title: A DNA motif related to the cAMP-responsive element and an exon-located activator protein-2 binding site in the human tissue-type plasminogen activator gene promoter cooperate in basal expression and convey activation by phorbol ester and cAMP.
abstract: Tissue-type plasminogen activator (t-PA) gene expression is regulated by the tumor-promoting phorbol ester, phorbol-12-myristate 13-acetate (PMA), by cyclic AMP analogues, and the cAMP agonist, forskolin. Based on nuclear "run-on" transcription assays, t-PA expression is modulated by PMA on the level of transcription. 8-Bromo-cyclic AMP and forskolin do not induce t-PA gene transcription alone but act synergistically with PMA. These effects are confirmed by transient expression assays in HeLa cells employing deletion mutants of the t-PA gene promoter fused to the chloramphenicol acetyltransferase (CAT) reporter gene. Constitutive expression and most of the PMA-mediated induction requires sequences downstream of position -145. DNase I protection ("footprint") analysis of this region reveals two protein-binding sites: one between position -102 and -115, differing from the consensus sequence of the cAMP-responsive element (CRE) by the substitution of an adenine for a guanine in the middle of the core motif (TGACATCA), and another, located in the first exon (between position +60 and +74), displaying homology to the consensus sequence of the activator protein 2- (AP-2) binding site (CCCCACCCCC). Base substitutions in the core of either the CRE-like element or the AP-2 site suppress constitutive CAT expression by over 80%, whereas the relative PMA- and PMA plus cAMP-mediated responses are retained. CAT expression is below the detection limit when both elements are mutagenized together. Hence, the CRE-like element and the exon-located AP-2-binding site have a cooperative impact on basal transcription, but each element can independently convey the effect of activators of the protein kinase C- and A-dependent pathways of signal transduction. The results of band-shift analysis and competition titration experiments demonstrate that the CRE-like element acts as a low affinity binding site for the same proteins which recognize the authentic CRE.
authors.affiliation: Central Hematology Laboratory, University of Lausanne Medical School, CHUV, Switzerland.
authors.forename: R L
authors.initials: RL
authors.lastname: Medcalf
authors.forename: M
authors.initials: M
authors.lastname: Rüegg
authors.forename: W D
authors.initials: WD
authors.lastname: Schleuning
mesh_terms: D000546:Amanitins / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000494:pharmacology*; D003513:Cycloheximide / Q000494:pharmacology; D004247:DNA / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003850:Deoxyribonuclease I; D005091:Exons; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D005796:Genes / Q000187:drug effects; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D013698:Templates, Genetic; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D010959:Tissue Plasminogen Activator / Q000235:genetics*; D050656:Transcription Factor AP-2; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection
chemical_list: D000546:Amanitins; D004268:DNA-Binding Proteins; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D050656:Transcription Factor AP-2; D014157:Transcription Factors; D004247:DNA; D003513:Cycloheximide; D000242:Cyclic AMP; D003850:Deoxyribonuclease I; D010959:Tissue Plasminogen Activator; D013755:Tetradecanoylphorbol Acetate