Genetic studies have revealed a critical role of Distal-homeobox (Dlx) genes in bone formation,and our previous study showed that Dlx2 overexpressing in neural crest cells leads to profound abnormalities of the cranio...Genetic studies have revealed a critical role of Distal-homeobox (Dlx) genes in bone formation,and our previous study showed that Dlx2 overexpressing in neural crest cells leads to profound abnormalities of the craniofacial tissues.The aim of this study was to investigate the role and the underlying molecular mechanisms of Dlx2 in osteogenic differentiation of mouse bone marrow stromal cells (BMSCs) and pre-osteoblast MC3T3-E1 cells.Initially,we observed upregulation of Dlx2 during the early osteogenesis in BMSCs and MC3T3-E1 cells.Moreover,Dlx2 overexpression enhanced alkaline phosphatase (ALP) activity and extracellular matrix mineralization in BMSCs and MC3T3-E1 cell line.In addition,micro-CT of implanted tissues in nude mice confirmed that Dlx2 overexpression in BMSCs promoted bone formation in vivo.Unexpectedly,Dlx2 overexpression had little impact on the expression level of the pivotal osteogenic transcription factors Runx2,Dlx5,Msx2,and Osterix,but led to upregulation of Alp and Osteocalcin (OCN),both of which play critical roles in promoting osteoblast maturation.Importantly,luciferase analysis showed that Dlx2 overexpression stimulated both OCN and Alp promoter activity.Through chromatin-immunoprecipitation assay and site-directed mutagenesis analysis,we provide molecular evidence that Dlx2 transactivates OCN and Alp expression by directly binding to the Dlx2-response cis-acting elements in the promoter of the two genes.Based on these findings,we demonstrate that Dlx2 overexpression enhances osteogenic differentiation in vitro and accelerates bone formation in vivo via direct upregulation of the OCN and Alp gene,suggesting that Dlx2 plays a crucial role in osteogenic differentiation and bone formation.展开更多
Eucommia ulmoides Oliv.(EuO),also known as Duzhong,native to China,has been reported to have antioxidative function,but its cellular mechanism is not fully examined yet.We investigated inhibitory effects of EuO leaf e...Eucommia ulmoides Oliv.(EuO),also known as Duzhong,native to China,has been reported to have antioxidative function,but its cellular mechanism is not fully examined yet.We investigated inhibitory effects of EuO leaf ethanol extracts on H2O2-induced apoptosis in rat osteoblastic MC3T3-E1 cells and underlying mechanisms.Locally-grown Duzhong leaves were extracted with ethanol.MC3T3-E1 cells were treated with EuO (6.25,12.5,25,50,and 100 μg/ml) for 24 h,and then H2O2 (800 μmol/L) for an additional 24 h.Cell survival rate,percentage of apoptosis,and expressions of caspases 3,6,7,and 9 were examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay,microscopic analysis,Western blotting,and reverse transcription polymerase chain reaction (RT-PCR).The final EuO leaf ethanol extract powder was detected to contain caffeotannic acid at 58 mg/g and geniposide at 3.45 mg/g by high performance liquid chromatography (HPLC).EuO remarkably restrained cell oxidative damage and increased cell survival rate in a dose-dependent manner: 0 μg/ml,0.21;6.25 μg/ml,0.28;12.5 μg/ml,0.31;25 μg/ml,0.48;50 μg/ml,0.54;and 100 μg/ml,0.66 (P<0.05),with the half-effective concentration being around 25 μg/ml.MTT results were confirmed by microscopic analysis.Western blotting and RT-PCR analyses showed that the expressions of caspases 3,6,7,and 9 were significantly decreased in the EuO-treated cells compared with the control (EuOand H2O2-free) (P<0.05),with the half-effective concentration of EuO ranging from 12.5 to 25 μg/ml.We conclude that the ethanol-extracted EuO leaf extracts promoted the growth of MC3T3-E1 cells,and suppressed the H2O2-induced apoptosis in a rat MC3T3-E1 osteogenic cell model,likely due to the inhibition of caspases’ activities.The results indicate that EuO is a potent antioxidant,which may contribute to its many cellular protective functions,including the promotion of bone growth.展开更多
基金supported by grant (81771036) from National Natural Science Foundation of China (to S.G.S.)grant (81741028) from National Natural Science Foundation of China (to J.D.)grant (17410710500) Shanghai International Scientific and Technological Cooperation Projects Laser Micromachine and Vascularization of TCP/PCL Scaffolds (to W.Z.)
