P2X7 receptors are expressed by primary human osteoblasts — A role in cell signaling?

Abstracts

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PP017 P2X7 receptors are expressed by primary human osteoblasts — A role in cell signaling? D. Aslana,⁎, S. Syberga, Z. Henriksena, P. Schwarza,b, T.K. Larsenc, N.R. Jørgensena a Research Center of Ageing and Osteoporosis, Depts. Clinical Biochemistry and Medicine, Glostrup Hospital, Glostrup, Denmark b Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark c Dept. of Orthopedic Surgery, Glostrup Hospital, Glostrup, Denmark

indicate that forced expression of sy2 results in reduced cell growth and increased apoptosis in proliferating non-differentiated osteoblastic precursor cells. Decreased alkaline phosphatase activity also suggests impaired commitment in the osteoblastic lineage. These effects of sy2 may be related to the alteration of Wnt signaling. Hence, sy2 appears as a new regulator of osteoblastic precursor fate through modulation of Wnt signaling. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared.

Abstract: Background: The ionotropic ATP-gated P2X7 receptor is not only primarily expressed in hematopoietic cells, but also in primary human osteoclasts (OC), where it is involved in signaling to OC from osteoblasts and among OC. Thus the receptor is involved in the regulation of osteoclast function, and subsequently in the regulation of bone metabolism. However, several studies have addressed the function and expression of the receptor in osteoblastic cells but with conflicting results. The aim of this study was to assess the expression of the P2X7 receptor in primary osteoblasts and to determine the function in relation to cell signaling and cell function in these cells. Method: Trabecular human osteoblasts (ThOB) were derived from human trabecular bone obtained from orthopedic surgical procedures. Bone chips were washed thoroughly in PBS, digested with collagenase and cultured for 3–4 weeks in Minimum Essential Medium without Phenol Red supplemented with 10% heat inactivated fetal calf serum and medium was changed every 7 days. For viability and mineralization assays, ThOB were seeded in 96-well plates with mineralization medium (50 μL/mL ascorbic acid, 10 mM b-glycerophosphate). Cells were daily treated with BzATP (1–100 μM) with and without preincubation with oxidized ATP (oATP — 300 μM). Mineralization and proliferation were detected by standard colorimetric analysis. Cultures treated for 1 and 3 h were used for RNA isolation, and following RealTime PCR determination of genes involved in osteoblast differentiation, matrix mineralization, apoptosis and osteoclast activation. Results: Short-term treatment with BzATP increased proliferation and long-term treatment increased mineralization. BzATP treatment inhibited RUNX2, ALP, CASP1 and 3, RANKL and GJA1, but increased expression of osteocalcin. Oxidized ATP inhibited mineralization and expression of ALP, osteocalcin, RANKL and increased RUNX2 and GJA1. Conclusion: Primary human osteoblasts require P2X7 signaling for proliferation and mineralization This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared.

PP019 The mechanical environment in bone marrow predicts osteoblast and osteoclast activities D. Webster⁎, F. Schulte, F. Lambers, G. Kuhn, R. Müller Institute for Biomechanics, ETH Zürich, Zürich, Switzerland

doi:10.1016/j.bone.2012.02.206

PP018 Syndecan-2 controls the fate of osteoblast precursors through modulation of Wnt signaling S. Devanadhan, R. Mansouri, F.-X. Dieudonné, C. Marty, A. Barbara, P.J. Marie, D. Modrowski⁎ Laboratory of Osteoblast Biology and Pathology, Unit 606, INSERM, Paris, France Abstract: Apoptosis of osteoblastic cells is an essential process that controls normal bone remodeling and contributes to bone loss occurring with age and in various pathological conditions. Understanding the mechanisms that control the maintenance of the osteoblastic precursor population is a major challenge in therapeutic strategies aiming to increase bone formation. We previously showed that a proteoglycan syndecan-2 (sy2) controls apoptosis and the response to cytotoxic stress in osteosarcoma cells. To determine the role of sy2 in normal osteoblastic cells, we analyzed the bone phenotype of transgenic (TG) mice with selective sy2 overexpression in osteoblastic cells driven by the 2.3 kb Col 1a1 promoter. Sy2 expression was increased in both mature osteoblasts and bone marrow mesenchymal stromal cells in TG compared to wild type mice. Sy2 overexpression decreased the trabecular bone volume and trabecular thickness and increased trabecular separation. Ex-vivo studies showed that sy2 markedly reduced the number of bone marrow (BM)derived colony-forming unit fibroblasts in long term cultures. Consistently, proliferation was decreased in sy2 overexpressing cells, as shown by crystal violet and BrDU incorporation. Similarly, murine C3H10T1/2 mesenchymal cells infected with a lentiviral vector coding sy2 displayed a significantly reduced cell growth after 7 days of culture. Sy2 also increased cell death, enhanced number of cells binding annexin-V in response to serum starvation and caspase activity. TUNEL staining revealed that the number of apoptotic cells was also increased in vivo in the bone marrow of TG mice. In contrast, in confluent cultures of C3H101/2 cells, sy2 did not modify cell number or caspase activity. Sy2 did not alter in vitro osteogenic capacity of BM-derived and C3H10T1/2 cells but decreased alkaline phosphatase activity. Moreover, sy2 overexpression resulted in reduced activation of TCF reporter and alkaline phosphatase activity induced by recombinant Wnt3a in C3H10T1/2 cells. Altogether, our results

