FIGURE 7.

PPARγ binds to the TRAIL promoter. A, PEDF enhances human TRAIL promoter activity. THP-1 macrophages were transfected with the indicated reporter construct. After 48 h post-transfection, cells were stimulated with PEDF or PEDF combined with 10 μm GW9662 for a further 24 h prior to reporter gene activity assays as described above. *, p < 0.05 versus. TRAILp. #, p < 0.05 versus PEDF + TRAILp. ##, p < 0.05 versus PEDF + TRAILp. B, ChIP assay. THP-1 macrophages were treated with PEDF (P) or PEDF solvent (S) for 24 h or pretreated with 10 μm GW9662 for 1 h and then incubated with PEDF for an additional 24 h. PPARγ-DNA complexes were immunoprecipitated with the anti-PPARγ antibody or IgG. Data are normalized to IgG immunoprecipitated DNA and input DNA. The binding of PPARγ to the TRAIL promoter in PEDF-stimulated THP-1 macrophages was measured and quantified by real-time PCR. C and D, identification of PPARγ and PPRE association by DNA pull-down assay. The 5′-biotinylated double-stranded oligonucleotides containing a consensus PPRE (positive control (PC)) or TRAILp PPRE or its mutant, as indicated in Fig. 6A, were used to precipitate PPARγ and RXRα from nuclear protein extracts of THP-1 macrophages treated with PEDF or its solvent. The mixtures were mixed with or without PPARγ antagonists (reaching a final concentration of 20 μm) and then pulled down by streptavidin beads. The proteins in the complex were analyzed by Western blotting using antibodies against PPARγ or RXR isoforms. Aliquots of nuclear extracts were also analyzed before the DNA pull-down assay (input). Shown are representative experiments that were repeated four times with similar observations. Error bars, S.E.