Functional linkage of NHE1 and calpain in IPAH

描述（由申请人提供）：我们的长期目标是了解参与肺动脉高压发病机制的细胞机制。以往的研究表明，在特发性肺动脉高压（IPAH）的发展过程中，发生在肺血管的形态和功能的变化，但这些变化背后的细胞机制仍然知之甚少。使用动物模型，我们最近证明了改变肺动脉内皮细胞（EC）和平滑肌细胞（SMC）细胞内pH值（pHi）的稳态，由于增加Na+/H+交换异构体1（NHE 1）的表达的作用。此外，我们还表明，这些细胞表现出升高的细胞内Ca 2+浓度和钙蛋白酶，一个家庭的胞质，Ca 2+激活，中性半胱氨酸蛋白酶的表达增加。pHi、Ca 2+稳态和钙蛋白酶活性的变化对细胞功能具有深远的影响，尽管这些途径在IPAH期间是否改变以及它们调节细胞生长和迁移的机制知之甚少。在肺动脉高压的动物模型中，我们发现抑制钙蛋白酶可以减少右心室肥大和肺血管重构。最近的研究表明，NHE 1可以作为膜锚独立于其离子转运特性。NHE 1与ezrin共定位并结合，ezrin是一种含有多个结合结构域的蛋白质，是蛋白质-蛋白质相互作用的重要介体。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。如果是真的，IPAH期间增加的NHE 1和/或埃兹蛋白表达/相互作用可以创造有利于膜/细胞骨架相互作用的条件。此外，钙蛋白酶的激活受pHi的影响，并导致Rho激酶的激活，这反过来又促进ezrin的激活和与靶蛋白的结合。因此，我们假设在IPAH期间，NHE 1和钙蛋白酶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，这是疾病发展和进展的标志。为了检验这一假设，我们将使用包括共聚焦显微镜、FRET、敲低和过表达方法、免疫共沉淀以及肺血管EC和SMC和肺组织中的迁移和增殖测定以及来自IPAH和对照受试者的mRNA在内的技术组合来确定：第一章IPAH患者EC和SMC中钙蛋白酶活性是否增强，以及通过遗传或药理学手段抑制是否改变了这些细胞的增殖和迁移; 2）在来自IPAH患者的血管细胞中，NHE 1的表达、活性以及与埃兹蛋白和肌动蛋白的相互作用是否改变，如果是，这些变化与细胞功能的关系，以及3）在Ca 2 +/钙蛋白酶和NHE 1/NHE 1之间是否存在相互作用或联系。ezrin途径，有助于在IPAH患者的EC和SMC中观察到的功能变化。 公共卫生相关性：本提案中的实验将探索参与特发性肺动脉高压（IPAH）发展的细胞机制，IPAH是一种治疗选择有限的毁灭性疾病。IPAH患者的组织和细胞的可用性有望揭示新的信息，这将进一步加深我们对IPAH发展过程中肺血管系统细胞变化的理解。这些信息对于推进治疗和开发治疗这种致命疾病的新治疗方案至关重要。(End摘要）

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)