Functional linkage of NHE1 and calpain in IPAH

IPAH 中 NHE1 和钙蛋白酶的功能联系

• 批准号: 8354085
• 负责人: Larissa A. Shimoda
• 金额: $ 8.1万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-08 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8354085
• 关键词: Actins; Address; Animal Model; Animals; Apoptosis; Attenuated; Binding; Binding Sites; Biological Assay; Blood Platelets; Blood Vessels; C-terminal; Calcium; Calpain; Cell Cycle; Cell Proliferation; Cell membrane; Cell physiology; Cells; Cellular biology; Chronic; Cleaved cell; Co-Immunoprecipitations; Confocal Microscopy; Cysteine Protease; Cytoskeletal Modeling; Cytoskeleton; DNA Sequence Rearrangement; Data; Development; Disease; Endothelial Cells; Enzymes; Exhibits; F-Actin; Family; Fibroblasts; Figs - dietary; Fluorescence Resonance Energy Transfer; Focal Adhesions; G Actin; Gene Family; Genetic; Goals; Growth Inhibitors; Homeostasis; Hypoxia; Immigration; Ions; Laboratories; Lead; Link; Lung; Mediator of activation protein; Membrane; Messenger RNA; Microfilaments; Molecular; Molecular Biology; Monocrotaline; PTK2 gene; Pathogenesis; Pathway interactions; Patients; Phosphotransferases; Process; Property; Protein Family; Protein Isoforms; Proteins; Pulmonary Hypertension; Pulmonary artery structure; Rho-associated kinase; Right Ventricular Hypertrophy; Rodent; Role; Scaffolding Protein; Smooth Muscle Myocytes; Structure of parenchyma of lung; System; Techniques; Testing; Tissues; Up-Regulation; Vascular Endothelial Cell; Vascular remodeling; abstracting; cell growth; cell motility; cell type; ezrin; hypertension control; interest; member; migration; moesin; novel; novel therapeutics; overexpression; pressure; protein protein interaction; pulmonary arterial hypertension; radixin protein; research study; rho; vasoconstriction

DESCRIPTION (provided by applicant): Our long-term goal is to understand the cellular mechanisms involved in the pathogenesis of pulmonary hypertension. Previous studies have characterized morphologic and functional changes that occur in the pulmonary vasculature during the development of idiopathic pulmonary arterial hypertension (IPAH), but the cellular mechanisms underlying these changes remain poorly understood. Using animal models, we recently demonstrated a role for alterations in pulmonary arterial endothelial (EC) and smooth muscle cell (SMC) intracellular pH (pHi) homeostasis, due to increased Na+/H+ exchanger isoform 1 (NHE1) expression. In addition, we have also shown that these cells exhibit elevated intracellular Ca2+ concentration and increased expression of calpains, a family of cytosolic, Ca2+-activated, neutral cysteine proteases. Changes in pHi, Ca2+ homeostasis and calpain activity have profound influences on cell function, although whether these pathways are altered during IPAH and the mechanism by which they modulate cell growth and migration are poorly understood. In animal models of pulmonary hypertension, we found that inhibition of calpain reduced right ventricular hypertrophy and pulmonary vascular remodeling. Recently, 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 protein which contains multiple binding domains and is important mediator 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. If true, increased NHE1 and/or ezrin expression/interactions during IPAH could create conditions favorable for membrane/cytoskeleton interactions. Moreover, calpain activation is influenced by pHi, and results in Rho kinase activation, which in turn facilitates ezrin activation and binding with targe proteins. Thus, we hypothesize that during IPAH, upregulation of NHE1 and calpain, and interactions between these pathways, lead to enhanced EC and SMC migration and proliferation, hallmarks of disease development and progression. To test this hypothesis, we will use a combination of techniques including confocal microscopy, FRET, knockdown and overexpression approaches, co-immunoprecipitation and migration and proliferation assays in pulmonary vascular ECs and SMCs and lung tissue and mRNA from IPAH and control subjects to determine: 1) whether calpain activity is enhanced in EC and SMCs from patients with IPAH and whether inhibition through genetic or pharmacologic means alters proliferation and migration in these cells; 2) whether NHE1 expression, activity and interactions with ezrin and actin are altered in vascular cells from IPAH patients and, if so, the involvement of these changes in cell function and 3) whether there is an interaction or linkage between the Ca2+/calpain and NHE1/ezrin pathways that contributes to the functional changes observed in ECs and SMCs from IPAH patients. PUBLIC HEALTH RELEVANCE: The experiments in this proposal will explore cellular mechanisms involved in the development of idiopathic pulmonary arterial hypertension (IPAH), a devastating disease with limited treatment options. The availability of tissues and cells from IPAH patients promises to reveal novel information that will further our understanding the cellular changes that occur in the pulmonary vasculature with development of IPAH. Such information is crucial to advancing treatment and developing new therapeutic options to treat this deadly disease. (End of Abstract)

描述（由申请人提供）：我们的长期目标是了解肺动脉高压发病机制中的细胞机制。先前的研究已经描述了特发性肺动脉高压（IPAH）发展过程中肺血管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。通过动物模型，我们最近证明了由于Na+/H+交换物异构体1 （NHE1）表达增加，导致肺动脉内皮（EC）和平滑肌细胞（SMC）细胞内pH （pHi）稳态的改变。此外，我们还表明，这些细胞表现出细胞内Ca2+浓度升高和钙蛋白酶的表达增加，钙蛋白酶是细胞质、Ca2+激活的中性半胱氨酸蛋白酶家族。pHi、Ca2+稳态和钙蛋白酶活性的变化对细胞功能有深远的影响，尽管这些途径是否在IPAH期间发生改变以及它们调节细胞生长和迁移的机制尚不清楚。在肺动脉高压动物模型中，我们发现抑制钙蛋白酶可减少右心室肥厚和肺血管重构。最近，研究表明NHE1可以独立于其离子转运特性而作为膜锚。NHE1与ezrin共定位并结合，ezrin是一种包含多个结合结构域的蛋白质，是蛋白质相互作用的重要介质。特别有趣的是，ezrin的c端包含一个丝状肌动蛋白的结合位点，提供了NHE1和细胞骨架重排之间的可能联系。如果这是真的，IPAH期间NHE1和/或ezrin表达/相互作用的增加可能为膜/细胞骨架相互作用创造有利条件。此外，calpain活化受pHi影响，导致Rho激酶活化，进而促进ezrin活化并与靶蛋白结合。因此，我们假设在IPAH期间，NHE1和calpain的上调以及这些途径之间的相互作用导致EC和SMC迁移和增殖增强，这是疾病发生和进展的标志。为了验证这一假设，我们将使用包括共聚焦显微镜、FRET、敲低和过表达方法、肺血管内皮细胞、SMCs和肺组织的共免疫沉淀、迁移和增殖试验以及来自IPAH和对照受试者的mRNA的组合技术来确定：1) IPAH患者的EC和SMCs中calpain活性是否增强，通过遗传或药物手段抑制是否会改变这些细胞的增殖和迁移；2) IPAH患者血管细胞中NHE1的表达、活性以及与ezrin和actin的相互作用是否发生改变，如果发生改变，这些变化是否涉及细胞功能；3)Ca2+/calpain和NHE1/ezrin通路之间是否存在相互作用或连锁，导致IPAH患者ECs和SMCs中观察到的功能改变。