MKL1 mediates TGF- (cid:1) 1-induced (cid:2) -smooth muscle actin expression in human renal epithelial cells

MA. mediates TGF- (cid:1) 1-induced (cid:2) -smooth muscle actin expression in human renal cells. Am growth factor- 1 (TGF- (cid:1) 1) is known to induce epithelial-mesenchymal transition in the kidney, a process involved in tubulointerstitial ﬁbrosis. We hypoth-esized that a coactivator of the serum response factor (SRF), megakaryoblastic leukemia factor-1 (MKL1), stimulates (cid:2) -smooth muscle actin ( (cid:2) -SMA) transcription in primary cultures of renal tubular epithelial cells (RTC), which convert into myoﬁbroblasts on treatment with TGF- (cid:1) 1. Herein, we study the effect of MKL1 expression on (cid:2) -SMA in these cells. We demonstrate that TGF- (cid:1) 1 stimulation of (cid:2) -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 (cid:2) -SMA transcription in serum-free media. MKL1 fused to green ﬂuorescent protein localizes to the nucleus and induces (cid:2) -SMA expression regardless of treatment with TGF- (cid:1) 1. Using proteasome inhibitors, we also demonstrate that the proteolytic ubiquitin pathway regulates MKL1 expression. These data indicate that MKL1 overexpression is sufﬁcient to induce (cid:2) -SMA expression. Inhibition of endogenous expression of MKL1 by small interfering RNA abolishes TGF- (cid:1) 1 stimulation of (cid:2) -SMA expression. Therefore, MKL1 is also absolutely required for TGF- (cid:1) 1 stimulation of (cid:2) -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- (cid:1) 1 induces binding of endogenous SRF and MKL1 to the (cid:2) -SMA promoter in chromatin. Since MKL1 constitutes a potent factor regulating (cid:2) -SMA expression, modulation of endogenous MKL1 expression or activity may have a profound effect on myoﬁbroblast formation and function in the kidney. (cid:6) g/ml transferrin, 6.25 ng/ml selenous acid (Biowhittaker, Walkersville, MD), and 10% (vol/vol) heat-inactivated FBS (Atlanta Biologicals, Norcross, GA), penicillin, and streptomycin in a 37°C-5% CO 2 incubator. Cell cultures were split 1:3 or 1:4 when conﬂuent using a 0.05% Trypsin-EDTA solution (Invitrogen) in T75 ﬂasks. RTC were used for experiments between passages 3 and 5 at 80–100% conﬂuence in six-well polystyrene tissue culture plates. EMT was induced by incubation in MEM (cid:2) medium (Invitrogen) supplemented with 10 ng/ml recombinant hu- man TGF- (cid:1) 1 (R&D Systems, Minneapolis, MN), which was changed every 2–3 days (9). Inhibitors E64, MG132, Z-LLF, and chloroquine were purchased from Sigma. Our study protocols were approved by the Institutional Review Board of The University of Oklahoma Health Sciences Center. Chromatin immunoprecipitation assay. Chromatin immunoprecipitation (CHIP) assay was performed using ChIP-IT kit from Active Motive (Carlsbad, CA) and analyzed using real-time PCR. Brieﬂy, cells treated with or without TGF- (cid:1) 1 for 3 days were ﬁxed with 1% formaldehyde and homogenized using a Dounce homogenizer. After centrifugation, the nuclear pellet was sheared by enzymatic digestion for 15 min to (cid:7) 200-bp DNA fragments and submitted to immunoprecipitation. We used rabbit polyclonal antibody against MKL1 and SRF (Santa Cruz Biotechnology), rabbit IgG (rIgG as negative control, Jackson), and mouse monoclonal RNA pol II antibody and mouse IgG (mIgG) that were provided with the kit as positive and negative controls, respectively. Antibodies and control IgGs were used at a concentration of 2 (cid:6) g/reaction. Recovered and input DNA was quantiﬁed by real-time PCR to amplify the proximal CArG-containing region and upstream distal region (used as negative control) of the (cid:2) -SMA promoter. The primers were designed using PrimerExpress Software (Applied Biosystems) as follows: (cid:3)