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Chondroadherin prevents bone loss and inhibits tumor growth in animal models of osteoporosis and cancer-induced bone disease
Abstracts two hormones' phosphate metabolism, glucose metabolism, energy expenditure and male fertility. One of the osteoblast-derived hormones is osteocalcin that promotes insulin expression and whose activity is itself regulated by insulin signaling. These observations raise novel questions. One of them is to determine the role, if any, of bone in the development of whole body insulin resistance in the case of type 2 diabetes. A second one is to identify extracellular signals regulating osteocalcin expression. In broader terms, another question is to determine whether osteocalcin has additional functions.
doi:10.1016/j.bone.2012.08.037
O37 Bone microarchitecture and local gene expression in elderly men with osteoporotic hip fractures U. Föger-Samwalda, J.M. Patscha,b, J. Deutschmanna,b, L. Hornekc, S. Salemd, P. Beckere, P. Paila, D. Schamalla, M. Mousavic, S. Beckere, F.M. Kainbergerb, H. Reschd, P. Pietschmanna a Department of Pathophysiology and Allergy Research, Medical University of Vienna, Austria b Department of Radiodiagnostics, Medical University of Vienna, Austria c Department of Trauma Surgery, Danube Hospital, Vienna, Austria d Department of Orthopaedics, St. Vincent Hospital, Vienna, Austria e Department of Spine Surgery, Orthopaedic Hospital Speising, Vienna, Austria Abstract: Osteoporosis is extremely frequent in post-menopausal women; nevertheless, also in men osteoporosis is a severe and frequent disease. Increased osteoclast activity is thought to be the underlying pathophysiological mechanism of postmenopausal osteoporosis. In a previous study we found evidence of osteoblast dysfunction in middle aged men with idiopathic osteoporosis. The aim of this study was to investigate gene expression and bone architecture in bone samples derived from elderly osteoporotic men with hip fractures in comparison to bone samples from age matched non-osteoporotic controls. Femoral heads and adjacent neck tissue were collected from 10 men with low-trauma hip fractures (mean age 82 ± 7) and consecutive surgical hip replacement. 14 bone samples from age matched patients with osteoarthritis served as controls. One half of each bone sample was subjected to RNA extraction, reverse transcription, and real-time polymerase chain reactions. The second half of each bone sample was analyzed by dual X-ray absorptiometry and by high resolution peripheral micro-computed tomography. We could show a significantly decreased expression of the osteoblast related genes runx2, osterix, sclerostin and osteocalcin in bone samples from hip fracture patients compared to controls (− 65.9%, − 89.3%, − 66%, − 89.5%). Areal bone mineral density was significantly lower in fracture samples. Comparing local bone microstructure, the femoral head displayed significantly lower BV/TV and trabecular thickness in the fractured when compared to the osteoarthritic bone samples. Therefore, decreased local gene expression of runx2, osterix and osteocalcin in men with hip fractures strongly supports the concept of osteoblast dysfunction in male osteoporosis.
doi:10.1016/j.bone.2012.08.038
S13
downregulation of Nitric Oxide Synthase (NOS)2. Consistently, treatment with the NO donor S-nitroso-N-acetyl-D,L-penicillamine blocked the effect of cCHAD rescuing normal preosteoclast motility, while the NOS2 inhibitor N5-(1-iminoethyl)-l-ornithine mimicked the outcome of cCHAD. Healthy mice treated with cCHAD showed dosedependent reduction of osteoclast number and surface (− 40% and − 50%, respectively) and increase of trabecular bone volume (1.55-fold), with no adverse effects. Daily treatment of ovariectomized (OVX) mice with cCHAD starting 3 days (preventive protocol) or 5 weeks (curative protocol) after OVX prevented bone loss and rescued bone mass, respectively, by inhibiting osteoclast increase. In vivo antiresorptive effect of cCHAD was also observed in mouse models of breast cancer-induced bone metastases obtained by intracardiac injection of MDA-MB-231 cells, with a strong decrease of incidence of osteolytic lesions. Moreover, cCHAD reduced orthotopic tumor growth, impaired in vitro tumor cell migration and invasion, and potentiated the effects of chemotherapy. In conclusion, we provided evidence that cCHAD safely counteracts bone loss and tumor expansion, and propose that it could represent a new regulator of bone resorption with translational relevance for therapy.
