Effects of Hypoxia on Alveolar Epithelial Cytoskeleton

描述（由申请人提供）：肺泡上皮直接暴露于肺泡氧张力的变化中，以响应许多生理或病理条件。例如，肺泡缺氧可能是阻塞性气道疾病的结果，或心力衰竭或急性肺损伤引起的肺水肿。虽然肺泡上皮在缺氧时直接暴露，但关于低氧张力对肺泡上皮细胞功能的影响的信息有限。在本研究的前一个周期中，我们报道了缺氧导致肺泡上皮细胞骨架的显著重塑。特别是，我们观察到缺氧启动角蛋白中间丝网络的快速和局部重组，并与多种信号转导途径相互作用，这使得角蛋白网络与细胞行为的许多方面（包括迁移、增殖和凋亡）密切相关。这一提议将提出一个假设，即角蛋白中间丝可以通过启动由角蛋白8和18磷酸化状态变化介导的网络的细胞保护性重组来动态响应低氧张力。我们制定了三个相互关联的具体目的来研究缺氧诱导的肺泡上皮角蛋白干扰素的调节。明确目标#1。确定缺氧介导的肺泡上皮细胞中角蛋白中间丝网络重组中角蛋白8和18的体内磷酸化位点。具体目标2。确定在肺泡上皮细胞和角蛋白敲除小鼠中重组野生型或磷酸化突变型角蛋白是否能恢复肺泡上皮功能。具体目标#3。确定缺氧诱导因子是否在缺氧介导的肺泡上皮细胞角蛋白8和/或角蛋白18基因的转录调控中起作用。本研究的目的是验证缺氧调节K8和K18蛋白磷酸化状态，从而介导KIF网络的组装动力学和微力学特性的假设。KIF网络对肺泡上皮细胞的完整性起着重要的促进作用。该实验将确定调节缺氧介导的角蛋白IFs重组和/或拆卸的分子机制。这种重组对肺泡上皮功能的影响将在体外和体内用原代ATII细胞、野生型和角蛋白缺陷小鼠进行研究。这些研究的完成将为角蛋白IF在缺氧诱导肺泡上皮功能障碍发病机制中的作用提供新的见解，这在肺水肿患者中具有生物学和生理学的重要性。

DESCRIPTION (provided by applicant): The alveolar epithelium is directly exposed to variations in alveolar O2 tension in response to many physiological or pathological conditions. For example, alveolar hypoxia may be the consequence of obstructive airway diseases, or pulmonary edema from heart failure or acute lung injury. Although the alveolar epithelium is directly exposed during hypoxia, limited information has been obtained about the effects of low O2 tension on alveolar epithelial cell functions. In the previous cycle of this proposal, we reported that hypoxia causes significant remodeling of the alveolar epithelial cytoskeleton. In particular, we observed that hypoxia initiates rapid and localized restructuring of the keratin intermediate filament network and interacted with multiple signal transduction pathways which permits the keratin network to be involved in an intimate crosstalk with many aspects of cell behavior including migration, proliferation, and apoptosis. This proposal will develop the hypothesis that keratin intermediate filaments can dynamically respond to low O2 tension by initiating a cytoprotective reorganization of the network that is mediated by changes in the phosphorylation state of keratin 8 and 18. We have formulated three interrelated specific aims to study the hypoxia-induced regulation of keratin IFs in the alveolar epithelium. Specific Aim #1. To identify the in vivo phosphorylation sites in keratin 8 and 18 involved in the hypoxia-mediated restructuring of the keratin intermediate filament network in alveolar epithelial cells. Specific Aim #2. To determine whether reconstituting wild-type or phospho-mutant keratin proteins in alveolar epithelial cells and in keratin knockout mice restores alveolar epithelial function. Specific Aim #3. To determine whether hypoxia inducible factor is required for the hypoxia-mediated transcriptional regulation of keratin 8 and/or keratin 18 genes in alveolar epithelial cells. The purpose of this proposal is to test the hypothesis that hypoxia regulates the state of phosphorylation of K8 and K18 proteins, which mediates the assembly dynamics and micromechanical properties of the KIF network. The KIF network plays an important contributory role to the cellular integrity of alveolar epithelial cells. The proposed experiments will determine the molecular mechanisms that regulate the hypoxia mediated reorganization and/or disassembly of keratin IFs. The consequences of this reorganization on alveolar epithelial function will be examined both in vitro and in vivo using primary ATII cells, wild-type and keratin-deficient mice. Completion of the proposed studies will provide novel insights on the role of keratin IF in the pathogenesis of hypoxia-induced alveolar epithelial dysfunction, which is of biological and physiological importance in patients with pulmonary edema.