Final Report Snap ‘n Dose David Xue (Programmer) Niraj Mistry (Apper) Pooja Viswanathan (Programmer) Total Report Word Count (excluding title page & sample projects page): 2485 (Penalty - 0) Total Apper Context Word Count: 499 (Penalty - 0) Final Report Introduction Fever is the most common and concerning reason for which parents bring their children
Ita-suomenbiopankki.fiCopyright 2002 The American Society for Pharmacology and Experimental Therapeutics Mol Pharmacol 62:366–378, 2002 Printed in U.S.A. Modulation of Mouse and Human Phenobarbital-ResponsiveEnhancer Module by Nuclear Receptors JANNE M ¨AKINEN, CHRISTIAN FRANK, JOHANNA JYRKK ¨ARINNE, JUKKA GYNTHER, CARSTEN CARLBERG, andPAAVO HONKAKOSKI Departments of Pharmaceutics (J.K., J.J., P.H.), Biochemistry (C.F., C.C.), and Pharmaceutical Chemistry (J.G.), University of Kuopio,Kuopio, Finland Received October 24, 2001; accepted May 1, 2002 This article is available online at http://molpharm.aspetjournals.org ABSTRACT
The constitutive androstane receptor (CAR) regulates mouse
suppress PBREM function via a coactivator-dependent pro- and human CYP2B genes through binding to the direct re- cess that may have relevance in vivo. In competition experi- peat-4 (DR4) motifs present in the phenobarbital-responsive ments, mouse PBREM is clearly more selective for CAR than enhancer module (PBREM). The preference of PBREM ele- human PBREM. Pregnane X, vitamin D, and thyroid hormone ments for nuclear receptors and the extent of cross-talk be- receptors can potentially compete with human CAR on human tween CAR and other nuclear receptors are currently unknown.
PBREM. In contrast to the selective nature of PBREM, CYP3A Our transient transfection and DNA binding experiments indi- enhancers are highly and comparably responsive to CAR, preg- cate that binding to DR4 motifs does not correlate with the nane X receptor, and vitamin D receptor. In addition, the ligand activation response and that mouse and human PBREM are specificities of human and mouse CAR were defined by mam- efficiently ‘insulated' from the effects of other nuclear receptors malian cotransfection and yeast two-hybrid techniques. Our despite their substantial affinity for DR4 motifs. Certain nuclear results provide new mechanistic explanations to several previ- receptors that do not bind to DR4 motifs, such as peroxisome ously unresolved aspects of CYP2B and CYP3A gene regula- proliferator-activated receptor-␣ and farnesoid X receptor, can Phenobarbital (PB) and many structurally unrelated xeno- Cyp2b10, human CYP2B6, and rat CYP2B1 and CYP2B2 biotics induce same drug- and carcinogen-metabolizing cyto- genes (reviewed by Honkakoski and Negishi, 2000). The chrome P450 and other genes as a protective response di- PBREM contains two CAR/RXR heterodimer binding sites, rected toward elimination of these xenobiotics from the body.
NR1 and NR2, that conform to the direct repeat-4 (DR4) Among tens of PB-inducible genes, CYP2B genes are the motif. Successive mutations of DR4 motifs result in gradual most efficiently activated (reviewed by Waxman, 1999; loss and, finally, abolition of trans-activation by CAR in Honkakoski and Negishi, 2000). Recent studies have estab- HEK293 cells and induction in primary hepatocytes. It is lished that the constitutive androstane receptor (CAR, known that NR1 sites alone are sufficient for CAR respon- NR1I3) is crucial for induction of CYP2B genes by PB and siveness (Sueyoshi et al., 1999), whereas the nuclear factor 1 (NFI) binding site between NR1 and NR2 may contribute to cause CYP2B mRNA inducibility is lost in CAR null mice the full inducibility (Honkakoski et al., 1998; Kim et al., (Wei et al., 2000). After forming a heterodimer with retinoid X receptor (RXR, NR2B), the xenobiotic-activated CAR binds Several nuclear receptors (NRs), such as vitamin D recep- to the phenobarbital-responsive enhancer module (PBREM) tor (VDR, NR1I1), thyroid hormone receptors ␣/␤ (TR, or unit located in the upstream regions of the mouse NR1A1/2), retinoic acid receptors ␣/␤/␥ (RAR, NR1B1/2/3),liver X receptors ␣/␤ (LXR, NR1H3/2), pregnane X receptor This study was supported by Academy of Finland grants 44040 and 51610 (to P.H.) and 50331 (to C.C.).
