MKL1 mediates TGF-beta1-induced alpha-smooth muscle actin expression in human renal epithelial cells.
Transforming growth factor-beta1 (TGF-beta1) 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 alpha-smooth muscle actin (alpha-SMA) transcription in primary cultures of renal tubular epithelial cells (RTC), which convert into myofibroblasts on treatment with TGF-beta1. Herein, we study the effect of MKL1 expression on alpha-SMA in these cells. We demonstrate that TGF-beta1 stimulation of alpha-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 alpha-SMA transcription in serum-free media. MKL1 fused to green fluorescent protein localizes to the nucleus and induces alpha-SMA expression regardless of treatment with TGF-beta1. Using proteasome inhibitors, we also demonstrate that the proteolytic ubiquitin pathway regulates MKL1 expression. These data indicate that MKL1 overexpression is sufficient to induce alpha-SMA expression. Inhibition of endogenous expression of MKL1 by small interfering RNA abolishes TGF-beta1 stimulation of alpha-SMA expression. Therefore, MKL1 is also absolutely required for TGF-beta1 stimulation of alpha-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-beta1 induces binding of endogenous SRF and MKL1 to the alpha-SMA promoter in chromatin. Since MKL1 constitutes a potent factor regulating alpha-SMA expression, modulation of endogenous MKL1 expression or activity may have a profound effect on myofibroblast formation and function in the kidney.
Elberg
Gerard
G
Section of Nephrology, Department of Pediatrics, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma, USA . gerard-elberg@ouhsc.edu
Chen
Lijuan
L
Elberg
Dorit
D
Chan
Michael D
MD
Logan
Charlotte J
CJ
Turman
Martin A
MA
D000199:Actins / Q000096:biosynthesis*; D015153:Blotting, Western; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D004847:Epithelial Cells / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D007684:Kidney Tubules / Q000166:cytology / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009130:Muscle, Smooth / Q000378:metabolism*; D015514:Oncogene Proteins, Fusion / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D011401:Promoter Regions, Genetic / Q000235:genetics; D034741:RNA, Small Interfering / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015534:Trans-Activators; D053773:Transforming Growth Factor beta1 / Q000502:physiology*
D000199:Actins; D004268:DNA-Binding Proteins; C431575:MRTFA protein, human; D015514:Oncogene Proteins, Fusion; D034741:RNA, Small Interfering; D015534:Trans-Activators; D053773:Transforming Growth Factor beta1; D049452:Green Fluorescent Proteins; D004247:DNA