Cooperation of two PEA3/AP1 sites in uPA gene induction by TPA and FGF-2

• Published in: Gene 1997-11, Vol.201 (1), p.179-187
• Main Authors: D'Orazio, Daniel, Besser, Daniel, Marksitzer, René, Kunz, Christina, Hume, David A, Kiefer, Birgitta, Nagamine, Yoshikuni
• Format: Article
• Language: English
• Subjects: 3T3 Cells; Animals; AP1; Base Sequence; Binding Sites; Cis-acting element; Deoxyribonuclease I - metabolism; DNA; DNA-Binding Proteins; Fibroblast Growth Factor 2 - pharmacology; Gene Expression Regulation - drug effects; Mice; Molecular Sequence Data; PEA3; Plasminogen activator; Promoter Regions, Genetic; Proto-Oncogene Protein c-ets-2; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins c-fos - metabolism; Repressor Proteins; Sequence Analysis, DNA; Signal transduction; Tetradecanoylphorbol Acetate - pharmacology; Trans-Activators - metabolism; Transcription Factor AP-1 - metabolism; Transcription Factors - metabolism; Transcriptional Activation; Urokinase-Type Plasminogen Activator - genetics
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/S0378-1119(97)00445-9

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 c i s-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 2.4 kb upstream of the transcription start site (−2.4 kb), where a known PEA3/AP1 (A G G A A A T G A G G T C A T) element is located. The other was located in a previously undefined far upstream region. Sequencing of this region revealed a similar AP1/PEA3 (G T G A T T C A C T T C C T) 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.