Mechanisms and pathways of trans-tight junction conductance

跨密封连接电导的机制和途径

• 批准号: 8076178
• 负责人: Christopher Weber
• 金额: $ 14.66万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8076178
• 关键词: Actomyosin; Affect; American; Behavior; Biological Assay; Celiac Disease; Cells; Characteristics; Charge; Colitis; Cytoskeleton; Data; Development; Diarrhea; Disease; Electrodes; Electrophysiology (science); Environment; Epithelial; Event; Exposure to; Figs - dietary; Functional disorder; Gastrointestinal Diseases; Goals; Health; Human; Inflammatory; Inflammatory Bowel Diseases; Integumentary system; Interleukin-13; Intestinal Diseases; Intestines; Ion Channel; Ions; Kidney; Knowledge; Lamina Propria; Lung; Measurement; Measures; Methods; Microscopic; Molecular; Morbidity - disease rate; Myosin Light Chain Kinase; Neuraxis; Nutrient; Organ; Patch-Clamp Techniques; Pathologist; Pathway interactions; Patients; Process; Proteins; Publishing; Qualifying; Regulation; Reporting; Research; Research Personnel; Resolution; Role; Skin; Symptoms; Therapeutic; Tight Junctions; Time; Training; Tumor Necrosis Factor-alpha; Voltage-Clamp Technics; base; cytokine; gastrointestinal; improved; innovation; intestinal epithelium; novel strategies; occludin; patch clamp; public health relevance; skills; small molecule; submicron; therapy development

DESCRIPTION (provided by applicant): Intestinal barrier function is reduced in inflammatory bowel disease (IBD) and other intestinal disorders. Alterations of tight junctions, which form the major paracellular barrier, contribute to barrier loss by allowing increased paracellular flux of ions and molecules. Published reports and my preliminary data, which assess the role of different inflammatory cytokines in the lamina propria of IBD patients, support the hypothesis that at least two mechanisms of tight junction barrier regulation are activated by inflammatory cytokines. TNF induces tight junction dysfunction via a mechanism involving occludin internalization that alters tight junction size selectivity and permits increased macromolecular flux. In contrast IL-13 activates a functionally distinct pathway involving expression of claudin proteins that affects ion selectivity but does not increase macromolecular flux. Despite advances in understanding the processes involved in cytokine-induced barrier regulation, the molecular events that define size- or ion-selective changes are unclear. This, in part, represents the technical obstacle posed by traditional time and spatially averaged measurements of barrier function. I hypothesize that the tight junction barrier is highly dynamic at the local, sub-micron level, and that regulation of paracellular flux occurs through modulation of opening and closing "events" involving more than one class of tight junction pore or other conductance pathway. Because the tight junction spans two cells, it has not been amenable to molecular and biophysical analyses and development of specific pharmacologic modulators, such as those that exist for transmembrane ion channels. In order to eliminate this gap in available methods and molecular understanding, I have developed a novel approach to analyze tight junction barrier function at the local sub-micron level, using a high resolution single electrode patch clamp technique. While this approach is extensively used in the study of transmembrane ion channels and transporters, it has not been applied to the study of tight junction function. My preliminary recordings using this approach show previously unrecognized barrier dynamics, and I hypothesize that this behavior underlies normal tight junction function as a selectively-permeable barrier and is also necessary for understanding the molecular mechanisms and basis for multiple pathways of barrier dysfunction in disease. These local measurements will be performed alongside traditional assays of tight junction function (Aim 1) to study tight junction dynamics at steady state (Aim 2) and after exposure to inflammatory cytokines IL-13 and TNF (Aim 3). The data will have significant positive effects on human health by improving the understanding of barrier function and dysfunction, which may provide better therapeutic approaches for intestinal disorders such as IBD, celiac disease, and infectious colitis. Furthermore, both the knowledge gained and the technical approaches developed will be easily adapted to the study of tight junctions in other organs, including integument, central nervous system, vasculature, lung, kidney, and skin, and, therefore, may have broad impact beyond gastrointestinal disease. PUBLIC HEALTH RELEVANCE: Narrative Intestinal barrier function is reduced in inflammatory bowel disease and other intestinal disorders. Alterations of tight junctions, which form the major paracellular barrier, contribute to barrier loss by allowing increased paracellular flux of ions and molecules. Barrier dysfunction involves separate mechanisms for ionic and macromolecular flux, but the molecular basis for these different pathways is unclear. The research outlined will study these mechanisms using high resolution patch clamp recordings of tight junction barrier dynamics.

描述（由申请人提供）：肠道屏障功能在炎症性肠病（IBD）和其他肠道疾病中降低。形成主要细胞旁屏障的紧密连接的改变通过增加离子和分子的旁细胞通量而导致屏障丧失。已发表的报告和我的初步数据评估了 IBD 患者固有层中不同炎症细胞因子的作用，支持这样的假设：炎症细胞因子激活至少两种紧密连接屏障调节机制。 TNF 通过涉及 occludin 内化的机制诱导紧密连接功能障碍，从而改变紧密连接尺寸选择性并允许增加大分子通量。相比之下，IL-13 激活一条功能独特的途径，涉及密蛋白的表达，影响离子选择性，但不会增加大分子通量。尽管在了解细胞因子诱导的屏障调节过程方面取得了进展，但定义尺寸或离子选择性变化的分子事件尚不清楚。这在一定程度上代表了屏障功能的传统时间和空间平均测量所带来的技术障碍。我假设紧密连接屏障在局部、亚微米水平上是高度动态的，并且细胞旁通量的调节是通过调节涉及不止一类紧密连接孔或其他电导途径的打开和关闭“事件”而发生的。由于紧密连接跨越两个细胞，因此它不适合分子和生物物理分析以及特定药理调节剂的开发，例如跨膜离子通道中存在的调节剂。为了消除现有方法和分子理解方面的差距，我开发了一种新方法，使用高分辨率单电极膜片钳技术在局部亚微米水平上分析紧密连接势垒功能。虽然这种方法广泛用于跨膜离子通道和转运蛋白的研究，但尚未应用于紧密连接功能的研究。我使用这种方法进行的初步记录显示了以前未被识别的屏障动力学，并且我假设这种行为是作为选择性渗透屏障的正常紧密连接功能的基础，并且对于理解疾病中屏障功能障碍的多种途径的分子机制和基础也是必要的。这些局部测量将与传统的紧密连接功能测定（目标 1）一起进行，以研究稳定状态下的紧密连接动力学（目标 2）以及暴露于炎症细胞因子 IL-13 和 TNF 后（目标 3）。这些数据将通过提高对屏障功能和功能障碍的理解对人类健康产生显着的积极影响，这可能为IBD、乳糜泻和传染性结肠炎等肠道疾病提供更好的治疗方法。此外，所获得的知识和开发的技术方法将很容易适应其他器官紧密连接的研究，包括体皮、中枢神经系统、脉管系统、肺、肾和皮肤，因此可能具有胃肠道疾病以外的广泛影响。 公众健康相关性：叙述 炎症性肠病和其他肠道疾病会导致肠道屏障功能降低。形成主要细胞旁屏障的紧密连接的改变通过增加离子和分子的旁细胞通量而导致屏障丧失。屏障功能障碍涉及离子和大分子通量的不同机制，但这些不同途径的分子基础尚不清楚。概述的研究将使用紧密连接势垒动力学的高分辨率膜片钳记录来研究这些机制。