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Post-transcriptional regulation of Transforming Growth Factor Beta-1 by microRNA-744.

ABSTRACT: Transforming Growth Factor Beta-1 (TGF-?1) is a pleiotropic cytokine that is of central importance in wound healing, inflammation, and in key pathological processes including cancer and progressive tissue fibrosis. TGF-?1 is post-transcriptionally regulated, but the underlying mechanisms remain incompletely defined. Previously, we have extensively delineated post-transcriptional regulation of TGF-?1 synthesis in the kidney, with evidence for relief of translational repression in proximal tubular cells in the context of diabetic nephropathy. In this study, we have investigated the role of the TGF-?1 3'Untranslated Region (3'UTR). Two different 3'UTR lengths have been reported for TGF-?1, of 543 and 137 nucleotides. Absolute quantification showed that, while both UTR lengths were detectable in various human cell types and in a broad range of tissues, the short form predominated in the kidney and elsewhere. Expression of both forms was up-regulated following auto-induction by TGF-?1, but the short:long UTR ratio remained constant. Incorporation of the short UTR into a luciferase reporter vector significantly reduced reporter protein synthesis without major effect on RNA amount, suggesting post-transcriptional inhibition. In silico approaches identified multiple binding sites for miR-744 located in the proximal TGF-?1 3'UTR. A screen in RNA from human tissues showed widespread miR-744 expression. miR-744 transfection inhibited endogenous TGF-?1 synthesis, while direct targeting of TGF-?1 was shown in separate experiments, in which miR-744 decreased TGF-?1 3'UTR reporter activity. This work identifies miR-744-directed post-transcriptional regulation of TGF-?1 which, given the pleiotropic nature of cellular responses to TGF-?1, is potentially widely significant.

SUBMITTER: Martin J

PROVIDER: S-EPMC3186795 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Post-transcriptional regulation of Transforming Growth Factor Beta-1 by microRNA-744.

Martin John J Jenkins Robert H RH Bennagi Rasha R Krupa Aleksandra A Phillips Aled O AO Bowen Timothy T Fraser Donald J DJ

PloS one 20111004 10

Transforming Growth Factor Beta-1 (TGF-β1) is a pleiotropic cytokine that is of central importance in wound healing, inflammation, and in key pathological processes including cancer and progressive tissue fibrosis. TGF-β1 is post-transcriptionally regulated, but the underlying mechanisms remain incompletely defined. Previously, we have extensively delineated post-transcriptional regulation of TGF-β1 synthesis in the kidney, with evidence for relief of translational repression in proximal tubular ...[more]

PMID: 21991303

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Project description:The importance of microRNA (miRNA) to vascular biology is becoming increasingly evident; however, the function of a significant number of miRNA remains to be determined. In particular, the effect of growth factor regulation of miRNAs on endothelial cell morphogenesis is incomplete. Thus, we aimed to identify miRNAs regulated by pro-angiogenic vascular endothelial growth factor (VEGF) and determine the effects of VEGF-regulated miRNAs and their targets on processes important for angiogenesis. Human umbilical vein endothelial cells (HUVECs) were thus stimulated with VEGF and miRNA levels assessed using microarrays. We found that VEGF altered expression of many miRNA, and for this study focused on one of the most significantly down-regulated miRNA in HUVECs following VEGF treatment, miR-30b. Using specific miRNA mimics, we found that overexpression of miR-30b inhibited capillary morphogenesis in vitro, while depletion of endogenous miR-30b resulted in increased capillary morphogenesis indicating the potential significance of down-regulation of miR-30b as a pro-angiogenic response to VEGF stimulation. MiR-30b overexpression in HUVEC regulated transforming growth factor beta 2 (TGFβ2) production, which led to increased phosphorylation of Smad2, indicating activation of an autocrine TGFβ signaling pathway. Up-regulation of TGFβ2 by miR-30b overexpression was found to be dependent on ATF2 activation, a transcription factor known to regulate TGFβ2 expression, as miR-30b overexpressing cells exhibited increased levels of phosphorylated ATF2 and depletion of ATF2 inhibited miR-30b-induced TGFβ2 expression. However, miR-30b effects on ATF2 were indirect and found to be via targeting of the known ATF2 repressor protein JDP2 whose mRNA levels were indirectly correlated with miR-30b levels. Increased secretion of TGFβ2 from HUVEC was shown to mediate the inhibitory effects of miR-30b on capillary morphogenesis as treatment with a neutralizing antibody to TGFβ2 restored capillary morphogenesis to normal levels in miR-30b overexpressing cells. These results support that the regulation of miR-30b by VEGF in HUVEC is important for capillary morphogenesis, as increased miR-30b expression inhibits capillary morphogenesis through enhanced expression of TGFβ2.

Dataset Information

Post-transcriptional regulation of Transforming Growth Factor Beta-1 by microRNA-744.

Publications

Post-transcriptional regulation of Transforming Growth Factor Beta-1 by microRNA-744.

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