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

描述（由申请人提供）：在肠道中，有效的上皮屏障形成需要存在紧密连接，其调节离子和分子通过细胞旁空间。与紧密连接相关的缺陷导致屏障功能障碍、腹泻以及肠道疾病中的大量营养和蛋白质损失。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，已知在炎症性肠病中升高的两种细胞因子IL-13和TNF通过功能不同的孔和泄漏途径诱导屏障功能障碍。IL-13诱导紧密连接紧密连接蛋白-2孔的形成，导致小阳离子而不是大离子或大分子的渗透性增加。相反，TNF通过诱导闭合蛋白内化增加非选择性紧密连接泄漏到小和大离子和分子。此外，我已经表明，这两个途径的差异调节酪蛋白激酶II依赖性调节claudin- 2孔的稳定性和MLCK依赖性的occludin的内化。在我的K 08研究中，我已经开发出一种方法来研究局部紧密连接功能的紧密连接的亚微米段。这使我能够以前所未有的分子分辨率定义紧密连接功能。虽然我的K 08奖继续支持我正在进行的研究，这些途径是如何在培养的上皮单层调节，这些R 03研究将定义在一个更复杂的体内设置孔和泄漏途径功能的调节。我的中心假设是，孔隙和泄漏途径渗透性的相对贡献取决于不同类别的动态紧密连接通道的开口和关闭与不同的大小和电荷选择性的相互作用。为了在体内验证这一假设，我打算研究在完整的小鼠粘膜孔和泄漏途径的调节。使用全球测量，我将确定的机制，调节紧密连接claudin-2孔功能，以及occludin和tricellulin依赖的泄漏渗透性。此外，我将调整我的局部膜片钳方法来研究claudin-2，occludin和tricellulin在小鼠粘膜局部紧密连接开放事件中的作用。总之，这些研究将确定体内孔隙和渗漏通路功能的相对贡献和分子方面。这些结果有望直接用于了解TNF和IL-13在IBD等肠道疾病中的作用，并有望在不久的将来为研究患者屏障功能障碍提供新的方法。这将有助于针对负责屏障功能障碍的分子改变的特定疗法的合理开发。

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.