Wildey Gary M, Howe Philip H
Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
J Biol Chem. 2009 Jul 24;284(30):20227-39. doi: 10.1074/jbc.M109.027201. Epub 2009 Jun 3.
Transforming growth factor beta (TGFbeta) regulates essential cellular functions such as cellular proliferation, differentiation, and apoptosis. The Bcl-2 family of proteins has been implicated as mediators of TGFbeta-induced apoptosis. We demonstrated previously that TGFbeta induces the expression of Bim (Bcl-2-interacting mediator of cell death), a member of the BH3-only family of pro-apoptotic Bcl-2 proteins, to induce cell death in B-lymphocytes. Here, we investigated the mechanism of TGFbeta-mediated Bim expression in two hepatocyte cell lines that undergo apoptosis with TGFbeta, AML-12 and Hep3B. We show that TGFbeta induces Bim protein and mRNA levels, and its expression is sufficient to induce cell death. Gene array results revealed that Runx1, a member of the Runx family of transcription factors, was induced by TGFbeta, and this induction was confirmed at the mRNA and protein levels. Interestingly, TGFbeta specifically induced the expression of Runx1 protein from an internal ribosome entry site (IRES)-dependent, cap-independent, mRNA transcript, and its overexpression was sufficient to induce hepatocyte apo pto sis. Deletion and mutation analyses of the murine Bim promoter identified a putative forkhead binding element, at position -174 to -168 from the transcription start site, as the mediator of Runx1 induction. Co-immunoprecipitation, electrophoretic mobility shift assays, and chromatin immunoprecipitation assays demonstrated that Runx1 does not bind directly to the identified forkhead binding element but rather binds the transcriptional regulator FOXO3, which occupies this site. Finally, small interfering RNA knockdown of Runx1 or FOXO3 decreased TGFbeta-induced Bim expression. Our results support a mechanism in which TGFbeta stimulates Bim transcription by up-regulating Runx1 expression, which binds FOXO3, and the two cooperate in the transcriptional induction of Bim.
转化生长因子β（TGFβ）调节细胞增殖、分化和凋亡等重要细胞功能。Bcl-2家族蛋白被认为是TGFβ诱导凋亡的介质。我们先前证明，TGFβ诱导仅含BH3结构域的促凋亡Bcl-2蛋白家族成员Bim（细胞死亡的Bcl-2相互作用介质）的表达，从而诱导B淋巴细胞死亡。在此，我们研究了TGFβ介导的Bim在两种经TGFβ诱导发生凋亡的肝细胞系AML-12和Hep3B中的表达机制。我们发现，TGFβ诱导Bim蛋白和mRNA水平升高，其表达足以诱导细胞死亡。基因芯片结果显示，转录因子Runx家族成员Runx1受TGFβ诱导，且在mRNA和蛋白水平均得到证实。有趣的是，TGFβ特异性地从依赖内部核糖体进入位点（IRES）、不依赖帽结构的mRNA转录本诱导Runx1蛋白表达，其过表达足以诱导肝细胞凋亡。对小鼠Bim启动子的缺失和突变分析确定，转录起始位点上游-174至-168位置的一个假定叉头结合元件是Runx1诱导的介质。免疫共沉淀、电泳迁移率变动分析和染色质免疫沉淀分析表明，Runx1并不直接结合已鉴定的叉头结合元件，而是结合占据该位点的转录调节因子FOXO3。最后，Runx1或FOXO3的小干扰RNA敲低降低了TGFβ诱导的Bim表达。我们的结果支持一种机制，即TGFβ通过上调Runx1的表达来刺激Bim转录，Runx1与FOXO3结合，二者协同作用于Bim的转录诱导。