(PXR, NR1I2) and farnesoid X receptor (FXR, NR1H4) dis- ABBREVIATIONS: PB, phenobarbital; CAR, constitutive androstane receptor; TCPOBOP, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene; RXR,
retinoid X receptor; PBREM, phenobarbital-responsive enhancer module; DRn, direct repeat with n base-pair spacing; ERn, everted repeat with
n base-pair spacing; HEK, human embryonic kidney; NR, nuclear receptor; NFI, nuclear factor 1; VDR, vitamin D receptor; TR, thyroid hormone
receptor; RAR, retinoic acid receptor; LXR, liver X receptor; PXR, pregnane X receptor; FXR, farnesoid X receptor; ERn, everted repeat with n
base-pair spacing; WY-14,643, [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid; tk, thymidine kinase promoter; LBD, ligand-binding do-
main; NCoR, nuclear receptor corepressor; VD3, 1␣,25-dihydroxycholecalciferol; RIF, rifampicin; RU486, mifepristone; ANDR, 3␣-androstenol;
CLOTR, clotrimazole; T3, tri-iodothyronine; COUP-TFI, chicken ovalbumin upstream promoter-transcription factor I; AF2, activation function-2;
trVD3, 1␣,25-dihydroxycholecalciferol; XREM, xenobiotic-responsive enhancer module; EE2, 17␣-ethynyl-3,17␤-estradiol.
Nuclear Receptor Cross-Talk on PB-Responsive CYP2B Enhancers
play considerable in vitro binding and activation of DR4-type Steven Kliewer (GlaxoSmithKline, Research Triangle Park, NC).
sites (Mangelsdorf and Evans, 1995; Laffitte et al., 2000; The mouse (mNR1) -tk-luc, human PBREM-tk-luc, and (hNR1) -tk- Quack and Carlberg, 2000; Xie et al., 2000b). Therefore, it luc plasmids have been described previously (Sueyoshi et al., 1999).
has been proposed that additional NRs could bind to PBREM The human XREM-3A4-luc reporter containing the proximal 362 and modulate its activity (Waxman, 1999). This idea is in line base pairs of CYP3A4 gene promoter and the distal enhancer (Good- with evidence that CYP2B mRNA induction is influenced by win et al., 1999) was a kind gift from Dr. Chris Liddle (University ofSydney at Westmead Hospital, Westmead, Australia). The UAS -tk- sex, steroid and thyroid hormones, sterol metabolites, and luc (Janowski et al., 1996) and rat CYP3A23[⫺1360/⫹82] reporters retinoids, all of which are known NR ligands (Honkakoski (Xie et al., 2000b) were donated by Dr. Ronald Evans (Salk Institute and Negishi, 2000). Several inducers such as PB and pesti- for Biological Studies, La Jolla, CA). Other luciferase reporter plas- cides activate not only CAR (Sueyoshi et al., 1999) but also mids for nuclear receptors were generated by inserting multiple PXR, a receptor important for CYP3A gene regulation (re- copies of their cognate DNA sites into BglII site of pGL3-Basic viewed by Quattrochi and Guzelian, 2001). CAR and PXR plasmid. All plasmids were purified with QIAGEN columns (Hilden, recognize similar DNA motifs that range from DR2 to DR5 Germany) and verified by restriction mapping, functional testing and everted repeat-6 (ER6), and both receptors are expressed and, when necessary, by sequencing.
in the liver and intestine (Honkakoski and Negishi, 2000; Expression Plasmids. The sources of expression vectors for
Quattrochi and Guzelian, 2001). Collectively, these data sug- mRAR␣ and hRAR␣ (Zelent et al., 1989), cTR␣ (Harbers et al., 1996), gest that other NRs might well affect the PBREM enhancer hLXR␤ (Teboul et al., 1995), mCOUP-TFI (NR2F1; Cooney et al., and influence CYP2B gene regulation through cross-talk 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 Surprisingly, there is very little information or systematic and mPXR (Lehmann et al., 1998) have been described previously.
studies on PBREM binding or cross-talk with CAR by other The expression plasmid for coactivator hTIF2 (Voegel et al., 1996) NRs. Such studies are much needed for detailed understand- was donated by Dr. Hinrich Gronemeyer (IGBMC, Illkirch, France).
