Role of claudin 18 in regulation of lung stem/progenitor cell homeostasis

 描述（由申请人提供）：必须严格调节干/祖细胞增殖和分化，以维持正常器官功能所需的适当细胞数量，同时预防肿瘤发生。阐明调节体干/祖细胞稳态的机制是理解这些群体如何维持和激活以满足损伤后组织再生需求的关键。细胞间连接是细胞间粘附的重要部位，包括紧密连接（TJ）、粘附连接（AJ）和桥粒。紧密连接蛋白是调节细胞旁通透性的整合TJ蛋白。Claudin 18（C18）是肺泡上皮细胞中表达最高的Claudin家族成员之一。我们最近产生的C18基因敲除（KO）小鼠表现出增加的肺上皮细胞的渗透性离子和溶质。有趣的是，C18 KO小鼠显示推定的肺干/祖细胞（包括肺泡上皮II型（AT 2）细胞）的扩增和增殖增加以及肺（和胃）大小增加，暗示整合TJ蛋白（特别是C18）作为肺干/祖细胞的新型调节剂。 上皮干/祖细胞稳态和器官大小。Hippo信号通路通过对增殖和凋亡的相反作用调节干/祖细胞功能、器官大小和再生。Hippo信号传导介导体外细胞增殖的接触抑制，并通过抑制上游激酶的磷酸化来限制体内组织的过度生长，所述上游激酶抑制正下游转录共激活因子、yes相关蛋白（雅普）和具有PDZ结合基序的转录共激活因子（TAZ）的活性。Hippo信号传导的上游调节因子尚未得到很好的表征，但包括通过G-蛋白偶联受体、肌动蛋白细胞骨架、顶端-基底外侧极性复合物以及与TJ相关和AJ蛋白的相互作用的细胞外刺激。整合TJ蛋白（例如，claudin）以前没有显示调节Hippo信号传导或雅普/TAZ活性。初步研究表明，雅普/TAZ激活C18基因敲除小鼠和雅普与C18在野生型小鼠，使我们假设，C18是一种新的调节肺干/祖细胞稳态通过调节雅普/TAZ亚细胞定位/活性。本项目的总体目标是研究C18在调节肺干/祖细胞稳态中的作用，具体目的如下：1）探索C18 KO小鼠肺表型的细胞机制; 2）研究C18 KO小鼠调节干/祖细胞稳态的信号传导机制; 3）表征C18调节雅普/TAZ信号传导的分子机制。阐明调节肺干/祖细胞功能的机制和鉴定将生长促进信号从TJ转导至细胞核的新途径对于调节干/祖细胞功能和增强肺损伤后的再生具有重要意义。

 DESCRIPTION (provided by applicant): Stem/progenitor cell proliferation and differentiation must be tightly regulated to maintain appropriate cell numbers for normal organ function while preventing tumorigenesis. Elucidation of mechanisms that regulate somatic stem/progenitor cell homeostasis is key to understanding how these populations are maintained and activated to meet demands for tissue regeneration following injury. Intercellular junctions, comprised of tight junctions (TJ), adherens junctions (AJ) and desmosomes, are sites of intercellular adhesion. Claudins are integral TJ proteins that regulate paracellular permeability. Claudin 18 (C18) is one of the most highly expressed Claudin family members in lung alveolar epithelium. We recently generated C18 knockout (KO) mice that exhibit increased lung epithelial permeability to ions and solutes. Intriguingly, C18 KO mice show expansion and increased proliferation of putative lung stem/progenitor cells (including alveolar epithelial type II (AT2) cells) and increased lung (and stomach) size, implicating integral TJ proteins (and C18 in particular) as novel regulators of epithelial stem/progenitor cell homeostasis and organ size. The Hippo signaling pathway regulates stem/progenitor cell function, organ size and regeneration through opposing effects on proliferation and apoptosis. Hippo signaling mediates contact inhibition of cell proliferation in vitro and limits tissue overgrowth in vivo via phosphorylation of upstream kinases that inhibit activity of orthologous downstream transcriptional co-activators, yes-associated protein (YAP) and transcriptional coactivator with PDZ binding motif (TAZ). Upstream regulators of Hippo signaling are not well characterized but include extracellular stimuli via G-protein coupled receptors, actin cytoskeleton, apical-basolateral polarity complexes and interactions with TJ-associated and AJ proteins. Integral TJ proteins (e.g., claudins) have not previously been shown to regulate Hippo signaling or YAP/TAZ activity. Preliminary studies demonstrate YAP/TAZ activation in C18 KO mice and association of YAP with C18 in wild type mice, leading us to hypothesize that C18 is a novel regulator of lung stem/progenitor cell homeostasis via modulation of YAP/TAZ subcellular localization/activity. The overall goal of this project is to investigate the role of C18 in regulating lung stem/progenitor cell homeostasis by addressing the following Specific Aims: 1) explore cellular mechanisms underlying lung phenotype of C18 KO mice: 2) investigate signaling mechanisms regulating stem/progenitor cell homeostasis in C18 KO mice; and, 3) characterize molecular mechanisms whereby C18 regulates YAP/TAZ signaling. Elucidation of mechanisms regulating lung stem/progenitor cell function and identification of novel pathways transducing growth-promoting signals from TJ to the nucleus have important implications for modulating stem/progenitor cell function and augmenting regeneration following lung injury.