The Experts below are selected from a list of 267 Experts worldwide ranked by ideXlab platform

Chi Zhang - One of the best experts on this subject based on the ideXlab platform.

  • iTRAQ quantitative proteomic study in patients with thoracic ossification of the ligamentum flavum
    Biochemical and Biophysical Research Communications, 2017
    Co-Authors: Bingxiang Wang, Zhongqiang Chen, Xiangyu Meng, Mengtao Li, Xiaoxi Yang, Chi Zhang
    Abstract:

    Thoracic ossification of the ligamentum flavum (TOLF) is a unique disease with ectopic ossification, and is a major cause of thoracic spinal stenosis and myelopathy. However, the underlying etiology remains largely unknown. In this study, the ligamentum flavum was systematically analyzed in TOLF patients by using comprehensive iTRAQ labeled quantitative proteomics. Among 1285 detected proteins, there were 282 proteins identified to be differentially expressed. The Gene Ontology (GO) analysis regarding functional annotation of proteins consists of the following three aspects: the biological process, the molecular function, and the cellular components. The function clustering analysis revealed that ten of the above proteins are related to inflammation, such as tumor necrosis Factor (TNF). This finding was subsequently validated by ELISA, which indicated that serum TNF-α of TOLF patients was significantly higher compared with the control group. To address the effect of TNF-α on ossification-related gene expression, we purified and cultured primary cells from thoracic ligamentum flavum of patients with TOLF. TNF-α was then used to stimulate cells. RNA was isolated and analyzed by RT-PCR. Our results showed that TNF-α was able to induce the expressions of osteoblast-specific Transcription Factor Osterix (Osx) in ligamentum flavum cells, suggesting that it can promote osteoblast differentiation. In addition, as the Osx downstream osteoblast genes OCN and ALP were also activated by TNF-α. This is the first proteomic study to identify inflammation Factors such as TNF-α involved in ossified ligamentum flavum in TOLF, which may contribute to a better understanding of the cause of TOLF.

  • synergistic inhibition of wnt pathway by hif 1α and osteoblast specific Transcription Factor Osterix osx in osteoblasts
    PLOS ONE, 2012
    Co-Authors: Dafu Chen, Yang Li, Zhiyu Zhou, Yonggang Xing, Yu Zhong, Wei Tian, Chi Zhang
    Abstract:

    Osterix (Osx) is an osteoblast-specific Transcription Factor required for osteoblast differentiation. Inhibition of Wnt pathway by Osx highlights the potential for feedback control mechanisms involved in bone formation. Hypoxia-inducible Factor-1α (HIF-1α) is a master regulator of hypoxia. HIF-1α has been reported to couple angiogenesis to osteogenesis. Our recent study has demonstrated that Osx and HIF-1α cooperatively regulate VEGF expression in osteoblasts. Effects of hypoxia/HIF-1α on osteoblast proliferation and related mechanisms are not well understood. In this study, osteoblast growth under hypoxia was examined. We observed that osteoblast growth was inhibited under hypoxia. To explore possible mechanisms for hypoxia/HIF-1α to inhibit osteoblast proliferation, we tested the effect of hypoxia/HIF-1α on Wnt pathway. Quantitative RT-PCR results revealed that Wnt target genes such as cyclin D1 and c-Myc were downregulated under hypoxia while HIF-1α was upregulated. Treatment of desferrioxamine, a HIF-1α activator, led to further downregulation of expressions of cyclin D1 and c-Myc in osteoblasts. On the contrary, the inhibition of HIF-1α by siRNA in osteoblasts led to the expression increase of cyclin D1 and c-Myc. These data suggest that HIF-1α inhibits Wnt pathway in osteoblasts. To examine the effect of HIF-1α on Wnt pathway, HIF-1α was cotransfected with β-catenin along with Topflash reporter in transient transfection assay. Our results showed that HIF-1α inhibited β-catenin-induced Topflash reporter activity. Interestingly, a synergistic interplay was observed between Osx and HIF-1α in the inhibition of β-catenin-induced Topflash expression. Our findings indicate that Osx and HIF-1α cooperatively inhibit Wnt pathway. This study revealed additional new information of the cooperation between HIF-1α and Osx in osteoblasts.

  • matrix metalloproteinase 13 mmp13 is a direct target of osteoblast specific Transcription Factor Osterix osx in osteoblasts
    PLOS ONE, 2012
    Co-Authors: Chi Zhang, Wanjin Tang, Yang Li
    Abstract:

    Osterix (Osx) is an osteoblast-specific Transcription Factor required for bone formation and osteoblast differentiation from mesenchymal stem cells. In Osx-null mice, no bone formation occurs. Matrix metalloproteinase 13 (MMP13) is a member of the matrix metalloproteinase family and plays an important role in endochondral ossification and bone remodeling. Transcriptional regulation of MMP13 expression in osteoblasts is not well understood. Here, we provide several lines of evidence which show that MMP13 is a direct target of Osx in osteoblasts. Calvaria obtained from Osx-null embryos displayed dramatic reductions in MMP13 expression compared to wild-type calvaria. Stable overexpression of Osx stimulated MMP13 expression in C2C12 mesenchymal cells. Inhibition of Osx expression by siRNA led to downregulation of MMP13 expression. Mechanistic approaches using transient transfection assays showed that Osx directly activated a 1 kb fragment of the MMP13 promoter in a dose-dependent manner. To define the region of the MMP13 promoter that was responsive to Osx, a series of MMP13 promoter deletion mutants were examined and the minimal Osx-responsive region was refined to the proximal 80 bp of the MMP13 promoter. Additional point mutant analysis was used to identify one GC-rich region that was responsible for MMP13 promoter activation by Osx. Gel Shift Assay showed that Osx bound to MMP13 promoter sequence directly. Chromatin immunoprecipitation assays demonstrated that endogenous Osx was associated with the native MMP13 promoter in primary osteoblasts in vivo. Taken together, these data strongly support a direct regulatory role for Osx in MMP13 gene expression in osteoblasts. They further provide new insight into potential mechanisms and pathways that Osx controls bone formation.

