Chronic Hypoxia and pH Homeostasis in Pulmonary Myocytes

描述（由申请人提供）：我们的长期目标是了解慢性缺氧（CH）导致肺动脉高压的机制。以前的研究已经描述了CH引起的肺血管系统的形态和功能变化，但这些变化背后的细胞机制仍然知之甚少。我们证明，CH诱导肺动脉平滑肌细胞（PASMC）细胞内pH值（pHi）的动态平衡的改变，由于增加Na+/H+交换异构体1（NHE 1）的表达。pHi的变化对细胞功能有着深远的影响，尽管在CH期间pHi调节PASMC生长、迁移和收缩的机制尚不完全清楚。研究表明，NHE 1可以作为膜锚独立于其离子转运特性。NHE 1与埃兹蛋白共定位并结合，埃兹蛋白是ERM（埃兹蛋白/根蛋白/膜突蛋白）蛋白家族的成员，其含有多个结合结构域并且是蛋白质-蛋白质相互作用的重要介质。特别令人感兴趣的是，ezrin的C-末端含有丝状肌动蛋白的结合位点，提供了NHE 1和细胞骨架重排之间的可能联系。Ezrin还结合50 kDa的Ezrin/根蛋白/膜突蛋白结合磷蛋白（EBP 50）或Na+/H+交换调节因子1（NHERF 1）。NHERF 1作为连接细胞内信号复合物的支架。尽管其名称暗示了其含义，但NHERF 1不结合NHE 1。我们发现PASMCs表达NHERF 1，在CH期间，NHERF 1表达减少，而NHE 1表达增加。我们还发现PASMCs表达ezrin; CH对ezrin表达的影响尚不清楚。NHE 1利用与NHERF 1相同的ezrin上的结合基序表明NHE 1和NHERF 1之间竞争ezrin结合。如果是真的，在CH过程中NHERF 1表达减少，NHE 1和/或ezrin表达增加，可能会创造有利于膜/细胞骨架相互作用的条件。因此，我们假设在CH期间，NHERF/ezrin相互作用的减少和ezrin/NHE 1结合的伴随增加导致NHE 1-肌动蛋白丝束缚增强，导致PASMC迁移、肥大、增殖和收缩。为了验证这一假设，我们将使用包括共聚焦FRET荧光显微镜、敲低和过表达方法、免疫共沉淀、迁移和增殖测定以及PASMCs中的牵引显微镜的技术的组合来确定：1）CH是否改变NHERF 1和ezrin的表达、定位和/或缔合; 2）NHE 1和肌动蛋白之间的相互作用在CH期间改变，如果是这样，这是否需要ezrin和3）ezrin介导的NHE 1-肌动蛋白结合是CH诱导的PASMC功能变化所必需的。 公共卫生相关性：本提案中的实验将探索参与缺氧性肺动脉高压发展的细胞机制，缺氧性肺动脉高压是一种治疗选择有限的毁灭性疾病。在美国，估计有600，000名患有慢性肺病的患者由于低氧血症而处于肺动脉高压发展的风险中，其中大约一半的这些患者可能已经患有肺动脉高压。了解随着缺氧性肺动脉高压的发展而发生在肺血管中的细胞变化是推进治疗和治疗选择以及减少与这种疾病相关的医疗保健成本负担的关键。

DESCRIPTION (provided by applicant): Our long-term goal is to understand the mechanism by which chronic hypoxia (CH) causes pulmonary hypertension. Previous studies have characterized the morphologic and functional changes that occur in the pulmonary vasculature in response to CH but the cellular mechanisms underlying these changes remain poorly understood. We demonstrated that CH induces alterations in pulmonary arterial smooth muscle cell (PASMC) intracellular pH (pHi) homeostasis, due to increased Na+/H+ exchanger isoform 1 (NHE1) expression. Changes in pHi have profound influences on cell function, although the mechanism by which pHi modulates PASMC growth, migration and contraction during CH are incompletely understood. Studies have suggested that NHE1 could act as a membrane anchor independent of its ion translocation properties. NHE1 co-localizes with and binds to ezrin, a member of the ERM (ezrin/radixin/moesin) family of proteins, which contain multiple binding domains and are important mediators of protein-protein interactions. Of particular interest, the C-terminal of ezrin contains a binding site for filamentous actin, providing a possible link between NHE1 and cytoskeletal rearrangement. Ezrin also binds ezrin/radixin/ moesin-binding phosphoprotein of 50 kDa (EBP50), or Na+/H+ exchange regulatory factor 1 (NHERF1). NHERF1 acts as a scaffold linking together intracellular signaling complexes. Despite the connotation inferred by its name, NHERF1 does not bind NHE1. We found that PASMCs express NHERF1 and that during CH, NHERF1 expression decreases whereas NHE1 expression increases. We also found that PASMCs express ezrin; the effect of CH on ezrin expression is unknown. That NHE1 utilizes the same binding motif on ezrin as NHERF1 suggests competition between NHE1 and NHERF1 for ezrin binding. If true, a decrease in NHERF1 expression with increased NHE1 and/or ezrin expression during CH could create conditions favorable for membrane/cytoskeleton interactions. Thus, we hypothesize that during CH, decreased NHERF/ezrin interactions and a concomitant increase in ezrin/NHE1 binding results in enhanced NHE1-actin filament tethering, resulting in PASMC migration, hypertrophy, proliferation and contraction. To test this hypothesis, we will use a combination of techniques including confocal FRET fluorescent microscopy, knockdown and overexpression approaches, co-immunoprecipitation, migration and proliferation assays and traction microscopy in PASMCs to determine whether: 1) CH alters NHERF1 and ezrin expression, localization and/or association; 2) the interaction between NHE1 and actin is altered during CH and, if so, whether this requires ezrin and 3) ezrin-mediated NHE1-actin binding is required for CH-induced changes in PASMC function. PUBLIC HEALTH RELEVANCE: The experiments in this proposal will explore cellular mechanisms involved in the development of hypoxic pulmonary hypertension, a devastating disease with limited treatment options. In the United States, there are an estimated 600,000 patients with chronic lung disease that are at risk for the development of pulmonary hypertension due to hypoxemia, with approximately one half of these patients likely to already have pulmonary hypertension. Understanding the cellular changes that occur in the pulmonary vasculature with development of hypoxic pulmonary hypertension is key to advancing treatment and therapeutic options and reducing the health care cost burden associated with this disease.