Hepatic microsomal cytochromes P450 (CYPs) display diverse functions in the metabolism of biological signaling molecules such as steroid hormones and xenochemicals, including pharmaceutical drugs and environmental contaminants. Inducible gene transcription by exposure to xenochemicals is characteristic for CYPs and increases the organism’s metabolic capabilities against chemical toxicity and carcinogenicity (3
). Phenobarbital (PB) is the prototype for a large number of structurally diverse xenochemicals that induce CYPs and other xenochemical-metabolizing enzymes (3
). The PB-responsive enhancer module (PBREM), a versatile enhancer capable of responding to numerous PB-type inducers, regulates PB induction of the CYP2B
genes in mouse, rat, and human cells (8
). PBREM contains two DR-4 nuclear receptor-binding motifs, NR1 and NR2. Acting as a retinoid X receptor (RXR) heterodimer, the liver-enriched constitutively active receptor (CAR) increases its binding to NR1 in PB-treated mice. Moreover, CAR can stimulate transcriptional activity from a cis
-linked PBREM-containing reporter plasmid transfected into HepG2 cells (11
). CAR-mediated transactivation, however, is constitutive in HepG2 cells, and the remaining key question is how CAR responds to PB in inducing the transcription of the CYP2B
gene in the liver.
CAR was originally characterized as a constitutive activator of an empirical set of retinoic acid response elements (1
). Forman et al. have recently shown that this activity can be repressed by 3α-androstenol in transfected CV-1 and HepG2 cells (7
). These results suggest the presence of ligands which may act positively to confer a regulatory capability to CAR (7
). We have demonstrated that 3α-androstenol represses expression of the endogenous CYP2B6
gene in stable HepG2 cells transfected with a CAR-expressing plasmid. Moreover, PB activates (i.e., induces) the repressed CYP
). Similarly, 3α-androstenol-repressed PBREM can be reactivated by PB in HepG2 cells. An activation by PB of repressed CAR may be a mechanism regulating the induction of CYP2B
genes. Despite the fact that binding of CAR to PBREM depends on PB treatment in vivo, an in vitro-translated CAR-RXR heterodimer binds to PBREM without the presence of PB (11
). Moreover, 3α-androstenol does not directly interfere with the ability of CAR to form a dimer with RXR or to bind to the elements (7
). Thus, an additional or alternative mechanism that confers PB responsiveness to CAR may remain undetected.
Binding of CAR to NR1 occurs only after PB induction in liver in vivo (11
), indicating that the function of CAR in the liver may differ from that in HepG2 cells. To explore this possibility, we examined the intracellular localization of CAR in mouse liver and primary hepatocytes. Nuclear receptors generally reside in nuclei and can be activated upon ligand binding. Constitutively active CAR may be excluded from nuclei to suppress unwanted gene activation in nontreated mice. If, in fact, CAR localizes to liver nuclei following PB treatment, the induction of CYP2B
genes may be regulated through a nuclear translocation process. Western blot and immunohistochemistry studies have been applied to demonstrate the cytoplasmic localization of CAR in nontreated mice. CAR undergoes nuclear translocation in livers of PB-treated mice. PB-induced nuclear translocation appears to be regulated through a phosphorylation-dephosphorylation pathway. Moreover, various PB-type inducers have been tested to see whether nuclear translocation of CAR is a general mechanism involved in CYP2B induction.