ing of CYP2B gene regulation, modulating factors, and spe- GAL4-LBD Fusion Plasmids. The ligand binding domains (LBD)
cies differences. So far, we know that CAR can activate the of mCAR (residues 118–358), hCAR (residues 108–348), mPXR (resi- PXR-responsive ER6 and DR3 motifs in CYP3A genes (Su- dues 104–431), and hPXR (residues 107–434) were amplified with Pfu eyoshi et al., 1999; Moore et al., 2000; Xie et al., 2000b; DNA polymerase from mouse and human liver RNAs and cloned into 5⬘ Smirlis et al., 2001). This is consistent with the report that EcoRI and 3⬘ BamHI or KpnI sites of CMX-GAL4 plasmid (Janowski et PB can induce CYP3A mRNA in PXR null mice (Xie et al., al., 1996) donated by Dr. Ronald Evans. GAL4-mCAR⌬8 plasmid coding 2000a). Recently, hPXR and mPXR were shown to bind to for a truncated mCAR lacking eight amino acids at the C terminus DR4 motifs and activate PBREM elements from various spe- (Choi et al., 1997) was donated by Dr. David Moore (Baylor College ofMedicine, Houston, TX).
cies by 3- to 6-fold. This effect is roughly comparable with Ligand Specificities of mCAR and hCAR. Ligand specificities of
that of CAR-mediated activation (Xie et al., 2000b; Goodwin mCAR and hCAR were assessed for PBREM preference studies (Figs.
et al., 2001; Smirlis et al., 2001). None of these studies, 6-8) because CAR and PXR are reported to share some ligands (Moore however, could address the preference of PBREM for CAR et al., 2000) and to assess the effect of other NR ligands on mCAR and and PXR. The data are also mostly based on in vitro DNA hCAR activity. The ligand specificities were measured first by chemical- binding assays with simple DR4 motifs (Sueyoshi et al., 1999; dependent modulation of GAL4 fusion protein-driven reporter activity Xie et al., 2000b; Smirlis et al., 2001) instead of functional in HEK293 cells (Table 1) according to Honkakoski et al. (2001) and assays with receptors and PBREM elements. Finally, there is then by yeast two-hybrid assays as described below.
practically no data on the modulation of PBREM by other Human and mouse CAR LBDs were inserted between EcoRI and NRs. Therefore, our aim was to gain more insight to the BamHI sites in pGBKT7 plasmid. The NR interaction domains from mouse and human PBREM function and its specificity for mouse (residues 1988–2304) and human (residues 1972–2290) core- CAR by evaluating the effects of several NRs on PBREM pressor NCoR (Hu and Lazar, 1999) were cloned from liver RNAs activity by functional and DNA binding assays. To help in and inserted between EcoRI and BamHI sites in pGADT7 plasmid(Matchmaker GAL4 System 3, BD Clontech). All the manipulations this effort, the ligand specificities of human and mouse CAR were done essentially according to manufacturer's instructions. Ran- were also defined.
dom yeast colonies selected on SD/Leu⫺/Trp⫺ plates were picked,amplified, and aliquots of cells were then treated with vehicle or testchemicals for 3.5 h before measurement of ␤-galactosidase activities Materials and Methods
and cell densities according to Nishikawa et al. (1999).
Chemicals. TCPOBOP was synthesized and purified according to
In Vitro Translation and Gel Shift Assays. NRs were produced
Honkakoski et al. (1996) to more than 98% purity as assessed by in vitro by first transcribing linearized expression vectors with T7 1H-NMR spectra and elemental analysis (observed: N, 6.7%; C, 46.9%; RNA polymerase and then translating these RNAs in vitro using H, 2.0%; expected: N, 6.8%; C, 46.8%; H, 2.2%). Steroids were from rabbit reticulocyte lysate as recommended by the supplier (Pro- Steraloids, Inc. (Newport, RI) or Sigma-Aldrich Chemical Co. (St. Louis, mega). Nuclear receptor heterodimers with RXR (approximately 10 MO). WY-14,643 was bought from ChemSyn, Inc. (Lenexa, KS). Other ng of specific protein; equal protein amounts verified by a parallel chemicals were at least analytical grade from Sigma, Fluka translation in the presence of [35S]methionine) were incubated with (Ronkonkoma, NY), or Calbiochem (La Jolla, CA).
