ABSTRACT
OBJECTIVE: Cardiovascular effects of angiotensin II are primarily mediated via the angiotensin II type 1 receptor (AT1R). Krüppel-like factor 4 (KLF4), a transcription factor that binds to the transforming growth factor (TGF)-ß control element (TCE), regulates a variety of receptor expression in vascular smooth muscle cells. In the present study, we investigated the mechanisms of TGF-ß-mediated KLF4 regulation of AT1R expression. METHODS AND RESULTS: Coimmunoprecipitation, chromatin immunoprecipitation, and luciferase assays were performed, with the results suggesting that Sp1 forms a complex with KLF4 bound to the TCE of the AT1R promoter and cooperatively activates AT1R transcription in vascular smooth muscle cells under basal conditions. On activation of TGF-ß1 signaling, Sp1 is dissociated from the KLF4-Sp1 complex through PKC-δ-mediated KLF4 phosphorylation at Thr401, downregulating AT1R expression. Simultaneously, TGF-ß1 facilitates KLF4-PPAR-γ complex formation and its binding to the TCE of the AT1R promoter through Smad-mediated KLF4 phosphorylation at Ser470, subsequently leading to inhibition of AT1R transcription. CONCLUSIONS: KLF4 functions as a protein platform that is able to bind to the TCE of the AT1R promoter. On activation of TGF-ß signaling, KLF4 mediates Sp1 dissociation from, and PPAR-γ association with, the AT1R promoter, leading to downregulation of AT1R expression in VSMCs.
Subject(s)
Kruppel-Like Transcription Factors/metabolism , Muscle, Smooth, Vascular/enzymology , Myocytes, Smooth Muscle/enzymology , PPAR gamma/metabolism , Protein Kinase C-delta/metabolism , Receptor, Angiotensin, Type 1/metabolism , Smad Proteins/metabolism , Sp1 Transcription Factor/metabolism , Transforming Growth Factor beta1/metabolism , Animals , Binding Sites , Cell Proliferation , Chromatin Immunoprecipitation , Down-Regulation , Genes, Reporter , HEK293 Cells , Humans , Immunoprecipitation , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , Male , Phosphorylation , Promoter Regions, Genetic , Protein Kinase C-delta/genetics , RNA Interference , RNA, Messenger/metabolism , Rats , Rats, Sprague-Dawley , Receptor, Angiotensin, Type 1/genetics , Serine , Sp1 Transcription Factor/genetics , Time Factors , Transcriptional Activation , TransfectionABSTRACT
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.
