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Functional analysis of leukemia inhibitory factor in osteoblast differentiation of bone marrow stromal cells
S6
Abstracts
O08 Bone material properties in hypoparathyroidism E.P. Paschalisa, P. Roschgera, M.R. Rubinb, E. Shaneb, S. Gamsjaegera, B. Hofstettera, R. Zoehrera, B.M. Misofa, D. Dempsterc, J. Slineyb, C. Compitob, H. Zhoub, S.J. Silverbergb, J.P. Bilezikianb, K. Klaushofera 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 Columbia University, New York, USA c Helen Hayes Hospital, West Haverstraw, New York, USA Abstract: Hypoparathyroidism (HypoPT) due to deficient or absent parathyroid hormone is characterized by markedly reduced bone turnover. The purpose of this study was to establish the intrinsic bone material properties from HypoPT subjects (n= 19, 52.6 +/− 10.0 years, range 34 to 69 years, 12 women, 7 men, HypoPT duration 3– 45 years) in comparison to those from healthy premenopausal women (CTRL, n = 38), and untreated, low-turnover postmenopausal osteoporotics (OP; n = 32). Transiliac bone samples were analyzed for trabecular bone mineralization density distribution (BMDD) based on quantitative backscattered electron imaging (qBEI), and mineral/matrix ratio, proteoglycan content, crystallinity, and collagen cross-links of actively bone forming areas based on Raman and Fourier transform infrared imaging (FTIRI). Mean calcium concentrations (CaMean) showed large variations, but on average similar to normal. The heterogeneity of mineralization (CaWidth −9.5%, p b 0.001) and the percentage of low mineralized bone areas (CaLow −25%, p = 0.003) were both decreased compared to normal. In actively forming trabecular surfaces, HypoPT subjects exhibited a lower mineral/ matrix ratio compared to CTRL and similar to postmenopausal osteoporotic (OP) subjects, a lower proteoglycan content compared to either CTRL or OP, mineral crystallites of lower maturity/crystallinity compared to CTRL and OP, and a significantly higher pyridinoline/ divalent collagen cross-link ratio compared to CTRL subjects. These data suggest that mineralization in hypoparathyroidism can have wide variations, despite low bone turnover, but that collagen maturity is consistently increased, while mineral maturity/crystallinity is decreased in newly formed bone compared to controls. These changes may contribute to our understanding of fracture risk in HypoPT.
doi:10.1016/j.bone.2012.08.009
O09 Functional analysis of leukemia inhibitory factor in osteoblast differentiation of bone marrow stromal cells K. Matsushitaa, S. Itoha, S. Ikedaa, Y. Yamamotoa, Y. Yamauchia, J. Aubinb, M. Hayashia a Department of Restorative Dentistry and Endodontology, Osaka University Graduate School of Dentistry,Japan b Faculty of Medicine, Department of Molecular Genetics, University of Toronto,Canada Abstract: Objectives: Leukemia Inhibitory Factor (LIF) belongs to IL-6 family cytokine. The effect of LIF on murine bone marrow stromal cells (BMSCs) is unclear. The purpose of this study is to clarify the effect of LIF on osteoblast differentiation of murine BMSCs. Methods: BMSCs were obtained from the femurs and tibiae of C57BL/6J mice, and seeded in alpha-MEM supplemented with antibiotics and 10% FBS. At 3 days after seeding, nonadherent cells were removed by washing 3 times with PBS. Approximately 2 weeks after seeding, when the adherent cells had expanded to ~ 80% sub-confluence, they were detached with trypsin-EDTA. BMSCs were cultured in osteogenic induction medium with or without LIF for 3 weeks and cells were double-stained for ALP activity and mineral deposition (von Kossa). Colony-forming units-osteoblast (CFU-O) defined as colonies with ALP-positive cells associated with von-Kossa-positive were counted. mRNA was extracted and synthesized cDNA to determine the quantity of the expression level of ALP, Col1a, BSP, OCN, Runx2, and Osx with real-time PCR. The phosphorylation level of STAT3 induced by the stimulation of LIF was detected by western blotting. Results: The number of CFU-Os cultured with osteogenic induction medium and LIF was significantly lower than that cultured with only osteogenic induction medium. The suppressive effect of LIF was confirmed by the lower expression levels of osteoblast differentiation markers: ALP, Col1a, BSP, OCN, Runx2, and Osx. STAT3 was phosphorylated from 5 min to 30 min after LIF stimulation. Thus these data imply that the suppressive effect of LIF is transmitted through LIF-STAT3 signaling pathway.
