LPA Induced Activation of NHE3 via LPA5 Receptor is Apical EGF Receptor Dependent

Abstract

G A A b st ra ct s the mechanisms by which polyamines regulate RBPs remain elusive. microRNAs (miRNAs) generally base-pair to sequences in the 3'-untranslated regions (UTR) of labile mRNAs and regulate gene expression posttranscriptionally. This study determined if polyamines regulate CUGBP1 expression through miRNA-503 (miR-503), thereby contributing to the control of intestinal mucosal growth. Methods: Studies were conducted in mice and cultured intestinal epithelial cells (IEC-6 line). CUGBP1 and miR-503 expression was examined by real-time PCR andWestern blotting analyses; CUGBP1 translation was measured by chimeric CUGBP1 3'-UTR luciferease reporter and newly synthesized CUGBP1 protein assays. Interaction of miR-503 with the CUGBP1 mRNA was examined by pmir-Glo reporter system and biotin labeled miR-503 pull-down assays. Polyamine levels were depleted by DFMO (a specific inhibitor of polyamine biosynthesis) but increased by ODC gene overexpression. The functions of miRNA-503 were examined by miR-503-directed siRNA and ectopic overexpression of its precursor. Results: Polyamine depletion by DFMO and fasting increased CUGBP1 protein expression (by ~2.6-fold) but decreased miR-503 in the small intestinal mucosa, which was associated with an inhibition of intestinal mucosal growth as indicated by a decrease in DNA synthesis and villous height. In cultured IECs, polyamine depletion also increased CUGBP1 protein (by ~3.5-fold) but decreased miR-503 (by ~70%), whereas elevated polyamines reduced CUGBP1 (by >85%) and increased miR-503 (by ~2-fold), neither intervention changed CUGBP1 mRNA levels. miR-503 directly bound the CUGBP1 mRNA via CUGBP1 coding region but not its 3'-UTR; this interaction was increased by increasing polyamines but it was repressed by polyamine depletion. miR-503 silencing reduced [miR-503/CUGBP1 mRNA] complex, stimulated CUGBP1 translation, increased its expression (by ~1.5-fold), and inhibited IEC proliferation. Conversely, miR-503 overexpression repressed CUGBP1 translation, decreased its protein levels (by >80%), and enhanced IEC growth. Ectopic CUGBP1 overexpression also repressed cell proliferation by ~65% when measured 6 days after the transfection. Conclusions: These results indicate that 1) polyamines repress CUGBP1 mRNA translation by increasing miR-503 and 2) induced CUGBP1 plays an important role in inhibition of intestinal mucosal growth after polyamine depletion.