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Late expression of c-Fos during osteoclast differentiation determines osteoclast survival and bone mass
Abstracts / Bone 44 (2009) S131–S141
location. The bone microenvironment during inflammatory bone disease is greatly altered and in response to these conditions the in vivo osteoclast precursor population may change. In adjuvantinduced arthritis mononuclear transition cells expressing both F4/ 80 and TRAP were associated with sites of resorption indicating that in this pathological setting macrophages contribute to the osteoclast precursor pool. Finally, we identified a canopy structure encapsulating mouse bone remodelling units, similar to that reported in human bone, and demonstrated that the canopy cell is an F4/80+ OsteoMac. These data suggest OsteoMacs, like other tissue macrophages, are highly adaptive and their roles in bone biology change depending on the local microenvironment. Specifically during pathological conditions OsteoMacs may function as osteoclast precursors. However, during physiologic conditions they may be important coordinators of bone remodelling through regulation of osteoclast recruitment, formation and activity as well as osteoblast function. doi:10.1016/j.bone.2009.01.300
387 Late expression of c-Fos during osteoclast differentiation determines osteoclast survival and bone mass Y. Takadaa, N. Iriea, L. Greshb, T. Nakamurac, S. Katoc, E.F. Wagnerb, K. Matsuoa a Department of Microbiology and Immunology, School of Medicine, Keio University, Tokyo, Japan b Research institute for Molecular Pathology (IMP), Vienna, Austria c Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo, Japan The transcription factor c-Fos is essential for osteoclast differentiation. c-Fos-deficient mice lack osteoclasts and show severe osteopetrotic phenotype due to impaired expression of the c-Fos target gene Nfatc1. c-Fos expression in osteoclastgenesis is biphasic: early expression is observed within 30 min and late expression 48 h after RANKL treatment. However, distinct roles of early and late expression of c-Fos are unknown. In this study, we examined the role of c-Fos late expression in osteoclastgenesis. First, we prepared bone marrow-derived macrophages from Fosflox/-mice, and treated these cells with RANKL. Twenty-four hours after treatment, we infected cells with adenovirus expressing Cre-recombinase to remove the late expression of c-Fos. As a result, expression of Nfatc1, Acp5 and Mmp9 was reduced, the number of multinuclear TRAP-positive cells was decreased, and resorption activity was diminished. We then removed c-Fos late expression in vivo by generating Fosflox/-mice carrying the Cathepsin K-Cre recombinase knock-in locus. During osteoclastgenesis using bone marrowderived macrophages prepared from these mice, we confirmed that late expression of c-Fos was removed while early expression of c-Fos was intact. Removal of c-Fos late expression by Cathepsin K-driven Cre-recombinase reduced expression of Nfatc1, number of multinuclear TRAP-positive cells, and resorption activity during RANKLinduced osteoclastgenesis. Unexpectedly, however, removal of c-Fos late expression prolonged survival of osteoclasts in vitro. Analyzing tibiae of these mice using micro-computed tomography and histomorphometry revealed that the mouse lacking late expression of c-Fos decreased bone mass. These findings suggest that late expression of c-Fos during osteoclast differentiation enhances full maturation and limits survival of osteoclasts. doi:10.1016/j.bone.2009.01.301
S137
388 The combination of calcium with vitamin D is a more effective suppressor of parathyroid hormone than either given alone D. Thomasa, A.G. Needa, P.D. O' Loughlina,b, P.S. Coatesa,b, M. Horowitzb,c, B.E.C. Nordina,b,c a Clinical Biochemistry, Institute of Medical and Veterinary Science, Adelaide, SA, Australia b Endocrine and Metabolic Unit, Royal Adelaide Hospital, Adelaide, SA, Australia c Discipline of Medicine, University of Adelaide, Adelaide, SA, Australia Introduction: Calcium with vitamin D is more effective in fracture prevention than either D alone. We postulated that the combination was a more effective suppressor of parathyroid hormone (PTH) than either alone and test this hypothesis in 20 volunteer women aged 59–75 with initial serum 25OHD below 60 nmol/L. Methodology: Because vitamin D takes weeks to exert its actions whereas the effect of