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(由申请者提供)：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肺血管对CH反应的形态和功能变化，但这些变化背后的细胞机制仍然知之甚少。我们证明，CH通过增加Na+/H+交换异构体1(NHE1)的表达，改变了肺动脉平滑肌细胞(PASMC)的细胞内pH(Phi)动态平衡。Phi的变化对细胞功能有深远的影响，尽管Phi在CH过程中调节PASMC生长、迁移和收缩的机制尚不完全清楚。研究表明，NHE1可以作为不依赖于其离子转运特性的膜锚。NHE1与Ezrin共定位并与Ezrin结合，Ezrin是ERM(Ezrin/Radisin/Moesin)家族的成员，包含多个结合结构域，是蛋白质相互作用的重要介质。特别令人感兴趣的是，Ezrin的C-末端包含一个丝状肌动蛋白的结合位点，为NHE1和细胞骨架重排之间提供了可能的联系。Ezrin还与50 kDa的Ezrin/Radioxin/Moesin结合的磷酸蛋白(EBP50)或Na+/H+交换调节因子1(NHERF1)结合。NHERF1作为连接细胞内信号复合体的支架。尽管NHERF1的名字暗示了它的内涵，但它并不绑定NHE1。我们发现PASMC表达NHERF1，而在CH过程中，NHERF1表达减少，而NHE1表达增加。我们还发现PASMC表达Ezrin；CH对Ezrin表达的影响尚不清楚。NHE1与NHERF1在Ezrin上利用相同的结合基序，表明NHE1和NHERF1之间竞争Ezrin结合。如果是真的，在CH过程中NHERF1表达减少而NHE1和/或Ezrin表达增加可能会为膜/细胞骨架相互作用创造有利条件。因此，我们假设在CH过程中，NHERF/Ezrin相互作用减少和Ezrin/NHE1结合增加导致NHE1-肌动蛋白细丝捆绑增强，导致PASMC迁移、肥大、增殖和收缩。为了验证这一假说，我们将使用联合技术，包括共聚焦FRET荧光显微镜、敲除和过表达方法、免疫共沉淀、迁移和增殖分析以及牵引显微镜，以确定：1)CH是否改变了NHERF1和Ezrin的表达、定位和/或结合；2)NHE1和肌动蛋白之间的相互作用在CH过程中发生了变化，如果是，这是否需要Ezrin；3)Ezrin介导的NHE1-肌动蛋白结合是CH诱导的PASMC功能变化所必需的。 与公共卫生相关：本提案中的实验将探索与缺氧性肺动脉高压发展有关的细胞机制，缺氧性肺动脉高压是一种治疗选择有限的破坏性疾病。在美国，估计有60万慢性肺病患者因低氧血症而面临发展为肺动脉高压的风险，其中约一半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在肺血管中发生的细胞变化是推进治疗和治疗选择并减少与这种疾病相关的医疗费用负担的关键。