Actomyosin cytoskeleton and the regulation of intestinal eipithelial barrier

肌动球蛋白细胞骨架与肠上皮屏障的调节

• 批准号: 9606158
• 负责人: Andrei Ivanovich Ivanov
• 金额: $ 29.55万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9606158
• 关键词: Actomyosin; cytoskeleton; regulation; intestinal; eipithelial

 DESCRIPTION (provided by applicant): Disruption of the intestinal epithelial barrier is a crucial manifestation of gastrointestinal disorders including inflammatory bowel disease, celiac disease, and infectious colitis. Integrity of the epithelial barrier is mediated by specialized adhesive structures known as tight junctions (TJ) and adherens junctions (AJ). Evidence suggests that AJ and TJ become disassembled in inflamed intestinal mucosa creating a leakiness of the gut barrier. Understanding mechanisms that regulate junctional integrity in healthy gut and drive AJ/TJ disassembly during mucosal inflammation is the major goal of the proposed study. Integrity and remodeling of TJ and AJ depend on the actomyosin cytoskeleton composed of actin filaments and a specialized motor protein non-muscle myosin (NM) II. NM II works as a molecular ensemble of different heavy chains and light chains. The heavy chains are responsible for all functional activities of this motor including actin binding, ATP hydrolysis and myofilament assembly. Surprisingly, little is known about the role and regulations of NM II heavy chains in normal and inflamed intestinal epithelium. The central innovative hypothesis of this proposal proposes that NM II heavy chains (motors) are critical regulators of the establishment and maintenance of the epithelial barrier in healthy gut and that impaired expression/assembly of these motors results in barrier disruption and inhibited epithelial restitution during mucosal inflammation. This hypothesis will be tested in the following Aims: (1) to determine the roles of different NM II heavy chains in maintenance, disruption, and restitution of the intestinal epithelil barrier in vivo; (2) to investigate the involvement of NM II chaperon, UNC-45A, in the regulation of epithelial barrier integrity and restitution; 3) to analyze the roles of the septin cytoskeletonin NM II assembly and remodeling of the intestinal epithelial barrier. These aims will be accomplished using in vitro intestinal epithelial cells exposed to inflammatory mediators and in vivo using murine models of colitis. The functions of different NM II heavy chains and NM II-targeting chaperons and septins will be analyzed via a combination of functional (permeability measurements, wound healing), biochemical (actin co-sedimentation, immunoblotting, detergent fractionation), immunocytochemical, and genetic (siRNA-mediated knock-downs, dominant-negative mutants, knockout mice) approaches. Significance: the proposed study will yield new insights into fundamental mechanisms that regulate normal epithelial barriers and mediate intestinal mucosal injury and restitution 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.

 描述（由申请人提供）：肠上皮屏障的破坏是胃肠道疾病的重要表现，包括炎症性肠病、乳糜泻和感染性结肠炎。上皮屏障的完整性是由称为紧密连接（TJ）的特殊粘合结构介导的。有证据表明，AJ 和 TJ 在发炎的肠粘膜中分解，导致肠道屏障渗漏。调节健康肠道的连接完整性并在粘膜炎症期间驱动 AJ/TJ 解体是本研究的主要目标 TJ 和 AJ 的完整性和重塑取决于由肌动蛋白丝和特殊运动蛋白非肌肉肌球蛋白组成的肌动球蛋白细胞骨架。 (NM) II。NM II 作为不同重链和轻链的分子整体，负责该马达的所有功能活动，包括肌动蛋白结合、ATP 水解。和 令人惊讶的是，人们对 NM II 重链在正常和发炎肠上皮中的作用和调节知之甚少，该提案的核心创新假设提出，NM II 重链（马达）是肌丝组装的关键调节因子。健康肠道中的上皮屏障以及这些马达的表达/组装受损导致屏障破坏并在粘膜炎症期间抑制上皮恢复，该假设将在下面进行检验。目的：(1) 确定不同 NM II 重链在体内肠上皮屏障维持、破坏和恢复中的作用；(2) 研究 NM II 伴侣 UNC-45A 在调节中的作用；上皮屏障的完整性和恢复；3) 分析隔膜细胞骨架在肠上皮屏障的 NM II 组装和重塑中的作用这些目标将通过体外肠上皮来实现。暴露于炎症介质的细胞和体内使用结肠炎小鼠模型将通过功能（渗透性测量、伤口愈合）、生化（肌动蛋白co）的组合来分析不同NM II重链和NM II靶向分子伴侣和败血症的功能。 -沉淀、免疫印迹、洗涤剂分级）、免疫细胞化学和遗传（siRNA 介导的敲除、显性失活突变体、敲除小鼠）方法 意义：拟议的研究。将对调节正常上皮屏障和介导炎症过程中肠粘膜损伤和恢复的基本机制产生新的见解，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增强修复提供新的治疗靶点。