Chronic Hypoxia and pH Homeostasis in Pulmonary Myocytes

肺肌细胞的慢性缺氧和 pH 稳态

• 批准号: 8287004
• 负责人: Larissa A. Shimoda
• 金额: $ 36.53万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8287004
• 关键词: Actins; Acute; Adhesions; Alveolar; Animals; Attenuated; Binding; Binding Sites; Biological Assay; C-terminal; Cell Adhesion Molecules; Cell Hypoxia; Cell Proliferation; Cell membrane; Cell physiology; Chronic; Chronic lung disease; Co-Immunoprecipitations; Complex; Cystic Fibrosis Transmembrane Conductance Regulator; Cytoskeleton; Cytosol; DNA Sequence Rearrangement; Data; Development; Disease; Epithelial Cells; Exhibits; Exposure to; F-Actin; Fibroblasts; Figs - dietary; Fluorescence Resonance Energy Transfer; Funding; Genes; Goals; Head; Health Care Costs; Homeostasis; Hypertrophy; Hypoxemia; Hypoxia; Integral Membrane Protein; Ions; Link; Lung; Lung diseases; Mediating; Mediator of activation protein; Membrane; Membrane Potentials; Membrane Transport Proteins; Microfilaments; Microscopy; Molecular Conformation; Muscle Cells; Muscle Contraction; Names; Oxygen measurement, partial pressure, arterial; Patients; Phosphoproteins; Phosphorylation; Play; Process; Property; Protein Family; Protein Isoforms; Proteins; Pulmonary Hypertension; Regulation; Reporting; Rest; Rho-associated kinase; Risk; Role; Scaffolding Protein; Signal Transduction; Signaling Molecule; Smooth Muscle; Smooth Muscle Myocytes; Tail; Techniques; Testing; Therapeutic; Traction; United States; Vascular remodeling; Voltage-Gated Potassium Channel; base; cell growth; cell motility; ezrin; hypoxia inducible factor 1; interest; kinase inhibitor; member; migration; moesin; mortality; overexpression; pH Homeostasis; pressure; protein protein interaction; public health relevance; radixin protein; receptor; research study; response; scaffold; sodium-hydrogen exchanger regulatory factor; transcription factor; vasoconstriction

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.

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。先前的研究已经描述了肺血管系统响应CH而发生的形态和功能变化，但这些变化背后的细胞机制仍然知之甚少。我们证明，由于 Na+/H+ 交换异构体 1 (NHE1) 表达增加，CH 会诱导肺动脉平滑肌细胞 (PASMC) 细胞内 pH (pHi) 稳态的改变。尽管 CH 期间 pHi 调节 PASMC 生长、迁移和收缩的机制尚不完全清楚，但 pHi 的变化对细胞功能具有深远的影响。研究表明，NHE1 可以充当膜锚，与其离子易位特性无关。 NHE1 与 ezrin 共定位并结合，ezrin 是 ERM (ezrin/radixin/moesin) 蛋白质家族的成员，该家族包含多个结合域，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 NHE1 和细胞骨架重排之间可能的联系。 Ezrin 还结合 50 kDa (EBP50) 的 ezrin/radixin/ moesin 结合磷蛋白或 Na+/H+ 交换调节因子 1 (NHERF1)。 NHERF1 充当将细胞内信号复合物连接在一起的支架。尽管 NHERF1 从其名称可以推断出其含义，但它并不与 NHE1 结合。我们发现 PASMC 表达 NHERF1，并且在 CH 期间，NHERF1 表达减少，而 NHE1 表达增加。我们还发现 PASMC 表达埃兹蛋白 (ezrin)； CH 对埃兹蛋白表达的影响尚不清楚。 NHE1 在埃兹蛋白上利用与 NHERF1 相同的结合基序，表明 NHE1 和 NHERF1 之间对埃兹蛋白结合的竞争。如果属实，CH 期间 NHERF1 表达的减少以及 NHE1 和/或 ezrin 表达的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 相互作用减少，同时 ezrin/NHE1 结合增加，导致 NHE1-肌动蛋白丝束缚增强，从而导致 PASMC 迁移、肥大、增殖和收缩。为了检验这一假设，我们将结合使用多种技术，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 PASMC 中的牵引显微镜，以确定是否：1) CH 是否改变 NHERF1 和 ezrin 表达、定位和/或关联； 2) NHE1 和肌动蛋白之间的相互作用在 CH 过程中发生改变，如果是这样，这是否需要埃兹蛋白，3) CH 诱导的 PASMC 功能变化需要埃兹蛋白介导的 NHE1-肌动蛋白结合。 公共健康相关性：本提案中的实验将探讨缺氧性肺动脉高压发生的细胞机制，这是一种治疗选择有限的破坏性疾病。在美国，估计有 600,000 名慢性肺病患者因低氧血症而面临肺动脉高压的风险，其中大约一半的患者可能已经患有肺动脉高压。了解肺血管系统中随着缺氧性肺动脉高压的发展而发生的细胞变化是推进治疗和治疗方案以及减少与该疾病相关的医疗费用负担的关键。