FIGURE 6.

PAI-1 gene promoter harbors a functional TBE that is responsible for β-catenin-mediated PAI-1 induction. A, diagram shows the construction of various luciferase reporter plasmids containing different lengths of the promoter region of human PAI-1 gene linked to the coding sequence of luciferase gene. Putative TBE, SBE, Sp1, and AP1 sites are indicated. B, β-catenin activates PAI-1 promoter in tubular epithelial cells. HKC-8 cells were cotransfected with various PAI-1 promoter-luciferase reporter plasmids as shown in A and stabilized β-catenin expression vector (pDel-β-cat) or empty vector (pcDNA3). R. reniformis luciferase vector (pRL-TK) was used as an internal control for transfection efficiency. Luciferase activities (arbitrary unit) were calculated, reported after normalizing for transfection efficiency, and presented as the means ± S.E. of three experiments. *, p < 0.05; **, p < 0.01 versus pcDNA3 controls. C, PAI-1 promoter harbors a functional TBE (0.42PAI-1-Luc) that is responsive to β-catenin and TGF-β1 stimulation. HKC-8 cells were cotransfected with 0.42PAI-1-Luc reporter plasmid with pcDNA3 or pDel-β-cat, followed by incubation without or with TGF-β1 (2 ng/ml) for 48 h. D–F, site-directed mutation in the TBE of PAI-1 promoter abolishes its responsiveness to β-catenin and TGF-β1 stimulation. The construction of wild-type and mutant TBE reporter plasmids (D) and luciferase activities (E and F) are presented. *, p < 0.05 versus controls; †, p < 0.05 versus pDel-β-cat alone.