Introduction

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Phenobarbital (PB) and many structurally unrelated xenobiotics induce same drug- and carcinogen-metabolizing cytochrome P450 and other genes as a protective response directed toward elimination of these xenobiotics from the body. Among tens of PB-inducible genes, CYP2B genes are the most efficiently activated (reviewed by Waxman, 1999; Honkakoski and Negishi, 2000). Recent studies have established that the constitutive androstane receptor (CAR, NR1I3) is crucial for induction of CYP2B genes by PB and 1,4-bis[2-(3,5-dichloropyridoxy)]benzene (TCPOBOP) because CYP2B mRNA inducibility is lost in CAR null mice (Wei et al., 2000). After forming a heterodimer with retinoid X receptor (RXR, NR2B), the xenobiotic-activated CAR binds to the phenobarbital-responsive enhancer module (PBREM) or unit located in the upstream regions of the mouse Cyp2b10, human CYP2B6, and rat CYP2B1 and CYP2B2 genes (reviewed by Honkakoski and Negishi, 2000). The PBREM contains two CAR/RXR heterodimer binding sites, NR1 and NR2, that conform to the direct repeat-4 (DR4) motif. Successive mutations of DR4 motifs result in gradual loss and, finally, abolition of trans-activation by CAR in HEK293 cells and induction in primary hepatocytes. It is known that NR1 sites alone are sufficient for CAR responsiveness (Sueyoshi et al., 1999), whereas the nuclear factor 1 (NFI) binding site between NR1 and NR2 may contribute to the full inducibility (Honkakoski et al., 1998; Kim et al., 2001).

Several nuclear receptors (NRs), such as vitamin D receptor (VDR, NR1I1), thyroid hormone receptors / (TR, NR1A1/2), retinoic acid receptors // (RAR, NR1B1/2/3), liver X receptors / (LXR, NR1H3/2), pregnane X receptor (PXR, NR1I2) and farnesoid X receptor (FXR, NR1H4) display considerable in vitro binding and activation of DR4-type sites (Mangelsdorf and Evans, 1995; Laffitte et al., 2000; Quack and Carlberg, 2000; Xie et al., 2000b). Therefore, it has been proposed that additional NRs could bind to PBREM and modulate its activity (Waxman, 1999). This idea is in line with evidence that CYP2B mRNA induction is influenced by sex, steroid and thyroid hormones, sterol metabolites, and retinoids, all of which are known NR ligands (Honkakoski and Negishi, 2000). Several inducers such as PB and pesticides activate not only CAR (Sueyoshi et al., 1999) but also PXR, a receptor important for CYP3A gene regulation (reviewed by Quattrochi and Guzelian, 2001). CAR and PXR recognize similar DNA motifs that range from DR2 to DR5 and everted repeat-6 (ER6), and both receptors are expressed in the liver and intestine (Honkakoski and Negishi, 2000; Quattrochi and Guzelian, 2001). Collectively, these data suggest that other NRs might well affect the PBREM enhancer and influence CYP2B gene regulation through cross-talk with CAR.

Surprisingly, there is very little information or systematic studies on PBREM binding or cross-talk with CAR by other NRs. Such studies are much needed for detailed understanding of CYP2B gene regulation, modulating factors, and species differences. So far, we know that CAR can activate the PXR-responsive ER6 and DR3 motifs in CYP3A genes (Sueyoshi et al., 1999; Moore et al., 2000; Xie et al., 2000b; Smirlis et al., 2001). This is consistent with the report that PB can induce CYP3A mRNA in PXR null mice (Xie et al., 2000a). Recently, hPXR and mPXR were shown to bind to DR4 motifs and activate PBREM elements from various species by 3- to 6-fold. This effect is roughly comparable with that of CAR-mediated activation (Xie et al., 2000b; Goodwin et al., 2001; Smirlis et al., 2001). None of these studies, however, could address the preference of PBREM for CAR and PXR. The data are also mostly based on in vitro DNA binding assays with simple DR4 motifs (Sueyoshi et al., 1999; Xie et al., 2000b; Smirlis et al., 2001) instead of functional assays with receptors and PBREM elements. Finally, there is practically no data on the modulation of PBREM by other NRs. Therefore, our aim was to gain more insight to the mouse and human PBREM function and its specificity for CAR by evaluating the effects of several NRs on PBREM activity by functional and DNA binding assays. To help in this effort, the ligand specificities of human and mouse CAR were also defined.

