Regulation of H/K-ATPase in Kidney and Colon

肾脏和结肠中 H/K-ATP 酶的调节

• 批准号: 7270663
• 负责人: BRUCE C. KONE
• 金额: $ 30.98万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-04-01 至 2010-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7270663
• 关键词: 5' Flanking Region; Abbreviations; Acids; Address; Aldosterone; Binding; Biological Assay; Biology; CREB1 gene; Calcium-Binding Proteins; Cells; Cellular biology; Chronic; Colon; Complex; Condition; Cyclic AMP; Data; Deoxyribonuclease I; Disruption; Distal; Duct (organ) structure; EEF1A2 gene; Elements; Enhancers; Epithelial Cells; Equilibrium; Exhibits; Exposure to; Funding; GKLF protein; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic Transcription; Genus Cola; Glucocorticoid Receptor; Glucocorticoids; Goals; H(+)-K(+)-Exchanging ATPase; HDAC6 gene; Histone Deacetylase; Histones; Homeostasis; Hyperaldosteronism; Hypokalemia; In Vitro; Intestines; Introns; Kidney; Knockout Mice; Link; Maintenance; Mediating; Messenger RNA; Mineralocorticoid Receptor; Mineralocorticoids; Modeling; Modification; Molecular; Monitor; Mus; NF-kappa B; Nuclear Protein; Nuclear Proteins; Nuclear Receptors; Nucleosomes; PCAF gene; Pattern; Phosphotransferases; Physiology; Play; Proteins; RNA Interference; Range; Reagent; Regulation; Regulatory Element; Reporter; Reporter Genes; Research; Research Personnel; Role; Serum; Sgk protein; Signal Transduction; Silencing Mediator of Retinoid Thyroid Receptor; Site; Specificity; Stimulus; Stretching; Structure; Surface; Testing; Thyroid Hormone Receptor; Tissues; Transcriptional Activation; Transcriptional Regulation; Transfection; Transgenic Mice; Transgenic Organisms; YWHAQ gene; Zinc Fingers; base; chromatin immunoprecipitation; clinically relevant; combinatorial; deprivation; extracellular; glucocorticoid receptor-interacting protein 1; histone acetyltransferase; hormone response element; human NCOR1 protein; human RIPK1 protein; human TIF2 factor; in vivo; inhibitor/antagonist; insight; novel; nuclear receptor coactivator 1; p300/CBP-Associated Factor; programs; promoter; protein inhibitors of activated STAT; research study; response; selective expression; stressor; tool; transcription factor; transgene expression; young adult

DESCRIPTION (provided by applicant): The broad, long-term objectives of this research are to identify the molecular mechanisms underlying transcriptional regulation of the H+-K+-ATPase alpha2 (HKalpha2) gene in kidney and colon. HKalpha2 plays a critical role in the maintenance of body K+ balance, and it has also been implicated in Na+ and acid-base homeostasis. Although there is ample evidence for cell specificity and differential inducibility of HKalpha2 expression during chronic hypokalemia and hyperaldosteronism, the molecular mechanisms for this control are unknown. In the previous funding period, we cloned and characterized the murine HKalpha2 gene, demonstrated that the proximal 177 bp of the 5'-flanking region confers collecting duct-selective transcriptional activity, and identified a novel NF-kappaB-histone deacetylases (HDAC)-6 complex in this region that suppresses HKa2 transcription in mIMCD3 cells. We also determined in transgenic mice that the 7.2 kb 5'-flanking region of the murine HKalpha2 gene directs EGFP expression to collecting duct principal cells, but surprisingly not to distal colon, the site of highest endogenous HKa2 expression. We now propose to use quantitative chromatin immunoprecipitation assays and promoter-reporter transient transfection assays to follow association of specific transcription factors and coregulatory proteins with the promoter, to define patterns of binding, to test hypotheses regarding interactions among these factors, and to monitor changes in covalent histone modifications associated with cell-specific transcriptional activation of the HKalpha2 gene under basal conditions and in response to K+ deprivation and to aldosterone. The ability of defined nuclear proteins to alter the HKalpha2 promoter in trans will be tested in coexpression and RNA interference experiments. Studies in transgenic mice will test whether candidate regulatory elements identified in vitro faithfully mirror the cell- and stimulus-specific responses of the endogenous HKalpha2 gene. Aim 1 will test the hypothesis that chronic K+ deprivation promotes sequential and combinatorial recruitment and dismissal of coregulatory proteins to the -104/-94 NF-kappaB element locus, which dictates the collecting duct cell-specific transcriptional activation of the HKalpha2 gene in this setting. Aim 2 will test the hypothesis that cell-specific, high-level expression of the HKalpha2 gene in surface epithelial cells of the distal colon is mediated by the action of KLF4 at an intron 1 enhancer and disruption of the basal -104/-94 NF-kappaB-HDAC6 repressor mechanism. Aim 3 will test the hypothesis that the -1071/-1056 hormone response element serves as the platform for an enhanceosome that confers mineralocorticoid receptor-specific trans-activation of the HKalpha2 gene selectively in distal colon. These studies should provide important molecular insights into HKalpha2 gene regulation and its unique roles in renal and intestinal cell biology and pathobiology, new insights into collecting duct-specific gene expression, and aldosterone regulation of target genes.

