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

Abstract

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)-α-induced intestinal epithelial barrier injury. Caco-2 cell monolayers were pre-transfected with an AGR2 plasmid and then exposed to TNF-α. 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)-κB p65 were determined by western blotting. In addition, the cellular distributions of ZO-1, occludin, F-actin, and NF-κB 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-α-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-α stimulation. Collectively, our study suggests that AGR2 inhibits TNF-α-induced Caco-2 cell hyperpermeability by regulating TJ and that this protective mechanism may be promoted by inhibition of NF-κB p65-mediated activation of the MLCK/p-MLC signaling pathway.

Cytokine Tuning of Intestinal Epithelial Function

“…In contrast to the barrier reinforcing properties of the cytokines described earlier, a handful of cytokines can also disrupt the intestinal epithelium and promote barrier permeability (Figure 4 ) ( 29 , 30 , 79 , 80 ).…”

“…Various effects of TNF-α on the intestinal epithelium discussed herein could disrupt the epithelial barrier; however, TNF-α stimulation of intestinal epithelial cells has also been specifically demonstrated to decrease the protein expression of the tight junction proteins claudin-1, occludin, and zonula occludens protein-1 (ZO-1), as well as to induce cytoskeletal F-actin rearrangement and the mislocalization of occludin and ZO-1 ( 29 , 30 ). Multiple studies have identified mechanisms to reduce TNF-α-induced epithelial barrier compromise, including the overexpression of anterior gradient protein 2 homolog, rebeccamycin treatment, and the stimulation of muscarinic cholinoceptor-mediated signaling ( 29 , 30 , 81 ).…”

“…In addition, cytokines can directly alter intestinal epithelial permeability ( 27 , 28 ). The permeability of epithelial tight junctions may be increased or decreased by cytokine modification of the expression or localization of their protein components ( 11 , 12 , 27 , 29 , 30 ). Cytokines can also drive phosphorylation of myosin light chains, resulting in contraction and opening of tight junctions ( 11 ).…”

Activation of the Nuclear Factor-kappa B Signaling Pathway Damages the Epithelial Barrier in the Human Pancreatic Ductal Adenocarcinoma Cell Line HPAF-II

“…20 Tumor necrosis factor-alpha can activate the NF-κB signaling pathway, causing increased NF-κB-mediated MLCK expression and MLC phosphorylation, resulting in altered permeability and TJ disruption in the intestinal epithelium. 16,21,22 To investigate whether impairment of pancreatic duct epithelium is consistent with the mechanism above, this study was carried out.…”

Total Flavonoid Extract from Hawthorn (Crataegus pinnatifida) Improves Inflammatory Cytokines-Evoked Epithelial Barrier Deficit

“…The structural integrity of the intestinal barrier depends on a complex network of cytoskeletal structures and intercellular tight junctions (TJs) [5]. TJs proteins are located at the top of intestinal epithelial cells, which are made up of cytoplasmic zonula occludens (ZO) proteins, junctional adhesion molecule, claudins, and occludin [6].…”