Increased Sp1-dependent transactivation of the LAMγ1 promoter in hepatic stellate cells co-cultured with HepG2 cells overexpressing cytochrome P450 2E1
Increased Sp1-dependent transactivation of the LAMγ1 promoter in hepatic stellate cells co-cultured with HepG2 cells overexpressing cytochrome P450 2E1
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DOI：
10.1074/jbc.m206790200
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发表时间：
2003-04-25
影响因子：
4.8
通讯作者：
Cederbaum, AI
中科院分区：
文献类型：
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作者：
Nieto, N;Cederbaum, AI
层粘连蛋白是一种基底膜蛋白，会增加肝纤维化。为了研究氧化应激对层粘连蛋白表达的作用，将肝星状细胞 (HSC) 与表达或不表达 CYP2E1（一种有效的氧自由基发生器）的 HepG2 细胞（分别为 E47 或 C34 细胞）共培养。与与 C34 细胞共孵育相比，HSC 与 E47 细胞共孵育增加了层粘连蛋白 beta1 和 gamma1 蛋白；抗氧化剂和 CYP2E1 抑制剂可以阻止这种增加。在与来自盐水或吡唑处理（具有高水平 CYP2E1）的大鼠的原代肝细胞共培养中观察到类似的结果。在任何系统的 HSC 中均未检测到层粘连蛋白 α1 链；然而，在与 E47 细胞共培养的 HSC 中，层粘连蛋白 α2 链增加。在 E47 共培养物中，HSC 中层粘连蛋白 beta1 和 gamma1 蛋白的合成增加，但周转率没有增加。 E47 的 HSC 中层粘连蛋白 beta1 和 gamma1 mRNA 上调。由于两个基因的转录激活而共培养。使用层粘连蛋白 gamma1 启动子驱动的报告构建体在 HSC 中进行的转染实验显示，E47 系统中的 -230/+106 和 -1400/+106 构建体具有最大响应性。凝胶位移测定表明，当与 E47 细胞共孵育时，Sp1 与 HSC 中层粘连蛋白 gamma1 启动子的结合增加，但可被抗 Sp1 抗体阻断。 Sp1 表达载体的共转染进一步增加了 HSC/E47 系统中 -330LAMgamma1-CAT 报告载体在 HSC 中的响应性。这些结果表明，可扩散的 CYP2E1 衍生的氧化应激介质通过 HSC 中的转录机制诱导层粘连蛋白的合成。肝细胞和造血干细胞之间的这种相互作用在肝纤维化过程中可能很重要。
Laminin is a basement-membrane protein that increases in liver fibrosis. To study the role of oxidative stress on laminin expression, hepatic stellate cells (HSC) were co-cultured with HepG2 cells that do or do not express (E47 or C34 cells, respectively) CYP2E1, a potent generator of oxygen radicals. Co-incubation of HSC with E47 cells increased laminin beta1 and gamma1 proteins compared with co-incubation with C34 cells; this increase was prevented by antioxidants and CYP2E1 inhibitors. Similar results were observed in co-culture with primary hepatocytes from saline- or pyrazole-treated (with high levels of CYP2E1) rats. Laminin alpha1 chain was not detectable in the HSC in any of the systems; however, laminin alpha2 chain increased in HSC co-cultured with E47 cells. Synthesis but not turnover of laminin beta1 and gamma1 proteins was increased in HSC in the E47 co-culture. Laminin beta1 and gamma1 mRNAs were up-regulated in HSC in the E47. co-culture because of transcriptional activation of both genes. Transfection experiments in HSC with reporter constructs driven by the laminin gamma1 promoter showed maximal responsiveness with the -230/+106 and the -1400/+106 constructs in the E47 system. Gelshift assays demonstrated an increase in Sp1 binding to the laminin gamma1 promoter in HSC when co-incubated with E47 cells, which was blocked by an anti-Sp1 antibody. Co-transfection of a Sp1 expression vector further increased the responsiveness of the -330LAMgamma1-CAT reporter vector in HSC in the HSC/E47 system. These results show that diffusable CYP2E1-derived oxidative-stress mediators induce synthesis of laminins by a transcriptional mechanism in HSC. Such interactions between hepatocytes and HSC may be important during liver fibrosis.