FIGURE 3.

Identification of functional cis-acting elements in PlGF-induced PAI-1 promoter activation. A, schematics of PAI-1 promoter (−806/+19 bp, relative to transcription start site), indicating the location of HRE-1 to -5, proximal and distal AP-1, C/EBP, and Egr-1 binding sites. Luc, luciferase. B, HPMVEC were co-transfected with either WT or individual HRE mutant (mut) constructs of PAI-1 promoter and β-galactosidase plasmid prior to PlGF treatment for 6 h. C, illustrations of an overlapping region (−158/−147 bp) containing the HRE-1 and AP-1 binding sites in the PAI-1 promoter. The mutated oligonucleotides corresponding to HRE-1, AP-1, and the overlapping region of HRE-1 and AP-1 are underlined. D, HPMVEC were co-transfected with either WT or HRE-1 mutant or AP-1 mutant or HRE-1/AP-1 mutant of PAI-1 promoter and β-galactosidase plasmid prior to PlGF treatment for 6 h. The luciferase and β-galactosidase activities were estimated. E, nuclear extracts from untreated and PlGF-treated HPMVEC (10 μg) were incubated either with a biotinylated double-stranded oligonucleotide probe corresponding to an overlapping region containing HRE-1 and AP-1 sites or with a probe containing mutations in an overlapping four shared nucleotides or mutation only in the AP-1 site. Data are representative of three independent experiments. ns, not significant.