. However, the expression of ERβ varied between patients and controls, and results suggest the presence of various isoforms in human osteoblasts. We also identified by sequencing an insert specific to isoform h-ERβ Sl, in 6/10 AIS patients and 8/8 controls. Immunohistochemistry revealed high levels of ERα and low levels of ERβ. Finally, osteoblasts of AIS patients exposed to 17-β-Estradiol show increased nuclear expression of ERα, which was not observed in control osteoblasts. It is already known that ERα and ERβ have overlapping roles in regulating growth and differentiation of bone tissues. However, ERβ is the dominant receptor in many ER-dependent signaling pathways in bone cells, and some isoforms exhibit an antagonistic effect on ER-mediated transcriptional activity. Surprisingly, the level of ERβ was very low in AIS cells, compared to ERα. In addition, the lack of the isoform h-ERβ Sl in some AIS patients, as well as the altered balance between ERα and ERβ, appears to be critical for estrogen-dependent functions of osteoblasts, which could impact bone homeostasis and lead to decreased bone mineral density and osteopenia in AIS.

Disclosures: D. Leboeuf. None.

This study received funding from: La Fondation Yves Cotrel-Institut de France.

T190

The Unliganded ERα or β, but not the AR, Potentiates BMP-induced Transcription and Osteoblastogenesis. M. Martin-Millan, L. Han, A. Warren*, V. Lowe*, C. A. O'Brien, M. Almeida, 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.

Whereas estrogens suppress osteoblastogenesis and estrogen loss increases it, female or male mice with ubiquitous ERα deletion (ERα−/−) exhibit decreased osteoblast number and bone formation rate. Based on this and recent evidence for distinct roles of the unliganded versus liganded ER in the transcriptional repression of the TNF gene, we have tested the hypothesis that the ER may have two opposite effects in osteoblast generation: attenuation in the presence of 17β-estradiol (E2) and potentiation in the absence of E2. We report that E2 at 10−7 to 10−8 M suppressed BMP-2-induced osteoblast progenitor commitment and differentiation in two cell lines, the uncommitted mesenchymal C2C12 and the pre-osteoblasts 2T3, as well as in primary cultures of murine bone marrow, as measured by alkaline phosphatase (AP) activity. Consistent with a suppressive effect on osteoblastogenesis, E2 also attenuated BMP-2-induced osteocalcin secretion, as well as the expression of the BMP-2 responsive gene Smad6. Importantly. ERα co-immunoprecipitated with Smad1 and Smad4 in all these cell models. Further, treatment of C2C12 or 2T3 cells with ICI182,780 (10−7M), an antagonist of ER that promotes its degradation, also attenuated BMP-2-induced AP activity, osteocalcin secretion and the expression of Smad6. In support of the contention that the inhibitory effect of ICI on osteoblastogenesis was indeed due to ER degradation, silencing ERα in C2C12 cells using specific siRNA oligonucleotides abrogated AP activity induced by BMP-2. In agreement with these observations, transfection of C2C12 cells with increasing amounts of plasmids encoding the ERα or the ERβ, dose-dependently increased BMP-induced transcription, as measured by a Smad6-luciferase reporter construct. This effect was also evident in cultures not treated with BMP, indicating that the unliganded ERα or β potentiated the effect of both endogenous as well as exogenous BMPs. Transfection with the same amounts of a plasmid expressing the androgen receptor had no effect on basal or BMP-stimulated transcription of the reporter construct. These findings strongly support the hypothesis that the unliganded ER stimulates BMP target genes, perhaps by acting as a co-activator of Smads, and that binding of E2 antagonizes this effect. Opposite effects of the unliganded versus the liganded ER on osteoblastogenesis can explain why, in spite of the well-known suppressive effect of estrogens on osteoblastogenesis as well as osteoclastogenesis, and thereby on remodeling, bone formation is decreased in mice with ERα deletion.

Disclosures: M. Martin-Millan, None.

T191

Serum Testosterone in Elderly Men Measured by Liquid Chromatography-Tandem Mass Spectrometry and Radioimmunoassay: Concordance and Effect on Epidemiologic Association. T. V. Nguyen1, C. Meier2, D. J. Handelsman*3, M. Kraenzlin2, M. M. Kushnir*4, A. L. Rockwood*4, A. W. Meikle*5, J. R. Center6, J. A. Eisman6, M. J. Seibel2, 1Bone and Mineral Research Program, Garvan Institute of Medical Research, St Vincent's Hospital and UNSW, Darlingurst, NSW, Australia, 2Bone Research Program, ANZAC Research Institute, Concord, NSW, Australia, 3Andrology, ANZAC Research Institute, Concord, NSW, Australia, 4ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT, USA, 5Department of Medicine and Pathology, University of Utah, Salt Lake City, UT, USA, 6Bone and Mineral Research Program, Garvan Institute of Medical Research, St Vincent's Hospital and UNSW, Darlinghurst, NSW, Australia.

Serum testosterone (T) is used as a measure to define androgenic status and its association with health outcomes. Established radioimmunoassays (RIA) have been used to measure T; however, liquid chromatography-tandem mass spectrometry (MS) has increasingly become a reference method for measuring T. The present study assessed the concordance between T measured by RIA and MS, and the effect of this concordance on the association between serum T and fracture risk in men.

T was measured by direct RIA (RIA-T; Delphia, Perkin-Elmar) and liquid chromatography-tandem mass spectrometry (MS-T) in 593 men aged 60+ years, who had participated in the Dubbo Osteoporosis Epidemiology Study between 1989 and 2005. Low-trauma fractures were recorded during the study period. The concordance between the two methods of measurement was assessed by the coefficient of concordance and the limit of agreement method. The association between each measured T and fracture risk was analyzed within the framework of the Cox's proportional hazards model.

The coefficient of concordance between RIA-T and MS-T was 0.67 (p18 pmol/L), RIA-T tended to underestimate MS-T by 25%. In the Cox's model, each SD of log MS-T was significantly associated with fracture risk (hazard ratio [HR] 1.37; 95% CI: 1.15, 1.6), which was slightly higher than the association between log RIA-T and fracture risk (HR: 1.19; 95% CI: 1.02, 1.40).

These data suggest that there was a modest concordance between MS-T and RIA-T, with minor effects on the assessment of associations between serum T and fracture in epidemiologic studies in men.

Disclosures: T. V. Nguyen, None.

T192

Down-Regulation of Klotho Protein Expression by Estrogen. O. K. Oz1, A. Hajibeigi*1, W. Siyambalapitiyage*1, K. Korach*2, M. Kuro-o*3, 1Radiology, UT Southwestern Medical Center at Dallas, Dallas, TX, USA, 2NIEHS, NIH, Research Triangle Park, NC, USA, 3Pathology, UT Southwestern Medical Center at Dallas, Dallas, TX, USA.

Klotho is a glycoprotein predominantly expressed in distal tubules cells of kidney. Low klotho gene expression in mice leads to multiple disorders, such as arteriosclerosis, skin atrophy, abnormal calcium homeostasis and shortened life span. More recently klotho has been shown to regulate activity of the calcium channel TRPV5 in the kidney. Estrogens are also regulators of calcium homeostasis. In this study we investigated a potential regulatory role for estradiol in klotho expression. The effect of estradiol treatment on klotho expression in vitro was tested in the renal tubule cell line MDCK grown in estrogen depleted (charcoal stripped) or repleted serum. Lysates of the treated cells were prepared and klotho expression quantified by western blot (WB). To determine the effects of estrogen in vivo, we used wild type (WT), aromatase deficient mice (ArKO), and ArKO mice treated with estradiol (20ug/mouse 3x/week) or vehicle for 3 weeks. RNA and protein were prepared from the kidneys for real time PCR and WB analysis, respectively. MDCK cells grown in charcoal stripped FBS (csFBS) showed higher klotho protein expression than cells grown in 10%FBS. However, klotho expression in cells grown in 10% csFBS supplemented with 10(-5)M and 10(-8)M estradiol was decreased to the same level as cells grown in 10%FBS. Kidneys from ArKO mice showed significantly higher expression of klotho, both at the mRNA and protein levels in ArKO animals compared to WT and estrogen treated ArKO mice. Similarly, protein extracts from estrogen receptor alpha knockout mice kidneys showed higher klotho levels than WT extracts. Based on these observations we conclude that estrogen, acting through ERalpha, down-regulates the klotho protein in the mouse kidney.

Disclosures: O.K. Oz, None.

T193

High Dose Estrodial Treatment Causes Destruction of Cortical Bone and Is Associated with an Increased Death Rate in Old Wild Type and ERKO Male Mice. Z. Peng1, P. Härkönen*2, K. Väänänen*3, 1Pharmatest Services Ltd, Turku, Finland, 2Department of Laboratory Medicine, Tumor Biology, Lund University, Malmö, Sweden, 3Department of Anatomy, University of Turku, Turku, Finland.

Estrogen plays an important role in maintaining bone mass in males as well as in females. The purpose of this study was to learn how estradiol and testosterone treatments influence bone metabolism in very old ERKO male mice. Sham-operated or orchidectomized (ORC) 10-25 mo old ERKO mice and their wild type (WT) littermates were implanted with a pellet of testosterone (Te) (0.21 mg/mouse/day), or estradiol (E2) (3μg/mouse/day or 12μg/mouse/day) and sacrificed after 4 weeks. Bone analysis of the animals showed that ORC decreased the tibial and femoral ash weight of WT mice, which was reversed by Te treatment. The lower dose E2 treatment increased bone ash weight, TBV, BMD and bending strength of both tibia and femur in wild type but not in ERKO mice. In contrast, a high dose E2 treatment caused a very high bone turnover rate with enhanced bone formation and an increased number of osteoclasts both in WT and in ERKO mice. TBV was clearly elevated but cortical bone revealed local areas with enhanced bone resorption leading to foci with an almost complete local loss of cortical structures. The mechanical strength of cortical bone was clearly decreased due to the local destruction of cortical bone. T treatment of ORC animals reversed the ORC-induced bone changes both in WT and ERKO mice. However, in intact old animals T treatment did not significantly improve measured bone parameters. Generally, all sham-operated animals, both WT and ERKO, survived the whole 4-week experiment whereas 4 out of 22 ORC WT mice (19.2%) and 16 out of 21 ORC ERKO mice (76.2%) died, and most of them were at 5 to 19 days after operation. However, in Te-treated ORC mouse groups, only 3 out of 21 WT (14,3%) and 2 out of 15 ERKO (13,3%) died. Both low and high dose E2 treatment of ORC WT and ORC ERKO male mice was associated with an increased death rate. Only 3 out of 39 WT (7.7%) and 5 out of 35 ERKO (14%) survived in E2-treated groups until the end of the study. Similarly, in E2-treated intact old WT and ERKO mice groups 5/14 and 4/16 animals died during the experiment, respectively. No deaths occurred in the control or Te-treated groups during the same time period. In conclusion, E2 treatment was associated with an increased death rate both in intact and ORC old WT and ERKO mice. ORC also increased death rate in old ERKO males which could be decreased by Te treatment of ORC mice. The reason for increased, delayed deaths after ORC or E2 treatments remains currently unknown since the routine histological examination of major organs did not reveal any obvious cause of death.

Disclosures: Z. Peng, None.

T194

Activation of MAPKs by 17 beta-Estradiol Is Mediated by PKC and c-Src in Skeletal Muscle Cells. A. C. Ronda*1, C. Buitrago*1, E. Roldan2, R. Boland1, 1Biologia, Bioquimica & Farmacia, Universidad Nacional del Sur, Bahia Blanca, Argentina, 2Gador S.A., Buenos Aires, Argentina.

The classical mechanism of action of 17β-estradiol (E2) involves its binding to the intracellular estrogen receptors (ER) α or β. These ligand-activated receptors stimulate mRNA synthesis and de-novo protein expression, for hours to days. Additionally, E2 is also known to exert rapid non-genomic effects on target tissues. Some of these effects involve activation of intracellular signalling pathways. We have previously shown in C2C12 skeletal muscle cells that 10−8M E2 stimulates ERK1/2 and p38 MAPK at 15 min and that the hormone promotes phosporylation of CREB and Elk-1 transcription factors in an ERK1/2 and p38-dependent manner. In the present work, we demonstrate that E2 activates c-Src in C2C12 cells within the same time interval as ERK1/2 and p38 MAPK phosphorylation. E2-induced ERK1/2 and p38 activation was abolished by the c-Src specific inhibitor PP2, involving c-Src in hormone stimulation of MAPKs. Treatment of the muscle cells with the specific PKC inhibitor Ro318220 demonstrated that activation of ERK 1/2 and p38 in response to the estrogen is also mediated by PKC. Moreover, E2 modulates Src activation in a PKC-dependent manner, possibly through a Src protein tyrosine phosphatase. The data altogether suggest that the steroid hormone 17β-estradiol triggers upstream at PKC/Src the signalling MAPK cascades leading then to phosphorylation of CREB and Elk-1 transcription factors in the C2C12 skeletal muscle line.

Disclosures: A.C. Ronda, None.

T195

Estrogen Preferentially Suppresses Osteogenic Gene Expression in Mice Lacking Classical ERE Signaling. V. Rudnik*, F. A. Syed, D. G. Fraser*, D. G. Monroe, S. Khosla. Mayo Clinic, Rochester, MN, USA.

Studies with female mice in whom the only functional estrogen receptor α (ERα) is one which cannot bind DNA (ERα-/NERKI) have shown that these mice gained cortical bone mass following ovariectomy (ovx), and E dose-dependently suppressed this increase; these changes were the opposite of those seen in wild type (WT) mice. These paradoxical effects of ovx and E on cortical bone in ERα-/NERKI mice could be due to an alteration of the balance between classical (through EREs) and non-classical (through protein-protein interactions) signaling of ERα in osteoblastic cells. To identify the potential signaling pathways involved in these effects of the NERKI receptor, 3 month old female WT and ERα-/NERKI mice were ovx'd or sham operated (n = 3 per group) for 1 week and then euthanized and the femurs extracted. The metaphyses were removed and bone marrow was flushed in order to isolate RNA purely from cortical bone. Following synthesis of cDNA, QPCR arrays were performed using mouse osteogenesis platforms (SuperArray). Fold differences (following normalization to 5 different housekeeping genes) were compared within the WT and ERα-/NERKI groups between E+ (sham) and ovx (E-) animals. Of particular interest, mRNA levels of TWIST1, which is a known inhibitor of osteoblast differentiation and of runx2 activity, were lower in the cortical bones of E+ as compared to E- WT and ERα-/NERKI mice (by 2 fold for both, P = 0.06 and 0.006, respectively). By contrast, mRNA levels for BMP1, BMP3, and LEF1 (a downstream target gene in the Wnt signaling pathway) were similar in the bones of the E+ and E- WT mice, but were lower (by 2, 2, and 4-fold, respectively, all P < 0.05) in the bones of the E+ as compared to the E-ERα-/NERKI mice. These in vivo data thus demonstrate that (I) TWIST 1 may be a novel E-regulated target gene and suggest that suppression of TWIST 1 may play a role in the stimulation of osteoblast differentiation by E; and (2) suppression by E of BMP1, BMP3, and Wnt signaling in ERα-/NERKI mice may explain their paradoxical cortical bone response (i.e., gain in bone mass) following ovx.

Disclosures: V. Rudnik, None.

T196

17beta-Estradiol Abrogates Apoptosis in Skeletal Muscle Cells Through Extra-Nuclear Estrogen Receptors. A. Vasconsuelo*, L. Milanesi*, A. Russo de Boland*, R. Boland. Biologia, Bioquimica & Farmacia, Universidad Nacional del Sur, Bahia Blanca, Argentina.

Although there is evidence showing that estrogens regulate apoptosis in various cellular systems, the underlying molecular mechanism is not well understood. The present study demonstrates that 17β-estradiol, at physiological concentrations, abrogates apoptosis in mouse skeletal muscle C2CI2 cells through estrogen receptors (ERs) with non-classical localization. Specific antibodies against ER α or β and silencing of ERs with ER α and β short interference RNAs (siRNAs) inhibited this protective action, which involved PI3K/ Akt activation and BAD phosphorylation. Apoptosis inhibition by 17β-estradiol was stronger when ER β was blocked, suggesting that the β isoform mediates to a greater extent than the α isoform the antiapoptotic effects of the steroid hormone. Expression and subcellular distribution of ER α and β in extra nuclear compartments (mitochondria, endoplasmic reticulum and Golgi) of C2C12 cells were confirmed by competitive binding assays, conventional and confocal fluorescence microscopy, silencing of ERs with siRNAs and RT-PCR using specific primers.

These results indicate that 17β-estradiol exerts antiapoptotic effects in skeletal muscle cells mediated by estrogen receptors with extra nuclear localization.

Disclosures: A. Russo de Boland, None.

T197

Bone Formation Is Predicted by Resting Metabolic Rate and Leptin in Exercising Women with Hypothalamic Amenorrhea. J. L. Scheid*, S. L. West*, J. D. Vescovi, S. Awdishu*, M. J. De Souza. Exercise Science, University of Toronto, Toronto, ON, Canada.

The purpose of this study was to assess metabolic factors that impact bone formation and bone resorption in exercising women with hypothalamic amenorrhea. For this observational study, subjects were divided according to menstrual status into one of three groups: 1) Ovulatory (OV, n = 21), 2) Anovulatory (ANOV, n = 9), 3) Amenorrheic (AMEN, n = 21). Menstrual status was assessed by daily urinary ovarian steroid measurements for 2-3 menstrual cycles, or 30-day monitoring periods if AMEN. Serum was analysed for type I procollagen amino-terminal propeptide (PINP), triiodothyronine (TT3), ghrelin, peptide YY (PYY), leptin, and 24 hour urinary samples for C-terminal telopeptide (CTX). Resting metabolic rate (RMR) was assessed by indirect calorimetry and bone density by dual-energy x-ray absorptiometry. As an indicator of energy deficiency, a measured RMR:Predicted RMR (pRMR) was defined as less than 0.90. All groups were similar (p>0.05) with respect to age (23.3±0.5yr), height (1.65±3.22m), weight (58.0±1.0kg), and BMI (21.2±0.3kg/m2). As expected, Z-scores were lower (p = 0.002) in the AMEN (-0.614±0.175) compared to both the OV (0.747±0.352) and ANOV (0.500±0.389) groups at the lumbar spine L2-L4. RMR was suppressed (p = 0.023; 28.7±0.7, 31.4±0.8, and 31.1±0.8kcal/day*kg FFM) in the AMEN compared to the OV and ANOV groups, respectively. RMR:pRMR was below 0.90 in the AMEN group, indicative of energy deficiency. Suppressed TT3 (p = 0.030), elevated ghrelin (p = 0.038) and elevated PYY concentrations (p = 0.039) were observed in the AMEN compared to the OV group, but similar to that observed in the ANOV group. Leptin concentrations were similar (p = 0.667) among groups (4.83±0.45ng/ml). PINP was similar (p = 0.440) among groups (121.5±7.8 ug/L), while CTX was increased (p = 0.034) in the AMEN (291.1±39.7ng/ml) compared to the OV (174.8±18.7ng/ml) group, and similar to that observed in the ANOV (224.2±31.4ng/ml) group. Variables in a regression model predicting PINP included RMR:pRMR and leptin which accounted for 28.6% (R2 = 0.327, p = 0.001) of the variance in PINP. However, in the AMEN group, 55.8% (R2 = 0.626, p = 0.004) of the variance in PINP was accounted for by RMR:pRMR and leptin. Variables in a model predicting CTX included the duration of amenorrhea, leptin and PYY concentrations and accounted for 39.9% (R2 = 0.454, p<0.001) of the variance in CTX. Exercising women with hypothalamic amenorrhea are energy deficient, as depicted by suppressed RMR, RMR:pRMR, TT3, elevated ghrelin, and PYY levels compared to their ovulatory counterparts, and their bone marker profile is predicted by their energy status.

Disclosures: J.L. Scheid, None.

T198

The Role of Androgen Receptor in the Lineage Commitment of Bone Marrow Stromal Cells. C. Shyr1, T. Meng-Yin*2, H. Ko-En*2, C. Chang*3, 1Chinese Medicine, Chang-Gung University, Niao-Sung, Taiwan, 2Obstetrics and Gynaecology, Chang-Gung Hospital, Niao-Sung, Taiwan, 3Pathology, University of Rochester, Rochester, NY, USA.

Androgen receptor (AR) mediates androgen action to affect bone, muscle and fat tissue metabolism through controlling cellular events like differentiation process. However, the molecular control by AR on the making of specialized cells such as osteocytes and adipocytes from progenitor cells and more primitive stem cell remain unclear. To determine the role of AR in stem cell fate decision and differentiation, we conducted experiments on bone marrow stromal cells (BMSC) obtained from wild type (WT) and AR knockout (KO) mice, which contain mensenchymal stem cells with pluripotency to give rise all types of cells. We found that ARKO mice had higher numbers of colony formation unit-fibroblast, (cfu-f) and colony formation unit-osteoblast, (cfu-o) than WT mice did. Gene expression profile determined by oligoarray chips revealed that the genes related to osteogenic differentiation decreased, but genes involved in adipogenic differentiation increased in ARKO mice compared to those in wt mice. The Q-RT-PCR results verified the findings obtained from microarray analysis with lower expression of genes linked to osteogenesis and higher expression of adipogensis-related genes in ARKO mice. Furthermore, cell surface epitope analysis by flow assay demonstrated that ARKO BMSCs exhibited different characteristics to WT BMSCs.

The study on ARKO mice is a direct and evident approach to investigate the role of AR in stem cell fate determination and lineage commitment. Our findings in this study imply that AR may accelerate the use of stem cells from their pool and direct them into differentiation because the loss of AR caused the increased progenitor cell numbers. For stem cell differentiation, we suggest that AR plays a role in promoting the stem cell differentiation decision for osteogenesis other than adipogenesis based on the gene expression profile. By understanding the role of AR on adult stem cell biology, we can find a precise and efficient way to control stem cell self-renewal and differentiation, resulting in a therapuetic application in treating illnesses such osteoporosis and obesity through the regulatory role of AR on stem cells.

Disclosures: C. Shyr, None.

T199

The UGT2B7 H268Y Polymorphism Is Associated with Serum Sex Steroid Levels and Cortical Bone Size in Young Adult Men. C. Swanson1, M. Lorentzon1, L. Vandenput1, F. Labrie*2, A. Rane*3, J. Jakobsson*3, S. Chouinard*2, A. Belanger*2, C. Ohlsson1, 1Center for Bone Research at the Sahlgrenska Academy, Department of Internal Medicine, Gothenburg University, Göteborg, Sweden, 2Laboratory of Molecular Endocrinology and Oncology, Laval University Hospital Research Center and Laval University, Quebec, PQ, Canada, 3Department of Laboratory Medicine, Division of Clinical Pharmacology, Karolinska Institutet at Karolinska University Hospital, Stockholm, Sweden.

Androgens stimulate periosteal bone expansion in males during sexual maturation and the androgen receptor (AR) is required for this effect. The bio-active androgens testosterone (T) and dihydrotestosterone (DHT) can be inactivated directly by conjugation with glucuronic acid. An alternative pathway is the transformation of DHT in two main metabolites, namely androstane-3α,17β-diol (3α-diol) and androsterone (ADT). Conjugation of 3α-diol and ADT with polar cofactors, such as glucuronic acid, is an irreversible step, abolishing its affinity for the AR. Conjugation of androgens with glucuronic acid has been suggested to play a role in the regulation of the intracellular levels of unconjugated steroids as well as their biological activities in tissues. It is now well established that UDP glucuronosyltransferase (UGT) 2B7 is one of three major enzymes responsible for the glucuronidation of all androgens and their metabolites in humans. The in vivo role of UGT2B7 for the regulation of circulating, as well as local, levels of androgens, estrogens and their metabolites is unknown. A H268Y polymorphism has been described in the UGT2B7 gene.

Our aim was to determine the impact of the UGT2B7 H268Y polymorphism on serum levels of sex steroids and cortical bone dimensions in the population-based GOOD-study, which includes 1068 young adult men at the age of peak bone mass.

Serum levels (measured by GC-MS) of T (YY 9% over HH, p<0.01), DHT (YY 10% over HH, p<0.01) and estradiol (YY 8% over HH, p<0.01) were associated with the UGT2B7 H268Y polymorphism (genotyping performed by Real-Time PCR). The polymorphism was associated with the metabolites (measured by LC-MS/MS) 3α-diol-17-glucuronide and 3α-diol-3-glucuronide (p<0.01) but not with ADT-glucuronide levels in serum. In addition, the UGT2B7 H268Y polymorphism was an independent predictor of cortical bone size (measured by pQCT) as reflected by periosteal circumference and cortical moment of inertia (p<0.01) in both the weight bearing tibia and the non-weight bearing radius.

In conclusion, the UGT2B7 H268Y polymorphism is independently associated with cortical bone size and serum sex steroid levels in young adult men. Subjects homozygous for the Y-allele had higher serum T and larger cortical bone size than subjects homozygous for the H-allele.

Disclosures: C. Swanson, None.

T200

Increased Caloric Intake Is Associated with Reversal of Amenorrhea and Favorable Changes in Metabolic and Bone Markers: A Case Study Report. S. L. West*1, J. L. Scheid*1, N. I. Williams2, J. D. Vescovi1, S. A. Jamal1, G. A. Hawker1, S. Awdishu*1, M. J. De Souza1, 1University of Toronto, Toronto, ON, Canada, 2Penn State University, State College, PA, USA.

Physically active women can develop exercise associated menstrual cycle disturbances (EAMD) such as amenorrhea secondary to a chronic energy deficiency. EAMD is frequently associated with low bone mass and stress fractures. Here we report data from an ongoing 12 month randomized controlled trial to determine if increased caloric intake would reverse amenorrhea, and improve energy and bone marker status. Subjects were categorized by ovulatory status into either an ovulatory control or EAMD group. After a one month run in period subjects in the EAMD group were randomly assigned to an EAMD control or EAMD+calories group that was required to increase daily caloric intake (20-30%) above their baseline energy needs. Resting energy expenditure (REE), dietary intake, daily energy expenditure, body composition (by DXA), serum markers of bone formation (PINP) and resorption (CTX), metabolic status (ghrelin and triiodothyronine [TT3]), and daily urinary ovarian steroids (EIG and PdG) were assessed over the study period. Presented is a case study of one participant randomized to the EAMD+calories group who has completed 6 months of the study protocol. The 24 year old subject had been amenorrheic for 2 years. She participated in over 300 minutes of exercise per week (primarily dance and aerobic exercise), and had a V02 max of 43.5 ml/kg/min. At study entry she weighed 54.5 kg, her BMI was 19.8 kg/m2 and she had 20% body fat. REE at baseline was 1171 kcal/day and represented 87% of predicted REE, indicative of an energy deficiency. Supplemental caloric intake averaged 300 kcal/day over the study period. After 6 months of increased calories the subject reported resumption of menses, confirmed by daily ovarian steroid profile (EIG cycle area under the curve increased 79.6% from 435.6 to 782.4). There was a 1.9 kg increase in body mass, a 0.7 kg/m2 increase in BMI, and endocrine alterations indicative of a move toward energy surplus including a 5.0% increase in REE, a 2.0% increase in TT3, and a 1.7% decrease in ghrelin. PINP increased 26.4% from 73.89 to 93.39 ug/L, while CTX remained unchanged (0.64 ng/ml vs. 0.69 ng/ml). Our data suggest that, in this subject, increased caloric intake results in resumption of menses, as well as more favourable metabolic, energy, and bone marker profiles.

Disclosures: S.L. West, None.

This study received funding from: United States Army Medical Research and Matreial Command Peer Reviewed Medical Research Program (Award Number: PR054531).

T201

Deficiency of the G protein-coupled Estrogen Receptor GPR30 Leads to Reduced Bone Growth and Bone Mineral Density in Female Mice. S. H. Windahl1, N. Andersson1, U. E. A. Mårtensson*2, C. Owman*2, C. R. Rosen3, M. L. Adamo*4, B. Olde*2, F. Leeb-Lundberg*2, C. Ohlsson1, 1Center for Bone Research at the Sahlgrenska Academy, Gothenburg, Sweden, 2Unit of Drug Target Discovery at Lund University, Lund, Sweden, 3The Jackson Laboratory, Bar Harbor, ME, USA, 44. Department of Biochemistry, The University of Texas Health Science Center, San Antonio, TX, USA.

Estrogens are important regulators of bone growth and adult bone metabolism. There are two known nuclear receptors for estrogen (ERα and ERβ). ERα is the most important nuclear ER for bone in both males and females while the role of ERβ mainly is to modulate ERα activity in female but not male mice. Recent in vitro studies have suggested that the G-protein-coupled receptor GPR30 is a functional ER but the impact of GPR30 in vivo is unknown. The aim of the present study was to investigate the in vivo role of GPR30 for the skeleton and for reproductive tissues. We therefore developed a mouse model in which the GPR30 gene locus was disrupted. GPR30−/− mice are viable and serum levels of estradiol and testosterone as well as the uterine weight were unchanged in GPR30−/− mice compared with wild type mice. Interestingly, after sexual maturation, the female GPR30−/− mice displayed a reduced body weight (22.6±0.4g, n = 25) compared to WT mice (24.7±0.6g, n = 26; p<0.01). The reduced body weight in female GPR30−/− mice was associated with a reduced skeletal growth, including both the axial skeleton (crown-rump length, p< 0.05) and the appendicular skeleton (femur length, p< 0.05). The magnitude of the skeletal growth disturbance was similar in the appendicular and the axial skeleton, indicating that it was a proportional skeletal growth disturbance. In contrast, male GPR30−/− mice displayed a normal skeletal growth. Because the growth disturbance became significant after sexual maturation and only was seen in female mice, one may speculate that sex steroid-dependent pubertal growth is affected in female GPR30−/− mice. The GH/IGF-I axis is a major determinant of skeletal growth. Serum IGF-I levels were reduced in female (-11%, p < 0.05) but not male GPR30−/− mice, indicating that reduced serum IGF-I levels might be involved in the growth disturbance seen in female GPR30−/− mice. Initial screening of BMD using total body DXA showed that the female but not the male GPR30−/− mice displayed reduced BMD (-3%, p < 0.05) compared with wild type mice.

In conclusion, deficiency of the proposed novel ER GPR30 results in reduced bone growth and total body BMD associated with reduced serum IGF-I levels in female mice.

Disclosures: S.H. Windahl, None.

