Hypoxia-inducible factor 1 (HIF-1) is a transcription factor involved in normal mammalian development and in the pathogenesis of several disease states. It consists of two subunits, HIF-1␣, which is degraded during normoxia, and HIF-1␤, which is constitutively expressed.

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MicroRNAs (miRNAs) are short non-coding RNAs that have been implicated in fine-tuning gene regulation, although the precise roles of many are still unknown. Pancreatic development is characterized by the complex sequential expression of a gamut of transcription factors. We have performed miRNA expression profiling at two key stages of mouse embryonic pancreas development, e14.5 and e18.5. miR-124a2 expression was strikingly increased at e18.5 compared with e14.5, suggesting a possible role in differentiated ␤-cells. Among the potential miR124a gene targets identified by biocomputation, Foxa2 is known to play a role in ␤-cell differentiation. To evaluate the impact of miR-124a2 on gene expression, we overexpressed or down-regulated miR-124a2 in MIN6 ␤-cells. As predicted, miR-124a2 regulated Foxa2 gene expression, and that of its downstream target, pancreatic duodenum homeobox-1 (Pdx-1). Foxa2 has been described as a master regulator of pancreatic development and also of genes involved in glucose metabolism and insulin secretion, including the ATP-sensitive K ؉ (K ATP ) channel subunits, Kir6.2 and Sur-1. Correspondingly, miR-124a2 overexpression decreased, and anti-miR-124a2 increased Kir6.2 and Sur-1 mRNA levels. Moreover, miR-124a2 modified basal and glucose-or KCl-stimulated intracellular free Ca 2؉ concentrations in single MIN6 and INS-1 (832/13) ␤-cells, without affecting the secretion of insulin or co-transfected human growth hormone, consistent with an altered sensitivity of the ␤-cell exocytotic machinery to Ca 2؉ . In conclusion, whereas the precise role of microRNA-124a2 in pancreatic development remains to be deciphered, we identify it as a regulator of a key transcriptional protein network in ␤-cells responsible for modulating intracellular signaling.Studies implicating small regulatory RNAs in the control of gene expression have demonstrated that transcriptional regulation is controlled not only by protein factors, but also by small endogenous RNA molecules of ϳ19 -23 nucleotides in length called microRNAs (miRNAs or miRs) 3 (1, 2). The first miRNAs discovered were lin-4 and let-7, which are crucial for regulating the developmental timing in the nematode, Caenorhabditis elegans (1, 3). Since this initial report, several hundred miRNAs have been identified in plants and animals that regulate diverse biological processes ranging from cell metabolism to cell differentiation and growth, apoptosis, and immune responses (4 -8). Moreover, it has been shown that miRNAs are characterized by differential spatial and temporal expression patterns supporting their role in such processes (3, 9). miRNAs serve as regulators of gene expression by binding to complementary sites on their target transcripts and, by an ill-defined mechanism, significantly induce the cleavage of mRNA or the repression of translation, depending on the partial or complete sequence homology, respectively (2, 10 -12). It has been estimated that miRNA genes represent ϳ1% of the genome of complex organisms. It appears that they share a certain...

The SOCS proteins are induced by several cytokines and are involved in negative feedback loops. Here we demonstrate that in 3T3-L1 adipocytes, insulin, a hormone whose receptor does not belong to the cytokine receptor family, induces SOCS-3 expression but not CIS or SOCS-2. Using transfection of COS-7 cells, we show that insulin induction of SOCS-3 is enhanced upon Stat5B expression. Moreover, Stat5B from insulin-stimulated cells binds directly to a Stat element present in the SOCS-3 promoter. Once induced, SOCS-3 inhibits insulin activation of Stat5B without modifying the insulin receptor tyrosine kinase activity. Two pieces of evidence suggest that this negative regulation likely results from competition between SOCS-3 and Stat5B binding to the same insulin receptor motif. First, using a yeast two-hybrid system, we show that SOCS-3 binds to the insulin receptor at phosphotyrosine 960, which is precisely where Stat5B binds. Second, using confocal microscopy, we show that insulin induces translocation of SOCS-3 from an intracellular compartment to the cell membrane, leading to colocalization of SOCS-3 with the insulin receptor. This colocalization is dependent upon phosphorylation of insulin receptor tyrosine 960. Indeed, in cells expressing an insulin receptor mutant in which tyrosine 960 has been mutated to phenylalanine, insulin does not modify the cellular localization of SOCS-3. We have thus revealed an insulin target gene of which the expression is potentiated upon Stat5B activation. By inhibiting insulin-stimulated Stat5B, SOCS-3 appears to function as a negative regulator of insulin signaling.