Functional linkage of NHE1 and calpain in IPAH

描述(由申请者提供)：我们的长期目标是了解参与肺动脉高压发病的细胞机制。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，我们最近证实了由于Na/H交换异构体1(NHE1)表达增加而导致的肺动脉内皮细胞(EC)和平滑肌细胞(SMC)细胞内pH(Phi)动态平衡的改变。此外，我们还发现，这些细胞表现出细胞内钙浓度的升高和钙激活的中性半胱氨酸蛋白酶家族--钙蛋白酶的表达增加。PHI、钙稳态和钙蛋白酶活性的变化对细胞功能有深远的影响，尽管这些途径在IPAH过程中是否发生了变化以及它们调控细胞生长和迁移的机制尚不清楚。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室肥厚和肺血管重构。最近的研究表明，NHE1可以作为一种不依赖于其离子转运特性的膜锚。NHE1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。如果是真的，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，钙蛋白酶的激活受到phi的影响，导致Rho激酶的激活，进而促进Ezrin的激活和与目标蛋白的结合。因此，我们假设在IPAH期间，NHE1和Calain上调，以及这些通路之间的相互作用，导致EC和SMC迁移和增殖增强，这是疾病发展和进展的标志。为了验证这一假说，我们将使用包括共聚焦显微镜、FRET、基因敲除和过表达方法、免疫共沉淀以及肺组织和肺组织的迁移和增殖分析在内的技术组合来确定：1)IPAH患者的EC和SMCs中的Calain活性是否增强，以及通过遗传或药物手段抑制是否改变了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，如果是，这些改变与细胞功能的关系；3)在IPAH患者的ECs和SMC中，是否存在与钙/钙蛋白和NHE1/Ezrin通路的相互作用或联系。 公共卫生相关性：本提案中的实验将探索特发性肺动脉高压(IPAH)发生的细胞机制，这是一种治疗选择有限的破坏性疾病。来自IPAH患者的组织和细胞的可获得性有望揭示新的信息，这些信息将进一步加深我们对IPAH发生在肺血管中的细胞变化的理解。这些信息对于推进治疗和开发新的治疗方案来治疗这种致命疾病至关重要。(摘要结束)

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)