	Materials and Methods

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Chemicals. TCPOBOP was synthesized and purified according to Honkakoski et al. (1996) to more than 98% purity as assessed by ¹H-NMR spectra and elemental analysis (observed: N, 6.7%; C, 46.9%; H, 2.0%; expected: N, 6.8%; C, 46.8%; H, 2.2%). Steroids were from Steraloids, Inc. (Newport, RI) or Sigma-Aldrich Chemical Co. (St. Louis, MO). WY-14,643 was bought from ChemSyn, Inc. (Lenexa, KS). Other chemicals were at least analytical grade from Sigma, Fluka (Ronkonkoma, NY), or Calbiochem (La Jolla, CA).

Reporter Plasmids. pCMV was purchased from BD Clontech Inc. (Palo Alto, CA). The mPBREM-tk-luc reporter was constructed by insertion of the PBREM element plus the thymidine kinase promoter (tk) from the mouse PBREM-tk-CAT (Honkakoski et al., 1998) into BglII site of pGL3-Basic luciferase plasmid (Promega, Madison, WI). The rat (rER6)₃-tk-luc reporter was constructed similarly from (ER6)₃-tk-CAT plasmid (Lehmann et al., 1998) donated by Dr. Steven Kliewer (GlaxoSmithKline, Research Triangle Park, NC). The mouse (mNR1)₃-tk-luc, human PBREM-tk-luc, and (hNR1)₅-tk-luc plasmids have been described previously (Sueyoshi et al., 1999). The human XREM-3A4-luc reporter containing the proximal 362 base pairs of CYP3A4 gene promoter and the distal enhancer (Goodwin et al., 1999) was a kind gift from Dr. Chris Liddle (University of Sydney at Westmead Hospital, Westmead, Australia). The UAS₄-tk-luc (Janowski et al., 1996) and rat CYP3A23[1360/+82] reporters (Xie et al., 2000b) were donated by Dr. Ronald Evans (Salk Institute for Biological Studies, La Jolla, CA). Other luciferase reporter plasmids for nuclear receptors were generated by inserting multiple copies of their cognate DNA sites into BglII site of pGL3-Basic plasmid. All plasmids were purified with QIAGEN columns (Hilden, Germany) and verified by restriction mapping, functional testing and, when necessary, by sequencing.

Expression Plasmids. The sources of expression vectors for mRAR and hRAR (Zelent et al., 1989), cTR (Harbers et al., 1996), hLXR (Teboul et al., 1995), mCOUP-TFI (NR2F1; Cooney et al., 1993), hPPAR and mPPAR (NR1C1; Sher et al., 1993), hFXR (Forman et al., 1995), hCAR and mCAR (Honkakoski et al., 1998; Sueyoshi et al., 1999), hVDR (Quack and Carlberg, 2000) and hPXR and mPXR (Lehmann et al., 1998) have been described previously. The expression plasmid for coactivator hTIF2 (Voegel et al., 1996) was donated by Dr. Hinrich Gronemeyer (IGBMC, Illkirch, France).

