Synthesis and deposition of extracellular matrix (ECM) within the glomerulus and interstitium characterizes renal fibrosis, but the mechanisms underlying this process are incompletely understood. The profibrotic cytokine TGF-b1 modulates the expression of certain microRNAs (miRNAs), suggesting that miRNAs may have a role in the pathogenesis of renal fibrosis. Here, we exposed proximal tubular cells, primary mesangial cells, and podocytes to TGF-b1 to examine its effect on miRNAs and subsequent collagen synthesis. TGF-b1 reduced expression of the miR-29a/b/c/family, which targets collagen gene expression, and increased expression of ECM proteins. In both resting and TGF-b1-treated cells, ectopic expression of miR-29 repressed the expression of collagens I and IV at both the mRNA and protein levels by targeting the 39untranslated region of these genes. Furthermore, we observed low levels of miR-29 in three models of renal fibrosis representing early and advanced stages of disease. Administration of the Rho-associated kinase inhibitor fasudil prevented renal fibrosis and restored expression of miR-29. Taken together, these data suggest that TGF-b1 inhibits expression of the miR-29 family, thereby promoting expression of ECM components. Pharmacologic modulation of these miRNAs may have therapeutic potential for progressive renal fibrosis.

OBJECTIVEProgressive fibrosis in the diabetic kidney is driven and sustained by a diverse range of profibrotic factors. This study examines the critical role of microRNAs (miRNAs) in the regulation of the key fibrotic mediators, TGF-β1 and TGF-β2.RESEARCH DESIGN AND METHODSRat proximal-tubular epithelial cells (NRK52E) were treated with TGF-β1 and TGF-β2 for 3 days, and expression of markers of epithelial-to-mesenchymal transition (EMT) and fibrogenesis were assessed by RT-PCR and Western blotting. The expression of miR-141 and miR-200a was also assessed, as was their role as translational repressors of TGF-β signaling. Finally, these pathways were explored in two different mouse models, representing early and advanced diabetic nephropathy.RESULTSBoth TGF-β1 and TGF-β2 induced EMT and fibrogenesis in NRK52E cells. TGF-β1 and TGF-β2 also downregulated expression of miR-200a. The importance of these changes was demonstrated by the finding that ectopic expression miR-200a downregulated smad-3 activity and the expression of matrix proteins and prevented TGF-β–dependent EMT. miR-200a also downregulated the expression of TGF-β2, via direct interaction with the 3′ untranslated region of TGF-β2. The renal expression of miR-141 and miR-200a was also reduced in mouse models representing early and advanced kidney disease.CONCLUSIONSmiR-200a and miR-141 significantly impact on the development and progression of TGF-β–dependent EMT and fibrosis in vitro and in vivo. These miRNAs appear to be intricately involved in fibrogenesis, both as downstream mediators of TGF-β signaling and as components of feedback regulation, and as such represent important new targets for the prevention of progressive kidney disease in the context of diabetes.

