Junctional exocytosis and breakdown of the intestinal barrier in inflammation

炎症中的连接胞吐作用和肠道屏障的破坏

• 批准号: 8465630
• 负责人: Andrei Ivanovich Ivanov
• 金额: $ 30.71万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8465630
• 关键词: Adherens Junction; Affinity Chromatography; Aging; Alcoholic Liver Diseases; Biochemical; Biotinylation; Celiac Disease; Cell membrane; Chemicals; Colitis; Complex; Development; Digestive System Disorders; Disease; Endocytosis; Epithelial; Epithelial Cells; Epithelium; Event; Excision; Exocytosis; Extracellular Space; Free Radicals; Functional disorder; Gastrointestinal Diseases; Genes; Genetic; Goals; Healed; Immunoprecipitation; In Vitro; Inflammation; Inflammation Mediators; Inflammatory; Inflammatory Bowel Diseases; Inflammatory Response; Inflammatory disease of the intestine; Injury; Insulin-Dependent Diabetes Mellitus; Intestinal Mucosa; Intestines; Link; Maintenance; Mediating; Mediator of activation protein; Membrane; Membrane Fusion; Missense Mutation; Modeling; Modification; Molecular; Morbidity - disease rate; Mucositis; Mucous Membrane; Mus; Mutagenesis; N-ethylmaleimide-sensitive protein; Nitric Oxide; Nitric Oxide Donors; Organ; Pathogenesis; Patients; Permeability; Phosphorylation; Play; Prevention; Protein Isoforms; Protein Overexpression; Proteins; RNA Interference; Reactive Oxygen Species; Recovery; Regulation; Research; Resistance; Risk; Role; S-nitro-N-acetylpenicillamine; SNAP receptor; Septic Shock; Small Interfering RNA; Structure; Testing; Tight Junctions; Tissues; Ulcerative Colitis; Vesicle; cohort; cytokine; design; healing; in vivo; injured; innovation; insight; intestinal epithelium; knock-down; microbial; monolayer; mortality; mutant; new therapeutic target; novel; overexpression; pathogen; prevent; protein complex; protein expression; public health relevance; receptor; research study; restoration; seal; syntaxin-2; trafficking

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.

描述（由申请人提供）：肠上皮的渗透性增强是肠道炎症性疾病的关键机制。这种肠泄漏性暴露于腔内微生物产物中，因此夸大了粘膜炎症并增加了系统性炎症反应的风险。肠屏障的崩溃是由专业上皮结构，紧密连接（TJ）和粘附连接（AJS）拆卸引起的。已知许多炎症介质，包括细胞因子，一氧化氮和活性氧，都会破坏AJ和TJ结构。理解肠道炎症期间上皮连接式拆卸的机制是拟议研究的主要目标。一个新兴的观点是，TJ和AJS经历了连续的重塑，该重塑包括通过内吞作用和通过胞胞吞作用递送新的TJ/AJ蛋白从质膜中去除衰老的连接成分。该提案的中心创新假设意味着，肠道炎症通过阻断囊泡介导的连接蛋白的胞吐作用，从而打断上皮AJ和TJ的稳态重塑。 AJ/TJ胞吐作用的这种抑制是通过抑制与质膜调节囊泡融合的蛋白质的表达和/或活性发生的，即N-乙基美酰胺敏感因子（NSF），可溶性NSF受体（SENRES）和NSF-附着蛋白（1SNAP）。 SNARE/NSF/1SNAP介导的连接蛋白的运输功能障碍可能会在有缺陷的AJ/TJ结构中发生，并增加肠道屏障的渗透性。我们将在以下目的中检验这一假设：（1）：研究军鼓介导的胞吐作用参与体外和体内的上皮结构结构和功能的调节； （2）确定NSF氧化修饰在自由基诱导的上皮连接中的拆卸中的作用； 3）分析1SNAP在粘膜损伤和恢复过程中拆卸和上皮连接的恢复中的作用。这些目标将使用暴露于促炎性介体的体外肠上皮细胞单层以及肠炎的体内鼠模型。囊泡融合机械将通过生化（生物素化，免疫沉淀，亲和力色谱法），免疫细胞化学和遗传学（snare蛋白的siRNA敲低，NSF和1SNAP突变体的过表达）的组合来分析。意义：拟议的研究将为炎症期间肠粘膜损伤的基本机制提供新的见解。了解这些机制将有可能提供新的治疗靶标，以防止消化疾病患者的肠壁障碍分解。 公共卫生相关性：拟议的研究旨在了解破坏和恢复肠上皮屏障的基础机制。障碍分解是不同胃肠病学疾病，包括溃疡性结肠炎，Chron病，腹腔疾病和感染性结肠炎的常见表现。此外，肠道障碍的功能障碍有助于其他疾病的发展，例如败血性休克，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引发和/或夸大的新型上皮相关机制，为了解胃肠道疾病的发病机理提供新的见解。此外，它可能为预防肠道屏障分解和加速损伤肠粘膜愈合的药理预防提供新的靶标。这可能导致大量炎症性疾病患者的发病率和死亡率降低。