The Epithelial Barrier Is Maintained by In Vivo Tight Junction Expansion During Pathologic Intestinal Epithelial Shedding

Abstract

BACKGROUND & AIMS 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. METHODS We studied wild-type and transgenic mice that express the fluorescent-tagged proteins enhanced green fluorescent protein–occludin or monomeric red fluorescent protein1–ZO-1. After injection of high doses of TNF (7.5µg, i.p.), 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. RESULTS Changes detected included redistribution of the tight junction proteins ZO-1 and occluding 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. CONCLUSIONS 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.

PI3-kinase activation is critical for host barrier permissiveness to Listeria monocytogenes

“…Perez et al (2008) proposed that in stable and cohesive epithelia, PI3-K activity is downregulated to allow the maintenance of Ecad interactions at cell-cell junctions, whereas in more dynamic epithelia, actin cytoskeleton remodeling requires PI3-K activity to disrupt adherens junctions and allow the formation of new sites of Ecad homophilic interactions. Our in vivo findings fit with this model: luminal accessibility of Ecad correlates with high PI3-K activity in intestinal GCs, ECs, and EEFs, where adherens junctions are dynamic (Madara et al, 1980;Madara and Trier, 1982;Kölsch et al, 2007;Marchiando et al, 2011). Indeed, and found, as we previously reported , a significant role for InlA and InlB in third term placental villous explant invasion (Fig.…”

Control of creatine metabolism by HIF is an endogenous mechanism of barrier regulation in colitis

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