calcium on PTH immediate, we randomly allocated eleven to one week of calcium 1000 mg daily followed by 7 weeks of Ca and D3 1000 i.u. daily and nine to 7 weeks of D3 followed by 1 week of D3 and Ca. Fasting blood samples were obtained at baseline, after one week of Ca or 7 weeks of D3 and at the end of 8 weeks. We measured Total Ca, Phosphate, PTH and CTX in addition to albumin, globulins, anion gap and bicarbonate. Ionised calcium was calculated from total calcium and the four ligands. Results: The two subgroups did not differ significantly from each other at baseline in any measured variable. Basal values and effects of treatments are shown in the table. Calcium (whether given first or second) significantly raised Ca, Ca++ and P and lowered PTH and CTX. D3 (first or second) did qualitatively the same but only the suppression of PTH and CTX was significant. Both treatments had greater and generally more significant effect than either alone. 25OHD rose from 48.1 nmol/L (SD 9.8) to 85.0 (SD 23.0) after 7 or weeks. Initial values of measured variables, change on three regimens and final values in 20 postmenopaussal women Variable
Total Ca (mmol/L) Ionised Ca (mmol/L) Phosphate (mmol/L) PTH (ρmol/L) CTX (ng/L)
Basal
Change After Ca
Change 2.36 0.055** 1.22 0.020* 1.11 0.019** 6.12 −0.65* 439 −114***
Final value After D3
After both
0.010 0.018 0.027 −0.75* −39*
0.070*** 0.028 0.060* −1.53** −161***
2.43*** 1.25* 1.69* 4.60** 278***
***P < 0.001, **P < 0.01, *P < 0.05 for significance fron baseline.
Conclusions: The combination of calcium and vitamin D was a more effective and significant suppressor of PTH than either alone. doi:10.1016/j.bone.2009.01.302
389 The calcitonin receptor on osteoclasts plays a physiological role to protect against induced hypercalcaemia in mice A.G. Turnera, F.A. Tjahyonoa, W.S.M. Chiua, A.J. Mooreb, D.M. Findlayc, H.A. Morrisb, J.D. Zajaca, R.A. Daveya a Medicine (AH/NH), University of Melbourne, Heidelberg, VIC, Australia b Hanson Institute, IMVS, Adelaide, SA, Australia c Dept of Orthopaedics and Trauma, University of Adelaide, Adelaide, SA, Australia We have previously demonstrated that the calcitonin receptor (CTR) plays a biological role to protect against induced hypercalcaemia in mice (1). The aim of the present study was to investigate the mechanism by
location. The bone microenvironment during inflammatory bone disease is greatly altered and in response to these conditions the in vivo osteoclast precursor population may change. In adjuvantinduced arthritis mononuclear transition cells expressing both F4/ 80 and TRAP were associated with sites of resorption indicating that in this pathological setting macrophages contribute to the osteoclast precursor pool. Finally, we identified a canopy structure encapsulating mouse bone remodelling units, similar to that reported in human bone, and demonstrated that the canopy cell is an F4/80+ OsteoMac. These data suggest OsteoMacs, like other tissue macrophages, are highly adaptive and their roles in bone biology change depending on the local microenvironment. Specifically during pathological conditions OsteoMacs may function as osteoclast precursors. However, during physiologic conditions they may be important coordinators of bone remodelling through regulation of osteoclast recruitment, formation and activity as well as osteoblast function. doi:10.1016/j.bone.2009.01.300
387 Late expression of c-Fos during osteoclast differentiation determines osteoclast survival and bone mass Y. Takadaa, N. Iriea, L. Greshb, T. Nakamurac, S. Katoc, E.F. Wagnerb, K. Matsuoa a Department of Microbiology and Immunology, School of Medicine, Keio University, Tokyo, Japan b Research institute for Molecular Pathology (IMP), Vienna, Austria c Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo, Japan The transcription factor c-Fos is essential for osteoclast differentiation. c-Fos-deficient mice lack osteoclasts and show severe osteopetrotic phenotype due to impaired expression of the c-Fos target gene Nfatc1. c-Fos expression in osteoclastgenesis is biphasic: early expression is observed within 30 min and late expression 48 h after RANKL treatment. However, distinct roles of early and late expression of c-Fos are unknown. In this study, we examined the role of c-Fos late expression in osteoclastgenesis. First, we prepared bone marrow-derived macrophages from Fosflox/-mice, and treated these cells with RANKL. Twenty-four