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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

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