GAL4-LBD Fusion Plasmids. The ligand binding domains (LBD) of mCAR (residues 118-358), hCAR (residues 108-348), mPXR (residues 104-431), and hPXR (residues 107-434) were amplified with Pfu DNA polymerase from mouse and human liver RNAs and cloned into 5' EcoRI and 3' BamHI or KpnI sites of CMX-GAL4 plasmid (Janowski et al., 1996) donated by Dr. Ronald Evans. GAL4-mCAR8 plasmid coding for a truncated mCAR lacking eight amino acids at the C terminus (Choi et al., 1997) was donated by Dr. David Moore (Baylor College of Medicine, Houston, TX).

Ligand Specificities of mCAR and hCAR. Ligand specificities of mCAR and hCAR were assessed for PBREM preference studies (Figs. 6-8) because CAR and PXR are reported to share some ligands (Moore et al., 2000) and to assess the effect of other NR ligands on mCAR and hCAR activity. The ligand specificities were measured first by chemical-dependent modulation of GAL4 fusion protein-driven reporter activity in HEK293 cells (Table 1) according to Honkakoski et al. (2001) and then by yeast two-hybrid assays as described below.

View this table: [in this window] [in a new window]   TABLE 1 Ligand specificities of CAR and PXR with selected xenobiotics Data are expressed as mean ± S.D. (n = 3) of fold activation of normalized luciferase activity. Other NR ligands (WY-14643, arotinoic acid, 25OH-cholesterol, VD3, and T3) were without any significant effect ( 15%) on hCAR or mCAR. Only chenodeoxycholic acid was a weak reactivator of ANDR-suppressed mCAR (2-fold). ANDR and EE2 did not have significant effects on VDR-, PPAR-, or FXR-dependent activities.

In Vitro Translation and Gel Shift Assays. NRs were produced in vitro by first transcribing linearized expression vectors with T7 RNA polymerase and then translating these RNAs in vitro using rabbit reticulocyte lysate as recommended by the supplier (Promega). Nuclear receptor heterodimers with RXR (approximately 10 ng of specific protein; equal protein amounts verified by a parallel translation in the presence of [³⁵S]methionine) were incubated with ligand for 15 min at room temperature in a total volume of 20 µl of binding buffer [10 mM HEPES, pH 7.9, 1 mM dithiothreitol, 0.2 µg/µl poly(dI-dC), and 5% glycerol], which was adjusted to 150 mM KCl. Approximately 1 ng of the ³²P-labeled human CYP2B6 or mouse Cyp2b10 DR4-type NR1 motif (50,000 cpm) was then added, and incubation was continued for 20 min. Protein-DNA complexes were resolved through 8% nondenaturing poly-acrylamide gels in 0.5× Tris/borate/EDTA (45 mM Tris, 45 mM boric acid, 1 mM EDTA, pH 8.3) and were quantified on a FLA3000 reader (Fuji, Tokyo, Japan) using Image Gauge software (Fuji).

Cell Culture and Nuclear Receptor Cotransfection. Mouse primary hepatocytes were isolated, transfected, and assayed as described previously (Honkakoski and Negishi, 1998; Honkakoski et al., 1998). HEK293 cells (American Type Culture Collection, Manassas, VA) were grown in phenol red-free Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum and 100 U/ml penicillin-100 µg/ml streptomycin (Invitrogen, Gaithersburg, MD). One day before transfection, the cells were seeded on 48-well plates in medium containing delipidated serum (Sigma) to remove potential NR-activating substances. After an overnight incubation, the medium was changed and the cells were transfected using a calcium phosphate method with pCMV (50 ng), various luciferase reporter plasmids (25 ng; 100 ng for XREM-3A4-luc and CYP3A23-luc), and variable amounts of expression vectors for NRs (varied from zero to 250 ng). In activation and suppression experiments, the amount of CAR expression plasmid that produced maximal activity from the reporter plasmid was 12.5 ng, and other NRs were titrated from zero to 20-fold excess (250 ng) over CAR so as to reach the effect plateau. In preference experiments, the total amount of NR expression vector was only 50 ng, much below levels that produced any unspecific squelching (200 ng). The balance of DNA was kept constant by addition of empty expression vector.

