MKL1 mediates TGF-β1-induced α-smooth muscle actin expression in human renal epithelial cells

Abstract

Elberg G, Chen L, Elberg D, Chan MD, Logan CJ, Turman MA. MKL1 mediates TGF-␤1-induced ␣-smooth muscle actin expression in human renal epithelial cells. Am J Physiol Renal Physiol 294: F1116-F1128, 2008. First published March 12, 2008 doi:10.1152/ajprenal.00142.2007.-Transforming growth factor-␤1 (TGF-␤1) is known to induce epithelial-mesenchymal transition in the kidney, a process involved in tubulointerstitial fibrosis. We hypothesized that a coactivator of the serum response factor (SRF), megakaryoblastic leukemia factor-1 (MKL1), stimulates ␣-smooth muscle actin (␣-SMA) transcription in primary cultures of renal tubular epithelial cells (RTC), which convert into myofibroblasts on treatment with TGF-␤1. Herein, we study the effect of MKL1 expression on ␣-SMA in these cells. We demonstrate that TGF-␤1 stimulation of ␣-SMA transcription is mediated through CC(A/T) 6-rich GG elements known to bind to SRF. These elements also mediate the MKL1 effect that dramatically activates ␣-SMA transcription in serum-free media. MKL1 fused to green fluorescent protein localizes to the nucleus and induces ␣-SMA expression regardless of treatment with TGF-␤1. Using proteasome inhibitors, we also demonstrate that the proteolytic ubiquitin pathway regulates MKL1 expression. These data indicate that MKL1 overexpression is sufficient to induce ␣-SMA expression. Inhibition of endogenous expression of MKL1 by small interfering RNA abolishes TGF-␤1 stimulation of ␣-SMA expression. Therefore, MKL1 is also absolutely required for TGF-␤1 stimulation of ␣-SMA expression. Western blot and immunofluorescence analysis show that overexpressed and endogenous MKL1 are located in the nucleus in non-stimulated RTC. Chromatin immunoprecipitation assay demonstrates that TGF-␤1 induces binding of endogenous SRF and MKL1 to the ␣-SMA promoter in chromatin. Since MKL1 constitutes a potent factor regulating ␣-SMA expression, modulation of endogenous MKL1 expression or activity may have a profound effect on myofibroblast formation and function in the kidney.epithelial-mesenchymal transition; myocardin; ubiquitin; transcription; myofibroblast RENAL FIBROSIS IS A COMMON feature of various kidney diseases leading to end-stage renal failure (15,44). This process is characterized by the accumulation of myofibroblasts defined by the expression of ␣-smooth muscle actin (␣-SMA). These cells are major contributors to the increased extracellular matrix deposition seen in kidney fibrosis (16,69). A number of studies demonstrate that renal tubular cells (RTC) can convert to myofibroblasts on epithelial-mesenchymal transition (EMT) stimulated by transforming growth factor-␤ (TGF-␤) (9,11,24,45,69).The regulation of ␣-SMA transcription has been extensively studied in smooth muscle cells and in cells from the myocardium and skeletal muscle, which express ␣-SMA in adults and embryos, respectively (66). Studies on the ␣-SMA promoter from chickens, rats, mice, and humans highlight the importance of cell context and species differences for ␣-SMA transcriptional regulat...

Activation of MRTF-A–dependent gene expression with a small molecule promotes myofibroblast differentiation and wound healing

“…Levels of MRTF-A were significantly higher in isoxazole-treated fibroblasts, which led to a greater proportion of cells displaying nuclear accumulation of MRTF-A. A previous study reported regulation of MRTF-A protein stability by the proteasome degradation pathway, suggesting the possibility that isoxazole might inhibit the ubiquitin-proteasome system (19).…”

“…Situations of stress that locally alter actin dynamics and promote F-actin polymerization promote nuclear accumulation of MRTFs (18)(19)(20)(21)(22)(23)(24). SRF/MRTF complexes bind to consensus CArG elements [CC(A/T) 6 GG] within the promoters of contractile and SMC-specific target genes, most notably smooth muscle α-actin (SMA, ACTA2) and transgelin (TAGLN, SM22) (25).…”

RhoA/ROCK Signaling Regulates TGFβ-Induced Epithelial-Mesenchymal Transition of Lens Epithelial Cells through MRTF-A

“…G-actin binds to the RPEL motifs on MRTF-A and, when bound, G-actin has been proposed to inhibit nuclear import, enhance nuclear export, and repress transcription of MRTF-A target genes (56). Thus far, work in proximal tubular epithelial cells has been pivotal in determining the involvement of MRTF-A subcellular localization in the context of TGFβ-induced EMT (34,37,57). Morita et al (2007) demonstrated that the presence of constitutively active MRTF-A was sufficient to induce expression of αSMA, in contrast to dominant negative MRTF-A, the pathophysiology of fibrosis of the lens and other tissues.…”

MKL1-184C>T gene polymorphism is associated with coronary artery disease in the Chinese Han population

“…Also, approximately 40% of MKL1 null mice embryos suffered from lethal cardiac cell necrosis with mitochondrial dysfunction (Parmacek, 2007). In addition, it has been reported that the expression of MKL1 in the smooth muscle cells may be associated with RhoA and TGF-beta-dependent channels, and that these two channels have an important role in the process of atherosclerosis (Elberg et al, 2008). Hence, we hypothesized that MKL1 may also play an important role in coronary atherosclerosis.…”

“…Human MKL1 gene is located on chromosome 22q13 (Ma et al, 2001). According to reports, MKL1 may have effects on the formation of the cardiovascular system and maintenance processes (Cen et al., 2004) through the regulation of abnormal proliferation of smooth muscle cells (Sata et al, 2002;Selvaraj and Prywes, 2003;Du et al, 2004;Parmacek, 2007;Kihara et al, 2008), as well as through RhoA and TGF-beta-dependent channels (Elberg et al, 2008) participating in coronary heart disease and its development. Hinohara et al (2009) first discovered the -184C> T polymorphism of MKL1 involved in the pathogenesis of coronary heart disease of Japanese and Koreans.…”