Figure 5. The −56/−63 bp and −105/−95 bp sequences mediate c-Jun-dependent activation of the CYP2J2 proximal promoter.

(A) Mutation of the −56/−63 bp and −105/−95 bp elements reduces c-Jun-dependent activation of the CYP2J2 promoter. The constructs 2J2(−152/+98; mt −56/−63) and 2J2(−152/+98; mt −106/−96) are mutated respectively at the previously identified c-Jun-binding site at −56 to −63 bp, and the newly identified c-Jun-binding sequence spanning −105 to −95 bp. In the construct 2J2(−152/+98; mt −56/−63, mt −106/−96), both the −56 to −63 bp and −105 to −95 bp sequences are mutated. CYP2J2 reporter constructs (1 μg per well) were co-transfected into HepG2 cells with a β-galactosidase expression plasmid (0.5 μg per well). A c-Jun expression plasmid was co-transfected at 0.5 μg per well as indicated. Luciferase activity was normalized as described in Figure 1(A). The results are expressed as the means±S.E.M. for at least three independent experiments. Significantly different, in the presence of c-Jun, from: *wild-type construct 2J2(−152/+98) (P<0.02), **wild-type construct 2J2(−152/+98) (P<0.0001), #mutated construct 2J2(−152/+98; mt −56/−63) (P<0.0001), and +mutated construct 2J2(−152/+98; mt −106/−96) (P<0.005). (B) Mutagenesis of the −56/−63 bp and −105/−95 bp elements reduces basal transcriptional activity of the CYP2J2 promoter. CYP2J2 reporter constructs or the empty pGL3basic vector were co-transfected (1 μg per well) into HepG2 cells with a β-galactosidase expression plasmid (0.5 μg per well). Luciferase activity was normalized to β-galactosidase activity and presented relative to the normalized activity of the empty pGL3basic vector (set to 1-fold). The results are expressed as the means±S.E.M. for at least three independent experiments. Significantly different from: * 2J2(−152/+98) (P<0.002), **2J2(−152/+98) (P<0.0005), #2J2(−152/+98; mt −56/−63) (P<0.005), and +2J2(−152/+98; mt −106/−96) (P<0.01).