Reporter Assays. Transfected HEK293 cells were cultured for 40 h, washed with PBS, and lysed. Luciferase and -galactosidase activities (Honkakoski et al., 2001) were determined from 20 µl of lysates in 96-well plates using the Victor2 multiplate reader (PerkinElmer Wallac, Turku, Finland). All luciferase activities were normalized to -galactosidase expression and expressed as mean ± standard deviation from three to four independent experiments.

	Results

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Ligand Specificities of mCAR and hCAR. With GAL4 fusion proteins in HEK293 cells (Table 1), we found that GAL4-mCAR activity was suppressed by ANDR, as expected, and that ANDR-suppressed activity could be reactivated by 0.5 µM TCPOBOP, 10 µM EE2, and 2 µM CLOTR to varying degrees. With GAL4-hCAR, a reproducible partial deactivation (50-60%) by EE2 and about 2-fold activation by CLOTR was seen. Furthermore, the partial deactivation by EE2 could be overcome by addition of CLOTR and, to a lesser extent, by 5-pregnanedione (Table 1). The known ligand profiles of mPXR and hPXR (Moore et al., 2000) were also reproduced: both receptors were activated by CLOTR, RU486, and 5-pregnanedione, but RIF activated only hPXR.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Ligand-dependent association of mouse and human CAR LBD with NR corepressor. Aliquots of yeast cells transformed with GAL4-mCAR LBD (top) or GAL4-hCAR LBD (bottom) plus NR interaction domain from NCoR plasmids were treated for 3.5 h with vehicle or test chemicals at indicated concentrations (micromolar) before cell lysis and -galactosidase assays as described under Materials and Methods. For mCAR-mNCoR association, the reporter activity with 10 µM ANDR was set to 100 (top, ). In TCPOBOP displacement experiment (top, ), the activity with 2 µM ANDR was set to 100 (same concentration as in mammalian GAL4 assays in Table 1). For hCAR-hNCoR association, the reporter activity with 10 µM EE2 was set to 100 (bottom, , ). The data shown are mean ± standard deviation from triplicate samples. The experiments were repeated independently two (mCAR) or three times (hCAR) with similar results. Activities with either GAL4-CAR or NCoR plasmid alone were below detection limit.

DNA Binding to NR1 Sites by NRs. PBREM elements are known to confer about 10-fold activation by CAR in HEK293 cells and about 10-fold induction by TCPOBOP in primary hepatocytes (Honkakoski et al., 1998; Sueyoshi et al., 1999). When organization of the mouse PBREM (5' NR1-NFI-NR2 3') was changed to NR1-NFI-NR1 or to NR2-NFI-NR2, the original and NR1-containing PBREM elements retained >10-fold activation. PBREM containing NR2 motifs only conferred much lower 3-fold activation by mCAR and TCPOBOP inducibility (Fig. 2). This indicates that NR1 is the stronger site for PBREM function.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Activation of mutated mouse PBREM elements. Reporter plasmids containing the wild-type mouse PBREM sequence, NR1 sites only, NR2 sites only, or no enhancer (tk only) was cotransfected in the presence of either empty or CAR expression vector into HEK293 cells (). Mouse hepatocytes were electroporated with the same reporters and treated with DMSO or 0.5 µM TCPOBOP (). Reporter activities from 3-4 independent experiments were measured as described under Materials and Methods.

View larger version (31K): [in this window] [in a new window]   Fig. 3.   RXR-Heterodimer complex formation of various NRs on the human (left) and mouse (right) NR1 sites. Gel shift experiments were performed with in vitro translated heterodimers of equal amounts of the indicated NRs with RXR that were preincubated at room temperature with saturating concentrations of activators () [5-pregnanedione (hCAR), TCPOBOP (mCAR), RIF (hPXR), RU486 (mPXR), VD3 (hVDR), T3 (cTR), 25OH-cholesterol (hLXR), all-trans-retinoic acid (hRAR), chenodeoxycholic acid (hFXR)] or solvent () and the 32P-labeled hNR1 or mNR1 site. Protein-DNA complexes were separated from the free probe through 8% nondenaturing polyacrylamide gels. Representative experiments are shown. The amount of heterodimer-DNA complexes in relation to free probe was quantified by bioimaging. Columns and bars indicate mean and S.D., respectively, from three experiments. NS, nonspecific complex.

