Actin dynamics and regulation of epithelial barrier in intestinal inflammation

描述（由申请人提供）：肠上皮屏障的破坏是包括炎症性肠病在内的各种胃肠道疾病的早期和重要表现。上皮屏障的完整性是由称为顶端连接复合物（AJC）的特殊粘附结构调节的，该结构由紧密连接（TJ）和粘附连接（AJ）组成。上皮连接的破坏在炎症肠粘膜通透性增加中起关键作用。了解粘膜炎症期间AJC分解的机制是本研究的主要目标。TJ和AJ与肌动蛋白细胞骨架相关，肌动蛋白被认为是连接完整性和重塑的关键调节剂。肌动蛋白丝是高度动态的聚合物，经历不断的周转（解聚和聚合）。丝状(F)肌动蛋白的这种转换对多种细胞功能很重要，但其参与AJ/TJ调节的研究仍然很少。该提议的一个核心创新假设表明，f -肌动蛋白的转换在调节肠上皮AJC的结构和重塑中起关键作用。特别是，我们提出f -肌动蛋白周转减少是维持健康肠道稳定连接和紧密上皮屏障的先决条件，而在肠道炎症期间，结周肌动蛋白丝的加速周转介导AJ/TJ的分解。这一假设将在以下目的中得到验证：(1)研究肌动蛋白丝的加速更新在上皮细胞顶端连接解体中的作用；(2)探讨肌动蛋白解聚因子(ADF)/cofilin蛋白在肠道炎症过程中对AJC的调控作用；3)分析F-actin交联蛋白在上皮顶端连接维持和拆卸中的作用。这些目标将通过暴露于不同炎症介质的体外肠上皮细胞单层和体内肠道炎症小鼠模型来实现。肌动蛋白丝的转换和肌动蛋白解聚和交联蛋白的功能将通过生化（肌动蛋白单体隔离、免疫印迹、洗涤剂分离）、免疫细胞化学和遗传学（siRNA介导的敲除、显性阴性或构成活性突变体的表达、敲除小鼠）方法的组合进行分析。意义：本研究将对炎症期肠黏膜损伤的基本机制提供新的认识。了解这些机制将为消化系统疾病患者提供新的治疗靶点，以防止肠道屏障的破坏和增强肠道屏障的修复。

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.