Regulation of ATF-2 mRNA Stability by RNA-Binding Protein HuR Following Polyamine Depletion in Intestinal Epithelial Cells.

Normal intestinal mucosal growth requires polyamines that regulate expression of various genes through distinct mechanisms. We have recently found that polyamines modulate subcellular distribution of HuR, a critical regulator of post-transcriptional fate of target transcripts, and that decreasing cellular polyamines increases cytoplasmic levels of HuR, leading to stabilization of NPM and p53 mRNAs. ATF-2 is a transcription factor and known to regulate epithelial cell proliferation and apoptosis. Present study tests the hypothesis that polyamines regulate ATF-2 expression through HuR in normal intestinal epithelial cells (IEC-6). Methods: Expression of the ATF-2 gene was examined by measurements of its promoter activity and mRNA and protein levels in the presence or absence of cellular polyamines. HuR binding to 3'-untranslated region (UTR) of ATF-2 mRNA was identified by RNA pull-down and HuR-immunoprecipitation assays. Functions of HuR were investigated by using siRNA that specifically targets HuR (siHuR). Results: Decreasing cellular polyamines by inhibiting ODC (key enzyme for polyamine biosynthesis) increased levels of ATF-2 mRNA and protein, while increasing polyamines through stable transfection of the ODC gene repressed ATF-2 expression. Polyamine depletion stabilized ATF-2 mRNA as indicated by an increase in its mRNA half-life, but had not effect on ATF-2 gene transcription. Increased cytoplasmic HuR specially bound to 3'-UTR of ATF-2 mRNA following polyamine depletion. HuR silencing by siHuR rendered the ATF-2 mRNA unstable and prevented increases in ATF-2 mRNA and protein in polyamine-deficient cells. Conclusions: These results indicate that polyamines destabilized ATF-2 mRNA through modulation of cytoplasmic HuR levels in IECs. [ABSTRACT FROM AUTHOR]