  • osteoblast specific Transcription Factor Osterix osx and hif 1α cooperatively regulate gene expression of vascular endothelial growth Factor vegf
    Biochemical and Biophysical Research Communications, 2012
    Co-Authors: Dafu Chen, Yang Li, Wanjin Tang, Wei Tian, Chi Zhang
    Abstract:

    Abstract Bone formation is a highly regulated process involving the differentiation of mesenchymal stem cells to osteoblasts. Angiogenesis and osteogenesis are tightly coupled during bone formation. Vascular endothelial growth Factor (VEGF) is involved in both processes. Relatively little is known about VEGF gene regulation in osteoblasts. Osterix (Osx) is a bone morphogenetic protein 2 (BMP-2) inducible osteoblast-specific Transcription Factor required for osteoblast differentiation and bone formation. Our recent study has demonstrated that Osx controls VEGF expression in osteoblasts. Here, we further characterized Osx regulation of VEGF. To address which domain of Osx is responsible for VEGF regulation, the deletion mutant analysis and transfection assay were carried out to show that proline-rich region (PRR) is required for Osx activation of VEGF promoter activity. Hypoxia-inducible Factor-1α (HIF-1α) has been reported to couple angiogenesis to osteogenesis, and to upregulate VEGF. Effect of Osx on HIF-1α expression was examined in this study. Quantitative RT-PCR results revealed that HIF-1α remained unchanged between wild type and Osx knockout calvaria at E18.5 in mouse embryos. Overexpression of Osx in stable C2C12 mesenchymal cells using Tet-off system did not affect HIF-1α expression. HIF-1α level did not change after Osx inhibition by siRNA in osteoblasts. Moreover, BMP-2 stimulation led to upregulation of Osx and VEGF, but not HIF-1α. These results demonstrate that HIF-1α is not a downstream target of Osx in osteoblasts, suggesting that Osx regulation of VEGF is independent of HIF-1α expression level. Interestingly, synergistic interplays were observed between Osx and HIF-1α in VEGF promoter activation in transfection assay. Our findings indicate that Osx and HIF-1α cooperatively regulate VEGF expression.

  • Transcriptional regulation of vascular endothelial growth Factor vegf by osteoblast specific Transcription Factor Osterix osx in osteoblasts
    Journal of Biological Chemistry, 2012
    Co-Authors: Wanjin Tang, Benoit De Crombrugghe, Fan Yang, Yang Li, Hongli Jiao, Guozhi Xiao, Chi Zhang
    Abstract:

    Abstract Osterix (Osx) is an osteoblast-specific Transcription Factor required for bone formation and osteoblast differentiation. The critical step in bone formation is to replace the avascular cartilage template with vascularized bone. Osteogenesis and angiogenesis are associated with each other, sharing some essential regulators. Vascular endothelial growth Factor (VEGF) is involved in both angiogenesis and osteogenesis. Transcriptional regulation of VEGF expression is not well known in osteoblasts. In this study, quantitative real-time RT-PCR results revealed that VEGF expression was down-regulated in Osx-null calvarial cells and that osteoblast marker osteocalcin expression was absent. Overexpression of Osx in stable C2C12 mesenchymal cells using a Tet-off system resulted in up-regulation of both osteocalcin and VEGF expression. The inhibition of Osx by siRNA led to repression of VEGF expression in osteoblasts. These results suggest that Osx controls VEGF expression. Transfection assays demonstrated that Osx activated VEGF promoter activity. A series of VEGF promoter deletion mutants were examined and the minimal Osx-responsive region was defined to the proximal 140-bp region of the VEGF promoter. Additional point mutants were used to identify two GC-rich regions that were responsible for VEGF promoter activation by Osx. Gel shift assay showed that Osx bound to the VEGF promoter sequence directly. Chromatin immunoprecipitation assays indicated that endogenous Osx associated with the native VEGF promoter in primary osteoblasts. Moreover, immunohistochemistry staining showed decreased VEGF protein levels in the tibiae of Osx conditional knock-out mice. We provide the first evidence that Osx controlled VEGF expression, suggesting a potential role of Osx in coordinating osteogenesis and angiogenesis.

Benoit De Crombrugghe - One of the best experts on this subject based on the ideXlab platform.

  • Mesenchymal deletion of histone demethylase NO66 in mice promotes bone formation
    Journal of Bone and Mineral Research, 2015
    Co-Authors: Qin Chen, Krishna M Sinha, Hideyo Yasuda, Jian Min Deng, Richard R Behringer, Ralf Krahe, Benoit De Crombrugghe
    Abstract:

    Our previous studies indicated that the Jumonji C (JmjC)-domain-containing NO66 is a histone demethylase with specificity for methylated histone H3K4 and H3K36. NO66 binds to the Transcription Factor Osterix (Osx) and inhibits its Transcriptional activity in promoter assays. However, the physiological role of NO66 in formation of mammalian bones is unknown. Here, using a genetically engineered mouse model, we show that during early skeletal development, Prx1-Cre dependent mesenchymal deletion of NO66 promotes osteogenesis and formation of both endochondral as well as intramembranous skeletal elements, leading to a larger skeleton and a high bone mass phenotype in adult mice. The excess bone formation in mice where NO66 was deleted in cells of mesenchymal origin is associated with an increase in the number of preosteoblasts and osteoblasts. Further analysis revealed that in the embryonic limbs and adult calvaria of mice with deletion of NO66 in cells of mesenchymal origin, expression of several genes including bone morphogenetic protein 2, insulin-like growth Factor 1 and osteoclast inhibitor osteoprotegerin was increased, concurrent with an increase in expression of bone formation markers such as Osx, type I collagen and bone sialoprotein. Taken together, our results provide the first in vivo evidence that NO66 histone demethylase plays an important role in mammalian osteogenesis during early development as well as in adult bone homeostasis. We postulate that NO66 regulates bone formation, at least in part, via regulating the number of bone-forming cells and expression of multiple genes that are critical for these processes.