doi:10.1016/j.bone.2012.02.207

Abstract: It is widely hypothesized that mechanical strains sensed by osteocytes dictate the magnitude of load-induced bone formation, i.e. osteoblastic activity. However, despite attempts, quantitative experimental verification is still missing. The mechanical environment in bone marrow has also been implicated in bone's response to mechanical stimulation. Here, we hypothesize that the mechanical signals generated in the bone marrow determine not only osteoblastic but also osteoclastic activity. Recently, using the mouse tail loading model in combination with in vivo microcomputed tomography (μCT) we tracked load induced changes in trabecular bone in the sixth caudal vertebrae (C6) of C57BL/6 mice (n = 9). With image registration techniques we quantified the amount of newly formed and resorbed bone volumes (fBV and rBV) [1]. To indentify the mechanical signals responsible for adaptation, local morphometric changes were compared to micro-finite element (μFE) models of C6 vertebrae prior to loading. To create the μFE models, bone voxels from μCT images of the C6 vertebrae prior to loading were mapped to brick elements (size ≈ 1,800,000 elements). The Young's moduli assigned to bone and marrow voxels were 14.8 GPa and 2.0 MPa, respectively. Similarly their Poisson's ratios were 0.3 and 0.16. The mechanical parameters calculated were: strain energy density (SED) on trabeculae at bone forming and resorbing surfaces (fTb.SED and rTb.SED), along with SED in the trabecular bone and marrow volumes (vTb.SED and vMr.SED). The gradients of each parameter were also calculated. Regression analysis showed mean SED gradients in the trabecular bone matrix to correlate well with fBV and rBV (R2 = 0.66 and 0.65). Nevertheless, SED gradients in the marrow were shown to be the best predictor of osteoblastic and osteoclastic activities (R2 = 0.93 & 0.93). The data presented here support our hypothesis that mechanical signals generated in the bone marrow govern osteoblastic and osteoclastic activities. Funding was provided by the European Union (VPHOP FP7-ICT2008-223865). [1] Lambers, F.M et al. Bone, 2011. 49(6): p. 1340–50. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared.

doi:10.1016/j.bone.2012.02.208

PP020 Prolonged effect of moderate and severe hypo- and hyperthermia on Rbm3 and Hsp70 in osteoblast cells G.R.A. Froemminga,⁎, A. Mohd Dinb, N.A. Mohd Khana, M. Kapitonovaa, S. Abd Rahimc, H. Mohd Nawawid a IMMB, Faculty of Medicine, Malaysia b IMMB, Universiti Teknologi MARA, Sungai Buloh, Malaysia c Faculty of Medicine, Universiti Teknologi MARA, Shah Alam, Malaysia d CPDRL, Faculty of Medicine, Universiti Teknologi MARA, Sungai Buloh, Malaysia Abstract: Temperature is an important physical factor for cell growth in vitro and in vivo. Cells respond to cold and heat stress by expressing cold and heat shock proteins respectively to facilitate correct translation of specific mRNAs. Moreover, cellular response to high temperature can induce morphological changes leading to cell detachment by disrupting adhesion molecules found on the cell surface hence interfering with cell signalling pathways for normal bone metabolism. This study looked at the association between temperature, cell spreading and the induction of cold and heat shock protein expression in Normal Human Osteoblasts (NHOst) cells during prolonged hypo- and hyperthermia. NHOst cells were cultured at moderate (35 °C) and severe (27 °C) hypothermia as well as moderate (39 °C) and severe (45 °C) hyperthermia and control at (37 °C) for 12 h, 24 h, 72 h and 5 days in a water-jacketed incubator. Rate of NHOst proliferation was measured by the production of formazan