Regulation of CYP2C11 by dehydroepiandrosterone and peroxisome proliferators: identification of the negative regulatory region of the gene.

Treatment of rats with peroxisome proliferators is known to affect gene expression, including suppression of CYP2C11. The current study examined the mechanism of negative regulation of CYP2C11, comparing the effects of a classic peroxisome proliferator, nafenopin, with those of the steroid dehydroepiandrosterone (DHEA). In vivo dose-response experiments for DHEA were carried out with rats. Only the highest dose of DHEA in the diet (0.45%), a dose previously shown to produce peroxisome proliferation, caused suppression of CYP2C11 expression. Lower doses of DHEA (0.012 to 0.20% in diet) had little effect on CYP2C11 expression. In HepG2 cells, negative regulation of a CYP2C11 reporter gene by nafenopin required coexpression of PPARalpha, whereas negative regulation by DHEA did not. Deletion analysis revealed that the responsive region for both DHEA and nafenopin was between -108 and -60 relative to the transcription start site. Mutations in several putative transcription factor binding sites in the 5'-flanking region of CYP2C11 were produced. A mutation at -121 bp significantly diminished basal expression of CYP2C11 but did not affect negative regulation by DHEA or nafenopin. A mutation at -75 bp had only a small effect on basal expression but completely abolished negative regulation by DHEA and nafenopin. Gel shift experiments indicated that PPARalpha/RXRalpha heterodimers do not bind DNA in this region. Therefore, the sequence at -75 bp of CYP2C11 is necessary for negative regulation by both DHEA and nafenopin. However, the upstream events leading to suppression at this site must differ for DHEA and nafenopin.