ligand for 15 min at room temperature in a total volume of 20 l of Reporter Plasmids. pCMV␤ was purchased from BD Clontech
binding buffer [10 mM HEPES, pH 7.9, 1 mM dithiothreitol, 0.2 Inc. (Palo Alto, CA). The mPBREM-tk-luc reporter was constructed g/l poly(dI-dC), and 5% glycerol], which was adjusted to 150 mM by insertion of the PBREM element plus the thymidine kinase pro- KCl. Approximately 1 ng of the 32P-labeled human CYP2B6 or mouse moter (tk) from the mouse PBREM-tk-CAT (Honkakoski et al., 1998) Cyp2b10 DR4-type NR1 motif (50,000 cpm) was then added, and into BglII site of pGL3-Basic luciferase plasmid (Promega, Madison, incubation was continued for 20 min. Protein-DNA complexes were WI). The rat (rER6) -tk-luc reporter was constructed similarly from resolved through 8% nondenaturing poly-acrylamide gels in 0.5⫻ (ER6) -tk-CAT plasmid (Lehmann et al., 1998) donated by Dr.
Tris/borate/EDTA (45 mM Tris, 45 mM boric acid, 1 mM EDTA, pH Ma¨kinen et al.
8.3) and were quantified on a FLA3000 reader (Fuji, Tokyo, Japan) 0.5 M TCPOBOP, 10 M EE2, and 2 M CLOTR to varying using Image Gauge software (Fuji).
degrees. With GAL4-hCAR, a reproducible partial deactiva- Cell Culture and Nuclear Receptor Cotransfection. Mouse
tion (50–60%) by EE2 and about 2-fold activation by CLOTR primary hepatocytes were isolated, transfected, and assayed as de- was seen. Furthermore, the partial deactivation by EE2 scribed previously (Honkakoski and Negishi, 1998; Honkakoski et could be overcome by addition of CLOTR and, to a lesser al., 1998). HEK293 cells (American Type Culture Collection, Manas-sas, VA) were grown in phenol red-free Dulbecco's modified Eagle extent, by 5␤-pregnanedione (Table 1). The known ligand medium supplemented with 10% fetal bovine serum and 100 U/ml profiles of mPXR and hPXR (Moore et al., 2000) were also penicillin-100 g/ml streptomycin (Invitrogen, Gaithersburg, MD).
reproduced: both receptors were activated by CLOTR, One day before transfection, the cells were seeded on 48-well plates RU486, and 5␤-pregnanedione, but RIF activated only hPXR.
in medium containing delipidated serum (Sigma) to remove potential Yeast two-hybrid assays supported the finding that ANDR NR-activating substances. After an overnight incubation, the me- and EE2 can deactivate mCAR and hCAR, respectively, be- dium was changed and the cells were transfected using a calcium cause they could dose-dependently increase association be- phosphate method with pCMV␤ (50 ng), various luciferase reporter tween the CAR LBD and the NR interaction domain of the plasmids (25 ng; 100 ng for XREM-3A4-luc and CYP3A23-luc), andvariable amounts of expression vectors for NRs (varied from zero to corepressor NCoR by 25- to 50-fold (Fig. 1, left, 䡺, u). This 250 ng). In activation and suppression experiments, the amount of association could be reversed by TCPOBOP and CLOTR (Fig.
CAR expression plasmid that produced maximal activity from the 1, right, 3), whereas these activators themselves had little if reporter plasmid was 12.5 ng, and other NRs were titrated from zero any effect on the CAR LBD-NCoR interaction. These results to 20-fold excess (250 ng) over CAR so as to reach the effect plateau.
obtained from two independent systems strongly suggest In preference experiments, the total amount of NR expression vector that the EE2 and CLOTR are true, reciprocally acting hCAR was only 50 ng, much below levels that produced any unspecific squelching (ⱖ200 ng). The balance of DNA was kept constant by DNA Binding to NR1 Sites by NRs. PBREM elements
addition of empty expression vector.
After a 4-h transfection period, the medium was changed. The are known to confer about 10-fold activation by CAR in fresh medium additionally contained an established NR ligand/acti- HEK293 cells and about 10-fold induction by TCPOBOP in vator at concentration sufficient for maximal or near-maximal NR primary hepatocytes (Honkakoski et al., 1998; Sueyoshi et response: 20 M WY-14643 for h/mPPAR␣, 0.1 M 1␣,25-dihydroxy- al., 1999). When organization of the mouse PBREM (5⬘ NR1- cholecalciferol (VD3) for hVDR, 10 M rifampicin (RIF) for hPXR, 10 NFI-NR2 3⬘) was changed to NR1-NFI-NR1 or to NR2-NFI- M mifepristone (RU486) for mPXR, 10 M 3␣-androstenol (ANDR) NR2, the original and NR1-containing PBREM elements re- or 0.5 M TCPOBOP for mCAR, 10 M 5␤-pregnanedione, 2 M tained ⬎10-fold activation. PBREM containing NR2 motifs clotrimazole (CLOTR), or 10 M 17␣-ethynyl-3,17␤-estradiol (EE2) only conferred much lower ⬇3-fold activation by mCAR and for hCAR (see Table 1), 10 M arotinoid acid for h/mRAR␣, 50 Mchenodeoxycholic acid for hFXR, 10 M 25OH-cholesterol for hLXR␤, TCPOBOP inducibility (Fig. 2). This indicates that NR1 is and 0.1 M tri-iodothyronine (T3) for cTR␣.
the stronger site for PBREM function.
