Regulation of alveolar barrier function by claudins

描述(申请人提供)：急性呼吸窘迫综合征(ARDS)，是一种毁灭性的呼吸衰竭综合征，在美国每年导致近75,000人死亡。急性肺损伤的治疗主要是支持性的，目前还没有可用的药物治疗方法。急性肺损伤的病理生理特征是炎症和肺泡-毛细血管屏障的破坏。急性肺损伤的恢复需要重建肺泡上皮屏障，包括在上皮细胞之间形成低通透性紧密连接。为了推动这一领域的发展，需要对肺泡上皮屏障的调节和修复有更全面的了解。克拉丁蛋白是屏障功能的核心。Claudins是紧密连接形成所需的跨膜蛋白，但可能参与其他重要的细胞修复过程。因此，我们研究了Claudins在肺泡上皮细胞中的功能和调节。我们的工作表明，肺泡上皮细胞中表达最丰富的两个Claudin在肺损伤过程中受到选择性调节。这份提案将调查具体的职能 以及使用包括肺损伤实验模型、原代细胞培养和体外灌流人肺研究在内的综合方法来调节Claudins-18和-4的机械细节。在目标1中，我们将确定肺特异的claudin-18.1的独特功能，以及它在急性肺损伤中选择性下调的机制。我们是否会检验这样一种假设，即Claudin-18是肺内大分子通透性屏障所必需的，并且Claudin-18的缺失会导致更严重的肺损伤。我们认为，claudin-18形成同型的紧密连接链，独特地限制了大分子的渗透性。在损伤过程中，肿瘤坏死因子-β通过对细胞骨架的影响，部分地调节了完整紧密连接中Claudin-18的选择性丢失。在目标2中，我们将继续我们先前的发现，即在急性肺损伤中持续诱导Claudin-4。我们将确定在肺损伤的EGFR介导的上皮修复中是否需要claudin-4。我们假设，claudin-4是由EGFR诱导的，以应对上皮细胞损伤和细胞-细胞接触的丧失。我们认为claudin-4通过与活性Rap1的一种未知的相互作用来加速修复，从而加速紧密连接的封闭和促进细胞的扩散。初步数据显示，Claudin-4在几种肺损伤模型和人类肺中持续表达，并与更多保存的屏障功能有关。Claudin-4是修复所必需的，并且优先与激活的Rap1结合。在目标3中，我们将利用体外灌流的人肺模型来确定claudin表达的差异对上皮屏障功能的临床意义。我们假设Claudin-18是人类1型细胞中主要的Claudin，在体外模型中，Claudin-18的特异性丢失导致肺泡上皮通透性增加，并对供者造成更大的肺损伤。我们将在体外系统中使用细菌性肺炎模型来研究claudin-18和claudin-4的调节机制。这些研究将对Claudin蛋白在急性肺损伤中肺泡上皮屏障功能丧失和恢复中的作用提供新的见解。

DESCRIPTION (provided by applicant): Acute respiratory distress syndrome (ARDS), is a devastating syndrome of respiratory failure that contributes to nearly 75,000 deaths annually in the United States. Therapy for acute lung injury is primarily supportive and currently no pharmacologic therapies are available. The pathophysiology of acute lung injury is characterized by inflammation and disruption of alveolar-capillary barrier. Recovery from acute lung injury requires re- establishment of the alveolar epithelial barrier, including the formation f low-permeability tight junctions between epithelial cells. To advance the field, a more complete understanding of alveolar epithelial barrier regulation and repair is needed. Claudin proteins are central to barrier function. Claudins are transmembrane proteins that are required for tight junction formation, but likely participate in other cellular processes important to repair. Therefoe we have investigated the functions and regulation of claudins in the alveolar epithelium. Our work has shown that two of the most abundantly expressed claudins in the alveolar epithelium are selectively regulated during lung injury. This proposal will investigate the specific functions and regulation of claudins-18 and -4 in mechanistic detail using an integrative approach that includes experimental models of lung injury, primary cell culture, and studies in ex vivo perfused human lungs. In Aim 1 we will determine the unique functions of lung-specific claudin-18.1, and the mechanisms for its selective down regulation in acute lung injury. Will we test the hypothesis that claudin-18 is required for the macromolecule permeability barrier in the lung and that the loss of claudin-18 contributes to more severe lung injury. We propose that claudin-18 forms homotypic tight junction strands that uniquely limit macromolecule permeability. During injury, TNF-¿ mediates the selective loss of claudin-18 from intact tight junctions, in part through effects on the cytoskeleton. In Aim 2 we will follow up on our previous finding that claudin-4 is consistently induced in acute lung injury. We will determine if claudin-4 is required for EGFR-mediated epithelial repair in lung injury. We hypothesize that claudin-4 is induced by EGFR in response to epithelial cell injury and the loss of cell-cell contact. We propose claudin-4 acts to accelerate repair through a previously unknown interaction with active Rap1 that speeds tight junction sealing and promotes cell spreading. Preliminary data show that claudin-4 is consistently induced in several models of lung injury and in human lungs in association with more preserved barrier function. Claudin- 4 is required for repair and preferentially associates with active Rap1. In Aim 3 we will determine the clinical significance of differences in claudin expression to epithelial barrier function using the ex vivo perfused human lung model. We hypothesize that claudin-18 is the predominant claudin in human type 1 cells and the specific loss of claudin-18 results in increased alveolar epithelial permeability in the ex vivo model and greater lung injury in donors. We will investigate the mechanism for the regulation of claudin-18 and claudin-4 using a bacterial pneumonia model in the ex vivo system. These studies will provide novel insights into the contributions of claudin proteins to the loss and recovery of alveolar epithelial barrier function in acute lung injury.