T202

DHT Treatment Reverses Gonadectomy-induced Changes in Fat and Lean Mass in Male but Not Female Mice. A. A. Semirale, X. Zhang, K. M. Wiren. VA Medical Center, Oregon Health & Science Univ, Portland, OR, USA.

Androgens have pervasive effects on target tissues including muscle, fat and bone. To characterize the role of androgen receptor (AR) signaling on body composition, an experimental paradigm of protracted hormone ablation followed by steroid replacement was employed. B6D2F2 control mice were sham operated or gonadectomized at 3 months of age. The effect of nonaromatizable dihydrotestosterone (DHT) was determined after an 8 week delay, to provide time for gonadectomy-induced changes in body composition to develop before intervention. The effect of androgen was assessed by dual energy x-ray absorptiometry (DXA) following 6 weeks of treatment with either DHT or placebo pellets. In control males (n = 8-11), lean mass as a % of body weight was significantly reduced (7.7 %, p < 0.001) following orchiectomy (ORX) while conversely, % fat mass increased (7.6 %, p < 0.001) vs. sham controls. DHT treatment was beneficial, improving or fully restoring body composition changes induced by ORX compared to placebo. In females (n = 4-10) a similar trend was observed with increased fat (2.2 %) and reduced lean mass (2.3 %) after ovariectomy (OVX). Surprisingly, females were resistant to the effects of DHT to restore body composition. To determine whether changes in bone metabolism contribute to body composition differences, transgenic mice with skeletally-targeted AR overexpression in mature osteoblasts (driven by the 2.3 kb fragment of type I αl collagen promoter) were characterized. AR2.3-tg mice demonstrated changes in body composition after gonadectomy that mirrored wild types (n = 3-9). Again similar to wild-type controls, DHT improved ORX-induced alterations in % fat and % lean mass in AR2.3-tg males but not females. In contrast to gonadectomized models, DHT treatment in intact wild type mice had a negative impact on body composition in both males and females. Males (n = 14-23) lost 4.4 % lean mass (p < 0.05) and increased fat mass by 4.3 % (p < 0.05) compared to placebo, with similar but less dramatic changes observed in females (n = 14-26). These results show both males and females increase fat and lose lean mass following gonadectomy. In the bone-targeted AR-overexpression model, enhanced androgen signaling does not influence the response to DHT suggesting that circulating factors derived from bone likely do not play a role in the body composition response to androgen therapy. Combined, these data indicate that systemic DHT positively influences body composition changes after a prolonged hypogonadal state in males but is ineffective in females over the same time frame. Furthermore, DHT treatment in the intact animal results in detrimental changes in body composition and should be approached with caution.

Disclosures: K.M. Wiren, None.

This study received funding from: DOD and NIH/NIDDK.

T203

Dissection of Androgen Receptor Signaling: Reconsideration of Direct Anabolic Action in Mature Bone. X. Zhang, A. A. Semirale, K. M. Wiren. VA Medical Center, Oregon Health & Science Univ, Portland, OR, USA.

The effects of androgen on bone remain poorly characterized and understudied. To develop insight into the cell types important in mediating androgen action, we constructed and compared two distinct transgenic lines employing different αl(I)-collagen promoter fragments to control skeletally-targeted AR overexpression. The col 3.6 AR-transgenic (AR3.6-tg) mice demonstrate AR overexpression throughout the osteoblast lineage including the periosteum, while col2.3 (AR2.3-tg) mice have more restricted overexpression in mature osteoblasts and osteocytes. Complex skeletal analysis using morphological characterization by μCT, dynamic and static histomorphometric analysis, dual-energy x-ray absorptiometry (DXA), biomechanical testing and gene expression studies all indicate that androgen signaling in mature osteoblasts during growth produces a low turnover state, and the consequences are detrimental to overall matrix quality and bone strength. To determine the consequences of androgen treatment in the adult and whether nonaromatizble dihydrotestosterone (DHT) may be effective for treatment of post-menopausal bone loss in an animal model, 3 month old male and female B6D2F2 control mice were gonadectomized. Six weeks of treatment with DHT or placebo pellets was delayed until age 5 months to allow rapid bone turnover to stabilize, thus minimizing the impact of anti-resorptive effects of androgen. DXA demonstrated that systemic DHT administration significantly increased BMD and BMC in both sexes, to reverse loss sustained after a prolonged hypogonadal state (n = 14-26). To test the consequences of enhanced androgen signaling targeted to bone in the adult, male and female AR3.6-tg and AR2.3-tg mice were treated in the same manner. In both AR-tg families and in contrast to wild-type mice, DHT replacement did not improve either measure versus placebo pellet (n = 3-14). Taken together, these results indicate that improvements in bone mass with androgen treatment after gonadecomy in wild-type mice are likely mediated through effects on extra-skeletal tissues, not osteoblasts. Consistent with detrimental effects of enhanced androgen signaling on bone quality, DHT treatment in intact controls significantly reduced bone mass in both males and females. These findings demonstrate that after a sustained hypogonadal period, androgen effectively treats bone loss but that enhanced androgen signaling directly in bone is not anabolic in either male or female adults. The data suggests that targeting androgen response to mature osteoblasts is not beneficial for bone formation, and raise concerns regarding androgen administration or anabolic steroid abuse in healthy individuals in both sexes.

Disclosures: K.M. Wiren, None.

This study received funding from: DOD and NIH/NIDDK.

T204

Genetic Variation in Post-Natal Skeletal Growth Defines Functional Interactions among Adult Bone Traits and Fragility. K. J. Jepsen1, B. Hu*1, M. A. Cordova*1, S. M. Tommasini2, C. Price1, H. W. Courtland1, J. H. Nadeau*3, 1Mt Sinai School of Medicine, NY, NY, USA, 2CUNY Graduate Center, NY, NY, USA, 3Case Western Reserve University, Cleveland, OH, USA.

Prior work revealed that several adult bone traits are functionally related, such that the small cross-sectional size of slender bones was compensated by increased cortical thickness and tissue-mineral density. The set of traits for slender bones, although sufficiently stiff for daily activities, performed poorly under extreme load conditions. Because slenderness is a key determinant of fracture risk, identifying the biological controls responsible for functional interactions among adult traits should advance our understanding of how genetic background influences fracture risk. Femora from 20 female AXB/BXA Recombinant Inbred (RI) mouse strains were analyzed to test the hypothesis that functional interactions among adult traits are determined during early post-natal growth. Femoral mid-diaphyseal traits analyzed at 1 and 28 days of age were compared to adult traits measured at 112 days. Z-transformed data were analyzed using Path Analysis, which uses conditional covariances to establish causal relationships among multiple traits. Path Analysis (Fig 1) revealed a significant correlation between the rate of increase in total area (periosteal expansion) and marrow area (endosteal expansion) from 1 to 28 days, indicating that expansion of the periosteal surface relative to the endosteal surface was biologically controlled. Further, the rate of increase in total area (periosteal expansion) showed a net negative relationship with adult TMD and CtTh, such that variable post-natal periosteal expansion rates defined the degree of matrix mineralization and the relative rate of marrow expansion (and thus cortical thickness). The functional interactions between post-natal surface expansions and the set of adult traits indicated there are adaptive processes, superimposed on allelic variation, that co-adapt traits to match loading demands. These adaptive processes appear to accommodate allelic variation affecting periosteal expansion (i.e., slenderness) by allowing mechanically functional structures to be constructed in different ways. This study provided important new evidence that the set of adult traits which define bone strength and fragility are determined in large part by genetic factors affecting post-natal skeletal growth patterns. 

Figure Figure 1..

Path Analysis showing causal (straight arrows) and non-causal (curved arrow) relationships among post-natal body weight gain (ΔBW1-28), periosteal expansion (ΔTtAr1-28), marrow expansion (ΔMaAr1-28), and adult tissue-mineral density (TMD112) and cortical thickness (CtTh112). Path coefficients calculated based on Z-transformed data.

Disclosures: K.J. Jepsen, None.

This study received funding from: N1H, DOD.

T205

Cells Cultured from Bone Lesions of Patients with Paget's Disease Show No Evidence of Measles Virus RNA or Somatic Mutations in SQSTM1. B. G. Matthews*1, U. Bava*1, K. Callon*1, M. A. Afzal*2, J. Cornish1, I. R. Reid1, D. Naot1, 1Medicine, University of Auckland, Auckland, New Zealand, 2Division of Virology, National Institute for Biological Standards & Control, Potters Bar, United Kingdom.

Paget's disease is a focal condition of bone of uncertain etiology. The disease appears to be associated with a combination of genetic and environmental factors. SQSTM1 mutations have been identified in familial Paget's and in a minority of sporadic cases, and several groups have suggested long-term infection with a paramyxovirus is associated with the disease. However, the mechanisms for the focal nature of Paget's disease are not understood. In this study, we used cells obtained from pagetic bone lesions to investigate local changes that could possibly account for the characteristic focal pathology.

We have collected RNA from cultured osteoblasts and bone marrow samples taken from both pagetic lesions and unaffected bone from a group of 22 patients with Paget's disease, and from control patients. Differential gene expression, analyzed by real-time PCR, identified several changes in these samples, including increased Dkkl and IL-6 and a reduced RANKL/OPG ratio in pagetic cells compared with controls.

Somatic mutations within the pagetic lesion have been suggested to be responsible for the focal nature of the disease. In order to investigate the possibility of somatic SQSTM1 mutations, exons 7 and 8 of the SQSTM1 cDNA were sequenced in all the patients with Paget's disease, in some cases using samples from both pagetic and unaffected tissue. The wild-type sequence was found in all but one patient, who was heterozygous for the P392L mutation. DNA from peripheral blood in this subject had an identical sequence of SQSTM1, indicating that this was a germ-line mutation. We conclude that somatic mutations for SQSTM1 are not commonly present in Paget's disease.

The RNA was also used to look for evidence of measles virus involvement in Paget's disease. Nested RT-PCR analysis at the National Institute for Biological Standards and Control in England did not detect measles virus nucleocapsid or matrix genes in any of the Pagetic or control samples. This suggests that long term measles virus infection is not commonly present in New Zealand patients with Paget's disease.

The results indicate that pagetic lesions are different from normal bone in terms of gene expression, but do not commonly contain somatic SQSTM1 mutations or measles virus infection.

Disclosures: B.G. Matthews, None.

This study received funding from: Health Research Council of New Zealand.

T206

A Mouse Model for Diabetes-mediated Osteoporosis. Z. Wang*1, K. Ding*2, M. W. Hamrick3, H. He*1, S. Yan*4, L. Zhou*1, C. M. Isales5, O. Mi1, 1Center for Biotechnology and Genomic Medicine/Dept. of Pathology, Medical College of Georgia, Augusta, GA, USA, 2Dept. of Medicine, Medical College of Georgia, Augusta, GA, USA, 3Dept. of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, GA, USA, 4Dept. of Endocrinology, The Medical School Hospital of Qingdao University, Qingdao, China, 5Dept. of Orthopedic Surgery, Medical College of Georgia, Augusta, GA, USA.

Decreased bone mass, osteoporosis, and increased fracture rates are common skeletal complications in patients with type 1 diabetes (T1D). There is a considerable amount of clinical data to link osteoporosis with T1D. However, very little basic research has delved into this phenomenon, and few have directly addressed the influence of diabetes on the skeletal system of mice, an animal model that is amendable to genetic manipulation. Recently, a streptozotocin (STZ)-induced T1D mouse model was used to examine the influence of T1D on bone. However, STZ-induced diabetes causes some diversity among individual animals in terms of the extent of severity and the onset of diabetes. In addition, STZ may directly affect bone function. Here, we employed a novel genetic approach by which T1D would be most stably induced in RIP-iNOStg mice as early as 1 week after birth due to the iNOS-mediated selective destruction of insulin-producing pancreatic beta cells. Pixus densitometric analysis showed a significant decrease in bone mineral density of whole bones in 3-month-old male RIP-iNOStg mice compared with that of age- and strain-matched wild-type mice. The radiographic analysis also showed a significant lower bone mass in RIP-iNOStg mice. However, the body weight was invariant between the two groups during the 3-month observation period. Our study demonstrated that diabetic RIP-iNOStg mice can spontaneously develop osteoporosis, and could be severed as a novel mouse model for studying T1D-related osteoporosis.

Disclosures: Q. Mi, None.

This study received funding from: Juvenile Diabetes Research Foundation International (19-2006-1065) to W. Mi.

T207

GLP-1 Action on Bone Turnover in Normal and Type 2 Diabetic State. B. Nuche-Berenguer*1, V. Sancho*1, J. Cancelas*1, P. Lozano*2, P. Esbrit2, M. L. Villanueva-Peñacarrillo*1, 1Metabolism, Nutrition & Hormones, Fundación Jiménez Díaz, Madrid, Spain, 2Laboratory of Bone & Mineral Metabolism, Fundación Jiménez Díaz, Madrid, Spain.

An insulin-independent antidiabetic action of GLP-1 (glucagon-like peptide 1) is widely documented, as well as its insulin-like effect upon glucose metabolism in liver, muscle and fat, and neurotrophic and anoretic properties. It was suggested that incretins could participate in bone turnover changes occurring after absorption of nutrients. We have explored the possible in vivo effect of GLP-1 on bone remodeling in normal and diabetic state. Type 2 diabetic model was developed by streptozotocin injection in neonatal Wistar rats (STZ-T2D). Adult rats, normal (n = 6) and STZ-T2D (n = 6), were treated −3 days through an osmotic pump- with GLP-1 (0.86 nmol/kg/h). In fed conditions, plasma samples were taken before (basal) and by the end of the treatment, when also tibias were collected. MC3T3-E1 osteoblasts were incubated for 24 hours in α-MEM with 5 or 25mM D-glucose (or mannitol), and in absence and presence of 10−9M GLP-1. Measurements: in plasma, osteocalcin (OC), tartrate-resistant acid phosphatase (TRAP) -ELISA-, insulin - RIA- and glucose; in bone and cells, gene expression of OC, osteoprotegerin (OPG) and RANK ligand (RANKL) -RT-PCR-. Untreated rats of both groups (n = 6, each) were included as respective controls. In plasma: GLP-1 did not modify normal basal OC (440.5±6.0 ng/ml), while in STZ-T2D group, initially showing an apparently lower than normal level, GLP-1 induced a reduction (-10.9±0.9%Δ of basal, p<0.001); TRAP was slightly increased by GLP-1 in normal (2.87±0.24 U/I vs basal 2.64±0.27) and also in STZ-T2D (2.88±0.29 U/l vs basal 2.45±0.27); glucose and insulin were not significantly affected in either group. In bone: OC mRNA level in STZ-T2D was 1.65±0.26 times that of normal rats; treatment with GLP-1 induced an increase in normal (2.35±0.50 times control) and also in STZ-T2D (1.99±0.44 times control); OPG mRNA was higher in STZ-T2D (4.87±0.30 times that of normal), but while GLP-1 raised the value in normal (2.73±0.30 times control, p<0.005), it reduced that in STZ-T2D (0.44±0.05 times control); RANKL mRNA was slightly higher in STZ-T2D (1.63±0.19 times that of normal), and was similarly increased by GLP-1 in both groups (normal: 1.97±0.29 times control; STZ-T2D: 1.38±0.13 times control). In control STZ-T2D, RANKL/OPG mRNA ratio was 0.326, and GLP-1 treatment raised it to 2.98, similar to the 2.20 value obtained in MC3T3-E1 cells incubated with GLP-1 and high glucose. In conclusion, these novel findings show that GLP-1 directly modulates bone turnover markers in both normal and type 2 diabetic rats, and suggest that this hormone might favor bone resorption.

Disclosures: B. Nuche-Berenguer, None.

This study received funding from: Research Grant from Ministerio de Sanidad y Consumo (FIS:06/0076).

T208

Investigating the Role of Mecp2 in the Epigenetic Regulation of Bone Mineral Density. R. D. O'Connor1, A. L. Ham*1, A. M. Wolff*1, M. Zayzafoon2, N. C. Schanen3, M. C. Farach-Carson1, 1Department of Biological Sciences, University of Delaware, Newark, DE, USA, 2Department of Pathology, University of Alabama at Birmingham, Birmingham, AL, USA, 3Laboratory for Human Genetics, Nemours/AI duPont Hospital for Children, Wilmington, DE, USA.

Rett Syndrome (RTT), a neurodevelopmental disorder, is most often caused by inactivating mutations in the X-linked gene encoding a regulator of epigenetic gene expression, methyl CpG binding protein, MeCP2. Clinical data show that, along with neurological defects, females with RTT frequently have marked decrease in Bone Mineral Density (BMD) beyond that expected from disuse atrophy. Preliminary studies with a Mecp2 knock-out mouse model reveal a difference between the wild-type and knock-out mice, with the knock-out mice having a reduced skeletal size and altered bone mineral content compared to their wild type littermates. Histological analyses revealed a noticeable morphological difference in femur and tibia growth plate apparent by 3 weeks of age, prior to the onset of neurological symptoms. We speculate that Mecp2 deficiency leads to a primary dysregulation of genes critical for regulation of bone growth, differentiation and mineral homeostasis. To test this hypothesis, we have utilized a chromatin immunoprecipitation assay (ChIP) on chip strategy to identify specific targets of Mecp2-regulated expression in bone. Several genes essential to proper bone formation and maintenance of bone and calcium homeostasis have been identified as likely targets of Mecp2 in a differentiating osteoblast cell system. Current studies are underway to determine the specific regions of Mecp2 binding, and the functional significance of Mecp2 deficiency on candidate gene expression and function.

Disclosures: R.D. O'Connor, None.

This study received funding from: Nemours.

T209

Dwarfism: Body Composition and Bone Mineral Density Analysis. M. G. B. Pippa*1, D. C. Andrade*2, S. R. Eis3, C. A. Brandao4, C. F. Zerbini1, 1Rheumatology, Hospital Heliopolis, Sao Paulo, Brazil, 2Clinical Research, Centro Paulista de Investigação Clinica, Sao Paulo, Brazil, 3Clinical Research, Centro de Diagnóstico e Pesquisa da Osteoporose do Espirito Santo, Vitoria, Brazil, 4Bone Densitometry, Fleury Medicina e Saude, Sao Paulo, Brazil.

Dwarfism is a medical condition which result in short stature. Characteristically, the arms are shorter than the forearms and the thighs are shorter than the legs. Little is known about bone mass adjusted for body size and body composition (BC) compartments in dwarfism. Our objective was to evaluate bone mineral density (BMD), BC and X- Ray alterations in this population. 24 females and males dwarfs were studied. All of them were submitted to dual-energy X-ray absorptiometry (DXA) in order to analyze BMD of lumbar spine, proximal femur(PF), total body BMD(TBBMD),radio 33%.(R33%) and BC as well. Total Lean mass (TLM), Total Fat Mass (TFM), percentage of adiposity (% adp), arms lean mass (ALM), legs lean mass (LLM), and body fat distribution (BFD) were also determined. Areal BMD was measured and reported in g/cm2 Adjusted BMD(Adj DMD) was calculated by the relation between bone mineral content (BMC)/ height and results were given in g/m2. Apparent BMD was calculed according to Bachrach. Sarcopenia was also determined according to Baumgartner classification. AP and lateral Plain X-Ray of dorsal / lombar spine were performed. Variables were evaluated as descriptive patterns. The adherence to normal curve was evaluated by Kolmogorov Smirnov test. Data were analysed using the statistical computer program Minitab vertion 15.0%. Twelve male and 12 female dwarfs from 6 to 53 years old were studied. Average age was 34,5 years (SD = 11,9),average weight 18 Kg (SD = 12.1),average BMI 33.9 Kg / m2 (SD = 9.2) and average height 1,20 m (SD = 0.12). Areal BMD L1-L4 mean was 0.991 g/cm2, App L1-L4 BMD 0.304 g/cm3 and adjusted L1-L4 BMD was 0.0274 g/cm2.TBBMD mean was 1.047 (SD = 0.20) BMD, and AdjBMDT mean was 0.616 (SD = 0.52). R33% BMD mean was 0.616 and its Z- score −1.92 (SD = 1.02). According Z- score areal L1-L4 BMD, low bone mass was found in 5 dwarfs (20.8%). When we used Z-score TBBMD no low bone mass was shown. Using Z-score R 33% BMD, low bone mass was present in 52.6%. Five male presented sarcopenia (20.8%) and 3 female (12.5%). Mean age sarcopenic dwarfs was 26.7 (SD = 13.2). All sample had 1 or more X-Ray alterations (scalloping, scoliosis, hyper lordosis,platispondily, narrow AP spinal canal, squared iliacs wings, hypoplastic (“bullet nose”) T-L vertebrae, mild and severe vertebral crush). Low bone mass was very frequent, particularly when R 33% Z- score was used. We concluded that dwarfism should be submitted to DXA analysis earlier than normal height people.

Disclosures: M.G.B. Pippa, None.

T210

Skeletal Development Analysis by Two-Dimensional Electrophoresis (2-DE) in White Bream (Diplodus sargus), Fed with Different Diets. P. M. L. Rodrigues*, O. Cordeiro*, T. S. Silva*, L. E. C. Conceição*, FCMA, Universidade do Algarve, Faro, Portugal.

Although high larval survival rates are commonly observed in aquaculture production of D. sargus, skeleton deformities are one of the main constrains. Diets with poor protein content and amino acid deficiencies have been related to the development of skeletal deformities. The central objective of this study is to evaluate the possibility of minimizing the skeletal deformity problems commonly found when D. sargus are cultured, through the use of amino acid supplements. It is also intended to verify how the expression of key proteins involved in skeletal development are affected, in order to better understand the mechanisms present in development of skeletal deformities. The fish used in this study were previously fed since the larval stage with diets containing different amino acid profiles. The effect on skeletal deformities and bone proteins expression of larval D. sargus of a well balanced diet in the different indispensable amino acids (group 1 - control), or the same diet supplemented with amino acids involved in skeletal formation (group 2 - trp supplement and group 3 - lys supplement), was studied. Bone and cartilage were removed from D. sargus and proteins extracted. Protein expression changes in the three groups for normal and deformed fish were characterized by 2D gels analysis. Triplicates were done for each different condition with a total of eighteen gels. Gels with separated protein spots were analyzed on an imaging densitometer. Qualitative and quantitative analysis was done with PDQuest 2-D analysis software, which enables detection, matching and quantification of protein spots. After spot detection and matching, the intensity volume of each spot was normalized by comparison with a reference set of non-differentially expressed spots. Evaluation of the statistical significance of spot variation between groups was performed using a non-parametric test (Mann-Whitney U test, p<0.05). The above figure representing the spots differentially expressed shows that the 2-DE technique allows the identification the key proteins involved in skeletal development and deformities. This information may be used for the development of better balanced diets, leading to fewer individuals with skeletal deformities in fish culture.

original image

Disclosures: P.M.L. Rodrigues, None.

This study received funding from: Fundação para a Ciěncia e Tecnologia.

T211

An ENU Mutation Mapped to a Distal Region of Chromosome 11 Is Major Determinant of Bone Size Independent of Body Growth. V. Chest*, S. Mohan, J. E. Wergedal, A. K. Srivastava. JLP VA Medical Center & Loma Linda University, Loma Linda, CA, USA.

Using a phenotype driven N-ethyl-nitrosourea (ENU) screen in growth hormone (GH) deficient mice, we have identified a mutant (named 14104) that exhibited 40% decreased bone size independent of body weight. The mutation is inherited as an autosomal dominant trait in a C57BL/6J background and magnitude of the bone size phenotype is modulated by GHRHR gene (7% lower bone size, p<0.05 by post Hoc analysis). Measurement of bone size in tibia, femur, and humerus by pQCT, μCT (Figure-1A), and histology showed 20% low (p<0.001) periosteal circumference (PC) in mutant mice as compared to control mice. The unique feature of the 14104 mutant mice is that the cortical thickness is not decreased in the same proportion as cross-sectional area due to a greater (40-50%, p<0.01) decrease in endosteal circumference (EC). This opposing movement of PC and EC is due to 25% decreased bone formation (p<0.05) at the periosteal surface and a much larger 45% decreased bone resorption (p<0.05) at the endosteal surface (Figure-1B), determined by histomorphometry. The osteoblasts from 14104 mice showed 40-50% (p<0.05) reduced proliferation rate and lower induction of ALP activity (p<0.05) in response to differentiating agents. To identify the chromosomal location of the mutation, we backcrossed the mutant 14104 mice with DBA mice and generated about 140 F2 mice. Employing a selective genotyping and phenotype based DNA pooling method we scanned the genome for linkage using 60 markers. We identified linked markers on chromosome 11 with peak LOD scores of 9.8 and 10.5 (p<0.000001) for PC and EC, respectively. The 95% confidence interval of the locus involves 52-66 cM region of Chr 11. The allelic distribution of B6 (n = 18) and DBA alleles (n = 15) confirmed that linked locus was contributed by mutant gene because homozygous B6 allele contributed to 21% low PC (p<0.001) and 53% low EC (p<0.001) as compared to homozygotes for DBA alleles. In conclusion, we report mutant allele that dramatically reduced bone size independent of bone size by interacting with GH/IGF signaling pathway. The mutation is mapped to a Chr 11 region that includes several important QTLs regulating bone density and size. Since disruption of none of the known genes in this region (GH, Igf2bpl, Collal, Integrin, Noggin) produced phenotype similar to 14104 mutant mice, we believe that mutation is in a novel gene.

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Disclosures: A.K. Srivastava, None.

T212

Serum Alkaline Phosphatase QTLs Provide Evidence that Genes on Chromosome 5 and 11 Regulate Bone Formation and Affect Bone size and Strength. J. E. Wergedal1, C. L. Ackert-Bicknell2, W. G. Beamer2, S. Mohan1, D. J. Baylink3, A. K. Srivastava1, 1JLP VA Medical Center & Loma Linda University, Loma Linda, CA, USA, 2The Jackson Laboratory, Bar Harbor, ME, USA, 3Dep. of Med., Loma Linda University, Loma Linda, CA, USA.

We have previously shown that the RF/J (RF) inbred mouse line has a greater femur cross sectional size associated with a higher bone formation rate than the NZB/B1NJ inbred mouse line. We sought to test the hypothesis that genetic loci that regulate bone formation contribute to variation in bone size and strength in the RF and NZ/B intercross. We analyzed serum alkaline phosphatase (ALP), as a surrogate marker for bone formation phenotype, to obtain evidence for the concordant loci regulating bone strength parameters and bone formation marker. Surprisingly, the mean serum ALP level was higher in the low bone formation mouse strain, NZB (n = 8, Mean±SD 151±39 U/L) as compared to RF (n = 16, 119±29 U/L, P<0.05 vs NZB). A genome-wide scan was carried out using 94 polymorphic markers and serum from 542 F2 female mice (Fig. 1). Total ALP levels in serum were measured by kinetic assay using a fully automated clinical chemistry analyzer. Interval mapping for co-segregation of genetic marker data with ALP activity revealed five significant QTL on chromosome (Chr) 3 (LOD 4.74, peak location 36 cM), Chr 5 (LOD 3.82, 64 cM), Chr 9 (LOD 5.1, 48 cM), Chr 11 (LOD 3.48,40 cM), and Chr 12 (LOD 5.04, 4 cM). A suggestive QTL was observed on Chr-17 (LOD 3.00, 56 cM). Combined together, these QTLs explain 29% of the F2 variance in ALP levels. Three of these loci on Chr 5, Chr 9, and Chr 12 represent novel ALP loci that have not been identified in any previously published crosses. The allelic distribution of ALP at the peak marker was compared with values for bone size (periosteal perimeter) and bone bending strength. Two of the ALP QTL (Chr 5 and 11 ) corresponded with size and strength QTL and had the allele from the RF strain as the high allele. Two additional ALP QTLs corresponded with size or strength QTL but did not have the same strain as the high allele. It is possible that these loci regulate ALP levels by influencing the ALP metabolism. In conclusion, five Chr loci have genes that affect ALP and may affect bone formation. Two Chr loci on 5 and 11 have genes that affect femur size and strength, possibly by regulating bone formation.

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Disclosures: A.K. Srivastava, None.

T213

FDPS, GGPS1 and FDFT1 Gene Polymorphisms as Pharmacogenetic Markers for the Response to Bisphosphonates. S. Carbonell Sala*1, V. Martineti*1, F. Del Monte*1, I. Tognarini*1, S. Silvestri*1, F. Marini*1, L. Masi1, A. Tanini1, A. Falchetti2, M. Brandi2, 1Internal Medicine, University of Florence, Florence, Italy, 2Internal Medicine, University of Florence, Florence, Italy.

The therapy of metabolic bone diseases is characterized by variability in the response with limited prediction on a patient-by-patient basis. This is relevant in osteoporosis, where therapy is required long-term before outcomes' evaluation. Our aim was to study how genetic traits influence efficacy and acute response to amino-bisphosphonates (N-BPs), key components for the therapy of high turnover metabolic bone diseases. N-BPs can specifically inhibit mevalonate pathway enzymes required for protein prenylation. As the response to N-BPs is highly variable among treated patients, the individual genetic variability could be an explanation.

In this study we analyzed the farnesyl pyrophosphate-synthase (FDPS), the geranilgeranil pyrophosphate-synthase (GGPS1) and the squalene synthase (FDFT1) gene polymorphisms to assess genetic aspects of N-BPs response. The genotype distribution for FDPS A/C polymorphism (intron 1) in the Italian population corresponded to an A-allele frequency of 0.82, and C-allele frequency is 0.18, with a heterozygosity index of 0.2981 (X2Test;p = 0.29). The Polymorphism Information Content of this marker is informative (PIC = 0.29). After the evaluation of the PIC value of GGPS1 and FDFT1 selected polymorphisms, our future goal is to perform a cohort study with patients involved in clinic trials with N-BPs, searching for correlations between genotype/haplotype and response to therapy. In a cohort study including a group of 200 Caucasian postmenopausal women was observed no statistically significant association between FDPS polymorphism and baseline-BMD, with a tendency of lowest response to therapy in patients with CC-genotype.

Our final goal is to identify the segregation of clinical response to N-BPs with genetic profile of their targets in chronic metabolic bone syndromes; and to provide models for N-BPs pharmacogenetic functionality studies. Some studies demonstrate the presence of osteoclast precursors in peripheral blood in the mononuclear cell fraction. Future studies will attempt to isolate peripheral human osteoclast precursors obtained from patients with FDPS, GGPS1 and FDFT1 opposite genotypes to perform “in vitro” functionality studies. Altogether these data can contribute to the selection of optimal therapeutic guidelines in patients treated with N-BPs.

Disclosures: S. Carbonell Sala, None.

