Elberg G, Chen L, Elberg D, Chan MD, Logan CJ, Turman MA. MKL1 mediates TGF-␤1-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 factor-␤1 (TGF-␤1) 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-␤1. Herein, we study the effect of MKL1 expression on ␣-SMA in these cells. We demonstrate that TGF-␤1 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-␤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 ␣-SMA expression. Inhibition of endogenous expression of MKL1 by small interfering RNA abolishes TGF-␤1 stimulation of ␣-SMA expression. Therefore, MKL1 is also absolutely required for TGF-␤1 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-␤1 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 RENAL FIBROSIS IS A COMMON feature of various kidney diseases leading to end-stage renal failure (15,44). This process is characterized by the accumulation of myofibroblasts defined by the expression of ␣-smooth muscle actin (␣-SMA). These cells are major contributors to the increased extracellular matrix deposition seen in kidney fibrosis (16,69). A number of studies demonstrate that renal tubular cells (RTC) can convert to myofibroblasts on epithelial-mesenchymal transition (EMT) stimulated by transforming growth factor-␤ (TGF-␤) (9,11,24,45,69).The regulation of ␣-SMA transcription has been extensively studied in smooth muscle cells and in cells from the myocardium and skeletal muscle, which express ␣-SMA in adults and embryos, respectively (66). Studies on the ␣-SMA promoter from chickens, rats, mice, and humans highlight the importance of cell context and species differences for ␣-SMA transcriptional regulat...

In previous studies, tungstate and molybdate were found to mimic the biological actions of insulin. It was suggested that these metallooxides initially inhibit vanadate-sensitive protein phosphotyrosine phosphatase (PTPase). This, in turn, stimulates a staurosporine-sensitive cytosolic protein tyrosine kinase (cytPTK), which activates several insulin bioeffects via insulin-independent pathways (Shisheva & Shechter, 1991, 1993; Elberg et al., 1994). Tungstate and molybdate, however, facilitate bioeffects in rat adipocytes only at high (millimolar) concentrations (Goto et al., 1992). We report here that incubations of tungstate or molybdate with hydrogen peroxide (H2O2) result in the formation of pertungstate (pW, peroxide of tungstate) or permolybdate (pMo, peroxide of molybdate). Pertungstate and permolybdate were found to stimulate all or most of the insulin bioeffects in rat adipocytes. Moreover, these permetallooxides are 80-180-fold more potent stimulators than the corresponding metallooxides. This shift in potency resembles that of pervanadate relative to vanadate in stimulating the same effect in rat adipocytes (Fantus et al., 1989). pW and pMo are also active in normalizing blood glucose levels in streptozotocin-induced diabetic rats. Further studies aimed at understanding the higher efficacy of this permetallooxide revealed the following: (a) All three permetallooxides (pV, pW, pMo) are oxidizing agents relative to reduced glutathione (GSH). They oxidize stoichiometric amounts of GSH to GSSG. (b) All three metallooxides do not oxidize GSH to GSSG. (c) Both metallooxides and permetallooxides inhibit rat adipocytic PTPase at micromolar quantities (IC50 = 3-10 microM). Permetallooxides, however, inhibited a larger PTPase fraction (80-100%) compared to metallooxides (40-70% of the total).(ABSTRACT TRUNCATED AT 250 WORDS)

Both exogenously added vanadate (oxidation state +5) and vanadyl (oxidation state +4) mimic the rapid responses of insulin through alternative signaling pathways, not involving insulin receptor activation [reviewed in Shechter et al. (1995) Mol. Cell. Biochem. 153, 39-47]. Vanadium exhibits complex chemistry, fluctuating between vanadate(+5) and vanadyl(+4), according to the prevailing conditions. Using several experimental approaches, we report here on a distinct vanadate(+5)-independent, vanadyl(+4)-dependent activating pathway. The key components of this pathway are membrane protein phosphotyrosine phosphatases (PTPases) and a cytosolic (nonreceptor) protein-tyrosine kinase (CytPTK). We further suggest that vanadate(+5) is not reduced rapidly to vanadyl(+4) inside the cell, and entered vanadyl sulfate(+4) is capable of undergoing spontaneous oxidation to vanadate(+5) in vivo. Finally, we show that the promotion and full expression of a downstream bioeffect such as lipogenesis requires both activation of CytPTK and prolonged stability of vanadyl(+4) against oxidation.

Peroxisome proliferator-activated receptor gamma (PPARgamma) and CCAAT/enhancer-binding proteins (C/EBPs) are transcriptional regulators essential for adipocyte differentiation and function. Previous findings indicate that PPARgamma2 transcription is regulated by members of the C/EBP family. We demonstrate here that C/EBPalpha and C/EBPdelta, but not C/EBPbeta, induce the activity of the PPARgamma2 promoter in transiently transfected 3T3-L1 preadipocytes and bind to two juxtaposed low affinity C/EBP binding sites. Results obtained with chimeras containing interchanged C/EBPalpha-C/EBPbeta N-terminal transactivation domain and C-terminal DNA binding dimerization domain indicate that the N-terminal part of C/EBPbeta prevents it from binding to the PPARgamma2 promoter. Indeed, deletion mutants of C/EBPbeta lacking the N-terminal part of the molecule are able to bind to the PPARgamma2 promoter. We further demonstrate that deletion of a region located between amino acids 184-212, upstream of the DNA binding domain, permits C/EBPbeta binding to the PPARgamma2 promoter, implicating an inhibitory region in C/EBPbeta for modulating DNA binding specificity to the PPARgamma2 promoter. In summary, this study indicates that C/EBPbeta but not C/EBPalpha or C/EBPdelta is unable to bind to C/EBP binding sites in the mouse PPARgamma2 promoter. The lack of binding is due to a region N-terminal of the C/EBPbeta DNA binding domain. Our findings illustrate a mechanism by which C/EBP isoforms differentially modulate the transactivation of the PPARgamma2 promoter.