Regulation of H/K-ATPase in Kidney and Colon

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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.