Cell-specificity and signaling pathway of endothelin-1 gene regulation by hypoxia.
OBJECTIVE
Endothelin-1 (ET-1) is a potent vasoconstrictor that is expressed in endothelial cells and in many other cells and tissues. Increased plasma levels of the peptide have been associated with ischemic heart disease, atherosclerosis, and myocardial infarction. The objectives of the current study were (1) to determine the tissue specificity for induction of the ET-1 gene by hypoxia, (2) to determine whether the hypoxia regulatory pathway is the same as that in other hypoxia regulated genes and (3) to analyze the contributions of protein kinases for basal and induced expression of ET-1.


METHODS
ET-1 transcript levels were measured by Northern blot and quantitative polymerase chain reaction in endothelial and non-endothelial cells following exposure to hypoxia. Regulatory steps within the pathway were identified by treating aerobic or hypoxic cultures with cycloheximide, PMA, a series of selective protein kinase inhibitors, and transition metals. The effects on ET-1 transcripts were compared with the ubiquitous hypoxia inducible pyruvate kinase gene.


RESULTS
The induction of ET-1 by hypoxia in vitro occurred exclusively in early passage endothelial cells. This induction was prevented by treatment with the protein synthesis inhibitor cycloheximide and was at least partially mimicked by treatment with transition metals. Induction by hypoxia was not effected by inhibitors of protein kinase C, protein kinase A, calcium-calmodulin dependent protein kinase, or cyclic GMP dependent protein kinase. The basal expression was decreased and hypoxic induction was eliminated by treating cells with tyrosine kinase-selective inhibitors.


CONCLUSIONS
Et-1 induction by hypoxia requires endothelial cell-specific factor(s) or steps, new protein synthesis, and may involve a haeme protein-containing pathway in oxygen sensing. A protein tyrosine kinase step is implicated for both basal and induced expression of the ET-1 gene.
Bodi
I
I
Department of Cell and Molecular Biology, SRI International, Menlo Park, CA 94025, USA.
Bishopric
N H
NH
Discher
D J
DJ
Wu
X
X
Webster
K A
KA
D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002478:Cells, Cultured; D003035:Cobalt / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D017931:DNA Primers / Q000235:genetics; D016232:Endothelins / Q000235:genetics* / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D000860:Hypoxia / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D012333:RNA, Messenger / Q000032:analysis / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015290:Second Messenger Systems / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014469:Umbilical Arteries; D014471:Umbilical Veins
D017931:DNA Primers; D016232:Endothelins; D012333:RNA, Messenger; D003035:Cobalt; D003513:Cycloheximide; D011505:Protein-Tyrosine Kinases; D013755:Tetradecanoylphorbol Acetate