PPARα Agonists Suppress Osteopontin Expression in Macrophages and Decrease Plasma Levels in Patients With Type 2 Diabetes

PPARα agonists inhibit OPN promoter activity. RAW 264.7 macrophages were transiently transfected with a full-length OPN promoter construct. Transfected macrophages were pretreated for 12 h with vehicle (DMSO) or the indicated PPARα agonist and stimulated with PMA (100 nmol/l) in the presence of the agonist. After 24 h, luciferase activities were analyzed as described in research design and methods. Data are expressed as normalized luciferase activity and presented as means ± SEM from three independently performed experiments (*P < 0.05 vs. vehicle).

PPARα agonists suppress OPN promoter activity by negative interference with AP-1 signaling pathways. A: RAW 264.7 macrophages were transiently transfected with the full-length wild-type or AP-1–mutated OPN promoter. Following transfection, macrophages were pretreated for 12 h with 50 μmol/l WY14643 and stimulated for 24 h with 100 nmol/l PMA as indicated. B: RAW 264.7 macrophages were transfected with the OPN promoter construct alone or cotransfected with the empty pCMV vector (400 ng) or pCMV-c-Fos (200 ng) and pCMV-c-Jun (200 ng) expression vectors. After transfection, cells were treated as described in A. C: RAW 264.7 macrophages were transfected with an AP-1–driven heterologous promoter. Transfected macrophages were pretreated for 12 h with vehicle (DMSO) or the indicated PPARα agonist and stimulated for 24 h with 100 nmol/l PMA in the presence of the agonist. Following stimulation, luciferase activities were analyzed. Data are expressed as normalized luciferase activity and presented as means ± SEM from three independently performed experiments (*P < 0.05 vs. vehicle).

PPARα agonists inhibit AP-1 binding to the proximal OPN promoter. RAW 264.7 macrophages were pretreated for 24 h with vehicle (DMSO), 50 μmol/l WY14643, or 250 μmol/l bezafibrate and stimulated with 100 nmol/l PMA for 24 h. ChIP assays were performed using c-Fos and phospho–c-Jun antibodies. Total extract (input) and rabbit IgG were used as controls. Ethidium bromide–stained agarose gels shown are representative of three independently performed experiments.

PPARα agonists inhibit c-Fos and phospho–c-Jun expression. RAW 264.7 macrophages were left untreated (—) or incubated with either vehicle (DMSO) or the indicated PPARα agonist for 24 h before stimulation with 100 nmol/l PMA in the presence of the ligand. Following stimulation for 3 h, nuclear extracts were isolated and analyzed for c-Fos (A) and phospho–c-Jun (B) protein expression. Quantification was performed by densitometry and normalization to Histone H3 of three independently performed experiments. Results are presented as means ± SEM (*P < 0.05 vs. vehicle). c-Fos (C) and c-Jun (D) mRNA expression levels were analyzed by real-time RT-PCR following stimulation with PMA for 30 min. Quantification was performed from three independently performed experiments and normalization to the housekeeping gene TATA-binding protein. Data are expressed as percentage of increase over unstimulated cells and presented as means ± SEM (*P < 0.05 vs. vehicle).

The inhibition of OPN by PPARα ligands is mediated through a receptor-dependent mechanism. Peritoneal macrophages from wild-type (A) and PPARα-deficient (B) mice were pretreated with the PPARα ligands bezafibrate (250 μmol/l), fenofibric acid (250 μmol/l), or GW9578 (500 nmol/l) for 24 h before stimulation with 100 nmol/l PMA. mRNA was isolated after 24 h and analyzed for OPN expression by real-time RT-PCR. Quantification was performed from three independently performed experiments and normalization to the housekeeping gene cyclophilin. Data are expressed as percentage of increase over unstimulated cells and presented as means ± SEM (*P < 0.05 vs. vehicle).