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Regulation of H/K-ATPase in Kidney and Colon

肾脏和结肠中 H/K-ATP 酶的调节

基本信息

批准号：
7686219
负责人：
BRUCE C. KONE
金额：
$ 30.66万
依托单位：
UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
依托单位国家：
美国
项目类别：
财政年份：
1994
资助国家：
美国
起止时间：
1994-04-01 至 2011-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7686219
关键词：
5&apos Flanking Region Abbreviations Acids Address Aldosterone Binding Biological Assay Biology CREB1 gene Calcium-Binding Proteins Cells Cellular biology Chronic Colon Complex Cyclic AMP Data Deoxyribonuclease I Distal Duct (organ) structure 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 Molecular Monitor Mus NF-kappa B Nuclear Protein Nuclear Proteins Nuclear Receptors Nucleosomes PCAF gene Pattern Phosphotransferases Physiology Play Proteins RNA Interference 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 Zinc Fingers base chromatin immunoprecipitation clinically relevant combinatorial deprivation extracellular glucocorticoid receptor-interacting protein 1 histone acetyltransferase histone modification 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 complex protein inhibitors of activated STAT research study response selective expression stressor tool transcription factor transgene expression young adult

项目摘要

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(申请人提供)：这项研究的广泛、长期目标是确定肾脏和结肠中H+-K+-ATPase alpha2(HKalpha2)基因转录调控的分子机制。HKalpha2在维持体内K+平衡方面起着关键作用，也参与了Na+和酸碱平衡的维持。尽管有充分的证据表明，在慢性低钾血症和高醛固酮血症中，HKAlpha2的细胞特异性和差异性诱导表达，但这种控制的分子机制尚不清楚。在之前的资助期间，我们克隆并鉴定了小鼠HKalpha2基因，证明了5‘侧翼区近端的177个碱基具有收集导管选择性转录活性，并在该区域发现了一个新的抑制mIMCD3细胞HKa2转录的核因子-kappaB-组蛋白去乙酰基酶(HDAC)-6复合体。我们还在转基因小鼠中确定了小鼠HKalpha2基因的7.2kb 5‘侧翼区将EGFP表达引导到集合管主细胞，但令人惊讶的是，并没有引导到远端结肠，这是内源性HKa2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-报告基因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关联，确定结合模式，测试关于这些因素之间相互作用的假设，并监测与基础条件下以及K+剥夺和醛固酮反应中HKalpha2基因细胞特异性转录激活相关的共价组蛋白修饰的变化。确定的核蛋白在反式中改变HKalpha2启动子的能力将在共表达和RNA干扰实验中进行测试。转基因小鼠的研究将测试在体外确定的候选调控元件是否忠实地反映了内源性HKalpha2基因的细胞和刺激特异性反应。目的1验证慢性K+缺乏促进共调控蛋白向-104/-94 NF-kappaB元件位点的顺序和组合募集和释放的假设，该基因决定了在这种情况下收集管细胞特异性转录激活HKalpha2基因。目的2验证HKalpha2基因在远端结肠表面上皮细胞中的细胞特异性高水平表达是由KLF4作用于内含子1增强子和破坏基本的-104/-94 NF-kappaB-HDAC6阻遏机制所介导的假说。目的3验证以下假设：-1071/-1056激素反应元件作为增强小体的平台，在远端结肠选择性地给予盐皮质激素受体特异性的反式激活HKalpha2基因。这些研究将为HKalpha2基因的调控及其在肾和肠细胞生物学和病理生物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以及醛固酮对靶基因的调控提供新的见解。