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: 7089798
• 负责人: CHRISTIE P. THOMAS
• 金额: $ 28.81万
• 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/7089798
• 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）：出生时气道和肺泡上皮的Na+重吸收至关重要，并在出生后和整个生命中继续发挥重要作用，以调节肺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+和液体重吸收可能有助于新生儿呼吸窘迫综合征（RDS）和肺部疾病的病理生理学，这些疾病以肺水肿或中毒性、感染性或炎症性肺损伤后出现的气道液体过多为特征。在本研究计划中，我们建议研究围产期肺中Na+运输的两个重要调节因子，环AMP和糖皮质激素，重点研究sgk1的作用，sgk1是一种激酶，似乎是刺激Na+运输的许多信号通路的会聚点。我们的假设是，通过sgk1作用的环AMP和GC刺激远端肺上皮和胎儿肺中的Na+运输，并且sgk1表达的调节主要发生在sgk1基因转录水平上。我们将首先研究环amp和gc调节的Na+在人胎儿肺和胎儿远端肺上皮细胞中的转运是否部分通过刺激sgk1发生。Na+运输将通过在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德敏感的管腔容积变化来测量。sgk1的作用将通过显性阴性sgk1或反义寡核苷酸的表达来研究。我们还将验证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+运输的假设。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中sgk1的表达，并确定其调控途径。拟议的实验将测量sgk1 mRNA和蛋白质；确定响应循环AMP刺激的调节机制并绘制调节通路。最后，我们将通过瞬时转染、凝胶迁移转移试验和染色质免疫沉淀试验，鉴定远端肺上皮细胞中环amp刺激的sgk1基因增强元件。了解通过激素介导的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上皮内Na+转运提供了可能。