doi:10.1016/j.bone.2012.08.010
O10 Minerals on the move: From new ion transporters to novel concepts R.J.M. Bindels Department of Physiology, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands Abstract: Ca2 + and Mg2 + are of great physiological importance in their function in neural excitability, muscle contraction, blood coagulation, bone formation, hormone secretion and cell adhesion. The human body is equipped with an efficient negative feedback system counteracting variations of the Ca2 + and Mg2 + balance. These divalents are maintained within a narrow range by the small intestine and kidney which both increase their fractional (re)absorption under conditions of deprivation. Rapid progress has recently been made in identification and characterization of the Ca2 + and Mg2 + transport proteins contributing to the delicate balance of divalent cations. Expression cloning approaches in combination with knockout mice models and genetic studies in families with a disturbed Mg2 + balance revealed novel gatekeeper proteins that belong to the super family of the transient receptor potential (TRP) channels. These epithelial Ca2 + (TRPV5 and TRPV6) and Mg2 + channels (TRPM6 and TRPM7) form prime targets for hormonal control of the active Ca2 + and Mg2 + flux from the urine space or intestinal lumen to the blood compartment. The characteristics of the newly identified transporters will be discussed and in particular the distinctive molecular regulation of these new epithelial Ca2 + and Mg2 + channels in (patho)physiological situations will be highlighted.
doi:10.1016/j.bone.2012.08.011
O11 RANKL and osteoimmunology H. Takayanagi Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Japan ERATO, JST, Japan Abstract: The immune and skeletal systems share various molecules, including cytokines, transcription factors, signaling molecules and membrane receptors [1]. RANKL is a key cytokine for osteoclast differentiation and function but accumulating evidence indicates the diverse roles of RANKL in many biological systems including the immune system. RANKL is expressed by mesenchymal cells including osteoblastic cells as well as hematopoietic cells like T cell, thus it has been difficult to precisely characterize RANKL-expressing cells under physiological and pathological settings [2]. The source of RANKL in bone has been thought to be osteoblasts or bone marrow stromal cells, but osteocyte-specific RANKL knockout mice revealed the crucial role of osteocytes in supporting osteoclastogenesis in adult bone remodeling [3]. In autoimmune arthritis, RANKL is expressed by joint mesenchymal cells like synovial fibroblasts stimulated with IL-17 and other inflammatory cytokines. RANKL signal transduction is mediated by numerous immune-related molecules including NFkappaB, NFATc1, ITAM and Btk, which shed light on the mechanisms shared by the bone and the immune systems [2]. Here I will review the history of osteoimmunology and discuss the emerging topics in osteoimmunology including regulation of bone formation by osteoclast-derived Sema4D [4] and osteoprotection by osteoblast-derived Sema3A [5]. References [1] [2] [3] [4] [5]
Nat Rev Immunol 7, 292-304, 2007. Nat Rev Rheumatol 5, 667–76, 2009. Nat Med 17, 1231–34, 2011. Nat Med. 17, 1473–80, 2011. Nature 485, 69–74, 2012.
