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CREB induces BMP2 transcription in osteoblasts and CREB knockout reduces bone mass in mice
Abstracts / Bone 44 (2009) S18–S55
Thus, our data demonstrate that increased bone mass can be observed in lean mice. Indeed, in addition to promoting osteoblastogenesis, Fra-1 inhibits adipocyte maturation, this latter being caused by C/EBP (alpha) and PPAR (gamma2) down-regulation. Moreover, we show that Rsk2 is regulating Fra-1 function in fat tissue but not in the bone. Understanding AP-1 function in mesenchymal cell fate decision may provide valuable insights into mesenchymal tissue regeneration and metabolic diseases. doi:10.1016/j.bone.2009.01.061
024 CREB induces BMP2 transcription in osteoblasts and CREB knockout reduces bone mass in mice M. Zhaoa, J. Edwardsa, S. Koa, R. Parlatoc, S. Harrisb, G. Mundya a Medicine/Clinical Pharmacology, Vanderbilt University, Nashville, Tennessee, United States b Periodontics, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States c Molecular Biology of the Cell I, German Cancer Research Center, Heidelberg, Germany Transcription factor CREB (cAMP-response element binding protein) plays an essential role in osteoblasts to mediate the anabolic signaling of intermittent dosage of PTH in bone. However, the downstream mechanisms of CREB in osteoblasts have to be demonstrated. Here, we have characterized the skeletal phenotype of CREB knockout mice and identified the potential transcriptional target of CREB in osteoblasts. The results of m CT measurement and alizarin red/alcian blue, ALP and von Kossa staining, have shown that global knockout of CREB in mice caused a dwarfism phenotype and a significant reduction of bone volume and mineralization in the embryonic skeleton. Since the global CREB KO mice do not survive after birth, we have generated the osteoblast-specific CREB KO mice (CREBfl;2.3ColCre) and examined their skeletal phenotype. m CT results have shown that bone volume of two-month-old CREBfl;2.3ColCre KO mice substantially reduced, along with decreased trabecular number and thickness and increased trabecular separation, compared with that of control mice. These results suggest that CREB is critical for both normal skeletal development and postnatal bone mass. Interestingly, we found that expression of BMP2, an important factor for osteoblast differentiation and bone formation, was reduced in the bones of CREB KO mice. Since there exists a similar osteopenic phenotype between CREB KO CREBfl;2.3ColCre) and BMP2 KO (BMP2fl;3.6ColCre) mice, and the BMP2 promoter contains multiple cAMP response elements (CRE), we hypothesized that the function of CREB in bone is mediated at least in part through BMP2. In cell culture, we found that both CREB and PTH stimulated BMP2 gene expression in osteoblasts. We also found that pharmacological manipulation of CREB phosphorylation by cAMP/PKA activator IBMX or inhibitor KT5720 affected CREB transactivation of the BMP2 expression. Furthermore, through promoter mutation studies, we demonstrated that CREB transactivated BMP2 gene by directly interacting with a specific CRE in the BMP2 promoter. Lastly, we have shown that overexpression of CREB promoted osteoblast differentiation and this action was blocked by addition of noggin in the cultures. Together, these results suggest that CREB plays an important role in osteogenesis embryonically and postnatally, and this function is mediated by up-regulation of BMP2 transcription in osteoblasts. doi:10.1016/j.bone.2009.01.062
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025 Osteoblast IL-33 mRNA expression is regulated by PTH, and IL-33 treatment causes both increased osteoblastic matrix mineralisation and reduced osteoclast formation in vitro H. Saleha,b,c, J.M.W. Quinna,b,c, T. Martinc, M.T. Gillespiea a Prince Henry's Institute, Clayton, VIC, Australia b Department of Medicine, University of Melbourne, Fitzroy, VIC, Australia c St. Vincent's Institute, Fitzroy, VIC, Australia IL-33 is a Th2 stimulating pro-inflammatory cytokine related to IL-1 and IL-18, its actions mediated by receptor ST2L. We have previously found that IL-33, like IL-18, indirectly inhibits osteoclast formation via T lymphocytes but does not directly affect osteoclast formation from RANKL-stimulated bone marrow macrophages (BMM). In mouse bone sections, anti-IL-33 antibody clearly immunostained osteoblasts and chondrocytes and some osteoclasts but not osteocytes or most bone marrow cells. DNA microarray array studies also showed ST2L mRNA expression in matured osteoblastic Kusa 4b10 cells is increased by PTH treatment, observations confirmed by further RT-PCR analysis. PTH