Regulation of H/K-ATPase in Kidney and Colon

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 （HKalpha2）基因转录调控的分子机制。HKalpha2在维持机体K+平衡中起关键作用，也与Na+和酸碱平衡有关。虽然有充分的证据表明慢性低钾血症和高醛固酮增多症中HKalpha2表达的细胞特异性和差异诱导性，但这种控制的分子机制尚不清楚。在之前的资助期内，我们克隆并鉴定了小鼠HKalpha2基因，证明了5'-侧翼区域近177 bp具有收集管选择性转录活性，并在该区域鉴定了一种新的nf - kappab -组蛋白去乙酰化酶(HDAC)-6复合物，该复合物可抑制mIMCD3细胞中HKa2的转录。我们还在转基因小鼠中确定，小鼠HKalpha2基因的7.2 kb 5'侧区域将EGFP表达引导到收集管主细胞，但令人惊讶的是，没有将EGFP表达到结肠远端，而结肠远端是内源性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测定和启动子-报告子瞬时转染测定来跟踪特定转录因子和协调节蛋白与启动子的关联，以确定结合模式，以测试关于这些因子之间相互作用的假设。并监测在基础条件下、K+剥夺和醛固酮反应下，与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。确定的核蛋白改变反式中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体外鉴定的候选调控元件是否忠实地反映内源性HKalpha2基因的细胞特异性和刺激特异性反应。目的1将验证慢性K+剥夺促进-104/-94 NF-kappaB元件位点的协同调节蛋白的顺序和组合募集和释放的假设，这决定了在这种情况下HKalpha2基因的收集管特异性转录激活。Aim 2将验证一种假设，即在结肠远端表面上皮细胞中，HKalpha2基因的细胞特异性高水平表达是由KLF4在内含子1增强子上的作用和基础-104/-94 NF-kappaB-HDAC6抑制机制的破坏介导的。目的3将验证-1071/-1056激素反应元件作为增强体平台的假设，该增强体可选择性地在结肠远端对HKalpha2基因进行矿物皮质激素受体特异性反式激活。这些研究将为HKalpha2基因调控及其在肾脏和肠道细胞生物学和病理生物学中的独特作用提供重要的分子认识，为收集管道特异性基因表达和靶基因的醛固酮调控提供新的见解。

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.