doi:10.1016/j.bone.2012.08.012
O12 RANKL derived from mesenchymal but not hematopoietic cellular sources is relevant for bone turnover in mice C. Streichera, A. Heynya, P. Kostenuikb, R.G. Erbena a University of Veterinary Medicine, Vienna, Austria b Amgen Inc., Thousand Oaks, CA, USA Abstract: Receptor activator of NFκB ligand (RANKL) is an essential cytokine for osteoclast differentiation and activity. RANKL can be produced by a variety of hematopoietic and mesenchymal cell types. However, it is still controversial to what extent RANKL derived from hematopoietic vs. mesenchymal cells contributes to the physiological regulation of
Abstracts
O08 Bone material properties in hypoparathyroidism E.P. Paschalisa, P. Roschgera, M.R. Rubinb, E. Shaneb, S. Gamsjaegera, B. Hofstettera, R. Zoehrera, B.M. Misofa, D. Dempsterc, J. Slineyb, C. Compitob, H. Zhoub, S.J. Silverbergb, J.P. Bilezikianb, K. Klaushofera 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 Columbia University, New York, USA c Helen Hayes Hospital, West Haverstraw, New York, USA Abstract: Hypoparathyroidism (HypoPT) due to deficient or absent parathyroid hormone is characterized by markedly reduced bone turnover. The purpose of this study was to establish the intrinsic bone material properties from HypoPT subjects (n= 19, 52.6 +/− 10.0 years, range 34 to 69 years, 12 women, 7 men, HypoPT duration 3– 45 years) in comparison to those from healthy premenopausal women (CTRL, n = 38), and untreated, low-turnover postmenopausal osteoporotics (OP; n = 32). Transiliac bone samples were analyzed for trabecular bone mineralization density distribution (BMDD) based on quantitative backscattered electron imaging (qBEI), and mineral/matrix ratio, proteoglycan content, crystallinity, and collagen cross-links of actively bone forming areas based on Raman and Fourier transform infrared imaging (FTIRI). Mean calcium concentrations (CaMean) showed large variations, but on average similar to normal. The heterogeneity of mineralization (CaWidth −9.5%, p b 0.001) and the percentage of low mineralized bone areas (CaLow −25%, p = 0.003) were both decreased compared to normal. In actively forming trabecular surfaces, HypoPT subjects exhibited a lower mineral/ matrix ratio compared to CTRL and similar to postmenopausal osteoporotic (OP) subjects, a lower proteoglycan content compared to either CTRL or OP, mineral crystallites of lower maturity/crystallinity compared to CTRL and OP, and a significantly higher pyridinoline/ divalent collagen cross-link ratio compared to CTRL subjects. These data suggest that mineralization in hypoparathyroidism can have wide variations, despite low bone turnover, but that collagen maturity is consistently increased, while mineral maturity/crystallinity is decreased in newly formed bone compared to controls. These changes may contribute to our understanding of fracture risk in HypoPT.
doi:10.1016/j.bone.2012.08.009
O09 Functional analysis of leukemia inhibitory factor in osteoblast differentiation of bone marrow stromal cells K. Matsushitaa, S. Itoha, S. Ikedaa, Y. Yamamotoa, Y. Yamauchia, J. Aubinb, M. Hayashia a Department of Restorative Dentistry and Endodontology, Osaka University Graduate School of Dentistry,Japan b Faculty of Medicine, Department of Molecular Genetics, University of Toronto,Canada Abstract: Objectives: Leukemia Inhibitory Factor (LIF) belongs to IL-6 family cytokine. The effect of LIF on murine bone marrow stromal cells (BMSCs) is unclear. The purpose of this study is to clarify the effect of LIF on osteoblast differentiation of murine BMSCs. Methods: BMSCs were obtained from the femurs and tibiae of C57BL/6J mice, and seeded in alpha-MEM supplemented with antibiotics and 10% FBS. At 3 days after seeding, nonadherent cells were removed by washing 3 times with PBS. Approximately 2 weeks after seeding, when the adherent cells had expanded to ~ 80% sub-confluence, they were detached with trypsin-EDTA. BMSCs were cultured in osteogenic induction medium with or without LIF for 3 weeks and cells were double-stained for ALP activity and mineral deposition (von Kossa). Colony-forming units-osteoblast (CFU-O) defined as colonies with ALP-positive cells associated with von-Kossa-positive were counted. mRNA was extracted and synthesized cDNA to determine the quantity of the expression level of ALP, Col1a, BSP, OCN, Runx2, and Osx with real-time PCR. The phosphorylation level of STAT3 induced by the stimulation of LIF was detected by western blotting. Results: The number of CFU-Os cultured with osteogenic induction medium and LIF was significantly lower than that cultured with only osteogenic induction medium. The suppressive effect of LIF was confirmed by the lower expression levels of osteoblast differentiation markers: ALP, Col1a, BSP, OCN, Runx2, and Osx. STAT3 was phosphorylated from 5 min to 30 min after LIF stimulation. Thus these data imply that the suppressive effect of LIF is transmitted through LIF-STAT3 signaling pathway.