During embryonic development, progenitor cells arising from the somitic mesoderm commit to a program of differentiation that facilitates the formation of skeletal muscle fibers (termed myogenesis). Under the control of a number of extracellular cues, these myogenic precursors adhere to an orchestrated process of mobilization, proliferation, differentiation, and fusion to create the multi-nucleated myotubes that ultimately become mature fibers (1-3). Many of the most important early changes in gene expression that direct the muscle cell lineage are driven by a family of basic helix-loop-helix transcription factors that includes MyoD, myogenin, MRF4, and Myf5, which are therefore commonly referred to as the muscle regulatory factors (MRFs) 2 (4 -6).TGF-␤ is well characterized as a potent inhibitor of muscle cell differentiation that acts by repressing the transcriptional activity of MRFs (7-9). Interaction between extracellular TGF-␤ and its membrane-bound receptor complex engages a cascade of intracellular signal transduction that promotes nuclear retention of Smad proteins 2 and 3 in complex with Smad4, which subsequently activates or represses hundreds of TGF-␤ target genes (10). Of particular relevance to skeletal muscle cell differentiation, the TGF-␤ signaling protein Smad3 has been shown to physically interact with MRFs in a manner that can inhibit differentiation (9). microRNAs (or miRs) are single-stranded 21-22-nucleotide noncoding RNAs that are capable of controlling gene expression at a post-transcriptional level by stalling the translation of the cognate mRNA or promoting its degradation in a process referred to as RNA interference (RNAi). Here, individual miRs that have been loaded into a specialized collection of interacting proteins referred to as the RNA-induced silencing complex identify and bind to highly specific sequences featuring within exons or the 3Ј-untranslated regions of target mRNAs. The degree of pairing complementarity between a microRNA and its target (as well as target location in the transcript) determine whether translation is subsequently repressed or the transcript is degraded (11,12). In skeletal muscle, specific miRs are increasingly being implicated as key regulators of differentiation, because of their predicted selectivity for genes that are involved in facilitating the myogenic program. Chief among these microRNAs are the so-called "myomiRs," or muscle-enriched microRNAs (including miR-1/206, -133a, and -133b) that are themselves transcribed as targets of MRF activity. As an example, increased miR-206 levels promote myogenic differentiation in vitro (13,14) and in vivo (15, 16), whereas inhibiting miR-206 appears capable of delaying or even preventing myogenic differentiation. Ongoing examination is establishing that additional miRs that are expressed in a variety of cell types including but not exclusive to skeletal muscle may also influence the events of differentiation. For instance, the miR-29 family regulates myogenesis by targeting proteins within the NF-B-YY1 sig...

OBJECTIVEIncreased deposition of extracellular matrix (ECM) within the kidney is driven by profibrotic mediators including transforming growth factor-β (TGF-β) and connective tissue growth factor (CTGF). We investigated whether some of their effects may be mediated through changes in expression of certain microRNAs (miRNAs).RESEARCH DESIGN AND METHODSProximal tubular cells, primary rat mesangial cells, and human podocytes were analyzed for changes in the expression of key genes, ECM proteins, and miRNA after exposure to TGF-β (1–10 ng/μl). Tubular cells were also infected with CTGF-adenovirus. Kidneys from diabetic apoE mice were also analyzed for changes in gene expression and miRNA levels.RESULTSTGF-β treatment was associated with morphologic and phenotypic changes typical of epithelial-mesenchymal transition (EMT) including increased fibrogenesis in all renal cell types and decreased E-cadherin expression in tubular cells. TGF-β treatment also modulated the expression of certain miRNAs, including decreased expression of miR-192/215 in tubular cells, mesangial cells, which are also decreased in diabetic kidney. Ectopic expression of miR-192/215 increased E-cadherin levels via repressed translation of ZEB2 mRNA, in the presence and absence of TGF-β, as demonstrated by a ZEB2 3′-untranslated region luciferase reporter assay. However, ectopic expression of miR-192/215 did not affect the expression of matrix proteins or their induction by TGF-β. In contrast, CTGF increased miR-192/215 levels, causing a decrease in ZEB2, and consequently increased E-cadherin mRNA.CONCLUSIONSThese data demonstrate the linking role of miRNA-192/215 and ZEB2 in TGF-β/CTGF–mediated changes in E-cadherin expression. These changes appear to occur independently of augmentation of matrix protein synthesis, suggesting that a multistep EMT program is not necessary for fibrogenesis to occur.

Epithelial-to-mesenchymal transition (EMT) of tubular cells contributes to the renal accumulation of matrix protein that is associated with diabetic nephropathy. Both TGF-␤1 and advanced glycation end products (AGE) are able to induce EMT in cell culture. This study examined the role of the prosclerotic growth factor connective tissue growth factor (CTGF) as a downstream mediator of these processes. EMT was assessed by the expression of ␣-smooth muscle actin, vimentin, E-cadherin, and matrix proteins and the induction of a myofibroblastic phenotype. CTGF, delivered in an adenovirus or as recombinant human CTGF (250 ng/ml), was shown to induce a partial EMT. This was not blocked by neutralizing anti-TGF-␤1 antibodies, suggesting that this action was TGF-␤1 independent. NRK-52E cells that were exposed to AGE-modified BSA (AGE-BSA; 40 M) or TGF-␤1 (10 ng/ml) also underwent EMT. This was associated with the induction of CTGF gene and protein expression. Transfection with siRNA to CTGF was able to attenuate EMT-associated phenotypic changes after treatment with AGE or TGF-␤1. These in vitro effects correlate with the in vivo finding of increased CTGF expression in the diabetic kidney, which co-localizes on the tubular epithelium with sites of EMT. In addition, inhibition of AGE accumulation was able to reduce CTGF expression and attenuate renal fibrosis in experimental diabetes. These findings suggest that CTGF represents an important independent mediator of tubular EMT, downstream of the actions of AGE or TGF-␤1. This interaction is likely to play an important role in progressive diabetic nephropathy and strengthens the rationale to consider CTGF as a potential target for the treatment of diabetic nephropathy.

