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: 6774733
• 负责人: CHRISTIE P. THOMAS
• 金额: $ 29.5万
• 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/6774733
• 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（由申请方提供）：气道和肺泡上皮中的Na+重吸收在出生时至关重要，并在出生后和整个生命过程中继续发挥重要作用，以调节离子组成和肺分泌物的体积。气道和肺泡中的不成熟或失调的Na+和液体重吸收可能导致新生儿呼吸窘迫综合征（RDS）和以过量气道液体为特征的肺部疾病的病理生理学，例如在肺水肿中或在毒性、感染性或炎性肺损伤之后所见。在这项研究计划中，我们建议研究两个重要的调节围产期Na+运输在肺，环磷酸腺苷和糖皮质激素，侧重于sgk 1的作用，激酶，似乎是一个点的收敛许多信号通路，刺激Na+运输。我们的假设是，环AMP和GC，通过Sgk 1刺激钠离子转运远端肺上皮细胞和胎肺和Sgk 1表达的调节主要发生在Sgk 1基因转录水平。我们将首先调查，如果环AMP和GC调节Na+运输在人胎肺和胎儿远端肺上皮细胞发生，部分，通过刺激sgk 1。将通过远端肺上皮细胞系中的短路电流和人胎肺中测量的管腔体积的阿米洛利敏感性变化来测量钠离子转运。将通过显性阴性sgk 1的表达或通过反义寡核苷酸研究sgk 1的作用。我们还将测试的假设，腺病毒转移的sgk 1可以增强环AMP和GC介导的钠离子转运在远端肺上皮细胞和胎儿肺。 第二，我们将确定环磷酸腺苷的升高是否刺激胎儿肺和远端肺上皮细胞中sgk 1的表达，并确定调节途径。拟议的实验将测量sgk 1 mRNA和蛋白质;确定响应于环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、凝胶迁移率改变试验和染色质免疫沉淀试验确定远端肺上皮细胞中sgk 1基因的环腺苷酸刺激增强子元件。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道上皮中的Na+转运提供了可能。