Role of Pen 1 and the mineralocorticoid receptor in the coordinate regulation of alpha ENaC in renal cortical collecting duct cells

Renal function and blood pressure (BP) exhibit a circadian pattern of variation, but the molecular mechanism underlying this circadian regulation is not fully understood. We have previously shown that the circadian clock protein Pen1 positively regulates the basal and aldosterone-mediated expression of the alpha subunit of the renal epithelial sodium channel (alpha ENaC). The mechanism of this regulation has not been determined however. To further elucidate the mechanism of mineralocorticoid receptor (MR) and Pen1 action, site-directed mutagenesis, DNA pull-down assays and chromatin immunoprecipitation (ChIP) methods were used to investigate the coordinate regulation of aENaC by Peni and MR. Mutation of two circadian response E-boxes in the human aENaC promoter abolished both basal and aldosterone-mediated promoter activity. DNA pull down assays demonstrated the interaction of both MR and Pen1 with the E-boxes from the aENaC promoter. These observations were corroborated by ChIP experiments showing increased occupancy of MR and Pen1 on an E-box of the aENaC promoter in the presence of aldosterone. This is the first report of an aldosterone-mediated increase in Pen1 on a target gene promoter. Taken together, these results demonstrate the novel finding that Pen1 and MR mediate the aldosterone response of aENaC through DNA/protein interaction in renal collecting duct cells.