treatment also increased IL-33 mRNA levels in osteoblasts. Long term osteoblastic cultures treated with ascorbate (which increases osteoblastic differentiation) also increased IL-33 mRNA levels. With long term pro-adipogenic treatment (dexamethasone, insulin and IBMX) of immature Kusa 4b10 cells IL33 mRNA levels increased but ST2L mRNA levels decreased. Since osteoblasts express ST2L we investigated IL-33 action on osteoblasts to identify possible autocrine actions. IL-33 promoted matrix mineralisation by primary osteoblasts. Furthermore, in long term ascorbate stimulated primary osteoblasts in which expression of osteocytic features are apparent (e.g. sclerostin and DMP-1 expression), IL-33 reduced sclerostin mRNA levels after 6 and 24 h of treatment, although other PTH regulated genes in these cells, such as ephrin B2 were not affected. We also investigated IL-33 effects on osteoblastic support of osteoclastogenesis. Osteoclast formation from BMM stimulated by 1,25 dihydroxyvitamin D3-treated Kusa O pre-osteoblastic cells was blocked in the presence of IL-33 (20 ng/ml), an action ablated by anti-GM-CSF antibody. GM-CSF mRNA was strongly upregulated by IL-33 treatment, as indeed was RANKL mRNA. However, Kusa O/BMM co-cultures treated with IL-33 and anti-GM-CSF antibody (without other stimulus) induced osteoclast formation only weakly. Thus we have found evidence that IL-33 stimulates osteoblastic function while indirectly, through two separate mechanisms, inhibiting osteoclast formation. IL-33 thus may play a role in maintaining bone mass, perhaps participating in the anabolic actions of PTH. doi:10.1016/j.bone.2009.01.063
026 A dairy-based protein, calcium and vitamin D supplement preserves trabecular bone and reduces falls in aged care residents S. Iuliano-Burnsa, K. Kinga, J. Woodsc, A. Evansb, A. Ghasem-Zadehb, E. Seemana a Endocrinology, Medicine, University of Melbourne/Austin Health, West Heidelberg, VIC, Australia b Bone and Mineral Research Unit, Austin Health, West Heidelberg, VIC, Australia c Nutrition and Dietetics, Monash University, Clayton, VIC, Australia Aged care residents are at higher risk of falls and fractures than elderly living in the community. Nutrient deficiencies may increase falls and fracture risk by contributing to bone loss and body sway and reducing muscle mass and strength. We aimed to determine if a dairybased protein, calcium and vitamin D supplement incorporated into
Thus, our data demonstrate that increased bone mass can be observed in lean mice. Indeed, in addition to promoting osteoblastogenesis, Fra-1 inhibits adipocyte maturation, this latter being caused by C/EBP (alpha) and PPAR (gamma2) down-regulation. Moreover, we show that Rsk2 is regulating Fra-1 function in fat tissue but not in the bone. Understanding AP-1 function in mesenchymal cell fate decision may provide valuable insights into mesenchymal tissue regeneration and metabolic diseases. doi:10.1016/j.bone.2009.01.061
024 CREB induces BMP2 transcription in osteoblasts and CREB knockout reduces bone mass in mice M. Zhaoa, J. Edwardsa, S. Koa, R. Parlatoc, S. Harrisb, G. Mundya a Medicine/Clinical Pharmacology, Vanderbilt University, Nashville, Tennessee, United States b Periodontics, University of Texas Health Science Center at San Antonio, San Antonio, Texas, United States c Molecular Biology of the Cell I, German Cancer Research Center, Heidelberg, Germany Transcription factor CREB (cAMP-response element binding protein) plays an essential role in osteoblasts to mediate the anabolic signaling of intermittent dosage of PTH in bone. However, the downstream mechanisms of CREB in osteoblasts have to be demonstrated. Here, we have characterized the skeletal phenotype of CREB knockout mice and identified the potential transcriptional target of CREB in osteoblasts. The results of m CT measurement and alizarin red/alcian blue, ALP and von Kossa staining, have shown that global knockout of CREB in mice caused a dwarfism phenotype and a significant reduction of bone volume and mineralization in the embryonic skeleton. Since the global CREB KO mice do not survive after birth, we have generated the osteoblast-specific CREB KO mice (CREBfl;2.3ColCre) and examined their skeletal phenotype. m CT results have shown that bone volume of two-month-old CREBfl;2.3ColCre KO mice substantially reduced, along with decreased trabecular number and thickness and increased trabecular separation, compared with that of control mice. These results suggest that CREB is critical for both normal skeletal development and postnatal bone mass. Interestingly, we found that expression of BMP2, an important factor for osteoblast differentiation and bone