Krüppel-like Factor 4 Promotes Differentiation by Transforming Growth Factor-β Receptor-mediated Smad and p38 MAPK Signaling in Vascular Smooth Muscle Cells

KLF4 (Krüppel-like factor 4) has been implicated in vascular smooth muscle cell (VSMC) differentiation induced by transforming growth factor beta (TGF-beta). However, the role of KLF4 and mechanism of KLF4 actions in regulating TGF-beta signaling in VSMCs remain unclear. In this study, we showed that TGF-beta1 inhibited cell cycle progression and induced differentiation in cultured rat VSMCs. This activity of TGF-beta1 was accompanied by up-regulation of KLF4, with concomitant increase in TbetaRI (TGF-beta type I receptor) expression. KLF4 was found to transduce TGF-beta1 signals via phosphorylation-mediated activation of Smad2, Smad3, and p38 MAPK. The activation of both pathways, in turn, increased the phosphorylation of KLF4, which enabled the formation of KLF4-Smad2 complex in response to TGF-beta1. Chromatin immunoprecipitation studies and oligonucleotide pull-down assays showed the direct binding of KLF4 to the KLF4-binding sites 2 and 3 of the TbetaRI promoter and the recruitment of Smad2 to the Smad-responsive region. Formation of a stable KLF4-Smad2 complex in the promoter's Smad-responsive region mediated cooperative TbetaRI promoter transcription in response to TGF-beta1. These results suggest that KLF4-dependent regulation of Smad and p38 MAPK signaling via TbetaRI requires prior phosphorylation of KLF4 through Smad and p38 MAPK pathways. This study demonstrates a novel mechanism by which TGF-beta1 regulates VSMC differentiation.