Interaction of the peroxisome proliferator-activated receptor α with the retinoid X receptor α unmasks a cryptic peroxisome proliferator response element that overlaps an ARP-1-binding site in the CYP4A6 promoter

P450 4A6 is highly induced by peroxisome proliferators in vivo. Gene transfer experiments indicate that this induction can be mediated by the mouse peroxisome proliferator-activated receptor α (PPARα) and that it is dependent on upstream enhancer elements in the CYP4A6 gene. However, as has been seen for other peroxisome proliferator response elements (PPREs), PPARα does not bind directly to a previously characterized PPRE of the CYP4A6 gene in the absence of additional proteins such as the retinoid X receptor α (RXRα). When PPARα and RXRα are coexpressed, the overall transcription of the CYP4A6 reporter is increased, and a synergistic response to both retinoids and peroxisome proliferators is evident that is dependent on the presence of both receptors. In addition, a cryptic response element is unmasked in constructs lacking the upstream enhancers. DNase I protection assays indicate that when present together, but not singly, PPARα and RXRα bind to a site located within 29 base pairs upstream of the CYP4A6 transcription start site. This region contains a sequence similar to that found in the apolipoprotein CIII gene that has been shown to bind RXRα and the orphan nuclear receptor, ARP-1. The corresponding sequence in the CYP4A6 gene also binds ARP-1. A similar sequence found in the promoter region of the rat CYP4A1 gene does not, however, bind either PPARα/RXRα or ARP-1. Transfection of increasing amounts of the ARP-1 expression vector blocks the PPARα/RXRα-mediated induction of transcription from the CYP4A6 promoter. Mutations that prevent the binding of either PPARα/RXRα or ARP-1 to a double-stranded oligonucleotide corresponding to the proximal enhancer eliminate the peroxisome proliferator-induced transcriptional response observed for the promoter construct in the presence of PPARα/RXRα, but these mutations do not eliminate the response seen when the upstream enhancers are present. These results indicate that the PPREs of the CYP4A6 gene are recognized by multiple members of the nuclear receptor family that are likely to contribute to the regulation of CYP4A6 expression in both an agonistic (RXRα) and an antagonistic (ARP-1) manner.