Chen W, Woodruff T K, Mayo K E
Department of Biochemistry, Center for Reproductive Science, Northwestern University, Evanston, Illinois 60208, USA.
Endocrinology. 2000 Mar;141(3):1263-72. doi: 10.1210/endo.141.3.7361.
A balance between cell proliferation and apoptosis is important for regulating normal liver function. Proteins of the transforming growth factor-beta superfamily are known to be important mediators of apoptosis in the liver. In this study we demonstrate that activin A potently induces apoptotic cell death in a hepatoma cell line, HepG2 cells. To determine the roles of activin receptors and downstream signaling proteins in activin A-induced apoptosis in these cells, the activin signaling pathway was analyzed using the transcription of an activin-responsive reporter gene, p3TP-Lux, as an assay. Although individual activin receptors had little effect on transcriptional activity, coexpression of an activin type I receptor and a type II receptor significantly increased both basal and activin-induced transcriptional activation, with the combination ofreceptors IB and IIB being the most potent. Similarly, expression of individual Smad proteins had only a modest effect on reporter gene activity, but the combination of Smad2 and Smad4 strongly stimulated transcription. Activin signaling induced a rapid relocation of Smad2 to the nucleus, as determined using a green fluorescence protein-Smad2 fusion protein. In contrast, green fluorescence protein-Smad4 remained localized to the cytoplasm unless it was coexpressed with Smad2. In agreement with the transcriptional response assays, overexpression or suppression of activin signaling components in HepG2 cells altered apoptosis. Overexpression of receptors IB and IIB or Smad proteins 2 and 4 stimulated apoptosis, whereas dominant negative mutant forms of the activin type IIB receptor or Smad2 blocked activin-stimulated apoptosis. These studies suggest that signaling from the cell surface to the nucleus through Smad proteins is a required component of the activin A-induced cell death process in liver cells.
细胞增殖与凋亡之间的平衡对于调节正常肝功能至关重要。已知转化生长因子-β超家族的蛋白质是肝脏中凋亡的重要介质。在本研究中，我们证明激活素A能有效诱导肝癌细胞系HepG2细胞发生凋亡性细胞死亡。为了确定激活素受体和下游信号蛋白在激活素A诱导这些细胞凋亡中的作用，我们使用激活素反应性报告基因p3TP-Lux的转录作为检测方法来分析激活素信号通路。尽管单个激活素受体对转录活性影响很小，但激活素I型受体和II型受体的共表达显著增加了基础转录激活和激活素诱导的转录激活，其中受体IB和IIB的组合最为有效。同样，单个Smad蛋白的表达对报告基因活性只有适度影响，但Smad2和Smad4的组合强烈刺激转录。使用绿色荧光蛋白-Smad2融合蛋白测定发现，激活素信号诱导Smad2迅速重新定位到细胞核。相反，绿色荧光蛋白-Smad4仍定位于细胞质，除非它与Smad2共表达。与转录反应检测结果一致，HepG2细胞中激活素信号成分的过表达或抑制改变了细胞凋亡。受体IB和IIB或Smad蛋白2和4的过表达刺激了细胞凋亡，而激活素IIB型受体或Smad2的显性负突变形式则阻断了激活素刺激的细胞凋亡。这些研究表明，从细胞表面通过Smad蛋白向细胞核的信号传导是激活素A诱导肝细胞死亡过程的必要组成部分。
