Effects of Hypoxia on Alveolar Epithelial Cytoskeleton

描述（由申请人提供）：肺泡性缺氧在许多生理和病理条件下观察到，包括慢性阻塞性肺疾病、心源性肺水肿、急性呼吸窘迫综合征和上升到高海拔。 然而，缺氧对肺泡上皮细胞功能和细胞骨架的影响尚未阐明。 细胞骨架主要负责细胞的结构支撑，并且已知角蛋白中间丝（IF）在维持上皮细胞的完整性中起重要作用。在肺泡上皮细胞（AEC）中，角蛋白IF是主要的结构蛋白。 完整的角蛋白IF网络对肺泡上皮功能的重要性通过我们在角蛋白8缺陷小鼠中发现肺泡液体清除严重受损而得到证实。 这项建议的重点是确定是否缺氧引起的角蛋白IF网络的变化导致AEC功能障碍。 我们假设缺氧在AEC中产生线粒体活性氧（ROS），其激活蛋白激酶，使角蛋白磷酸化并调节角蛋白IF的组织、分解和降解。 肺泡上皮细胞中角蛋白IFs的修饰可能导致肺泡上皮功能受损。我们制定了四个相互关联的具体目标，研究缺氧诱导的调节角蛋白IFs在肺泡上皮细胞。 具体目标1。探讨缺氧是否通过线粒体活性氧影响肺泡上皮细胞角蛋白中间丝网络的组装状态。 具体目标2。确定低氧诱导的角蛋白IFs变化是否受肺泡上皮细胞中蛋白激酶C依赖的角蛋白8和18磷酸化的调节。具体目标3。 探讨低氧是否通过泛素-蛋白酶体途径诱导角蛋白8和18的降解。 具体目标4。确定低氧诱导的角蛋白IFs变化是否调节角蛋白8基因敲除小鼠的肺泡上皮功能。 所提出的实验将确定调节缺氧介导的角蛋白IFs重组和/或分解的分子机制。 将使用AEC、大鼠和K8缺陷小鼠在体外和体内检查这种重组对肺泡上皮功能的影响。完成拟议的研究将提供新的见解角蛋白IF在缺氧诱导的肺泡上皮功能障碍的发病机制中的作用，这是肺水肿患者的生物学和生理学的重要性。

DESCRIPTION (provided by applicant): AIveolar hypoxia is observed in many physiological and pathological conditions including chronic obstructive pulmonary disease, cardiogenic pulmonary edema, acute respiratory distress syndrome and ascent to high altitude. The effect(s) of hypoxia on the function and cytoskeleton of the alveolar epithelium, however, have yet to be elucidated. The cytoskeleton is largely responsible for a cell s structural support, and keratin intermediate filaments (IFs) are known to play an important role in maintaining the integrity of epithelial cells. In alveolar epithelial cells (AEC) keratin IFs are the major structural proteins. The importance of an intact keratin IF network for alveolar epithelial function is demonstrated by our finding of profoundly impaired alveolar fluid clearance in keratin 8-deficient mice. This proposal is focused on determining whether hypoxia-induced changes in the keratin IF network leads to AEC dysfunction. We hypothesize that hypoxia generates mitochondrial reactive oxygen species (ROS) in AEC, which activate protein kinases that phosphorylate keratin proteins and regulate the organization, disassembly and degradation of the keratin IF. Modifications to keratin IFs in the alveolar epithelial cell may contribute to impaired alveolar epithelial function. We have formulated four interrelated specific aims to study the hypoxia-induced regulation of keratin IFs in the alveolar epithelium. Specific Aim 1. To determine whether hypoxia, via mitochondrial reactive oxygen species, affects the assembly state of keratin intermediate filament network in alveolar epithelial cells. Specific Aim 2. To determine whether hypoxiainduced changes in keratin IFs are regulated by protein kinase C-dependent phosphorylation of keratin 8 and 18 in alveolar epithelial cells. Specific Aim 3. To determine whether hypoxia-induces the degradation of keratin 8 and 18 via the ubiqutin-proteasome pathway. Specific Aim 4. To determine whether hypoxiainduced changes in keratin IFs modulate alveolar epithelial function in keratin 8 knockout mice. 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 AEC, rats and K8-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.