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
Oestrogen is essential for maintaining bone mass, and it has been demonstrated to induce osteoblast proliferation and bone formation.In this study, complementary DNA（cDNA） microarrays were used to identify and study...Oestrogen is essential for maintaining bone mass, and it has been demonstrated to induce osteoblast proliferation and bone formation.In this study, complementary DNA（cDNA） microarrays were used to identify and study the expression of novel genes that may be involved in MC3T3-E1 cells’ response to 17-b estradiol. MC3T3-E1 cells were inoculated in minimum essential media alpha（a-MEM）cell culture supplemented with 17-b estradiol at different concentrations and for different time periods. MC3T3-E1 cells treated with1028mol？L2117-b estradiol for 5 days exhibited the highest proliferation and alkaline phosphatase（ALP） activity; thus, this group was chosen for microarray analysis. The harvested RNA was used for microarray hybridisation and subsequent real-time reverse transcription polymerase chain reaction（RT-PCR） to validate the expression levels for selected genes. The microarray results were analysed using both functional and pathway analysis. In this study, microarray analysis detected 5 403 differentially expressed genes,of which 1 996 genes were upregulated and 3 407 genes were downregulated, 1 553 different functional classifications were identified by gene ontology（GO） analysis and 53 different pathways were involved based on pathway analysis. Among the differentially expressed genes, a portion not previously reported to be associated with the osteoblast response to oestrogen was identified. These findings clearly demonstrate that the expression of genes related to osteoblast proliferation, cell differentiation, collagens and transforming growth factor beta（TGF-b）-related cytokines increases, while the expression of genes related to apoptosis and osteoclast differentiation decreases, following the exposure of MC3T3-E1 cells to a-MEM supplemented with 17-b estradiol. Microarray analysis with functional gene classification is critical for a complete understanding of complementary intracellular processes. This microarray analysis provides large-scale gene expression data that require further confirmatory studies.展开更多
The essential effect of vitamin A on immune function occurs through various mechanisms including direct effect on ThloTh2 balance modulation. However, it is unclear whether or not vitamin A can regulate Thl-Th2 balanc...The essential effect of vitamin A on immune function occurs through various mechanisms including direct effect on ThloTh2 balance modulation. However, it is unclear whether or not vitamin A can regulate Thl-Th2 balance under a strong Thl-polarizing condition. Therefore, the purpose of our study was to examine the effect of vitamin A metabolite allotrans retinoic acid （ATRA） on ThloTh2 differentiation in CD4~ T cells under GATA-3 deficiency, which can induce Thl-polarizing condition. In the present study, GATA-3 deficiency T cells were induced by siRNA and checked by real-time quantitative PCR and western blot. GATA-3 deficiency CD4＋ T cells and normal CD4＋ T were treated for 48 h with or without ATRA.展开更多
Objective： To investigate the role of peroxisome proliferator-activated receptors δ （PPARδ） in inflammatory reaction and its possible mechanism in adipocyte. Methods：Lentivirus-mediated RNA interference （RNAi）...Objective： To investigate the role of peroxisome proliferator-activated receptors δ （PPARδ） in inflammatory reaction and its possible mechanism in adipocyte. Methods：Lentivirus-mediated RNA interference （RNAi） was used to block the expression of PPARδ in 3T3-L1 cells. In order to induce inflammation in 3T3-L1, cells were stimulated with tumor necrosis factor-α（TNFα, 20 ng/ml） for 4 h. The expression of PPARδ, nuclear factor κB （NFκB） and C reactive protein （CRP） were determined by Western blot analysis. Results：The expression of PPARδ was reduced by 80% after RNAi. Blockage of PPARδ promoted the expression of CRP and NFκB in cells stimulated with TNFα but had no effect on normal cells. Conclusion： PPARδ is involved in inflammatory reaction in adipocyte. Blockage of PPARδ can promote the inflammation mediated by inflammatory factors and increase the expression of NFκB and CRP in 3T3-L1 cells stimulated with TNFα.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supported by grants from the Natural Science Fund (ZR2010HM035) of Shandong Provincethe Shandong Provincial Health Development Project Fund (2011WSB19002) in China
文摘Oestrogen is essential for maintaining bone mass, and it has been demonstrated to induce osteoblast proliferation and bone formation.In this study, complementary DNA（cDNA） microarrays were used to identify and study the expression of novel genes that may be involved in MC3T3-E1 cells’ response to 17-b estradiol. MC3T3-E1 cells were inoculated in minimum essential media alpha（a-MEM）cell culture supplemented with 17-b estradiol at different concentrations and for different time periods. MC3T3-E1 cells treated with1028mol？L2117-b estradiol for 5 days exhibited the highest proliferation and alkaline phosphatase（ALP） activity; thus, this group was chosen for microarray analysis. The harvested RNA was used for microarray hybridisation and subsequent real-time reverse transcription polymerase chain reaction（RT-PCR） to validate the expression levels for selected genes. The microarray results were analysed using both functional and pathway analysis. In this study, microarray analysis detected 5 403 differentially expressed genes,of which 1 996 genes were upregulated and 3 407 genes were downregulated, 1 553 different functional classifications were identified by gene ontology（GO） analysis and 53 different pathways were involved based on pathway analysis. Among the differentially expressed genes, a portion not previously reported to be associated with the osteoblast response to oestrogen was identified. These findings clearly demonstrate that the expression of genes related to osteoblast proliferation, cell differentiation, collagens and transforming growth factor beta（TGF-b）-related cytokines increases, while the expression of genes related to apoptosis and osteoclast differentiation decreases, following the exposure of MC3T3-E1 cells to a-MEM supplemented with 17-b estradiol. Microarray analysis with functional gene classification is critical for a complete understanding of complementary intracellular processes. This microarray analysis provides large-scale gene expression data that require further confirmatory studies.
基金supported by the National Natural Science Foundation of China(No.30671761)
文摘The essential effect of vitamin A on immune function occurs through various mechanisms including direct effect on ThloTh2 balance modulation. However, it is unclear whether or not vitamin A can regulate Thl-Th2 balance under a strong Thl-polarizing condition. Therefore, the purpose of our study was to examine the effect of vitamin A metabolite allotrans retinoic acid （ATRA） on ThloTh2 differentiation in CD4~ T cells under GATA-3 deficiency, which can induce Thl-polarizing condition. In the present study, GATA-3 deficiency T cells were induced by siRNA and checked by real-time quantitative PCR and western blot. GATA-3 deficiency CD4＋ T cells and normal CD4＋ T were treated for 48 h with or without ATRA.
文摘Objective： To investigate the role of peroxisome proliferator-activated receptors δ （PPARδ） in inflammatory reaction and its possible mechanism in adipocyte. Methods：Lentivirus-mediated RNA interference （RNAi） was used to block the expression of PPARδ in 3T3-L1 cells. In order to induce inflammation in 3T3-L1, cells were stimulated with tumor necrosis factor-α（TNFα, 20 ng/ml） for 4 h. The expression of PPARδ, nuclear factor κB （NFκB） and C reactive protein （CRP） were determined by Western blot analysis. Results：The expression of PPARδ was reduced by 80% after RNAi. Blockage of PPARδ promoted the expression of CRP and NFκB in cells stimulated with TNFα but had no effect on normal cells. Conclusion： PPARδ is involved in inflammatory reaction in adipocyte. Blockage of PPARδ can promote the inflammation mediated by inflammatory factors and increase the expression of NFκB and CRP in 3T3-L1 cells stimulated with TNFα.
