Actin dynamics and regulation of epithelial barrier in intestinal inflammation

描述(申请人提供)：肠道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。上皮屏障的完整性由特殊的黏附结构调节，称为尖端连接复合体(AJC)，由紧密连接(TJ)和黏附连接(AJ)组成。上皮连接的破坏在炎症性肠黏膜通透性增加中起关键作用。了解AJC在黏膜炎症过程中的分解机制是拟议研究的主要目标。TJ和AJ与肌动蛋白细胞骨架有关，肌动蛋白细胞骨架被认为是连接完整性和重塑的关键调节因子。肌动蛋白细丝是一种高度动态的聚合物，经历着持续的周转(解聚和聚合)。丝状(F)肌动蛋白的这种转换对于多种细胞功能是重要的，然而它参与AJ/TJ调节的研究仍然很少。这一提议的一个中心创新假说表明，F-肌动蛋白周转在调节肠上皮AJC的结构和重塑中发挥关键作用。特别是，我们认为，F-肌动蛋白转换率的降低是维持健康肠道稳定连接和紧密上皮屏障的先决条件，而连接周围肌动蛋白细丝的加速转换率介导了肠道炎症过程中AJ/TJ的分解。这一假说将在以下目的得到验证：(1)检测肌动蛋白细丝翻转加速在上皮尖连接解体中的作用；(2)研究肌动蛋白解聚因子(ADF)/cofilin蛋白在肠道炎症过程中对AJC的调节作用；3)分析F-肌动蛋白交联蛋白在上皮尖连接的维持和解体中的作用。这些目标将使用暴露于不同炎症介质的体外肠道上皮细胞单层和体内小鼠肠道炎症模型来实现。肌动蛋白细丝的周转以及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、洗涤剂分级)、免疫细胞化学和遗传学(siRNA介导的敲除、显性阴性或结构性活性突变体的表达、基因敲除小鼠)相结合的方法来分析。意义：这项拟议的研究将为炎症过程中肠粘膜损伤的基本机制提供新的见解。了解这些机制将为预防消化系统疾病患者肠道屏障的破坏和促进修复提供新的治疗靶点。 与公共卫生相关：拟议的研究旨在了解肠道上皮屏障破坏的潜在机制。屏障破坏是各种消化系统疾病的常见表现，包括溃疡性结肠炎、克罗恩病、乳糜泻和感染性结肠炎。此外，肠道屏障功能障碍还会导致其他疾病的发展，如败血症休克、酒精性肝病和I型糖尿病。该项目将通过探索一种涉及粘膜炎症的启动和/或夸大的与上皮相关的新机制，为理解胃肠道疾病的发病机制提供新的见解。此外，它可能为药物预防肠屏障的破坏和加速损伤的肠粘膜愈合提供新的靶点。这可能会降低一大批炎症性疾病患者的发病率和死亡率。

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.