Reporter Assays. Transfected HEK293 cells were cultured for
Gel shift assays were performed to compare the ability of 40 h, washed with PBS, and lysed. Luciferase and ␤-galactosidase NR/RXR␣ heterodimers to form complexes with NR1 sites activities (Honkakoski et al., 2001) were determined from 20 l of (Fig. 3). The human and mouse NR1 sites were similar in lysates in 96-well plates using the Victor2 multiplate reader their binding patterns. In the absence of specific activating (PerkinElmer Wallac, Turku, Finland). All luciferase activities were ligands, the ranking of complex formation was found to be normalized to ␤-galactosidase expression and expressed as mean ⫾standard deviation from three to four independent experiments.
cTR␣ ⬎⬎ mCAR ⬎ hCAR ⬇ hVDR ⬎ hPXR ⬎ mPXR ⬇hCOUP-TFI ⬎ hRAR␣ ⬎ hLXR␤. Heterodimers of hFXR andhPPAR␣ showed no binding to NR1 sites but were demon- strated to bind to consensus DR1-type motifs (data not Ligand Specificities of mCAR and hCAR. With GAL4
shown). Addition of specific ligands enhanced complex forma- fusion proteins in HEK293 cells (Table 1), we found that tion only for mCAR, hCAR, and hVDR. However, the VD3- GAL4-mCAR activity was suppressed by ANDR, as expected, induced complex formation of hVDR was so strong that it and that ANDR-suppressed activity could be reactivated by practically equalled that of cTR␣. Thus, cTR␣ and hVDR TABLE 1Ligand specificities of CAR and PXR with selected xenobioticsData 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 (ⱕ 15%) on hCAR or mCAR. Only chenodeoxycholic acid was a weak reactivator of ANDR-suppressed mCAR (2-fold). ANDR and EE2 didnot have significant effects on VDR-, PPAR␣-, or FXR-dependent activities.
12.52 ⫾ 1.20 a 1.70 ⫾ 0.15 a 0.70 ⫾ 0.11 a 1.41 ⫾ 0.12 a 8.18 ⫾ 0.23 a 0.41 ⫾ 0.05 a 1.97 ⫾ 0.02 a 1.50 ⫾ 0.13 a 1.83 ⫾ 0.23 a 1.73 ⫾ 0.06 a 2.26 ⫾ 0.23 a 3.21 ⫾ 0.58 aa 2.19 ⫾ 0.03 a 4.02 ⫾ 0.05 a 2.28 ⫾ 0.32 a 2.70 ⫾ 0.15 a N.D., not done.
a Statistically different from vehicle (p ⬍ 0.05).
Nuclear Receptor Cross-Talk on PB-Responsive CYP2B Enhancers
surpass both CAR and PXR isoforms in their ability to bind to First, the maximal effect of NRs on simple DR4 motifs was mouse and human NR1 sites.
tested (Fig. 4, top, 䡺, u). Mouse (NR1) -tk-luc was activated, Activation of NR1 Sites and PBREM Enhancers by
in descending order, by mCAR (11.2-fold) ⬎⬎ hVDR (3.4- NRs. Binding to NR1 may indicate a potential influence on
fold) ⬎ cTR␣ (2.6-fold) ⬎ mPXR (2.0-fold) ⬇ mRAR␣ (1.9- PBREM activity. To compare between NRs, increasing fold). Addition of hLXR␤ resulted in a slight 30% increase in amounts of NR expression vectors were cotransfected with activity, whereas COUP-TFI suppressed it by 25%. Human NR1- or PBREM-driven reporter genes. The NRs were then (NR1) -tk-luc was activated by hCAR (8.9-fold) ⬎⬎ hPXR activated by established ligands, and the reporter activities (3.5-fold) ⬎ hVDR (2.2-fold) ⬇ cTR␣ (2.1-fold). Human LXR␤ were measured. The results shown below are the maximal and mRAR␣ increased and hFXR and COUP-TFI decreased effects observed for each NR, usually at the same concentra- the human NR1-driven activity slightly. Control experiments tion as the optimal CAR concentration. The NRs themselves with tk-luc plasmid lacking any enhancers established the could ligand-dependently activate reporters driven by their specificity of NR effects (data not shown). In addition, control consensus response elements (data not shown), demonstrat- experiments with activating NR ligands (Fig. 4, top, o, p) ing that the constructs were functional.
