Phosphorylated bone sialoprotein increases resorption by osteoclasts through the NFATc1 pathway

ABSTRACTS / Bone 43 (2008) S38–S75

associations with total hip BMD and troancher BMD (p = 0.0540.006). Interestingly, mutations in the MATN3 gene have been reported in a variety of skeletal diseases and a functional knockout of the MATN3 gene increases BMD in mice (van der Weyden et al., 2006). Conclusion: MATN3 and ZIC2 genes were identified as novel osteoporosis susceptibility genes by computational disease gene identification strategy.

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A21 Single Nucleotide Polymorphism T(861-20)C in TGFbeta1 Gene Affects its Expression in Peripheral Blood Mononuclear Cells in Russian Postmenopausal Women Elena Tchetina, Mikhail Krylov, Oksana Nikitinskaya, Nikolai Demin, Tatiana Korotkova, Natalia Toroptsova, Karina Maslova, Lidiya Benevolenskaya, Valerii Myakotkin Institute of Rheumatology, Russian Academy of Medical Sciences, Moscow, Russia

doi:10.1016/j.bone.20 08.08.021

A20 Implication of cytosolic phospholipase A2 in the control of osteoclast metabolism Hugues Allard-Chamard, Artur José de Brum Fernandes Division of Rheumatology, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Québec, Canada Objective: In order to investigateif cytosolic phospholipase A2 (cPLA2) is implicated in the control of osteoclast (OC) metabolism, we decided to study its expression in mature human OCs and the effect of its inhibition on OCs physiology (osteoclastogenesis, apoptosis and cytoskeleton). We also studied the molecular regulation of cPLA2 by RANKL, an important mediator of OCs function. Methods: In this study, immunohistochemistry with the avidin/ biotin system was performed on bone slices from biopsies of human healthy or pathologic bone and on human OCs culture to determine the presence of cPLA2. The impact of phospholipase A2 inhibition on actin remodeling, apoptosis rate and osteoclastogenesis was studied using a model of OC differentiated from peripheral mononuclear blood cells incubated in the presence of M-CSF 10 μg/ ml and RANKL 30 μg/ml for 21 days. The actin structure of mature OCs was assessed under epifluorescence microscopy after staining with rhodamine-phalloidine, tartrate-resistant acid phosphatase positive (TRAP+) and Hoesch 33342 to reveal the OC phenotype. OCs were counted as TRAP+, multinucleated cells. Osteoclastogenesis was assessed after exposition of OC precursors to cPLA2 inhibitors for 21 days in the presence of RANKL and M-CSF. Th effect of cPLA2 inhibitors on mature OCs apoptosis rate was assessed using terminal transferase-mediated DNA end labeling and JC-1 mitochondrial membrane potential assay. Phosphorylation of cPLA2 following stimulation of OCs with RANKL was observed using immunofluorescence. Results: Immunohistochemistry showed that cPLA 2 was expressed in OCs from both normal or pathologic (osteoporosis, osteoarthritis, Paget's disease) bone and in in vitro-differentiated human OCs. Inhibition of cPLA2 in human OCs promoted the formation fillipodia and disruption of actin rings, it also increased OC formation and decreased apoptosis rate of mature OCs. Stimulation of starved OCs with RANKL increased cPLA2 phosphorylation in the nucleus. Conclusion: cPLA2 is present in OCs where it is linked to specific functions. It participates in the maintenance of actin ring structures. It also increases osteoclastogenesis and decreases the apoptosis rate of mature OCs. RANKL, a major regulator of osteoclastogenesis and OCs function, induce cPLA2 phosphorylation in the nuclear compartment of OCs; although the exact physiological meaning of this phosphorylation is not know yet it suggests that cPLA2 could act a regulator of OCs functions. doi:10.1016/j.bone.20 08.08.022