描述（由申请人提供）：本研究的广泛、长期目标是确定肾脏和结肠中H+-K+-ATP酶α 2（HK α 2）基因转录调控的分子机制。HK α 2在维持身体K+平衡中起关键作用，并且它还涉及Na+和酸碱平衡。虽然有充分的证据表明，在慢性低钾血症和高醛固酮血症的细胞特异性和差异诱导HK α 2的表达，这种控制的分子机制是未知的。在之前的资助期间，我们克隆并鉴定了小鼠HK α 2基因，证明了5 '侧翼区的近端177 bp赋予了收集管选择性转录活性，并在该区域鉴定了一种新型NF-κ B-组蛋白脱乙酰酶（HDAC）-6复合物，该复合物抑制了mIMCD 3细胞中的HKa 2转录。我们还在转基因小鼠中确定，鼠HK α 2基因的7.2 kb 5 '侧翼区指导EGFP表达到集合管主细胞，但令人惊讶的是不指导远端结肠，即内源性HKa 2表达最高的部位。我们现在建议使用定量染色质免疫沉淀分析和启动子-报告子瞬时转染分析来跟踪特定转录因子和共调节蛋白与启动子的结合，以确定结合模式，测试关于这些因子之间相互作用的假设，并监测与细胞相关的共价组蛋白修饰的变化，HK α 2基因在基础条件下以及对K+剥夺和醛固酮的响应中的特异性转录激活。将在共表达和RNA干扰实验中测试确定的核蛋白改变HK α 2启动子的反式能力。在转基因小鼠中的研究将测试体外鉴定的候选调控元件是否忠实地反映内源性HK α 2基因的细胞特异性和刺激特异性反应。目的1将测试的假设，慢性K+剥夺促进连续和组合的招聘和解雇的共调节蛋白的-104/-94 NF-κ B元件位点，这决定了集合管细胞特异性的转录激活的HK α 2基因在这种情况下。目的2将检验以下假设：远端结肠表面上皮细胞中HK α 2基因的细胞特异性高水平表达是由KLF 4在内含子1增强子处的作用和基础-104/-94 NF-κ B-HDAC 6阻遏物机制的破坏介导的。目的3将检验假设，即-1071/-1056激素反应元件作为增强体的平台，增强体在远端结肠中选择性地赋予HK α 2基因的盐皮质激素受体特异性反式激活。这些研究将为HK α 2基因调控及其在肾脏和肠道细胞生物学和病理学中的独特作用提供重要的分子见解，为收集导管特异性基因表达和靶基因的醛固酮调控提供新的见解。