title: Regulation of type 1 plasminogen activator inhibitor gene expression in cultured bovine aortic endothelial cells. Induction by transforming growth factor-beta, lipopolysaccharide, and tumor necrosis factor-alpha.
abstract: Cultured bovine aortic endothelial cells (BAEs) synthesize and secrete type 1 plasminogen activator inhibitor (PAI-1), an Mr 50,000 glycoprotein which inhibits both urokinase and tissue-type plasminogen activators. The synthesis of PAI-1 in BAEs is positively regulated by a variety of agents. To elucidate the mechanisms which govern expression of the PAI-1 gene, total cytoplasmic RNA was prepared from BAEs and analyzed by Northern blotting using a 1.3-kilobase (kb) human PAI-1 cDNA probe. Hybridization under conditions of high stringency revealed two bovine PAI-1 RNA species, 3.0 and 1.6 kb in length. The ratio of the two species was approximately 4:1. The 3.0-kb mRNA was bound by oligo(dT)-cellulose, whereas the 1.6-kb form was not, suggesting that the latter form lacked a poly(A) terminus. Treatment of BAEs with transforming growth factor beta (TGF-beta), bacterial lipopolysaccharide (LPS), or tumor necrosis factor alpha (TNF-alpha) markedly enhanced the steady-state levels of both RNA species. In each case, increases were detectable within 1 h, and maximal effects (i.e. greater than 30-fold increase) were observed between 6 and 18 h of treatment, followed by a decline to near-basal levels by 48 h. The response to each of these agents was dose-dependent, with maximal induction observed at concentrations of 10 ng/ml TGF-beta, 10 ng/ml LPS, and 25 ng/ml TNF-alpha. Induction of PAI-1 mRNA by these agents was not blocked by the protein synthesis inhibitor cycloheximide, suggesting that de novo protein synthesis was not required. In fact, treatment with cycloheximide (2 micrograms/ml) alone also increased PAI-1 mRNA levels. Treatment with cycloheximide in combination with TGF-beta, LPS, or TNF-alpha further enhanced the accumulation of PAI-1 mRNA. Nuclear transcription run-on experiments indicated that these agents elevated the rate of PAI-1 gene transcription 20-30-fold and that gene template activity was temporally correlated with the accumulation of PAI-1 mRNA. These data are consistent with the conclusion that the observed increases in PAI-1 steady-state mRNA levels result from primary effects of these agents on the rate of PAI-1 gene transcription.
authors.affiliation: Research Institute of Scripps Clinic, La Jolla, California 92037.
authors.forename: M
authors.initials: M
authors.lastname: Sawdey
authors.forename: T J
authors.initials: TJ
authors.lastname: Podor
authors.forename: D J
authors.initials: DJ
authors.lastname: Loskutoff
mesh_terms: D000818:Animals; D001011:Aorta; D002417:Cattle; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D015342:DNA Probes; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects*; D005796:Genes / Q000187:drug effects*; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics*; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology*; D010960:Plasminogen Activators / Q000037:antagonists & inhibitors; D015849:Plasminogen Inactivators; D012333:RNA, Messenger / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects*; D015291:Transforming Growth Factors / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D015342:DNA Probes; D006023:Glycoproteins; D008070:Lipopolysaccharides; D015849:Plasminogen Inactivators; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D015291:Transforming Growth Factors; D010960:Plasminogen Activators