Activation of NR1 Sites and PBREM Enhancers by NRs. Binding to NR1 may indicate a potential influence on PBREM activity. To compare between NRs, increasing amounts of NR expression vectors were cotransfected with NR1- or PBREM-driven reporter genes. The NRs were then activated by established ligands, and the reporter activities were measured. The results shown below are the maximal effects observed for each NR, usually at the same concentration as the optimal CAR concentration. The NRs themselves could ligand-dependently activate reporters driven by their consensus response elements (data not shown), demonstrating that the constructs were functional.

View larger version (38K): [in this window] [in a new window]   Fig. 4.   Activation and suppression of mouse and human NR1- and PBREM-driven reporter genes by various NRs. Activation by NRs (top) was assessed by cotransfection into HEK293 cells of increasing amounts of indicated NR expression vectors (0-250 ng) and reporter genes (25 ng) driven by mNR1 (), hNR1 (), mPBREM (), or hPBREM () elements and addition of NR-specific ligands. , , ligand controls (empty vector + activating NR ligand). Representative results at optimal 12.5 ng of NR expression vectors (37.5 ng for RAR) are shown, with columns and bars denoting mean and S.D., respectively. Suppression by NRs (bottom) was assessed by cotransfection of increasing amounts of indicated NR expression vectors (0-250 ng) together with saturating amount of mouse or human CAR expression vector (12.5 ng) and reporter genes (25 ng) driven by mNR1 (), hNR1 (), mPBREM (), or hPBREM () elements. Transfected HEK293 cells were treated with NR-specific ligands. Activity with empty vector was set to 100. Representative results at maximal effect (125 ng of NR expression vectors) are shown, with columns and bars denoting mean and S.D., respectively. No CAR, basal reporter activity in the presence of empty expression vector substituted for both CAR and the competing NRs.

Suppression of CAR-Activated NR1 Sites and PBREM Enhancers by NRs. Because NRs may influence PBREM function by competing for DNA binding sites or for common NR coregulators, NRs were cotransfected in the presence of CAR and the maximal NR-mediated suppression of CAR-dependent NR1- or PBREM-driven activities were analyzed. Increasing amounts of NR expression vectors (0- to 20-fold excess over CAR) were used and effects at plateau only (typically 10-fold excess) are shown for clarity.

Suppressive Effects of NRs Occurring through CAR LBD. Expression vectors for GAL4-m/hCAR and UAS₄-tk-luc reporter were used in the above suppression assay to determine whether suppression could be attributed to competition for factors associated with the CAR LBD. This approach would eliminate any competition at the level of DNA binding, which was prominent for cTR, hVDR, and PXR isoforms. Figure 5, top, indicates that with both GAL4-mCAR and GAL4-hCAR as activators, the strongest suppressors were full-length CAR and cTR (<20% of control activity), followed by COUP-TFI  PXR isoforms  hFXR (25-30%), whereas hVDR, RAR, and PPAR isoforms were weaker suppressors (35-55%). No suppression was seen with GAL4-mCAR8 that lacks the AF2 core sequence (data not shown), indicating that NR-mediated suppression of CAR depends on the presence of intact AF2 domain. Therefore, suppression of CAR LBD probably reflects competition for NR coactivators. Indeed, cotransfection of TIF2 vector in this suppression assay (Fig. 5, bottom) resulted in partial restoration of mCAR-dependent and especially hCAR-dependent reporter activity. Differences in the extent of suppression and restoration of reporter activity further imply that NRs may have different affinities for various NR coactivators. In summary, NR1-binding cTR and hVDR showed a remarkable difference in their ability to suppress CAR LBD. PPAR isoforms and hFXR that do not bind to NR1 sites could inhibit PBREM through an AF2- and coactivator-dependent mechanism.

