Stabilization of Cx43 mRNA by RNA-binding protein HuR enhances intestinal epithelial barrier function

Abstract

BACKGROUND & AIMS: Connexin 43 (Cx43) acts as a gap junction protein and is involved in many aspects of different cellular processes including intercellular communication. The levels of cellular Cx43 change markedly in response to various pathobiological stresses, but the exact mechanism underlying Cx43 expression in the intestinal epithelium remains largely unknown. The RNA-binding protein HuR regulates the stability and translation of target mRNAs and is identified as a biological regulator of the intestinal epithelium homeostasis. In this study, we tested if HuR regulates Cx43 expression at the posttranscription level and further examined the influence of HuR/Cx43 interaction on the intestinal epithelial barrier function. METHODS: Studies were conducted in differentiated IEC-Cdx2L1 and Caco-2 cells. HuR binding to the Cx43 mRNA was determined by mRNP/IP assays, and the stability of Cx43 mRNA was examined by its half-life with qPCR analysis. Functions of HuR and its regulator Chk2 kinase were examined by their gene silencing or overexpression. Epithelial barrier function was assayed by transepithelial electrical resistance (TEER) and membrane-impermeable trace FITC-dextran. RESULTS: HuR directly bound to the Cx43 mRNA through its 3’-untranslated region (UTR) rather than 5’-UTR and coding region. HuR silencing decreased Cx43 levels by destabilizing Cx43 mRNA and caused the epithelial barrier dysfunction, as evidenced by a decrease in TEER and an increase in paracellular flux of FITC-dextran. In contrast, Cx43 overexpression rescued the epithelial barrier function in HuR-silenced cells. HuR mediated-Cx43 expression was regulated by ChK2 dependent HuR phosphorylation since decreasing the levels of phosphorylated HuR by silencing ChK2 lowered Cx43 levels and disrupted the barrier function. Reduction in phosphorylated HuR levels by polyamine depletion also inhibited HuR binding to the Cx43 mRNA and decreased Cx43 protein by destabilizing the Cx43 mRNA, which was associated with the epithelial barrier dysfunction. Ectopic overexpression of ChK2 in polyamine-deficient cells not only rescued Cx43 expression but also restored the barrier function. CONCLUSIONS: These results indicate that the Cx43 mRNA is a novel target of HuR and that HuR-induced expression of Cx43 via stabilization of its mRNA plays a critical role in the maintenance of intestinal epithelial barrier function. US Department of Veterans Affairs and NIH This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.