PBREM, the phenobarbital-responsive enhancer module of the cytochrome P-450 Cyp2b10 gene, contains two potential nuclear receptor binding sites, NR1 and NR2. Consistent with the finding that anti-retinoid X receptor (RXR) could supershift the NR1-nuclear protein complex, DNA affinity chromatography with NR1 oligonucleotides enriched the nuclear orphan receptor RXR from the hepatic nuclear extracts of phenobarbital-treated mice. In addition to RXR, the nuclear orphan receptor CAR was present in the same enriched fraction. In the phenobarbital-treated mice, the binding of both CAR and RXR was rapidly increased before the induction of CYP2B10 mRNA. In vitro-translated CAR bound to NR1, but only in the presence of similarly prepared RXR. PBREM was synergistically activated by transfection of CAR and RXR in HepG2 and HEK293 cells when the NR1 site was functional. A CAR-RXR heterodimer has thus been characterized as a trans-acting factor for the phenobarbital-inducible Cyp2b10 gene.Cytochromes P-450 (CYPs) comprise a superfamily of heme-thiolate proteins. They function as monooxygenases that are activated by accepting electrons from NADPH-CYP reductase (19). The CYP enzymes display diverse functions, from the synthesis and degradation of biological signaling molecules such as steroid hormones and fatty acid derivatives to the metabolism of xenobiotic chemicals including pharmaceutical drugs and environmental contaminants and carcinogens. Phenobarbital (PB) is the prototype of a large group of structurally diverse xenobiotic chemicals that induce the subset of the CYP genes within the CYP2A, CYP2B, CYP2C, and CYP3A subfamilies, with the CYP2B genes being the most effectively induced (3,4,8,14,24). PB-type inducers regulate mainly at the transcription level. Compared with the well-known Ah receptormediated regulation of the CYP1A1 gene (4, 6), the mechanism by which PB induces transcription of the CYP2B genes has been elusive.Recently, PB-responsive enhancer activity has been associated with DNA sequences found approximately -2.3 kbp upstream of the initiation site of the rat CYP2B2 and mouse Cyp2b10 genes (8,9,18,23). The core enhancer sequence is the 51-bp DNA sequence located at bp -2339 to -2289 of the Cyp2b10 gene and was designated phenobarbital-responsive enhancer module (PBREM) (8-10). PBREM seems to be a general PB-responsive enhancer since it responds to numerous PB-type inducers including 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), polychlorinated biphenyls, chlorinated pesticides, organic solvents, and some plant products such as camphor (10). The PBREM sequences are conserved and functional in the PB-inducible rat CYP2B genes but are mutated and nonfunctional in the noninducible mouse Cyp2b9 gene (8). The nuclear factors that regulate the PBREM activity have not been identified.PBREM contains putative nuclear receptor binding sites, NR1 and NR2, that flank a nuclear factor 1 (NF1) binding site. Specific mutations of these NR sites resulted in a complete loss of the responsiveness of PBREM to PB-ty...

The endogenous CYP2B6 gene becomes phenobarbital (PB) inducible in androstenol-treated HepG2 cells either transiently or stably transfected with a nuclear receptor CAR expression vector. The PB induction mediated by CAR is regulated by a conserved 51-base pair element called PB-responsive enhancer module (PBREM) that has now been located between ؊1733 and ؊1683 bp in the gene's 5-flanking region. An in vitro translated CAR acting as a retinoid X receptor ␣ heterodimer binds directly to the two nuclear receptor sites NR1 and NR2 within PBREM. In a stably transfected HepG2 cell line, both PBREM and NR1 are activated by PB and PB-type compounds such as chlorinated pesticides, polychlorinated biphenyls and chlorpromazine. In addition to PBREM, CAR also transactivates the steroid/rifampicin-response element of the human CYP3A4 gene in HepG2 cells. Thus, activation of the repressed nuclear receptor CAR appears to be a versatile mediator that regulates PB induction of the CYP2B and other genes.

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