Pengyuan Wang

Stabilization of XIAP mRNA through the RNA binding protein HuR regulated by cellular polyamines

The X chromosome-linked inhibitor of apoptosis protein (XIAP) is the most potent intrinsic caspase inhibitor and plays an important role in the maintenance of intestinal epithelial integrity. The RNA binding protein, HuR, regulates the stability and translation of many target transcripts. Here, we report that HuR associated with both the 3′-untranslated region and coding sequence of the mRNA encoding XIAP, stabilized the XIAP transcript and elevated its expression in intestinal epithelial cells. Ectopic HuR overexpression or elevated cytoplasmic levels of endogenous HuR by decreasing cellular polyamines increased [HuR/XIAP mRNA] complexes, in turn promoting XIAP mRNA stability and increasing XIAP protein abundance. Conversely, HuR silencing in normal and polyamine-deficient cells rendered the XIAP mRNA unstable, thus reducing the steady state levels of XIAP. Inhibition of XIAP expression by XIAP silencing or by HuR silencing reversed the resistance of polyamine-deficient cells to apoptosis. Our findings demonstrate that HuR regulates XIAP expression by stabilizing its mRNA and implicates HuR-mediated XIAP in the control of intestinal epithelial apoptosis.

Chk2-dependent HuR phosphorylation regulates occludin mRNA translation and epithelial barrier function

Occludin is a transmembrane tight junction (TJ) protein that plays an important role in TJ assembly and regulation of the epithelial barrier function, but the mechanisms underlying its post-transcriptional regulation are unknown. The RNA-binding protein HuR modulates the stability and translation of many target mRNAs. Here, we investigated the role of HuR in the regulation of occludin expression and therefore in the intestinal epithelial barrier function. HuR bound the 3′-untranslated region of the occludin mRNA and enhanced occludin translation. HuR association with the occludin mRNA depended on Chk2-dependent HuR phosphorylation. Reduced HuR phosphorylation by Chk2 silencing or by reduction of Chk2 through polyamine depletion decreased HuR-binding to the occludin mRNA and repressed occludin translation, whereas Chk2 overexpression enhanced (HuR/occludin mRNA) association and stimulated occludin expression. In mice exposed to septic stress induced by cecal ligation and puncture, Chk2 levels in the intestinal mucosa decreased, associated with an inhibition of occludin expression and gut barrier dysfunction. These results indicate that HuR regulates occludin mRNA translation through Chk2-dependent HuR phosphorylation and that this influence is crucial for maintenance of the epithelial barrier integrity in the intestinal tract.

c-Jun enhances intestinal epithelial restitution after wounding by increasing phospholipase C-γ1 transcription

c-Jun is an activating protein 1 (AP-1) transcription factor and implicated in many aspects of cellular functions, but its exact role in the regulation of early intestinal epithelial restitution after injury remains largely unknown. Phospholipase C-γ1 (PLCγ1) catalyzes hydrolysis of phosphatidylinositol 4,5 biphosphate into the second messenger diacylglycerol and inositol 1,4,5 triphosphate, coordinates Ca2+ store mobilization, and regulates cell migration and proliferation in response to stress. Here we reported that c-Jun upregulates PLCγ1 expression and enhances PLCγ1-induced Ca2+ signaling, thus promoting intestinal epithelial restitution after wounding. Ectopically expressed c-Jun increased PLCγ1 expression at the transcription level, and this stimulation is mediated by directly interacting with AP-1 and CCAAT-enhancer-binding protein (C/EBP) binding sites that are located at the proximal region of the rat PLCγ1 promoter. Increased levels of PLCγ1 by c-Jun elevated cytosolic free Ca2+ concentration and stimulated intestinal epithelial cell migration over the denuded area after wounding. The c-Jun-mediated PLCγ1/Ca2+ signal also plays an important role in polyamine-induced cell migration after wounding because increased c-Jun rescued Ca2+ influx and cell migration in polyamine-deficient cells. These findings indicate that c-Jun induces PLCγ1 expression transcriptionally and enhances rapid epithelial restitution after injury by activating Ca2+ signal.

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

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.

481 The RNA-Binding Protein HuR Regulates Occludin Translation Through Chk2-Dependent HuR Phosphorylation Modulating Intestinal Epithelial Barrier Function

Zonula occludens proteins direct assembly of epithelial tight junctions. While crucial functions have been assigned for N-terminal PDZ domains, a central SH3-GUK module, and the C-terminal actin-binding region (ABR) of ZO-1, the remainder of the C-terminus, including a putative ZU5 domain, is poorly characterized. The aim of this study was to determine the roles of the ZO-1 C-terminus in tight junction assembly and maintenance. METHODS: Cultured epithelial (MDCK) cells were stably transfected with full-length human ZO-1 tagged with enhanced green fluorescent protein (EGFP) at the N-terminus (EGFP-ZO-1); a deletion mutant lacking the ABR (EGFP-ZO-1ΔABR); a truncation mutant lacking the ABR and remaining C-terminus (EGFP-ZO-11-1152); a mutant containing the ABR but lacking the remaining C-terminus (EGFP-ZO-11-1372); and a truncation mutant lacking only the ZU5 domain (EGFP-ZO-11-1620). Tight junction assembly was induced by addition of extracellular Ca2+ and barrier function was monitored as transepithelial resistant (TER). Endogenous proteins were detected by immunofluorescence microscopy and behavior of tagged proteins at tight junctions was determined by fluorescent recovery after photobleaching (FRAP). RESULTS: EGFP-ZO-11-1152 expression severely delayed epithelial barrier development as well as peak TER achieved. In contrast, ZO-1ΔABR expression did not delay assembly and only slightly diminished peak TER, suggesting that C-terminal sequence other than the ABR is required for tight junction assembly and function. Neither EGFP-ZO-11-1152 nor EGFPZO-1ΔABR bound directly to F-actin, but EGFP-ZO-11-1152 disrupted interactions between endogenous ZO-1 and F-actin. EGFP-ZO-11-1152 was recruited to tight junctions inefficiently and interfered with trafficking of endogenous ZO-1 and β-catenin, consistent with a dominant negative effect. EGFP-ZO-11-1372 and EGFP-ZO-11-1620 were also recruited inefficiently, but EGFP-ZO-1 and EGFP-ZO-1ΔABR were recruited normally and did not impair endogenous protein trafficking, supporting the hypothesis that the effect of EGFP-ZO-11-1152 expression reflects loss of C-terminal ZO-1 domains beyond the ABR. To define these domains, ZO-1 stability at the tight junction was assessed by FRAP. EGFP-ZO-11-1152 was less stable than EGFP-ZO-1, with immobile fractions of 16±1% and 35±2%, respectively. EGFP-ZO-11-1372 and EGFP-ZO-11-1620 behaved similarly to EGFP-ZO-11-1152, while EGFP-ZO-1ΔABR was comparable to EGFP-ZO-1. CONCLUSION: C-terminal sequence including the ZU5 domain, but not the ABR, of ZO-1 is required for tight junction assembly and function. These data are the first to identify the essential role of the ZU5 domain.