T214

Genome-wide Haplotype Association Mapping (HAM) in Mice Leads to an Identification of a Genetic Variant in CER1 Associated with Bone Mineral Density in Premenopausal Women. C. L. Cheung1, P. L. Tang*2, P. C. Sham*3, P. McClurg*4, S. Chan*2, D. K. Smith*2, A. I. Su*4, K. S. Cheah*2, A. W. Kung1, Y. Song*2, 1Medicine, The University of Hong Kong, Hong Kong, Hong Kong, 2Biochemistry, The University of Hong Kong, Hong Kong, Hong Kong, 3Genome Research Centre, The University of Hong Kong, Hong Kong, Hong Kong, 4Genomics Institute of the Novartis Research Foundation, San Diego, CA, USA.

Bone Mineral Density (BMD) is a complex trait likely determined by multiple genes. We attempted to identify the quantitative trait loci (QTL) for BMD in mouse genome and to replicate the findings in human. Information on single nucleotide polymorphisms (SNPs) (1) and whole body BMD of 18 weeks female mice (2) were gathered from 30 mouse strains. The Haplotype Association Mapping (HAM) program which utilized a sliding window of 3 SNPs in the grouping of a haplotype block was applied (1). A modified F-test was used to query for the existence of some haplotypes structure that can partition mice with high BMD and low BMD. The positional candidate gene was then replicated in 1,083 young southern Chinese women aged 20-40 years having extreme high (BMD Z score > +1 at either the spine or hip) and low BMD (Z score = < −1.28, equivalent to the lowest 10th percentile of the population). The association was examined using binary logistic regression with adjustment of age, height and weight. 22 blocks in the female mice genome were identified to contain genes for BMD variation. Chromosome 4,82.2-87.9 Mb and Chromosome 12, 26.9-28.6 Mb had two peaks in close proximity. No genes can be found in the gap in Chromosome 12, 26.9-28.6 Mb. 27 genes were found in that of Chromosome 4, 82.2-87.9 Mb. Examination of the gene list identified Cerl as a positional candidate gene in chromosome 4 QTL. Cerl is a homolog of Cerberus in Xenopus, which belongs to a cystine knot superfamily containing a cystine knot motif in a C-terminal cysteine-rich region. Genotyping of 10 SNPs in human CER1 gene in 1,083 high and low BMD subjects revealed a non-synonymous SNP (rs3747532) was associated with an increased risk of low BMD in premenopausal women (odds ratio 2.2; 95% confidence interval: 1.0 – 4.6; p < 0.05). Our successful identification of an association of CER1 with BMD variation in both young mature female mice and humans suggested that CER1 is one of the genes associated with peak bone mass. Our study highlights the utility of publicly available databases for rapidly surveying the genome for QTL.

References:

1. Pletcher, M. T., McClurg, P., Batalov, S., Su, A. I., Barnes, S. W., Lagler, E., et al. (2004). Use of a dense single nucleotide polymorphism map for in silico mapping in the mouse. PLoS Biol 2:e393

2. www.jax.org/phenome

Disclosures: C.L. Cheung, None.

T215

Association Study of CLCN7 Polymorphisms and Areal Bone Mineral Density in White or Black Women or White Men. K. Chu1, S. Ichikawa*1, D. L. Koller*2, R. Snyder*1, L. Curry*1, X. Xuei*3, H. J. Edenberg*3, T. M. Foroud*2, M. Peacock*1, M. J. Econs1, 1Medicine, Medical School of Indiana University, Indianapolis, IN, USA, 2Medical and Molecular Genetics, Medical School of Indiana University, Indianapolis, IN, USA, 3Biochemistry and Molecular Biology, Medical School of Indiana University, Indianapolis, IN, USA.

Mutations in the Chloride Channel 7 (CLCN7) gene result in autosomal dominant osteopetrosis (ADO) or autosomal recessive infantile osteopetrosis. Previously, we found that polymorphisms in CLCN7 were associated with disease severity of ADO. A SNP (single nucleotide polymorphism) in CLCN7 (rs 12926089; V418M) was also found to be associated with femoral neck BMD in Scottish women. To determine whether CLCN7 SNPs are associated with normal variation in peak areal BMD we genotyped 6 SNPs distributed throughout CLCN7 in a sample of 1704 premenopausal white women, 517 premenopausal black women or 717 white men. The SNPs were tested for association with lumbar spine and femoral neck BMD in these three samples. We found SNP rs2235579 to be associated with femoral neck BMD (P = 0.03) in white women; however, only a small proportion (0.52%) of the total variation in femoral neck BMD was explained by rs2235579. No significant association was found in white men or black women. We did not detect significant evidence of association (P >0.92) with the SNP rs 12926089 (V418M) in our samples. In conclusion, variation in CLCN7 is not a major contributor to the observed variability in peak BMD at either the femoral neck or lumber spine in white or black women or white men.

Disclosures: K. Chu, None.

This study received funding from: NIH.

T216

Influence of the Gene Polymorphisms of Collagenase-1 in Osteogenesis Imperfecta. P. Román-García*1, C. Gomez1, M. Rodríguez-García*1, D. Alvarez-Hernández*1, I. Rodríguez-García*2, M. Muñoz-Torres*3, N. Guanyabens-Gay*4, J. Cannata-Andia1, 1Bone and mineral research unit., Hospital Universitario Central de Asturias., Oviedo, Spain, 2Molecular Genetics Laboratory., Hospital Universitario Central de Asturias., Oviedo, Spain, 3Endocrinology, Hospital Universitario San Cecilio de Granada, Granada, Spain, 4Reumatology, Hospital Clinic de Barcelona, Barcelona, Spain.

The osteogenesis imperfecta (OI) is an inheritable disease, characterized by extreme bone fragility, and several extraosseous clinical manifestations. Recent results have revealed that mutations in genes no related with collagen synthesis are the cause of the new OI types. Thus, mechanisms not related to collagen synthesis might explain the different severity of the disease. The matrix metalloproteinases (MMPs) are implicated in the collagen digestion in the bone turnover process.

The aim of this study was to investigate if the functional polymorphism +1519 T/- in the MMP-1 gene (Collagenase I) was associated to the development of the disease and to the number of fractures. We studied the new T/T > T/- polimorphism, at the +1519 position in the MMP-1 gene in 30 patients with clinical diagnosis of OI and healthy controls using PCR-SSCP technique. The OI patients were 10 males and 20 females, 37.08 ± 16.4 years old (8 to 70). As reference population we used 450 people, age and sex matched from a large sample of our community hematological donors.

The frequency of the T/- genotype in OI was 3 fold higher than the observed in control population. Table 1. The clinical picture in patients with OI and T/- genotype was not different with regard T/T patients in mean number of fractures (11.3 ± 5 vs. 11.7 ± 9), presence of blue esclerae. By contrast in T/- patients we did not observed any vertebral fractures whereas in T/T patients, the prevalence of vertebral fractures was 95%. None of T/- patients had familiar history of OI versus 61% in the T/T patients. In terms of BMD we found a trend to have a higher BMD in lumbar spine and total hip in T/- patients.

There is an association between the genotype T/- and the disease. The allele (-) implicates a “frameshit reading” change and the patients with the (-) allele may have a modified collagenolitic activity. These data supports the theory that there are other molecules are affecting the disease or its manifestations or even the possibility of a misclassification in patients with clinical diagnosis.

Table Table 1. (* p<0.05 compared to control)
inline image

Disclosures: C. Gomez, None.

T217

Common Variant of SOSTDC1 Is Associated with Increased Risks of Fractures and Osteoporosis–A Novel Candidate Gene Revealed by Fine Mapping from Anhui Genome-Wide Scan. Y. H. Hsu1, T. Niu*2, H. Terwedow*2, C. Rosen3, X. Xu*2, J. Brain*2, 1Hebrew SeniorLife and Harvard Sch Public Health, Boston, MA, USA, 2Harvard Sch Public Health, Boston, MA, USA, 3Maine Center for Osteoporosis Research and Education, St. Joseph Hospital, Bangor, ME, USA.

Previously, we have revealed three novel QTLs (LOD >3.65) on Chr 7p21 for femoral neck (FN) BMD, Chr 2q24 for total hip BMD and Chr 5q21 for BMDs combined at different skeletal sites in a genome-wide scan of 3093 adult Chinese siblings selected based on their extreme hip BMD. To narrow down the QTL region, we first genotyped 10-20 microsatellite markers (∼2 cM per marker) in each of the QTLs in the same 3093 siblings. Fine mapping of the Chr 7p21 QTL narrowed to a 8 cM region (LOD = 3.72). Among 23 known genes in this region, twist homolog 1 (TWIST 1) and sclerostin domain-containing protein 1 (SOSTDC1) have been known functionally relevant to bone metabolism. TWIST1 inactivation reduces the binding ability of CBFA1/RUNX2 to the osteocalcin promoter. SOSTDC1 belongs to a class of bone morphogenetic protein (BMP) antagonists. To test whether polymorphisms in the TWIST 1 and SOSTDC1 are associated with BMDs in our cohort, we genotyped tag SNPs in an independent set of 2392 extreme low FN BMD cases (T-score < −1) and extreme high FN BMD controls matched by age and sex, selected from a study of 23,327 Chinese men and women. Tag SNPs were chosen from HapMap dataset (5 tag SNPs with pairwise mean r2 = 0.98 to un-tag SNPs for SOSTDC1; 4 tag SNPs with r2 = 0.94 for TWIST1). Multiple logistic regression with additive genetic model was used, adjusted for age, height, weight, physical activity, smoking, and menopause (women). First, the adjusted odds ratios (ORs) for extreme low FN BMD was associated with three adjacent SNPs located in exon 1 and intron 1 of SOSTDC1 (ORs 1.4-1.6, p-values 0.004-4×10−5, p-values by permutation test 0.041- 5×10−4) in men. A weak association was found in women for the SNP located in exon 1 of SOSTDCI (p = 0.017). No association was found between TWIST1 SNPs and BMD. Second, the ORs (95%CI) for men carrying the polymorphic allele A for the strongest associated SNP (rs16878762, MAF: 28.6%) of SOSTDCI were 1.7 (1.3-2.4) for osteoporosis, and 2.0 (1.1-3.7) for osteoporotic fractures. Third, men carrying AA genotype had 54 and 80 mg/cm2 lower whole body and Lumbar Spine BMD, respectively. Finally, SNP rs 16878762 was significantly associated with SOSTDC1 gene expression in men (p = 0.004) and women (p = 0.049). Notable, despite the close homologue to sclerostin (SOST), no significant association was found for SOST polymorphism (SRP9) in our study population. In sum, results from linkage, population-based association, and gene expression studies all suggest SOSTDCI variants may causally reduce BMDs and increase risks of osteoporotic fractures.

Disclosures: Y.H. Hsu, None.

This study received funding from: NIH/NIAMS.

T218

An Alul Polymorphism in ESR2 Is Associated with Reduced Risk of Osteoporotic Fractures. L. B. Husted, N. Gonzalez-Bofill, L. Stenkjær*, M. Carstens*, B. L. Langdahl. Research Lab. C, Aarhus University Hospital, Århus, Denmark.

Estrogen is important for acquisition and maintenance of bone mineral density (BMD) and therefore also for the development of osteoporosis. It acts by binding to estrogen receptors, which are transcription factors belonging to the nuclear receptor hormone superfamily. Two types of estrogen receptors, ERα and ERβ, have been identified. We have previously found that a TA-repeat polymorphism in the gene encoding ERα, ESR1 is associated with osteoporotic fracture risk. In this study we investigated the effect of three polymorphisms in the ERβ gene (ESR2): a synonymous Rsal polymorphism (rsl256049), an Alul polymorphism in the 3′UTR (rs4986938) and a CA-repeat polymorphism (Dl4SI026) in intron 5 on BMD and risk of vertebral fractures in a case-control study including 466 osteoporotic patients and 336 normal controls.

Prevalence of the CC and CT + TT genotypes of the Rsal polymorphism was 94.5% and 5.5% in patients with vertebral fractures, respectively, and 95.0% and 5.0% in normal controls, respectively, x2 = 0.071, ns. BMD at the lumbar spine was 0.844±0.190 g/cm2 in carriers of the T allele vs 0.836±0.178 g/cm2 in individuals with the CC genotype, ns. Likewise, no differences in BMD between the genotypes were found at any of the hip sites. The Alul polymorphism was associated with reduced risk of vertebral fractures. Distribution of the CC, CT and TT genotypes in patients with vertebral fractures and normal controls was 44.3%, 45.3% and 10.4% and 34.9%, 49.1% and 16%, respectively, x2 = 7.05, p = 0.029. BMD at the lumbar spine was 0.828±0.179 g/cm2, 0.837±0.176 g/cm2 and 0.866±0.182 g/cm2 in individuals with the CC, CT and TT genotypes, respectively, ns. Similar results were found at the hip. In men, however, the difference in BMD at the total hip between the genotypes was significant (p = 0.003).

The distribution of the number of CA-repeats was bimodal and therefore the CA-repeat alleles were divided into a low (≤ 21 repeats) and a high repeat number group (> 22 repeats) for statistical analyses. The prevalence of the CA genotypes was similar between patients with vertebral fractures and normal controls, and there was no difference in BMD between the genotypes.

Because the CA-repeat polymorphism and the Rsal polymorphism were in linkage disequilibrium haplotypes could be estimated. We found no effect of the three common haplotypes on BMD or fracture risk.

By examining the TA-repeat polymorphism in ESR1 and the CA-repeat polymorphism in ESR2 there seemed to be an additive effect of the two polymorphisms on fracture risk. In conclusion, we found that the Alul polymorphism in ESR2 is associated with reduced risk of osteoprotic fractures.

Disclosures: L.B. Husted, None.

T219

ENPP1 as a Candidate Gene for Femoral Bone Geometry. Y. K. Cho1, S. Demissie*1, G. Livshits*2, J. B. Meigs*3, L. A. Cupples*1, J. C. Florez*3, J. McAteer*3, D. P. Kiel4, D. Karasik4, 1Biostatistics, BU Sch Public Health, Boston, MA, USA, 2Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel, 3Mass General Hospital and Broad Inst, Boston-Cambridge, MA, USA, 4Hebrew Senior Life and Harvard Med School, Boston, MA, USA.

Secondary mineralization in bone is a controlled process with a tight balance between the levels of extracellular phosphate ions and inorganic pyrophosphate (PPi), which inhibits hydroxyapatite crystal growth and therefore prevents excessive calcification. Ecto-nucleotide pyrophosphatase phosphodiesterase 1 (ENPPl) generates PPi. It is uncertain to what extent ENPPl may be involved in appendicular skeletal mineralization.

We examined the association of polymorphisms in the ENPPl gene with femoral geometry measured with Hip Structural Analysis (HSA), in 789 women and 704 men from the Framingham Offspring Cohort (mean age ±SD: 61.3 ± 9.1 years), who had DXA and DNA available. HSA measures included femoral neck length (FNL), neck-shaft angle (NSA), and subperiosteal width at the narrowest section of the femoral neck (NN_WID) and shaft (S_WID).

Thirty one single nucleotide polymorphisms (SNPs) were genotyped on an iPLEX Sequenom platform, these SNPs captured 69% of common variants in ENPPl (minor allele frequency >5%) with an r2 >0.8. We performed sex-specific ANCOVA using 2 models adjusted for covariates: age (model 1) and age, height, BMI, smoking status, as well as estrogen history in women (model 2).

In men, we found 6 SNPs with nominally significant associations with the age-adjusted bone geometry traits. Intronic SNP (rs7775386) was associated with FNL at p = 0.0009. Other SNPs (rs9493100, rs1044498, rs9493116, rs7768480, and rs1510) were also associated with bone geometry traits (p < 0.008). In women, no nominally significant associations were found. After adjustment for additional covariates, including body size, in general, magnitude and direction of the association did not change. To correct for multiple testing, we performed 10,000 random permutations of phenotypes and covariates while keeping the genotype LD and phenotype correlation information intact. The corrected p-value for rs7775386 and FNL was 0.10, suggesting that the observed association may be due to chance.

In conclusion, this is the first study which examined a fairly comprehensive set of common variants in ENPPl in relation to bone geometry. Nominally significant associations between ENPPl polymorphisms and femoral geometry in Caucasian males generate the hypothesis that the observed genetic associations are not mediated by body size. These results require improved coverage of ENPPl polymorphisms and confirmation in larger samples.

Disclosures: D. Karasik, None.

This study received funding from: NHLBL NIAMS/NIA.

T220

Association of ADIPOR1 and ADIPOR2 Gene Polymorphisms with Bone Mineral Density in Postmenopausal Korean Women. H. Y. Kim1, B. Oh*2, J. Y. Lee*2, B. L. Park*3, H. D. Shin*3, T. H. Kim*4, E. K. Park*4, S. Y. Kim*4, J. M. Koh5, G. S. Kim5, 1Division of Endocrinology and Metabolism, University of Wonkwang College Medicine, Sanbon Medical Center, Gunpo, Republic of Korea, 2National Genome Research Institute, National Institute of Health, Seoul, Republic of Korea, 3Department of Genetic Epidemiology, SNP Genetics, Inc., Seoul, Republic of Korea, 4Skeletal Disease Genome Research Center, Kyungpook National University Hospital, Daegu, Republic of Korea, 5Division of Endocrinology and Metabolism, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Republic of Korea.

Obesity is associated with both higher fat mass and higher bone mineral density(BMD). Adiponetin is circulating peptide derived from adipose tissue. It meditates its insulin sensitizing and anti-artherogenic effects on target tissue through two known receptor, adiponectin receptor 1 and 2 (ADIPOR1; ADIPOR2). Recently, the expression of both adiponectin and its receptors in the bone forming cells of humam was reported. The possibility was suggested that interaction between adiponectin and recetor might have en effect on bone metabolism. Therefore, we examined the associations between ADIPOR1 and ADIPOR2 gene polymorphisms and bone mineral density (BMD) in postmenopausal Korean women.

All exons, their boundaries, and the promoter region (approximately 1.5 kb) of ADIPOR1 and ADIPOR2 were sequenced in 24 individuals. Among identified polymorphisms, three polymorphisms in ADIPOR1 and three polymorphisms in ADIPOR2 were selected based on LDs and frequencies and genotyped in all study participants (n = 729). BMD at the lumbar spine and femur neck was measured using dual energy X-ray absorptiometry.

The mean age of the study subjects was 58.7 ± 7.5 years and the mean years since menopause was 9.5 ± 7.8 years. Mutivariate analysis showed an association of the + 5843 G> A in ADIPOR1 with lumbar spine BMD. Individual with + 5843 A allele had lower BMD at the lumbar spine compared with those without A allele(0.90±0.18 vs 0.86±0.16 g/cm2, P = 0.005) after adjustment age, years since menopause, weight, and height. There was significant association between the −64243T>G in ADIPOR2 with femur neck BMD. Individual with −64243 G allele showed lower BMD at femur neck than those without G allele(0.74±0.12 vs 0.71±0.13g/cm2, P = 0.005). However, we did not find any association with fractures.

These finding suggests that the + 5843 G> A in ADIPOR1 and −64243T>G in ADIPOR2 gene may be one of the genetic determinants of osteoporosis in Korean postmenopausal women.

Disclosures: H. Y. Kim, None.

T221

Associations of Paraoxonase Gene (PON1) Promoter Polymorphisms with Vertebral Fracture Risk Independently of Bone Mineral Density in Postmenopausal Women. B. Kim*1, S. H. Lee*1, Y. Chung*2, B. Oh*3, J. Lee*3, J. Lee*3, B. L. Park*4, H. D. Shin*4, T. Kim*5, E. K. Park*5, S. Kim*5, J. Koh1, G. S. Kim1, 1Division of Endocrinology and Metabolism, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea, 2Department of Internal Medicine, Seoul Veterans Hospital, Seoul, Republic of Korea, 3National Genome Research Institute, National Institute of Health, Seoul, Republic of Korea, 4Department of Genetic Epidemiology, SNP Genetics, Inc., Seoul, Republic of Korea, 5Skeletal Diseases Genome Research Center, Kyungpook National University Hospital, Daegu, Republic of Korea.

There is increasing evidence of a biochemical link between oxidative stress and bone metabolism. Paraoxonase (PON1) is associated with high density lipoprotein and has the antioxidant effect by both preventing the formation of oxidized low density lipoprotein (LDL) and inactivating LDL-derived oxidized phospholipids once they are formed. Therefore, PON1 could be an important candidate gene for the modulation of bone metabolism. In order to investigate the effects of PON1 polymorphisms, especially promoter polymorphisms, on the risk of osteoporotic fractures (OFs) and bone mineral density (BMD), we directly sequenced the PON1 gene in 24 Korean individuals, and identified 30 sequence variants. In the present study, we selected 3 promoter polymorphisms of them, and they were genotyped in a larger-scale study of postmenopausal women (n = 729). Areal BMD (g/cm2) of the anterior-posterior lumbar spine and the non-dominant proximal femur was measured using dual energy X-ray absorptiometry. Lateral thoracolumbar (T4-L4) radiographs were obtained in all subjects. Multivariate analyses showed that all three promoter polymorphisms were not associated with BMD at the both sites. However, we found that the PON1-1741A>G and −909G>C promoter polymorphisms were associated with vertebral fracture risk independently of BMD before and after adjustment for age, years since menopause, height, weight, and/or BMD. After the adjustments for all covariates, the subjects with the GG homozygote of - 1741A>G and the CC homozygote of −909G>C were significantly related with higher vertebral fracture risk (odds ratio = 2.37, 95% CI 1.36-4.15, P = 0.002 in the recessive model; and odds ratio = 2.35, 95% CI 1.35-4.07, P = 0.002 in the recessive model, respectively). When we categorized the subjects into four levels according to the number of vertebral fractures, we found that the rare alleles of PONl-1741A>G (P = 0.004) and −909G>C (P = 0.006) increased, as the number of OFs did. These findings suggest that PON1 could be one of the genetic predictive markers for osteoporotic fracture risk and severity in postmenopausal women.

Disclosures: B. Kim, None.

T222

The Association of Bone Mass with SNPs of Frizzled Genes in Wnt System and Circulating Osteoprotegerin (OPG) - Receptor Activator of NF-kB Ligand (RANKL) System. J. Kim, H. Kim*, S. Chae*, C. Suh*, S. Kim, Y. Choi, S. Moon. Dept. of Obsterics & Gynecology, Seoul National University Hospital, Seoul, Republic of Korea.

The aim of this study was to investigate the association between single nucleotide polymorphisms (SNPs) in frizzled (FZD)s gene in Wnt signal pathway, and circulating osteoprotegrin (OPG), soluble receptor activator of NF-kB ligand (sRANKL) levels, bone turnover markers, and bone mineral density (BMD) in postmenopausal Korean women, The SNPs in FZD1 (rs3752146), FZD5 (rs1056614), FZD6 (rs17855053; rs12549394), FZD7 (rs12546413), and FZD9 (rs17852398; rs17856756; rs17856757; rs17852397) gene were analyzed by direct sequencing in 371 Korean postmenopausal women. Levels of serum OPG, sRANKL, osteocalcin (OST), C-telopeptide of type I collagen (CTX), calcium (Ca), phosphorus (P), parathyroid hormone (PTH) and calcitonin, and BMD at the lumbar spine and proximal femur were measured. The SNPs in FZD1, FZD5, FZD7, and FZD9 gene, and in exon 2 of FZD6 gene were not observed. The distributions of C345A SNP (rs17855053) in the exon4 and A664C SNP (rs12549394) in the exon 8 of FZD6 gene were as follows: CC 36.4%, AC 43.7%, AA 19.9%, CC 95.7%, and CA 4.3%, No significant differences in the adjusted BMD of lumbar spine and proximal femur were noted among the genotypes of C345A and A664C SNPs in FZD 6 gene and the distributions of these genotypes were not different according to the status of bone mass. No significant differences in serum levels of OPG, and sRANKL, and their ratios, and bone markers such as OST, CTX, Ca, P, PTH and calcitonin were observed according to these genotypes of FZD 6 gene polymorphisms. In conclusion, the FZDs gene polymorphisms was not associated with BMD of of the lumbar spine and proximal femur, bone turnover markers, and circulating OPG-sRANKL levels in Korean women.

Disclosures: J. Kim, None.

This study received funding from: Seoul National University Hospital (#03-2006-0220)

T223

Association Study of Catalase Gene Polymorphisms with an Osteonecrosis of Femoral Head in Korean Population. J. Hong*1, X. Dai*1, T. Kim*1, J. Park*1, J. Jung*1, E. Park2, S. Kim3, 1Skeletal diseases Genome Research Center, Kyungpook National University Hospital, Daegu, Republic of Korea, 2Pathology and Regenerative Medicine, School of Dentistry, Kyungpook National University, Daegu, Republic of Korea, 3Department of Orthopedic Surgery, Kyungpook National University Hospital, Daegu, Republic of Korea.

Osteonecrosis of femoral head (ONFH) frequently leads to progressive collapse of the femoral head followed by a degenerative arthritis of the hip joint. Various theories have been proposed regarding the pathogenic mechanisms of osteonecrosis (ON). Oxidative stress, which has been implicated in many pathological conditions, including vascular injury, has been recently suggested to play a part in the development of ON. In addition, tissue oxidation is known to induce apoptosis, which has also been suggested to be involved ON. Recently, oxidative stress has been also suggested to participate in the development of osteoporosis. Some in vitro and animal studies have suggested that oxidative stress decrease bone formation by modulating the differentiation and survival of osteoblasts and stimulating bone resorption.

Catalase (CAT) is a major antioxidant enzyme that detoxifies hydrogen peroxide by converting it into water and oxygen, thereby preventing cellular injury by oxidative stress. Individuals with reduced catalase activity have an increased incidence of oxidative stress-related disease, such as atherosclerosis, diabetes, and dyslipidaemiaTo examine the associations between the CAT gene polymorphisms and ON in Korean population, we selected nine polymorphic sites of CAT from public databases (dbSNP, KSNP and HapMap), and genotyped in 460 ONFH patients and 300 control subjects. The CAT genotype did not deviate from Hardy-Weinberg equilibrium in any of the case or control groups. We classified the patients according to etiology: alcohol-induced, steroid-induced, and idiopathic ONFH subgroup. We found that −89A>T, −20T>C, +14539A>T, and +22348C>T polymorphisms of CAT gene were significantly associated with the risk of ONFH in all alternative analysis model (p range; 0.0003-0.047, OR; 0.56-3.57). However, the minor allele of −89A>T and −20T>C had protective effect on ONFH with significant p-values (p range; 0.004-0.047, OR; 0.56-0.75). Further analysis based on pathological etiology (alcohol-, steroid- or idiopathic) and sex showed that the genotype of −89A>T, −20T>C, +14539A>T, and +22348C>T were also associated with the risk of ONFH in each subgroup with significance. Our results provide, for the first time, evidence supporting the association of CAT gene polymorphisms with the risk of ONFH in Korean population, and suggest that oxidative stress may play an important role in the pathogenesis of ONFH.

Disclosures: S. Kim, None.

T224

Association of Lipoprotein Lipase Hind III Polymorphism with Lumbar Bone Mineral Density in Korean Premenopausal Women. S. Lee1, O. Kim*2, J. Kim*2, K. Lee*2, H. Baik*2, Y. Jo*3, H. Kim*3, B. Kim*3, K. Park*3, H. Choi4, 1Biochemistry-Molecular Biology/Internal Medicine, School of Medicine, Eulji University, Daejeon, Republic of Korea, 2Biochemistry-Molecular Biology, School of Medicine, Eulji University, Daejeon, Republic of Korea, 3Internal Medicine, School of Medicine, Eulji University, Daejeon, Republic of Korea, 4Family Medicine, School of Medicine, Eulji University, Daejeon, Republic of Korea.

The association between serum levels of lipid and bone mineral density (BMD) remains debatable. The lipoprotein lipase (LPL) plays a key role in lipid metabolism. Association of LPL Hind III polymorphism with BMD has not been studied. In the present study, we attempted to examine the association between LPL Hind III polymorphism and lumbar BMD in pre- and post-menopausal women, respectively. 201 Korean women (130, premenopausal women; 71, postmenopausal women) were recruited at the Eulji University Hospital, South Korea. We measured lumbar spine L1-L4 BMD using DXA. We determined LPL gene polymorphisms affecting Hind III polymorphism site in intron 8 using PCR techniques. The polymorphic allele displaying the restriction site was referred to as (+) and the allele without the site as (-). The (-) allele was less common. We compared the anthropometric parameters, blood lipid levels, and lumbar BMD between women with H(+/+) and H(+/-) genotype in pre- and post-menopausal women, respectively. Frequencies of H(+/+), H(+/-) and H(-/-) genotype were 60.8 %, 33.8 %, and 5.4 %, respectively, in premenopausal women. Frequencies of H(+/+), H(+/-) and H(-/-) genotype were 57.8 %, 39.4 %, and 2.8 %, respectively, in postmenopausal women. There were no differences in total cholesterol levels (184±31 vs 184±37 mg/dL), triglyceride levels (100±56 vs 106±91 mg/dL), HDL-cholesterol levels (54±10 vs 54±11 mg/dL), LDL-cholesterol levels (111±28 vs 109±32 mg/dL), waist circumference (74±7 vs 74±7 cm), and body mass index (22.5±2.5 vs 22.7±2.6 kg/m2) between women with H(+/+) and H(+/-) genotype in premenopausal women. However, there was a difference in lumbar BMD (0.970±0.111 g/cm2 vs 1.015±0.108 g/cm2) between women with H(+/+) and H(+/-) genotype in premenopausal women. In postmenopausal women, there were no differences in blood lipid levels, obesity and lumbar BMD between women with H(+/+) and H(+/-) genotype. Our data indicate an association between LPL Hind III polymorphism and lumbar BMD in premenopausal women and suggest that the effect of LPL Hind III polymorphism on lumbar BMD is different between pre- and post-menopausal women at Korea.

Disclosures: S. Lee, None.

This study received funding from: Korea Research Foundation Grant funded by the Korean Government (KRF-2006-331-E00051).

T225

Associations of TSH Receptor Gene (TSHR) Polymorphisms with Bone Mineral Density (BMD) in Postmenopausal Women. S. H. Lee*1, B. Kim*1, W. G. Kim*1, Y. Chung*2, B. Oh*3, J. Lee*3, J. Lee*3, B. L. Park*4, H. D. Shin*4, T. Kim*5, E. K. Park*5, S. Kim*5, J. Koh1, G. S. Kim1, 1Division of Endocrinology and Metabolism, Asan Medical Center, University of Ulsan College of Medicine, Seoul, Republic of Korea, 2Department of Internal Medicine, Seoul Veterans Hospital, Seoul, Republic of Korea, 3National Genome Research Institute, National Institute of Health, Seoul, Republic of Korea, 4Department of Genetic Epidemiology, SNP Genetics, Inc., Seoul, Republic of Korea, 5Skeletal Diseases Genome Research Center, Kyungpook National University Hospital, Daegu, Republic of Korea.

