Physiological Regulation of Intestinal Epithelial Transport and Barrier Function

描述（由申请人提供）：肠道运输缺陷是许多肠道疾病的核心组成部分。这种运输以营养物质、离子和水分吸收的形式，通过特定的跨细胞转运蛋白以及被动的跨上皮紧密连接的细胞旁运动来完成。一种新兴的观点认为，个体转运蛋白可以相互调节，也可以调节细胞旁通透性，从而协调调节肠道运输。我们对Na+-葡萄糖共转运依赖的紧密连接调节的研究支持了这一概念，研究表明，顶端Na+-葡萄糖共转运蛋白SGLT1激活信号转导级联，依次触发顶端Na+-H+交换剂NHE3传递到质膜，增加顶端Na+-H+交换，肌球蛋白轻链（MLC）激酶激活，MLC磷酸化，肌动球蛋白收缩，增加紧密连接通透性。尽管有这些进展和其他进展，但泡泡运输、蛋白质相互作用和肌动球蛋白收缩调节肠上皮运输和屏障功能的机制在很大程度上仍不明确。这个关键的差距限制了我们理解肠道运输和屏障功能障碍疾病的机制，包括感染性、炎症性和吸收不良腹泻疾病。因此，本应用的目的是确定激活膜运输和修饰蛋白质相互作用的信号转导途径参与调节肠道运输和屏障功能的机制。我们将通过三个具体目标来实现这些目标：为了明确ezrin在蛋白向质膜传递的急性调控中的作用，2 .确定确定紧密连接处蛋白质动态行为的机制；明确肌动球蛋白依赖性紧密连接维持和调控的机制和意义。这些研究将在体外和体内模型中进行，包括对活细胞和组织中表达的荧光融合蛋白的成像，以及对紧结通透性转基因操作的免疫激活的体内分析。因此，我们将显著推进我们对细胞膜、蛋白质和细胞骨架动力学对转运和屏障功能调节的机制的理解。除了提供新的基础知识外，预计这将对人类健康产生重大的积极影响，因为它将允许合理地开发新的治疗策略，以治疗运输或屏障功能不足的疾病。

DESCRIPTION (provided by applicant): Defective intestinal transport is a central component of many intestinal diseases. Such transport, in the form of nutrient, ion, and water absorption, is accomplished by specific transcellular transporters as well as passive paracellular movement across the epithelial tight junction. An emerging view is that individual transporters can regulate one another as well as paracellular permeability, resulting in coordinate regulation of intestinal transport. This concept is supported by our studies of Na+-glucose cotransport-dependent tight junction regulation showing that SGLT1, the apical Na+-glucose cotransporter, activates a signal transduction cascade that sequentially triggers delivery of NHE3, an apical Na+-H+ exchanger, to the plasma membrane, increased apical Na+-H+ exchange, myosin light chain (MLC) kinase activation, MLC phosphorylation, actomyosin contraction, and increased tight junction permeability. Despite these and other advances, the mechanisms by which vesicular transport, protein interactions, and actomyosin contraction effect regulation of intestinal epithelial transport and barrier function remain largely undefined. This critical gap limits our ability to understand the mechanisms of diseases with intestinal transport and barrier dysfunction, including infectious, inflammatory, and malabsorptive diarrheal diseases. Thus, the objectives of this application are to define the mechanisms by which signal transduction pathways that activate membrane traffic and modify protein interactions are involved in regulation of intestinal transport and barrier function. We will accomplish these objectives through three specific aims: 1. To define the role of ezrin in acute regulation of protein delivery to the plasma membrane, 2. To identify the mechanisms that define the dynamic behavior of proteins at the tight junction, and 3. To define the mechanisms and significance of actomyosin-dependent tight junction maintenance and regulation. These studies will be performed using in vitro and in vivo models that include imaging of fluorescent fusion proteins expressed in living cells and tissues and in vivo analysis of immune activation secondary to transgenic manipulation of tight junction permeability. As a result we will significantly advance our understanding of the mechanisms by which membrane, protein, and cytoskeletal dynamics contribute to regulation of transport and barrier function. In addition to providing new fundamental knowledge this is expected to have significant positive effects on human health because it will allow the rational development of new therapeutic strategies for diseases with deficient transport or barrier function.