Cadherin Regulation of Epithelial Barriers

描述（由申请方提供）：上皮在组织隔室之间形成动态屏障，并在生理和病理生理过程中调节其渗透性。上皮细胞旁屏障的不适当维持或控制有助于许多疾病过程，特别是炎症。钙粘蛋白对于调节上皮细胞和内皮细胞中的细胞旁通透性特别重要，并且钙粘蛋白-连环蛋白系统已直接涉及炎性疾病。本实验室的新发现为上皮细胞中E-钙粘蛋白的调节机制提供了重要的新见解，并为实验性地操纵钙粘蛋白活性提供了新的方法。我们发现了一种机制，控制钙粘蛋白的构象和粘附活性在细胞表面的表达水平或相关的连环蛋白的量的任何变化无关。这包括发现E-钙粘蛋白激活单克隆抗体（mAb）和p120-连环蛋白磷酸化和微管在控制E-钙粘蛋白活性状态中的作用。我假设，调节的粘合剂homophilic键本身是一个关键的事件，在细胞连接调节，使连环蛋白相关的细胞骨架拉除了一旦粘合剂键被打破，并在炎症过程中，这种机制在上皮和内皮细胞的屏障调节中发挥作用。这项建议的总体目标是确定上皮屏障功能的控制中钙粘蛋白细胞表面调节的作用，并更深入地了解钙粘蛋白在细胞表面调节的机制。具体目标是：A.在细胞培养和动物模型中研究E-钙粘蛋白活性状态在炎症过程中对上皮屏障的调节作用。可溶性炎症介质的调节，中性粒细胞迁移的控制，以及树突状细胞与上皮细胞相互作用的调节将被研究。将在肺部炎症的动物模型中评估E-钙粘蛋白调节在体内的作用。B。确定调节细胞表面钙粘蛋白粘附结合活性的机制。对活性相关单克隆抗体识别的表位结构的分析，嗜同性粘附键性质的生物物理变化，以及粘附位点中钙粘蛋白寡聚化、聚集和定位的作用，都将提供见解 细胞外粘附结构域是如何调节的。C.研究E-cadherin激活和细胞表面调节的细胞质机制。确定控制p120-catenin磷酸化的酶，微管在控制E-钙粘蛋白活性和p120-catenin磷酸化中的作用，以及p120-catenin磷酸化控制粘附活性的效应物，将为粘附调节的分子机制提供深入了解。这些研究的发现可能使我们能够开发基于钙粘蛋白的方法，甚至治疗方法，以操纵炎症和相关疾病过程中的屏障功能。

DESCRIPTION (provided by applicant): Epithelia form dynamic barriers between tissue compartments and modulate their permeability properties during physiological and pathophysiological processes. Inappropriate maintenance or control of epithelial paracellular barriers contributes to many disease processes, especially inflammation. Cadherins are particularly important for the regulation of paracellular permeability in epithelia as well as endothelia, and the cadherin-catenin system has been directly implicated in inflammatory disease. New findings from my laboratory provide important new insights into the mechanisms of E-cadherin regulation in epithelia as well as novel approaches to manipulate cadherin activity experimentally. We discovered a mechanism that controls cadherin conformation and adhesive activity at the cell surface independent of any changes in levels of expression or amounts of associated catenins. This included the discovery of E-cadherin activating monoclonal antibodies (mAbs) and a role for p120-catenin phosphorylation and microtubules in the control of E-cadherin activity state. I hypothesize that regulation of the adhesive homophilic bond itself is a key event in cell junction regulation, allowing the catenin-associated cytoskeleton to pull the junctions apart once the adhesive bond is broken, and that this mechanism plays a role in barrier regulation in both epithelia and endothelia during inflammatory processes. The overall goals of this proposal are to determine the roles of cadherin cell surface regulation in the contro of epithelial barrier function and to understand in greater depth the mechanisms by which cadherins are regulated at the cell surface. The specific aims are: A. Investigate the role of E-cadherin activity state in the regulation of the epithelial barrier during inflammatory processes i cell culture and animal models. Regulation by soluble inflammatory mediators, control of neutrophil transmigration, and regulation of the interactions of dendritic cells with epithelium wil be investigated. The role of E-cadherin regulation in vivo will be assessed in an animal model of lung inflammation. B. Determine the mechanisms regulating cadherin adhesive binding activity at the cell surface. Analysis of the structure of epitopes recognized by activity associated mAbs, the biophysical changes in the properties of the homophilic adhesive bond, and the role of cadherin oligomerization, clustering, and localization in adhesion sites, all will provide insights into how the extracellular adhesive domain is regulated. C. Investigate cytoplasmic mechanisms of E-cadherin activation and cell surface regulation. Determining the enzymes that control p120-catenin phosphorylation, the role of microtubules in controlling E-cadherin activity and p120-catenin phosphorylation, and the effectors through which p120-catenin phosphorylation controls adhesion activity, will provide insights into the molecular mechanisms underlying adhesion regulation. Findings from these studies may allow us to develop cadherin- based approaches, or even therapeutics, to manipulate barrier function during inflammation and related disease processes.