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Abstract：
OBJECTIVE: Multiple transcription factors (TFs) have previously been shown to control hypertrophic chondrocyte-specific mouse type X collagen gene (Col10a1) expression via interaction with Col10a1 promoters. This study aims to investigate the role and mechanism of the potential binding factor signal transduction and transcription activator 5a (Stat5a) of Col10a1 cis-enhancer, in controlling Col10a1 gene expression and chondrocyte hypertrophic differentiation.
METHODS: The potential Col10a1 regulator was predicted by the transcription factor affinity prediction (TRAP) analysis of the 150-bp Col10a1 cis enhancer. Stat5a was screened and verified by qRT-PCR, western blot and IHC analyses. Transfection of Stat5a siRNA or expression plasmid into MCT and ATDC5 cells was performed to either knockdown or over-express Stat5a and to investigate the influence of Stat5a on Col10a1 gene expression during the chondrocyte hypertrophy. Dual-luciferase reporter assay was performed to explore the mechanism of Stat5a affecting Col10a1 transcription. Alcian blue, alkaline phosphatase, and alizarin red staining, as well as qRT-PCR analyses of related marker genes were performed to investigate the effect and possible mechanism of Stat5a on chondrocyte differentiation.
RESULTS: The potential binding factor of Col10a1 cis-enhancer Stat5a and Col10a1 were both highly expressed and positively correlated within hypertrophic chondrocytes in vitro and in situ. Knockdown of Stat5a reduced Col10a1 expression, while overexpression of Stat5a enhanced Col10a1 expression in hypertrophic chondrocytes, suggesting Stat5a as a positive Col10a1 regulator. Mechanistically, Stat5a was shown to potentiate the reporter activity mediated by Col10a1 promoter/enhancer. In addition, Stat5a increased the intensity of alkaline phosphatase staining of ATDC5 cells and the expression of relevant hypertrophic marker genes including Runx2, which was consistent with the expression of Stat5a and Col10a1.
CONCLUSIONS: Our results support that Stat5a promoted Col10a1 expression and chondrocyte hypertrophic differentiation, possibly via interaction with the 150-bp Col10a1 cis-enhancer.
METHODS: The potential Col10a1 regulator was predicted by the transcription factor affinity prediction (TRAP) analysis of the 150-bp Col10a1 cis enhancer. Stat5a was screened and verified by qRT-PCR, western blot and IHC analyses. Transfection of Stat5a siRNA or expression plasmid into MCT and ATDC5 cells was performed to either knockdown or over-express Stat5a and to investigate the influence of Stat5a on Col10a1 gene expression during the chondrocyte hypertrophy. Dual-luciferase reporter assay was performed to explore the mechanism of Stat5a affecting Col10a1 transcription. Alcian blue, alkaline phosphatase, and alizarin red staining, as well as qRT-PCR analyses of related marker genes were performed to investigate the effect and possible mechanism of Stat5a on chondrocyte differentiation.
RESULTS: The potential binding factor of Col10a1 cis-enhancer Stat5a and Col10a1 were both highly expressed and positively correlated within hypertrophic chondrocytes in vitro and in situ. Knockdown of Stat5a reduced Col10a1 expression, while overexpression of Stat5a enhanced Col10a1 expression in hypertrophic chondrocytes, suggesting Stat5a as a positive Col10a1 regulator. Mechanistically, Stat5a was shown to potentiate the reporter activity mediated by Col10a1 promoter/enhancer. In addition, Stat5a increased the intensity of alkaline phosphatase staining of ATDC5 cells and the expression of relevant hypertrophic marker genes including Runx2, which was consistent with the expression of Stat5a and Col10a1.
CONCLUSIONS: Our results support that Stat5a promoted Col10a1 expression and chondrocyte hypertrophic differentiation, possibly via interaction with the 150-bp Col10a1 cis-enhancer.
摘要：
目的：先前已显示多种转录因子（TF）通过与Col10a1启动子的相互作用来控制肥大性软骨细胞特异性小鼠X型胶原基因（Col10a1）的表达。本研究旨在探讨Col10a1顺式增强子的潜在结合因子信号转导和转录激活因子5a(Stat5a)的作用和机制,控制Col10a1基因表达和软骨细胞肥大分化。
方法：通过150-bpCol10a1顺式增强子的转录因子亲和力预测（TRAP）分析来预测潜在的Col10a1调节因子。通过qRT-PCR筛选和验证Stat5a，蛋白质印迹和IHC分析。将Stat5asiRNA或表达质粒转染到MCT和ATDC5细胞中以敲低或过表达Stat5a，并研究Stat5a在软骨细胞肥大期间对Col10a1基因表达的影响。通过双荧光素酶报告基因实验探讨Stat5a影响Col10a1转录的机制。阿尔辛蓝，碱性磷酸酶,和茜素红染色，同时对相关标记基因进行了qRT-PCR分析，以研究Stat5a对软骨细胞分化的影响和可能的机制。
结果：Col10a1顺式增强子Stat5a和Col10a1的潜在结合因子在体外和原位肥大软骨细胞中均高表达并呈正相关。敲除Stat5a降低Col10a1表达，而Stat5a的过表达增强了Col10a1在肥大软骨细胞中的表达，建议Stat5a为Col10a1阳性调节剂。机械上,Stat5a显示增强由Col10a1启动子/增强子介导的报告活性。此外,Stat5a增加了ATDC5细胞碱性磷酸酶染色的强度和包括Runx2在内的相关肥大标记基因的表达，与Stat5a和Col10a1的表达一致。
结论：我们的结果支持Stat5a促进Col10a1表达和软骨细胞肥大分化，可能通过与150bpCol10a1顺式增强子相互作用。
方法：通过150-bpCol10a1顺式增强子的转录因子亲和力预测（TRAP）分析来预测潜在的Col10a1调节因子。通过qRT-PCR筛选和验证Stat5a，蛋白质印迹和IHC分析。将Stat5asiRNA或表达质粒转染到MCT和ATDC5细胞中以敲低或过表达Stat5a，并研究Stat5a在软骨细胞肥大期间对Col10a1基因表达的影响。通过双荧光素酶报告基因实验探讨Stat5a影响Col10a1转录的机制。阿尔辛蓝，碱性磷酸酶,和茜素红染色，同时对相关标记基因进行了qRT-PCR分析，以研究Stat5a对软骨细胞分化的影响和可能的机制。
结果：Col10a1顺式增强子Stat5a和Col10a1的潜在结合因子在体外和原位肥大软骨细胞中均高表达并呈正相关。敲除Stat5a降低Col10a1表达，而Stat5a的过表达增强了Col10a1在肥大软骨细胞中的表达，建议Stat5a为Col10a1阳性调节剂。机械上,Stat5a显示增强由Col10a1启动子/增强子介导的报告活性。此外,Stat5a增加了ATDC5细胞碱性磷酸酶染色的强度和包括Runx2在内的相关肥大标记基因的表达，与Stat5a和Col10a1的表达一致。
结论：我们的结果支持Stat5a促进Col10a1表达和软骨细胞肥大分化，可能通过与150bpCol10a1顺式增强子相互作用。