文摘Genetic studies have revealed a critical role of Distal-homeobox (Dlx) genes in bone formation,and our previous study showed that Dlx2 overexpressing in neural crest cells leads to profound abnormalities of the craniofacial tissues.The aim of this study was to investigate the role and the underlying molecular mechanisms of Dlx2 in osteogenic differentiation of mouse bone marrow stromal cells (BMSCs) and pre-osteoblast MC3T3-E1 cells.Initially,we observed upregulation of Dlx2 during the early osteogenesis in BMSCs and MC3T3-E1 cells.Moreover,Dlx2 overexpression enhanced alkaline phosphatase (ALP) activity and extracellular matrix mineralization in BMSCs and MC3T3-E1 cell line.In addition,micro-CT of implanted tissues in nude mice confirmed that Dlx2 overexpression in BMSCs promoted bone formation in vivo.Unexpectedly,Dlx2 overexpression had little impact on the expression level of the pivotal osteogenic transcription factors Runx2,Dlx5,Msx2,and Osterix,but led to upregulation of Alp and Osteocalcin (OCN),both of which play critical roles in promoting osteoblast maturation.Importantly,luciferase analysis showed that Dlx2 overexpression stimulated both OCN and Alp promoter activity.Through chromatin-immunoprecipitation assay and site-directed mutagenesis analysis,we provide molecular evidence that Dlx2 transactivates OCN and Alp expression by directly binding to the Dlx2-response cis-acting elements in the promoter of the two genes.Based on these findings,we demonstrate that Dlx2 overexpression enhances osteogenic differentiation in vitro and accelerates bone formation in vivo via direct upregulation of the OCN and Alp gene,suggesting that Dlx2 plays a crucial role in osteogenic differentiation and bone formation.
基金Project (No.2007C33030) supported by the Science and Technology Program of Zhejiang Province,China
文摘Eucommia ulmoides Oliv.(EuO),also known as Duzhong,native to China,has been reported to have antioxidative function,but its cellular mechanism is not fully examined yet.We investigated inhibitory effects of EuO leaf ethanol extracts on H2O2-induced apoptosis in rat osteoblastic MC3T3-E1 cells and underlying mechanisms.Locally-grown Duzhong leaves were extracted with ethanol.MC3T3-E1 cells were treated with EuO (6.25,12.5,25,50,and 100 μg/ml) for 24 h,and then H2O2 (800 μmol/L) for an additional 24 h.Cell survival rate,percentage of apoptosis,and expressions of caspases 3,6,7,and 9 were examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay,microscopic analysis,Western blotting,and reverse transcription polymerase chain reaction (RT-PCR).The final EuO leaf ethanol extract powder was detected to contain caffeotannic acid at 58 mg/g and geniposide at 3.45 mg/g by high performance liquid chromatography (HPLC).EuO remarkably restrained cell oxidative damage and increased cell survival rate in a dose-dependent manner: 0 μg/ml,0.21;6.25 μg/ml,0.28;12.5 μg/ml,0.31;25 μg/ml,0.48;50 μg/ml,0.54;and 100 μg/ml,0.66 (P<0.05),with the half-effective concentration being around 25 μg/ml.MTT results were confirmed by microscopic analysis.Western blotting and RT-PCR analyses showed that the expressions of caspases 3,6,7,and 9 were significantly decreased in the EuO-treated cells compared with the control (EuOand H2O2-free) (P<0.05),with the half-effective concentration of EuO ranging from 12.5 to 25 μg/ml.We conclude that the ethanol-extracted EuO leaf extracts promoted the growth of MC3T3-E1 cells,and suppressed the H2O2-induced apoptosis in a rat MC3T3-E1 osteogenic cell model,likely due to the inhibition of caspases’ activities.The results indicate that EuO is a potent antioxidant,which may contribute to its many cellular protective functions,including the promotion of bone growth.