doi:10.1016/j.bone.2012.08.039
O39 The LIM-only protein FHL2 controls mesenchymal cell osteogenic differentiation and bone formation through Wnt5a and Wnt10b J. Bruna,b, O. Fromiguéa,b, F.-X. Dieudonnéa,b, C. Martya,b, J. Chenc, J. Dahand, Y. Weid, P.J. Mariea,b a INSERM, U606, Paris, France b Université Paris Diderot, Sorbonne Paris Cité, UMR-606, Paris, France c Department of Medicine, Univ. of California San Diego, La Jolla, CA, USA d Department of Virology, Institut Pasteur, Paris, France Abstract: We investigated the implication of Wnt proteins in mesenchymal stromal cells (MSC) osteoblast and adipocyte differentiation by FHL2, a LIM-only protein with four and a half LIM domains. We found that Wnt3a increased FHL2 expression in murine C3H10T1/2 MSC. Silencing FHL2 using short hairpin (sh) RNA attenuated β-catenin transcriptional activity, Runx2, ALP and COL1A1 expression and osteogenic differentiation induced by Wnt3a. In addition, FHL2 silencing reduced the expression of Wnt5a and Wnt10b. Wnt10b, but not Wnt5a, overcome the negative effect of FHL2 knockdown on osteoblast gene expression. Histomorphometric analysis in 15-month old FHL2 knockout mice showed decreased trabecular number and thickness and reduced bone mass. This phenotype was associated with decreased Wnt5a and Wnt10b associated with lower than normal c-myc, cyclin D1 mRNA levels expression in the bone marrow and reduced phenotypic osteoblast gene expression. In support of in vitro data, ex vivo analysis showed decreased basal and Wnt3a-induced Wnt5a and Wnt10b mRNA expression in FHL2deficient bone marrow cells. In addition, FHL2 deficiency reduced adipogenic differentiation of C3H10T1/2 mesenchymal cells and decreased bone marrow adipogenesis in FHL2 knockout mice, indicating that FHL2 is a positive modulator of adipocyte differentiation. This effect was related to increased expression of Foxo1, a factor that negatively regulates adipocyte differentiation. Collectively, the results indicate that FHL2 deficiency compromises MSC allocation to the osteoblast or adipocyte lineage by altering Wnt10b and Foxo1, respectively. This reveals a previously unrecognised mechanism by which FHL2 positively controls mesenchymal cell fate, bone formation and bone mass.
doi:10.1016/j.bone.2012.08.040 O38 Chondroadherin prevents bone loss and inhibits tumor growth in animal models of osteoporosis and cancer-induced bone disease N. Ruccia, M. Capullia, O.K. Olstadb,c, K.M. Gautvikb,c, L. Haglundd, D. Heinegårdd, A. Tetia a Department of Biotechnological and Applied Clinical Sciences, University of L'Aquila, L'Aquila, Italy b Department of Clinical Chemistry, Ullevaal University Hospital, Oslo, Norway c Institute of Biochemistry, University of Oslo, Oslo, Norway d Department of Experimental Medical Science, Lund University, Lund, Sweden Abstract: Chondroadherin (CHAD) is a leucine-rich protein promoting cell attachment through binding to integrin alpha2beta1. We tested a cyclic peptide, representing this integrin binding sequence (cCHAD), and found a marked inhibition of osteoclastogenesis with no effect on osteoblasts. Potency was greater at low cellular density, suggesting an effect on preosteoclast motility, confirmed by a scratch-healing assay showing an 88% reduction of migration in cCHAD-treated preosteoclasts vs. control. This effect was mediated by a cCHAD-dependent transcriptional
O40 Sclerosteosis patients have altered intrinsic bone material properties as assessed by Raman spectroscopy E.P. Paschalisa, A. van Lieropb, R. van Bezooijenb, S. Gamsjaegera, B. Hofstettera, A. Roschgera, P. Roschgera, K. Klaushofera, S.E. Papapoulosb a Ludwig Boltzmann Institute of Osteology at the Hanusch Hospital of WGKK and AUVA Trauma Centre Meidling, 1st Medical Department, Hanusch Hospital, Vienna, Austria b Department of Endocrinology & Metabolic Diseases, Leiden University Medical Center, Leiden, The Netherlands Abstract: Sclerosteosis (SC) is a rare, autosomal recessive, bone sclerosing dysplasia characterized by osteosclerosis, caused by loss-of-function mutations in the SOST gene encoding for sclerostin, a protein produced in bone by osteocytes that decreases bone formation by inhibiting the Wnt signaling pathway in osteoblasts, resulting in unrestrained bone formation. SC patients do not sustain fractures suggesting that their
