Actin dynamics and regulation of epithelial barrier in intestinal inflammation

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DESCRIPTION (provided by applicant): Breakdown of the intestinal epithelial barrier represents an early and important manifestation of various gastrointestinal disorders including inflammatory bowel disease. The integrity of the epithelial barrier is regulated by specialized adhesive structures known as the apical junctional complex (AJC), which is composed by tight junctions (TJ) and adherens junctions (AJ). Disruption of epithelial junctions plays a key role in the increased permeability of inflamed intestinal mucosa. Understanding mechanisms of AJC disassembly during mucosal inflammation represents the major goal of the proposed study. TJ and AJ associate with the actin cytoskeleton which is considered as crucial regulator of junctional integrity and remodeling. Actin filaments are highly dynamic polymers undergoing a constant turnover (depolymerization and polymerization). Such a turnover of filamentous (F) actin is important for a variety of cellular functions, however its involvement in AJ/TJ regulation remains poorly investigated. A central innovative hypothesis of this proposal implies that F-actin turnover plays a key role in regulating structure and remodeling of the intestinal epithelial AJC. Particularly, we propose that the decreased F-actin turnover is a prerequisite for maintenance of stable junctions and tight epithelial barrier in healthy gut, whereas accelerated turnover of perijunctional actin filaments mediates AJ/TJ disassembly during intestinal inflammation. This hypothesis will be tested in the following Aims: (1) To examine the role of accelerated actin filament turnover in disassembly of epithelial apical junctions; (2) to investigate the involvement of actin-depolymerizing factor (ADF)/cofilin proteins in regulation of the AJC during intestinal inflammation; 3) to analyze the role of F-actin cross-linking proteins in maintenance and disassembly of epithelial apical junctions. These aims will be accomplished using in vitro intestinal epithelial cell monolayers exposed to different inflammatory mediators and in vivo murine models of intestinal inflammation. Turnover of actin filaments and functions of actin-depolymerizing and cross-linking proteins will be analyzed by a combination of biochemical (actin-monomer sequestration, immunoblotting, detergent fractionation), immunocytochemical and genetic (siRNA- mediated knock-downs, expression of dominant-negative or constitutively active mutants, knockout mice) approaches. Significance: the proposed study will give new insights into fundamental mechanisms of intestinal mucosal injury during inflammation. Understanding these mechanisms will provide new therapeutic targets to prevent breakdown and enhance reparation of the gut barrier in patients with digestive diseases. PUBLIC HEALTH RELEVANCE: The proposed research is aimed to understand mechanisms underlying disruption of the intestinal epithelial barrier. The barrier breakdown is a common manifestation of different gastroenterological disorders including ulcerative colitis, Crohn's disease, celiac diseases and infectious colitis. Furthermore, dysfunctions of the gut barrier contribute to the development of other diseases such as septic shock, alcoholic liver disease and type I diabetes. This project will provide new insights into understanding the pathogenesis of gastrointestinal disorders by exploring a novel epithelium- related mechanism involving in initiation and/or exaggeration of mucosal inflammation. Furthermore, it may provide novel targets for pharmacological prevention of the intestinal barrier breakdown and for accelerated healing of the injured gut mucosa. This may result in decreased morbidity and mortality of a large cohort of patients with inflammatory disorders.