formation, was reduced in the bones of CREB KO mice. Since there exists a similar osteopenic phenotype between CREB KO CREBfl;2.3ColCre) and BMP2 KO (BMP2fl;3.6ColCre) mice, and the BMP2 promoter contains multiple cAMP response elements (CRE), we hypothesized that the function of CREB in bone is mediated at least in part through BMP2. In cell culture, we found that both CREB and PTH stimulated BMP2 gene expression in osteoblasts. We also found that pharmacological manipulation of CREB phosphorylation by cAMP/PKA activator IBMX or inhibitor KT5720 affected CREB transactivation of the BMP2 expression. Furthermore, through promoter mutation studies, we demonstrated that CREB transactivated BMP2 gene by directly interacting with a specific CRE in the BMP2 promoter. Lastly, we have shown that overexpression of CREB promoted osteoblast differentiation and this action was blocked by addition of noggin in the cultures. Together, these results suggest that CREB plays an important role in osteogenesis embryonically and postnatally, and this function is mediated by up-regulation of BMP2 transcription in osteoblasts. doi:10.1016/j.bone.2009.01.062
S27
025 Osteoblast IL-33 mRNA expression is regulated by PTH, and IL-33 treatment causes both increased osteoblastic matrix mineralisation and reduced osteoclast formation in vitro H. Saleha,b,c, J.M.W. Quinna,b,c, T. Martinc, M.T. Gillespiea a Prince Henry's Institute, Clayton, VIC, Australia b Department of Medicine, University of Melbourne, Fitzroy, VIC, Australia c St. Vincent's Institute, Fitzroy, VIC, Australia IL-33 is a Th2 stimulating pro-inflammatory cytokine related to IL-1 and IL-18, its actions mediated by receptor ST2L. We have previously found that IL-33, like IL-18, indirectly inhibits osteoclast formation via T lymphocytes but does not directly affect osteoclast formation from RANKL-stimulated bone marrow macrophages (BMM). In mouse bone sections, anti-IL-33 antibody clearly immunostained osteoblasts and chondrocytes and some osteoclasts but not osteocytes or most bone marrow cells. DNA microarray array studies also showed ST2L mRNA expression in matured osteoblastic Kusa 4b10 cells is increased by PTH treatment, observations confirmed by further RT-PCR analysis. PTH treatment also increased IL-33 mRNA levels in osteoblasts. Long term osteoblastic cultures treated with ascorbate (which increases osteoblastic differentiation) also increased IL-33 mRNA levels. With long term pro-adipogenic treatment (dexamethasone, insulin and IBMX) of immature Kusa 4b10 cells IL33 mRNA levels increased but ST2L mRNA levels decreased. Since osteoblasts express ST2L we investigated IL-33 action on osteoblasts to identify possible autocrine actions. IL-33 promoted matrix mineralisation by primary osteoblasts. Furthermore, in long term ascorbate stimulated primary osteoblasts in which expression of osteocytic features are apparent (e.g. sclerostin and DMP-1 expression), IL-33 reduced sclerostin mRNA levels after 6 and 24 h of treatment, although other PTH regulated genes in these cells, such as ephrin B2 were not affected. We also investigated IL-33 effects on osteoblastic support of osteoclastogenesis. Osteoclast formation from BMM stimulated by 1,25 dihydroxyvitamin D3-treated Kusa O pre-osteoblastic cells was blocked in the presence of IL-33 (20 ng/ml), an action ablated by anti-GM-CSF antibody. GM-CSF mRNA was strongly upregulated by IL-33 treatment, as indeed was RANKL mRNA. However, Kusa O/BMM co-cultures treated with IL-33 and anti-GM-CSF antibody (without other stimulus) induced osteoclast formation only weakly. Thus we have found evidence that IL-33 stimulates osteoblastic function while indirectly, through two separate mechanisms, inhibiting osteoclast formation. IL-33 thus may play a role in maintaining bone mass, perhaps participating in the anabolic actions of PTH. doi:10.1016/j.bone.2009.01.063
026 A dairy-based protein, calcium and vitamin D supplement preserves trabecular bone and reduces falls in aged care residents S. Iuliano-Burnsa, K. Kinga, J. Woodsc, A. Evansb, A. Ghasem-Zadehb, E. Seemana a Endocrinology, Medicine, University of Melbourne/Austin Health, West Heidelberg, VIC, Australia b Bone and Mineral Research Unit, Austin Health, West Heidelberg, VIC, Australia c Nutrition and Dietetics, Monash University, Clayton, VIC, Australia Aged care residents are at higher risk of falls and fractures than elderly living in the community. Nutrient deficiencies may increase falls and fracture risk by contributing to bone loss and body sway and reducing muscle mass and strength. We aimed to determine if a dairybased protein, calcium and vitamin D supplement incorporated into