TGF-β1通过RUNX2调控人肝星状细胞系LX－2中骨桥蛋白表达

目的：探讨人肝星状细胞系LX-2中促纤维化因子TGF-β1对骨桥蛋白（Osteopontin,OPN）的转录调控作用,研究其潜在关系及作用通路。方法：经重组因子TGF-β1刺激后,westernblot检测人肝星状细胞系LX-2中OPN和RUNX2的表达水平。通过生物信息学分析软件预测RUNX2在OPN启动子序列上的结合位点。构建基于pGL3-Basic的人OPN启动子载体和相应截短体,与RUNX2共转染HEK293细胞,结合双荧光素酶报告基因实验分析RUNX2对OPN启动子的转录调控作用。结果：经TGF－61刺激后,LX-2细胞系中OPN与RUNX2的蛋白表达水平均升高,提示二者均为TGF-81下游效应分子。TESS生物信息学分析显示OPN启动子序列-78-73存在RUNX2的结合位点ACCACA。通过双荧光素酶报告基因实验结果显示：RUNX2对OPN启动予具有正向调控作用,且通过截短删除之前预测的结合位点后,该调控作用消失。结论：TGF-β1可促进人肝星状细胞系中OPN的表达,该作用部分通过其下游转录因子RUNX2作用于OPN启动子区域的ACCACA序列而实现。     

Intracellular redox equilibrium is essential for the constitutive expression of AP-1 dependent genes in resting cells: studies on TGF-β1 regulation

The mechanisms involved in the continuous expression of constitutive genes are unclear. We hypothesize that steady state intracellular reactive oxygen species (ROS), which their levels are tightly maintained, could be regulating the expression of these constitutive genes in resting cells. We analyzed the regulation of an important constitutive gene, TGF-β1, after decreasing intracellular ROS concentration in human mesangial cells. Decreased intracellular hydrogen peroxide by catalase addition reduced TGF-β1 protein, mRNA expression and promoter activity. Furthermore, catalase decreased the basal activity of Activated Protein-1 (AP-1) that regulates TGF-β1 promoter activity. This effect disappeared when AP-1 binding site was removed. Similar results were observed with another protein containing AP-1 binding sites in its promoter, such as eNOS, but it was not the case in other constitutive genes without any AP-1 binding site, as COX1 or PKG1. The pharmacological inhibition of the different ROS synthesis sources by blocking NADPH oxidase, the mitochondrial respiratory chain or xanthine oxidase, or the use of human fibroblasts with genetically deficient mitochondrial activity, induced a similar, significant reduction of steady state ROS concentration as the one observed with catalase. Moreover, there was decreased TGF-β1 expression in all the cases excepting the xanthine oxidase blockade. These findings suggest a novel role for the steady state intracellular ROS concentration, where the compartmentalized, different systems involved in the intracellular ROS production, could be essential for the expression of constitutive AP1-dependent genes, as TGF-β1.