AGR2 ameliorates tumor necrosis factor-α-induced epithelial barrier dysfunction via suppression of NF-κB p65-mediated MLCK/p-MLC pathway activation

Intestinal epithelial barrier dysfunction plays a critical role in the pathogenesis of inflammatory bowel disease (IBD). Anterior gradient protein 2 homologue ( AGR2) assists in maintaining intestinal homeostasis in dextran sulphate sodium-induced mouse ileocolitis; however, it is unclear whether it modulates intestinal barrier function. Our study aimed to investigate the protective role of AGR2 in tumor necrosis factor (TNF)-alpha-induced intestinal epithelial barrier injury. Caco-2 cell monolayers were pre-transfected with an AGR2 plasmid and then exposed to TNF-alpha. Epithelial permeability was assessed by detecting transepithelial electrical resistance and fluorescein isothiocyanate-dextran (40 kDa) flux. The protein expression levels of zonula occludens-1 (ZO-1), occludin, claudin-1, myosin light chain kinase (MLCK)/p-MLC, and nuclear factor (NF)-kappa B p65 were determined by western blotting. In addition, the cellular distributions of ZO-1, occludin, F-alpha ctin, and NF-kappa B p65 were evaluated by immunofluorescence staining. The results showed that the AGR2 mRNA and protein expression levels were both decreased in the Caco-2 cell monolayers, while AGR2 overexpression significantly ameliorated TNF-alpha-induced epithelial barrier hyperpermeability, increased the expression of tight junction (TJ) proteins and stabilized the cytoskeletal structure. Furthermore, AGR2 inhibited the changes in MLCK, MLC and p-MLC expression in response to TNF-alpha stimulation. Collectively, our study suggests that AGR2 inhibits TNF-alpha-induced Caco-2 cell hyperpermeability by regulating TJ and that this protective mechanism may be promoted by inhibition of NF-kappa B p65-mediated activation of the MLCK/p-MLC signaling pathway.