hours after treatment, we infected cells with adenovirus expressing Cre-recombinase to remove the late expression of c-Fos. As a result, expression of Nfatc1, Acp5 and Mmp9 was reduced, the number of multinuclear TRAP-positive cells was decreased, and resorption activity was diminished. We then removed c-Fos late expression in vivo by generating Fosflox/-mice carrying the Cathepsin K-Cre recombinase knock-in locus. During osteoclastgenesis using bone marrowderived macrophages prepared from these mice, we confirmed that late expression of c-Fos was removed while early expression of c-Fos was intact. Removal of c-Fos late expression by Cathepsin K-driven Cre-recombinase reduced expression of Nfatc1, number of multinuclear TRAP-positive cells, and resorption activity during RANKLinduced osteoclastgenesis. Unexpectedly, however, removal of c-Fos late expression prolonged survival of osteoclasts in vitro. Analyzing tibiae of these mice using micro-computed tomography and histomorphometry revealed that the mouse lacking late expression of c-Fos decreased bone mass. These findings suggest that late expression of c-Fos during osteoclast differentiation enhances full maturation and limits survival of osteoclasts. doi:10.1016/j.bone.2009.01.301
S137
388 The combination of calcium with vitamin D is a more effective suppressor of parathyroid hormone than either given alone D. Thomasa, A.G. Needa, P.D. O' Loughlina,b, P.S. Coatesa,b, M. Horowitzb,c, B.E.C. Nordina,b,c a Clinical Biochemistry, Institute of Medical and Veterinary Science, Adelaide, SA, Australia b Endocrine and Metabolic Unit, Royal Adelaide Hospital, Adelaide, SA, Australia c Discipline of Medicine, University of Adelaide, Adelaide, SA, Australia Introduction: Calcium with vitamin D is more effective in fracture prevention than either D alone. We postulated that the combination was a more effective suppressor of parathyroid hormone (PTH) than either alone and test this hypothesis in 20 volunteer women aged 59–75 with initial serum 25OHD below 60 nmol/L. Methodology: Because vitamin D takes weeks to exert its actions whereas the effect of calcium on PTH immediate, we randomly allocated eleven to one week of calcium 1000 mg daily followed by 7 weeks of Ca and D3 1000 i.u. daily and nine to 7 weeks of D3 followed by 1 week of D3 and Ca. Fasting blood samples were obtained at baseline, after one week of Ca or 7 weeks of D3 and at the end of 8 weeks. We measured Total Ca, Phosphate, PTH and CTX in addition to albumin, globulins, anion gap and bicarbonate. Ionised calcium was calculated from total calcium and the four ligands. Results: The two subgroups did not differ significantly from each other at baseline in any measured variable. Basal values and effects of treatments are shown in the table. Calcium (whether given first or second) significantly raised Ca, Ca++ and P and lowered PTH and CTX. D3 (first or second) did qualitatively the same but only the suppression of PTH and CTX was significant. Both treatments had greater and generally more significant effect than either alone. 25OHD rose from 48.1 nmol/L (SD 9.8) to 85.0 (SD 23.0) after 7 or weeks. Initial values of measured variables, change on three regimens and final values in 20 postmenopaussal women Variable
Total Ca (mmol/L) Ionised Ca (mmol/L) Phosphate (mmol/L) PTH (ρmol/L) CTX (ng/L)
Basal
Change After Ca
Change 2.36 0.055** 1.22 0.020* 1.11 0.019** 6.12 −0.65* 439 −114***
Final value After D3
After both
0.010 0.018 0.027 −0.75* −39*
0.070*** 0.028 0.060* −1.53** −161***
2.43*** 1.25* 1.69* 4.60** 278***
***P < 0.001, **P < 0.01, *P < 0.05 for significance fron baseline.
Conclusions: The combination of calcium and vitamin D was a more effective and significant suppressor of PTH than either alone. doi:10.1016/j.bone.2009.01.302
389 The calcitonin receptor on osteoclasts plays a physiological role to protect against induced hypercalcaemia in mice A.G. Turnera, F.A. Tjahyonoa, W.S.M. Chiua, A.J. Mooreb, D.M. Findlayc, H.A. Morrisb, J.D. Zajaca, R.A. Daveya a Medicine (AH/NH), University of Melbourne, Heidelberg, VIC, Australia b Hanson Institute, IMVS, Adelaide, SA, Australia c Dept of Orthopaedics and Trauma, University of Adelaide, Adelaide, SA, Australia We have previously demonstrated that the calcitonin receptor (CTR) plays a biological role to protect against induced hypercalcaemia in mice (1). The aim of the present study was to investigate the mechanism by