Objective: Transforming growth factor beta1 (TGFbeta1) is essential in regulation of bone remodelling. It is responsible for osteoblast differentiation, matrix growth as well as inhibition of osteoclast differentiation and resorptive activity. Therefore this protein is considered as a candidate gene controlling bone mineral density (BMD). Here we hypothesized that TGFbeta1 single nucleotide polymorphism (SNP) associated with lower bone mass may affect its gene expression thus predisposing osteoporosis (OP) development. Methods: T(861-20)C SNP of TGFbeta1 gene located in the 5th intron was detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of DNA of 185 postmenopausal osteoporotic (OP) women and 186age-matched healthy women from Russia. BMD was examined by DXA. mRNA was isolated from peripheral blood of 28 OP patients and 17 healthy women and used in gene expression studies performed with Real-time PCR. Results: No significant difference in the frequency of individual alleles and genotypes between OP sample and control patients has been noted. The minor allele (A) frequency was 0.28. Our studies have shown significant association of (861-20)CC TGFbeta1 genotype with lower lumbar spine BMD (p = 0.03) in Russian OP women. This was accompanied by lower TGFbeta1 gene expression in peripheral blood mononuclear cells (PBMC) in (861-20)CC (n = 10) genotype carriers compared to combined (861-20)TT and (861-20)TC (n = 12) genotype carriers in OP sample. Healthy women carrying (861-20)CC genotype (n = 13) have also shown lower TGFbeta1 gene expression in PBMC compared to combined (861-20)TT and (861-20)TC (n = 4) genotype carriers (p b 0.0001). Conclusion: Our studies have shown significant downregulation of TGFbeta1 expression in PBMC of osteoporotic patients versus healthy sample, which may account for their decreased bone forming capacity. C-allele dosage of TGFbeta1 gene T(861-20)C SNP can affect its expression in PBMC of Russian postmenopausal women. Lower TGFbeta1 expression associated with lower bone mass in healthy carriers with (861-20)CC TGFbeta1 genotype may indicate their predisposition to osteoporosis while in osteoporotic (861-20)CC genotype carriers excessive bone mass loss could be expected. doi:10.1016/j.bone.20 08.08.023

A22 Phosphorylated bone sialoprotein increases resorption by osteoclasts through the NFATc1 pathway Hong Hong Chen, Jonathan Gordon, Alexey Pereverzev, Stephen Sims, Graeme Hunter, Jeffrey Dixon, Harvey Goldberg CIHR Group in Skeletal Development and Remodeling, The University of Western Ontario, London, ON, Canada Objective: Extracellular matrix proteins such as bone sialoprotein (BSP) have been shown to affect osteoclastogenesis and osteoclastic activity. Furthermore, post-translational modifications, specifically phosphates, on BSP are apparently involved in the activation of osteoclastic bone-resorption activity. However, the mechanism underlying osteoclast activation has remained elusive. NFATc1 is a

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ABSTRACTS / Bone 43 (2008) S38–S75

transcription factor that plays a critical role in osteoclast differentiation. Here we study whether the effects of BSP on osteoclastic resorption involve NFATc1. Methods: The following proteins were purified to homogeneity using FPLC: post-translationally modified rat bone native BSP (nBSP); rat recombinant BSP (rBSP) containing no modifications; protein kinase CK2-treated rBSP (CK2-rBSP); and protein kinase CK2-treated rBSP Ser/Thr137,170,171,180,192Ala [CK2-rBSP(S/T5A)]. Phosphate content was determined by MALDI-TOF mass spectrometry. Osteoclasts were isolated from the long bones of neonatal rabbits. To quantify resorption, osteoclasts were incubated for 24 h on dentin slices coated with protein. Slices were then stained for tartrate-resistant acid phosphatase (TRAP) activity to determine osteoclast number and with toluidine blue to determine total area resorbed. In parallel studies, osteoclast attachment and nuclear localization of NFATc1 were quantified 3 h after plating on coverslips coated with protein. Results: Coating of substrate with nBSP, rBSP, CK2-rBSP or CK2rBSP(S/T5A) did not alter osteoclast attachment. However, nBSP and CK2-rBSP promoted resorption (nBSP, 2.3 ± 0.3 fold of control; CK2rBSP, 2.5 ± 0.9), whereas rBSP had no effect (1.1 ± 0.1). Similarly, nuclear translocation of NFATc1 was enhanced in osteoclasts plated on coverslips coated with nBSP (1.68 ± 0.05 fold of control) or CK2-rBSP (1.48 ± 0.10), but not with rBSP (1.05 ± 0.03) or CK2-rBSP(S/T5A) (1.09 ± 0.24 fold). To investigate the role of NFAT in mediating BSP-induced resorption, we used 11R-VIVIT (a cell-permeable peptide inhibitor of NFAT activation) and 11R-VEET (inactive control peptide). 11R-VIVIT, but not 11R-VEET, decreased NFATc1 translocation in osteoclasts bound to nBSP and blocked the stimulatory effect of nBSP on resorptive activity Conclusion: These findings demonstrate that phosphorylated BSP, both nBSP and CK2-rBSP, enhances NFATc1 activation in osteoclasts, which in turn stimulates resorption. Furthermore, specific phosphorylation sites in BSP are critical for the stimulation of NFATc1 translocation. This study was supported by the Canadian Institutes of Health Research (CIHR). doi:10.1016/j.bone.20 08.08.024

