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Proteolytically processed repressor Gli3 inhibits bone formation by repressing BMP2 transcription in osteoblasts
Abstracts / Bone 44 (2009) S18–S55
Bones' ability to withstand the loads of everyday activity without fracture is achieved through resident bone cells using loadinginduced strains in the bone tissue as a stimulus to adjust bone mass and architecture to achieve and maintain a “target strain environment”. Only short exposure to dynamic strain is necessary to stimulate the cascade of events that prevent bone resorption and/ or stimulate bone formation. Early events following exposure to dynamic strain in resident bone cells include calcium fluxes; stimulation of the ERK pathway; production of prostaglandins and nitric oxide; activation of the canonical Wnt pathway; altered expression of a large number of non-bone specific genes; depression of sclerostin production and increased secretion of insulin-like growth factors. Estrogen receptor α, independently of estrogen, is involved up and down stream of ERK activation; in the production of nitric oxide but not prostaglandins; translocation of β-catenin to the nucleus; the regulation of a large number of genes and activation of the IGFR. The conservational and potentially osteogenic response to bone loading is substantially reduced in animals lacking ERα, but not ERβ. The most widespread failure of bone's mechanically-adaptive response to maintain the skeleton sufficiently robust to withstand everyday loading without fracture occurs in post-menopausal osteoporosis in women and age-related osteoporosis in men. In both sexes the level of bone loss is associated with reduced bioavailable estrogen. Estrogen, but not strain, appears to regulate the ER number in osteoblasts and osteocytes, which in the estrogen replete state is only some 200–300/cell. We hypothesise that the etiology of osteoporosis is substantially due to declining estrogen levels leading to insufficient ERα in resident bone cells to adequately respond to strain. This produces a similar downstream situation as the absence of strain, as in disuse. The result is also similar—bone loss towards a genetically determined minimum. Since this bone loss is accompanied by continued loading the consequence is increased incidence of fragility fracture. doi:10.1016/j.bone.2009.01.056
019 Winning the battle against childhood physical inactivity: The key to bone strength? H.A. McKay Centre for Hip Health and Mobility, Vancouver, BC, Canada The British Columbia (Canada) Provincial Health Officer recently published a report that stated “Habits of living, nutrition and exercise patterns become established during the growing years. It is clear from the global experience that opportunities exist within the school setting to significantly and positively influence many domains of youth health.” Childhood and adolescence are also key times for the development of a healthy skeleton and the contributions of physical activity to bone health are well known. Given that children spend 30 of their waking hours in school each week, schools may provide the ideal vehicle to deliver physical activity models that target overall health — including bone health. In this presentation I will introduce effective school-based bone health intervention models and strategies for dissemination. Our understanding of the complex nature of bone's adaptation to physical activity has evolved over the years. Traditional methods of assessing bone mass at any age – such as dual energy x-ray absorptiometry (DXA) – fail to represent the complex, hierarchical 3-dimensional structure of bone. Novel imaging techniques such as peripheral quantitative computed tomography and extreme CT technology can safely and precisely achieve this and assess bone
S25
structure and strength. Thus, I will also introduce innovative techniques for evaluating the 3-D structure of bone and bone's adaptation to physical activity interventions. doi:10.1016/j.bone.2009.01.057
