MKL1 mediates TGF- 1-induced -smooth muscle actin expression in human renal epithelial cells

Elberg G, Chen L, Elberg D, Chan MD, Logan CJ, Turman MA. MKL1 mediates TGF1-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 factor1 (TGF1) 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 TGF1. Herein, we study the effect of MKL1 expression on -SMA in these cells. We demonstrate that TGF1 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 TGF1. 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 TGF1 stimulation of -SMA expression. Therefore, MKL1 is also absolutely required for TGF1 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 TGF1 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.