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器官暴露于肠道微生物产物，从而加重粘膜炎症，增加全身炎症反应的风险。肠道屏障的破坏是由特殊上皮结构、紧密连接（TJs）和粘附连接（AJs）的破坏引起的。许多炎症介质，包括细胞因子、一氧化氮和活性氧，已知会破坏AJ和TJ结构。了解肠道炎症期间上皮连接层解体的机制是本研究的主要目标。一种新兴的观点认为，TJ和AJ经历了一个持续的重塑过程，包括通过内吞作用从质膜上去除老化的连接成分，并通过胞吐作用传递新的TJ/AJ蛋白。该提议的一个核心创新假设认为，肠道炎症通过阻断囊泡介导的连接蛋白的胞吐作用，中断了上皮AJs和TJs的稳态重塑。这种AJ/TJ胞外分泌的抑制是通过抑制调节囊泡与质膜融合的蛋白的表达和/或活性发生的，即n -乙基丙烯酰亚胺敏感因子（NSF）、可溶性NSF受体（SNAREs）和NSF-附着蛋白（1SNAP）。SNARE/NSF/ 1snap介导的连接蛋白运输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。我们将在以下目的中验证这一假设：(1)：在体外和体内研究snare介导的胞吐作用对上皮连接结构和功能的调节；(2)确定NSF氧化修饰在自由基诱导的上皮连接断裂中的作用；3)分析1SNAP在粘膜损伤和恢复过程中上皮连接的拆卸和恢复中的作用。这些目标将通过暴露于促炎介质的体外肠上皮细胞单层以及体内肠道炎症小鼠模型来实现。囊泡融合机制将通过生化（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）方法的组合来分析。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。了解这些机制将有可能提供新的治疗靶点，以防止消化系统疾病患者肠道屏障的破坏。

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.