Fibrates increase human REV-ERBalpha expression in liver via a novel peroxisome proliferator-activated receptor response element

Abstract

Fibrates are widely used hypolipidemic drugs that act by modulating the expression of genes involved in lipid and lipoprotein metabolism. Whereas the activation of gene transcription by fibrates occurs via the nuclear receptor peroxisome proliferator-activated receptor-alpha (PPARalpha) interacting with response elements consisting of a direct repeat of the AGGTCA motif spaced by one nucleotide (DR1), the mechanisms of negative gene regulation by fibrates and PPARalpha are largely unknown. In the present study, we demonstrate that fibrates induce the expression of the nuclear receptor Rev-erbalpha, a negative regulator of gene transcription. Fibrates increase Rev-erbalpha mRNA levels both in primary human hepatocytes and in HepG2 hepatoblastoma cells. In HepG2 cells, fibrates furthermore induce Rev-erbalpha protein synthesis rates. Transfection studies with reporter constructs driven by the human Rev-erbalpha promoter revealed that fibrates induce Rev-erbalpha expression at the transcriptional level via PPARalpha. Site-directed mutagenesis experiments identified a PPAR response element that coincides with the previously identified Rev-erbalpha negative autoregulatory Rev-DR2 element. Electromobility shift assay experiments indicated that PPARalpha binds as heterodimer with 9-cis-retinoic acid receptor to a subset of DR2 elements 5' flanked by an A/T-rich sequence such as in the Rev-DR2. PPARalpha and Rev-erbalpha bind with similar affinities to the Rev-DR2 site. In conclusion, these data demonstrate human Rev-erbalpha as a PPARalpha target gene and identify a subset of DR2 sites as novel PPARalpha response elements. Finally, the PPARalpha and Rev-erbalpha signaling pathways cross-talk through competition for binding to those response elements.