  • Mesenchyme-specific overexpression of nucleolar protein 66 in mice inhibits skeletal growth and bone formation
    The FASEB Journal, 2015
    Co-Authors: Qin Chen, Benoit De Crombrugghe, Liping Zhang, Ralf Krahe
    Abstract:

    Previous studies showed that nucleolar protein 66 (NO66), the Jumonji C-domain-containing histone demethylase for methylated histone H3K4 and H3K36 (H3K36me), negatively regulates osteoblast differentiation in vitro by inhibiting the activity of Transcription Factor Osterix (Osx). However, whether NO66 affects mammalian skeletogenesis in vivo is not yet known. Here, we generated transgenic (TG) mice overexpressing a flag-tagged NO66 transgene driven by the Prx1 (paired related homeobox 1) promoter. We found that NO66 overexpression in Prx1-expressing mesenchymal cells inhibited skeletal growth and bone formation. The inhibitory phenotype was associated with >50% decreases in chondrocyte/osteoblast proliferation and differentiation. Moreover, we found that in bones of NO66-TG mice, expression of Igf1, Igf1 receptor (Igf1r), runt-related Transcription Factor 2, and Osx was significantly down-regulated (P 50% reduction in levels of phosphorylated protein k...

  • structural insights into histone demethylase no66 in interaction with osteoblast specific Transcription Factor Osterix and gene repression
    Journal of Biological Chemistry, 2013
    Co-Authors: Krishna M Sinha, Benoit De Crombrugghe, Minhao Wu, Xing Zhou, Bin Hu, Jianye Zang
    Abstract:

    Abstract Osterix (Osx) is an osteoblast-specific Transcriptional Factor and is required for osteoblast differentiation and bone formation. A JmjC domain-containing protein NO66 was previously found to participate in regulation of Osx Transcriptional activity and plays an important role in osteoblast differentiation through interaction with Osx. Here, we report the crystal structure of NO66 forming in a functional tetramer. A hinge domain links the N-terminal JmjC domain and C-terminal winged helix-turn-helix domain of NO66, and both domains are essential for tetrameric assembly. The oligomerization interface of NO66 interacts with a conserved fragment of Osx. We show that the hinge domain-dependent oligomerization of NO66 is essential for inhibition of Osx-dependent gene activation. Our findings suggest that homo-oligomerization of JmjC domain containing proteins might play a physiological role through interactions with other regulatory Factors during gene expression.

  • identification and characterization of micrornas controlled by the osteoblast specific Transcription Factor Osterix
    PLOS ONE, 2013
    Co-Authors: Qin Chen, Krishna M Sinha, Hideyo Yasuda, Benoit De Crombrugghe
    Abstract:

    Osterix (Osx) is an osteoblast-specific Transcription Factor which is essential for bone formation. MicroRNAs (miRNAs) have been previously shown to be involved in osteogenesis. However, it is unclear whether Osx is involved in the regulation of miRNA expression. In this study, we have identified groups of miRNAs that are differentially expressed in calvaria of the E18.5 Osx−/− embryos compared to wild type embryos. The correlation between the levels of miRNAs and Osx expression was further verified in cultured M-Osx cells in which over-expression of Osx is inducible. Our results suggest that Osx down-regulates expression of a group of miRNAs including mir-133a and -204/211, but up-regulates expression of another group of miRNAs such as mir-141/200a. Mir-133a and -204/211 are known to target the master osteogenic Transcription Factor Runx2. Further assays suggest that Sost, which encodes the Wnt signaling antagonist Sclerostin, and alkaline phosphatase (ALP) are two additional targets of mir-204/211. Mir-141/200a has been known to target the Transcription Factor Dlx5. Thus, we postulate that during the process of Osx-controlled osteogenesis, Osx has the ability to coordinately modulate Runx2, Sclerostin, ALP and Dlx5 proteins at levels appropriate for optimal osteoblast differentiation and function, at least in part, through regulation of specific miRNAs. Our study shows a tight correlation between Osx and the miRNAs involved in bone formation, and provides new information about molecular mechanisms of Osx-controlled osteogenesis.

  • Transcriptional regulation of vascular endothelial growth Factor vegf by osteoblast specific Transcription Factor Osterix osx in osteoblasts
    Journal of Biological Chemistry, 2012
    Co-Authors: Wanjin Tang, Benoit De Crombrugghe, Fan Yang, Yang Li, Hongli Jiao, Guozhi Xiao, Chi Zhang
    Abstract:

    Abstract Osterix (Osx) is an osteoblast-specific Transcription Factor required for bone formation and osteoblast differentiation. The critical step in bone formation is to replace the avascular cartilage template with vascularized bone. Osteogenesis and angiogenesis are associated with each other, sharing some essential regulators. Vascular endothelial growth Factor (VEGF) is involved in both angiogenesis and osteogenesis. Transcriptional regulation of VEGF expression is not well known in osteoblasts. In this study, quantitative real-time RT-PCR results revealed that VEGF expression was down-regulated in Osx-null calvarial cells and that osteoblast marker osteocalcin expression was absent. Overexpression of Osx in stable C2C12 mesenchymal cells using a Tet-off system resulted in up-regulation of both osteocalcin and VEGF expression. The inhibition of Osx by siRNA led to repression of VEGF expression in osteoblasts. These results suggest that Osx controls VEGF expression. Transfection assays demonstrated that Osx activated VEGF promoter activity. A series of VEGF promoter deletion mutants were examined and the minimal Osx-responsive region was defined to the proximal 140-bp region of the VEGF promoter. Additional point mutants were used to identify two GC-rich regions that were responsible for VEGF promoter activation by Osx. Gel shift assay showed that Osx bound to the VEGF promoter sequence directly. Chromatin immunoprecipitation assays indicated that endogenous Osx associated with the native VEGF promoter in primary osteoblasts. Moreover, immunohistochemistry staining showed decreased VEGF protein levels in the tibiae of Osx conditional knock-out mice. We provide the first evidence that Osx controlled VEGF expression, suggesting a potential role of Osx in coordinating osteogenesis and angiogenesis.

Yang Li - One of the best experts on this subject based on the ideXlab platform.

  • synergistic inhibition of wnt pathway by hif 1α and osteoblast specific Transcription Factor Osterix osx in osteoblasts
    PLOS ONE, 2012
    Co-Authors: Dafu Chen, Yang Li, Zhiyu Zhou, Yonggang Xing, Yu Zhong, Wei Tian, Chi Zhang
    Abstract:

    Osterix (Osx) is an osteoblast-specific Transcription Factor required for osteoblast differentiation. Inhibition of Wnt pathway by Osx highlights the potential for feedback control mechanisms involved in bone formation. Hypoxia-inducible Factor-1α (HIF-1α) is a master regulator of hypoxia. HIF-1α has been reported to couple angiogenesis to osteogenesis. Our recent study has demonstrated that Osx and HIF-1α cooperatively regulate VEGF expression in osteoblasts. Effects of hypoxia/HIF-1α on osteoblast proliferation and related mechanisms are not well understood. In this study, osteoblast growth under hypoxia was examined. We observed that osteoblast growth was inhibited under hypoxia. To explore possible mechanisms for hypoxia/HIF-1α to inhibit osteoblast proliferation, we tested the effect of hypoxia/HIF-1α on Wnt pathway. Quantitative RT-PCR results revealed that Wnt target genes such as cyclin D1 and c-Myc were downregulated under hypoxia while HIF-1α was upregulated. Treatment of desferrioxamine, a HIF-1α activator, led to further downregulation of expressions of cyclin D1 and c-Myc in osteoblasts. On the contrary, the inhibition of HIF-1α by siRNA in osteoblasts led to the expression increase of cyclin D1 and c-Myc. These data suggest that HIF-1α inhibits Wnt pathway in osteoblasts. To examine the effect of HIF-1α on Wnt pathway, HIF-1α was cotransfected with β-catenin along with Topflash reporter in transient transfection assay. Our results showed that HIF-1α inhibited β-catenin-induced Topflash reporter activity. Interestingly, a synergistic interplay was observed between Osx and HIF-1α in the inhibition of β-catenin-induced Topflash expression. Our findings indicate that Osx and HIF-1α cooperatively inhibit Wnt pathway. This study revealed additional new information of the cooperation between HIF-1α and Osx in osteoblasts.

  • matrix metalloproteinase 13 mmp13 is a direct target of osteoblast specific Transcription Factor Osterix osx in osteoblasts
    PLOS ONE, 2012
    Co-Authors: Chi Zhang, Wanjin Tang, Yang Li
    Abstract:

    Osterix (Osx) is an osteoblast-specific Transcription Factor required for bone formation and osteoblast differentiation from mesenchymal stem cells. In Osx-null mice, no bone formation occurs. Matrix metalloproteinase 13 (MMP13) is a member of the matrix metalloproteinase family and plays an important role in endochondral ossification and bone remodeling. Transcriptional regulation of MMP13 expression in osteoblasts is not well understood. Here, we provide several lines of evidence which show that MMP13 is a direct target of Osx in osteoblasts. Calvaria obtained from Osx-null embryos displayed dramatic reductions in MMP13 expression compared to wild-type calvaria. Stable overexpression of Osx stimulated MMP13 expression in C2C12 mesenchymal cells. Inhibition of Osx expression by siRNA led to downregulation of MMP13 expression. Mechanistic approaches using transient transfection assays showed that Osx directly activated a 1 kb fragment of the MMP13 promoter in a dose-dependent manner. To define the region of the MMP13 promoter that was responsive to Osx, a series of MMP13 promoter deletion mutants were examined and the minimal Osx-responsive region was refined to the proximal 80 bp of the MMP13 promoter. Additional point mutant analysis was used to identify one GC-rich region that was responsible for MMP13 promoter activation by Osx. Gel Shift Assay showed that Osx bound to MMP13 promoter sequence directly. Chromatin immunoprecipitation assays demonstrated that endogenous Osx was associated with the native MMP13 promoter in primary osteoblasts in vivo. Taken together, these data strongly support a direct regulatory role for Osx in MMP13 gene expression in osteoblasts. They further provide new insight into potential mechanisms and pathways that Osx controls bone formation.

