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...

Autosomal-dominant polycystic kidney disease (ADPKD) is characterized by formation of cysts from tubular epithelial cells. Previous studies indicate that secretion of prostaglandin E2 (PGE2) into cyst fluid and production of cAMP underlie cyst expansion. However, the mechanism by which PGE2 directly stimulates cAMP formation and modulates cystogenesis is still unclear, because the particular E-prostanoid (EP) receptor mediating the PGE2 effect has not been characterized. Our goal is to define the PGE2 receptor subtype involved in ADPKD. We used a three-dimensional cell-culture system of human epithelial cells from normal and ADPKD kidneys in primary cultures to demonstrate that PGE2 induces cyst formation. Biochemical evidence gathered by using real-time RT-PCR mRNA analysis and immunodetection indicate the presence of EP2 receptor in cystic epithelial cells in ADPKD kidney. Pharmacological evidence obtained by using PGE2-selective analogs further demonstrates that EP2 mediates cAMP formation and cystogenesis. Functional evidence for a role of EP2 receptor in mediating cAMP signaling was also provided by inhibiting EP2 receptor expression with transfection of small interfering RNA in cystic epithelial cells. Our results indicate that PGE2 produced in cyst fluid binds to adjacent EP2 receptors located on the apical side of cysts and stimulates EP2 receptor expression. PGE2 binding to EP2 receptor leads to cAMP signaling and cystogenesis by a mechanism that involves protection of cystic epithelial cells from apoptosis. The role of EP2 receptor in mediating the PGE2 effect on stimulating cyst formation may have direct pharmacological implications for the treatment of polycystic kidney disease.

Background/Aims: Progressive renal fibrotic disease is accompanied by the massive accumulation of myofibroblasts as defined by α smooth muscle actin (αSMA) expression. We quantitated gene expression using real-time RT-PCR analysis during conversion of primary cultured human renal tubular cells (RTC) to myofibroblasts after treatment with transforming growth factor-β₁ (TGF-β₁). We report herein the limitations of commonly used reference genes for mRNA quantitation. Methods: We determined the expression of αSMA and megakaryoblastic leukemia-1 (MKL1), a transcriptional regulator of αSMA, by quantitative real-time PCR using three common internal controls, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), cyclophilin A and 18S rRNA. Results: Expression of GAPDH mRNA and cyclophilin A mRNA, and to a lesser extent, 18S rRNA levels varied over time in culture and with exposure to TGF-β₁. Thus, depending on which reference gene was used, TGF-β₁ appeared to have different effects on expression of MKL1 and αSMA. Conclusions: RTC converting to myofibroblasts in primary culture is a valuable system to study renal fibrosis in humans. However, variability in expression of reference genes with TGF-β₁ treatment illustrates the need to validate mRNA quantitation with multiple reference genes to provide accurate interpretation of fibrosis studies in the absence of a universal internal standard for mRNA expression.

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BETA2/NeuroD, a basic helix-loop-helix transcription factor, is expressed in pancreatic endocrine cells during development and regulates insulin gene expression. We demonstrated previously that the endocrine pancreas of BETA2/NeuroD-deficient mice undergoes massive apoptosis and, consequently, animals die of diabetes shortly after birth. Here we show that a significant fraction of BETA2-deficient mice in a new genetic background can survive diabetes and live to adulthood through the process of beta-cell neogenesis. Morphometric examination indicates that pancreatic beta-, but not alpha-cell mass, was restored to a level comparable to that of wild-type animals. However, the newly formed islet cells cannot form mature islets of Langerhans, indicating an indispensable role of BETA2 in morphogenesis of normal islet structure. Furthermore, immunohistochemical examinations revealed that newly formed beta-cells of BETA2/NeuroD-deficient mice come from two sources: either directly budding from the pancreatic ductal tree or from the preexisting beta-cells in the residual endocrine pancreas. Our results indicate that beta-cell neogenesis in our BETA2/NeuroD-deficient mice contributes to their survival, and these mice may provide a useful model for studying the mechanism of beta-cell regeneration.