Na, K-ATPase Regulation by Glucocorticoids in Lung

糖皮质激素对肺中 Na、K-ATP 酶的调节

• 批准号: 6724638
• 负责人: CHRIS H WENDT
• 金额: $ 33.41万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-12-15 至 2007-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6724638
• 关键词: DNA; SDS polyacrylamide gel electrophoresis; cell line; chromatin immunoprecipitation; epithelium; gel mobility shift assay; genetic transcription; glucocorticoids; heat shock proteins; laboratory rat; lung; mass spectrometry; molecular chaperones; nucleic acid sequence; polymerase chain reaction; site directed mutagenesis; sodium potassium exchanging ATPase; tissue /cell culture; transcription factor; western blottings

DESCRIPTION (provided by applicant): To allow for normal gas exchange at birth, the ion transport functions of the lung must change dramatically as the fetus transits into postnatal life. Late in gestation, as the fetus approaches birth, the lung changes from a chloride secreting to a sodium-absorbing organ. This results in the net resorption of salt and water from the alveolar airspace and prepares the fetus for the successful transition to the postnatal period in which the alveolus is dry and normal gas exchange takes place. This resorptive process occurs through the active transport of sodium via the basolateral Na, K-ATPase (sodium pump) and the passive transport of sodium, primarily through the apical amiloride-sensitive sodium channel. During late gestation, glucocorticoids influence the expression of the Na, K-ATPase gene in a manner that is dependent on the gestational age at the time of glucocorticoid administration, and the duration and dose of glucocorticoid therapy. The major effect of glucocorticoids is on the beta1 subunit, demonstrating a concomitant increase in gene expression, protein levels and activity as measured by wet to dry weights. Glucocorticoids regulate the Na, K-ATPase both transcriptionally and translationally depending on cell type. We found that glucocorticoids increase Na, K-ATPase gene expression via transcriptional regulation in fetal and adult lung epithelial cells, however, the regulatory elements and specific transcription factors involved in this upregulation remain unknown. Although the Na, K-ATPase beta1 promoter contains partial glucocorticoid receptor elements (GRE), few of these elements are classical GRE. In addition, upregulation by glucocorticoids occurs over hours, suggesting regulation may be through a unique delayed primary response that involves secondary proteins. The main objective of this grant is to extend our previous studies of pump transcriptional regulation by glucocorticoids and explore in vitro mapping of DNA regulatory elements and transcription factor identification that are critical for Na, K-ATPase beta1 regulation comparing fetal to adult lung epithelial cells. I have chosen to study the Na, K-ATPase beta1, since: 1) glucocorticoids increase beta1 protein levels concomitant to increased mRNA levels; 2) the Na, K-ATPase beta1 subunit is the rate limiting subunit in lung epithelial cells; and 3) the Na, K-ATPase beta1 subunit is necessary for the assembly of alpha-beta heterodimers and plasma membrane targeting.

描述(申请人提供)：为了在出生时进行正常的气体交换，随着胎儿进入出生后生命，肺的离子转运功能必须发生巨大变化。在妊娠后期，随着胎儿接近出生，肺从一个氯化物分泌变成一个吸收钠的器官。这导致从肺泡空隙净吸收盐分和水，并为胎儿成功过渡到出生后肺泡干燥和进行正常气体交换的时期做好准备。这种吸收过程通过钠泵主动转运钠离子和被动转运钠离子，主要是通过心尖阿米洛利敏感的钠离子通道。在妊娠晚期，糖皮质激素影响Na，K-ATPase基因表达的方式取决于使用糖皮质激素时的胎龄，以及糖皮质激素治疗的持续时间和剂量。糖皮质激素的主要作用是在β1亚基上，表现出伴随的基因表达、蛋白质水平和活性的增加，以湿重到干重衡量。糖皮质激素根据细胞类型在转录和翻译上调节Na，K-ATPase。我们发现糖皮质激素通过转录调控增加了胎儿和成人肺上皮细胞Na，K-ATPase基因的表达，然而，参与这种上调的调控元件和特异性转录因子尚不清楚。尽管Na，K-ATPase Beta1启动子含有部分糖皮质激素受体元件(GRE)，但这些元件很少是典型的GRE。此外，糖皮质激素的上调作用持续数小时，这表明调节可能是通过涉及次级蛋白质的独特的延迟初级反应来实现的。这笔赠款的主要目的是扩展我们之前对糖皮质激素对Pump转录调控的研究，并探索DNA调控元件的体外定位和转录因子鉴定，这些调控元件和转录因子对Na，K-ATPase Beta1调控至关重要，比较胎儿和成人肺上皮细胞。我之所以选择研究Na，K-ATPaseβ1，是因为：1)糖皮质激素增加了β1蛋白水平，同时伴随着mRNA水平的增加；2)Na，K-ATPaseβ1亚基是肺上皮细胞的限速亚单位；以及3)Na，K-ATPaseβ1亚单位是组装α-β异源二聚体和质膜靶向所必需的。