Junctional exocytosis and breakdown of the intestinal barrier in inflammation

描述(申请人提供)：肠上皮通透性增强是肠道炎症性疾病的关键机制。这种肠道渗漏使内脏暴露于管腔微生物产物，因此夸大了粘膜炎症，增加了全身炎症反应的风险。肠道屏障的破坏是由特殊的上皮结构、紧密连接(TJ)和黏附连接(AJ)的分解引起的。已知许多炎症介质，包括细胞因子、一氧化氮和活性氧物种，可以破坏AJ和TJ的结构。了解肠道炎症过程中上皮细胞连接解离的机制是拟议研究的主要目标。一种新的观点认为，TJs和AJs经历了一种持续的重塑，包括通过内吞作用将老化的连接成分从质膜上移除，并通过胞吞作用输送新的TJ/AJ蛋白。这一提议的一个中心创新假说表明，肠道炎症通过阻断小泡介导的结合蛋白的胞吐来干扰上皮AJ和TJ的稳态重构。这种对AJ/TJ胞吐的抑制是通过抑制调控囊泡与质膜融合的蛋白的表达和/或活性来实现的，这些蛋白是N-乙基马来酰亚胺敏感因子(NSF)、可溶性NSF受体(SNARs)和NSF附着蛋白(1SNAP)。SNARE/NSF/1SNAP介导的连接蛋白转运功能障碍可能最终导致AJ/TJ结构缺陷和肠屏障通透性增加。我们将在以下目的验证这一假说：(1)研究SNARE介导的胞吐作用在体内外上皮连接结构和功能调节中的作用；(2)确定NSF的氧化修饰在自由基诱导的上皮连接解离中的作用；3)分析1SNAP在黏膜损伤和修复过程中上皮连接解离和修复中的作用。这些目标将使用暴露于促炎介质的体外肠道上皮细胞单层以及体内小鼠肠道炎症模型来实现。囊泡融合机制将通过生物化学(生物素化、免疫沉淀、亲和层析)、免疫细胞化学和遗传学(SNARE蛋白的siRNA敲除、NSF和1SNAP突变体的过表达)相结合的方法进行分析。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。了解这些机制可能会为预防消化系统疾病患者肠道屏障的破坏提供新的治疗靶点。 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、慢性疾病、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): Enhanced permeability of intestinal epithelium is a key mechanism of inflammatory diseases of the gut. Such intestinal leakiness exposes internal organs to luminal microbial products, therefore exaggerating mucosal inflammation and increasing the risk of systemic inflammatory responses. Breakdown of the intestinal barrier is caused by disassembly of specialized epithelial structures, tight junctions (TJs) and adherens junctions (AJs). Many inflammatory mediators, including cytokines, nitric oxide and reactive oxygen species, are known to disrupt AJ and TJ structure. Understanding mechanisms of epithelial junctional disassembly during intestinal inflammation represents the major goal of the proposed study. An emerging view is that TJs and AJs undergo a continuous remodeling consisting of the removal of aging junctional components from the plasma membrane by endocytosis and delivery of new TJ/AJ proteins via exocytosis. A central innovative hypothesis of this proposal implies that intestinal inflammation interrupts a steady-state remodeling of epithelial AJs and TJs by blocking vesicle-mediated exocytosis of junctional proteins. This suppression of AJ/TJ exocytosis occurs via inhibition of expression and/or activity of proteins regulating vesicle fusion with the plasma membrane, namely the N-ethylmaleimide sensitive factor (NSF), soluble NSF receptors (SNAREs) and NSF- attachment protein (1SNAP). Dysfunction of SNARE/NSF/1SNAP-mediated trafficking of junctional proteins is likely to eventuate in the defective AJ/TJ structure and increased intestinal barrier permeability. We will test this hypothesis in the following Aims: (1): to investigate the involvement of SNARE-mediated exocytosis in regulation of epithelial junctional structure and functions in vitro and in vivo; (2) to determine the role of oxidative modification of NSF in free-radical induced disassembly of epithelial junctions; 3) to analyze the role of 1SNAP in disassembly and recovery of epithelial junctions during mucosal damage and restitution. These aims will be accomplished using in vitro intestinal epithelial cell monolayers exposed to proinflammatory mediators as well as in vivo murine models of intestinal inflammation. Vesicle fusion machinery will be analyzed by a combination of biochemical (biotinylation, immunoprecipitation, affinity chromatography), immunocytochemical and genetic (siRNA knock-down of SNARE proteins, overexpression of NSF and 1SNAP mutants) approaches. Significance: the proposed study will provide new insights into fundamental mechanisms of intestinal mucosal injury during inflammation. Understanding these mechanisms will potentially provide new therapeutic targets to prevent breakdown of the intestinal 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, Chron'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.