showed that NR1-elements were not activated in the absence Fig. 1. Ligand-dependent associa-
tion 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 concentra-
tions (micromolar) before cell lysis
and ␤-galactosidase assays as de-
scribed under Materials and Meth-
ods. For mCAR-mNCoR associa-
tion, the reporter activity with 10
M ANDR was set to 100 (top, 䡺).
In TCPOBOP displacement experi-ment (top, 3), the activity with 2 M ANDR was set to 100 (sameconcentration as in mammalianGAL4 assays in Table 1). ForhCAR-hNCoR association, the re-porter activity with 10 M EE2 wasset to 100 (bottom, u, 3). The datashown are mean ⫾ standard devia-tion from triplicate samples. Theexperiments were repeated inde-pendently two (mCAR) or threetimes (hCAR) with similar results.
Activities with either GAL4-CAR orNCoR plasmid alone were below de-tection limit.
Fig. 2. Activation of mutated mouse PBREM elements.
PBREM sequence, NR1 sites only, NR2 sites only, or noenhancer (tk only) was cotransfected in the presence ofeither empty or CAR expression vector into HEK293 cells(䡺). Mouse hepatocytes were electroporated with the samereporters and treated with DMSO or 0.5 M TCPOBOP(f). Reporter activities from 3– 4 independent experimentswere measured as described under Materials and Meth-ods.
Ma¨kinen et al.
of NR expression vectors. In summary, almost all NRs capa- 20-fold excess over CAR) were used and effects at plateau ble of NR1 binding in vitro were able to activate NR1-driven only (typically 10-fold excess) are shown for clarity.
gene transcription to varying degrees, whereas COUP-TFI Figure 4, bottom, indicates that mCAR-activated NR1- inhibited it.
driven activity (Fig. 4, 䡺) was suppressed most efficiently by When the same experiment was done with PBREM-driven COUP-TFI (to 11% of control activity), followed by cTR␣ reporters (Fig. 4, top, f), a more restricted and attenuated (32%) and hVDR (44%). Mouse PPAR␣, hFXR, and mPXR response to NRs was noted. Mouse CAR was by far the displayed a comparable 50 to 60% decrease, followed by strongest activator of the mPBREM (10.1-fold), followed by mRAR␣ and hLXR␤. Human CAR (Fig. 4, u) was suppressed, ⱕ2-fold activation by cTR␣, hVDR, and mPXR. The human in descending order, by COUP-TFI (to 16% of control activ- PBREM was activated, in descending order, by hCAR (7.9- ity) ⬎ cTR␣ ⬇ hVDR (about 30%) ⬎ hPXR ⬇ hPPAR␣ (about fold) and ⱕ2.1-fold by hPXR, hVDR, and cTR␣. Human RAR␣ 50%), followed by hFXR (65%). Again, hRAR␣ and hLXR␤ did not affect human PBREM, even though the NR1 element had little or no effect. A less prominent suppression by the was modestly but reproducibly activated. In addition, the NRs was found on PBREM-driven reporters (Fig. 4, bottom, extent of activation by other NRs was always less on PBREM , f). Instead of the 50 to 90% decrease in activity that was than on NR1 sites. For instance, the activation of NR1 by observed on NR1 sites with COUP-TFI, cTR␣, hVDR, PXR, or hVDR or PXR reached 28 and 40% of that by CAR, respec- PPAR␣ isoforms, PBREM was inhibited by only 20 to 60% by tively. On PBREM, hVDR and PXR reached only 18 and 27% the same receptors. There was also a tendency for human of CAR-dependent activity. PBREM seems to be activated NR1 and PBREM to be inhibited more than corresponding preferentially by CAR and then by similar efficiency (ⱕ2- mouse elements by NR1-binding PXR isoforms, hVDR, and fold) by cTR␣, hVDR, and PXR. Mouse PXR was a poorer cTR␣. In line with activation results, mPXR was a weaker activator of both NR1 and PBREM elements than hPXR.