doi:10.1016/j.bone.2012.08.037
O37 Bone microarchitecture and local gene expression in elderly men with osteoporotic hip fractures U. Föger-Samwalda, J.M. Patscha,b, J. Deutschmanna,b, L. Hornekc, S. Salemd, P. Beckere, P. Paila, D. Schamalla, M. Mousavic, S. Beckere, F.M. Kainbergerb, H. Reschd, P. Pietschmanna a Department of Pathophysiology and Allergy Research, Medical University of Vienna, Austria b Department of Radiodiagnostics, Medical University of Vienna, Austria c Department of Trauma Surgery, Danube Hospital, Vienna, Austria d Department of Orthopaedics, St. Vincent Hospital, Vienna, Austria e Department of Spine Surgery, Orthopaedic Hospital Speising, Vienna, Austria Abstract: Osteoporosis is extremely frequent in post-menopausal women; nevertheless, also in men osteoporosis is a severe and frequent disease. Increased osteoclast activity is thought to be the underlying pathophysiological mechanism of postmenopausal osteoporosis. In a previous study we found evidence of osteoblast dysfunction in middle aged men with idiopathic osteoporosis. The aim of this study was to investigate gene expression and bone architecture in bone samples derived from elderly osteoporotic men with hip fractures in comparison to bone samples from age matched non-osteoporotic controls. Femoral heads and adjacent neck tissue were collected from 10 men with low-trauma hip fractures (mean age 82 ± 7) and consecutive surgical hip replacement. 14 bone samples from age matched patients with osteoarthritis served as controls. One half of each bone sample was subjected to RNA extraction, reverse transcription, and real-time polymerase chain reactions. The second half of each bone sample was analyzed by dual X-ray absorptiometry and by high resolution peripheral micro-computed tomography. We could show a significantly decreased expression of the osteoblast related genes runx2, osterix, sclerostin and osteocalcin in bone samples from hip fracture patients compared to controls (− 65.9%, − 89.3%, − 66%, − 89.5%). Areal bone mineral density was significantly lower in fracture samples. Comparing local bone microstructure, the femoral head displayed significantly lower BV/TV and trabecular thickness in the fractured when compared to the osteoarthritic bone samples. Therefore, decreased local gene expression of runx2, osterix and osteocalcin in men with hip fractures strongly supports the concept of osteoblast dysfunction in male osteoporosis.
doi:10.1016/j.bone.2012.08.038
S13
downregulation of Nitric Oxide Synthase (NOS)2. Consistently, treatment with the NO donor S-nitroso-N-acetyl-D,L-penicillamine blocked the effect of cCHAD rescuing normal preosteoclast motility, while the NOS2 inhibitor N5-(1-iminoethyl)-l-ornithine mimicked the outcome of cCHAD. Healthy mice treated with cCHAD showed dosedependent reduction of osteoclast number and surface (− 40% and − 50%, respectively) and increase of trabecular bone volume (1.55-fold), with no adverse effects. Daily treatment of ovariectomized (OVX) mice with cCHAD starting 3 days (preventive protocol) or 5 weeks (curative protocol) after OVX prevented bone loss and rescued bone mass, respectively, by inhibiting osteoclast increase. In vivo antiresorptive effect of cCHAD was also observed in mouse models of breast cancer-induced bone metastases obtained by intracardiac injection of MDA-MB-231 cells, with a strong decrease of incidence of osteolytic lesions. Moreover, cCHAD reduced orthotopic tumor growth, impaired in vitro tumor cell migration and invasion, and potentiated the effects of chemotherapy. In conclusion, we provided evidence that cCHAD safely counteracts bone loss and tumor expansion, and propose that it could represent a new regulator of bone resorption with translational relevance for therapy.
