Physiological Regulation of Intestinal Epithelial Transport and Barrier Function

描述(申请人提供)：肠道运输缺陷是许多肠道疾病的中心组成部分。这种转运，以营养、离子和水分吸收的形式，通过特定的细胞转运蛋白和被动的细胞旁运动穿过上皮紧密连接来完成。一种新的观点认为，单个转运体可以相互调节，也可以调节细胞旁的通透性，从而导致对肠道运输的协调调节。我们对钠-葡萄糖共转运依赖的紧密连接调控的研究表明，钠-葡萄糖共转运体SGLT1激活了一个信号转导级联，从而触发了顶端Na+-H+交换蛋白NHE3的传递到质膜，增加了顶端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，并增加了紧密连接通透性。尽管有这些和其他方面的进展，但囊泡运输、蛋白质相互作用和肌动球蛋白收缩对肠上皮运输和屏障功能的调节作用的机制在很大程度上仍不清楚。这一关键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的能力，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改变蛋白质相互作用的信号转导通路参与肠道转运和屏障功能调节的机制。我们将通过三个特定的目标来实现这些目标：1.确定Ezrin在蛋白质转运到质膜的急性调节中的作用；2.确定定义紧密连接蛋白质动态行为的机制；3.确定肌动球蛋白依赖的紧密连接维持和调节的机制和意义。这些研究将使用体外和体内模型进行，包括对活细胞和组织中表达的荧光融合蛋白进行成像，以及对转基因操作紧密连接通透性的继发免疫激活进行体内分析。因此，我们将极大地促进我们对膜、蛋白质和细胞骨架动力学对运输和屏障功能调节的机制的理解。除了提供新的基本知识外，预计这将对人类健康产生重大积极影响，因为它将使人们能够合理地制定新的治疗策略，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.