Figure 1
PPARδ expression and activity is increased by phorbol ester. A: Time course of induction of PPARδ message in response to phorbol ester. THP-1 cells were plated in RPMI medium supplemented with 10% FCS and 1 nM PMA at a density of 1 × 106 per well. At each time point, cells were lyzed and RNA prepared, followed by cDNA synthesis. QPCR analysis was performed using PPARδ-specific primers and probe to determine the amount of message. Results are expressed as total relative to 18S and were performed in triplicate. B: Expression of PPARδ protein by THP-1 cells is up-regulated in response to phorbol ester. THP-1 cells were plated in RPMI medium supplemented with 10% FCS and either 1 nM PMA or vehicle and incubated for 48 h. Cells were then lyzed directly into SDS-PAGE sample buffer for Western blotting. Blots were probed using a rabbit polyclonal anti-serum raised against a GST-PPARδ AB domain fusion protein. C: COS-1 cells were transiently co-transfected with pFABPLuc and pCLDNhPPARδ. Transfection efficiency was controlled for by co-transfection with a plasmid encoding β-galactosidase. Cells thus transfected were treated with increasing concentrations of PMA for 24 h after which luciferase activity was measured. D: PMA-induced δ-activation is mediated through the ligand binding domain. COS-1 cells were transfected with an expression vector encoding a Gal-4/PPARδ ligand-binding domain chimera. Cells were then treated for 24 h with compound F, PMA or vehicle only and luciferase readings obtained.