Novel Regulation of Endothelin-1 Promoter Activity by Protein Kinase C

Abstract

Endothelin-1 (ET-1) is produced in unusually large amounts by the renal collecting duct and acts locally to control renal salt and water excretion and arterial pressure; disorders of collecting duct ET-1 activity can cause marked hypertension. The mechanisms regulating collecting duct ET-1 synthesis are, however, poorly understood. In this study, we investigated the role of protein kinase C (PKC), a known regulator of ET-1 production in endothelial cells, in (1) the control of collecting duct ET-1 production; and (2) the modulation of ET-1 promoter region activity. Cultured rat inner medullary collecting duct (IMCD) cells were studied. Calphostin C, a PKC inhibitor, greatly reduced IMCD ET-1 release. Sustained exposure to phorbol myristate acetate (PMA) also decreased ET-1 secretion. PKC inhibition decreased steady-state ET-1 mRNA content. A brief exposure (15 min) to PMA augmented ET-1 mRNA levels, while prolonged PMA exposure (120 min) reduced ET-1 mRNA content, PKC inhibition did not affect ET-1 mRNA stability. Transfection of ET-1 promoter-luciferase reporter constructs into IMCD cells demonstrated that PKC inhibition reduced activity of only the larger promoter fragments (containing at least 1,725 bp 5' to the ET-1 gene transcription start site). Mutation of a previously identified AP-1 site at -186 in the ET-1 promoter greatly reduced activity of transfected ET-1 promoter-reporter constructs (containing 366 or 1,725 bp 5' to the transcription start site); however, this region appears not to be regulated by PKC in IMCD cells. In summary, PKC stimulates collecting duct ET-1 synthesis via transcriptional activation of the ET-1 promoter. Such transcriptional activation occurs at a heretofore undescribed PKC-regulated region of the ET-1 promoter.