  • osteoblast specific Transcription Factor Osterix osx and hif 1α cooperatively regulate gene expression of vascular endothelial growth Factor vegf
    Biochemical and Biophysical Research Communications, 2012
    Co-Authors: Dafu Chen, Yang Li, Wanjin Tang, Wei Tian, Chi Zhang
    Abstract:

    Abstract Bone formation is a highly regulated process involving the differentiation of mesenchymal stem cells to osteoblasts. Angiogenesis and osteogenesis are tightly coupled during bone formation. Vascular endothelial growth Factor (VEGF) is involved in both processes. Relatively little is known about VEGF gene regulation in osteoblasts. Osterix (Osx) is a bone morphogenetic protein 2 (BMP-2) inducible osteoblast-specific Transcription Factor required for osteoblast differentiation and bone formation. Our recent study has demonstrated that Osx controls VEGF expression in osteoblasts. Here, we further characterized Osx regulation of VEGF. To address which domain of Osx is responsible for VEGF regulation, the deletion mutant analysis and transfection assay were carried out to show that proline-rich region (PRR) is required for Osx activation of VEGF promoter activity. Hypoxia-inducible Factor-1α (HIF-1α) has been reported to couple angiogenesis to osteogenesis, and to upregulate VEGF. Effect of Osx on HIF-1α expression was examined in this study. Quantitative RT-PCR results revealed that HIF-1α remained unchanged between wild type and Osx knockout calvaria at E18.5 in mouse embryos. Overexpression of Osx in stable C2C12 mesenchymal cells using Tet-off system did not affect HIF-1α expression. HIF-1α level did not change after Osx inhibition by siRNA in osteoblasts. Moreover, BMP-2 stimulation led to upregulation of Osx and VEGF, but not HIF-1α. These results demonstrate that HIF-1α is not a downstream target of Osx in osteoblasts, suggesting that Osx regulation of VEGF is independent of HIF-1α expression level. Interestingly, synergistic interplays were observed between Osx and HIF-1α in VEGF promoter activation in transfection assay. Our findings indicate that Osx and HIF-1α cooperatively regulate VEGF expression.

  • Transcriptional regulation of vascular endothelial growth Factor vegf by osteoblast specific Transcription Factor Osterix osx in osteoblasts
    Journal of Biological Chemistry, 2012
    Co-Authors: Wanjin Tang, Benoit De Crombrugghe, Fan Yang, Yang Li, Hongli Jiao, Guozhi Xiao, Chi Zhang
    Abstract:

    Abstract Osterix (Osx) is an osteoblast-specific Transcription Factor required for bone formation and osteoblast differentiation. The critical step in bone formation is to replace the avascular cartilage template with vascularized bone. Osteogenesis and angiogenesis are associated with each other, sharing some essential regulators. Vascular endothelial growth Factor (VEGF) is involved in both angiogenesis and osteogenesis. Transcriptional regulation of VEGF expression is not well known in osteoblasts. In this study, quantitative real-time RT-PCR results revealed that VEGF expression was down-regulated in Osx-null calvarial cells and that osteoblast marker osteocalcin expression was absent. Overexpression of Osx in stable C2C12 mesenchymal cells using a Tet-off system resulted in up-regulation of both osteocalcin and VEGF expression. The inhibition of Osx by siRNA led to repression of VEGF expression in osteoblasts. These results suggest that Osx controls VEGF expression. Transfection assays demonstrated that Osx activated VEGF promoter activity. A series of VEGF promoter deletion mutants were examined and the minimal Osx-responsive region was defined to the proximal 140-bp region of the VEGF promoter. Additional point mutants were used to identify two GC-rich regions that were responsible for VEGF promoter activation by Osx. Gel shift assay showed that Osx bound to the VEGF promoter sequence directly. Chromatin immunoprecipitation assays indicated that endogenous Osx associated with the native VEGF promoter in primary osteoblasts. Moreover, immunohistochemistry staining showed decreased VEGF protein levels in the tibiae of Osx conditional knock-out mice. We provide the first evidence that Osx controlled VEGF expression, suggesting a potential role of Osx in coordinating osteogenesis and angiogenesis.

  • osteoblast specific Transcription Factor Osterix increases vitamin d receptor gene expression in osteoblasts
    PLOS ONE, 2011
    Co-Authors: Chi Zhang, Fan Yang, Wanjin Tang, Yang Li, Diane R Dowd, Paul N Macdonald
    Abstract:

    Osterix (Osx) is an osteoblast-specific Transcription Factor required for osteoblast differentiation from mesenchymal stem cells. In Osx knock-out mice, no bone formation occurs. The vitamin D receptor (VDR) is a member of the nuclear hormone receptor superfamily that regulates target gene Transcription to ensure appropriate control of calcium homeostasis and bone development. Here, we provide several lines of evidence that show that the VDR gene is a target for Transcriptional regulation by Osx in osteoblasts. For example, calvaria obtained from Osx-null embryos displayed dramatic reductions in VDR expression compared to wild-type calvaria. Stable overexpression of Osx stimulated VDR expression in C2C12 mesenchymal cells. Inhibition of Osx expression by siRNA led to downregulation of VDR. In contrast, Osx levels remained unchanged in osteoblasts in VDR-null mice. Mechanistic approaches using transient transfection assays showed that Osx directly activated a 1 kb fragment of the VDR promoter in a dose-dependent manner. To define the region of the VDR promoter that was responsive to Osx, a series of VDR promoter deletion mutants were examined and the minimal Osx-responsive region was refined to the proximal 120 bp of the VDR promoter. Additional point mutants were used to identify two GC-rich regions that were responsible for VDR promoter activation by Osx. Chromatin immunoprecipitation assays demonstrated that endogenous Osx was associated with the native VDR promoter in primary osteoblasts in vivo. Cumulatively, these data strongly support a direct regulatory role for Osx in VDR gene expression. They further provide new insight into potential mechanisms and pathways that Osx controls in osteoblasts and during the process of osteoblastic cell differentiation.

Wanjin Tang - One of the best experts on this subject based on the ideXlab platform.