020 Proteolytically processed repressor Gli3 inhibits bone formation by repressing BMP2 transcription in osteoblasts M. Zhaoa, S. Koa, I. Garrettb, G. Mundya a Medicine/Clinical Pharmacology, Vanderbilt University, Nashville, Tennessee, United States b Bone and Drug Delivery, Zimmer, Austin, Texas, United States Zinc finger Gli proteins mediate hedgehog signaling which is required for osteoblast differentiation. C′ terminal truncated Gli3, a proteolytically processed fragment of Gli3, is a predominant and functional form of Gli3 and in general acts as gene transcriptional repressor. Here, we have investigated the role of repressor Gli3 (rGli3) in osteoblasts. We found in osteoblast precursor cells that full-length Gli3 underwent proteolytic processing which resulted in such a truncated rGli3 through the PKA–ubiquitination–proteasome pathway. We determined the effects of rGli3 on osteoblast differentiation and bone formation. Ex vivo calvarial organ culture has shown that overexpression of rGli3 significantly decreased proteasome inhibitor-induced calvarial ALP activity and new bone formation over the calvarial surface. These results suggest that rGli3 is an inhibitor of bone formation and this action is likely mediated through BMP2 since we have previously demonstrated that proteasome inhibitors are powerful enhancers of BMP2 expression in osteoblasts. By BMP2 promoter and mRNA assays, we found that rGli3 overexpression markedly inhibited BMP2 transcription in osteoblasts. Interestingly, we also found that rGli3 not only reduced the basal level of BMP2 expression, also antagonized the levels enhanced by Gli2, Smad1, b-catenin/TCF4, all of which have been previously shown to activate BMP2 expression in osteoblasts. To determine the interaction of rGli3 with BMP2 promoter, we performed promoter deletion and mutation studies, and identified three Gli binding elements in the BMP2 promoter responsible for rGli3 repression. These data suggest that rGli3 is a strong transcriptional repressor of BMP2 gene in osteoblasts. Lastly, we determined the role of rGli3 in bone in vivo. We generated osteoblast-specific rGli3 transgenic mice using 2.3Col1a1 promoter. m CT and histomorphometric results have shown that bone volume of rGli3 transgenic mice was substantially reduced along with decreased trabecular bone number and thickness and increased trabecular bone separation, compared with those of wild-type mice, suggesting that rGli3 has an inhibitory effect on normal bone mass. Together, our data provide evidence that C′ terminal shortened Gli3, the naturally processed product of full-length Gli3, is a strong repressor of osteoblastic bone formation and this function is due to its inhibition of BMP2 expression in osteoblasts. doi:10.1016/j.bone.2009.01.058
021 Cthrc1 is a positive regulator of osteoblastic bone formation H. Kimuraa, K. Kwanb, Z. Zhangc, J. Dengc, B.G. Darnayd, R.R. Behringerc, T. Nakamuraa, B. De Crombrugghec, H. Akiyamaa a Department of Orthopaedics, Kyoto University, Kyoto, Japan b Department of Biology, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China
Bones' ability to withstand the loads of everyday activity without fracture is achieved through resident bone cells using loadinginduced strains in the bone tissue as a stimulus to adjust bone mass and architecture to achieve and maintain a “target strain environment”. Only short exposure to dynamic strain is necessary to stimulate the cascade of events that prevent bone resorption and/ or stimulate bone formation. Early events following exposure to dynamic strain in resident bone cells include calcium fluxes; stimulation of the ERK pathway; production of prostaglandins and nitric oxide; activation of the canonical Wnt pathway; altered expression of a large number of non-bone specific genes; depression of sclerostin production and increased secretion of insulin-like growth factors. Estrogen receptor α, independently of estrogen, is involved up and down stream of ERK activation; in the production of nitric oxide but not prostaglandins; translocation of β-catenin to the nucleus; the regulation of a large number of genes and activation of the IGFR. The conservational and potentially osteogenic response to bone loading is substantially reduced in animals lacking ERα, but not ERβ. The most widespread failure of bone's mechanically-adaptive response to maintain the skeleton sufficiently robust to withstand everyday loading without fracture occurs in post-menopausal osteoporosis in women and age-related osteoporosis in men. In both sexes the level of bone loss is associated with reduced bioavailable estrogen. Estrogen, but not strain, appears to regulate the ER number in osteoblasts and osteocytes, which in the estrogen replete state is only some 200–300/cell. We hypothesise that the etiology of osteoporosis is substantially due to declining estrogen levels leading to insufficient ERα in resident bone cells to adequately respond to strain. This produces a similar downstream situation as the absence of strain, as in disuse. The result is also similar—bone loss towards a genetically determined minimum. Since this bone loss is accompanied by continued loading the consequence is increased incidence of fragility fracture. doi:10.1016/j.bone.2009.01.056
