577 Reduced HuR Association With Occludin mRNA Plays an Important Role in Pathogenesis of Gut Barrier Dysfunction During Septic Stress

Abstract

Gut barrier dysfunction occurs in various critical illnesses, leading to the translocation of luminal toxic substances and bacteria to the blood stream. Occludin in the tight junctions (TJs) is an integral membrane protein that forms the sealing element of TJs and its normal expression is critical for maintaining epithelial barrier function during stress and is tightly regulated at multiple levels. The RNA-binding protein HuR binds to many labile mRNAs bearing Uor AU-rich elements and modulates their stability and translation. Our previous studies show that HuR directly interacts with occludin mRNA and regulates occludin expression in cultured intestinal epithelial cells (IECs) and that HuR silencing represses occludin translation In Vitro. This study further determines whether HuR-mediated occludin expression plays a role in the pathogenesis of gut barrier dysfunction during septic stress. Methods: Studies were conducted in A/J mice, and septic stress was induced by the method of cecal ligation and puncture (CLP). Gut permeability was detected by using the fluorescent tracer FITC-dextran; and levels of occludin mRNA and protein were examined by quantitative real-time PCR and Western blotting analyses. HuR binding to occludin mRNA was examined by ribonucleoprotein immunoprecipiatation assays. Results: Exposure to CLP for 8 h decreased occludin protein levels, and maximal reduction in occludin (by ~85%) occurred 24 and 48 h, then it began to recover gradually thereafter. Consistently, gut barrier dysfunction as indicated by an increase (by >2-fold) in the mucosal permeability to FITC-dextran also occurred 8 h after CLP and maintained for additional 40 h. CLP failed to reduce total occludin mRNA levels, but it repressed occludin mRNA association with HuR (by ~80%). At 72 h after CLP, the levels of [HuR/occludin mRNA] complex increased partially (by ~33%) as compared with those observed in 24 and 48 h, which was associated with partial recovery of gut barrier function. In addition, depletion of cellular polyamines by treatment with DFMO (a specific inhibitor of polyamine biosynthesis) not only repressed HuR binding to occludin mRNA but also delayed the recovery of gut barrier function in mice exposed to CLP. In cultured IECs, polyamine depletion inhibited HuR phosphorylation, reduced HuR association with occludin mRNA, and repressed occludin translation, thus disrupting the barrier function. Conclusions: CLP-induced repression of occludin expression occurs at the posttranscriptional level and that reduced HuR association with occludin mRNA plays an important role in gut barrier dysfunction during critical stress.