title: 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.
authors.affiliation: Division of Nephrology, University of Utah Health Sciences Center, Salt Lake City, UT 84132, USA.
authors.forename: Peter K
authors.initials: PK
authors.lastname: Stricklett
authors.forename: Kevin A
authors.initials: KA
authors.lastname: Strait
authors.forename: Donald E
authors.initials: DE
authors.lastname: Kohan
mesh_terms: D000818:Animals; D019332:Endothelin-1 / Q000235:genetics* / Q000378:metabolism; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011493:Protein Kinase C / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D014158:Transcription, Genetic
chemical_list: D019332:Endothelin-1; D012333:RNA, Messenger; D011493:Protein Kinase C