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TGF-β is a potent pleiotropic factor that promotes small intestinal cell differentiation. The role of microRNAs in the TGF-β induction of intestinal epithelial phenotype is largely unknown. We hypothesized that microRNAs are functionally involved in TGF-β-induced intestinal cell growth. In this study, TGF-β caused a morphological change of IEC-6 cells and stimulated expression of the epithelial cell markers alkaline phosphatase, villin, and aminopeptidase N. By global microRNA profiling during TGF-β-induced intestinal crypt cell (IEC-6) differentiation, we identified 19 differentially expressed microRNAs. We showed by real-time Q-PCR that miR-146b expression increased rapidly after TGF-β treatment; sequence analysis and in vitro assays revealed that miR-146b targets SIAH2, an E3 ubiquitin ligase, with decreased protein expression upon IEC-6 cell differentiation. Transfection of miR-146b inhibitor before TGF-β treatment blocked the down-regulation of SIAH2 in response to TGF-β. Moreover, SIAH2 over-expression during TGF-β treatment caused a significant decrease in Smad7 protein expression in IEC-6 cells. Furthermore, activation of the ERK1/2 pathway is active in the up-regulation of miR-146b by TGF-β. These findings suggest a novel mechanism whereby TGF-β signaling during IEC-6 cell differentiation may be modulated in part by microRNAs, and we propose a key role for miR-146b in the homeostasis of growth factor TGF-β signaling through a negative feedback regulation involving down-regulation of SIAH2 repressed Smad7 activities.
Moderate reductions in peroxisome proliferator-activated receptor (PPAR)γ levels control insulin sensitivity as effectively as activation of PPARγ in adipocytes by the thiazolidinediones. That observation suggests that PPARγ activity can be regulated by modulating the amount of PPARγ protein in adipocytes. Activation of PPARγ in adipocytes is linked to changes in PPARγ protein levels via increased degradation of PPARγ proteins by the ubiquitin proteasome system. Identification of the ubiquitin ligase or ligases that recognize ligand bound PPARγ is an essential step in determining the physiological significance of the relationship between activation and ubiquitin-dependent degradation of PPARγ. Using an RNA interference-based screen, we identified five RING (really interesting new gene)-type ubiquitin ligases that alter PPARγ protein levels in adipocytes. Here, we demonstrate that Drosophila seven-in-absentia homolog 2 (Siah2), a mammalian homolog of Drosophila seven-in-absentia, regulates PPARγ ubiquitylation and ligand-dependent activation of PPARγ in adipocytes. We also demonstrate that Siah2 expression is up-regulated during adipogenesis and that PPARγ interacts with Siah2 during adipogenesis. In addition, Siah2 is required for adipogenesis. These data suggest that modulation of PPARγ protein levels by the ubiquitin ligase Siah2 is essential in determining the physiological effects of PPARγ activation in adipocytes.
