title: Induction of PAI-1 expression by tumor necrosis factor alpha in endothelial cells is mediated by its responsive element located in the 4G/5G site.
abstract: Plasminogen activator inhibitor type 1 (PAI-1) is induced by many proinflammatory and pro-oxidant factors. Among them, tumor necrosis factor alpha (TNFalpha), a pivotal early mediator that regulates and amplifies the development of inflammation, is one of the strongest PAI-1 synthesis activators. Location of the TNFalpha response element in the PAI-1 promoter is still ambiguous. In this study, we attempted to evaluate the significance of the element located in the 4G/5G site of the PAI-1 promoter in the TNFalpha stimulation of PAI-1 expression in endothelial cells. PAI-1 expression was monitored at: (a) the level of mRNA using real-time PCR, (b) PAI-1 gene transcription by transfection reporter assays, and (c) protein synthesis using the enzyme immunoassay. NF-kappaB activity was monitored using the electrophoretic mobility shift assay. Its activity was modified by either antisense oligonucleotides or transfection of endothelial cells with the wild-type or mutated IkappaBalpha. We have shown that TNFalpha-induced expression and gene transcription of PAI-1 involves a regulatory region present in segment -664/-680 of the PAI-1 promoter. This reaction involves the TNFalpha-induced generation of superoxide leading to activation of NF-kappaB, and can be abolished by antioxidants and by overexpression of a super-suppressor phosphorylation-resistant IkappaBalpha. Stimulation of PAI-1 under these conditions involves the motif of the PAI-1 promoter adjacent to the 4G/5G site, which can directly interact with NF-kappaB. We show that activation of PAI-1 gene by TNFalpha and reactive oxygen species is mediated by interaction of NF-kappaB with the cis-acting element located in the -675 4G/5G insertion/deletion in the PAI-1 promoter.
authors.affiliation: Department of Molecular and Medical Biophysics, Medical University in Lodz, Poland.
authors.forename: Maria
authors.initials: M
authors.lastname: Swiatkowska
authors.forename: Janusz
authors.initials: J
authors.lastname: Szemraj
authors.forename: Czeslaw S
authors.initials: CS
authors.lastname: Cierniewski
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D024202:Electrophoretic Mobility Shift Assay; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005452:Fluoresceins; D017353:Gene Deletion; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics / Q000378:metabolism; D008156:Luciferases / Q000378:metabolism; D016254:Mutagenesis, Insertional; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010544:Peroxidases; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D020218:Response Elements; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D014471:Umbilical Veins / Q000166:cytology
chemical_list: D005452:Fluoresceins; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606204:NFKBIA protein, human; D016376:Oligonucleotides, Antisense; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; C051321:SERPINE1 protein, human; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; D011092:Polyethylene Glycols; D010544:Peroxidases; C437331:dichlorofluorescin peroxidase; D008156:Luciferases; D000111:Acetylcysteine