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。上皮屏障的完整性受称为紧密连接(TJ)和黏附连接(AJ)的细胞结构的调节。TJs和AJs的破坏是炎症性肠上皮通透性增加的基础，而连接重组对于粘膜重建过程中上皮屏障的恢复至关重要。了解肠上皮损伤和修复过程中AJ和TJ重塑的机制是本研究的主要目标。TJS和AJs与肌动蛋白细胞骨架有关，肌动蛋白细胞骨架是连接重组和完整性的重要调节因子。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至关重要的，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-肌动蛋白翻转在粘膜损伤和修复过程中上皮性AJ和TJ的重组中发挥关键作用。特别是，我认为交界周围肌动蛋白细丝的加速翻转既介导了AJ/TJ的解体，也介导了新生的AJ样接头的重塑，而F-肌动蛋白翻转的减少是TJ的建立和屏障重新密封的先决条件。这一假说将在以下目的得到验证：(1)检测肌动蛋白细丝转换在上皮性AJ和TJs分解和重建中的作用；(2)研究肌动蛋白解聚因子(ADF)/cofilin蛋白在肠上皮损伤和修复过程中连接重建中的作用；3)分析F-肌动蛋白交联蛋白在上皮连接解离和重建中的作用。这些目标将使用暴露于炎症介质的体外肠上皮细胞单层以及活体小鼠结肠炎模型和人类患者的组织活检来实现。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破坏和修复肠道上皮屏障的潜在机制。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 cellular structures known as tight junctions (TJs) and adherens junctions (AJs). Disruption of TJs and AJs underlies increased permeability of inflamed intestinal epithelium, whereas junctional reassembly is crucial for recovery of the epithelial barrier during mucosal restitution. Understanding mechanisms of AJ and TJ remodeling during intestinal epithelial damage and restitution represents the major goal of the proposed study. TJs and AJs associate with the actin cytoskeleton which is a crucial regulator of junctional reorganizations and integrity. Actin filaments are highly dynamic polymers undergoing a constant turnover (depolymerization and polymerization). Such a turnover of filamentous (F) actin is critical 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 reorganizations of epithelial AJs and TJs during mucosal damage and restitution. Particularly, I propose that accelerated turnover of perijunctional actin filaments mediates both AJ/TJ disassembly and reformation of nascent AJ-like junctions, whereas decreased F-actin turnover is prerequisite for the establishment of TJs and barrier resealing. This hypothesis will be tested in the following Aims: (1) To examine the role of actin filament turnover in disassembly and reformation of epithelial AJs and TJs; (2) to investigate the involvement of actin-depolymerizing factor (ADF)/cofilin proteins in junctional remodeling during intestinal epithelial damage and restitution; 3) to analyze the role of F-actin cross-linking proteins in disassembly and reestablishment of epithelial junctions. These aims will be accomplished using in vitro intestinal epithelial cell monolayers exposed to inflammatory mediators as well as in vivo mouse model of colitis and tissue biopsies of human patients. 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 provide 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 and restoration 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.