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

Abstract

KLF4 (Krüppel-like factor 4) has been implicated in vascular smooth muscle cell (VSMC) differentiation induced by transforming growth factor ␤ (TGF-␤). However, the role of KLF4 and mechanism of KLF4 actions in regulating TGF-␤ signaling in VSMCs remain unclear. In this study, we showed that TGF-␤1 inhibited cell cycle progression and induced differentiation in cultured rat VSMCs. This activity of TGF-␤1 was accompanied by up-regulation of KLF4, with concomitant increase in T␤RI (TGF-␤ type I receptor) expression. KLF4 was found to transduce TGF-␤1 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-␤1. Chromatin immunoprecipitation studies and oligonucleotide pull-down assays showed the direct binding of KLF4 to the KLF4-binding sites 2 and 3 of the T␤RI 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 T␤RI promoter transcription in response to TGF-␤1. These results suggest that KLF4-dependent regulation of Smad and p38 MAPK signaling via T␤RI requires prior phosphorylation of KLF4 through Smad and p38 MAPK pathways. This study demonstrates a novel mechanism by which TGF-␤1 regulates VSMC differentiation. Proliferation of vascular smooth muscle cells (VSMCs)2 plays a key role in the pathogenesis of a variety of proliferative vascular diseases, such as atherosclerosis, restenosis, and hypertension (1, 2). Several lines of evidence have shown that the factors controlling VSMC proliferation and growth inhibition, including various growth factors, signal transduction molecules, and transcription factors, constitute a complex functional network. Krüppel-like factors (KLFs), retinoic acid receptor-␣, platelet-derived growth factor (PDGF) receptor, and T␤RI and T␤RII (transforming growth factor-␤ type I and II receptor, respectively) are expressed in VSMCs and are components of such a network (3-8). Our recent study demonstrates that KLF4 (GKLF) inhibits proliferation by PDGF receptor-mediated but not by retinoic acid receptor-␣-mediated phosphatidylinositol 3-kinase and ERK signaling in VSMCs (4). However, the actual relationship between KLF4 and T␤R in the regulation of VSMC proliferation and growth inhibition is not fully understood.KLF4 is a member of the Sp1 transcription factor family characterized by three C2H2 zinc finger motifs, mainly involved in regulating cell growth, differentiation, proliferation, and apoptosis, by controlling the expression of a large number of genes with GC/GT-rich promoters. KLF4 has been identified in VSMCs (9) and is induced by shear stress (10, 11), transforming growth factor-␤ (TGF-␤) (12), and PDGF-BB (13). Studies from the Owens laboratory (13) suggest that KLF4 not only mediates the repressive effects of PDGF-BB on VSMC gene expression but also represses the TGF-␤1-dependent increase...

Regulation of Krüppel-like Factor 4 by the Anaphase Promoting Complex Pathway Is Involved in TGF-β Signaling

“…Although it has been demonstrated that KLF4 is regulated by various post-translational modifications, including phosphorylation, acetylation, and sumoylation, less is known if KLF4 is regulated by UPS (19,27,28,53). The present study has demonstrated that KLF4 is regulated by the proteolysis depending on ubiquitin.…”

“…In addition to transcriptional regulation, KLF4 is also known to be subjected to multiple post-translational modifications, including acetylation (27), sumoylation (28,54), and phosphorylation (53). Our findings have provided another layer to our understanding of the KLF4 regulatory circuitry, which in addition could enhance our understanding about the pivotal role of KLF4 in stem cell biology and carcinogenesis.…”

Krüppel-like factor 4 is involved in cell scattering induced by hepatocyte growth factor

“…Our discovery therefore adds HGF as a novel member of the few stimuli reported to regulate KLF4 expression , Feinberg et al, 2005Li et al, 2010;Liu et al, 2012). Additionally, it is noteworthy that we identified KLF4 as its own transcriptional activator (Fig.…”

“…For instance, the role of KLF4 as an oncogene or a tumor suppressor largely depends on the functional status of p21 CIP1/WAF1 in the cells examined (Rowland et al, 2005). Additionally, KLF4 is responsible for TGF-b1-induced differentiation of vascular smooth muscle cells (Li et al, 2010), whereas it is a negative regulator of the prostatic EMT triggered by TGF-b1 (Liu et al, 2012). More strikingly, an opposite conclusion was described even in the same cell line MDA-MB-231 regarding the role of KLF4 in cell migration and invasion (Yori et al, 2010;Yu et al, 2011).…”

Extracellular-Superoxide Dismutase Expression in COS7 Cells Exposed to Cadmium Chloride

“…It has been reported that the activation of MAPK induces the activation of transcriptional factor, krüppel like-factor, which acts as a transcriptional repressor of EC-SOD. 35,36) From these observations, we speculated that the reduction of EC-SOD might be not directly regulated by p38-MAPK, but, in part, by the activation and/or inactivation of transcriptional factors.…”