PPAR alpha Induces the Expression of CAR That Works as a Negative Regulator of PPAR alpha Functions in Mouse Livers

The nuclear receptor peroxisome proliferator-activated receptor alpha (PPAR alpha) is a transcription factor that controls the transcription of genes responsible for fatty acid metabolism. We have recently reported a possible drug-drug interaction mechanism via the interaction of PPAR alpha with the xenobiotic nuclear receptor constitutive androstane receptor (CAR). Drug-activated CAR competes with the transcriptional coactivator against PPAR alpha and prevents PPAR alpha-mediated lipid metabolism. In this study, to elucidate the crosstalk between CAR and PPAR alpha, we focused on the influence of PPAR alpha activation on CAR's gene expression and activation. Male C57BL/6N mice (8-12 weeks old, n = 4) were treated with PPAR alpha and CAR activators (fenofibrate and phenobarbital, respectively), and hepatic mRNA levels were determined using quantitative reverse transcription PCR. Reporter assays using the mouse Car promoter were performed in HepG2 cells to determine the PPAR alpha-dependent induction of CAR. CAR KO mice were treated with fenofibrate, and the hepatic mRNA levels of PPAR alpha target genes were determined. Treatment of mice with a PPAR alpha activator increased Car mRNA levels as well as genes related to fatty acid metabolism. In reporter assays, PPAR alpha induced the promoter activity of the Car gene. Mutation of the putative PPAR alpha-binding motif prevented PPAR alpha-dependent induction of reporter activity. In electrophoresis mobility shift assay, PPAR alpha bound to the DR1 motif of the Car promoter. Since CAR has been reported to attenuate PPAR alpha-dependent transcription, CAR was considered a negative feedback protein for PPAR alpha activation. Treatment with fenofibrate induced the mRNA levels of PPAR alpha target genes in Car-null mice more than those in wild-type mice, suggesting that CAR functions as a negative feedback factor for PPAR alpha.