CELL-SPECIFICITY AND SIGNALING PATHWAY OF ENDOTHELIN-1 GENE-REGULATION BY HYPOXIA

Objectives: Endothelin-1 (ET-1) is a potent vasoconstrictor that is ex pressed in endothelial cells and in many other cells and tissues. Incr eased plasma levels of the peptide have been associated with ischemic heart disease, atherosclerosis, and myocardial infarction. The objecti ves of the current study were (1) to determine the tissue specificity for induction of the ET-1 gene by hypoxia, (2) to determine whether th e hypoxia regulatory pathway is the same as that in other hypoxia regu lated genes and (3) to analyze the contributions of protein kinases fo r basal and induced expression of ET-1. Methods: ET-1 transcript level s were measured by Northern blot and quantitative polymerase chain rea ction in endothelial and non-endothelial cells following exposure to h ypoxia. Regulatory steps within the pathway were identified by treatin g aerobic or hypoxic cultures with cycloheximide, PMA, a series of sel ective protein kinase inhibitors, and transition metals. The effects o n ET-1 transcripts were compared with the ubiquitous hypoxia inducible pyruvate kinase gene. Results: The induction of ET-1 by hypoxia in vi tro occurred exclusively in early passage endothelial cells. This indu ction was prevented by treatment with the protein synthesis inhibitor cycloheximide and was at least partially mimicked by treatment with tr ansition metals. Induction by hypoxia was not effected by inhibitors o f protein kinase C, protein kinase A, calcium-calmodulin dependent pro tein kinase, or cyclic GMP dependent protein kinase. The basal express ion was decreased and hypoxic induction was eliminated by treating cel ls with tyrosine kinase-selective inhibitors. Conclusions: Et-1 induct ion by hypoxia requires endothelial cell-specific factor(s) or steps, new protein synthesis, and may involve a haeme protein-containing path way in oxygen sensing. A protein tyrosine kinase step is implicated fo r both basal and induced expression of the ET-1 gene.