It is well known that hyperthyroidism is associated with lower bone mass as well as higher fracture risk. Not only high thyroid hormone level but also low thyroid stimulating hormone (TSH), is associated with lower bone mass. We selected TSH receptor gene (TSHR) as a candidate gene and investigated the genetic effects of selected TSHR polymorphisms on the bone mineral density (BMD) in postmenopausal Korean women with normal thyroid function. Four SNPs were selected in the Oriental SNP data, and genotypes for the selected SNPs were determined in 729 healthy postmenopausal women. Areal BMD (g/cm2) of the anterior-posterior lumbar spine and the femoral neck was measured using dual energy X-ray absorptiometry. Serum concentrations of TSH and free thyroxin were measured using immunoluminometry and radioimmunoassay, respectively. Mean age of the studied subjects was 58.7 ± 7.5 year old, and years since menopause was 9.5 ± 7.8 years. We found that TSHR +140974T>C polymorphism was significantly associated with BMDs at the lumbar spine and the femoral neck. After adjustment for age, year since menopause, weight and height, the subjects with the TT homozygotes had lower BMD at the lumbar spine (p = 0.01 and 0.01, respectively) and femoral neck (p = 0.003 and 0.001, respectively) in co-dominant and dominant models. When we divided the subjects into the two groups according to the serum TSH concentrations, the significant associations of the polymorphism with BMDs were persistent at the lumbar spine (p = 0.019) and femoral neck (p = 0.007) in the higher TSH group (1.8-5.0 μU/ml), but not in the lower TSH group (0.4-1.7 μU/ml) (p = 0.491 and 0.667, respectively). These results suggest + 140974T>C polymorphism in TSHR is a possible genetic factors for BMD values in the postmenopausal women with normal thyroid function, and the association was more prominent in those with higher serum TSH concentration.

Disclosures: S.H. Lee, None.

T226

Multiplex SNP Genotyping and Data Analysis on 360 Hungarian Postmenopausal Women. G. Speer*, Á. Lazáry, J. Kósa*, B. Tóbiás*, T. Mez*, K. Bácsi*, B. Balla*, I. Takács, Z. Nagy, P. Lakatos. 1st Departement of Internal Medicine, Semmelweis University, Budapest, Hungary.

Many single nucleotid polymorphisms were described as risk factors for osteoporosis (OP) but there are only a few reports in the literature about more complex genomic analyses such as haplotypes and gene-gene interactions. The aim of this study was to test the associations between SNPs or haplotypes of OP-related genes and the quantitative and qualitative traits of bone loss in Hungarian postmenopausal women. Thirty-eight SNPs found in OP-related genes were genotyped with GenomeLab SNPstream Genotyping system (Beckman Coulter) from DNA isolated from blood samples of 360 unrelated postmenopausal women. Associations between individual SNPs and traits of OP in three measured skeletal site (hip, spine and radius) were tested by MANOVA adjusted for BMI and years since menopause. We applied Multifactor Dimensionality Reduction (MDR) software for detecting gene-gene interactions and THESIAS software for validating them. Among individual SNPs showing the strongest association with OP were those found in RANKL, OPG and COLIA1 genes, respectively. We could not confirm the association with OP in cases of some previously reported SNPs, however, some new individual relationships have been determined. MDR analysis has shown the best models built by 2, 3, 4 or 5 SNPs for the estimation of OP that have been validated later. “TA” haplotype of RANKL (rs9533156) and VDR (rs1544410) was associated with OP risk on lumbar site (p<0.05) while “AC” haplotype of CYP19 (rs12901187) and IL1B (rs1143634) was associated with total femoral T-score and OP at the femoral site (p<0.05). Two models of three SNPs were determined; the “TAA” haplotype of COLIA1 (rs2857396), IL6 (rs1800797) and VDR (rs1544410) has strong association with the lumbar BMD (p<0.01) and T-score (p<0.05). The “GCA” haplotype of CYP19 (rs12901187), RANKL (rs3742257) and CASR (rs9740) was also associated with OP at the lumbar site (p<0.05). Our results suggest that multiplex SNP analysis can increase the force of genetic studies and can reveal new contexts of the genetic background of OP.

Disclosures: G. Speer, None.

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

T227

Relationship between Variation in the PTH, PTH Related Peptide and the PTH Receptors 1 and 2 Genes and BMD and Fracture in Elderly Women. M. Tenne*1, P. Gerdhem*1, H. Luthman*2, K. Åkesson1, F. Mc Guigan*1, 1Orthopaedics and Clinical Sciences, Malmö University Hospital, Lund University, Malmö, Sweden, 2Medical Genetics, Clinical Sciences, Malmö University Hospital, Lund University, Malmö, Sweden.

The aim of this study was to evaluate the relationship between genetic variations in 4 key genes of the PTH regulatory pathway and fracture and BMD in 75-year old women.

Parathyroid hormone (PTH) plays a key role in calcium homeostasis and skeletal integrity whereas parathyroid hormone related peptide (PTHRP) contributes to skeletal development. Secretion of PTHRP has also been observed in tumors. PTH and PTHRP act through the same receptor, the PTH/PTHRP receptor (PTHR1). In addition, a second receptor, PTHR2 has been identified. We have previously reported on variation in the PTH and PTHRP genes in this cohort. However, we have extended the analyses to also include the receptor genes; PTHR1 and PTHR2.

The study group included 1044 elderly women, all 75-yrs old at baseline, from the Malmö Osteoporosis Prospective Risk Assessment study (OPRA). This is an extensively phenotyped cohort which has been followed for up to 9 years. Three single nucleotide polymorphisms (SNPs) at the PTHR1 locus on chromosome 3 and three at the PTHR2 locus on chromosome 2 have been analysed, in addition to six SNPs at the PTH locus on chromosome 11 and three at the PTHRP locus on chromosome 12. Furthermore, for the PTH gene, after verifying LD between the SNPs, we analysed 5 common haplotypes identified using the PHASE program. We found no association between BMD, fracture or serum-PTH and PTHR1 SNPs. Neither did we find any association with PTHR2, a gene relatively unexplored in relation to osteoporosis. There was no association between SNPs in the PTH and PTHRP genes after adjusting for confounders.

Analysis revealed an association between a common haplotype of the PTH gene; Hap 5 occurring in 37% of the population and risk of fracture between age 50 and 75 (p = 0.018), an association that was strengthened by adding prospectively occurring fractures i.e. from age 75 onwards (p = 0.011). For Hap 1 (13%) the association was lower (p = 0.02) for fractures age 50 to 75. In addition, PTH Hap 9 (19%) was associated with risk for any fracture sustained since childhood (p = 0.011). We did not find any association between PTH haplotypes and BMD.

A shift towards increasing PTH levels in the elderly may lead to fracture and genetic variation in the components of the PTH pathway may play a role. The absence of an association with the receptor genes could reflect that they are less important in the elderly. In this study the association between variation in PTH genes and fracture is evaluated for the first time. In our population of elderly women the effect of PTH gene variation, however modest, appears to be independent of BMD.

Disclosures: M. Tenne, None.

T228

No Role for Polymorphisms in the Gene Encoding RANKL in the Development of Sporadic Paget's Disease of Bone in Contrast to Some OPG Polymorphisms. G. Beyens*1, F. de Freitas*1, F. Vanhoenacker*2, L. Verbruggen*3, H. G. Zmierczak*4, R. Westhovens*5, J. Van Offel*6, J. Devogelaer7, W. Van Hul1, 1Department of Medical Genetics, University of Antwerp, Antwerp, Belgium, 2Department of Radiology, University of Antwerp, Antwerp, Belgium, 3Department of Rheumatology, University Hospital of Brussels, Brussels, Belgium, 4Unit of Osteoporosis and Metabolic Bone Diseases, University Hospital of Ghent, Ghent, Belgium, 5Department of Rheumatology, Catholic University of Leuven, Leuven, Belgium, 6Department of Rheumatology, University of Antwerp, Antwerp, Belgium, 7Department of Rheumatology, Saint-Luc University Hospital, Brussels, Belgium.

Paget's disease of bone (PDB) is a metabolic bone disease characterized by focal lesions of increased bone turnover and affects between 1% and 5% of individuals older than 55 years in Western countries. Epidemiological studies have shown that PDB patients can be divided into two groups, a familial patient group and a sporadic group, with at least partially divergent pathogenic mechanisms. In a previous study, we have shown that polymorphisms in the TNFRSF1 IB gene encoding OPG have a gender-specific influence on the development of sporadic PDB in both Belgian and UK study populations [1]. With the current research we wanted to investigate whether a similar effect could be observed for SNPs in TNFSF11 encoding RANKL.

Based on HapMap data and using an r2-based tagging strategy, we selected 10 SNPs and 2 multi-marker tests that cover all known common (minor allele frequency > 5%) genetic variation in a 44.57kb region surrounding TNFSF11. These SNPs were genotyped in a population of 159 sporadic Belgian PDB patients and 186 Belgian control individuals. After genotyping one SNP, rs633137, turned out not to be in Hardy-Weinberg equilibrium in controls and was excluded. The remaining 9 SNPs in combination with the 2 multi-marker tests fail to cover 6 informative SNPs in the selected region (resulting in 85.4% coverage). However, these SNPs are all located intronic or at the 3′end of the gene. Genotyping and testing the selected SNPs and multi-marker tests in our Belgian study population indicated that none of them is associated with PDB and no gender-specific effects were observed.

This indicates that in contrast to OPG polymorphisms, RANKL polymorphisms do not play a role in the development of sporadic PDB. Analysis of other populations is necessary to confirm this result.

[1] Beyens et al. JBMR, in press

Disclosures: W. Van Hul, None.

T229

Multifactor Dimensionality Reduction Reveals Antagonistic Epistasis Between ESR1 and ESR2 with BMD Variation in Southern Chinese Women. S. M. Xiao, C. L. Cheung, A. W. C. Kung. The Department of Medicine, The University of Hong Kong, Hong Kong, China.

Osteoporosis is a complex disease, which is affected by both genetic and environmental factors. Epistasis is thought to be a critical genetic architecture underlying complex disease, which allows genetic buffering, i.e. to stabilize the trait through the interaction of polymorphisms in different loci in the gene network. A recent study demonstrated that multifactor dimensionality reduction (MDR) is one of the best ways to study epistasis. In this study, we applied MDR in studying the epistasis between two closely related candidate genes for BMD variation, ESR1 and ESR2.

To study the relation of ESR1 and ESR2 polymorphisms in hip BMD variation in southern Chinese women, 827 subjects with either low BMD (defined by a BMD Z score less than −1.28 at either the femoral neck or total hip) or high BMD (score greater than +1) were studied. A novel CA repeat polymorphism (D6S440) in ESR1 and 8 SNPs in ESR2 were genotyped. We previously reported CA repeat size of 18 was most discriminative for hip BMD in a population cohort. Multifactor dimensionality reduction was applied to test the epistatic effect, with subsequent confirmed by binary logistic regression (at least one allele >18 CA repeats vs <18 repeats) with adjustment of age, height and weight.

Carriers of 18 CA repeats of ESR1 were associated with higher BMD at the femoral neck (OR: 2.4, p = 0.001) or hip (OR: 3.2, p <0.001). Variant allele of T-1213C of ESR2 was associated with lower BMD at the femoral neck (OR: 1.7, p = 0.042) and variant allele of T-1213C and rs3742641 was associated with low BMD at the total hip (OR: 2.2, p = 0.011 and OR: 2.2, p = 0.013 respectively). MDR revealed significant epistasis between 18 CA repeats of ESR1 with several polymorphisms of ESR2 for 2-way, 3-way and 4-way interactions (testing accuracy > 50%). These results were confirmed by binary logistic regression model. Interestingly, when 18 CA repeat interacted with rs944052, rs3742641 and rs928554 of ESR2, the OR resulted from gene-gene interaction was 1.0 (p = 0.038) at femoral neck and 1.1 (p = 0.004) at hip.

The epistasis between ESR1 and ESR2 significantly altered the single gene effect of either ESR1 or ESR2. We have demonstrated the epistasis between genes in the same gene network significantly altered the single gene effect. Testing for gene epistasis in association with BMD variation is a powerful approach to understand the pathophysiology of low bone mass in the general population.

Disclosures: S.M. Xiao, None.

This study received funding from: Grants from Hong Kong Research Grant Council, Osteoporosis Research Fund. Matching Grant and University Research Committee/ Committee on Research and Conference Grants of the University of Hong Kong.

T230

The Association between Peak Hip Axis Length and Polymorphisms of the Vitamin D Receptor and Estrogen Receptor 1 genes in Chinese Healthy Men. Z. Zhang, J. He*, J. Gu*, The Department of Osteoporosis, Osteoporosis Research Unit, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai, China.

To investigate the possible relationship between peak hip axis length (HAL) and polymorphisms of the vitamin D receptor (VDR) and estrogen receptor 1(ESR1) genes in Chinese healthy men. A total of 252 healthy men aged from 20 to 40 (29.9±5.9 years) of Han nationality in Shanghai were recruited and were measured for hip axis length (HAL, cm) using DXA (Lunar-Prodigy). The VDR Apa I and Fok I genotype and ESR1 Pvu II and Xba I genotype were determined by PCR-RFLP. The distribution of Apa I genotype was as follows: aa 54.4%, Aa 38.5%, AA 7.1%, respectively. Frequencies of Ff, FF, and ff genotype were 52.4%, 25.4%, and 22.2%, respectively. The distribution of Pvu II genotype was as follows: Pp 46.0%, pp 41.3%, PP 12.7%, respectively. Frequencies of xx, Xx and XX genotype were 71.0%, 25.4% and 3.6%, respectively. After phenotypes were adjusted for age, height, and weight, a significant association was found between VDR Apa I genotype and HAL (P = 0.04). The HAL of subjects with aa genotype (111.5±0.5) were significant higher than that of Aa genotype (109.8±0.5) (p = 0.015). Moreover, significant association was found between VDR Fok I genotype and HAL (P = 0.002), and The HAL of subjects with ff genotype (112.6±0.7) were significant higher than that of Ff genotype (110.8±0.5) (p = 0.031) and that of FF genotype (109.1±0.7) (p = 0.000). Neither Pvu II genotype nor Xba I genotype was significantly associated with HAL. The study suggested that Apa I and Fok I polymorphism in VDR gene may have influence on variation of peak HAL in Chinese men, but Pvu II and Xba I polymorphism in ESR1 gene is not the quantitative trait loci (QTL) underlying peak HAL variation.

Disclosures: Z. Zhang. None.

T231

Paclitaxel-loaded Hydroxyapatite-alginate Beads for Local Chemotherapy of Metastatic Spine Cancer in Rats. T. Abe*1, M. Sakane1, T. Ikoma*2, T. Yoshioka*1, J. Tanaka*3, N. Ochiai*1, 1Orhopedic Surgery, University of Tsukuba, Tsukuba, Japan, 2Biomaterials Center, National Institute for Materials Science, Tsukuba, Japan, 3Metallurgy and Ceramics Science, Tokyo Institute of Technology, Tokyo, Japan.

Breast cancer is one of the most common cancers to develop metastasis to bone. Of these, spine is the most frequent site. As therapy for systemic cancer improves, local control of metastatic site is becoming important. Among patients with breast cancer that had metastasized to bone, 5-10% did not respond to treatment and developed metastatic epidural spinal cord compression. Paralysis caused by spinal cord compression is directly associated with survival and quality of life of breast cancer patients. We have shown successful loading of the Hydroxyapatite (HAp)-alginate carrier with paclitaxel (PTX) previously. This study was made to clarify the effects of the PTX-loaded HAp-alginate beads administered therapeutically in a rat model of metastatic spine cancer. Rat's mammary adenocarcinoma (the CRL-1666 cell line) and female Fischer 344 rats (8 weeks old) weighing 130-150 grams were used. The PTX-loaded HAp-alginate beads were available by the spray-drying method and the gelation of alginate acid. A 7.4 wt% of PTX (0.13 mg) was contained in each HAp-alginate bead with a diameter of 2.2 mm. We made in vivo biocompatibility test by implantation in the paravertebral muscle, performed surgery to obtain metastatic spine cancer model. Thirty rats with metastatic spine cancer were divided into 3 groups. We checked hind-limb motor function and survival time using the BBB scale. The mean time before onset of paralysis of the local-treatment group (n = 12) was 17.45 days, which was significantly longer than 9.0 days of the control group (n = 8). The effect of the systemic-treatment group (n = 10) that administered intravenously was not evident despite 3 times of 6-fold higher dosage of PTX. In addition, the HAp-alginate carrier and local administration of the PTX-loaded HAp-alginate beads showed no toxicity, whereas intravenous administration of PTX showed severe complications including phlebitis of tail vein and sudden death. This report documents that the local administration of the PTX-loaded HAp-alginate beads showed significant delay in the mean time before onset of hind-limb paralysis. The results indicate local chemotherapy offers promise as less invasive treatment for metastatic spine cancer to delay the potential problem of onset of paralysis.

Disclosures: T. Abe. None.

T232

Both N- and C-terminal Fragments of PTHrP exert Pro-Survival Effects on Human Prostate Cancer Cells Through Transcription Factor Runx-2. V. Alonso*1, F. C. Pérez-Martinez*2, F. J. Calahorra*2, P. Esbrit1. 1Bone and Mineral Metabolism Laboratory, Fundación Jiménez Diaz, Madrid, Spain, 2Urology Department, Fundación Jiménez Diaz, Madrid, Spain.

The propensity of prostate cancer (PCa) to metastasize to bone seems to be related to the osteomimetic properties of PCa cells. In the present study, we analyzed the putative mechanisms whereby the osteoblast-like features of these cells can promote their survival. Human prostate cancer PC-3 cells were treated with a high dose (50 μM) of the phytoestrogens genistein and daidzein or 17β-estradiol for 24-48 h. Cell death was analyzed by trypan blue exclusion and flow cytometry (propidium iodide staining). Bone-related factors that are expressed by PC-3 cells: parathyroid hormone-related protein (PTHrP) and the PTHl receptor (PTHIR), osteoprotegerin (OPG), receptor activator of NF-kβ ligand (RANKL), and Runx2, as well as apoptosis-related proteins were evaluated by Western blot. Our results show that genistein induces apoptosis (2-fold vs basal) in PC-3 cells; this effect does not appear to be dependent on estrogen receptors, since the same dose of either daidzein or 17β-estradiol failed to affect PC-3 cell death. The pro-apoptotic effect of genistein was unchanged by a neutralizing anti-OPG antibody. However, PTHrP (7-34), a PTHIR antagonist, induced PC-3 cell death in the presence or absence of each phytoestrogen. Moreover, PTHIR protein expression was significantly increased (2-fold vs basal) by both genistein and daidzein in these cells. In addition, pre-treatment (1 h) with 100 nM of either PTHrP (1-36) or PTHrP (107-139) -which does not interact with the PTH1R- abrogated the pro-apoptotic effect of genistein in PC-3 cells. This effect of each PTHrP peptide was associated with an increase in RANKL protein expression. However, blockade with a neutralizing RANKL antibody or stimulation with exogenous RANKL (50 ng/ml) failed to modify genistein-induced PC-3 cell death. We found that each PTHrP fragment triggered Runx-2 nuclear protein internalization, and Bcl-2/Bax ratio (1.5-fold vs basal; at the expense of a Bcl-2 protein increase) in PC-3 cells. Interestingly, PC-3 cells transfected with a dominant negative Runx2 construct abolished these protective effects of both PTHrP peptides.

In conclusion, the present in vitro findings support the anti-apoptotic action of both N- and C-terminal PTHrP fragments in PCa cells. Our data also indicate the pivotal role of Runx-2 on this protective effect of these PTHrP fragments on these cells.

Disclosures: V. Alonso, None.

T233

Progression of Osteonecrosis of the Jaw in Breast Cancer Patients even with Discontinuation of Intravenous Bisphosphonate Therapy. C. Barragan-Adiemian*1., L. Lausten*2., M. L. Johnson1, J. O. Katz*3, L. F. Bonewald1, 1Oral Biology, University of Missouri Kansas City, kansas City, MO, USA, 2Special Patient Care, University of Missouri Kansas City, kansas City, MO, USA, 3 Oral Pathology, Medicine and Radiology, University of Missouri Kansas City, kansas City, MO, USA.

Intravenous bisphosphonate (BP) therapy has become the standard of care for the treatment of cancers that metastasize to bone. BPs have been associated with osteonecrosis of the alveolar bones, a condition known as bisphosphonate-related osteonecrosis of the jaw (BRONJ). The incidence or pathogenesis of BRONJ is largely unknown. The lesions are characterized by exposed necrotic bone with no evidence of healing after 8 weeks in the absence of radiation to the head and neck. BRONJ lesions have been recalcitrant to conventional therapies. The current recommendation is to proceed with non invasive dental treatment with patients on BP therapy. If an ONJ lesion has been diagnosed discontinuance of IV infusion of BPs is recommended if systemic conditions permit. We hypothesized that discontinuation of the intravenous BPs in breast cancer patients does not prevent further evolution of the lesions as detected using cone-beam computerized tomography (CBCT), although soft tissue may appear healed. Six subjects with breast cancer in remission that had discontinued BP therapy were followed for at least two years. Dental evaluations and CBCT were performed to follow the progression of the BRONJ lesions. BRONJ lesions were clinically observed and diagnosed during the period of 1 month to 2 years after discontinuation of BP. In all cases, there was exposed, necrotic bone. Two of the BRONJ lesions were associated with an extraction, one lesion was associated with an adjacent periradicular abscess and subsequent root canal and three lesions occurred spontaneously. All of the subjects received the appropriate treatment for the lesions. None of the lesions had completely healed even after discontinuation of BP therapy for up to 2 years. In one subject, the baseline CBCTs revealed a large sequestrum on the mandibular ridge with a surrounding sclerotic area. After vigorous treatment with antibiotics and local surgical debridement of the sequestrum, the gingival tissue healed leaving a small fistula. A CBCT taken one year after diagnosis demonstrating further progression of the diseased state; sclerotic bone and small sequestrums were still present 2 years on CBCT after cessation of the BP therapy. In conclusion, BRONJ lesions can persist for up to 2 years after cessation of BP treatment and CBCT is a technology that can identify and follow the progression of lesions in BRONJ patients allowing better diagnosis and assessment of disease status.

Disclosures: C. Barragan-Adjemian, None.

T234

PTHrP Regulation of Aldo-Keto Reductase 1C3 and Growth in Prostate Cancer Cells. T. M. Downs*1, R. H. Hastings*2, F. Araiza*3, D. Burton*4, A. Stolz*5, L. J Deftos4, 1Surgery, Veterans Administration San Diego Healthcare System and University of California, San Diego, CA, USA, 2 Anesthesiology, Veterans Administration San Diego Healthcare System and University of California, San Diego, CA, USA, 3Moore's Cancer Center, San Diego, CA, USA, 4Medicine, Veterans Administration San Diego Healthcare System and University of California, San Diego, CA, USA, 5Medicine, University of Southern California, San Diego, CA, USA.

Parathyroid hormone-related protein is involved in prostate tumor development, growth, progression and metastasis to bone, but the molecular mechanisms that PTHrP uses are not well defined. We previously demonstrated that DU 145 prostate carcinoma cells upregulated aldo-keto reductases (AKR1C1-3) mRNAs when stably transformed transfected to express PTHrP. The goal of this study was to explore the role of PTHrP on prostate cancer growth and its interactions with AKR1C3 and the regulation of androgen and prostaglandin metabolism. We studied the effects of PTHrP on AKR1C3 levels in DU 145 human prostate cancer cells with quantitative PCR, western, and enzymatic studies. We also evaluated the effects of PTHrP, androgens, and prostaglandins on cell proliferation and apoptosis. PTHrP treatment demonstrated significant increases in AKR1C3 mRNA levels in DU 145 cells, increased AKR1C3 protein expression up to 5-fold, and augmented cellular reductase activity. Furthermore, PTHrP promoted prostate cancer cell proliferation and protected the prostate cancer cells against UV and gamma irradiation induced apoptosis. 5α-dihydrotestosterone and 5-androstane-3α-17(3-diol had no stimulatory activity on cell proliferation in the androgen receptor-negative cells, but the androgens stimulated the androgen-dependent LNCaP cells. Prostaglandin D2, an AKR1C3 substrate, inhibited prostate cancer cell proliferation, but its metabolite, 9α-11β-prostaglandin F2, had no effect on growth. In summary, PTHrP stimulates the growth, apoptosis, and AKR1C3 expression of androgen-independent prostate cancer cells. These effects could be mediated through AKR1C3-catalyzed reductions in the levels of prostaglandin D2.

Disclosures: D. Burton, None.

This study received funding from: Moore's Cancer Center and the Department of Veterans Affairs.

T235

Wnts Increase Osteoblast Activity in Prostate Cancer Through Bone Morphogenetic Proteins. J. Dai, C. L. Hall*, J. Escara-Wilke*, E. T. Keller. Department of Urology, University of Michigan, Ann Arbor, MI, USA.

Our previous studies have shown that bone morphogenetic proteins (BMPs) promote prostate cancer (PCa) osteoblastic activity, but upstream inducers of BMP expression in PCa are unknown. Wnts are mediators of osteogenesis. In this study, we investigated a potential role of Wnts in PCa osteoblastic lesions.

To investigate whether Wnts regulate the expression of BMPs in PCa cells, C4-2B cells were treated with Wnt3a and Wnt5a. Wnt3a increased BMP4 mRNA and protein expression, whereas Wnt5a increased BMP6 mRNA and protein expression. To investigate whether Wnts activate BMP promoters, C4-2B cells were transfected with BMP4 or BMP6 promoter. Wnt3a increased mainly BMP4 promoter activity, whereas Wnt5a only increased BMP6 promoter activity, DKK-1 and sFRP-1, inhibitors of Wnt binding to Wnt receptors, blocked Wnt3a-induced BMP4 promoter activity. To elucidate whether Wnt canonical pathway or non-canonical JNK-dependent signaling pathway mediate Wnts-induced BMP promoter activity, C4-2B cells transfected with BMP4 or BMP6 promoter were treated with Axin-2, a negative regulator of the canonical Wnt pathway, or a JNK inhibitor. Axin-2 blocked Wnt3a-induced BMP4 promoter activity, and the JNK inhibitor diminished Wnt5a-induced BMP6 promoter activity. To define how Wnts mediate osteoblast activity, MC3T3-EI cells were treated with Wnt3a or Wnt5a. Wnt3a and Wnt5a increased alkaline phosphatase activity and osteocalcin level. Dkk-1 completely, whereas Noggin, a BMP inhibitor, partially blocked Wnt3a-mediated osteoblast activity, both together did not show further inhibition. DKK-1 and Noggin partially blocked Wnt5a-mediated osteoblast activity and both together showed further inhibition.

To investigate whether the secreted proteins from PCa cells mediate osteoblast activity, MC3T3-E1 cells were treated with C4-2B cells or LuCaP23.1 cells conditioned medium (CM). C4-2B cells and LuCaP23.1 CM increased ALP activity and osteocalcin level, Dkk-1 and sFRP-1 partially blocked CM-mediated pro-osteoblast activity, DKK-1 and Noggin together block CM-mediated osteoblast activity. To further elucidate whether C4-2B cells or LuCaP23.1 cells CM-mediated osteoblastic activity through BMPs, MC3T3-E1 cells were treated with CM from C4-2B or LuCaP23.1 cells pretreated with DKK-1 and/or Noggin. DKK-1 completely, whereas Noggin partially, blocked CM-mediated pro-osteoblast activity, DKK-1 and Noggin together had no further inhibition on the CM-mediated pro-osteoblast activity.

Taken together, we conclude that PCa cells promte osteoblastic activity through both direct Wnt3a and Wnt5a activity on pre-osteoblasts, as well as through induction of BMP4 and BMP6 expression that occurs through both the canonical and non-canonical pathways.

Disclosures: J. Dai, None.

T236

Urinary Excretion of 41Ca in Mice: Application of 41Ca in Cancer-induced Bone Disease. R. L. Fitzgerald1, D. W. Burton2, T. Griffin*1, L. J. Deftos2, D. A. Herold*1, D. J. Hillegonds3, 1Pathology, VAMC/UCSD, San Diego, CA, USA, 2Endocrinology, VAMC/UCSD, San Diego, CA, USA, 3AMS, Lawrence Livermore National Laboratories, Livermore, CA, USA.

Background: 41Ca is novel tracer technique that promises a direct measure of net bone calcium balance in both humans and in animal models.

Purpose: To establish a timeline for the excretion of 41Ca in normal mouse urine and to show the feasibility of monitoring prostate cancer induced bone disease.

Methods: 6 week old male mice were injected i.p. with 10 nCi 41Ca and 24 hour urines were collected once per week for 7 weeks. Feasibility of monitoring prostate cancer induced bone disease was demonstrated by dosing three groups of male immunocompromised mice (1 = saline/saline, 2 = tumor/saline, and 3 = tumor/bisphosphonate) with 10 nCi 41Ca. Three weeks later, the mice were injected i.p. with saline or zolendronate (5 ug/mouse). Four weeks after 41Ca dosing, the mice were injected intra-tibially with saline or 106PC-3-GFP cells. The saline or zolendronate injections were repeated every 4 weeks. Urine and fluorescent images were collected weekly from each mouse for 10 weeks.

Results: Steady-state 41Ca/Ca ratios were reached at 14 days after 41Ca injection and the 41Ca/Ca ratios were approximately 10,000-fold above baseline (see Figure 1). In the prostate cancer model, initial results demonstrate that the tumor can be visualized 2 weeks after prostate cancer cell injection and that zolendronate significantly reduces the severity of the bone lesion, as quantitated by x-ray scoring.

Conclusion: Successful implementation of a 41Ca assay in cancer patients with metastatic bone disease could have wide benefits, including improved risk assessment, monitoring treatment, and early detection of metastasis. 

Figure Figure 1..

Mean (+/-SEM) of urinary 41Ca/Ca following a 10 nCi dose of 41Ca (N = 2 or 3). Note y axis is logarithmic.

Disclosures: R.L. Fitzgerald, None.

This study received funding from: NIH.

