Figure 4

PPARs modulate human ADRP promoter activity via a PPRE located between -2361 and -2345 bp. A, A sequence corresponding to the -2366/-2339 region of the human adrp gene, was compared with a consensus PPRE and the analogous regions in the mouse adrp gene promoter (-2013/-1986). A human ADRPmut indicates a human ADRP promoter whose potential PPRE was mutated. Asterisks denote conserved bases and arrows represent response element half-sites. B, Schematic representation of the chimeric genes containing the human ADRP promoters; each wild type (hADRP-4K), point mutation (hADRP-mut), and deletion (hADRP-d1) of the ADRP promoter was cloned in front of the firefly luciferase reporter gene. Lowercase letters indicate mutations introduced in the human ADRP PPRE. C, D, E and F, HepG2 cells were co-transfected with a human ADRP reporter plasmid (50 ng), phRL-TK (50 ng) and either pcDNA3-hPPARα (5 ng) (C), pcDNA3-hPPARδ (5 ng) (D), pcDNA3-hPPARγ1 (5 ng) (E) or pcDNA3-hPPARγ2 (5 ng) (F). Transfected cells were treated with ligands (100 μM fenofibric acid (C), 100 nM GW501516 (D) or 10 μM ciglitizone (E and F)) for 24 h and the cells were used for reporter gene assays. Luciferase activities from reporter plasmids were normalized by internal Renilla luciferase activity. Values are expressed as fold induction of the control (the value when only reporter plasmid (ADRP-4K) was transfected) set at 1. Values represent the mean ± S.E.M. (n = 3).