Identification of a tightly regulated hypoxia-response element in the promoter of human plasminogen activator inhibitor-1.
Plasminogen activator inhibitor-1 (PAI-1) plays a key role in control of coagulation and tissue remodeling and has been shown to be regulated by a number of cell stimuli, among those hypoxia. In this study we characterize the hypoxia-mediated induction of PAI-1 in human hepatoma cell line HepG2. We found that PAI-1 is tightly regulated in a narrow oxygen gradient. After incubation at oxygen concentrations of 1% to 2%, a 60-fold increase in PAI-1 messenger RNA levels was observed, whereas mild hypoxic conditions of more than 3.5% did not appear to induce transcription. Moreover, increased levels of PAI-1 protein were observed after incubation at low oxygen tensions. Through sequence analysis, several putative hypoxia-response elements (HREs 1-5) were identified in the human PAI-I promoter. Reporter gene assays showed that the HRE-2 (-194 to -187) was necessary and sufficient for the hypoxia-mediated response. By electrophoretic mobility assay we observed hypoxia-dependent binding of a protein complex to the HRE-2 motif. Further analysis demonstrated that HRE-2 was specifically recognized by the hypoxia-inducible transcription factor 1alpha-arylhydrocarbon nuclear translocator complex. Taken together, our data demonstrate that hypoxia-induced transcription is mediated through HIF-1 interaction with the HRE-2 site of the human PAI-1 promoter.
Fink
Trine
T
Department of Virus and Cancer, Danish Cancer Society, Gustav Wieds Vej 10, 8000 Aarhus C, Denmark. trine@virus.au.dk
Kazlauskas
Arunas
A
Poellinger
Lorenz
L
Ebbesen
Peter
P
Zachar
Vladimir
V
D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D000860:Hypoxia / Q000378:metabolism*; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D010100:Oxygen / Q000494:pharmacology; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000187:drug effects; D018336:Receptors, Aryl Hydrocarbon; D020218:Response Elements / Q000235:genetics* / Q000502:physiology; D016415:Sequence Alignment; D014157:Transcription Factors / Q000378:metabolism; D014407:Tumor Cells, Cultured
C497396:ARNT protein, human; D004268:DNA-Binding Proteins; C497442:HIF1A protein, human; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; D018336:Receptors, Aryl Hydrocarbon; D014157:Transcription Factors; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator; D010100:Oxygen