T237

Breast Cancer Cells Inhibit Osteoblast Differentiation. J. E. Fong*1, D. Le Nihouannen*1, O. Hussein*1, P. M. Siegel*2, S. V. Komarova1, 1Dentistry, McGill University, Montreal, PQ, Canada, 2Medicine, McGill University, Montreal, PQ, Canada.

Skeletal metastasis is a major complication of advanced breast cancer which results in bone fractures and considerable pain burden. Osteoclasts recruited to the site of metastasis lead to the destruction of bone and formation of osteolytic lesions. Osteoblasts are known to contribute to osteoclast activation by producing a key pro-resorptive cytokine RANKL, however the effects of cancer cells on osteoblasts are not fully understood. We have studied the effects of MDA-MB-231 human breast carcinoma cells on proliferation and differentiation of primary osteoblast precursors. MDA-MB-231 cells were cultured until 80% confluent, and conditioned medium was collected after 24h of culturing. Bone marrow cells were isolated from the long bones of C57BL/6J mice, and treated for 5 days with ascorbic acid (50 μg/ml) in the presence or absence of MDA-MB-231 conditioned medium (10%). As a control, we used medium conditioned by MC3T3-E1 cells. Cell numbers were assessed in samples labeled with nuclear stain DAPI, and processed using image analysis. Treatment with ascorbic acid alone led to formation of 390 +/-90 cells/mm2. Addition of MDA-MB-231 conditioned medium to osteoblastic cultures led to a 2-3 fold increase in cell number, whereas treatment of cultures with MC3T3 conditioned medium had no effect, indicating that MDA-MB-231 cells produce factors that stimulate proliferation of osteoblastic cells. To assess osteoblast differentiation, the cultures were stained for alkaline phosphatase (ALP) and the labeled area was quantified using image analysis. Even at this early cultures, treatment with ascorbic acid alone led to significant increase in APL-positive cells, compared to untreated cultures. Addition of MDA-MB-231 conditioned medium to ascorbic acid-treated cultures resulted in a 5-fold decrease in ALP expression. Thus, our data indicate that soluble factors produced by breast cancer cells stimulate proliferation and inhibit differentiation of native osteoblasts. Since it has been previously shown that immature osteoblast precursors represent a major source of RANKL, compared to mature osteoblasts, our findings suggest a novel mechanism for osteoblast-mediated stimulation of osteoclasts that potentially contributes to the establishment and progression of metastatic lesions in bone.

Disclosures: J.E. Fong, None.

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

T238

TGF-β Increases Osteolytic Prostate Cancer Bone Metastases and Expression of Pro-metastatic Genes. P. G. J. Fournier, G. A. Clines, J. M. Chirgwin, T. A. Guise. Internal Medicine, University of Virginia, Charlottesville, VA, USA.

Prostate cancers commonly metastasize to bone, where high concentrations of TGF-β are released by osteoclastic resorption. TGF-β stimulates production of PTHrP, IL-11 and CTGF, which are central factors in bone metastases due to breast cancer and melanoma. We hypothesized that TGF-β would also promote prostate cancer bone metastases.

First, we showed that a specific inhibitor of the TGF-β type I receptor (TαRI) kinase, SD-208, inhibited TGF-β-dependent Smad2 phosphorylation in PC-3 prostate cancer cells in vitro. Mice were inoculated with PC-3 cells via the left cardiac ventricle. In both prevention and treatment protocols, SD-208 (50mg/kg/d) decreased osteolytic metastases and increased survival. To determine downstream targets of TGF-β in prostate cancer, we analyzed PC-3 cells treated with TGF-β (24h, 5ng/mL) by Affymetrix gene array with DMT and dCHIP data analyses. Significantly upregulated genes included known TGF-β targets PTHrP, CTGF, MMP-13, TSP-1 and ADAM19, which function in bone remodeling or are dysregulated in cancer.

The most increased gene was PMEPA1 (23.2-fold, P<0.03), a protein highly expressed in breast, colon and prostate cancers. Using real-time qPCR of RNA from PC-3 cells treated with TGF-β (5ng/mL, for 0 to 48h), we confirmed that PMEPA1 mRNA was rapidly induced and peaked at 24h (16.7-fold, P<0.05). TGF-β also increased PMEPA1 mRNA in prostate, breast and lung cancer lines. PC-3 cells were treated with SD-208, the transcription inhibitor actinomycin D, or the translation inhibitor cycloheximide; the results showed that TGF-β directly activates PMEPA1 transcription. TGF-β also increased PMEPA1 protein production in PC-3 by Western blot; the induction was prevented by SD-208. We cloned and made deletion mutants in 3.7kb of the PMEPA1 promoter, which contains 5 putative Smad binding elements. Dual-luciferase assays and overexpression of Smad2, −3 and −4, indicated that PMEPA1 transcription is regulated by TGF-β via both Smad-dependent and independent pathways.

PMEPA1 interacts with the E3 ubiquitin ligase Nedd4, which is related to the Smurfs. These proteins inhibit TGF-β signaling by targeting Smads and TβRI for proteasomal degradation. PMEPA1 could prevent proteasomal inhibition of the TGF-β pathway by suppressing Nedd4/Smurf activity, leading to sustained TGF-β signaling in bone metastases. Our data indicate that TGF-β promotes osteolytic bone metastases by stimulating known prometastatic factors, as well as novel factors that may enhance TGF-β signaling in the tumor cell. Thus, TGF-β inhibitors should be effective treatments for osteolytic prostate cancer bone metastases.

Disclosures: P.G.J. Fournier, None.

This study received funding from: Department of Defense - Prostate Cancer Research Program.

T239

Serum TRACP 5b Increases with Advancement of Osteolysis in a Nude Mouse Model of Breast Cancer Bone Metastasis. M. I. Suominen, R. Käkönen*, J. P. Rissanen, S. Ylönen*, J. M. Halleen. Pharmatest Services Ltd, Turku, Finland.

Serum tartrate-resistant acid phosphatase isoform 5b (TRACP 5b), a marker of osteoclast number, is significantly elevated in breast cancer patients with bone metastases. We investigated if elevated TRACP 5b values would indicate the development of bone lesions in a mouse model of breast cancer bone metastasis. MDA-MB-231(SA) breast cancer cells (n = 19) or PBS (n = 11) were inoculated into the left cardiac ventricle of five-week-old female nude mice. Serum samples were collected before the inoculation (day 0), at days 6, 9, 13, 16, and at sacrifice (day 23). Bone lesions were quantitated by radiography at days 16 and 23. Bone samples were collected for histomorphometric analysis at day 23, and osteoclasts were counted from TRACP-stained sagittal sections. Statistical analysis was performed using Student's t-test with necessary transformations after checking the assumptions for normality and homogeneity of variances. A p-value <0.05 was considered statistically significant. Pearson's correlation coefficients were calculated for cancer-inoculated mice. Body weight of the tumor-bearing animals was significantly lower compared with the PBS-controls at sacrifice due to tumor-induced cachexia. Osteolytic foci were found in 47.4% of the cancer cell-inoculated mice already at day 16, and all animals had lesions at sacrifice. Because the animals were growing rapidly during the study, their TRACP 5b values decreased in both groups during the first two weeks. At day 16, the TRACP 5b values reached a plateau in the control mice inoculated with PBS. However, in the cancer cell-inoculated mice, TRACP 5b values were elevated on day 16, and a sharp increase was observed between days 16 and 23. The changes in TRACP 5b values were significantly different in the cancer cell-inoculated mice than in the control mice on days 16 and 23. The TRACP 5b values correlated with osteolytic area determined by radiography on day 23. Furthermore, the TRACP 5b values correlated reversibly with body weight in tumor-bearing mice at sacrifice, whereas no correlation was observed in control mice. The histomorphometrically determined number of osteoclasts was radically increased, as expected based on the increased TRACP 5b values, and bone volume was decreased in the cancer cell-inoculated mice. Of note, large numbers of osteoclasts were found residing inside the tumor, with no bone surface nearby. These results demonstrate that TRACP 5b correlates with osteolysis and tumor-induced cachexia and is therefore a useful marker in the nude mouse model of breast cancer bone metastasis.

Disclosures: J.M. Halleen, SBA-Sciences 5, 7.

T240

Calcium Enhances Anti-tumor Effects of Ibandronate in Breast Cancer Cells. F. Journe1, N. Kheddoumi*1, C. Chaboteaux*1, G. Laurent*2, J. J. Body1, 1Institut Bordet, Brussels, Belgium, 2Université de Mons-Hainaut, Mons, Belgium.

Bisphosphonates now constitute standard therapy for the management of breast cancer-induced skeletal complications. Although their therapeutic effects mainly result from an inhibition of osteoclastic bone resorption, in vitro data indicate that bisphosphonates may also act directly on breast cancer cells by inhibiting their proliferation and inducing their apoptosis. This study examined the effects of Ca++ on the growth inhibitory activity of ibandronate in MDA-MB-231 and MCF-7 breast cancer cell lines. In presence of 0.6 mM Ca++, 30 μM ibandronate had no effect on MDA-MB-231 cell growth, and only a borderline effect on MCF-7 cell growth (13.6±6.6% inhibition). By contrast, in presence of 2 mM Ca++, ibandronate dramatically inhibited cell culture growth by 55.5±7.8% and 76.1±4.6% (p<0.01) in MDA-MB-231 and MCF-7 cells, respectively. Moreover, dose-response curves revealed that increasing Ca++ concentrations from 0.6 to 1.6 mM decreased ibandronate IC50 values of from 100 to 30 μM in MDA-MB-231 cells and from 60 to 10 μM in MCF-7 cells. In addition, whereas 30 μM ibandronate did not markedly induce cell apoptosis in presence of 0.6 mM Ca++, it significantly increased the percentage of apoptotic cells (annexin V-PE-positive) in presence of 1.6 mM Ca++ (11.4 and 32.9% in MDA-MB-231 and MCF-7 cells, respectively). Of note, Ca++ chelation by EGTA at a concentration which did not affect cell growth (0.5 mM) significantly reduced the growth inhibitory effect of 30 μM ibandronate in culture medium containing 1.6 mM Ca++. To address the effects of Ca++ on cell drug accumulation, cells were cultured in medium containing 0.6 or 1.6 mM Ca++ and were exposed to [14C]-ibandronate for 4 hours. In presence of 1.6 mM Ca++, cells accumulated much more [14C]-ibandronate than cells cultured in presence of 0.6 mM Ca++ (about 4.6 and 11.4-fold increases in MDA-MB-231 and MCF-7 cells, respectively). Finally, when evaluating the inhibition of protein prenylation, we found that high Ca++ level increased the intracellular activity of ibandronate. Thus, in both cell lines, 10 μM ibandronate were sufficient to produce a detectable inhibition of Rap1A prenylation in presence of 2 mM Ca++, while 100 μM ibandronate were required to achieve a similar effect in presence of 0.6 mM Ca++. In conclusion, our data indicate that extracellular Ca++, likely through its binding to ibandronate, modifies its cellular pharmacokinetics and increases its inhibitory activity on tumor cells. Thus, Ca++ released during the process of tumor-induced osteolysis could enhance the anti-tumor effects of bisphosphonates.

Disclosures: F. Journe, Hoffmann-LaRoche, Basel, Switzerland 2.

T241

Heparin-like Polysaccharides Markedly Reduce Osteolytic Bone Destruction and Tumor Growth in a Mouse Model of Breast Cancer Bone Metastasis. R. S. Kakonen*1, S. Kakonen2, K. S. Mohammad*3, J. P. Rissanen1, J. M. Halleen1, A. Warri*4, L. Nissinen*4, M. Pihlavisto*4, J. M. Chirgwin*3, M. Salmivirta*5, A. Marjamaki*4, T. A. Guise3, 1Pharmatest Services Ltd, Turku, Finland, 2Department of Anatomy, University of Turku, Turku, Finland, 3University of Virginia, Charlottesville, VA, USA, 4BioTie Therapies Corp, Turku, Finland, 5Turku Centre for Biotechnology, Turku, Finland.

Heparin treatment effectively prevents venous thromboembolism, but may also prolong survival in patients with malignant disease. Experimental studies support the hypothesis that cancer progression can be influenced by heparin and heparin-like glycosaminoglycans (HLGAGs), but their widespread use is hindered by their high anticoagulant activity. We have evaluated the therapeutic potential of two HLGAGs with low anti-coagulant activity, a low-molecular-weight synthetic heparin (fragmin), and a high-molecular-weight K5-derived heparin-like polysaccharide (K5-NSOS), to inhibit osteolytic bone destruction and tumor growth in an experimental mouse model of breast cancer bone metastasis. MDA-MB-231 breast cancer cells were inoculated into the left cardiac ventricle of athymic nude mice, which were then administered with Fragmin, K5-NSOS or vehicle once daily for 4 weeks. HLGAG-treated animals had significantly increased body weight when compared to vehicle. In addition, osteolytic lesion area as measured by radiography was significantly decreased by both HLGAGs. Histomorphometric examination revealed that both HLGAGs significantly reduced tumor burden in bone. In vitro studies showed that both HLGAGs inhibited significantly cancer cell adhesion, and K5-NSOS inhibited significantly bone resorption activity of human osteoclasts. Our data demonstrate that HLGAGs can efficiently reduce osteolytic lesion area and tumor burden in bone. K5-NSOS HMW is a potential antimetastatic and antiresorptive agent with low anticoagulant activity, and it could be further optimized as an anti-tumor agent.

Disclosures: R.S. Kakonen, Pharmatest Services Ltd 3, 4.

T242

Human Breast Stem Cell Transformation and Invasiveness Is Mediated Upregulation of IL-8 by TWIST Interactions with NFkB. S. E. Kendall*1, K. S. Aboody*2, S. P. Flanagan*3, C. A. Glackin1, 1Molecular Medicine, City of Hope/Beckman Research Institute, Duarte, CA, USA, 2Hematopoietic Cell Transplantation/Neurosciences, City of Hope/Beckman Research Institute, Duarte, CA, USA, 3Neurosciences, City of Hope/Beckman Research Institute, Duarte, CA, USA.

Interleukin (IL-8), a prototypical chemokine has been shown to play an important role in tumor growth, angiogenesis and metastasis. Transcriptional activation of IL-8 is critically dependent on NFkB, but can be modulated by several transcription factors, i.e. AP-1, C/EBP and NRE. TWIST, a basic helix-loop-helix transcription factor has been shown to bind to NFkB and transcriptionally repress two pro-inflammatory cytokines, IL-1β and TNFα at E-box consensus sites on their respective promoters. We have previously demonstrated that TWIST over-expression in benign human breast cancer cells (MCF-7) significantly enhances their growth and invasiveness though an epithelial to mesenchymal transition (EMT) process. In our current studies, designed to delineate these early transformation events, we over-expressed TWIST in the MCF10A human breast luminal epithelial cell line. Results display a phenotypic change in their cell surface marker expression (ESA+, Mucl, CD49f+), reminiscent of breast stem cells. Affymetrix microarray analysis confirmed EMT associated differential gene expression where epithelial markers such as Keratins and E-cadherins were down regulated and mesenchymal markers such as vimentin were upregulated. Western blot analysis also reflected EMT related changes, such as the E- to N-cadherin switch. However, elevated levels of cytokines (IL-1α. IL-8, uPAR?) and their target genes (NFkB, MMP9, uPA) were observed, indicating that alterations in these inflammatory signaling pathways were being affected by TWIST overexpression. Protein array screening of conditioned media from TWIST over-expressing cells confirmed significantly elevated levels of IL-8. Moreover, TWIST and NFkB form a transcription factor complex that interacts to enhance IL-8 expression through the NFkB consensus site on the IL-8 promoter. The signals produced through CXCR1 and CXCR2 binding activate MMP9 gene expression. Importantly, TWIST over-expressing cells show a significant level of MMP9 activity that facilitates their invasion towards osteocytes. Collectively our results suggest that TWIST over-expression in breast epithelial cells promotes their transformation to a stem cell- like state and enhances their invasive capabilities through the creation of an IL-8 feedback loop. Thus, TWIST may play a significant role in the transformation of normal/benign breast cells towards malignancy.

Disclosures: S.E. Kendall, None.

This study received funding from: NIH/NCI.

T243

Zoledronic Acid Delayed the Wound Healing of Tooth Extraction Socket but Failed to Cause Osteonecrosis of the Jaw in Mice. Y. Kobayashi*1, T. Hiraga*2, A. Ueda1, R. Nishimura1, H. Yatani*3, T. Yoneda1, 1Biochem, Osaka Univ Grad Dent, Suita, Japan, 2Oral Anat, Matsumoto Dent Univ, Matsumoto, Japan, 3Fixed Prosth, Osaka Univ Grad Dent, Suita, Japan

Bisphosphonates (BPs), particularly nitrogen-containing BP such as zoledronic acid (ZOL) and pamidronate, have been widely- and beneficially-used for the treatment of cancer patients with bone metastases and/or hypercalcemia. However, observations are accumulating that cancer patients who have received these BPs occasionally show osteonecrosis of the jaw (ONJ) following dental treatments, especially tooth extraction. To understand the underlying mechanism of ONJ, we examined the effects of ZOL on the wound healing of tooth extraction socket. C57BL/J mice (6 week-old, male) were given ZOL (5μg/mouse, ip) once a day. After 7 days, the upper right first molar was extracted under anesthesia. Administration of ZOL was continued until 5 days after the tooth extraction. There was no marked histological difference in the wound healing of extraction sockets between control and ZOL-treated group until day 3. At day 5, histomorphometrical analysis revealed active new bone formation and substantial numbers of CD3-positive blood vessels in control mice. In contrast, the amount of new bones and the numbers of TRAP-positive osteoclasts and CD31 -positive blood vessels were significantly decreased in ZOL-treated mice, indicating the delay of the socket healing. The non-nitrogen-containing BP etidronate (ETI, 5μg/mouse, ip) showed no effects on the healing. In the dorsal air sac (DAS) assay in which Millipore chambers containing VEGF and/or ZOL were implanted in the skin at the back of mice, ZOL significantly inhibited the angiogenesis promoted by VEGF. Moreover, ZOL (5-50μM), but not ETI, inhibited the proliferation of bone marrow endothelial cells and human endothelial-like cells in monolayer and collagen gel in a dose-dependent manner. ZOL also inhibited the capillary tube formation of human umbilical vessel endothelial cells on matrigel. Finally, we examined the effects of ZOL combined with doxorubicin (100μg/mouse, ip) and/or dexamethazone (400μg/mouse, ip) as a clinically-relevant setting. Contrary to our expectation, however, combination of these agents did not further delay the healing than ZOL alone. Of note, we did not observe ONJ following the treatment with each or combination of these agents. In conclusion, our results suggest that ZOL delays the wound healing of tooth extraction sockets through inhibiting new bone formation and angiogenesis. However, ZOL alone or combined with steroid and/or anti-cancer agent failed to cause ONJ in our model. Additional unknown factors may be involved in the pathogenesis of ONJ seen in cancer patients who have been treated with BPs.

Disclosures: Y. Kobayashi, None.

T244

Oateoblast - Prostate Cancer Cell Signaling: Role of Hedgehog Pathway. M. L. G. Lamm, T. Douglas*, Pediatrics, Northwestern University Feinberg School of Medicine, and Children's Memorial Research Center, Chicago, IL., USA

The key to understanding the proclivity of metastatic prostate carcinoma to bone lies in unraveling unique interactions between invading prostate cancer cells and host bone stroma including bone cells and extracellular matrix. The purpose of the present study is to test the hypothesis that Sonic hedgehog signaling between prostate cancer cells and osteoblasts promotes osteoblast differentiation, a requisite step toward metastatic prostate tumor formation in bone. Sonic hedgehog (Shh) is a secreted ligand that mediates epithelial-mesenchymal interactions critical to normal morphogenesis of many developing organs including the prostate. Shh signaling activates the expression of genes for the transcription factor Glil and the Shh receptor Ptcl in target cells. The genes for Shh, Glil, and Ptcl are expressed in human prostate cancer specimens including prostate metastatic carcinoma. We have previously shown that human prostate cancer cells, LNCaP, which are genetically-engineered to express high levels of Shh (designated LNCaP-Shh cells) upregulate Glil and Ptcl expression in stromal cells and promote xenograft prostate tumor growth in a mouse model.

In the present study, LNCaP-Shh cells and vector-transfected control LNCaP cells were co-cultured with mouse calvaria-derived non-transformed osteoblast precursor cells MC3T3-E1. Cells were co-cultured for varying periods of time either as mixed populations within the same culture chamber or separate populations in two chambers that share the same culture medium. Gene expression analysis was performed by real time quantitative RT-PCR using species-specific primers to differentiate gene expression between human and mouse cells. Staining for alkaline phosphatase activity was used as a marker of osteoblast differentiation.

Data show that Shh-expressing prostate cancer cells upregulate Glil and Ptcl expression in osteoblasts and, this effect is inhibited by cyclopamine and dependent upon Gli transcriptional activity. Shh signaling in osteoblasts increases alkaline phosphatase activity and upregulates expression of osteopontin indicative of osteoblast differentiation. These results demonstrate that Shh signaling between prostate cancer cells and osteoblasts promotes osteoblast differentiation, and implicate the hedgehog signaling pathway in the development of osteoblastic prostate cancer metastasis.

Disclosures: M.L.G. Lamm, None.

This study received funding from: National Cancer Institute.

T245

Non-isomerized C-telopeptides of Type I Collagen (ALPHA CTX) for Early Detection and Treatment Monitoring of Bone Metastases Secondary to Prostate Cancer. P. J. Leeming1, A. Hegele*2, M. A. Karsdal1, P. Ovist1, P. Olbert*2, C. Christiansen*1, I. Byrjalsen1, 1Nordic Bioscience, Herlev, Denmark, 2Department of Urology, Philipps University, Medical School, Marburg, Germany.

Skeletal spread of metastasis represents a devastating event, which should be detected as early as possible prior to that of traditional imaging techniques. The objective of this study was to compare a novel biochemical marker of cancer mediated bone resorption, ALPHA CTX, to that of prostate specific antigen (PSA) and total alkaline phosphates (tALP) in patients with prostate cancer.

Two studies were performed. 1) Cross-sectional study of 170 prostate cancer patients without bone metastases (-BM) prior to radical prostatectomy, including 24 patients with lymph node metastases (+LN), compared to 68 controls. 2) Prospective study of 40 hormone refractory stage prostate cancer patients including 20 with bone metastases (+BM). Patients received four treatment cycles during four months of 35mg/m2 docetaxel in week 1, 2, and 3 (all patients) and 4mg zoledronate in week 1 (only +BM). Urine samples were collected at 0, 1, 2, 3 and 4 months and assessed in ALPHA CrossLaps (ααCTX) (Nordic Bioscience), PSA and tALP (Roche Diagnostics). Presence of bone metastases was determined by bone scans and verified by X-ray. Adjacent bone metastases sections from prostate cancer patients were stained for the presence of tumor cells (anti-cytokeratin antibody), osteoclasts (TRAP activity) and αCTX (anti-αCTX antibody).

PSA was elevated in both -LN and +LN patients compared to controls (p<0.001), ααCTX was only elevated in +LN patients (p<0.01); in contrast no difference in tALP was detected. In the prospective study, only ααCTX decreased significantly, −52% (p<0.05) and 62% (p<0.01) in the combined zoledronate/docetaxel group at week 4 and 16, respectively. The suppression of PSA and tALP in the +BM group was similar to the suppression induced by docetaxel in the-BM group. Immunohistochemistry revealed accumulation of TRAP+ osteoclasts and intense staining for αCTX epitopes in the proximity of tumor cells.

ααCTX was increased in +LN, indicating that these patients already had sustained skeletal spread as detected prior to that of imaging techniques, representing a high-risk group that need to be closely monitored. ααCTX in these patients predicts presence of skeletal spread of the malignancy, presently undetected by bone scans, and may thus be of great benefit for early detection of metastasis. In the prospective study, ααCTX decreased in zoledronate/docetaxel treated +BM patients to levels below docetaxel-treated -BM patients, emphasizing the skeletal specificity of this markers in contrast to PSA and tALP.

Disclosures: D.J. Leeming, Nordic Bioscience 3.

T246

The αvβ3 Integrin Inhibitor MK-0429 Is Generally Safe and Decreases Bone Turnover in Men with Hormone-Refractory Prostate Cancer (HRPC) and Bone Metastases (MBD). M. Rosenthal*1, P. Davidson*2, E. Rolland*3, A. Lombardi4, Y. Berd*4, W. He*4, L. Wehren4, 1Melbourne Comprehensive Cancer Centre, Parkville, Australia, 2CURT Medical Trials Trust, Christchurch, New Zealand, 3Centre Gauducheau Medecine Oncologie, Saint Herblain, France, 4Merck & Co., Inc., Rahway, NJ, USA.

Background: Osteoclast adhesion to bone surface, essential for bone resorption, is mediated by αvβ3 integrin. Inhibition of this activity might slow the development or progression of MBD. MK-0429 is an oral αvβ3 inhibitor being evaluated in MBD in patients (pts) with HRPC.

Materials and methods: This multicenter Phase 1 study enrolled 21 pts with HRPC and MBD. Pts were randomized to receive either 200 mg (n = 5) or 1600 mg (n = 16) b.i.d. of MK-0429 for 4 weeks, with an optional open-label extension of 48 weeks. The study examined the toxicity and pharmacokinetics of MK-0429 in this population, as well as its effect on markers of bone turnover, urinary cross-linked N-telopeptides of type I collagen to creatinine ratio (uNTx), and bone-specific alkaline phosphatase (BSAP).

Results: Eighteen pts (86%) completed 4 weeks of treatment. All pts in the 200-mg group and 8 pts (50%) in the 1600-mg group subsequently entered the extension study. Nausea (Grade 1 or less commonly Grade 2) occurred in 11 pts in the 1600-mg group, and 1 pt in the 200-mg group. Only 1 Grade 3 event (anemia) occurred. Two pts in the 1600-mg group discontinued treatment (1 due to spinal cord compression, the other to nausea), and an additional pt withdrew consent. At 4 weeks, the mean AUC0-12hr and Cmax values were 210 μM*hr and 42 μM in the 200-mg group and 673 μM*hr and 154 μM in the 1600-mg group, respectively. Baseline and end-of-treatment values of the biochemical markers are displayed in the table.

Conclusions: In this Phase I study, MK-0429 was generally safe in pts with HRPC and MBD, with the most common side effect being nausea. There was evidence of an early reduction in biochemical markers of bone turnover. 

Table  .  
  1. *Presented as mean (standard deviation) at baseline; geometric mean % change from baseline (standard error) after treatment.

inline image

Disclosures: A. Lombardi, Merck & Co., Inc. 1, 3.

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

T247

PTHrP-induced MCP-1 Production by Human Bone Marrow Endothelial Cells and Osteoblasts Promotes Osteoclast Differentiation In Vitro: A Novel Role in Prostate Cancer Bone Metastasis. Y. Lu1, G. Xiao1, Z. Cai*1, X. Chen*1, D. L. Galson1, Y. Nishio*2, A. Mizokami*3, J. Zhang1, 1Medicine, University of Pittsburgh, Pittsburgh, PA, USA, 2Medicine, Shiga University of Medical Science, Shiga, Japan, 3Urology, Kanazawa University, Kanazawa, Japan.

Prostate cancer (PCa) preferentially metastasizes to bone resulting in osteoblastic lesions with underlying osteolytic activities. The mechanisms by which PCa cells promote osteolytic activities and subsequent osteoblastic bone formation remain poorly understood. Parathyroid hormone-related protein (PTHrP), produced by bone cells and PCa, binds to receptors on osteoblasts and stimulates bone formation and resorption. We have previously reported that MCP-1 acts as a paracrine and autocrine factor for PCa progression. However, the role of PTHrP in regulating MCP-1 expression in bone microenvironment, specifically by human bone marrow endothelial cells (HBME) and osteoblasts (hFOB), as well as by PCa cells, has not been studied. Accordingly, we determined the effect of PTHrP on MCP-1 expression by bone cells and PCa cells. PTHrP induced both MCP-1 protein and mRNA expression by HBME and hFOB cells, but not by PCa LNCaP and PC3 cells. To further determine the mechanisms of PTHrP-induced MCP-1 transcription, analysis of the MCP-1 promoter was performed. MCP-1 promoter activity was induced by PTHrP. Both C/EBPβ and NF-kB binding elements are required for PTHrP-induced MCP-1 transcription. Finally, when a constitutively-active PTH receptor construct was transfected into HBME and hFOB cells, MCP-1 production was increased. The conditioned media collected from these cells induced osteoclast differentiation and PC3 proliferation and invasion in vitro. These effects were partially inhibited by MCP-1 neutralizing antibody. We conclude that PTHrP-induced MCP-1 production by HBME and hFOB cells promotes osteoclast differentiation in vitro and such induction may play a critical role in PCa development in the bone microenvironment.

Disclosures: Y. Lu, None.

T248

Activation of MCP-1/CCR2 Axis Promotes Prostate Cancer Invasion and the Tumor-induced Osteoclast Activity In Vitro. Y. Lu1, Z. Cai*1, X. Chen*1, G. Xiao1, A. Mizokami*2, G. D. Roodman1, J. Zhang1, 1Medicine, University of Pittsburgh, Pittsburgh, PA, USA, 2Urology, Kanazawa University, Kanazawa, Japan.

When prostate cancer (PCa) metastasizes to the skeleton, bidirectional interactions between tumor cells and the bone microenvironment occur. Both the tumor cells and cells from the bone microenvironment produce growth factors, angiogenic factors, and chemotactic factors which enhance tumor growth. These factors may account for the increased frequency of bone metastasis in PCa. Monocyte chemotactic protein-1 (MCP-1) is a member of the CC chemokine superfamily that plays a critical role in recruitment and activation of monocytes during acute inflammation and angiogenesis. We previously showed that MCP-1, produced by PCa epithelial cells, stroma/osteoblasts, and bone marrow endothelial cells, is chemotactic for PCa cells. More recently, we reported that CCR2 expression correlates with Gleason score and clinical pathological stages as determined by immunohistochemistry. We hypothesized that the MCP-1/CCR2 axis plays a critical role in PCa invasion and the tumor-induced osteoclast activity. Accordingly, to determine whether the expression of CCR2 on PCa cells play a fundamental role in PCa invasion and proliferation, CCR2 or control shRNAs were transfected into human C4-2B or PC3 cells using lipofectamine reagents and individual clones were selected and evaluated. CCR2 knockdown in both C4-2B and PC3 cells significantly diminished the MCP-1 -induced cell invasion in matrigel assays. Furthermore, to determine whether MCP-1 knockdown in PCa cells alters the PCa conditioned media (CM)-induced osteoclast formation, MCP-1 or control shRNA were generated and transfected into C4-2B or PC3 cells. CM were collected from 4 clones and evaluated for their capacity to induce osteoclast formation. Non-adherent mouse bone marrow cells were cultured for 7 days with M-CSF (10ng/ml) and/or RANKL (50ng/ml) or 10% CM collected from the stable clones. We found that MCP-1 knockdown significantly diminished the capacity of PCa CM to induce osteoclast formation. Taken together, we have demonstrated that activation of MCP-1/CCR2 axis promotes prostate cancer invasion and the tumor-induced osteoclast activity in vitro. Our data suggest that MCP-1/CCR2 axis may serve as a therapeutic target.

