Cadherin Regulation of Epithelial Barriers

描述(申请人提供)：在生理和病理生理过程中，上皮细胞在组织间形成动态屏障，并调节其渗透性。不适当地维持或控制上皮细胞旁屏障会导致许多疾病过程，尤其是炎症。钙粘附素在调节内皮细胞和上皮细胞旁通透性方面尤为重要，而钙粘附素-连环蛋白系统与炎症性疾病有直接的关系。来自我的实验室的新发现为E-钙粘蛋白在上皮细胞中的调节机制提供了重要的新见解，以及从实验上操纵钙粘附素活性的新方法。我们发现了一种机制，可以控制细胞表面钙粘附素的构象和黏附活性，而不依赖于相关连环蛋白表达水平或数量的任何变化。这包括E-钙粘蛋白激活型单抗的发现，以及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，粘附性亲和键本身的调节是细胞连接调节中的关键事件，允许连接素相关的细胞骨架在粘附键断裂后将连接分开，并且这一机制在炎症过程中对上皮和内皮细胞的屏障调节都起到了作用。该方案的总体目标是确定钙粘蛋白细胞表面调节在控制上皮屏障功能中的作用，并更深入地了解钙粘附素在细胞表面调节的机制。其具体目的是：1.研究E-钙粘附素活性状态在炎症过程中上皮屏障调节中的作用I、细胞培养和动物模型。可溶性炎性介质的调节，中性粒细胞迁移的控制，以及树突状细胞与上皮细胞的相互作用的调节将被研究。E-钙粘附素在体内的调节作用将在肺部炎症的动物模型中进行评估。B.确定调节细胞表面钙粘附素黏附结合活性的机制。对活性相关单抗识别的表位结构的分析，高亲和性粘附键性质的生物物理变化，以及钙粘蛋白寡聚、聚集和定位于粘附点的作用，都将提供见解。 细胞外粘附域是如何被调节的。C.研究E-钙粘附素激活和细胞表面调节的细胞质机制。确定控制p120-连环蛋白磷酸化的酶，微管在控制E-钙粘蛋白活性和p120-连环蛋白磷酸化中的作用，以及p120-连环蛋白磷酸化控制黏附活性的效应器，将为深入了解黏附调节的分子机制提供帮助。这些研究的发现可能会让我们开发基于钙粘附素的方法，甚至是治疗方法，来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.