Chronic Hypoxia and pH Homeostasis in Pulmonary Myocytes

肺肌细胞的慢性缺氧和 pH 稳态

• 批准号: 8505017
• 负责人: Larissa A. Shimoda
• 金额: $ 34.78万
• 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 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8505017
• 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.

描述（由申请人提供）：我们的长期目标是了解慢性缺氧（CH）引起肺动脉高压的机制。先前的研究表征了响应CH的肺脉管系统中发生的形态和功能变化，但是这些变化的细胞机制仍然很少了解。我们证明CH诱导肺动脉平滑肌细胞（PASMC）细胞内pH（PHI）稳态的改变，这是由于Na+/H+交换剂同工型1（NHE1）表达增加。 PHI的变化对细胞功能有深远的影响，尽管PHI调节CH期间PASMC生长，迁移和收缩的机制尚不完全了解。研究表明，NHE1可以充当膜锚，独立于其离子易位性能。 NHE1与Ezrin（EMER/Radixin/Moesin）家族的Ezrin共定位并与Ezrin结合，蛋白质（Ezrin/radixin/Moesin）家族蛋白包含多个结合结构域，并且是蛋白质蛋白质相互作用的重要介体。特别值得一提的是，Ezrin的C末端包含丝状肌动蛋白的结合位点，提供了NHE1与细胞骨架重排之间的可能联系。 Ezrin还结合50 kDa（EBP50）或Na+/H+交换调控因子1（NHERF1）的Ezrin/radixin/Moesin结合磷酸蛋白（NHERF1）。 NHERF1充当脚手架，将细胞内信号传导复合物联系在一起。尽管由其名称推断出的含义，但NHERF1不结合NHE1。我们发现PASMC表达NHERF1，并且在CH期间，NHERF1表达降低，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利用Ezrin上的结合基序与NHERF1相同，表明NHE1和NHERF1之间的Ezrin结合竞争。如果是正确的话，在CH期间，NHEF1表达的降低可能会增加NHE1和/或Ezrin表达，这可能会产生有利于膜/细胞骨架相互作用的条件。因此，我们假设在CH期间，NHERF/Ezrin相互作用降低，Ezrin/NHE1结合的伴随增加会导致NHE1-肌动蛋白丝链接的增强，从而导致PASMC迁移，肥大，增殖，增殖和收缩。为了检验这一假设，我们将使用包括共焦特性荧光显微镜，敲低和过表达方法，共免疫沉淀，迁移和增殖测定和PASMC中的牵引显微镜的组合，以确定：1） 2）在CH期间，NHE1和肌动蛋白之间的相互作用发生了变化，如果是，是否需要Ezrin和3）Ezrin介导的NHE1-肌动蛋白结合是CH诱导的PASMC功能的变化所必需的。