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Sclerostin inhibits mineralisation by human osteoblasts
Abstracts / Bone 47 (2010) S72–S241
PP127 Sclerostin inhibits mineralisation by human osteoblasts H. Lim1, A. Wijenayaka1, K. Welldon1, G. Atkins1,⁎, D. Findlay2 1 Bone Cell Biology Group, Discipline of Orthopaedics and Trauma, University of Adelaide, Adelaide, Australia 2 University of Adelaide, Adelaide, Australia Formation of hydroxyapatite mineral in bone is a highly regulated process, although the details of this regulation remain to be fully elucidated. We have recently reported [1] that the TNF-family member, TWEAK, inhibits mineralisation by human osteoblasts. This action was accompanied by induction by TWEAK of SOST/sclerostin mRNA and protein. This finding prompted us to investigate the effect of recombinant exogenously applied sclerostin in human osteoblast mineralisation. Osteoblasts were derived from trabecular bone obtained at hip replacement surgery for osteoarthritis or fragility fracture of the hip. Cells were cultured in mineralisation aMEM-10 medium containing dexamethasone (10- 8 M) and KH2PO4 (1.8 mM) in the presence or absence of combinations of recombinant human sclerostin (R&D Systems) for up to 6 weeks before measurement of cell layer-associated Ca2+ levels. Mineral deposition was very sensitive to sclerostin, with concentrations as low as 10 ng/ml inhibiting cell layer-associated Ca2+. The inhibition of mineralisation by sclerostin was associated with reduced expression by the osteoblasts of mRNA encoding the bone formation markers osteocalcin and collagen I, and increased expression of the pre-osteocyte marker E11. This implies that sclerostin acts in part by inhibiting the progression of cells through to the mature osteocyte stage. Consistent with this, DMP1 expression was also reduced in response to sclerostin treatment. We interpret these results to indicate that sclerostin inhibits the differentiation of human osteoblasts and/or their ability to regulate mineral deposition and we are currently investigating the mechanisms of these actions. Conflict of Interest: None 1. Vincent C, Findlay DM, Welldon KJ, Wijenayaka AR, Zheng TS, Haynes DR, Fazzalari NL, Evdokiou A, Atkins GJ (2009) Proinflammatory cytokines TNF-related weak inducer of apoptosis (TWEAK) and TNFalpha induce the mitogen-activated protein kinase (MAPK)-dependent expression of sclerostin in human osteoblasts. J Bone Miner Res 24:1434-1449 Disclosure of Interest: None declared Keywords: human osteoblast, mineralisation, sclerostin, gene expression
S121
phenotype caused by a gain-of-function mutation of Lrp5 (T253I) as well as controls. The hospital registry was used to recruit patients with genetically verified HBM, whereas controls were recruited on basis of personal contact. All participants avoided food potentially influencing the level of serotonin as well as drugs known to affect serotonin levels (i.e. acetylsalicylic acid and vasodilators). After centrifugation and separation of platelet-rich plasma, a serotonin ELISA kit (Fitzgerald) was used to determine the level serotonin. The study population consisted of 27 including 9 HBM patients and 18 sex and age matched controls. The group of HBM patients consisted of 6 women and 3 men (DXA (Hologic): z-scores: total hip: mean ± SD: 6.79 ± 1.61, lumbar spine: 6.85 ± 1.37). The serotonin concentrations in platelet-poor plasma were significantly lower in HBM patients compared to the controls (Mean ± SEM: 2.16 ± 0.28 versus 3.51 ± 0.49, P < 0.05). Together with results previously published by Yaday et al (1), our data support the hypothesis that gut-derived serotonin levels cause the HBM phenotype resulting form gain of function mutations in Lrp5 in humans. (1) Yadav VK, Ryu JH, Suda N, Tanaka KF, Gingrich JA, Schutz G, et al. Lrp5 controls bone formation by inhibiting serotonin synthesis in the duodenum. Cell 2008 Nov 28;135(5):825-37. For the graph: A. Levels of serotonin individually. B. Comparison of serotonin levels in HBM patients and controls presented as mean ± standard error of the mean (SEM). ⁎P < 0.05 Student's t-test. Image/Graph:
