Cooperation of two PEA3/AP1 sites in uPA gene induction by TPA and FGF-2.
We have previously shown in NIH 3T3 fibroblasts that treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) or fibroblast growth factor-2 (FGF-2) activates the Ras/Erk signaling pathway in NIH 3T3 fibroblasts, leading to the induction of the urokinase-type plasminogen activator (uPA) gene. In this study, we characterize cis-acting elements involved in this induction. DNase I hypersensitive (HS) site analysis of the uPA promoter showed that two regions were enhanced after TPA and FGF-2 treatment. One was located 2.4kb upstream of the transcription start site (-2.4kb), where a known PEA3/AP1 (AGGAAATGAGGTCAT) element is located. The other was located in a previously undefined far upstream region. Sequencing of this region revealed a similar AP1/PEA3 (GTGATTCACTTCCT) element at -6.9 kb corresponding to the HS site. Deletion analysis of the uPA promoter in transient transfection assays showed that both PEA3/AP1 elements are required for full inducibility, suggesting a synergism between the two elements. When the two sites were inserted together upstream of a minimal promoter derived from the thymidine kinase gene, expression of the reporter gene was more strongly induced by TPA and FGF-2 than with either of the two elements alone. Alone, the -6.9 element was more potent than the -2.4 element. The involvement of AP1 as well as Ets transcription factors was confirmed by examining different promoter constructs containing deletions in either the AP-1 or the PEA3 element, and by using an expression plasmid for dominant negative Ets-2. Electromobility shift analyses using specific antibodies showed that c-Jun and, JunD bind to both elements with or without induction. In addition, ATF-2 binds to the -2.4-kb element even without induction and c-Fos to the -6.9-kb element only after induction. Accordingly, overexpression of c-Fos caused induction from the -6.9-kb element, but reduced induction from the -2.4-kb element. The involvement of the Ets-2 transcription factor was shown by using expression plasmids for wild-type and dominant negative Ets-2.
D'Orazio
D
D
Friedrich Miescher Institute, Basel, Switzerland.
Besser
D
D
Marksitzer
R
R
Kunz
C
C
Hume
D A
DA
Kiefer
B
B
Nagamine
Y
Y
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA; D004268:DNA-Binding Proteins; D003850:Deoxyribonuclease I / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000187:drug effects; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D050787:Proto-Oncogene Protein c-ets-2; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D012097:Repressor Proteins; D017422:Sequence Analysis, DNA; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D015534:Trans-Activators / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics*
D004268:DNA-Binding Proteins; C107465:ERF protein, human; C494070:Ets2 protein, mouse; D050787:Proto-Oncogene Protein c-ets-2; D011518:Proto-Oncogene Proteins; D016760:Proto-Oncogene Proteins c-fos; D012097:Repressor Proteins; D015534:Trans-Activators; D018808:Transcription Factor AP-1; D014157:Transcription Factors; C065788:transcription factor PEA3; D016222:Fibroblast Growth Factor 2; D004247:DNA; D003850:Deoxyribonuclease I; D014568:Urokinase-Type Plasminogen Activator; D013755:Tetradecanoylphorbol Acetate