Sgk1 in Na+ transport in fetal lung epithelia

Sgk1 在胎儿肺上皮细胞 Na 转运中的作用

• 批准号: 6679664
• 负责人: CHRISTIE P. THOMAS
• 金额: $ 28.79万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-11 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6679664
• 关键词: SDS polyacrylamide gel electrophoresis; biological signal transduction; cell line; clinical research; cyclic AMP; gel mobility shift assay; glucocorticoids; human fetus tissue; immunoprecipitation; ion transport; lung; messenger RNA; oligonucleotides; phosphotransferases; polymerase chain reaction; respiratory epithelium; sodium channel; sodium ion; tissue /cell culture; transcription factor; western blottings

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.

描述（由申请人提供）：气道和肺泡上皮细胞中的 Na+ 重吸收在出生时至关重要，并且在出生后和整个生命过程中继续发挥重要作用，以调节肺部分泌物的离子组成和体积。气道和肺泡中不成熟或失调的 Na+ 和液体重吸收可能会导致新生儿呼吸窘迫综合征 (RDS) 和肺部疾病的病理生理学，这些疾病的特征是气道液体过多，例如肺水肿或中毒性、感染性或炎性肺损伤后出现的情况。在本研究计划中，我们建议研究肺部围产期 Na+ 转运的两个重要调节因子，即环 AMP 和糖皮质激素，重点关注 sgk1 的作用，sgk1 是一种激酶，似乎是刺激 Na+ 转运的许多信号通路的汇聚点。我们的假设是，环 AMP 和 GC 通过 sgk1 作用，刺激远端肺上皮和胎儿肺中的 Na+ 转运，并且 sgk1 表达的调节主要发生在 sgk1 基因转录水平。我们将首先研究人胎肺和胎儿远端肺上皮细胞中环 AMP 和 GC 调节的 Na+ 转运是否部分通过刺激 sgk1 发生。 Na+转运将通过远端肺上皮细胞系中的短路电流和在人胎儿肺中测量的阿米洛利敏感的管腔体积变化来测量。 sgk1 的作用将通过显性失活 sgk1 的表达或反义寡核苷酸进行研究。我们还将测试以下假设：sgk1 的腺病毒转移可以增强远端肺上皮和胎儿肺中环 AMP 和 GC 介导的 Na+ 转运。 其次，我们将确定环 AMP 的升高是否会刺激胎儿肺和远端肺上皮细胞中 sgk1 的表达，并确定调节途径。拟议的实验将测量 sgk1 mRNA 和蛋白质；确定响应环 AMP 刺激的调节机制并绘制调节途径。最后，我们将通过瞬时转染、凝胶迁移率变化测定和染色质免疫沉淀测定来鉴定远端肺上皮细胞中 sgk1 基因的环 AMP 刺激的增强子元件。了解激素介导事件和第二信使系统调节 Na+ 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道上皮细胞中 Na+ 转运提供了潜力。