doi:10.1016/j.bone.2010.04.263
PP128 Patients with high bone mass phenotype due to Lrp5-T253i mutation have low plasma levels of serotonin M. Frost1,⁎, T. Andersen1, V. Yadav2, K. Brixen3, G. Karsenty2, M. Kassem1 1 Department of Endocrinology and Metabolism, Laboratory for Molecular Endocrinology (KMEB), University of Southern Denmark, Odense, Denmark 2 Department of Genetics and Development, Columbia University, New York, United States 3 Department of Endocrinology and Metabolism, University of Southern Denmark, Odense, Denmark The Lrp5 gene is of great importance to bone health. Mutations in Lrp5 may cause high bone mass (HBM) as well as osteoporosis pseudoglioma syndrome. Recently, the effect of Lrp5 on bone mass was demonstrated to be achieved by obstruction of the synthesis of gut-derived serotonin, which inhibits bone formation. In the present study, blood serotonin levels were analysed in patients with a HBM
Disclosure of Interest: None declared Keywords: LRP5, serotonin doi:10.1016/j.bone.2010.04.264
PP129 Stem cell transplantation promotes implant fixation in osteopenic FGFR3-/- mice C. Gao1,2,⁎, B. El Chaarani1,2, A. Carli1,3, L. Lim2, E. Harvey1,3, J. Seuntjens4,5, J.E. Henderson1,2,3 1 JTN Wong Labs, RI-MUHC, Montreal, Canada 2 Medicine, McGill University, Montreal, Canada 3 Surgery, McGill University, Montreal, Canada 4 Medical Physics Unit, RI-MUHC, Montreal, Canada 5 Oncology, McGill University, Montreal, Canada Relevance - Joint replacement is the most common major orthopaedic surgery whose success is critically dependent on early
PP127 Sclerostin inhibits mineralisation by human osteoblasts H. Lim1, A. Wijenayaka1, K. Welldon1, G. Atkins1,⁎, D. Findlay2 1 Bone Cell Biology Group, Discipline of Orthopaedics and Trauma, University of Adelaide, Adelaide, Australia 2 University of Adelaide, Adelaide, Australia Formation of hydroxyapatite mineral in bone is a highly regulated process, although the details of this regulation remain to be fully elucidated. We have recently reported [1] that the TNF-family member, TWEAK, inhibits mineralisation by human osteoblasts. This action was accompanied by induction by TWEAK of SOST/sclerostin mRNA and protein. This finding prompted us to investigate the effect of recombinant exogenously applied sclerostin in human osteoblast mineralisation. Osteoblasts were derived from trabecular bone obtained at hip replacement surgery for osteoarthritis or fragility fracture of the hip. Cells were cultured in mineralisation aMEM-10 medium containing dexamethasone (10- 8 M) and KH2PO4 (1.8 mM) in the presence or absence of combinations of recombinant human sclerostin (R&D Systems) for up to 6 weeks before measurement of cell layer-associated Ca2+ levels. Mineral deposition was very sensitive to sclerostin, with concentrations as low as 10 ng/ml inhibiting cell layer-associated Ca2+. The inhibition of mineralisation by sclerostin was associated with reduced expression by the osteoblasts of mRNA encoding the bone formation markers osteocalcin and collagen I, and increased expression of the pre-osteocyte marker E11. This implies that sclerostin acts in part by inhibiting the progression of cells through to the mature osteocyte stage. Consistent with this, DMP1 expression was also reduced in response to sclerostin treatment. We interpret these results to indicate that sclerostin inhibits the