Runx1 Is a Co-activator with FOXO3 to Mediate Transforming Growth Factor β (TGFβ)-induced Bim Transcription in Hepatic Cells

Transforming growth factor beta (TGF beta) regulates essential cellular functions such as cellular proliferation, differentiation, and apoptosis. The Bcl-2 family of proteins has been implicated as mediators of TGF beta-induced apoptosis. We demonstrated previously that TGF beta induces the expression of Bim (Bcl-2-interacting mediator of cell death), a member of the BH3-only family of proapoptotic Bcl-2 proteins, to induce cell death in B-lymphocytes. Here, we investigated the mechanism of TGF beta-mediated Bim expression in two hepatocyte cell lines that undergo apoptosis with TGF beta, AML-12 and Hep3B. We show that TGF beta induces Bimprotein 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 TGF beta, and this induction was confirmed at the mRNA and protein levels. Interestingly, TGF beta 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 apoptosis. 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 TGF beta-induced Bim expression. Our results support a mechanism in which TGF beta stimulates Bim transcription by up-regulating Runx1 expression, which binds FOXO3, and the two cooperate in the transcriptional induction of Bim.