Sgk1 in Na+ transport in fetal lung epithelia

描述(由申请人提供)：NA在出生时在呼吸道和肺泡上皮细胞中的重吸收是至关重要的，在出生后和整个生命过程中继续发挥重要作用，以调节肺分泌物的离子成分和体积。在新生儿呼吸窘迫综合征(RDS)和以呼吸道液体过多为特征的肺部疾病的病理生理学过程中，呼吸道和肺泡内未成熟或调节失调的钠和液体重吸收可能是导致新生儿呼吸窘迫综合征(RDS)的病理生理因素之一，如肺水肿或中毒、感染性或炎症性肺损伤。在这项研究计划中，我们建议研究肺内围产期钠转运的两个重要调节因子，环磷酸腺苷和糖皮质激素，重点是SGK1的作用，它似乎是刺激钠转运的许多信号通路的汇聚点。我们的假设是，cAMP和GC通过SGK1作用于远端肺上皮细胞和胎肺，刺激Na转运，SGK1表达的调节主要发生在SGK1基因转录水平。我们将首先研究AMP和GC是否部分通过刺激SGK1在人胎肺和胎肺远端上皮细胞中调节Na的循环转运。NA转运将通过远端肺上皮细胞系的短路电流和人胎肺对阿米洛利敏感的管腔体积变化来测量。SGK1的作用将通过显性阴性SGK1的表达或通过反义寡核苷酸来研究。我们还将验证腺病毒转导SGK1可以增强AMP和GC介导的远端肺上皮细胞和胎肺钠转运的假设。其次，我们将确定cAMP的升高是否会刺激胎肺和远端肺上皮细胞中SGK1的表达，并确定其调节途径。拟议的实验将测量SGK1的mRNA和蛋白；确定响应循环AMP刺激的调控机制，并绘制调控路径图。最后，我们将通过瞬时转染、凝胶迁移率改变分析和染色质免疫沉淀实验来鉴定远端肺上皮细胞中环AMP刺激的SGK1基因的增强子元件。了解激素介导的事件和第二信使系统调节钠转运的机制，为在包括RDS在内的各种病理生理条件下调节呼吸上皮钠转运提供了可能性。

DESCRIPTION (provided by applicant): Na+ reabsorption in airway and alveolar epithelia is critically important at the time of birth and continues to play a significant role after birth and throughout life to regulate the ionic composition, and volume of pulmonary secretions. Immature or dysregulated Na+ and fluid reabsorption in the airway and alveoli may contribute to the pathophysiology of respiratory distress syndrome (RDS) of the newborn and of lung disorders that are characterized by excess airway liquid such as that seen in pulmonary edema or following a toxic, infectious or inflammatory lung injury. In this research plan we propose to study two important regulators of perinatal Na+ transport in the lung, cyclic AMP and glucocorticoids, focusing on the role of sgk1, a kinase that appears to be a point of convergence for many signaling pathways that stimulate Na+ transport. Our hypothesis is that cyclic AMP and GC, acting through sgk1 stimulate Na+ transport in distal lung epithelia and in fetal lung and that regulation of sgk1 expression occurs primarily at the level of sgk1 gene transcription. We will first investigate if cyclic AMP-and GC-regulated Na+ transport in human fetal lung and in fetal distal lung epithelial cells occurs, in part, through stimulation of sgk1. Na+ transport will be measured by short-circuit current in a distal lung epithelial cell line and amiloride-sensitive changes in lumen volume measured in human fetal lung. The role of sgk1 will be investigated by expression of dominant negative sgk1 or by antisense oligonucleotides. We will also test the hypothesis that adenoviral transfer of sgk1 can enhance cyclic AMP- and GC-mediated Na+ transport in distal lung epithelia and in fetal lung. Second, we will determine if elevation of cyclic AMP stimulates sgk1 expression in fetal lung and in distal lung epithelia and identify the pathways of regulation. The proposed experiments will measure sgk1 mRNA and protein; determine the mechanism of regulation in response to cyclic AMP stimulation and map pathways of regulation. Finally, we will identify the cyclic AMP-stimulated enhancer elements of the sgk1 gene in distal lung epithelia by transient transfection, gel mobility shift assays and by chromatin immunoprecipitation assays. Understanding the mechanisms of regulation of Na+ transport by hormone mediated events and second messenger systems offers the potential for modulating Na+ transport in respiratory epithelia in a variety of pathophysiological conditions including RDS.