Actomyosin cytoskeleton and the regulation of intestinal eipithelial barrier

 描述（由申请方提供）：肠上皮屏障破坏是胃肠道疾病（包括炎症性肠病、乳糜泻和感染性结肠炎）的重要表现。上皮屏障的完整性由称为紧密连接（TJ）和粘附连接（AJ）的专门粘附结构介导。有证据表明，AJ和TJ在发炎的肠粘膜中分解，造成肠道屏障的泄漏。了解在健康肠道中调节连接完整性并在粘膜炎症期间驱动AJ/TJ分解的机制是所提出的研究的主要目标。TJ和AJ的完整性和重塑依赖于肌动球蛋白细胞骨架组成的肌动蛋白丝和一个专门的运动蛋白非肌肉肌球蛋白（NM）II。NM II作为不同重链和轻链的分子系综起作用。重链负责该马达的所有功能性活动，包括肌动蛋白结合、ATP水解和肌内营养。 肌丝组装令人惊讶的是，很少有人知道NM II重链在正常和发炎的肠上皮细胞中的作用和调节。该提议的核心创新假设提出NM II重链（马达）是健康肠道中上皮屏障的建立和维持的关键调节剂，并且这些马达的表达/组装受损导致屏障破坏并在粘膜炎症期间抑制上皮恢复。本研究的目的是：（1）确定不同NM Ⅱ重链在体内肠上皮屏障的维持、破坏和恢复中的作用;（2）研究NM Ⅱ伴侣蛋白，β-45 A，在上皮屏障完整性和恢复的调节中的作用;（3）分析Septin cytokinin NM Ⅱ在肠上皮屏障组装和重建中的作用。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小鼠模型的体内细胞来实现。将通过功能（渗透性测量、伤口愈合）、生物化学（肌动蛋白共沉降、免疫印迹、去污剂分级）、免疫细胞化学和遗传（siRNA介导的敲除、显性阴性突变体、敲除小鼠）方法的组合分析不同NM II重链和NM II靶向分子伴侣和隔膜蛋白的功能。重要性：这项研究将对调节正常上皮屏障和介导炎症期间肠粘膜损伤和恢复的基本机制产生新的见解。了解这些机制将提供新的治疗靶点，以防止消化系统疾病患者的肠道屏障破坏和增强修复。

 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.