019 Winning the battle against childhood physical inactivity: The key to bone strength? H.A. McKay Centre for Hip Health and Mobility, Vancouver, BC, Canada The British Columbia (Canada) Provincial Health Officer recently published a report that stated “Habits of living, nutrition and exercise patterns become established during the growing years. It is clear from the global experience that opportunities exist within the school setting to significantly and positively influence many domains of youth health.” Childhood and adolescence are also key times for the development of a healthy skeleton and the contributions of physical activity to bone health are well known. Given that children spend 30 of their waking hours in school each week, schools may provide the ideal vehicle to deliver physical activity models that target overall health — including bone health. In this presentation I will introduce effective school-based bone health intervention models and strategies for dissemination. Our understanding of the complex nature of bone's adaptation to physical activity has evolved over the years. Traditional methods of assessing bone mass at any age – such as dual energy x-ray absorptiometry (DXA) – fail to represent the complex, hierarchical 3-dimensional structure of bone. Novel imaging techniques such as peripheral quantitative computed tomography and extreme CT technology can safely and precisely achieve this and assess bone
S25
structure and strength. Thus, I will also introduce innovative techniques for evaluating the 3-D structure of bone and bone's adaptation to physical activity interventions. doi:10.1016/j.bone.2009.01.057
020 Proteolytically processed repressor Gli3 inhibits bone formation by repressing BMP2 transcription in osteoblasts M. Zhaoa, S. Koa, I. Garrettb, G. Mundya a Medicine/Clinical Pharmacology, Vanderbilt University, Nashville, Tennessee, United States b Bone and Drug Delivery, Zimmer, Austin, Texas, United States Zinc finger Gli proteins mediate hedgehog signaling which is required for osteoblast differentiation. C′ terminal truncated Gli3, a proteolytically processed fragment of Gli3, is a predominant and functional form of Gli3 and in general acts as gene transcriptional repressor. Here, we have investigated the role of repressor Gli3 (rGli3) in osteoblasts. We found in osteoblast precursor cells that full-length Gli3 underwent proteolytic processing which resulted in such a truncated rGli3 through the PKA–ubiquitination–proteasome pathway. We determined the effects of rGli3 on osteoblast differentiation and bone formation. Ex vivo calvarial organ culture has shown that overexpression of rGli3 significantly decreased proteasome inhibitor-induced calvarial ALP activity and new bone formation over the calvarial surface. These results suggest that rGli3 is an inhibitor of bone formation and this action is likely mediated through BMP2 since we have previously demonstrated that proteasome inhibitors are powerful enhancers of BMP2 expression in osteoblasts. By BMP2 promoter and mRNA assays, we found that rGli3 overexpression markedly inhibited BMP2 transcription in osteoblasts. Interestingly, we also found that rGli3 not only reduced the basal level of BMP2 expression, also antagonized the levels enhanced by Gli2, Smad1, b-catenin/TCF4, all of which have been previously shown to activate BMP2 expression in osteoblasts. To determine the interaction of rGli3 with BMP2 promoter, we performed promoter deletion and mutation studies, and identified three Gli binding elements in the BMP2 promoter responsible for rGli3 repression. These data suggest that rGli3 is a strong transcriptional repressor of BMP2 gene in osteoblasts. Lastly, we determined the role of rGli3 in bone in vivo. We generated osteoblast-specific rGli3 transgenic mice using 2.3Col1a1 promoter. m CT and histomorphometric results have shown that bone volume of rGli3 transgenic mice was substantially reduced along with decreased trabecular bone number and thickness and increased trabecular bone separation, compared with those of wild-type mice, suggesting that rGli3 has an inhibitory effect on normal bone mass. Together, our data provide evidence that C′ terminal shortened Gli3, the naturally processed product of full-length Gli3, is a strong repressor of osteoblastic bone formation and this function is due to its inhibition of BMP2 expression in osteoblasts. doi:10.1016/j.bone.2009.01.058
021 Cthrc1 is a positive regulator of osteoblastic bone formation H. Kimuraa, K. Kwanb, Z. Zhangc, J. Dengc, B.G. Darnayd, R.R. Behringerc, T. Nakamuraa, B. De Crombrugghec, H. Akiyamaa a Department of Orthopaedics, Kyoto University, Kyoto, Japan b Department of Biology, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, China