A novel PPAR response element in the murine iNOS promoter

The nuclear hormone receptor peroxisome proliferation activated receptor gamma (PPARgamma) is a modulator of inflammation including down-regulation of inducible nitric oxide synthase (iNOS) and nitric oxide (NO) production. PPARgamma agonists reduce iNOS expression and NO production in a dose-dependent manner in macrophages, mesangial cells and other inflammatory cells. However, the mechanisms involved in the inhibition of iNOS expression by PPARgamma and its agonists are not fully understood. Here we show that the PPARgamma agonist ciglitazone dose-dependently inhibited a murine iNOS-luciferase reporter construct by up to 50% in transfected mesangial cells. Blocking de novo protein synthesis in mesangial cells had no effect on PPARgamma agonist activity, indicating that ciglitazone acts directly to inhibit iNOS transcription. We identified a novel PPAR response element (PPRE) in the murine iNOS promoter that is homologous to the PPRE consensus sequence. In binding assays PPARgamma directly binds to this response element in vitro and can function as a positive element in response to PPARgamma agonists when placed in front of a reporter gene. Site-directed mutagenesis of this PPRE in a murine iNOS promoter/reporter construct did not block the inhibitory activity of a synthetic PPARgamma agonist on the iNOS promoter/reporter construct in transfected mesangial cells. However, the mutated construct exhibited lower basal expression, and higher expression in response to inflammatory stimuli compared to the intact construct. These data suggest that the iNOS PPRE contributes to positive basal expression and negative expression of iNOS in response to inflammatory stimuli. The PPRE is not necessary, however, for synthetic PPARgamma agonists to inhibit iNOS expression.