The epithelial barrier is maintained by in vivo tight junction expansion during pathologic intestinal epithelial shedding.

Abstract

Tumor necrosis factor (TNF) increases intestinal epithelial cell shedding and apoptosis, potentially challenging the barrier between the gastrointestinal lumen and internal tissues. We investigated the mechanism of tight junction remodeling and barrier maintenance as well as the roles of cytoskeletal regulatory molecules during TNF-induced shedding. We studied wild-type and transgenic mice that express the fluorescent-tagged proteins enhanced green fluorescent protein-occludin or monomeric red fluorescent protein 1-ZO-1. After injection of high doses of TNF (7.5 μg intraperitoneally), laparotomies were performed and segments of small intestine were opened to visualize the mucosa by video confocal microscopy. Pharmacologic inhibitors and knockout mice were used to determine the roles of caspase activation, actomyosin, and microtubule remodeling and membrane trafficking in epithelial shedding. Changes detected included redistribution of the tight junction proteins ZO-1 and occludin to lateral membranes of shedding cells. These proteins ultimately formed a funnel around the shedding cell that defined the site of barrier preservation. Claudins, E-cadherin, F-actin, myosin II, Rho-associated kinase (ROCK), and myosin light chain kinase (MLCK) were also recruited to lateral membranes. Caspase activity, myosin motor activity, and microtubules were required to initiate shedding, whereas completion of the process required microfilament remodeling and ROCK, MLCK, and dynamin II activities. Maintenance of the epithelial barrier during TNF-induced cell shedding is a complex process that involves integration of microtubules, microfilaments, and membrane traffic to remove apoptotic cells. This process is accompanied by redistribution of apical junctional complex proteins to form intercellular barriers between lateral membranes and maintain mucosal function.