suppressor than hPXR. In the context of PBREM, hFXR, and PPAR␣ isoforms suppressed CAR as efficiently as PXR iso- PBREM Enhancers by NRs. Because NRs may influence
forms hVDR and cTR␣, which bind to and inhibit NR1 more PBREM function by competing for DNA binding sites or for common NR coregulators, NRs were cotransfected in the Suppressive Effects of NRs Occurring through CAR
presence of CAR and the maximal NR-mediated suppression LBD. Expression vectors for GAL4-m/hCAR and UAS -tk-luc
of CAR-dependent NR1- or PBREM-driven activities were reporter were used in the above suppression assay to deter- analyzed. Increasing amounts of NR expression vectors (0- to mine whether suppression could be attributed to competition 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 (f) [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 (䡺) 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.
Nuclear Receptor Cross-Talk on PB-Responsive CYP2B Enhancers
for factors associated with the CAR LBD. This approach effect of NRs for PBREM enhancers. Furthermore, the activa- would eliminate any competition at the level of DNA binding, tion and suppression experiments yielded information on only which was prominent for cTR␣, hVDR, and PXR isoforms.
the maximal effect by an NR, not on the preference of PBREM Figure 5, top, indicates that with both GAL4-mCAR and for a particular NR. Therefore, detailed titrations with selected GAL4-hCAR as activators, the strongest suppressors were NR1-binding NRs were performed. The transfected cells were full-length CAR and cTR␣ (⬍20% of control activity), fol- treated with CAR-deactivating chemical and an activator spe- lowed by COUP-TFI ⬇ PXR isoforms ⬇ hFXR (25–30%), cific for the competing NR. We selected ANDR and EE2 for whereas hVDR, RAR␣, and PPAR␣ isoforms were weaker mCAR and hCAR, respectively, because ANDR can completely suppressors (35–55%). No suppression was seen with GAL4- deactivate mCAR (Forman et al., 1998; Sueyoshi et al., 1999), mCAR⌬8 that lacks the AF2 core sequence (data not shown), and EE2 is a partial deactivator of hCAR (Table 1, Fig. 1). In indicating that NR-mediated suppression of CAR depends on contrast, ANDR and EE2 had either a slight positive effect on the presence of intact AF2 domain. Therefore, suppression of PXR isoforms (Table 1) or did not affect other NRs at all (see CAR LBD probably reflects competition for NR coactivators.
below). These "reciprocal" effects on NR activity allowed us to Indeed, cotransfection of TIF2 vector in this suppression better assess the functional preference of PBREM.
assay (Fig. 5, bottom) resulted in partial restoration of It should be noted that in preference studies, much lower mCAR-dependent and especially hCAR-dependent reporter total amounts of NRs than in suppression assays (Fig. 4, activity. Differences in the extent of suppression and resto- bottom) were used (50 versus 250 ng), and therefore unspe- ration of reporter activity further imply that NRs may have cific squelching effects are not likely. In activation experi- different affinities for various NR coactivators. In summary, ment titrations (data not shown), we did not see any signif- NR1-binding cTR␣ and hVDR showed a remarkable differ- icant self-suppression by increasing amounts of CAR, PXR, ence in their ability to suppress CAR LBD. PPAR␣ isoforms or any other NR plasmid. This would happen if NR coregu- and hFXR that do not bind to NR1 sites could inhibit PBREM lators were a limiting factor and result in squelching. This through an AF2- and coactivator-dependent mechanism.
did not seem to be the case.
Preference of NRs for PBREM Enhancers. As shown
Figure 6, top, shows that in the presence of saturating above, DNA binding studies were not sufficient to assess the amounts of mPXR only, mPBREM was activated 2-fold by Fig. 4. Activation and suppression of
mouse and human NR1- and PBREM-
driven reporter genes by various NRs. Ac-
tivation by NRs (top) was assessed by
cotransfection into HEK293 cells of in-
creasing amounts of indicated NR expres-
sion vectors (0 –250 ng) and reporter
genes (25 ng) driven by mNR1 (䡺), hNR1
(u), mPBREM ( ), or hPBREM (f) ele-
ments and addition of NR-specific li-
gands. o, p, ligand controls (empty vector
⫹ activating NR ligand). Representativeresults at optimal 12.5 ng of NR expres-sion vectors (37.5 ng for RAR␣) areshown, with columns and bars denotingmean and S.D., respectively. Suppressionby NRs (bottom) was assessed by cotrans-fection of increasing amounts of indicatedNR expression vectors (0 –250 ng) to-gether with saturating amount of mouseor human CAR expression vector (12.5ng) and reporter genes (25 ng) driven bymNR1 (䡺), hNR1 (u), mPBREM ( ), orhPBREM HEK293 cells were treated with NR-spe-cific ligands. Activity with empty vectorwas set to 100. Representative results atmaximal effect (125 ng of NR expressionvectors) are shown, with columns andbars denoting mean and S.D., respec-tively. No CAR, basal reporter activity inthe presence of empty expression vectorsubstituted for both CAR and the compet-ing NRs.