  • matrix metalloproteinase 13 mmp13 is a direct target of osteoblast specific Transcription Factor Osterix osx in osteoblasts
    PLOS ONE, 2012
    Co-Authors: Chi Zhang, Wanjin Tang, Yang Li
    Abstract:

    Osterix (Osx) is an osteoblast-specific Transcription Factor required for bone formation and osteoblast differentiation from mesenchymal stem cells. In Osx-null mice, no bone formation occurs. Matrix metalloproteinase 13 (MMP13) is a member of the matrix metalloproteinase family and plays an important role in endochondral ossification and bone remodeling. Transcriptional regulation of MMP13 expression in osteoblasts is not well understood. Here, we provide several lines of evidence which show that MMP13 is a direct target of Osx in osteoblasts. Calvaria obtained from Osx-null embryos displayed dramatic reductions in MMP13 expression compared to wild-type calvaria. Stable overexpression of Osx stimulated MMP13 expression in C2C12 mesenchymal cells. Inhibition of Osx expression by siRNA led to downregulation of MMP13 expression. Mechanistic approaches using transient transfection assays showed that Osx directly activated a 1 kb fragment of the MMP13 promoter in a dose-dependent manner. To define the region of the MMP13 promoter that was responsive to Osx, a series of MMP13 promoter deletion mutants were examined and the minimal Osx-responsive region was refined to the proximal 80 bp of the MMP13 promoter. Additional point mutant analysis was used to identify one GC-rich region that was responsible for MMP13 promoter activation by Osx. Gel Shift Assay showed that Osx bound to MMP13 promoter sequence directly. Chromatin immunoprecipitation assays demonstrated that endogenous Osx was associated with the native MMP13 promoter in primary osteoblasts in vivo. Taken together, these data strongly support a direct regulatory role for Osx in MMP13 gene expression in osteoblasts. They further provide new insight into potential mechanisms and pathways that Osx controls bone formation.

  • osteoblast specific Transcription Factor Osterix osx and hif 1α cooperatively regulate gene expression of vascular endothelial growth Factor vegf
    Biochemical and Biophysical Research Communications, 2012
    Co-Authors: Dafu Chen, Yang Li, Wanjin Tang, Wei Tian, Chi Zhang
    Abstract:

    Abstract Bone formation is a highly regulated process involving the differentiation of mesenchymal stem cells to osteoblasts. Angiogenesis and osteogenesis are tightly coupled during bone formation. Vascular endothelial growth Factor (VEGF) is involved in both processes. Relatively little is known about VEGF gene regulation in osteoblasts. Osterix (Osx) is a bone morphogenetic protein 2 (BMP-2) inducible osteoblast-specific Transcription Factor required for osteoblast differentiation and bone formation. Our recent study has demonstrated that Osx controls VEGF expression in osteoblasts. Here, we further characterized Osx regulation of VEGF. To address which domain of Osx is responsible for VEGF regulation, the deletion mutant analysis and transfection assay were carried out to show that proline-rich region (PRR) is required for Osx activation of VEGF promoter activity. Hypoxia-inducible Factor-1α (HIF-1α) has been reported to couple angiogenesis to osteogenesis, and to upregulate VEGF. Effect of Osx on HIF-1α expression was examined in this study. Quantitative RT-PCR results revealed that HIF-1α remained unchanged between wild type and Osx knockout calvaria at E18.5 in mouse embryos. Overexpression of Osx in stable C2C12 mesenchymal cells using Tet-off system did not affect HIF-1α expression. HIF-1α level did not change after Osx inhibition by siRNA in osteoblasts. Moreover, BMP-2 stimulation led to upregulation of Osx and VEGF, but not HIF-1α. These results demonstrate that HIF-1α is not a downstream target of Osx in osteoblasts, suggesting that Osx regulation of VEGF is independent of HIF-1α expression level. Interestingly, synergistic interplays were observed between Osx and HIF-1α in VEGF promoter activation in transfection assay. Our findings indicate that Osx and HIF-1α cooperatively regulate VEGF expression.

  • Transcriptional regulation of vascular endothelial growth Factor vegf by osteoblast specific Transcription Factor Osterix osx in osteoblasts
    Journal of Biological Chemistry, 2012
    Co-Authors: Wanjin Tang, Benoit De Crombrugghe, Fan Yang, Yang Li, Hongli Jiao, Guozhi Xiao, Chi Zhang
    Abstract:

    Abstract Osterix (Osx) is an osteoblast-specific Transcription Factor required for bone formation and osteoblast differentiation. The critical step in bone formation is to replace the avascular cartilage template with vascularized bone. Osteogenesis and angiogenesis are associated with each other, sharing some essential regulators. Vascular endothelial growth Factor (VEGF) is involved in both angiogenesis and osteogenesis. Transcriptional regulation of VEGF expression is not well known in osteoblasts. In this study, quantitative real-time RT-PCR results revealed that VEGF expression was down-regulated in Osx-null calvarial cells and that osteoblast marker osteocalcin expression was absent. Overexpression of Osx in stable C2C12 mesenchymal cells using a Tet-off system resulted in up-regulation of both osteocalcin and VEGF expression. The inhibition of Osx by siRNA led to repression of VEGF expression in osteoblasts. These results suggest that Osx controls VEGF expression. Transfection assays demonstrated that Osx activated VEGF promoter activity. A series of VEGF promoter deletion mutants were examined and the minimal Osx-responsive region was defined to the proximal 140-bp region of the VEGF promoter. Additional point mutants were used to identify two GC-rich regions that were responsible for VEGF promoter activation by Osx. Gel shift assay showed that Osx bound to the VEGF promoter sequence directly. Chromatin immunoprecipitation assays indicated that endogenous Osx associated with the native VEGF promoter in primary osteoblasts. Moreover, immunohistochemistry staining showed decreased VEGF protein levels in the tibiae of Osx conditional knock-out mice. We provide the first evidence that Osx controlled VEGF expression, suggesting a potential role of Osx in coordinating osteogenesis and angiogenesis.

