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

Transforming growth factor-13 1 (TGF-131) 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-i (MKLi), stimulates a-smooth muscle actin (a-SMA) transcription in primary cultures of renal tubular epithelial cells (RTC), which convert into myofibroblasts on treatment with TGF-βl. Herein,we study the effect of MKLI expression on α-SMA in these cells. We demonstrate that TGF-β1 stimulation of α-SMA transcription is mediated through CC(AIF)6-rich GO elements known to bind to SRF. These elements also mediate the MKL1 effect that dramatically activates α-SMA transcription in serum-free media. MKL1 fused to green fluorescent protein localizes to the nucleus and induces α-SMA expression regardless of treatment with TGF-βI. Using proteasome inhibitors, we also demonstrate that the proteolytic ubiquitin pathway regulates MKL1 expression. These data indicate that MKLI overexpression is sufficient to induce a-SMA expression. Inhibition of endogenous expression of MKL1 by small interfering RNA abolishes TGF-131 stimulation of a-SMA expression. Therefore, MKL1 is also absolutely required for TGF-131 stimulation of α-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 MKLI to the a-SMA promoter in chromatin. Since MKL1 constitutes a potent factor regulating α-SMA expression, modulation of endogenous MKLI expression or activity may have a profound effect on myofibroblast formation and function in the kidney. [ABSTRACT FROM AUTHOR]