Inducible gene transcription as a result of exposure to xenobiotic chemicals is characteristic of the xenobiotic-metabolizing enzymes. PB alone can induce the genes encoding CYP, NADPH-CYP reductase, and different transferases involved in conjugation reactions, in addition to many other genes (9
). We have now identified the nuclear orphan receptors CAR and RXR as factors regulating the transcription of the PB-inducible Cyp2b10
genes. Presumably as the RXR heterodimer, CAR regulates the enhancer activity of the 51-bp PBREM through its binding to the NR1 site. The Cyp2b10
gene is the first target gene identified for the orphan receptor CAR.
hCAR (originally called MB67) was initially cloned by screening a human liver cDNA library with degenerate oligonucleotides corresponding to the DNA binding domain (P box) of the RAR/TR orphan receptor subfamily (1
). CAR is a liver-enriched nuclear receptor and, as a heterodimer with RXR, can activate a subset of retinoic acid response elements (RAREs) consisting of direct repeats related to the hexamer AGGTCA (1
). In the light of the fact that the CAR-RXR heterodimer can regulate the PBREM activity in the transformed cell lines, does CAR bind to NR1 in vivo in response to PB induction so as to regulate the PB-inducible transcription of the Cyp2b10
gene? Indeed, CAR binding to NR1, presumably as a heterodimer with RXR, increased sharply after PB treatment. The PB-induced increase in CAR binding occurred in accordance with that of the supershift band by anti-RXR and preceded the accumulation of CYP2B10 mRNA. Moreover, the mRNA level decreased as the level of CAR decreased at 24 h after PB induction. These results therefore provide compelling evidence that the CAR-RXR heterodimer is a trans
-acting factor responsible for NR1-mediated transcription activity from PBREM in the liver. PBREM is a composite regulatory element consisting of two NR sites with different sequences and NF1 site. It responds to the numerous structurally unrelated PB-type inducers (10
). Our present studies therefore do not limit orphan receptors involved in the induction of the CYP2B
gene by PB only to CAR.
Since none of the transformed cell lines, including HepG2 cells, responds to PB by inducing the CYP2B
genes, it is not possible at present to investigate directly whether and how PB can activate the binding of mCAR to PBREM. The constitutive (or intrinsic) activation by mCAR in cell lines in the absence of PB may have several explanations. mCAR may be activated by a potential ligand present in the cell culture medium. Alternatively, an unknown repressor of mCAR in the liver may not have been present in the cell lines. The repressors can be proteins, such as SHP, which represses the activation of RAREs by various orphan receptors including mCAR (20
), or they can be metabolites, such as geranylgeraniol, that repress the nuclear orphan receptor LXRα (5
). In addition, a role for protein phosphorylation and dephosphorylation in the PB induction of CYP2B
genes is evident (11
). CAR may be constitutively activated by the lack of proper signaling pathways in the transformed cell lines. However, each of these possible explanations may be a clue to uncovering the induction mechanism in the liver.
With respect to the scores of the PB-type inducers, many of the members of this immense group of structurally unrelated chemicals can activate transcription from the PBREM-CAT reporter gene in the mouse primary hepatocytes (10
). It would be interesting to see whether the CAR-RXR heterodimer can mediate most of these activations. Expression of the PB-inducible P-450 genes can also be affected by endocrine factors, such as glucocorticoid, sex, and thyroid hormones (reference 9
and references therein). Since these endocrine factors often control their target genes through nuclear steroid hormone receptors, these factors may interfere with PB induction signals by inducing cross talk between nuclear receptors. The CYP-dependent metabolism can, on the other hand, produce a practically unlimited number of potential ligands (both endogenous hormones and exogenous chemicals) for the nuclear receptors. It appears that the induction of the CYP
genes may depend on a tight interaction between members of the two superfamilies, those of the nuclear receptor and the CYP
genes themselves. Our present finding that the CAR-RXR heterodimer is a PB-responsive trans
-acting factor strengthens this view, and as an additional example, the CYP4A
gene is activated by exposure to peroxisome proliferators through binding of the PPAR-RXR heterodimer (12
). The PXR-RXR heterodimer may activate transcription of CYP3A
gene by synthetic glucocorticoids such as pregnenolone 16α-carbonitrile and dexamethasone t
). Finally, a large group of the nuclear orphan receptors may provide cells with the capability to induce the various CYP
genes and other genes responsive to unlimited numbers of xenobiotic chemicals.