doi:10.1016/j.bone.2012.08.039
O39 The LIM-only protein FHL2 controls mesenchymal cell osteogenic differentiation and bone formation through Wnt5a and Wnt10b J. Bruna,b, O. Fromiguéa,b, F.-X. Dieudonnéa,b, C. Martya,b, J. Chenc, J. Dahand, Y. Weid, P.J. Mariea,b a INSERM, U606, Paris, France b Université Paris Diderot, Sorbonne Paris Cité, UMR-606, Paris, France c Department of Medicine, Univ. of California San Diego, La Jolla, CA, USA d Department of Virology, Institut Pasteur, Paris, France Abstract: We investigated the implication of Wnt proteins in mesenchymal stromal cells (MSC) osteoblast and adipocyte differentiation by FHL2, a LIM-only protein with four and a half LIM domains. We found that Wnt3a increased FHL2 expression in murine C3H10T1/2 MSC. Silencing FHL2 using short hairpin (sh) RNA attenuated β-catenin transcriptional activity, Runx2, ALP and COL1A1 expression and osteogenic differentiation induced by Wnt3a. In addition, FHL2 silencing reduced the expression of Wnt5a and Wnt10b. Wnt10b, but not Wnt5a, overcome the negative effect of FHL2 knockdown on osteoblast gene expression. Histomorphometric analysis in 15-month old FHL2 knockout mice showed decreased trabecular number and thickness and reduced bone mass. This phenotype was associated with decreased Wnt5a and Wnt10b associated with lower than normal c-myc, cyclin D1 mRNA levels expression in the bone marrow and reduced phenotypic osteoblast gene expression. In support of in vitro data, ex vivo analysis showed decreased basal and Wnt3a-induced Wnt5a and Wnt10b mRNA expression in FHL2deficient bone marrow cells. In addition, FHL2 deficiency reduced adipogenic differentiation of C3H10T1/2 mesenchymal cells and decreased bone marrow adipogenesis in FHL2 knockout mice, indicating that FHL2 is a positive modulator of adipocyte differentiation. This effect was related to increased expression of Foxo1, a factor that negatively regulates adipocyte differentiation. Collectively, the results indicate that FHL2 deficiency compromises MSC allocation to the osteoblast or adipocyte lineage by altering Wnt10b and Foxo1, respectively. This reveals a previously unrecognised mechanism by which FHL2 positively controls mesenchymal cell fate, bone formation and bone mass.
doi:10.1016/j.bone.2012.08.040 O38 Chondroadherin prevents bone loss and inhibits tumor growth in animal models of osteoporosis and cancer-induced bone disease N. Ruccia, M. Capullia, O.K. Olstadb,c, K.M. Gautvikb,c, L. Haglundd, D. Heinegårdd, A. Tetia a Department of Biotechnological and Applied Clinical Sciences, University of L'Aquila, L'Aquila, Italy b Department of Clinical Chemistry, Ullevaal University Hospital, Oslo, Norway c Institute of Biochemistry, University of Oslo, Oslo, Norway d Department of Experimental Medical Science, Lund University, Lund, Sweden Abstract: Chondroadherin (CHAD) is a leucine-rich protein promoting cell attachment through binding to integrin alpha2beta1. We tested a cyclic peptide, representing this integrin binding sequence (cCHAD), and found a marked inhibition of osteoclastogenesis with no effect on osteoblasts. Potency was greater at low cellular density, suggesting an effect on preosteoclast motility, confirmed by a scratch-healing assay showing an 88% reduction of migration in cCHAD-treated preosteoclasts vs. control. This effect was mediated by a cCHAD-dependent transcriptional
O40 Sclerosteosis patients have altered intrinsic bone material properties as assessed by Raman spectroscopy E.P. Paschalisa, A. van Lieropb, R. van Bezooijenb, S. Gamsjaegera, B. Hofstettera, A. Roschgera, P. Roschgera, K. Klaushofera, S.E. Papapoulosb a Ludwig Boltzmann Institute of Osteology at the Hanusch Hospital of WGKK and AUVA Trauma Centre Meidling, 1st Medical Department, Hanusch Hospital, Vienna, Austria b Department of Endocrinology & Metabolic Diseases, Leiden University Medical Center, Leiden, The Netherlands Abstract: Sclerosteosis (SC) is a rare, autosomal recessive, bone sclerosing dysplasia characterized by osteosclerosis, caused by loss-of-function mutations in the SOST gene encoding for sclerostin, a protein produced in bone by osteocytes that decreases bone formation by inhibiting the Wnt signaling pathway in osteoblasts, resulting in unrestrained bone formation. SC patients do not sustain fractures suggesting that their