The Nuclear Orphan Receptor CAR-Retinoid X Receptor Heterodimer Activates the Phenobarbital-Responsive Enhancer Module of the CYP2B Gene

Abstract

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...

Regulatory network of lipid-sensing nuclear receptors: roles for CAR, PXR, LXR, and FXR

“…Classically, the barbiturate phenobarbital induces its own metabolism and excretion by elevating CYP levels [2]. However, the molecular mechanisms underlying this observation remained a conundrum until the discovery and subsequent characterization of the constitutive androstane receptor (CAR, official nomenclature NR1I3) and the pregnane X receptor (PXR, NR1I2, alternatively called PAR or SXR), two members of the superfamily of nuclear receptors [3][4][5][6][7]. Mice with genetic ablations of CAR and PXR have significantly reduced inducibility of CYPs by a variety of drugs [8,9].…”

“…Moreover, the steroid hormone receptors usually bind DNA as a homodimer [30][31][32]. In their initial characterization, ligands of the NR1I receptors were drugs and other xenobiotics for CAR and PXR, 1,25-dihydroxyvitamin D 3 for VDR [33], oxysterols for LXR [34] and bile acids for FXR [35][36][37]. However, later findings showed that a number of endogenous compounds are also able to influence PXR and CAR activity and that these xenosensors share an overlapping ligand pattern with other members of the NR1I and NR1H subfamilies (Fig.…”

Redundant enhancement of mouse constitutive androstane receptor transactivation by p160 coactivator family members

“…Constitutive androstane receptor (CAR) is the key regulatory factor that mediates regulation of the CYP2B transcription by PB [3,4]. CAR is unusual among nuclear receptors because it has substantial constitutive activity in the absence of ligand.…”

Nuclear receptors CAR and PXR in the regulation of hepatic metabolism

“…Similar to PXR (also known as steroid and xenobiotic receptor SXR), CAR heterodimerizes with RXR and binds to the phenobarbital responsive enhancer module (PBREM) to induce the transcription of CYP2B genes (Baes et al 1994;Choi et al 1997;Honkakoski and Negishi 1997). Closer examination of the PBREM and the other CARregulated promoters revealed that CAR preferentially binds to DR4 (direct repeat spaced by 4 nucleotides) binding sites (Honkakoski et al 1998). …”