No abstract

It is clear that the well-described phenomenon of epithelial-mesenchymal transition (EMT) plays a pivotal role in embryonic development, wound healing, tissue regeneration, organ fibrosis and cancer progression. EMTs have been classified into three subtypes based on the functional consequences and biomarker context in which they are encountered. This review will highlight findings on type II EMT as a direct contributor to the kidney myofibroblast population in the development of renal fibrosis, specifically in diabetic nephropathy, the signalling molecules and the pathways involved in type II EMT and changes in the expression of specific miRNA with the EMT process. These findings have provided new insights into the activation and development of EMT during disease processes and may lead to possible therapeutic interventions to suppress EMTs and potentially reverse organ fibrosis.

Overexpression of the human multidrug resistance gene 1 (MDR1) is a negative prognostic factor in leukemia. Despite intense efforts to characterize the gene at the molecular level, little is known about the genetic events that switch on gene expression in P-glycoprotein-negative cells. Recent studies have shown that the transcriptional competence of MDR1 is often closely associated with DNA methylation. Chromatin remodeling and modification targeted by the recognition of methylated DNA provide a dominant mechanism for transcriptional repression. Consistent with this epigenetic model, interference with DNA methyltransferase and histone deacetylase activity alone or in combination can reactivate silent genes. In the present study, we used chromatin immunoprecipitation to monitor the molecular events involved in the activation and repression of MDR1. Tumors become resistant to chemotherapy by a variety of mechanisms, including altered DNA repair, alterations in scavenging enzymes, and increased drug efflux. Although clinical drug resistance may be multifactorial, understanding these various mechanisms is important and may afford a means of improving treatment regimens. One of the most extensively studied mechanisms of multidrug resistance is associated with the overexpression of the MDR1 product, P glycoprotein (Pgp) (for reviews see references 27 and 55). A transmembrane protein, Pgp acts as an efflux pump, reducing intracellular drug levels and thus cytotoxic activity. We and others have demonstrated that the human MDR1 promoter is activated by changes in CpG methylation in B-cell chronic lymphocytic leukemia (26), acute myeloid leukemia (38), and tumor cell lines (12).DNA hypermethylation is associated with transcriptional silencing and is accompanied by the accumulation of deacetylated histones on heterochromatin (13,21). Methylation is often associated with the pathological silencing of tumor suppressor genes in human cancer and neurodevelopmental syndrome (20, 23). In contrast, transcriptionally active chromatin is characterized by the enrichment of hyperacetylated histone and is generally associated with hypomethylated chromatin (2, 34). The mechanisms underlying the correlation between DNA methylation and histone deacetylation in the control of gene expression have been established by recent biochemical experiments (24,42). Evidence has emerged that a family of methylCpG binding proteins binds heterochromatin to stably repress transcription (10, 18). The transcriptional repressor methylCpG binding protein 2 (MeCP2 ) is the best-characterized family member (18,41). MeCP2 is typically associated on heterochromatin in a methylation-dependent manner by its methyl-binding domain (MBD) and can displace histone H1 for nucleosome binding, indicating that it can dynamically interact with assembled chromatin (30,40). The transcriptional-repression domain of MeCP2 recruits the corepressors mSin3 and histone deacetylases (HDACs) (24, 42), causing transcriptional repression (41). Moreover, repression by MeCP2 is partial...