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Suppression of GAL4-CAR-activated reporter activities by various NRs. Top, suppression by NRs was assessed by cotransfection of increasing amounts of indicated NR expression vectors (0-250 ng) together with saturating amount of mouse () or human () GAL4-CAR LBD expression vector (12.5 ng) and UAS4-tk-luc reporter (25 ng) into HEK293 cells, followed by addition of NR-specific ligands. Reporter activity with empty vector was set to 100. Representative results at maximal effect (125 ng of NR expression vectors, 10-fold excess over CAR) are shown, with columns and bars denoting mean and S.D., respectively. GAL4 only, basal activity in the absence of any LBD in the construct. Bottom, reactivation of suppressed GAL4-mCAR- or GAL4-hCAR-dependent activity was performed as above with cotransfection (500 ng) of empty expression vector (, ) or hTIF2 plasmid (, ).

Preference of NRs for PBREM Enhancers. As shown above, DNA binding studies were not sufficient to assess the effect of NRs for PBREM enhancers. Furthermore, the activation and suppression experiments yielded information on only the maximal effect by an NR, not on the preference of PBREM for a particular NR. Therefore, detailed titrations with selected NR1-binding NRs were performed. The transfected cells were treated with CAR-deactivating chemical and an activator specific for the competing NR. We selected ANDR and EE2 for mCAR and hCAR, respectively, because ANDR can completely deactivate mCAR (Forman et al., 1998; Sueyoshi et al., 1999), and EE2 is a partial deactivator of hCAR (Table 1, Fig. 1). In contrast, ANDR and EE2 had either a slight positive effect on PXR isoforms (Table 1) or did not affect other NRs at all (see below). These "reciprocal" effects on NR activity allowed us to better assess the functional preference of PBREM.

View larger version (31K): [in this window] [in a new window]   Fig. 6.   Dominance of mCAR over other NRs on mPBREM. Activation of mPBREM- or mNR1-driven reporter genes (25 ng) was assessed by cotransfection of indicated amounts of NR expression vectors (0-25 ng) into HEK293 cells, with balance of DNA kept constant by addition of empty expression vector. The transfected cells were treated with vehicle (), mCAR-specific deactivator ANDR (), competitor NR-specific activating ligand (), or their combination (). Activity with empty vector plus vehicle only was set to 100. Columns and bars denote mean and S.D., respectively, from three independent experiments.

View larger version (30K): [in this window] [in a new window]   Fig. 7.   Dominance of hCAR over other NRs on hPBREM. Activation of hPBREM- or hNR1-driven reporter genes (25 ng) was assessed as in Fig. 6. The transfected cells were treated with vehicle (), hCAR-specific partial deactivator EE2 (), competitor NR-specific activating ligand () or their combination ().

View larger version (33K): [in this window] [in a new window]   Fig. 8.   Dominance of hPXR and mPXR over other NRs on CYP3A enhancers. Activation of XREM-3A4- or CYP3A23-driven reporter genes (100 ng) was assessed as in Fig. 6. The transfected cells were treated with vehicle (), hPXR-specific RIF or mPXR-specific RU486 (), competitor NR-specific ligand (), or their combination ().

	Discussion

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The preference of PBREM for various NRs is not known although many NRs can bind to DR4-type motifs contained in PBREM. Thus, assessment of NRs with respect to their PBREM-modulating activity is important for understanding of CYP2B gene regulation, mechanisms, and species differences therein. Our studies were aimed at resolving the functional interplay between several NRs expressed in the liver and the mouse and human PBREM elements. To help in this task, CAR ligand binding specificities had to be defined in more detail as well.

