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Constitutive Androstane Receptor

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The hormone nuclear receptor CAR (NR1I3) was isolated through screening of a cDNA library with the nuclear receptor DBD based oligonucleotide as a

probe [61]. The name CAR was originally referred to as constitutively activated receptor, since it forms a heterodimer with RXR that binds to retinoic acid response elements (RAREs) and transactivates target genes in the absence of ligands in transfection assays [61,62]. Belonging to the same subgroup of the orphan nuclear receptor superfamily as the farnesoid X receptor (FXR), the liver X receptor (LXR), and PXR, CAR is mainly expressed in liver with a minor presence in small intestine [61]. By screening several small hydrophobic compounds, two androstane metabolites, androstanol (5a-androstan-3a-ol) and androstenol (5a-androstan-16-en-3a-ol), were identified as endogenous CAR ligands. However, instead of activating CAR, both ligands act as antagonists by dissociating CAR from its coactivator and inhibiting the transactiva-tion of CAR [63]. Thus CAR also was referred to as constitutive androstane receptor.

CYP2B, another important CYP subfamily, is effectively induced by phenobarbital (PB) in most mammalian species. The CYP2B gene and cDNA originally were sequenced and cloned in the 1980s [64]. However, the real breakthrough in the understanding of the mechanism of PB induction of this gene was with the identification of a 163 bp DNA sequence located between -2318 and -2155 bp upstream from the CYP2B2 encoding region, which was linked with PB responsiveness [65]. Furthermore consecutive deletion analysis of a similar 163 bp sequence in the mouse CYP2b10 indicated that a 51 bp minimum sequence was required for PB induction, and was termed the Phenobarbital-response enhancer module (PBREM) [66]. Later this PBREM was identified in rat, rabbit, and human CYP2B genes [67]. Sequence analysis of the PBREM revealed that this element was composed of two nuclear receptor-binding sites (NR1 and NR2) and a nuclear factor 1 (NF1) binding site. Both NR1 and NR2 are DR-4 motifs. NR1 is identical in rat and mice, while only a base-pair difference is observed in the human NR1. This conserved NR1 site is critical for conferring PB responsiveness [59,68]. The function of the NF1 site is unknown, but it is believed to be required for full PBREM activity.

Using a cell-based transfection assay, Negishi and colleagues screened a number of known nuclear receptors, such as RXR, CAR, LXR, thyroid receptor-a, hepatocyte nuclear factor 4 (HNF4), and chicken ovalbumin upstream promoter-transcription factor (COUP-TF), for their capacity to bind and transactivate the PBREM in reporter assays. The results indicated that CAR alone was able to stimulate the PBREM reporter gene expression [60]. Further NR1-affinity chromatography was used to purify the protein that bound to PBREM, and both binding assays and Western blot analysis proved that CAR was the protein mediating the PB induction response. Thereafter a group of structurally diverse compounds was identified to induce CYP2B through activation of CAR and thus was referred to as "phenobarbital-type" inducers. The most potent member from among these compounds is 1,4-Ws[2-(3,5-dichlorpyridyloxy)]benzene (TCPOBOP), which was originally identified as a pesticide contaminant [69].

In contrast to PXR, CAR is located in the cytoplasm of hepatocytes in the absence of ligands, and it is translocated into the nucleus after treatment with PB-like CYP2B inducers [70]. It appears that nuclear accumulation of CAR is important and might be the first activation step in response to PB-type inducers. This translocation phenomenon is common among steroid receptors, such as GR, which are dissociated from their cytoplasmic complex with other proteins (e.g., heat shock proteins, immunophilins, and p23 proteins) and translocated into the nucleus upon binding to a ligand, such as DEX [71-73]. However, several lines of evidence indicate that ligand binding is not an absolute requirement for CAR translocation. First, in vitro binding assays showed that TCPOBOP can bind directly to mCAR but not hCAR, but in vivo transfection assays revealed that both hCAR and mCAR accumulate in the nucleus in mouse liver after TCPOBOP treatment [70]. Second, in the same in vitro binding assay, it was shown that PB does not bind directly to either mCAR or hCAR [3,74]. Finally, in HepG2 cells, transiently expressed CAR always accumulates in the nucleus even in the absence of CAR ligands or CYP2B inducers [60]. By contrast, transfected CAR behavior is more reflective of the in vivo condition in primary hepatocyte cultures. For example, in the absence of CAR ligands or CYP2B inducers, CAR is predominantly localized in the cell cytoplasm, while in the presence of CAR ligands or CYP2B inducers, marked activation of CAR is observed [75,76]. These results indicate that some unidentified cellular factor(s) plays an important role in the translocation and activation of CAR, and primary hepatocyte cultures may be an effective means to identify potential CAR activators.

CAR is the closest relative of PXR on the branch of the orphan nuclear receptor tree. Although they were originally recognized as the regulators of CYP2B and CYP3A, respectively, there is significant overlap in the inducers of these two gene subfamilies (Table 4.1). For example, PB can induce both