Tight Junction Barriers in the Gastrointestinal Tract

描述（由申请人提供）：肠道紧密连接屏障的选择性丧失和通透性增加导致炎症和腹泻。解释和纠正这些病理变化的一个障碍是，我们缺乏对屏障的分子结构及其大小和电荷选择性和渗透性的基础的理解。最近的数据支持通过结的两种不同途径的存在：一种是由小尺寸和电荷选择性孔形成的；第二种允许较大溶质的体积无关流动，可能是病理性“渗漏”的重要组成部分。我们的长期目标是了解紧密连接的特定蛋白质成分如何定义这些不同途径的选择性和渗透性，以便肠道屏障可以用于治疗目的，中心假设是特定的跨膜紧密连接和粘附蛋白控制选择性和渗透性的不同方面。强有力的初步研究支持这一假设，将在三个具体目标中进行测试。(1) claudin、occludin和tricellulin是否决定小细胞旁孔的数量和大小？根据我们已发表的和初步的研究，我们假设是克劳丁创造了孔隙，并可能赋予不同的孔隙大小和数量；其他粘附蛋白如occludin可能控制孔隙度。这些蛋白在调节过选择性中的作用将在培养的Caco-2和其他上皮单分子层的过表达、突变和siRNA敲低研究中进行测试。(2)在基线和生理变化过程中，非粘连蛋白粘附分子是否会影响大小无关的途径？我们的工作假设是，大小无关途径的有效性是由粘附分子如occludin， tricellulin， JAM-A和Ig- super家族的其他成员控制的。这些蛋白的作用将在体外培养的上皮模型（即激活肌球蛋白磷酸化途径）和小鼠肠道（在JAM-A缺失的小鼠中）通过生理刺激敲除和增强渗透性后进行测试。(3)确定claudin相互作用的结构基础。我们的工作假设是，胞苷单体在细胞膜内和细胞旁空间相互作用，形成孔和连续的屏障。实验将确定这些相互作用的氨基酸序列规则；以及Blue-Native PAGE中分离的650kDa含cludin复合物的组成。整体方法是创新的，因为分析分析可以量化孔隙的大小和数量以及非限制性路径的渗透率水平。这项工作的意义在于，它将确定每种蛋白质对控制屏障选择性和渗透性的贡献。这些信息可用于解释特定蛋白质变化在疾病中的作用，并开始设计选择性靶向策略，以保护和恢复肠屏障或增强经黏膜药物传递。公共卫生相关性：拟议的研究旨在从分子水平上了解肠上皮如何在肠内容物（包括营养物质和潜在有害细菌和抗原）与身体其他部分之间形成选择性屏障。该项目的公共卫生意义在于，选择性的丧失和通透性的增加会导致许多肠道疾病，包括腹泻和炎症。这些发现将指导保护和恢复肠屏障或增强经黏膜药物传递的渗透性的策略。

DESCRIPTION (provided by applicant): Loss of selectivity and increased permeability of the intestinal tight junction barrier contributes to inflammation and diarrhea. An impediment to interpreting and correcting these pathologic changes is that we lack an understanding of the molecular structure of the barrier and the basis for its size- and charge- selectivity and permeability. Recent data support the existence of two distinct pathways through the junction: one is formed by small size- and charge-selective pores; the second allows bulk size-independent flow of larger solutes and is likely to be an important component of pathologic "leakiness". Our long range goal is to understand how specific protein components of the tight junction define selectivity and permeability of these distinct pathways so that the intestinal barrier can be manipulated for therapeutic purposes, with the central hypothesis that specific transmembrane tight junction and adhesive proteins control different aspects of selectivity and permeability. Strong Preliminary Studies support this hypothesis, which will be tested in three Specific Aims. (1) Do claudins, occludin and tricellulin determine the number and size of the small paracellular pores? Based on our published and Preliminary Studies we hypothesize that claudins create the pores and may confer different pore sizes and numbers; other adhesion proteins like occludin may control porosity. The role of these proteins in regulating permselectivity will be tested in overexpression, mutational and siRNA knockdown studies in cultured Caco-2 and other epithelial monolayers. (2) Is the size-independent pathway influenced by non-claudin adhesion molecules at baseline and during physiologic changes? Our working hypothesis is that effectiveness of the size-independent pathway is controlled by adhesion molecules like occludin, tricellulin, JAM-A and other members of the Ig- super family. The role of these proteins will be tested after knockdown and after permeability enhancement by physiologic stimuli in cultured epithelial models (i.e. activating myosin phosphorylation pathways) and in mouse intestine (in JAM-A null mice). (3) Determine the structural basis for claudin interactions. Our working hypothesis is that claudin monomers interact both within one cell membrane and across the paracellular space to create the pores and the continuous barrier. Experiments will define the amino acid sequence rules for these interactions; and the composition of a 650kDa claudin-containing complex isolated in Blue-Native PAGE. The overall approach is innovative because of a profiling assay which allows quantification of the size and number of pores and level of permeability of the non-restrictive path. The significance of this work is that it will define the contribution of each protein to controlling barrier selectivity and permeability. This information can be used to interpret the role of specific protein changes in disease and to begin to design selective targeted strategies to preserve and restore the intestinal barrier or enhance transmucosal drug delivery. PUBLIC HEALTH RELEVANCE: The proposed studies are aimed at understanding at a molecular level how the intestinal epithelium creates a selective barrier between the bowel contents, which include both nutrients and potentially harmful bacteria and antigens, and the rest of the body. The public health significance of this project is that loss of selectivity and increased permeability contribute to a number of intestinal diseases, including diarrhea and inflammation. The findings will guide strategies to preserve and restore the intestinal barrier or enhance permeability for transmucosal drug delivery.