Disclosures: Y. Lu, None.

T249

The Constitutive Activity of the NF-kB p65/p50 Transcriptional Complex is Critical for Bone Metastasis in Breast Cancer. C. Menaa*1, A. Moscovitz*2, S. Kim*1, C. J. Froelich*1, S. M. Sprague1, 1Department of Medicine, Evanston Northwestern Healthcare, Northwestern University Feinberg School of Medicine, Evanston, IL, USA, 2Department of Medicine, Hadassah University Medical Center, Jerusalem, Israel.

Long-term breast cancer (BC) survival decreases dramatically in patients with bone metastases and represents a major challenge for treatment. This is due in large part to the capacity of tumor cells to induce osteoclastogenesis. How tumor cells acquire this capacity and the mechanism(s) involved are not yet defined. We previously reported that NF-kB is a key pathway for bone metastasis. Here we demonstrate that the NF-kB p65/p50 transcriptional complex controls the capacity of BC cells (MDA and MCF7) to support OC formation in vitro and tumor growth and bone metastasis in vivo. The over-expression of p65 DN significantly reduced the production of osteoclastogenic factors (OF), including PTHrP expression in vitro. Furthermore, the p50 subunit is necessary to form the active transcriptional complex with the p65 subunit in BC. The transdominant negative form of p50, where the DNA binding capacity has been disrupted, significantly reduced the constitutive activation of NF-kB in BC and the transcriptional activity of the p65 as measured by gel shift and reporter gene assays. Moreover, stable BC cells expressing p50 mutants where (A56/L-F57/A) or (Y59/A-V60/S) are unable to support osteoclastogenesis and PTHrP production in vitro. The intra-cardiac injection of the wild type cells into nude mice results in diffuse bone metastasis, whereas the injection of the p50 mutant cells do not lead to osteolytic lesions as assessed by X-Rays and histological analyses. This effect was not due to a reduction in cell viability and/or proliferation, since p50 mutant cells form tumors similarly to the WT cells when injected subcutaneously, but lack the ability to induce osteoclastogenesis. In addition, BC p50 mutant cells are less invasive and aggressive compared to the WT. We specifically found that the transcriptional factor snail is down-regulated in non-bone metastatic BC (MCF7) as well as MDA-p50 mutant cells compared to MDA WT cells. These data suggest that snail could be the down stream factor for NF-kB that is responsible for bone metastases.

In summary, we found that the NF-kB p65/p50 transcriptional complex is essential for the NF-kB constitutive activation and the capacity of BC to form osteolytic metastasis by controlling expression and secretion of osteoclastogenic factors such as PTHrP which could be mediated by snail transcription factor. In conclusion, these data further highlight the role of NF-kB in bone metastasis in breast cancer. Thus, NF-kB (p65/p50 complex) could be a suitable therapeutic target for bone destruction in breast cancer.

Disclosures: C. Menaa, None.

This study received funding from: NIH.

T250

High Extracellular Calcium Directly Stimulates MDA-MB 231 Human Breast Cancer Cell by a Mechanism Involving the Calcium Sensing Receptor. R. Mentaverri*, R. Abdoune*, J. Lion*, M. Brazier, S. Kamel*, Pharmacy, INSERM, ERI-12, Amiens, France.

Bone is the most common site of breast cancer metastasis and skeletal metastases greatly contribute to the morbidity and mortality associated to the the disease. Over the past several years, significant effort has been focused on elucidating the molecular mechanisms that govern bone metastasis. However, so far, the factors that regulate the preferential metastasis of breast cancer cells in bone are not well identified. Recently, it has been demonstrated that cancer cells invade preferentially the bone microenvironment subjected to high bone remodeling suggesting that some factors released during high bone turnover can accelerate the development of cancer cell in bone. Here we show that high extracellular calcium concentrations ([Ca]e) can trigger migration of human MDA-MB 231 breast cancer cells, in Boyden's chambers. Thus, when breast cancer cells were exposed to different concentrations of calcium (from 1.8 to 7.5 mM) the mobility of the cells was dose-dependently stimulated. The maximum effect was obtained at 5mM where the cell mobility was approximately three fold increased compared to cell submitted to control medium ([Ca]e = 1.8 mM). Using pharmacological inhibitors known to selectively block the ERK1/2 and phospholipase C pathway (i.e. U-0126 and U-73122), we completely abrogated the [Ca]e-induced effect, suggesting the implication of a G Protein Coupled Receptor. Because these two pathways are known to be triggered by the calcium sensing receptor (CaR), we first confirmed that beast cancer cell expressed both at transcriptional and at protein level the CaR. Finally, confirming the role played by the CaR in this process, we demonstrated that small interfering RNA (SiRNA) targeting of the CaR almost completely reversed the effects of calcium on cancer cell mobility. Taken together, these data demonstrate that high [Ca]e, such as those seen in the bone microenvironment, can directly stimulate the breast cancer cell mobility by a mechanism involving the CaR. From these results, it can be hypothesized, that high [Ca]e may participate to processes allowing tumor cells which express the CaR to invade and proliferate into the bone.

Disclosures: R. Mentaverri, None.

T251

Skeletal Effects of 2-Methoxyestradiol in Combination with other Prostate Cancer Therapies. K. S. Mohammad1, V. Duong*1, L. Kingsley1, C. R. McKenna*1, H. Walton*1, X. Peng*1, L. J. Suva2, T. A. Guise1, J. M. Chirgwin1, 1Internal Medicine, University of Virginia, Charlottesville, VA, USA, 2Orthopaedic surgery, University of Arkansas for Medical Sciences, Little Rock, AR, USA.

The majority of patients with hormone-refractory advanced prostate cancer develop bone metastases, in which the characteristic disorganized new bone formation results from the paracrine stimulation of osteoblasts by tumor cells. Many of the secreted proteins that activate osteoblasts are regulated by the hypoxic response pathway via induction of HIF-1α. We hypothesized that anti-hypoxic therapy would be effective against bone metastases and tested this idea with 2-methoxyestradiol (2ME2), an inhibitor of microtubule polymerization and the Hif-1α pathway that is currently in phase II clinical trials in cancer patients.

2ME2 was used alone and in combination with two established treatments for bone metastases: zoledronic acid (ZA), and the endothelin A receptor antagonist, atrasentan. We assessed the skeletal effects of treatments on tumor-free bones in 8 groups of nude mice: vehicle control, 3 single-treatment groups, 3 double-treatment groups and 1 triple-treatment group. Mice were treated for 20wks, followed by X-ray and Piximus for bone mineral density and analyzed after euthanasia by histomorphometry and micro-CT of selected bones. Atrasentan (20mg/kg/d) had no substantial effect on bone parameters when used alone or in combination with ZA or 2ME2 as determined by X-ray, histomorphometry and micro-CT. Histomorphometric analysis of mice treated with ZA (5ug/kg/3x/wk sc) showed an increase in trabecular bone volume fraction (BV/TV%). Micro-CT revealed a similar increase in BV/TV, with a parallel increase in trabecular number and a reduction in trabecular separation. Across all single, double and triple treatment groups, we observed that 2ME2 (150mg/kg ip) increased bone mass. Histomorphometric analysis showed increased BV/TV and more osteoclasts in 2ME2-treated mice. Micro-CT demonstrated a similar increase in BV/TV and a parallel increase in trabecular thickness and reduction in trabecular separation. The groups in which 2ME2 and ZA were combined showed significant additive effects on bone mass, with even greater increases in BV/TV and trabecular thickness and decreased trabecular separation and osteoclast number.

The data demonstrate that 2ME2 increased bone mass and osteoclast number. Inhibition of osteoclasts with ZA further increased bone mass. Preclinical studies are underway to see if these additive therapeutic effects also reduce prostate cancer tumor burden in bone. The experiments will test if 2ME2 is selectively active against bone metastases and if it is more effective in combination with ZA than either agent alone.

Disclosures: K.S. Mohammad, None.

T252

Tissue Engineered Bone: Components of Native Bone Environment Involved in Breast Cancer Metastasis. J. E. Moreau1, K. Anderson*1, M. Reagan*2, D. L. Kaplan*2, M. Rosenblatt1, 1Physiology, Tufts University School of Medicine, Boston, MA, USA, 2Biomedical Engineering, Tufts University, Medford, MA, USA.

Challenges in breast cancer (BrCa) treatment are due to the migration of primary tumor cells to the skeleton, initiating osteolysis, formation of metastases, and the entry of the disease into an incurable phase. Tissue engineered bone constructs afford a controllable microenvironment and have been confirmed as efficacious targets for metastatic spread using a mouse model of metastasis originating from the orthotopic site. We continued assessment of the contribution of individual components of engineered bone present upon initiation of metastasis cells, scaffold, and protein to begin characterizing the metastatic bone environment. Specimens of porous silk-based tissue engineered bone and native human bone (controls) were prepared for subcutaneous implantation in immunodeficient (NOD/SCID) mice. Orthotopic injection of the luciferin-expressing human epithelial breast cancer cell line, SUM1315, for assessment of cell migration was subsequently performed one month following implantation. Four groups of scaffolds untreated silk, bone morphogenic protein 2 (BMP2) coupled, bone marrow stromal cell (BMSC) seeded, and BMP2/BMSC treated were prepared and implanted subcutaneously over the left shoulder, with native human bone controls cored from discarded femoral heads over the right, BMSC were administered to scaffolds one day prior to implantation in their respective treatment groups. One month following implantation, SUM1315 cells were injected into mouse mammary fat pads and monitored over the course of 12 weeks. Luminescent imaging of implants at final harvest revealed the presence of metastases in 0 of 6 untreated scaffolds, 4 of 4 with BMSC treatment, 4 of 4 with BMP2 treatment, and 1 of 6 with BMP2/BMSC treatment. Metastatic spread to engineered scaffolds and the progression of bone remodeling was further confirmed through immunohistochemistry, labeling cytokeratin 5/6 for BrCa identification, trichrome for collagen I and early bone development, and von Kossa staining of calcium deposit. All metastatic spread was exclusive to the implants, with no invasion of the mouse skeleton, as confirmed via luminescent imaging. These findings provide impetus for continued research to explore the roles of bone proteins in breast cancer recruitment, the nature of the developing bone scaffold as a homing site, and the differentiation/influence of adult stem cells in creating a niche for metastatic spread.

Disclosures: J.E. Moreau, None.

T253

Metastasis-Associated Protein 1 (MTA1) was Highly Expressed and Localized in Nucleus in Prostate Cancer Bone Metastasis. J. Wang. A. Levenson*, J. Jarrett*, W. Laskin*, R. Satcher*, Northwestern University, Chicago, IL, USA.

Bone is the most frequent target site for prostate cancer metastasis. The preferential metastasis to bone suggests that bone microenvironment provides suitable conditions for tumor growth and survival. Bone marrow stromal cells (BMSC) play an active role in regulating colonization and progression of metastatic malignant epithelial cells through diffusible factors and cell-cell contact. The molecular mechanism responsible for stromal-epithelial crosstalk in bone microenvironment remains largely unknown.

We established a novel co-culture system with direct heterotypic cell-cell contact. Five different prostate cancer cell lines (PC3, DU145, LNCaP, C4-2, MDA-PCa2b) were labeled with fluorescence and co-cultured with BMSC respectively. Two types of cells were then separated by fluorescence-activated cell sorting. Gene expression profiles were analyzed using cDNA microarray. Among the most significantly modified genes, metastasis-associated protein 1 (MTA1) was found to be highly up-regulated in multiple prostate cancer cell lines during the co-culture. The expression of MTA1 was further studied in normal prostate tissue, primary prostate tumor, and bone metastasis tissue using tissue microarray and immunohistochemistry. The results showed that in normal prostate tissue (n = 15) there was weak staining in cytoplasm (Intensity = 0.8±0.4, Frequency = 1.1±0.7) but no staining in nucleus. In contrast, in primary prostate cancer tissue (n = 72) there was moderate staining both in cytoplasm (I = 1.8±0.7, F = 3.3±1.2, P = 0.000002 vs normal tissue) and in nucleus (I = 2.1±0.6, F = 3.6±0.8). There was no significant correlation between MTA1 expression level with Gleason score, TNM grade or PSA level. In bone metastasis tissue (n = 8), there was no staining in cytoplasm, but strong staining in nucleus (I = 2.6±0.5, F = 3.4±0.5, P = 0.04 vs primary tumor).

We concluded that MTA1 was differentially expressed in normal prostate tissue, primary tumor and bone metastasis tissue. The differential localization in cytoplasm and nucleus of MTA1 may indicate the degree of metastasis potential. As a member of nucleosome remodeling complex, the translocation of MTA1 between cytoplasm and nucleus in primary tumor and bone metastasis may conduct different function on transcription regulation and signaling transduction.

Disclosures: J. Wang, None.

T254

Regulation of Protein Synthesis Factors in Estrogen Metabolite-Mediated Inhibitions of Osteosarcoma Cells. K. L. Shogren*1, M. J. Yaszemski1, T. E. Hefferan1, M. C. Charlesworth*2, B. J. Madden*2, R. T. Turner3, A. Maran1, 1Orthopedic Research, Mayo Clinic, Rochester, MN, USA, 2Proteomics Research Center, Mayo Clinic, Rochester, MN, USA, 3Nutrition and Exercise Sciences, Oregon State University, Corvallis, OR, USA.

Protein biosynthesis is one of the fundamental processes involved in both the regulation of normal eukaryotic cell growth and tumorigenesis. Previous studies show that a natural metabolite of 17β-estradiol, 2-Methoxyestradiol (2-ME) can induce growth inhibition and cell death in human osteosarcoma cells. 2-ME treatment leads to the induction of apoptosis and the activation of RNA dependent protein kinase (PKR). Here, we have further investigated the molecular mechanisms associated with 2-ME-dependent cell death in human osteosarcoma cells. We employed 2-dimensional gel electrophoresis followed by trypsin digestion of the 2D spots. Subsequent analysis of the peptides by nano-LC-ESI tandem mass spectrometry combined with the Swiss-Prot database identified several proteins that participate in the mammalian protein synthesis. Western blot analysis using MG63, KHOS and SAOS2 osteosarcoma cells, further validated the proteomic results and revealed a significant down-regulation of proteins involved in translational regulation. Another protein synthesis initiation factor, EIF5A-2, which has been implicated in tumorigenesis was down-regulated by 2.5-fold within 24hrs of 2-ME treatment. These findings are consistent with the studies showing that 2-ME treatment results in the activation of PKR and subsequent phosphorylation of the initiation factor EIF-2. Taken together, these studies suggest that multiple translational control pathways are modulated by 2-ME treatment and the deregulation of factors catalyzing the various steps of polypeptide chain synthesis contributes to apoptosis and anti-osteosarcoma effects of 2-ME.

Disclosures: A. Maran, None.

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

T255

Tyrosine Kinase Inhibitors Slow Motility of Osteosarcoma Cells In Vitro. P. J. Messerschmitt*, E. M. Greenfield. Orthopaedics, CWRU, Cleveland, OH, USA.

We determined whether tyrosine kinases regulate osteosarcoma cell motility, a critical component of metastasis. Three genetically-related human osteosarcoma cell lines were used: TE85: non-tumorigenic and non-metastatic; MNNG: tumorigenic, non-metastatic; and 143B: tumorigenic, highly metastatic. Motility assays measured migration distance, the width between two converging cell fronts, in a scrape created in confluent cultures. All tyrosine kinase inhibitors were tested at maximally effective concentrations based on the literature. In vitro motility correlated with metastatic ability as the 143B cells migrated 65% (p<0.001) faster than the non-metastatic cells. These differences are not due to proliferation, since aphidicolin, a DNA polymerase inhibitor, did not affect migration, but completely blocked proliferation.

Inhibitors of HER-2, NGF-R, PDGF-R, and JAK had little effect on the motility of all cell lines. In contrast, the EGF-R inhibitor (Calbiochem #324674) slowed motility of all cell lines by 50-75% (p<0.001). The met inhibitor (SU11274) slowed motility of MNNG cells by 80% (p<0.001), while only slowing motility of the 143B and TE85 cells by 20% (p<0.001). This is consistent with the finding that tumorigenesis in the MNNG cells is due to a chromosomal translocation resulting in overexpression of met. Suramin slowed motility of the MNNG and TE85 cells by 60% (p<0.001) and 41% (p<0.001), but only slowed motility of the 143B cells by 11% (p = 0.012). Suramin prevents growth factor binding and thereby inhibits activation of many receptor tyrosine kinases, including activation of met by Hepatocyte Growth Factor (HGF). The preferential inhibition of MNNG motility suggests that met-dependent motility in these cells is due to autocrine production of HGF. The molecular basis for inhibition of TE85 motility remains to be identified. 3,4-Methylenedioxy-β-nitrostyrene (MNS), a newly identified tyrosine kinase inhibitor, preferentially slowed motility of the metastatic 143B cells by 53% (p<0.001), while only slowing motility of the MNNG cells by 25% (p<0.001) and not detectably affecting motility of the TE85 cells. MNS inhibits the tyrosine kinase acitivity of both Syk and Src. However, other inhibitors of Syk and Src, either alone or in combination, had no detectable effect on 143B motility. Thus, it is likely that MNS slows motility by inhibiting another, as yet unidentified, tyrosine kinase. Nonetheless, MNS is an attractive, potential therapeutic agent for further study as it preferentially slowed motility of the metastatic cell line. Our results also show that inhibitors of EGF-R and met are attractive therapeutic agents for patients with osteosarcoma that overexpress those receptors.

Disclosures: P.J. Messerschmitt, None.

T256

Fracture Risk Is Increased in Danish Men with Prostate Cancer. A Nationwide Register Study. M. F. Nielsen1, K. Brixen2, S. Walter*3, J. Andersen*4, P. Eskildsen*5, B. Abrahamsen6, 1Dept. of Endocrinology, Odense University Hospital, Odense, Denmark, 2Dept of Endocrinology, Odense University Hospital, Odense, Denmark, 3Urology, Odense University Hospital, Odense, Denmark, 4Dept. of Urology, Roskilde Hospital, Roskilde, Denmark, 5Dept. of Internal Medicine, Koege Hospital, Koege, Denmark, 6Dept. of Medicine, Copenhagen University Hospital Gentofte, Copenhagen, Denmark.

The purpose of this study was to examine the effect of prostate cancer on fracture risk and calculate the national impact of prostate cancer on hip fracture incidence, while taking into account any modifying effects of orchiectomy and androgen deprivation therapy (ADT). Data from the National Hospital Discharge Register, the National Bureau of Statistics, and the National Prescriptions Database were merged. The analysis covered 15,716 men aged 50+ years presenting with a fracture at any hospital in the country in the year 2000 and 47,149 age-matched controls. A prior diagnosis of prostate cancer had been recorded in 1.3% of controls and 2.5% of fracture cases, with a median time span between the first consultation for prostate cancer and the index fracture of three years. In 75 percent of the patients, the time span was less than five years. Prostate cancer was associated with significant increase in all-fracture risk, OR 1.8 (95% CI 1.6-2.1), and hip fracture risk, OR 3.7 (3.1-4.4) whereas no impact on vertebral fracture risk was found, OR 1.0 (0.7-1.5). The effects were undiminished by adjustment for age, prior fracture, living circumstances and income. In men aged 50-65 years, prostate cancer increased the risk of hip fracture by as much as nine-fold, OR 9.2 (4.8 – 17.5).

After adjustment for previous fracture, cancer diagnosis, and age, both ADT and orchiectomy significantly increased overall fracture risk, OR 1.7 (1.2-2.5, p<0.01) and 1.7 (1.2-2.4, p<0.01), respectively. ADT also increased hip-fracture risk (OR 1.9, 1.2-3.0, p<0.05), but prostate cancer therapy was not associated with demonstrable increases in vertebral fracture risk.

In conclusion, prostate cancer was associated with an increase in all-fracture risk as well as hip-fracture. The increased fracture risk became apparent early after diagnosis and remained pronounced even in long-term survivors. In total, 3.1% of hip fractures in men aged 50+ were attributable to prostate cancer. The risk of hip fracture was not confined to the very old, neither was fracture risk made negligible by the excess mortality in patients with advanced prostate cancer.

Disclosures: M.F. Nielsen, None.

This study received funding from: Cancer Foundation.

T257

Role of CXC Chemokine Ligand 13 in Squamous Cell Carcinoma Associated Osteolysis in Athymic Mice. S. N. M. Pandruvada*1, X. Liu*2, J. S. Norris*2, K. Sundaram1, S. Shanmugarajan1, W. L. Ries1, S. D. London*3, S. V. Reddy1, 1Charles P. Darby Children's Research Institute, Charleston, SC, USA, 2Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, SC, USA, 3College of Dental Medicine, Medical University of South Carolina, Charleston, SC, USA.

Head and neck squamous cell carcinoma is the most common malignant neoplasm estimated to be more than 40,000 cases per year in the US. These malignant tumors are known to have a potent activity of local bone invasion; however the molecular mechanisms of SCC associated osteolysis are unknown. In this study, we identified high level expression of chemokine ligand, CXCL13 and RANK ligand (RANKL) in squamous cell carcinoma (SCC) derived cell lines. SCC 14a cell conditioned media (20%) induced osteoclast differentiation which was inhibited by addition of OPG in human peripheral blood derived monocyte cultures indicating that SCC cells produce soluble RANKL. In addition, recombinant CXCL13 (10 ng/ml) significantly enhanced RANKL stimulated osteoclast differentiation in these cultures. Trans-well migration assay further identified that CXCL13 induces (7-fold) chemotaxis of peripheral blood monocytes in vitro which was inhibited by addition of anti-CXCR5 receptor antibody. Zymogram analysis of conditioned media obtained from RAW 264.7 macrophage cells stimulated with CXCL13 for 48 hr demonstrated a significant increase (4.0-fold) in the level of MMP-9 expression. Real-time PCR analysis of total RNA isolated from human bone marrow derived stromal cells stimulated with CXCL13 for 48 hr showed a significant increase (4-fold) in RANKL mRNA expression. Interestingly, CXCL13 treatment to SCC 14a cells induced CXCR5 receptor and MMP-9 expression suggesting an autocrine regulatory function in SCC cells. To further examine the molecular mechanisms associated with SCC tumor cell invasion and osteolysis of bone, we established an in vivo model for SCC by subcutaneous injection of SCC 14a cells (7×106 cells in PBS) onto the surface of calvaria in NCr-nu/nu athymic mice, which developed tumors in 4-5 weeks. Immunohistochemical analysis confirmed CXCL13 and MMP-9 expression in the tumor cells and invasion of bone/osteolysis in vivo. Histochemical staining further demonstrated a significant increase in the numbers of multinucleated TRAP positive osteoclasts at the tumor bone interface. Thus, our data implicate a functional role for CXCL13 in SCC tumor cells invasion/osteolysis of bone and may be a potential therapeutic target to prevent osteolysis associated with SCC in vivo.

Disclosures: S.N.M. Pandruvada, None.

T258

Balloon Kyphoplasty in the Treatment of Osteolytic Lesions in the Thoracic and Lumbar Spine Due to Cancer - 2 Years Prospective Follow-up. R. Pflugmacher*, Centrum für Muskuloskeletale Chirurgie, Charité-Universitätsmedizin Berlin, Berlin, Germany.

Purpose: Balloon Kyphoplasty is a minimally invasive stabilization procedure for osteoporotic and osteolytic vertebral fractures. The purpose of this prospective study was to evaluate this operative procedure in the treatment of osteolytic vertebral fractures in reduction of pain and functional improvement of these patients and further to evaluate the restoration of vertebral height postoperatively and over a time period of 24 months.

Materials and methods: In this prospective study, 96 patients (45 patients with multiple myeloma, 51 patients with metastatic disease) with osteolytic vertebral fractures (176 vertebrae) were treated with Balloon Kyphoplasty. We were able to collect 2 year follow up data for 82 patients. 38 patients (30 male, 8 female) with 83 osteolytic vertebral fractures due to multiple myeloma and 44 patients (26 male, 18 female) with 78 osteolytic vertebral fractures due to metastatic disease. Preoperatively conventional radiographs in lateral and a.p. view, CT and / or MRI were performed. Pre- and postoperatively, pain (Visual Analogue Scale) and disability (Oswestry disability index) were evaluated. Radiographs were performed pre- and postoperatively and after 3, 6, 12 and 24 months. Vertebral height and endplate angles were measured.

Results: Clinical results for the patients with multiple myeloma and metastatic disease improved significantly. The median pain scores (VAS) decreased significant from pre- to post-treatment (p < 0.001) as well as the Oswestry disability index (p < 0.001). Balloon Kyphoplasty led to a significant and sustained reduction of pain resulting in a significant functional improvement of the patients. In the radiological evaluation a significant restoration of vertebral height (p < 0.05) and reduction of the kyphotic angle (p < 0.05) could be achieved in the patients with multiple myeloma. In patients with metastatic disease only a slight restoration of vertebral height and reduction of the kyphotic angle was achieved. Balloon Kyphoplasty was able to stabilize the vertebral height and avoid a further kyphotic deformity in the long term for both patient groups. Radiation therapy and / or chemotherapy could be performed without any loss of time.

Conclusion: In the treatment of osteolytic vertebral fractures Balloon Kyphoplasty led to a quick and sustained reduction of pain and functional improvement. Balloon Kyphoplasty was able to stabilize the fractured vertebrae in the long-term and was able to prevent an increase of kyphotic deformity. Balloon Kyphoplasty is an outstanding alternative in comparison to the established therapeutic concepts in the treatment of osteolytic vertebral fractures.

Disclosures: R. Pflugmacher, None.

T259

Chondrostatin: A New Inhibitor of Angiogenesis, Tumor Invasion and Osteoclast Activity. L. J. Sandell1, Z. Wang*1, C. Franz*1, N. Havioglu*2, J. Bryan*1, B. Ell*3, A. Rapraeger*3, 1Orthopaedic Surgery, Washington University, St. Louis, MO, USA, 2Pathology, St. Louis University, St. Louis, MO, USA, 3Pathology and Laboratory Medicine, University of Wisconsin, Madison, WI, USA.

Cartilage tissue is avascular and resistant to tumor invasion: the molecule mechanism of is unknown. We have recently shown that a normally produced fragment of the predominant cartilage collagen, called chondrostatin, is responsible for exquisite targeting of inhibition of cell migration and for cell death. The cells targeted are endothelial cells, osteoclasts and tumor cells. The targeting is based on the specific binding of the chondrostatin to integrins αvβ3 and αvβ5, expressed on osteoclasts, endothelial and tumor cells but not on normal chondrocytes. The chondrosarcoma cell line, Ch-1 and the breast cancer cell line MDA-MB 231 were used to establish the proof of concept. First, we demonstrated that Ch-1 cells adhere to chondrostatin dependent on binding to an RGD sequence. Next, we established that Ch-1 and MDA cells express the αvβ3 and αvβ5, integrins and that these integrins are responsible for adhesion. Confocal microscopy studies showed that chondrostatin is internalized into the cells. Inside the cells, it is able to induce cell death in a dose and time-dependent manner. Inhibition of αv integrin by RNA interference eliminated adhesion and cell killing. In further studies, we examined the ability of chondrostatin to inhibit angiogenesis. Two well accepted assays were used: formation of tubes from HUVEC endothelial cells, and outgrowth of endothelial cells from explants of aorta. Chondrostatin inhibited angiogenesis in both of these assays reducing angiogenesis by 50% at a concentration of 100 nM. In conclusion, naturally occurring chondrostatin inhibits angiogenesis and kills tumor cells mediated by the αvβ3 and αvβ5 integrins. This molecule may be useful in the treatment of neo-vascular disease, cancers and osteoporosis.

Disclosures: L.J. Sandell, None.

This study received funding from: NIH.

T260

Development of Small Molecule Adrenomedullin Antagonists for Treatment of Bone Metastases. V. A. Siclari1, K. S. Mohammad2, A. Martinez*3, C. Gineste*4, H. M. Geysen*4, T. A. Guise2, J. M. Chirgwin2, 1Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA, USA, 2Internal Medicine, University of Virginia, Charlottesville, VA, USA, 3Institute Cajal, Madrid, Spain, 4Chemistry, University of Virginia, Charlottesville, VA, USA.

Adrenomedullin (AM) is a 52 amino acid peptide of the calcitonin/CGRP gene family secreted by cancers such as breast, lung, and prostate, where it can stimulate angiogenesis and autocrine signaling and may cause cancer-associated pain. AM also dose-dependently stimulates new bone formation at picomolar concentrations, by binding to the calcitonin-receptor-like receptor plus RAMP2 or 3 and stimulating adenyl cyclase. It stimulates osteoblast proliferation, although the molecular pathways activated in bone cells by AM are poorly understood. When we treated primary mouse osteoblasts with AM, the mRNAs encoding bone matrix proteins (bone sialoprotein, type I collagen, osteocalcin, and osteopontin) were not increased. However, AM stimulated IL-6 mRNA expression by primary osteoblasts. We previously reported increased or decreased bone metastases due to AM overexpression and siRNA knockdown in prostate and lung cancer models respectively. These observations identify AM as a significant target for therapeutic intervention against bone metastasis.

Small molecules have been identified that function as agonists or antagonists of the action of AM to increase cAMP, by binding to the AM ligand rather than the receptor. One of the antagonists, NC1 16311, binds with Kd = 8nM to AM without altering ligand:receptor binding affinity. We added 100nM 16311 to neonatal mouse calvarial organ cultures. Increases in new bone and osteoblast number caused by 1nM AM were completely blocked by 16311, without cellular toxicity or blockade of new bone formation due to IGF1 receptor activation. Two additional antagonists were tested in the same assays. NCI 28086 was ineffective, but the methylene-bis- benzoic acid derivative NCI 37133 was as effective as NCI 16311 at blocking AM-induced new bone formation. The two lead compounds may be effective bone-selective antagonists of tumor-secreted AM. NCI 16311 and 37133 are aromatic carboxylic acid derivatives that act extracellularly, offering attractive pharmacological properties without needing to be cell-permeant. Further development of these compounds into higher affinity, second-generation derivatives should be possible. Small molecule AM antagonists may provide improved treatment for osteoblastic bone metastasis and associated cancer bone pain.