differentiation of human osteoblasts and/or their ability to regulate mineral deposition and we are currently investigating the mechanisms of these actions. Conflict of Interest: None 1. Vincent C, Findlay DM, Welldon KJ, Wijenayaka AR, Zheng TS, Haynes DR, Fazzalari NL, Evdokiou A, Atkins GJ (2009) Proinflammatory cytokines TNF-related weak inducer of apoptosis (TWEAK) and TNFalpha induce the mitogen-activated protein kinase (MAPK)-dependent expression of sclerostin in human osteoblasts. J Bone Miner Res 24:1434-1449 Disclosure of Interest: None declared Keywords: human osteoblast, mineralisation, sclerostin, gene expression
S121
phenotype caused by a gain-of-function mutation of Lrp5 (T253I) as well as controls. The hospital registry was used to recruit patients with genetically verified HBM, whereas controls were recruited on basis of personal contact. All participants avoided food potentially influencing the level of serotonin as well as drugs known to affect serotonin levels (i.e. acetylsalicylic acid and vasodilators). After centrifugation and separation of platelet-rich plasma, a serotonin ELISA kit (Fitzgerald) was used to determine the level serotonin. The study population consisted of 27 including 9 HBM patients and 18 sex and age matched controls. The group of HBM patients consisted of 6 women and 3 men (DXA (Hologic): z-scores: total hip: mean ± SD: 6.79 ± 1.61, lumbar spine: 6.85 ± 1.37). The serotonin concentrations in platelet-poor plasma were significantly lower in HBM patients compared to the controls (Mean ± SEM: 2.16 ± 0.28 versus 3.51 ± 0.49, P < 0.05). Together with results previously published by Yaday et al (1), our data support the hypothesis that gut-derived serotonin levels cause the HBM phenotype resulting form gain of function mutations in Lrp5 in humans. (1) Yadav VK, Ryu JH, Suda N, Tanaka KF, Gingrich JA, Schutz G, et al. Lrp5 controls bone formation by inhibiting serotonin synthesis in the duodenum. Cell 2008 Nov 28;135(5):825-37. For the graph: A. Levels of serotonin individually. B. Comparison of serotonin levels in HBM patients and controls presented as mean ± standard error of the mean (SEM). ⁎P < 0.05 Student's t-test. Image/Graph:
doi:10.1016/j.bone.2010.04.263
PP128 Patients with high bone mass phenotype due to Lrp5-T253i mutation have low plasma levels of serotonin M. Frost1,⁎, T. Andersen1, V. Yadav2, K. Brixen3, G. Karsenty2, M. Kassem1 1 Department of Endocrinology and Metabolism, Laboratory for Molecular Endocrinology (KMEB), University of Southern Denmark, Odense, Denmark 2 Department of Genetics and Development, Columbia University, New York, United States 3 Department of Endocrinology and Metabolism, University of Southern Denmark, Odense, Denmark The Lrp5 gene is of great importance to bone health. Mutations in Lrp5 may cause high bone mass (HBM) as well as osteoporosis pseudoglioma syndrome. Recently, the effect of Lrp5 on bone mass was demonstrated to be achieved by obstruction of the synthesis of gut-derived serotonin, which inhibits bone formation. In the present study, blood serotonin levels were analysed in patients with a HBM
Disclosure of Interest: None declared Keywords: LRP5, serotonin doi:10.1016/j.bone.2010.04.264
PP129 Stem cell transplantation promotes implant fixation in osteopenic FGFR3-/- mice C. Gao1,2,⁎, B. El Chaarani1,2, A. Carli1,3, L. Lim2, E. Harvey1,3, J. Seuntjens4,5, J.E. Henderson1,2,3 1 JTN Wong Labs, RI-MUHC, Montreal, Canada 2 Medicine, McGill University, Montreal, Canada 3 Surgery, McGill University, Montreal, Canada 4 Medical Physics Unit, RI-MUHC, Montreal, Canada 5 Oncology, McGill University, Montreal, Canada Relevance - Joint replacement is the most common major orthopaedic surgery whose success is critically dependent on early