MNNG induces uPA transcription in C2C12 cells: requirement of an AP1 enhancer element.

(A) Transcriptional activity of uPA promoter constructs in response to MNNG in C2C12 and NIH3T3 cells. C2C12 and NIH3T3 cells were transiently transfected with p-8.2Luc and p-0.035Luc, containing 8.2 kb and 35 bp, respectively, of murine uPA 5′-flanking region ligated upstream of the luciferase reporter gene, and luciferase activity was determined after 8 hours stimulation with 70 μmol/L MNNG. Three independent experiments, showing less than 25% variability in luciferase activity, were performed. Luciferase activity corresponding to each plasmid in the absence of MNNG stimulation was arbitrarily assigned a value of 1. (B) Luciferase induction of uPA promoter-containing cell lines by MNNG. C2C12 cells were stably transfected with different uPA 5′-deletion mutants, containing −0.035 kb, −2.0 kb, −4.9 kb, −6.6 kb, and −8.2 kb, respectively. For each uPA-luciferase plasmid transfection, neomycin-resistant colonies were pooled and chimerical plasmid insertion was normalized by Southern blot analysis with a luciferase DNA probe, as described in Miralles et al,72 and the corresponding cell lines were treated or not with MNNG for 8 hours. Luciferase activities for each cell line are expressed relative to the activity found in the corresponding untreated cells, which was assigned a value of 1. Values are the mean value of 4 experiments. (C) The uPA AP1-enhancer is required for MNNG transcriptional induction. C2C12 cells stably transfected with different uPA promoter-luciferase constructs containing (as in panel B) or lacking the AP1-enhancer (p-8.2(ΔAP1)Luc, p-6.6(ΔAP1)Luc, p-4.9(ΔAP1)Luc,72were treated with MNNG as described in (A). Only results obtained with p-8.2Luc– and p-8.2(ΔAP1)Luc–containing cell lines are shown. Luciferase activity for each cell line is expressed relative to the activity found in the untreated cells, which was assigned a value of 1. All normalized activities represent a minimum of 4 experiments, showing less than 25% variability. (D) MNNG treatment induces AP1-binding activity to the uPA enhancer element. Left: Induction of uPA 5′-TRE- and 3′-TRE-binding activities by MNNG treatment. C2C12 cells were grown in 0.5% FBS for 16 hours and either treated (+) or not (−) with MNNG (70 μmol/L) for 2 hours. Nuclear extracts were prepared at 2 hours after treatment and incubated with 20 000 cpm of the indicated³²P-labeled probes, corresponding to the 5′-TRE (AP1_A) and 3′-TRE (AP1_B) elements of the uPA promoter, respectively. Right: Specificity of the induced uPA AP1-binding activity. Two-hour–induced C2C12 nuclear extract was incubated with the labeled AP1_A or AP1_Boligonucleotide, respectively, in the absence or presence of 150-fold molar excess of unlabeled competitors (described in the “Materials and methods” section): cold AP1_A and AP1_Bwere used as specific competitors for the corresponding labeled oligonucleotides, whereas cold IgκB (κB site of the Igκ enhancer) was used as unrelated competitor. The filled arrowhead indicates specific TRE-binding complexes.