Disclosures: V.A. Siclari, None.

T261

Synthesis and Biological Evaluation of Vitamin K2 Metabolites. Y. Suhara*, A. Murakami*, M. Kamao*, S. Mimatsu*, K. Nakagawa*, N. Tsugawa*, T. Okano. Department of Hygienic Sciences, Kobe Pharmaceutical University, Kobe, Japan.

Vitamin K is an essential nutrient and a cofactor for the carboxylation of specific glutamyl residues of proteins to gamma-glutamyl residues, which activates osteocalcin related to the bone formation. Recently, the metabolism of vitamin K has been studied in humans and rats, and urinary metabolites as glucuronides of K acid I, II, and omega-carboxylic acid have been detected. However, their biological activities have hardly been evaluated so far. We anticipated that if those metabolites were studied in detail, they would provide insight into the biological significance of vitamin K and valuable information for the development of new drugs. In this study, we synthesized six kinds of “predictable” vitamin K metabolites. They were introduced vitamin K2 (menaquinone-2, 3, and 4) molecule to omega-hydroxyl group or omega-aldehyde group instead of omega-carboxylic acid because the metabolites including omega-carboxylic acid were unstable and unable to use for assay. The requisite analogues were synthesized by coupling the naphthoquinone derivative and side-chain moiety. For the synthesis of side-chain part, we chose geraniol, farnesol and geranylgeraniol as the starting material. While the naphthoquinone part was prepared from 1,4-diacetoxy-2-methylnaphthoquinone. After coupling reaction, deprotection of protective group gave omega-hydroxyl derivative, and additional oxidative reaction yielded omega-aldehyde derivative. To investigate the biological activities of those analogues, we focused on the apoptosis-inducing activity against HL-60 cells (human leukemia cells). Samples of 5 and 10 μM analogues were successively added to HL-60 cells. After 72 h, the cells were collected and treated with propidium iodide, and then apoptosis activity was determined with a fluorescence-activated cell sorter (FACS). As a result of biological evaluation, omega-hydroxylation of the side-chain part of menaquinone-4 increased apoptosis-inducing activity for HL-60 cells compared to menaquinone-4. Our results indicated that metabolites of vitamin K might have potential as biologically active compounds and can provide useful information to develop new drugs based on vitamin K with modification of the terminal alkyl group. A more detailed examination is currently undergoing based on these findings.

Disclosures: Y. Suhara. None.

T262

Benign and Malignant Bone Tumors Express the Melatonin Receptor Isoform 1 (MT1). M. Svoboda*1, C. D. Toma*2, F. Arrich*2, Ekmekcioglu*1, O. Assadian*4, T. Thalhammer*1, 1Department of Pathophysiology, Medical University Vienna, Vienna, Austria, 2Department of Orthopaedic Surgery, Medical University Vienna, Vienna, Austria, 3Department of Physiology, Medical University Vienna, Vienna, Austria, 4Clinical Department of Hygiene and Medical Microbiology, Medical University Vienna, Vienna, Austria.

The indolamine melatonin is synthesized and secreted by the pineal gland during night time and is well known for its role in the regulation of circadian rhythms. Recent studies showed that melatonin exhibits potent anti-tumor activity in vitro as well as in vivo and it is produced in extrapineal tissues, e.g. the bone marrow. Melatonin acts by receptor independent and dependent pathways. The latter is mediated through high-affinity G-protein receptors (MT1 and MT2) signaling.

To account for a possible role of MT1/MT2 in bone homoestasis, we investigated whether MT1/MT2 are expressed in malignant and non-malignant human bone lesions and osteosarcoma cell lines (HOS, MG-63). By RT-PCR, Western blot and immunofluorescence we found that MT1, but not MT2 was detectable in the tumor cell lines. By TaqManPCR, we analyzed 30 specimens from osteosarcoma and 11 from benign bone tumors for MT1-mRNA expression and determined mRNA expression (relative to a control specimen) of the osteoclast activity stimulating receptor activator of nuclear factor-kB ligand (RANKL) and its opponent osteoprotegerin (OPG).

We found that MT1 was similarly expressed in malignant (4.39 ± 4.98-fold) and benign samples (4.64 ± 6.81-fold). Also, mean RANKL- and OPG-mRNA levels were comparable in malignant and benign samples (RANKL: 7.38 ± 9.61-fold vs. 3.57 ± 3.11-fold, p = 0.207; OPG: 23.45 ± 32.76 vs. 8.07 ± 7.23-fold, p = 0.13). Expression of MT1-mRNA was also found in normal human osteoblasts (hOB), and bone marrow stromal cells (BMSC). High expression levels of MT1-mRNA together with low OPG-mRNA were found in both cancer cell lines, while in normal hOB and BMSC, high OPG-mRNA levels were associated with low MT1-mRNA levels.

In summary, we found that MT1-mRNA is abundantly expressed in human malignant and benign bone tumors and in osteosarcoma cells lines. Despite our finding that the relative expression levels of MT1 mRNA in malignant and non-malignant bone tumors did not differ significantly, the highest MT1 levels were detected in individual osteosarcoma specimens, i.e., in two specimens from patients with a local recurrence of the tumor. Whether this may be a prognostic parameter remains to be established in further work.

Disclosures: M. Svoboda. None.

T263

TGF-beta Inhibition Suppresses Myeloma Cell Growth via Enhancement of Osteoblast Differentiation. K. Takeuchi*1, M. Abe1, A. Oda*1, H. Amou*1, M. Hiasa*2, O. Tanaka*1, S. Fujii*1, S. Nakamura*1, A. Mihara*1, H. Miki*1, L. Asano*1, K. Kagawa*1, K. Kitazoe*1, K. Yata*1, T. Hashimoto*1, S. Ozaki*1, S. Kido1, T. Matsumoto1, 1Dept of Medicine and Bioregulatory Sciences, Univ of Tokushima Graduate Sch of Med Sci, Tokushima, Japan, 2Dept of Orthodontics and Dentofacial Orthopedics, Univ of Tokushima Graduate Sch of Dent Sci, Tokushima, Japan.

Multiple myeloma (MM) enhances osteoclast formation and suppresses stromal cell differentiation into osteoblasts (OBs). Because both osteoclasts and undifferentiated stromal cells enhance MM cell growth and survival, such effects of MM create a microenvironment suitable for MM expansion in the bone marrow, which can be called as myeloma niche. TGF-beta, an inhibitor of terminal OB differentiation, is released from the bone matrix through enhanced bone resorption by MM. We previously demonstrated that TGF-beta inhibition is able to restore OB differentiation suppressed by MM. However, it is not known whether the restoration of OB differentiation by TGF-beta inhibition affects MM growth. We therefore aimed to clarify the role of TGF-beta inhibition on MM growth in relation to the induction of OB differentiation. Terminally differentiated MC3T3-E1 osteoblastic cells with mineralized nodules secreted a factor(s) with potent apoptosis-inducing activity in 5TGM1 and RPMI8226 MM cells. The anti-MM activity was elaborated exclusively by terminally differentiated MC3T3-E1 cells with mineralized nodule formation, whereas untreated MC3T3-E1 cells with pre-osteoblastic phenotypes enhanced MM growth. SB431542, a TGF-beta type 1 receptor kinase inhibitor, facilitated OB differentiation as well as the production of MM cell growth inhibitory activity in MC3T3-E1 cells. Although interaction of MM cells with stromal cells/undifferentiated OBs gives rise to drug resistance in MM cells, terminally differentiated OBs potentiated the cytotoxic effects of doxorubicin on MM cells. These results demonstrate that, in contrast to stromal cells/undifferentiated OBs, terminally differentiated OBs inhibit MM cell growth and survival, and that inhibition of TGF-beta actions enhances OB differentiation, suppresses MM cell growth and potentiates responsiveness to cytotoxic agents. It is suggested that suppression of OB differentiation by MM not only accelerates bone destruction but also enhances MM growth to create myeloma niche, and that induction of OB differentiation by TGF-beta inhibition may provide a novel approach to disrupt myeloma niche.

Disclosures: K. Takeuchi, None.

T264

Surgical Treatment for Benign Bone Tumors with Fully Interconnected Porous Hydroxyapatite Ceramics. N. Tamai*, A. Myoui*, T. Ueda*, H. Yopshikawa*, Dep. of Orthopaedics, Osaka University Graduated School of Medicine, Osaka, Japan.

PURPOSE: Large bone defects remaining after curettage of benign bone tumors should be filled with a substitute to restore mechanical strength. We have utilized several kinds of CHAs available in Japan for massive bone defect after curettage of benign bone tumors and have reported our follow-up study. In 2000, we have developed a fully interconnected porous calcium hydroxyapatite ceramics (NEOBONE) and have utilized them as bone substitute. The purpose of this study is to evaluate the clinical outcomes with the use of NEOBONE as bone substitute after curettage of benign bone tumors.

METHODS: We reviewed the results of 27 patients with benign bone tumors sequentially treated by curettage followed by implantation of NEOBONETM between 2000 and 2004. There were 8 women and 19 men, with the mean age of 29 years. The locations were as follows: phalanx (11 patients), femur (6), pelvis (3), tibia (2), radius (1), calcaneus (1), humerus (1), talus (1). The histological diagnoses included enchondroma (9patients), solitary bone cyst (6), chondroblastoma (4), giant cell tumor of bone (3), fibrous displagia (3), intraosseous ganglion (2). The mean follow-up period was 30 months. Assessment was based on plain radiography at the final follow-up. Radiographic findings were classified into four grades: Grade 1, slight bone formation in NEOBONE; Grade 2, moderate bone formation in NEOBONE and their openings; Grade3, consolidation phase; Grade4, absorption phase.

RESULTS: Radiographs at the final follow-up were graded as follows; Grade1 in 6 patients; Grade2 in 4; Grade3 in 13; Grade4 in 4. The mean periods required to reach each grade were as follows; Grade1 in 2.5 months (2-9 months); Grade2 in 6 months (2-12 months); Grade3 in 18 months (6-31 months); Grade4 in 41 months (27-55 months). There were two local recurrences, observed in each one case of solitary bone cyst and giant cell tumor of bone.

CONCLUSIONS: NEOBONE (pore size: 150-300μm, porosity: 75%) has a finely-organized, 3-dimensional interconnecting pore structure. The large interconnecting channels (average diameter: 40 μm) permit the easy penetration of tissue into the deep pores and, therefore, NEOBONE can itself induce local bone repair processes. In the present study, we utilized NEOBONE as a bone substitute after curettage of benign bone tumors, and demonstrated its superior osteoconductivity. We conclude that NEOBONE is an excellent next generational bone substitute in surgery for benign bone tumors.

Disclosures: N. Tamai. None.

T265

Mediation of Cell Proliferation and Tumorgenicity of Musculoskeletal Tumor Cells by an Interferon Inducible IFI16 Protein. Y. Zhang*, R. D. Howell*, L. Kong*, C. Liu.. Orthopaedic Surgery, New York University, New York, NY, USA.

IFI 16, a member of an interferon-inducible p200-protein family, modulates cell prolifération, differentiation, apoptosis and cellular senescence. It has also been implicated in the pathophysiology of autoimmunodiseases and antitumor activity of several kinds of cancer cells. In this study we showed that the level of IFI 16 was dramatically reduced in the primary human osteosarcoma compared to normal bone. Overexpression of functional IFI 16 in human osteosarcoma and chondrosarcoma cell lines significantly inhibited cell growth. This IFI16-mediated inhibition on musculoskeletal tumor cells is overcome by a siRNA that specifically knockdown the expression of IFI 16, suggesting that certain level of IFI 16 is critical for controlling musculoskeletal tumor growth. In addition, ectopic expression of IFI 16 in musculoskeletal tumor cell lines markedly inhibited colony formation and was associated with a senescence-like phenotype, production of senescence-associated beta-galactosidase. Further, upiegulation of p21 and inhibition of cyclin E, cyclin Dl, c-Myc and Ras accompanied inhibition of cell growth by IFI 16 in musculoskeletal tumor cell lines. Taken together, our observations provide insight into the process of musculoskeletal tumors regulated by IFI 16, and more importantly, our findings also provide evidence indicating that IFI 16 has great potential to be employed as a therapeutic target for treating musculoskeletal tumors, including both osteosarcoma and condrosarcoma.

Disclosures: Y. Zhang, None.

T266

Longitudinal Changes in Bone Density and Management in Men with Osteopenia. R. A. Adler, X. Samaropoulos*, M. I. Williams*, V. I. Petkov*, Endocrinology, McGuire VAMC, Richmond, VA, USA.

Until 10 year fracture risk can be easily calculated from DXA and clinical factors, patients with osteopenia (low bone mass but not osteoporosis) by DXA usually receive recommendations to take calcium, vitamin D, exercise and repeat DXA in two years. We identified 78 men who had participated in a screening program based on the OST (Osteoporosis Self-Assessment Tool) equation, which identifies risk for osteoporosis by DXA based on age and weight only. The men had at least 1 T-score in spine, total hip, femoral neck, total forearm, or distal 1/3 forearm between −1 and −2.5, except for 4 men who had a T-score of ≤ −2.5 but not given pharmacologic treatment. The men had a second DXA at least 2 years after the first. At first DXA, mean age was 70.7 yrs, mean weight 76.4 kg, mean OST score 1 (moderate risk). Low bone mass was found in 33% of spine, 46% of total hip, 78% femoral neck, 51% total forearm, and 40% distal 1/3 forearm DXA measurements. 37/78 men were prescribed calcium and 21/78 Vitamin D (beyond a multivitamin). After an average of 998 days, weight was down an average of 1.2 kg. The overall diagnosis worsened to osteoporosis in 3 men, but in only one man was the change in DXA statistically significant. Interestingly, after the second DXA, calcium and/or vitamin D supplementation was started in 17 men. Thus, while the second DXA only identified 3 more men with osteoporosis, an additional group had conservative treatment of osteopenia started for the first time. In conclusion, a second bone density test for men at risk of osteoporosis seldom changes the diagnosis. However, the second DXA may spur providers to start preventive methods.

Disclosures: R.A. Adler, None.

T267

Solid-State 31P MRI Quantifies Decreased Mineralization Density in OVX Rat Bone. S. Anumula*1, S. L. Wehrli*2, J. Magland*1, F. W. Wehrli1, 1Radiology, University of Pennsylvania, Philadelphia, PA, USA, 2NMR Core Facility, Children's Hospital of Philadelphia, Philadelphia, PA, USA.

Osteoporosis is characterized by rapid bone turnover resulting in decreased mineralization density of bone (MDB), a parameter that cannot currently be measured in vivo. Here we examine the hypothesis that 3D 31P solid-state magnetic resonance imaging (SS-PH) is able to quantify cortical bone phosphorus content in situ in a rat model of osteoporosis in view of possible future application of the technique to humans in vivo. The study involved six ovariectomized rats (4 months old, OVX) compared to six sham-operated rats of the same age (NO). After the surgery all the animals were given standard chow and water ad libitum for 50 days after which they were euthanized and cortical bones extracted from the right and left femurs. In order to optimize the imaging parameters, the spin lattice relaxation time T, was measured in the specimens using inversion-recovery sequence. 3D SS-PH was performed at 9.4 Tesla (∼ 162 MHz) using a sequence with non-selective excitation followed by sampling of the free induction decay in the presence of a readout gradient whose K-space data were reconstructed off-line yielding an isotropic resolution of 177 μm (Fig. 1). Specimens were co-imaged with a reference phantom of 2M K2HPO4 in order to quantify the mineral phosphorus. The sequence was implemented on a vertical-bore superconducting system (DMX-400, Bruker Instruments, USA) in conjunction with a home-built solenoid coil. Quantitative MRI results were compared with micro-CT derived MDB values using calibration samples made of various concentrations of K2HPO4. Phosphorus concentration obtained by SS-PH was lower in the OVX than in the NO group (6.7±0.9 vs 7.5±0.7%; p = 0.01) (Fig. 2a). This result was paralleled by lower MDB measured by μ-CT in the OVX group (1199±24 vs 1225 ±10; p = 0.02) (Fig. 2b). In conclusion our data suggest that 31P SS-PH may have potential for studying changes in mineral metabolism nondestructively and noninvasively, i.e. without the use of ionizing radiation. ,  ,  

Figure Figure 1.

Volume-rendered nP MR image of the rat femur with central reference sample.

Figure Figure 2..

a) phosphorus content (SS-PH MRI); b) mineralization density (μ-CT).

T268

Accuracy and Precision of Adipose Tissue Cross-sectional Area Measured Using Peripheral Quantitative Computed Tomography (pQCT). C. Gordon1, D. Inglis*2, L. Giangregorio3, J. M. Zmuda4, 1Radiology, McMaster University, Hamilton, ON, Canada, 2Civil Engineering, McMaster University, Hamilton, ON, Canada, 3Kinesiology, University of Waterloo, Waterloo, ON, Canada, 4Epidemiology, University of Pittsburgh, Pittsburgh, PA, USA.

Purpose: Using magnetic resonance images (MRI) as the gold standard to quantify adipose tissue cross-sectional area (CSA), this study assessed the accuracy of quantifying sub-cutaneous fat, marrow fat, and fat in and around muscle from pQCT images of the calf. The precision errors for each of the fat components were also determined.

Methods: Twenty-five subjects participated in this study. Ten subjects were scanned with MRI and pQCT at a calf location corresponding to 66% of the distance up from the medial maleolus to the medial condyle of the tibia. pQCT scans were acquired with a STRATEC XCT 2000L scanner (Orthometrix Inc., White Plains, NY) at a voxel size of 0.4 mm2 and scan speed of 10 mm/sec. The MRI images were acquired on a 1.0 T peripheral magnet (ONI Corp, Wilmington, MA) using a fast spin echo sequence that acquired 10 contiguous 2 mm slices around the 66% location. CSAs of the adipose tissue components in the pQCT image were determined using various edge tracking and threshold steps available within the pQCT analysis software. To reduce noise, median filtering was applied before segmentation. Custom software was used to extract the CSA of the adipose tissue components in the MRI calf images. Least squares analysis was used to compare pQCT and MRI fat areas. To assess precision, 15 subjects had pQCT scans done on the same day, with repositioning between scans. Precision error was calculated as percent coefficient of variation.

Results: There was good agreement between pQCT and MRI derived areas for total fat (R2 = 0.98, p<0.0001), subcutaneous fat (R2 = 0.96, p<0.0001) and marrow fat (R2 = 0.61, p<0.03). CSA of the fat in and around the calf muscles was not well defined with pQCT (R2 = 0.12, p = 0.8). pQCT defined adipose tissue at the calf with precision errors of 10% for total fat, 4% for subcutaneous fat, and 16% for marrow fat. The precision error for the area of fat in and around the calf muscle exceeded 100%.

Conclusion: Adipose tissue CSAs in the calf can be accurately derived from pQCT images. The fatty infiltration between and within the calf muscles may have to exceed a threshold to be accurately detected with pQCT or be best characterized by an index such as muscle density.

Disclosures: C. Gordon, Orthometrix Inc. 5.

T269

Comparison of QCT and DXA Derived Areal Bone Mineral Density. B. C. C. Khoo*1, S. Henzell*2, S. Gustafsson*2, K. Zhu2, R. I. Price1, R. L. Prince3, 1Medical Technology and Physics, Sir Charles Gairdner Hospital, Nedlands, Australia, 2Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Nedlands, Australia, 3School of Medicine and Pharmacology, University of Western Australia, Crawley, Australia.

WHO guidelines, promulgated in 1994, considered that a gold standard for diagnosis of bone fragility in the clinical setting should be areal bone mineral density (aBMD, gem2) measurements of the proximal femur using DXA. Recently there has been a return to the use of quantitative computed tomography (QCT) in clinical diagnosis to facilitate the understanding of bone fragility and structural analysis in individuals. aBMD is one of many variables that can be generated in such QCT studies. A critical question arises is how similar are the aBMD measurements derived from QCT and DXA?

This study compared the aBMD and T scores of 91 elderly female subjects aged 82.8 (2.5) years [mean (SD)], height 157.4 (6.0) cm, weight 63.6 (12.4) kg. All subjects were scanned on a Hologic Discovery DXA and a Phillips Brillance CT scanner. The DXA aBMD was measured using Hologic software version 12.6 and QCT aBMD using Mindways software version 4.1.3. Using the QCT data aBMD data a “Hologic aBMD equivalent” can be calculated using manufacturer data which then allows calculation of an NHANES equivalent T score. The data was compared using linear regression and Bland-Altman (B-A) plots.

In linear regression the R2 for total hip (TH), femoral neck (FN), inter-trochanter (IT) and trochanter (T) aBMD comparisons were 0.88, 0.84, 0.84 and 0.85 respectively, the regression coefficients were 0.87, 0.83, 0.90 and 0.83 respectively. The B-A plots showed that the QCT aBMD measurement under estimated the DXA aBMD (TH 0.13 (0.05) g/cm2, FN 0.09 (0.04) g/cm2, IT 0.14 (0.07) g/cm2 and T 0.10 (0.05) g/cm2) with no bias as the mean increased.

These data confirm that QCT hip areal structural values are highly correlated with DXA aBMD. However there is an offset which needs further consideration.

Disclosures: B.C.C. Khoo, None.

T270

A New CUSUM Method for Simultaneous Quality Control of BMD, BMC, and Area for DXA ScannersY. Lu, S. Zhao*, B. Fan, J. Shephard. Department of Radiology, University of California, San Francisco, San Francisco, CA, USA.

CUSUM method has been used in longitudinal quality control (QC) of bone mineral density (BMD) by dual X-ray absorptiometry (DXA) scanners. It identifies significant deviations from the baseline as breakpoints for further investigations. With an increase of pediatric and whole body studies, a new QC method is necessary to monitor BMD, bone mineral contents, and area simultaneously. In this paper, we presented a new multivariate CUSUM method. After a log-transformation of variables (In(BMD) = In(BMC)-In(Area)), we normalized and rotated variables In(BMD) and In(BMC) into two linearly unassociated variables × and Y based on their observed covariance matrix. A modified univariate CUSUM was then performed on × and Y separately in both increasing and decreasing directions. Multivariate (2-D) CUSUM parameters were selected to assure a false alarm rate similar to the univariate CUSUM of one variable. Simulation studies were conducted to evaluate performance comparing to our previously proposed 2-D Shewhart. In(BMD), In(BMC), and In(Area) were generated by multivariate normal distribution using mean and covariance matrix derived from a whole body QC study. We simulated the in-control condition with 365 scans. We then generated 365 off-control data in the following conditions: (1) Case I: both log BMD and BMC increased 1 SD and Area was relative stable; (2) Case 2: BMC increased in ISD but not BMD, and area increased accordingly; and (3) Case 3: BMC increased 1 SD but BMD decreased ISD. Each condition was repeated for 2000 times. Table 1 showed simulation results. When a scanner was in control, the median running length for the 1st false alarm was 213 and 31 scans, respectively, for the 2-D CUSUM and Shewhart methods. When a scanner was out of control, CUSUM identified the breakpoints within 5 to 20 scans on average similar to Shewhart. In conclusion, multivariate CUSUM method can be used for QC of BMD, BMC, and Area simultaneously for whole body phantom data with satisfactory false and true positive rates.

Table Table 1:. Simulation Results for Bivariate QC Methods (n = 2000)
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Disclosures: Y. Lu, NIH R01EB004079 2.

This study received funding from: R01EB004079.

T271

Vitamin D Status/insufficiency May Predict Physical Performance in a Group of Osteopenic/osteoporotic Women. Preliminary Study. S. R. Mastaglia*, M. Seijo*, D. M. Muzio*, B. Oliveri. Sección Osteopatias Médicas, Hospital de Clínicas. Universidad de Buenos Aires, Buenos Aires, Argentina.

Hypovitaminosis D is frequent in population over 65 years of age, producing a secondary hyperparathyroidism, increase of bone remodeling, bone loss and osteoporotic fractures, loss of the muscle force function. The aim of this study was to assess the vitamin D (25OHD) nutritional state in osteopenic/osteoporotic women >65 and the relationship with muscle force and function. To date, forty women were assessed, with an average age (X±DS) 71.2±5.3, body mass index (BMI) 27.5±4.2, with a bone mineral density (BMD) (DXA-LUNAR) of femoral neck (FN) 0.752±0.71 (T- 1.9). The exclusion criteria applied were: having received vitamin D during the year preceding the study, pathology or medication affecting mineral metabolism, neurological and/or motor-function disease. The physical performance of lower limbs (walking speed, balance, standing-up and sitting-down) and the hip force with a dynamometer was assessed. The population sample was divided into two groups, taking as cut point 20ng/ml of 25OHD (Lips P Endocr Rev 2001): Group 1 (G1): > 20 ng/ml, Group 2: (G2) ≤ 20 ng/ml. The results obtained are shown in the table.A lower score in the physical performance and a tendency to a lesser left hip abduction in women with 25OHD ≤ 20ng/ml was observed.

Preliminary data show that the levels of insufficient vitamin D could predict physical performance in osteopenic/osteoporotic women >65. 

Table Table.  
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Disclosures: S.R. Mastaglia, None.

T272

In Vivo and In Vitro Comparison of DXA Scanners. T. K. Omsland*1, N. Emaus*2, C. G Gjesdal*3, J. A. Falch*4, G. S. Tell*3, L. Forsen*5, G. Berntsen*2, H. E. Meyer1, 1University of Oslo, Oslo, Norway, 2University of Tromsø, Tromsø, Norway, 3University of Bergen, Bergen, Norway, 4Aker University Hospital, Oslo, Norway, 5Norwegian Institute of Public Health, Oslo, Norway.

When comparing BMD measured by different DXA scanners in multi-centre studies, the agreement between scanners is essential for the quality of the data obtained. In-vivo calibration is considered the “gold standard” when assessing agreement, but phantoms are frequently used instead. The European Spine Phantom (ESP) is an imitation of human vertebrae developed for comparison between scanners. The aim of this study was [1] to assess the agreement between in-vivo measurements (total femur) on three scanners (one GE Lunar DPX-IQ and two GE Lunar Prodigy scanners) and [2] to determine whether the ESP was able to reproduce the in-vivo variability in hip measurements on the different scanners.

The data were collected as part of the Norwegian Epidemiological Osteoporosis Studies (NOREPOS). The scanners compared in this study were located in three different cities, and the study subjects travelled between the cities. 6 men and 10 women aged 28-66 years had three repeated scans (with repositioning) on each machine. Body weight ranged from 58 to 100 kg. The ESP was scanned at least 40 times on each machine. Bland & Altman plots and multilevel linear regression analyses were used to analyse the data.

Mean difference between hip measurements on the two Prodigy scanners was small (<0.001 g/cm2) and insignificant (p = 0.97). The differences between the scanners were not dependent on BMD level. The ESP was able to reproduce the in-vivo findings.

Mean difference between hip measurements on Prodigy and DPX-IQ was also small (0.007 g/cm2) and insignificant (p = 0.10). However, the differences between the scanners changed over the range of BMD. The ESP did not fully reproduce the in-vivo difference between the scanners. The mean difference between ESP and measurements in humans was 0.010 g/cm2 (p = 0.02).

In conclusion, there were little overall differences between the in-vivo measurements. The ESP is a valid substitute when assessing agreement between Prodigy scanners. However, when assessing agreement between Prodigy and DPX-IQ, substitution of in-vivo with in-vitro measurements should be made with caution.

Disclosures: T.K. Omsland, None.

T273

The Effect of Bone Mineral Density (BMD) Measurement of Hip Bilateral in Clinical Practice. J. B. Lopes*, C. F. Danilevicius*, V. F. Caparbo*, L. Takayama*, R. M. Pereira. Rheumatology Division (Bone Metabolism Laboratory), University of Sño Paulo, Sño Paulo, Brazil.

The aim of this study was to determine the effect of adding BMD measurement of contralateral hip on osteoporosis (OP) treatment based on NOF (National Osteoporosis Foundation) criteria. Sixty hundred and five consecutive elderly people community-dwelling (374 women/ 231 men) with 65 years-old or more (73.28 ± 5.40 yr) were evaluated. Subjects with a stroke causing hemiplegia, confined to a wheelchair, or with conditions resulting in immobilization of one limb were also excluded from the study. Densitometry of both hips and lumbar spine was evaluated using dual-energy X-ray absorptiometry (DXA) with HOLOGIC Discovery scanner. Precision errors and least significant change (LSC) were determined for each site: left/right hip (femoral neck and total femur) and lumbar spine. Clinical and anthropometric data were obtained by specific questionnaire and physical examination. The lowest T-score analyzed by DXA was considered in three distinct circumstances: lumbar spine+right hip; only hips and lumbar spine+hips. The risk factors were associated to the lowest T-score in these 3 situations and NOF criteria were applied to point out pharmacological treatment. Person's coefficient was used to assess the correlation between the 2 hips and McNemar's test to assess the differences using the NOF treatment criteria adding BMD measurement of contralateral hip.

There was a highly significant correlation between BMD of the two hips at femoral neck and total femur (r = 0.93 and 0.95, respectively; p<0.0001). Using the lowest T-score in the sites (lumbar spine, right/left femoral neck, right/left total femur) and WHO classification, osteoporosis was found in: 48.6% (n = 294) analyzing lumbar spine+right hip; 31.2% (n = 189) analyzing only hips and 49.75% (n = 301) analyzing lumbar spine+hips. Similarly, pharmacological treatment using NOF criteria differed compare all analysis situations. Treatment indication occurred in 65.4% (396) analyzing lumbar spine+right hip versus 55.0% (333) analyzing only hips with a significant discordance treatment (p<0.0001). Comparing lumbar spine+right hip BMD analysis and lumbar spine+hips analysis, the pharmacological therapy was indicated in 65.4% vs. 69,3% respectively with a significant discordance in treatment suggestion (p = 0.028). This study indicate that the added of the both hips at the lumbar spine scan; can expand the number of people with the osteoporosis diagnosis and treatment indication. Furthermore, although of the artifacts such as osteoarthritis and osteophyte calcification influence in the lumbar spine BMD in elderly people, this site should be consider in the OP diagnosis and treatment.

Disclosures: R.M. Pereira, FAPESP # 03/09313-0 2.