doi:10.1016/j.bone.2012.08.010
O10 Minerals on the move: From new ion transporters to novel concepts R.J.M. Bindels Department of Physiology, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands Abstract: Ca2 + and Mg2 + are of great physiological importance in their function in neural excitability, muscle contraction, blood coagulation, bone formation, hormone secretion and cell adhesion. The human body is equipped with an efficient negative feedback system counteracting variations of the Ca2 + and Mg2 + balance. These divalents are maintained within a narrow range by the small intestine and kidney which both increase their fractional (re)absorption under conditions of deprivation. Rapid progress has recently been made in identification and characterization of the Ca2 + and Mg2 + transport proteins contributing to the delicate balance of divalent cations. Expression cloning approaches in combination with knockout mice models and genetic studies in families with a disturbed Mg2 + balance revealed novel gatekeeper proteins that belong to the super family of the transient receptor potential (TRP) channels. These epithelial Ca2 + (TRPV5 and TRPV6) and Mg2 + channels (TRPM6 and TRPM7) form prime targets for hormonal control of the active Ca2 + and Mg2 + flux from the urine space or intestinal lumen to the blood compartment. The characteristics of the newly identified transporters will be discussed and in particular the distinctive molecular regulation of these new epithelial Ca2 + and Mg2 + channels in (patho)physiological situations will be highlighted.
doi:10.1016/j.bone.2012.08.011
O11 RANKL and osteoimmunology H. Takayanagi Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Japan ERATO, JST, Japan Abstract: The immune and skeletal systems share various molecules, including cytokines, transcription factors, signaling molecules and membrane receptors [1]. RANKL is a key cytokine for osteoclast differentiation and function but accumulating evidence indicates the diverse roles of RANKL in many biological systems including the immune system. RANKL is expressed by mesenchymal cells including osteoblastic cells as well as hematopoietic cells like T cell, thus it has been difficult to precisely characterize RANKL-expressing cells under physiological and pathological settings [2]. The source of RANKL in bone has been thought to be osteoblasts or bone marrow stromal cells, but osteocyte-specific RANKL knockout mice revealed the crucial role of osteocytes in supporting osteoclastogenesis in adult bone remodeling [3]. In autoimmune arthritis, RANKL is expressed by joint mesenchymal cells like synovial fibroblasts stimulated with IL-17 and other inflammatory cytokines. RANKL signal transduction is mediated by numerous immune-related molecules including NFkappaB, NFATc1, ITAM and Btk, which shed light on the mechanisms shared by the bone and the immune systems [2]. Here I will review the history of osteoimmunology and discuss the emerging topics in osteoimmunology including regulation of bone formation by osteoclast-derived Sema4D [4] and osteoprotection by osteoblast-derived Sema3A [5]. References [1] [2] [3] [4] [5]
Nat Rev Immunol 7, 292-304, 2007. Nat Rev Rheumatol 5, 667–76, 2009. Nat Med 17, 1231–34, 2011. Nat Med. 17, 1473–80, 2011. Nature 485, 69–74, 2012.
doi:10.1016/j.bone.2012.08.012
O12 RANKL derived from mesenchymal but not hematopoietic cellular sources is relevant for bone turnover in mice C. Streichera, A. Heynya, P. Kostenuikb, R.G. Erbena a University of Veterinary Medicine, Vienna, Austria b Amgen Inc., Thousand Oaks, CA, USA Abstract: Receptor activator of NFκB ligand (RANKL) is an essential cytokine for osteoclast differentiation and activity. RANKL can be produced by a variety of hematopoietic and mesenchymal cell types. However, it is still controversial to what extent RANKL derived from hematopoietic vs. mesenchymal cells contributes to the physiological regulation of