Mechanisms of tight junction pore and leak pathway regulation in intestinal mucos

肠粘膜紧密连接孔和渗漏途径的调节机制

• 批准号: 8881173
• 负责人: Christopher Weber
• 金额: $ 7.9万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8881173
• 关键词: Accounting; Award; Biopsy; Cations; Celiac Disease; Charge; Colitis; Complex; Coupled; Defect; Development; Diarrhea; Disease; Electrophysiology (science); Epithelial; Epithelial Cells; Epithelium; Event; Functional disorder; Future; Goals; Health; Human; Immune; In Vitro; Inflammatory; Inflammatory Bowel Diseases; Interleukin-13; Intestinal Diseases; Intestines; Ion Channel; Ions; Lamina Propria; Link; Measurement; Mediating; Molecular; Molecular Target; Mucous Membrane; Mus; Myosin Light Chain Kinase; Nutrient; Pathologist; Pathway interactions; Patients; Permeability; Property; Proteins; Qualifying; Radial; Regulation; Relative (related person); Research; Resolution; Role; Stimulus; Testing; Therapeutic Intervention; Tight Junctions; Time; Tissues; Translating; Tumor Necrosis Factor-alpha; base; casein kinase II; cytokine; design; experience; gastrointestinal; in vivo; innovation; intestinal epithelium; macromolecule; monolayer; mouse model; novel strategies; occludin; patch clamp; submicron; targeted treatment; therapeutic target; therapy development

DESCRIPTION (provided by applicant): In the intestine, effective epithelial barrier formation requires the presence of tight junctions, which regulate the passage of ions and molecules through the paracellular space. Defects associated with the tight junction contribute to barrier dysfunction, diarrhea, and extensive nutrient and protein loss in intestinal diseases. In immune-mediated intestinal diseases, cytokines secreted in the lamina propria are thought to be a primary driver of barrier loss. My preliminary in vitro studies demonstrate that two cytokines known to be elevated in inflammatory bowel disease, IL-13 and TNF, induce barrier dysfunction through functionally distinct pore and leak pathways. IL-13 induces the formation of tight junction claudin-2 pores resulting in increased permeability of small cations, but not large ions o macromolecules. In contrast, TNF increases non-selective tight junction leak to small and large ions and molecules by inducing occludin internalization. Further, I have shown that these two pathways are differentially regulated by casein kinase II dependent regulation of claudin- 2 pore stability and MLCK dependent internalization of occludin. During my K08 studies, I have developed an approach to study local tight junction function in submicron segments of tight junction. This has permitted me to define tight junction function with unprecedented molecular resolution. While my K08 award continues to support my ongoing studies of how these pathways are regulated in cultured epithelial monolayers, these R03 studies will define regulation of pore and leak pathway function in a more complex in vivo setting. My central hypothesis is that the relative contribution of pore and leak pathway permeability depends on the interplay of different classes of dynamic tight junction channel openings and closings with varying size and charge selectivity properties. To test this hypothesis in vivo, I intend to study regulation of pore and leak pathways in intact mouse mucosa. Using global measurements I will determine the mechanisms that regulate tight junction claudin-2 pore function as well as occludin- and tricellulin-dependent leak permeability. In addition, I will adapt my local patch clamp approach to study the role of claudin-2, occludin, and tricellulin to local tight junction opening events in mouse mucosa. In summary, these studies will define the relative contribution and molecular aspects of pore and leak pathway function in vivo. The results are expected to be directly applicable to understanding the roles of TNF and IL-13 in intestinal diseases such as IBD, and are further expected to provide novel approaches to study barrier dysfunction in patients in the near future. This will aid in the rational development of specific therapies targeting molecular alterations responsible for barrier dysfunction.

描述(申请人提供)：在肠道中，有效的上皮屏障的形成需要存在紧密的连接，它调节离子和分子通过细胞旁空间。与紧密连接相关的缺陷会导致肠道疾病中的屏障功能障碍、腹泻和广泛的营养和蛋白质丢失。在免疫介导的肠道疾病中，固有层中分泌的细胞因子被认为是屏障丧失的主要驱动因素。我的初步体外研究表明，在炎症性肠病中已知的两种细胞因子IL-13和TNF升高，通过功能不同的毛孔和渗漏途径导致屏障功能障碍。IL-13诱导紧密连接的Claudin-2孔的形成，导致小阳离子的渗透性增加，但不能增加大分子离子的渗透性。相反，肿瘤坏死因子通过诱导封闭蛋白内化，增加了对小离子和大分子的非选择性紧密连接泄漏。此外，我已经证明这两条通路是由酪蛋白激酶II依赖的Claudin-2孔稳定性调节和MLCK依赖的封闭蛋白内化而不同地调节的。在我的K08研究中，我开发了一种方法来研究紧连接的亚微米段中的局部紧连接功能。这使得我能够以前所未有的分子分辨率来定义紧密连接功能。虽然我的K08奖继续支持我正在进行的关于这些通路如何在培养的上皮单层中调节的研究，但这些R03研究将在更复杂的活体环境中定义毛孔和渗漏通路的调节功能。我的中心假设是，孔道和渗漏通道渗透率的相对贡献取决于不同类别的动态紧密连接通道开闭的相互作用，这些通道具有不同的大小和电荷选择性特性。为了在活体内验证这一假说，我打算研究完整小鼠粘膜中毛孔和渗漏途径的调节。利用全球测量，我将确定调节紧密连接claudin-2孔功能的机制，以及依赖于封闭素和三纤维素的渗漏渗透性。此外，我将采用我的局部膜片钳方法来研究claudin-2、occludin和tricellin在小鼠粘膜局部紧密连接开放事件中的作用。综上所述，这些研究将确定体内毛孔和渗漏途径功能的相对贡献和分子方面。这一结果有望直接应用于了解肿瘤坏死因子和白介素13在肠病等肠道疾病中的作用，并有望在不久的将来为研究患者的屏障功能障碍提供新的方法。这将有助于针对导致屏障功能障碍的分子变化的特定治疗方法的合理开发。