  • osteoblast specific Transcription Factor Osterix increases vitamin d receptor gene expression in osteoblasts
    PLOS ONE, 2011
    Co-Authors: Chi Zhang, Fan Yang, Wanjin Tang, Yang Li, Diane R Dowd, Paul N Macdonald
    Abstract:

    Osterix (Osx) is an osteoblast-specific Transcription Factor required for osteoblast differentiation from mesenchymal stem cells. In Osx knock-out mice, no bone formation occurs. The vitamin D receptor (VDR) is a member of the nuclear hormone receptor superfamily that regulates target gene Transcription to ensure appropriate control of calcium homeostasis and bone development. Here, we provide several lines of evidence that show that the VDR gene is a target for Transcriptional regulation by Osx in osteoblasts. For example, calvaria obtained from Osx-null embryos displayed dramatic reductions in VDR expression compared to wild-type calvaria. Stable overexpression of Osx stimulated VDR expression in C2C12 mesenchymal cells. Inhibition of Osx expression by siRNA led to downregulation of VDR. In contrast, Osx levels remained unchanged in osteoblasts in VDR-null mice. Mechanistic approaches using transient transfection assays showed that Osx directly activated a 1 kb fragment of the VDR promoter in a dose-dependent manner. To define the region of the VDR promoter that was responsive to Osx, a series of VDR promoter deletion mutants were examined and the minimal Osx-responsive region was refined to the proximal 120 bp of the VDR promoter. Additional point mutants were used to identify two GC-rich regions that were responsible for VDR promoter activation by Osx. Chromatin immunoprecipitation assays demonstrated that endogenous Osx was associated with the native VDR promoter in primary osteoblasts in vivo. Cumulatively, these data strongly support a direct regulatory role for Osx in VDR gene expression. They further provide new insight into potential mechanisms and pathways that Osx controls in osteoblasts and during the process of osteoblastic cell differentiation.

  • osteoblast specific Transcription Factor Osterix osx is an upstream regulator of satb2 during bone formation
    Journal of Biological Chemistry, 2011
    Co-Authors: Wanjin Tang, Yang Li, Lindsey Osimiri, Chi Zhang
    Abstract:

    Osterix (Osx) is an osteoblast-specific Transcription Factor essential for osteoblast differentiation and bone formation. Osx knock-out mice lack bone completely. Satb2 is critical for osteoblast differentiation as a special AT-rich binding Transcription Factor. It is not known how Satb2 is Transcriptionally regulated during bone formation. In this study, quantitative real-time RT-PCR results demonstrated that Satb2 was down-regulated in Osx-null calvaria. In stable C2C12 mesenchymal cells using the tetracycline (Tet)-Off system, overexpression of Osx stimulated Satb2 expression. Moreover, inhibition of Osx by siRNA led to repression of Satb2 expression in osteoblasts. These results suggest that Osx controls Satb2 expression. Transient transfection assay showed that Osx activated 1kb Satb2 promoter reporter activity in a dose-dependent manner. To define the region of Satb2 promoter responsive to Osx activation, a series of deletion mutants of Satb2 constructs were made, and the minimal region was narrowed down to the proximal 130 bp of the Satb2 promoter. Further point mutation studies found that two GC-rich region mutations disrupted the Satb2 130bp promoter activation by Osx, suggesting that these GC-rich binding sites were responsible for Satb2 activation by Osx. Gel shift assay showed that Osx bound to the Satb2 promoter sequence directly. ChIP assays indicated that endogenous Osx associated with the native Satb2 promoter in osteoblasts. Importantly, Satb2 siRNA significantly inhibited Osx-induced osteoblast marker gene expressions. Taken together, our findings indicate that Osx is an upstream regulator of Satb2 during bone formation. This reveals a new additional link of the Transcriptional regulation mechanism that Osx controls bone formation.

Krishna M Sinha - One of the best experts on this subject based on the ideXlab platform.

  • Mesenchymal deletion of histone demethylase NO66 in mice promotes bone formation
    Journal of Bone and Mineral Research, 2015
    Co-Authors: Qin Chen, Krishna M Sinha, Hideyo Yasuda, Jian Min Deng, Richard R Behringer, Ralf Krahe, Benoit De Crombrugghe
    Abstract:

    Our previous studies indicated that the Jumonji C (JmjC)-domain-containing NO66 is a histone demethylase with specificity for methylated histone H3K4 and H3K36. NO66 binds to the Transcription Factor Osterix (Osx) and inhibits its Transcriptional activity in promoter assays. However, the physiological role of NO66 in formation of mammalian bones is unknown. Here, using a genetically engineered mouse model, we show that during early skeletal development, Prx1-Cre dependent mesenchymal deletion of NO66 promotes osteogenesis and formation of both endochondral as well as intramembranous skeletal elements, leading to a larger skeleton and a high bone mass phenotype in adult mice. The excess bone formation in mice where NO66 was deleted in cells of mesenchymal origin is associated with an increase in the number of preosteoblasts and osteoblasts. Further analysis revealed that in the embryonic limbs and adult calvaria of mice with deletion of NO66 in cells of mesenchymal origin, expression of several genes including bone morphogenetic protein 2, insulin-like growth Factor 1 and osteoclast inhibitor osteoprotegerin was increased, concurrent with an increase in expression of bone formation markers such as Osx, type I collagen and bone sialoprotein. Taken together, our results provide the first in vivo evidence that NO66 histone demethylase plays an important role in mammalian osteogenesis during early development as well as in adult bone homeostasis. We postulate that NO66 regulates bone formation, at least in part, via regulating the number of bone-forming cells and expression of multiple genes that are critical for these processes.