A23 Effect of glucose and pioglitazone on rat bone mesenchymal stem cells differentiation into adipocytes Haining Fang, Yuming Li, Zhiping Liu, Liping Deng Wuhan Union Hospital, Wuhan, China Objective: To examine the modulation of pioglitazone on rat bone mesenchymal stem cell (BMSCs) differentiation into adipocytes in different doses of glucose concentration and to investigate the effect of glucose and pioglitazone on bone metabolism. Methods: BMSCs were harvested from the femur and tibia bones of the rat, then separated, purified, proliferated and differentiated into adipocytes in the presense of an adipogenic medium (including dexamethasone, 3 - isobutyl - 1 - methyl - xanthine, insulin ) using different doses of glucose (25 mmol/l, 50 mmol/l) alone or with different doses of piogitazone (0.1 μg/ml, 1 μg/ml), differentiated adipocytes were identificated by Oil Red O, real time PCR were taken to assay the expression of adipose-specific mRNAs: LPL and PPAR gamma. Results: After adipocytic inducing for 21 days, the number of adipocyes increased in higher concentration glucose as detected by Oil Red O, the mRNA expression of LPL and PPAR gamma γincreased 1.40 and 1.63 folds in 50 mmol/l glucose than in 25 mmol/l glucose. The differentiation of adipocytes increased in a dose-dependent manner

with pioglitazone than without pioglitazone in both concentrations of glucose. In 25 mmol/l glucose group, the expression of LPL and PPAR gamma grew1.43and 1.50 fold in 0.1 μg/ml pioglitazone group comparing with pioglitazone-absence group .1 μg/ml pioglitazone group raised even more, and 50 mmol/l glucose group kept similar situation with 25 mmom/l glucose group. Conclusions: High concentration glucose stimulated the differentiation of BMSCs into adipocytes, this observation provides a potential mechanism of diabetes-induced osteoporosis. Pioglitazone dramaticly increased adipogensis of BMSCs, and this effect shows a dose-dependent argument, pioglitazone cause decreasing of bone mass by promoting adipogenesis and inhibiting osteoblastic diffen. doi:10.1016/j.bone.20 08.08.025

A24 Novel application of HA-TCP biomaterials in distraction osteogenesis shortened the lengthening time and promoted bone consolidation Yan Wang, Ming Ni, Peifu Tang, Gang Li Department of Orthpedics, General Military Hospital, China Department of Orthpedics, Queens University Medical School, Belfast, UK Introduction: Distraction osteogenesis (DO) is induction of osteogenesis by means of an osteotomy, followed by fixation with an external fixator and subsequent controlled gradual lengthening. Hydroxyapatite (HA) and Tri-calcium phosphates (TCP) are osteoconductive, porous HA-TCP biomaterials that have similar composition, structure and characteristics as native bone with good biocompatibility. This study tested the hypothesis that the use of biomaterials in distraction osteogenesis (DO) would reduce the treatment time and enhance bone formation quality. Methods: A 1.0 cm tibial shaft was removed from the left tibia of 36 rabbits. Rabbits were randomly divided into three groups: Group A, where the defect gap was reduced with the tibia shortened for 1.0-cm; Group B, where the defect gap was filled with 1.0-cm restorable porous hydroxyapatite and Tri-calcium phosphates cylindrical block (HA/TCP block, diameter is 0.5-cm); Group C, where the 1.0-cm defect gap was reduced to 0.5 cm and the remaining 0.5-cm defect gap was filled with 0.5-cm HA/TCP block. The tibia was then fixed with unilateral lengthener; for groups A and C, lengthening started 7 days after surgery at a rate of 1.0 mm/ day, in two steps. Group A received lengthening for 10 days and Group C for 5 days, there was no lengthening for Group B. All animals were terminated on Day 37 following surgery. The excised bone specimens were subject to micro-CT, mechanical testing and histological examinations. Results: Bone mineral density and content and tissue mineral density and content, as well as the mechanical properties of the regenerates were significantly higher in Group C compared to Groups A and B. Micro CT and histological examinations also confirmed that the regenerates in Group C had most advanced bone formation, consolidation and remodeling compared to other groups. Conclusion: In summary, we have demonstrated for the first time that a combination of biomaterials with distraction osteogenesis technique could be a new and cost effective means to reduce the treatment time and enhance bone consolidation in the management of larger bone defects. doi:10.1016/j.bone.20 08.08.026