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（由适用提供）：肠上皮屏障的破坏是胃肠道疾病的关键表现，包括炎症性肠病，腹腔疾病和感染性结肠炎。上皮屏障的完整性是由称为紧密连接（TJ）的专业粘合剂结构和粘附连接（AJ）介导的。有证据表明，AJ和TJ在发炎的肠粘膜中拆解，从而形成了肠道障碍的渗漏。理解在粘膜注射过程中调节健康肠道并驱动AJ/TJ拆卸的连接完整性的机制是拟议研究的主要目标。 TJ和AJ的完整性和重塑取决于肌动蛋白细胞骨架由肌动蛋白丝和专用运动蛋白非肌肉肌球蛋白（NM）II组成。 NM II是不同重链和轻链的分子集合。重链负责该电机的所有功能活动，包括肌动蛋白结合，ATP水解和 令人惊讶的是，关于NM II重链在正常和发炎的肠上皮上皮的作用和法规知之甚少。该提案的中心创新假设是，NM II重链（电动机）是健康肠道中上皮屏障的建立和维护的关键调节因子，并且这些电动机的表达/组装受损会导致屏障破坏并抑制粘膜注射过程中上皮约束。该假设将在以下目的中进行检验：（1）确定不同NM II重链在体内肠上皮屏障的维持，破坏和限制中的作用； （2）研究NM II链UNC-45A的参与，以调节上皮屏障完整性和约束； 3）分析septin cytoskeletin nm II组装和肠上皮屏障的重塑的作用。这些目标将使用暴露于炎症介质的体外肠上皮细胞和使用结肠炎的鼠模型在体内实现。 The functions of different NM II heavy chains and NM II-targeting chainers and septins will be analyzed via a combination of functional (permeability measurements, wound healing), biochemical (actin co-sedimentation, immunoblotting, determining fractionation), immunocytochemical, and genetic (siRNA-mediated knock-downs, dominant-negative mutants, knockout mice) approaches.意义：拟议的研究将对调节正常上皮屏障和中间肠粘膜损伤和炎症期间的基本机制产生新的见解。了解这些机制将提供新的治疗靶标，以防止消化疾病患者的肠道屏障分解和增强反应。