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This Article Full Text Full Text (PDF) Supplemental Figures All Versions of this Article: 294/5/F1116    most recent 00142.2007v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Elberg, G. Articles by Turman, M. A. PubMed PubMed Citation Articles by Elberg, G. Articles by Turman, M. A.

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

Section of Nephrology, Department of Pediatrics, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma

Submitted 27 March 2007 ; accepted in final form 6 March 2008

Transforming growth factor-β1 (TGF-β1) 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-β1. Herein, we study the effect of MKL1 expression on -SMA in these cells. We demonstrate that TGF-β1 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-β1. 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-β1 stimulation of -SMA expression. Therefore, MKL1 is also absolutely required for TGF-β1 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-β1 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