  • structural insights into histone demethylase no66 in interaction with osteoblast specific Transcription Factor Osterix and gene repression
    Journal of Biological Chemistry, 2013
    Co-Authors: Krishna M Sinha, Benoit De Crombrugghe, Minhao Wu, Xing Zhou, Bin Hu, Jianye Zang
    Abstract:

    Abstract Osterix (Osx) is an osteoblast-specific Transcriptional Factor and is required for osteoblast differentiation and bone formation. A JmjC domain-containing protein NO66 was previously found to participate in regulation of Osx Transcriptional activity and plays an important role in osteoblast differentiation through interaction with Osx. Here, we report the crystal structure of NO66 forming in a functional tetramer. A hinge domain links the N-terminal JmjC domain and C-terminal winged helix-turn-helix domain of NO66, and both domains are essential for tetrameric assembly. The oligomerization interface of NO66 interacts with a conserved fragment of Osx. We show that the hinge domain-dependent oligomerization of NO66 is essential for inhibition of Osx-dependent gene activation. Our findings suggest that homo-oligomerization of JmjC domain containing proteins might play a physiological role through interactions with other regulatory Factors during gene expression.

  • identification and characterization of micrornas controlled by the osteoblast specific Transcription Factor Osterix
    PLOS ONE, 2013
    Co-Authors: Qin Chen, Krishna M Sinha, Hideyo Yasuda, Benoit De Crombrugghe
    Abstract:

    Osterix (Osx) is an osteoblast-specific Transcription Factor which is essential for bone formation. MicroRNAs (miRNAs) have been previously shown to be involved in osteogenesis. However, it is unclear whether Osx is involved in the regulation of miRNA expression. In this study, we have identified groups of miRNAs that are differentially expressed in calvaria of the E18.5 Osx−/− embryos compared to wild type embryos. The correlation between the levels of miRNAs and Osx expression was further verified in cultured M-Osx cells in which over-expression of Osx is inducible. Our results suggest that Osx down-regulates expression of a group of miRNAs including mir-133a and -204/211, but up-regulates expression of another group of miRNAs such as mir-141/200a. Mir-133a and -204/211 are known to target the master osteogenic Transcription Factor Runx2. Further assays suggest that Sost, which encodes the Wnt signaling antagonist Sclerostin, and alkaline phosphatase (ALP) are two additional targets of mir-204/211. Mir-141/200a has been known to target the Transcription Factor Dlx5. Thus, we postulate that during the process of Osx-controlled osteogenesis, Osx has the ability to coordinately modulate Runx2, Sclerostin, ALP and Dlx5 proteins at levels appropriate for optimal osteoblast differentiation and function, at least in part, through regulation of specific miRNAs. Our study shows a tight correlation between Osx and the miRNAs involved in bone formation, and provides new information about molecular mechanisms of Osx-controlled osteogenesis.

  • Multiple functions of Osterix are required for bone growth and homeostasis in postnatal mice
    Proceedings of the National Academy of Sciences of the United States of America, 2010
    Co-Authors: Xin Zhou, Krishna M Sinha, Zhaoping Zhang, Jian Q. Feng, Vladmir M. Dusevich, Hua Zhang, Bryant G. Darnay, Benoit De Crombrugghe
    Abstract:

    The Transcription Factor Osterix (Osx) is required for osteoblast differentiation and bone formation during embryonic development, but it is not known whether Osx has an essential function in postnatal bone growth and in bone homeostasis. Conditional deletion of Osx at several time points postnatally revealed that Osx was essential for osteoblast differentiation and new bone formation in growing and adult bones. Additionally, inactivation of Osx in bones severely disrupted the maturation, morphology, and function of osteocytes. These findings identify Osx as having an essential role in the cell-specific genetic program of osteocytes. Interestingly, Osx inactivation also led to the massive accumulation of unresorbed calcified cartilage in a large area below the growth plate of endochondral bones. This specific area was also marked by an unanticipated almost complete lack of bone marrow cells and a marked decrease in the density and size of osteoclasts. This diminished density of osteoclasts could contribute to the lack of resorption of mineralized cartilage. In addition, we speculate that the abnormally accumulated, mainly naked cartilage represents an unfavorable substrate for osteoclasts. Our study identifies Osx as an essential multifunctional player in postnatal bone growth and homeostasis.

  • regulation of the osteoblast specific Transcription Factor Osterix by no66 a jumonji family histone demethylase
    The EMBO Journal, 2010
    Co-Authors: Krishna M Sinha, Hideyo Yasuda, Madelene M Coombes, Sharon Y R Dent, Benoit De Crombrugghe
    Abstract:

    Osterix (Osx) is an osteoblast-specific Transcription Factor required for osteoblast differentiation and bone formation. Osx null mice develop a normal cartilage skeleton but fail to form bone and to express osteoblast-specific marker genes. To better understand the control of Transcriptional regulation by Osx, we identified Osx-interacting proteins using proteomics approaches. Here, we report that a Jumonji C (JmjC)-domain containing protein, called NO66, directly interacts with Osx and inhibits Osx-mediated promoter activation. The knockdown of NO66 in preosteoblast cells triggered accelerated osteoblast differentiation and mineralization, and markedly stimulated the expression of Osx target genes. A JmjC-dependent histone demethylase activity was exhibited by NO66, which was specific for both H3K4me and H3K36me in vitro and in vivo, and this activity was needed for the regulation of osteoblast-specific promoters. During BMP-2-induced differentiation of preosteoblasts, decreased NO66 occupancy correlates with increased Osx occupancy at Osx-target promoters. Our results indicate that interactions between NO66 and Osx regulate Osx-target genes in osteoblasts by modulating histone methylation states.