Role of the Na K ATPase Beta 1 subunit in alveolar epithelial integrity

Na K ATPase Beta 1 亚基在肺泡上皮完整性中的作用

基本信息

批准号：
9069957
负责人：
Laura Andrea Dada
金额：
$ 35.04万
依托单位：
NORTHWESTERN UNIVERSITY AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-08-01 至 2017-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9069957
关键词：
Active Biological Transport Acute Lung Injury Adherens Junction Adhesions Alveolar Alveolus Amino Acids Cell Adhesion Molecules Cell Communication Cell membrane Cell-Cell Adhesion Cells Clinical Complication Cytoskeleton Data Detergents Diffusion Edema Endoplasmic Reticulum Enzymes Epithelial Epithelial Cells Epithelium Exposure to Family Gases Health Hypoxia Impairment In Vitro Integral Membrane Protein Intercellular Junctions Ion Transport Lead Lectin Lentivirus Vector Life Link Liquid substance Lung Mediating Molecular Molecular Chaperones Mus Na(+)-K(+)-Exchanging ATPase Outcome Pathway interactions Permeability Polysaccharides Process Proteins Pulmonary Edema Pump Resistance Resolution Role Site-Directed Mutagenesis Stimulus Structure Testing Tissues Up-Regulation alveolar epithelium biological adaptation to stress dimer dysadherin effective therapy improved in vivo inhibitor/antagonist insight lung injury monolayer overexpression prevent research study sodium ion

项目摘要

DESCRIPTION (provided by applicant): Lung edema is a life-threatening complication of lung injury. The active transepithelial Na+ ion transport performed in part by the alveolar epithelial Na,K-ATPase is critical for lung edema clearance. Edema impairs gas exchange, which leads to alveolar hypoxia. Hypoxia, in turn, impairs intercellular adhesion and decreases the amount of the Na,K-ATPase at the plasma membrane, worsening clinical outcomes. The minimal functional unit of the Na,K-ATPase is a dimer consisting of an α and a β subunit, which not only transports ions, but also acts as a cell adhesion molecule. FXYD5 is a one of the 7 tissue-specific regulatory subunits of the Na,K- ATPase activity, which is also implicated in the impairment of intercellular junctions. In preliminary experiments, we observed that hypoxia impairs intercellular adhesion and up-regulates FXYD5 in alveolar epithelial cells. We hypothesize that the Na,K-ATPase is important for modulating the integrity of the alveolar epithelium by strengthening cell-cell contacts due to the interaction between the Na,K-ATPase β1 subunits of neighboring alveolar epithelial cells and by weakening these contacts by FXYD5- mediated impairment of β1:β1 bridges during hypoxia. In specific aim # 1, we will determine whether the interactions between the Na,K-ATPase β1 subunits of neighboring alveolar epithelial cells are impaired by hypoxia We will determine whether modulating β1:β1 interactions by removing N-glycans or modifying their structure alters cell-cell adhesion during normoxic and hypoxic conditions in vitro or in vivo. In specific aim #2, we will determine whether FXYD5 up-regulation during hypoxia impairs β1:β1 interactions. By over-expressing or silencing of FXYD5 in lung epithelial cells, in both normal and hypoxic conditions, we will determine whether FXYD5 contributes to hypoxia-induced impairment of intercellular adhesion by disrupting the interaction between the Na,K-ATPase β subunits of neighboring cells in vitro or in vivo. In specific aim #3, we will investigate chaperone-assisted maturation pathways of the Na,K-ATPase in the endoplasmic reticulum (ER) of alveolar epithelial cells in normal conditions and during hypoxia. In experiments performed for this proposal, we observed that hypoxia results in significant retention of the Na,K-ATPase in the ER of alveolar epithelial cells and thus decreases its abundance in the plasma membrane, which would impair edema fluid clearance and barrier function. We will determine whether up-regulation of ER chaperones by N-glycan processing inhibitor, castanospermine, rescues the maturation of the Na,K-ATPase and whether application of this inhibitor prior to exposure of mice to hypoxia improves barrier function. The proposed studies will provide insights into non- canonical roles of the Na,K-ATPase in stabilization of cell cell contacts, which are crucial for normal function of alveolar epithelia. Understanding the mechanism(s) underlying chaperone-assisted maturation of the Na,K- ATPase in alveolar epithelial cells and finding the means to prevent ER retention of the enzyme during hypoxia may lead to more effective treatment for pulmonary edema and acute lung injury.

描述（通过应用程序提供）：肺水肿是肺部损伤生命并发症的一种并发症。肺泡上皮Na进行的一部分进行的活性旋转Na+离子转运，K-ATPase对于肺水肿清除至关重要。水肿会损害气体交换，从而导致肺泡缺氧。反过来，缺氧会损害细胞间粘合剂，并降低质膜上Na，K-ATPase的量，从而恶化临床结果。 Na，K-ATPase的最小功能单元是由α和β亚基组成的二聚体，不仅传输离子，而且还充当细胞粘合分子。 FXYD5是Na，K-ATPase活性的7个组织特异性调节亚基之一，这也与细胞间连接的损害有关。在初步实验中，我们观察到缺氧会损害细胞间粘合剂，并上调肺泡上皮细胞中的FXYD5。我们假设Na，K-ATPase对于通过增强Na，K-ATPaseβ1亚基之间的相互作用而在相邻肺泡上皮细胞的相互作用中加强细胞细胞接触来调节肺泡上皮细胞的完整性很重要，并通过FXYD55555-介导的这些接触在FXYD-righip impairs impair impair impair impair impair impairs hypox hypox hypox hypox hypox prid phimia弱化。在特定的目标＃1中，我们将确定邻近肺泡上皮细胞的Na，K-ATPaseβ1亚基之间的相互作用是否受到缺氧的损害，我们将确定是否通过删除N-糖果来调节β1：β1的相互作用是通过在正常氧和过度氧中的细胞粘附在VIRCO中的细胞细胞粘附而改变的β1：β1相互作用。在特定的目标＃2中，我们将确定缺氧期间的FXYD5上调是否会损害β1：β1相互作用。通过在正常和低氧条件下，通过在肺上皮细胞中过度表达FXYD5的过度表达或沉默，我们将确定FXYD5是否会通过破坏NA，K-ATPaseβ亚基之间的NA，K-ATPaseβ亚un的相互作用来破坏缺氧诱导的细胞间粘附损害。在特定的目标＃3中，我们将研究肺泡上皮细胞在正常条件下和缺氧期间的内质网（ER）中Na，K-ATPase（ER）中Na，K-ATPase的成熟途径。在该提案进行的实验中，我们观察到缺氧导致Na，K-ATPase在牙槽上皮细胞的ER中显着保留，从而降低了其在质膜中的丰度，这会损害水肿流体清除率和屏障功能。我们将确定通过N-聚糖加工抑制剂Castanospermine对ER伴侣的上调是否可以挽救Na，K-ATPase的成熟，以及在暴露于小鼠之前对这种抑制剂的应用是否可以改善屏障功能。拟议的研究将提供对Na，K-ATPase在稳定细胞中的非规范作用的见解细胞接触，这对于肺泡上皮的正常功能至关重要。了解肺泡上皮细胞中Na，K- ATPase的基础机制的成熟，并找到防止在缺氧期间保持ER保留酶的手段，可能会导致对肺水肿和急性肺损伤的更有效治疗。