T274

The Relationship Between the Knowledge on Osteoporosis in Females and Bone Mineral Density at Hip, Spine and Forearm. W. Pluskiewicz*1, B. Drozdzowska*2, A. Grodzki*3, 1Metabolic Bone Diseases Unit, Silesian School of Medicine, Zabrze, Poland, 2Silesian School of Medicine, Zabrze, Poland, 3Central Clinical Hospital, Medical University in Warsaw, Warsaw, Poland.

The study assessed the thesis that better knowledge on osteoporosis ought to be related with better results of bone mineral density (BMD) measurements. The study group included 859 females (aged 18-88 y.) without prior treatment for osteoporosis and fractures. The knowledge of osteoporosis and attitude towards methods for preventing the disease were assessed using a questionnaire consisting often questions. BMD was measured at femoral neck, spine or forearm.

The level of knowledge and its duration did not influence BMD of spine, femoral neck and forearm either in the whole group and in subgroups divided according to the level of education. Mean number of correct answers was 7.25+/-1.73, and was significantly higher in better than in less educated women (7.93+/-1.06 versus 6.39+/-2.01, p<0.0001). In the whole group, the majority of women (63.7%) declared an increase in calcium intake (Chi-square test = 74.5, df = 3, p<0.00001), and only 44.5% declared modification of physical activity (Chi-square test = 44.3, df = 3, p<0.00001).

In less educated women an increase in calcium intake declared 54.7% versus 71.3% for better educated women (Chi-square test = 15.0, df = 1, p<0.001). An increase in physical activity declared 37.6% versus 50.4%, respectively (Chi-square test = 8.34, df = 1, p<0.01). Concluding, the level of knowledge on osteoporosis is not related with skeletal status in female population. 

Table  .  
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Disclosures: W. Pluskiewicz, None.

T275

The Qualitative Method and the Method Based on the Concept of 10 Years Probability of Bone Fracture in Qualifying Patients for Pharmacological Treatment of Osteoporosis. J. Przedlacki, K. Ksiezopolska-Orlowska*, A. Grodzki*, T. Bartuszek*, D. Bartuszek*, A. Swirski*, J. Musial*, E. Luczak*, E. Loth*, P. Teter*, A. Lasiewicki*, A. Walkiewicz*, I. Drozdowska-Rusinowicz*, Krajowe Centrum Osteoporozy, Warsaw, Poland.

Introduction: The lack of generally accepted guidelines for osteoporosis in Poland obliges the specialist centers to perform free diagnostic procedures for each referred patient. The one worked out and used in Krajowe Centrum Osteoporozy (KCO) is a qualitative method based on the data from the specialist literature. It combines the assessment of the clinical risk factors with the results of DXA test. Another method, which considered for introduction to Poland, is that worked out by the Canadian authors derived from the concept of 10 years probability of bone fracture.

Purpose of study: The aim of the study was to compare both methods in terms of qualifying patients for the pharmacological treatment of osteoporosis.

Material and methods: The diagnostic tests were done on 908 patients (108 men and 800 women) aged over 50 years (64,7+/-8,3 years). They were referred to KCO mainly by GPs, for the diagnosis and treatment of osteoporosis from 21.03.2006 to 20.03.2007. The tests leading to the diagnosis were conducted separately according to each method. Results: According to the method used in KCO 250 patients were qualified for pharmacological treatment of osteoporosis (27,5% of all, 16 men and 234 women). According to the Canadian authors' method 334 patients would have been qualified for the pharmacological treatment (risk of bone fracture >20%). The decision to introduce pharmacological treatment was unanimous in both methods in the case of 243 patients. In the group of women <65 years old and men <70 years old according to the Canadian authors' method there would have been qualified 95 patients out of 483 (19,7%), and according to the KCO method 44 patients (9,1%) - conformity 46,3%. In the group of 55 patients that were not qualified for the pharmacological treatment 41 (74,5%) didn't have an indication for DXA test. In the group of men and women in older age according to the Canadian authors' method there would have been 239 patients out of 425 (56,2%) qualified for treatment, and according to KCO method 205 (48,6%)- conformity 84,5%. In the group of 37 patients not qualified for treatment 18 had a T-score>-2,0.

Conclusion: There was a moderate general agreement of implication for treatment in both methods, significant in older patients, smaller in younger ones. The authors think that the method currently used in KCO can be continued with. Interpretation of the differences in both methods is not explicit. The obtained results will be taken into account in preparing a periodical actualisation of our method.

Disclosures: J. Przedlacki, None.

T276

Evaluation of Bone Area in the BMIL QA/QC Phantom on the Norland System. T. V. Sanchez1, J. M. Wang2, G Ekker*3, K. M. Dudzek*4, 1Research and Development, Norland–a CooperSurgical Company, Socorro, NM, USA, 2Research and Development, Norland–a CooperSurgical Company, Beijing, China, 3Engineering, Norland–a CooperSurgical Company, Fort Atkinson, WI, USA, 4Customer Service, Norland–a CooperSurgical Company, Fort Atkinson, WI, USA.

Bone area assessment using DXA varies with bone edge detection algorithms and equipment. Phantoms offer a tool by which to evaluate equipment performance when examining bone area. The current study evaluated DXA-based measurement of bone area on the BMIL QA/QC Phantom using AP Spine, Research and Small Subject Software to determine instrument response.

The commercially available BMIL QA/QC Phantom was evaluated on the Norland XR-46 using AP Spine Software at 1.5 × 1.5 mm and scan speeds of 65mm/s or 130mm/s and both Research and Small Subject Software at 1.0 × 1.0 mm with a scan speed of 15mm/s. The Phantom was scanned 25 times on each setting and operator set region of interest were analyzed for the total individual vertebrae (L1, L2, L3 and L4) on each scan. Regression analysis evaluated instrument performance.

When analyzed the studies done using AP Spine at 130 mm/s resulted in Bone Area for L1, L2, L3 and L4 of 13.526 cm2, 17.484 cm2, 21.648 cm2 and 26.668 cm2, respectively. Regression analysis between AP Spine mode at 130 mm/s and AP Spine mode at 65 mm/s (y = 0.9928x + 0.3621, r = 0.9815), Research mode (y = 1.0812x - 6.399, r = 0.9909) or Small Subject mode (y = 0.9777x - 4.4908, r = 0.9991) indicated strong relationships. In summary, this study demonstrates that the Norland scanning system is able to assess the four segments of the BMIL QA/QC Phantom and that the operating software modes produce similar bone area results. Phantoms, like the BMIL QA/QC Phantom, can prove to be powerful tools for evaluating the performance of a DXA system.

Disclosures: T. V. Sanchez, None.

T277

Limits for Exclusion of Individual Vertebra from the L2-L4 AP Spine Assessment. J. M. Wang1, T. V. Sanchez2, 1Research and Development, Norland–a CooperSurgical Company, Beijing, China, 2Research and Development, Norland–a CooperSurgical Company, Socorro, NM, USA.

Evaluation of the AP Spine L2-L4 region assumes that the individual vertebrae are free of defects that will skew the L2-L4 result. When evaluating the Norland AP Spine study, methodology calls for examining the individual vertebrae for a spread of less than 10% to insure that group value is not skewed. The current study examines the effect of spread on the L2-L4 result to establish if the criteria are valid.

A population of 424 AP Spine studies were pulled from clinical achieves. T-score results of studies were divided into four groups based on the spread of the L2, L3 and L4 percent young normal value Group 1 (n = 115) with ≤ 5% variation, Group 2 (n = 167) with variation over 5% to 10%, Group 3 (n = 78) with variation over 10% to 15% and Group 4 (n = 64) with variation over 15%. To assess the effect of variation on the clinical findings, we examined discordance in T-score based diagnosis obtained from individual vertebrae and the L2-L4 region. The number of discordant diagnosis moved from 21% and 27.5% in Group 1 and Group 2 to a substantial 49% and 59% in Group 3 and Group 4. The study demonstrates that as spread of the percent young normal increases in individual vertebrae beyond ten percent the population experiences a substantial increase in the number of discordant diagnosis. A limit in spread of ten percent or less among individual vertebrae is justified.

Disclosures: T. V. Sanchez. None.

T278

Reliability of Two Consecutive Prodigy Densitometers. M. C. Schoeller*1, S. Mehle*2, C. Simonelli1, 1Internal Medicine, HealthEast Osteoporosis Care, Woodbury, MN, USA, 2HealthEast Research and Education, HealthEast Medical Research Institute, Woodbury, MN, USA.

Evaluation of long-term stability of DXA devices is important for assessing precision and maintaining quality control of bone mineral density measurements in longitudinal studies. Quality control procedures generally use spine phantoms with known values provided by manufacturer. We evaluated long-term precision over a 4-year and 9-month period of measurement of a single Lunar spine phantom on two consecutive Lunar Prodigy (GE Healthcare) scanners located in an osteoporosis clinic.

The phantom consisted of an aluminum spine with increasing density from L2 to L4 embedded in a plastic that mimics human tissue (%fat = 30%). The original Prodigy scanner was replaced 3.5 years into the study with a Prodigy Advance scanner. Six versions of enCORE operating software were used during the study. In addition to routine service on both scanners, two service interventions (tube head replacement) occurred on the first scanner. Both Prodigy scanners were used heavily (more than 160 scans/week on average). A total of 785 measurements of the spine phantom (L2-L4) were analyzed.

Long-term BMD precision was 0.4% and a regression of BMD on date showed no significant trend (p(t) = 0.08, R2 = 0.004). The average individual measurement change from baseline (first 5 measurements) was −0.31%. Minitab run chart evaluations of BMD for trends and oscillation around the mean were not significant (p = 0.20 and 0.80, respectively). The average change seen in phantom BMD measurements was insignificant at 0.0002 g/cm2 per year and there were no significant trends.

We conclude that spine phantom measurements over a 4-year 9-month period on Lunar densitometers involving service interventions, multiple software versions, and a complete scanner exchange revealed highly stable values with average BMD shifts of −0.31%. The excellent BMD precision error of 0.4% for these densitometers demonstrated reliable system performance, long-term stability of results, and enhanced confidence in longitudinal studies. 

Table  .  
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Disclosures: M.C. Schoeller, None.

T279

Phalangeal BMD Assessment Using Radiographic Absorptiometry From Hand X-rays Taken With Digital Mammography. S. L. Silverman1, X. Bi*2, L. Al-Daveh*2, 1Cedars-Sinai/UCLA, Beverly Hills, CA, USA, 2CompuMed, Inc, Los Angeles, CA, USA.

Osteoporosis is under diagnosed and under treated. Recent guidelines from the US Preventive Systems Task Force recommend bone density screening for women over age 65 or women above age 60 with risk factors. Yet only 22.9% of Medicare recipients have had a BMD test in the 3 years after Medicare reimbursement for osteoporosis screening began1. Breast cancer risk increases steadily with age. Mammography is recommended yearly for women over age 50.

We hypothesized that the availability of software to measure phalangeal BMD with digital mammography equipment would allow at risk postmenopausal women to have their BMD assessed when a routine mammogram is performed.

Radiographic Absorptiometry (RA) measures volumetric BMD of the middle phalanges from a standard AP x-ray of the non-dominant hand (with an aluminum reference wedge placed near the hand). We conducted a feasibility study to validate the use of Full Field Digital Mammography (FFDM) system to acquire the required hand x-ray for RA analysis, and to assess the accuracy of the results from the mammography system as compared to the results from standard x-ray films.

A customized module of the OsteoGram ® (CompuMed Inc., Los Angeles, CA) which uses RA, has been developed to accept digital images from a Hologic - LORAD Selenia digital mammography system. The module accounted for normalizing the radiographic optical density response of the mammography system to the standard radiographic response of standard x-ray equipment.

50 volunteers, age between 40 to 78 seen for routine mammography at University of California, San Diego (UCSD) Medical Center were recruited to assess their phalangeal BMD by taking two hand x-rays. One x-ray was taken with the Hologic FFDM system with the settings at 35 kVp, 5 mAs; and another using standard x-ray equipment with mammography films. The exclusion criterion was a history of diagnosed osteoporosis.

To analyze the two data sets of radiographs, the customized module was used for the FFDM data and a standard OsteoGram system was used for standard film data (using standard flatbed scanner). Regression analysis of the results showed a significant (p<0.001) Pearson correlation coefficient of 0.94 between the two methods validating the results from FFDM.

Of the 50 volunteers, 16 had a phalangeal BMD T-score of less than −1.0 and 4 had a T-score below −2.5 (T-score calculated using OsteoGram's normative database). This finding calls for assessing the prevalence of low bone density amongst females scheduled for mammogram.

We conclude that the availability of phalangeal BMD testing during breast cancer screening with FFDM may provide an opportunity for BMD screening for osteoporosis.

1. J Am Geriatr Soc. 2006 Mar;54(3):485-9.

Disclosures: S.L. Silverman. CompuMed 6.

This study received funding from: CompuMed.

T280

Clinical Evaluation of Jaw Bone Density with a Newly Developed “Jaw Bone Density Evaluation System” - Relationship with Osteoporosis Evaluation. Y. Takaishi1, A. Kamada2, T. Ikeo2, T. Miki3, Takaishi Dental Clinic, Himeji-city, Japan, 2Biochemistry, Osaka Dental University, Osaka, Japan, 3Geriatric medicine, Osaka City University, Osaka, Japan.

<Background> The relationship between osteoporosis and changes in alveolar bone has become a target of interest, as the formation and resorption processes of the bone structure and periodontal structure are analogous. The number of osteoporosis patients increases in accordance with age. The same trend is observed in patients with periodontal diseases or with teeth loss. Therefore, osteoporosis may be one of the risk factors of the teeth loss due to reduction of the alveolar bone. Unfortunately clinical methods to evaluate alveolar bone have yet to be established and medical proofs are still insufficient.

<Objective> We estimated alveolar bone density using a new “mandibular bone density evaluation system” and examined the relationship with osteoporosis to determine whether jaw bone evaluation is clinically useful in assessing osteoporosis.

<Method> Thirty four postmenopausal female subjects (age: 50-69, average age: 59.9) participated in the experiment. In a simple X-ray scan of mandibular bone, standard substance (aluminum step wedge) was also scanned. The bone density was estimated based on the difference in their rates of transmittance. The correlations among bone mineral density (BMD) by DXA, cortical bone of proper alveolar bone portion and cancellous bone of support alveolar bone portion in mandibular bone were examined.

<Results> Cancellous bone density of support alveolar bone (A) and cortical bone density of proper alveolar bone(B) were 87.0±18.1(SD) and 89.9±26.7, respectively. Significant correlation between (A) and (B) was observed (r = 0.736,p<0.01). The correlations of lumber BMD by DXA and BMD (A) and (B) were significant, respectively (r = 0.638,p<0.01, 0.414,p<0.05).

<Conclusion> Both cancellous bone density of support alveolar bone (A) and cortical bone density of proper alveolar bone (B) correlated with lumber BMD; however, the correlation with (A) was greater. These results suggest alveolar bone BMD by the jaw bone density evaluation system can be utilized as one of the screening tools for osteoporosis. This system may also utilized in a large scale screening of osteoporosis in the dental fields because of low cost and short period for the evaluation. This system may provide great contributions for osteoporosis patients.

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Disclosures: Y. Takaishi, None.

T281

Body Composition Measurements with Lunar iDXA: Precision Evaluation. E. Toussirot*1, C. Semon*2, F. Penfornis*2, D. Wendling1, 1Rheumatology, University Hospital, Besancon, France, 2Endocrinology, University Hospital, Besancon, France.

Dual-energy x-ray absorptiometry (DXA), widely used for osteoporosis assessment, is increasingly used for measurement of body composition. Body composition is an important issue in metabolic diseases, pediatric patients but also in rheumatological conditions such as inflammatory rheumatic diseases, osteoarthritis and osteoporosis.

Precision error is a measure of the ability of a DXA system to detect small changes in a patient's body composition and bone mineral density (BMD). Lower precision error reduces the least significant change (LSC), allowing a smaller change in BMD or fat and lean mass to be identified as biological rather than related to instrument variability. Precision error is related to technology as well as the subject population; precision may degrade in thicker subjects due to decreased x-ray flux and the effect of thicker tissue on edge detection.

We evaluated precision error of total body BMD, bone mineral content (BMC), %fat, fat mass, and lean mass.

We scanned 24 women and 7 men for a total of 31 subjects (mean age 56.9 yrs, SD 13.1; mean BMI 28.4, range 17.3 – 39.6). 13 subjects had BMI values in the obese category (above 30), with a mean total %fat of 38% for the entire group. Each subject's total body was scanned two times, with repositioning between scans. Precision (%CV) was calculated as the root-mean-square standard deviation.

Precision values for Lunar iDXA total body BMD and BMC were 0.56%and 0.57% respectively. Body composition precision values were 0.63% for %fat, 0.59% for fat mass, and 0.45% for lean mass.

We conclude that the Lunar iDXA provided excellent precision for total body measurements, including BMD, BMC and body composition. 

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Disclosures: E. Toussirot, None.

T282

An Evaluation of Forearm BMD Measurement for Diagnosis and Treatment Monitoring in Men. N. Vallarta-Ast, D. Krueger, N. Binkley. University of Wisconsin, Madison, WI, USA.

Degenerative disease commonly confounds lumbar spine (LS) DXA assessment in older men, as such, forearm measurements are often appropriate. The ISCD recommends that the 1/3rd or “mid” radius site be utilized as the forearm region of interest (ROI) for diagnosing osteoporosis. It is therefore logical that this site would also be utilized for monitoring therapy. However, as the 1/3rd radius ROI is small and comprised primarily of cortical bone, it is plausible that use of the “total” radius ROI, which contains a higher proportion of cancellous bone, would be preferable for monitoring response to therapy. This report investigates the utility of routine forearm measurement in men and explores the utility of using the 1/3rd vs. the total radius ROI in monitoring BMD change over time in men. Between November 2003 and September 2006, 1599 men had clinically-indicated DXA scans performed at the Middleton VAMC. LS, proximal femur and non-dominant forearm images were obtained in all. Thirty-two percent (516/1599) of these predominately white men (97%, 2% Black), of mean age 68 years and mean BMI 28.3 kg/m2, were osteoporotic by lowest T-score. The 1/3rd radius was equal to the spine (p = 0.9) in ability to detect low BMD in the group as a whole. However, in men age 80 and over, the 1/3rd radius was superior in low BMD detection (p = 0.007). Moreover, in men age 70+, osteoporosis was uniquely identified at the lumbar spine in only 2%, but at the 1/3rd radius in 14%.

From November 2003 to September 2006, 141 men had a repeat DXA scan performed, of whom 49 had initiated bisphosphonate (BP) therapy. As such, evaluation of non-response to BP therapy (defined here as a BMD decline greater than the LSC) is feasible. As might be expected, a BMD increase greater than the LSC was observed in 43%, 27%, 12% and 14% at the L1-4 spine, total femur, 1/3rd and total radius respectively. However, significant BMD declines were observed in none of these men at the LS, 4% at the total femur, 6% at the 1/3rd radius and 16% at the total radius.

In conclusion, the 1/3 radius is useful in identifying men with osteoporosis. However, “non-response” to BP therapy will be more common at the total radius than the 1/3rd radius, total femur or LS. DXA interpreters should be aware of this phenomenon when utilizing the total radius as a site to monitor therapy. Evaluation of the clinical significance, if any, of this “non-response” is indicated.

Disclosures: N. Vallarta-Ast, None.

T283

Skeletal Health between Two Different Groups of Hispanic American Women. J. Vargas-Jerez*, M. D. Walker, C. Gagel*, D. J. McMahon, R. A. Lantigua*, J. P. Bilezikian. College of Physicians and Surgeons, Columbia University, New York, NY, USA.

Osteoporosis is common in non-Caucasian women, particularly Hispanic Americans. Multiple studies suggest obvious racial differences in bone mineral density (BMD) and fracture risk. It is unclear whether fracture risk in non-Caucasian individuals should be assessed using race-specific referent BMD databases. A Hispanic referent BMD database for the hip, comprised of BMD values from Mexican Americans, is available from the third National Health and Nutrition Examination Survey (NHANES III). It is not known if this Mexican American database is applicable to other Hispanic groups. The purpose of this study is (1) to develop a local referent BMD database for Dominican American women who make up the majority of our local Hispanic population; and (2) to compare these data to referent values for Mexican and Caucasian American women.

560 women, 80 per decade from 20 to 90, are being recruited. Along with DXA of the total hip (TH), femoral neck (FN) lumbar spine (LS) and forearm, demographic, familial, medical, nutritional and behavioral information is being obtained. To date, 332 women have been recruited. Preliminary data suggest that Dominican American women have significantly higher mean BMD values at the TH for the decades beginning at age 50 (0.927 ± 0.113 vs. 0.859 ± 0.134 g/cm2; p<0.001), 60 (0.876 ± 0.131 vs. 0.815 ± 0.134 g/cm2; p = 0.04), and 70 (0.830 ± 0.109 vs. 0.720 ± 0.134 g/cm2; p<0.001) but not at the younger decades up to 40, or over 80 as compared with the NHANES III database for Mexican Americans. Femoral neck (FN) values were similar between the Dominican and Mexican American groups.

Dominican American women have higher bone density at the TH compared to Caucasian women for the decades beginning at age 40 (0.985 ± 0.125 vs. 0.922 ± 0.122; p<0.001), 50 (0.927 ± 0.113 vs. 0.886 ± 0.122 g/cm2; p = 0.04), 60 (0.876 ± 0.131 vs. 0.827 ± 0.122 g/cm2; p = 0.07). 70 (0.830 ± 0.109 vs. 0.759 ± 0.122 g/cm2; p<0.001), & 80 (0.780 ± 0.081 vs. 0.691 ± 0.122 g/cm2; p = 0.04) but not for the 2 younger decades (20-40). Mean FN values were also greater in the Dominican American group compared to Caucasians across most age ranges. Mean LS BMD values were similar between groups.

These data suggest major differences in BMD between two Hispanic American subgroups and in comparison to the standard Caucasian database. The data also suggest that a single Hispanic American referent database may not be applicable to all Hispanic American women. How these differences relate to fracture risk among different Hispanic American groups is an important next investigative step.

Disclosures: M.D. Walker, None.

T284

Comparison of pQCT-based Measures of Radial Bone Geometry and Apparent Trabecular Structure Acquired Using Different Algorithms. N. J. MacIntyre*1, D. Inglis2, 1School of Rehabilitation Science, McMaster University, Hamilton, ON, Canada, 2Department of Civil Engineering, McMaster University, Hamilton, ON, Canada.

The purpose of this study was to compare methods of analyzing peripheral quantitative computed tomography (pQCT) images using different software and/or algorithms aimed at evaluating similar bone characteristics in the distal radius. The sensitivity of variables to changes in positioning in the region of the 4% site was also assessed.

Seven left cadaveric forearms (mean age (SD) 74.13 (7.09) y) were imaged at 0.5 mm intervals around the 4% site using pQCT (10 slices, 0.2 mm × 0.2 mm, 2.5 mm thick; Stratec XCT2000L). We analyzed the images for total and cortical cross-sectional areas (B.Ar, Ct.Ar), mean cortical thickness (Ct.Th) and its variation in terms of SD using both commercial software (Stratec v6.00 B) and an in-house developed program (pQCT Pro). pQCT Pro not only quantifies a variety of bone geometry variables, but also provides estimates of apparent trabecular structure, including number (App.Tb.N), thickness (App.Tb.Th) and spacing (App.Tb.Sp) using two different stereological approaches: the parallel plate model and mean intercept length (MIL) analysis. Measures of similar bone characteristics (mean values for 10 slices) were compared using Bland Altman plots and the absolute mean differences were expressed as percentages. We assessed the effect of slice position using one-way repeated measures ANOVA (p <0.05).

The percent difference in measures varied from 3.5% (B.Ar) to 64.7% (App.Tb.Sp). As the mean value for App.Tb.Sp increased, the difference between the measures increased. The other measures did not differ systematically. Comparable methods produced mean values that ranked the seven specimens similarly with few exceptions (Ct.Th, specimen 3; App.Tb.Th, specimen 7; App.Tb.N, specimens 1, 3, 5, 6).

Measurements of B.Ar at the 4% site differed from those acquired more than 0.5 mm proximal and all more distal positions (p < 0.001). Ct.Ar at the 4% site differed only from positions that were at least 2 mm more proximal or distal (p < 0.001). Parallel plate model measurements of App.Tb.Sp at the 4% site differed from those 2 mm more proximal (p < 0.02). For the other variables, positioning did not have a significant effect.

Excellent agreement in B.Ar measures was observed. Differences between the other measures reflect differences in the algorithms used to derive them and these differences need to be considered when selecting outcome measures. Parallel plate derived measures are more influenced by anatomic variation than MIL based measures of apparent structure which makes the latter measure more desirable for cross sectional study designs. With the exception of B.Ar. measures acquired within 2 mm of the 4% site are consistent.

Disclosures: D. Inglis, None.

This study received funding from: NSERC RGPIN (NJM).

T285

Longitudinal Change in Bone Strength Indices: The Framingham Study. S. Menn, Y. H. Hsu, D. P. Kiel, D. Karasik. IFAR, Hebrew SeniorLife, Boston, MA, USA.

Bone strength is a valid predictor of bone's ability to resist fracture. Metacarpal bones have been routinely measured in previous research studies to predict fractures and quantify bone loss. Since change in metacarpal bones geometry over time may characterize the other long bones in the body, we evaluated effect of age, height and weight, as well as change in height and weight over a time interval of 22.5±0.8 yrs on change in radiographic metacarpal Bone Strength Indices (BSIs).

Convenience sample of digitized hand x-rays (208 out of ∼800) participants of the Framingham Study Original cohort (54±5 yrs old at baseline, BL), was used. At both BL (1967–1970) and follow-up, FU (1992–1994), the following indices as well as changes in them, Δ, were measured at the midshaft: Metacarpal Cortical Index (MCI), Cortical Thickness (MCT), and Section Modulus (MZ). Second (Met2) and third (Met3) metacarpals were studied.

All analyses were done by gender. We calculated correlation coefficients between ΔBSIs and BL age, height, weight, Δheight, and Δweight. Subsequently, we performed regression analysis to estimate effect of the above variables on ΔBSIs over time.

In final analysis, 79 men and 129 women were included. Changes in Met2 and Met3 BSIs were normally distributed and evident in both sexes, ranging from −45.6 to 38.5% in men and −63.6 to 42.4% in women.

In Met2, age at BL significantly negatively correlated with ΔMCI and ΔMCT in women only (r = −0.25 to −0.29, p<0.005). Height on BL significantly negatively correlated with μMCI and ΔMCT in men, while weight on BL and Δheight correlated with ΔMZ in women. Δweight positively correlated with ΔMCI, ΔMCT, and ΔMZ of both Met2 and Met3 in women (r = 0.17 to 0.24, p< = 0.05).

In regression analyses of Δheight and Δweight (with BL age, height, and weight in the same model), BL weight and Δheight were marginally significant predictors of Met2 ΔMCI in men, whereas in women, BL weight, height and Δheight predicted Met2 ΔMZ (beta = −1.17, 8.74 and 26.20, p<0.05). In women, Δweight was significant predictor of both Met2 and Met3 ΔMCI and ΔMCT (beta = 0.07 to 0.11, p< = 0.05).

In conclusion, in this pilot study, change in metacarpal bone geometry and strength was observed over an interval of 22 yrs, mostly in women. There was a wide range of changes with age, from loss to gain of bone strength indices. Weight and height at baseline, as well as change in weight and height, predicted the longitudinal change in BSIs in women, less strongly in men. Age-related change in metacarpal bone strength indices may reflect general behavior of long bones.

Disclosures: D. Karasik, None.

This study received funding from: NIAMS/NIA.

T286

Correlations Between HR-pQCT & High-Field MR In Vivo Bone Imaging. G. J. Kazakia1, B. Hyun1, A. Burghardt1, R. Krug1, D. Newitt1, A. DePapp2, T. Link1, S. Majumdar1, 1Radiology, UC San Francisco, San Francisco, CA, USA, 2Merck & Co., Inc., Bryn Mawr, PA, USA.

The purpose of this study was to compare bone structural measures obtained via high-resolution peripheral QCT (HR-pQCT) to those obtained by high-field MRI in a cohort of post-menopausal females enrolled in a longitudinal pilot study comparing the effects of alendronate to placebo on bone microarchitecture.

Fifty-two early post-menopausal women classified as osteopenic by lumbar and/or hip DXA T-score underwent HR-pQCT (XtremeCT, Scanco Medical AG) and MR imaging of the distal radius and tibia. For HR-pQCT imaging a 9 mm axial length was imaged, with isotropic 82 micron voxels. Bone volume fraction (BV/TV) was derived assuming a density of 1200 mg HA/cc for mineralized bone. The 3D distance transform (DT) method was used to assess trabecular number (Tb.N). Following cortical segmentation using a Gaussian blur and a fixed intensity threshold, cortical thickness (Ct.Th) was calculated as the cortical volume divided by the periosteal area.

MR imaging was performed at 3 Tesla (GE Medical) using a balanced steady state free precession sequence. Spatial resolution of the resulting images was 156 × 156 × 500 micron. Regions of interest corresponding to the HR-pQCT regions were used to calculate structural parameters. Each image was binarized using a global threshold, and BV/TV, apparent Tb.N and other structural parameters were calculated using 2D histomorphometric methods. Cortical boundaries were defined manually and Ct.Th was derived using the DT method.

Despite the distinct differences between analysis techniques, significant correlations between HR-pQCT and MR parameters were found. The correlation was strongest for Tb.N (R2 = 0.52) and Ct.Th (R2 = 0.43) and weaker for all other parameters. However, MR and HR-pQCT provide statistically different measures of structure parameters. BV/TV was significantly higher in the MR analyses (mean MR/pQCT = 3.2 radius; 3.9 tibia) while Tb.N was lower (mean MR/pQCT = 0.9 radius & tibia). Tb.Th and Tb.Sp values were therefore higher and lower, respectively, as calculated by MRI. MR analysis resulted in higher values of Ct.Th (mean MR/pQCT = 2.0 radius; 1.3 tibia). In terms of correlations with DXA measurements, significant relationships were found only between HR-pQCT parameters at the distal radius and radius T-score (R2 = 0.19-0.58).

The XtremeCT system uses a unique density derived analysis technique, necessitating the careful investigation of relationships between HR-pQCT and MR measures. Our results demonstrate discrepancies in absolute values and moderate correlations between modalities, suggesting that differences in the analysis techniques must be taken into account when comparing and interpreting the results of these imaging modalities.

Disclosures: G.J. Kazakia, Merck & Co., Inc. 2.

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

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