Contributions of Myosin Light Chain Kinase to Regulation of Epithelial Paracellular Permeability and Mucosal Homeostasis

Intestinal barrier function is required for the maintenance of mucosal homeostasis. Barrier dysfunction is thought to promote progression of both intestinal and systemic diseases. In many cases, this barrier loss reflects increased permeability of the paracellular tight junction as a consequence of myosin light chain kinase (MLCK) activation and myosin II regulatory light chain (MLC) phosphorylation. Although some details about MLCK activation remain to be defined, it is clear that this triggers perijunctional actomyosin ring (PAMR) contraction that leads to molecular reorganization of tight junction structure and composition, including occludin endocytosis. In disease states, this process can be triggered by pro-inflammatory cytokines including tumor necrosis factor-α (TNF), interleukin-1β (IL-1β), and several related molecules. Of these, TNF has been studied in the greatest detail and is known to activate long MLCK transcription, expression, enzymatic activity, and recruitment to the PAMR. Unfortunately, toxicities associated with inhibition of MLCK expression or enzymatic activity make these unsuitable as therapeutic targets. Recent work has, however, identified a small molecule that prevents MLCK1 recruitment to the PAMR without inhibiting enzymatic function. This small molecule, termed Divertin, restores barrier function after TNF-induced barrier loss and prevents disease progression in experimental chronic inflammatory bowel disease

Notch1 promotes survival of E2A-deficient T cell lymphomas through pre–T cell receptor–dependent and –independent mechanisms

Loss of E2A transcription factor activity or activation of the intracellular form of Notch1 (ICN) leads to the development of leukemia or lymphoma in humans or mice, respectively. Current models propose that ICN functions by suppressing E2A through a pre–T cell receptor (TCR)–dependent mechanism. Here we show that lymphomas arising in E2A–/– mice require the activation of Notch1 for their survival and have accumulated mutations in, or near, the Notch1 PEST domain, resulting in increased stability and signaling. In contrast, lymphomas arising in p53–/– mice show the activation of Notch1, but no mutations were identified in ICN. The requirement for Notch1 signaling in E2A–/– lymphomas cannot be overcome by ectopic expression of pTα; however, pTα is required for optimal survival and expansion of these cells. Our findings indicate that the activation of Notch1 is an important “second hit” for the transformation of E2A–/– T cell lymphomas and that Notch1 promotes survival through pre–TCR-dependent and -independent mechanisms

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

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 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. RESULTS: 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. 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