Ligand Specificities of mCAR and hCAR. The known ligand profiles of mCAR and PXR isoforms (Forman et al., 1998; Lehmann et al., 1998; Sueyoshi et al., 1999; Moore et al., 2000) were well reproduced in our GAL4 fusion protein assays. With respect to hCAR, we confirmed that 5-pregnanedione was a modest activator, TCPOBOP had no effect, and ANDR was a weak deactivator, as shown earlier by Moore et al. (2000). Intriguingly, EE2 proved to be an activator of mCAR but a partial deactivator of hCAR. Because HEK293 cells do not express estrogen receptors (Kahlert et al., 2000), EE2 cannot inhibit hCAR activity via estrogen receptor-dependent squelching. EE2 was found to strongly promote the interaction between hCAR LBD and a NR corepressor, lending strong support for direct inhibitory action of EE2 on hCAR. In contrast to the report by Moore et al. (2000), we did not detect any suppression by CLOTR of hCAR activity. Instead, CLOTR activated GAL4-hCAR on its own and could also overcome the inhibition by EE2. This finding was also supported by our yeast two-hybrid experiments. Moore et al. (2000) reported decreases by CLOTR in hCAR activity in CV-1 cells and in in vitro association between hCAR LBD and coactivator SRC-1 with a FRET-based assay. Perhaps cell- and assay-specific differences may explain these differences. For instance, the decrease by ANDR of mCAR-SRC1 interaction that was seen in a GST pulldown assay (Forman et al., 1998) could not be reproduced by Moore et al. (2000). In our view, deactivators may be studied best with corepressor association assays.

NR1 Binding and Activation Specificity. Although NR1 sites alone confer CAR responsiveness (Sueyoshi et al., 1999), the presence of both NR1 and NR2 sites in the natural PBREM enhancer seems crucial for optimal activation (Honkakoski et al., 1998; Goodwin et al., 2001). Despite previous observations that mouse CAR/RXR heterodimer binds to NR1 and NR2 sites with equal efficiency in vitro (Tzameli et al., 2000), the present functional studies indicated that NR1 site is the stronger of these DR4 motifs. Paquet et al. (2000) have also suggested that NR1 and NR2 in rat CYP2B2 gene are not identical. Among many NRs capable of DR4 binding, only hPXR and mPXR have been reported to bind to the NR1 site with affinity similar to CAR (Xie et al., 2000b; Goodwin et al., 2001; Smirlis et al., 2001). Here, many other NRs were assessed through in vitro translation and NR1 probe binding under optimized conditions. Human VDR and cTR bound to NR1 with greater efficiency than CAR, which in turn displayed better binding than PXR isoforms. If the binding efficiency to NR1 were the sole determinant of PBREM activation, then one would predict that cTR and hVDR would be strong activators of PBREM. Clearly, this was not the case. On simple NR1 sites, activation by CAR greatly surpassed that of cTR, VDR, or PXR, which showed a maximal 2- to 3.5-fold activation. On natural PBREM elements, these three receptors were even less efficient. This is in contrast with the results of Smirlis et al. (2001) and Xie et al. (2000b), who found similar or 40% smaller activation of rodent PBREMs by mPXR or hPXR than by mCAR, respectively. However, they found ~2-fold activation by PCN of PBREM in hepatocytes, which is similar to the 2- to 2.5-fold activation by RU486 seen in HEK293 cells.

NR Cross-Talk Is Attenuated on PBREM Elements. The activation potential of NRs was significantly weaker on PBREM enhancers compared with NR1 sites. This suggests that NR interaction with DR4 motifs imbedded in PBREM is restricted, resulting in increased specificity for CAR. Furthermore, PBREM enhancers are more `insulated' than simple DR4 motifs from the repressive effects, as shown by diminished suppression by, for example, cTR, hVDR, PXR, and PPAR isoforms. Only COUP-TFI, a well-known suppressor (Cooney et al., 1993), could bring the CAR-dependent PBREM activity below 50%. We observed that hPBREM is notably less selective for CAR and more prone to NR-mediated suppression than mPBREM. In hPBREM, the NFI site seems to be mutated (Sueyoshi et al., 1999); therefore, NFI might play a role in the high selectivity of mPBREM. Kim et al. (2001) have recently shown that NFI and CAR can bind simultaneously to rat PBREM in vitro and that NFI coexpression may enhance trans-activation by CAR. This attractive mechanism cannot yet explain the selectivity of PBREM for CAR because CAR and NFI bound independently of each other, at least in vitro, and other NRs could potentially substitute for CAR. It may be possible that specific cofactors, lacking from in vitro studies, mediate the interaction between NFI and CAR. Other possibilities include co-operation between CAR-bound NR1 and NR2 sites that cannot be reproduced on multimeric NR1 sites. This option is consistent with the earlier report that mutation of any NR half-site in mPBREM reduced the PB inducibility to a similar extent (Honkakoski et al., 1998). Further studies into these hypotheses are warranted.

Preference of PBREM and XREM for NRs. The NR preference studies indicated that mCAR predominates on mPBREM over weaker effectors such as mPXR. Therefore, only weak activation by pure NR ligands of Cyp2b10 gene might be expected in vivo. Indeed, hepatocytes transfected with a PBREM construct showed only 2-fold activation after PCN treatment (Xie et al., 2000b; Smirlis et al., 2001); hepatic CYP2B10 was induced 37-fold by PB but only 7-fold by PCN (Pellinen et al., 1994); CYP2B10 mRNA induction was not affected either by thyroid hormone or by retinoic acid in mouse hepatocytes (Honkakoski and Negishi, 1998); T3 does not seem to affect PBREM or its associated factors in rats (Ganem et al., 1999). The identity of 5'-flanking nucleotides in DR4 motifs is important for TR-mediated activation (Harbers et al., 1996; Zhang and Lazar, 2000) and this property may explain the discrepancy between the strong binding and inefficient function by TR on PBREM. Although TR isoforms are expressed in liver (Zhang and Lazar, 2000), and TR can inhibit CAR LBD, the levels of TR relative to CAR may be too low for significant suppression via competition for NR coregulators. On the other hand, hPBREM seems to allow some hVDR and especially hPXR interactions. This probably explains why RIF, a specific hPXR ligand, can efficiently induce CYP2B6 mRNA in human hepatocytes (e.g., Goodwin et al., 2001). To our knowledge, there are no data available on the response of CYP2B genes to VDR ligands or VDR status.

Interference of CAR Signaling without Significant NR1 Binding. PPAR and FXR that bind poorly if at all to NR1 sites can still significantly inhibit CAR-mediated signaling. This suppression seems to be caused by reversible competition for coactivators. Recent experiments with NR null mice suggest that the interference of CAR function, detected here by cotransfection assays, may have physiological relevance in the liver where all these NRs are predominantly expressed. For example, the lack of PPAR greatly enhances the mitogenic effects of TCPOBOP (Columbano et al., 2001) that are mediated by CAR (Wei et al., 2000). Because CAR/RXR heterodimer binding is important for both basal and inducible CYP2B gene expression (Wan et al., 2000; Wei et al., 2000), it is possible that PPAR suppresses CAR and exerts an effect on PBREM. The activation of CYP3A and CYP2B gene expression in the absence of FXR (Schuetz et al., 2001) is difficult to interpret similarly because bile acids that accumulate in FXR null mice are also activators for PXR (e.g., Schuetz et al., 2001) and possibly weak ligands for mCAR as well (see Table 1). An FXR-specific chemical probe should help resolve this question and further elucidate interactions between NRs and P450 gene expression.