Ma¨kinen et al.
RU486, regardless of the presence of ANDR, as expected from ratios of mCAR to cTR␣, the combined T3⫹ANDR treatment data in Table 1. In the presence of mCAR only, mPBREM was decreased activities to control levels, suggesting a strong mCAR activated 7-fold, which was completely abolished by ANDR.
dominance over cTR␣.
Already at 1:25 ratio of mCAR to mPXR expression vectors, the Human PBREM was activated 1.8-fold by EE2 and 2.5-fold combined RU486⫹ANDR treatment suppressed the mPBREM- by RIF in the presence of hPXR only. As predicted, hPBREM driven activity to control levels, suggesting that mCAR clearly activity was decreased to 40% by EE2 but not affected by RIF dominates mPXR on PBREM. Mouse PBREM was activated in the presence of hCAR only (Fig. 7, top). Compared with 2-fold by 0.1 M VD3 in the presence of hVDR (Fig. 6, middle).
results with mCAR-to-mPXR titration on mPBREM, hPXR When the ratio of mCAR to hVDR was increased stepwise, the predominated strikingly over hCAR at a 1:25 ratio, showed combined VD3⫹ANDR treatment gave slightly higher LUC substantial activity at a 5:25 ratio; hCAR predominated only activities than ANDR alone up to 1:25 ratio, suggesting that at a 25:25 ratio. Cotransfection of hVDR (Fig. 7, middle) that hVDR binds to mPBREM slightly more avidly than mPXR.
combined VD3⫹EE2 treatment reduced the activity below However, the hVDR-mediated suppression at 5:25 ratio (about those elicited by VD3 alone beginning at hCAR-to-hVDR 15%) was less than for mPXR. Both these results are well in line ratio of 5:25. In contrast to mPBREM, hPBREM activity was with DNA binding and GAL4-mCAR suppression studies (Figs.
substantially reduced (to 60%) by VD3, matching the similar 3 and 4, bottom). Because of very modest activation of mP- difference seen in suppression assay in Fig. 4. On human BREM by cTR␣ (see Fig. 3), the experiment was done with NR1 sites, cTR␣ was the dominant receptor up to 5:25 ratio mNR1 reporter (Fig. 6, bottom). Already at 1:25 and greater of hCAR to cTR␣, above which the combined T3⫹EE2 treat- 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) to-
gether with saturating amount of mouse (䡺) or hu-
man (f) GAL4-CAR LBD expression vector (12.5 ng)
and UAS -tk-luc reporter (25 ng) into HEK293 cells,
followed by addition of NR-specific ligands. Reporteractivity with empty vector was set to 100. Represen-tative results at maximal effect (125 ng of NR expres-sion vectors, 10-fold excess over CAR) are shown,with columns and bars denoting mean and S.D., re-spectively. GAL4 only, basal activity in the absence ofany LBD in the construct. Bottom, reactivation ofsuppressed GAL4-mCAR- or GAL4-hCAR-dependentactivity was performed as above with cotransfection(500 ng) of empty expression vector (䡺, ) or hTIF2plasmid ( , f).
Nuclear Receptor Cross-Talk on PB-Responsive CYP2B Enhancers
ment began to decrease the activity (Fig. 7, lower). These present. XREM activity driven by hCAR only was again results show that human PBREM was less selective for CAR decreased 50% by EE2 but not affected by RIF. Titration with than mouse PBREM.
increasing amounts of hPXR vector indicated that the de- creasing effect of EE2 on XREM-driven activity was lost only CYP3A23-luc reporters were used in similar preference stud- at 25:25 ratio of hPXR to hCAR. This suggests that hCAR has ies with hCAR, mCAR, and hVDR. Figure 8, top, shows that considerable affinity to XREM motifs ER6 and/or DR3. Ex- the human XREM enhancer was activated 2- to 3-fold by periments with (rER6) -tk-luc reporter proved that at least EE2, RIF, and their combination when hPXR only was ER6-driven activity could be enhanced comparably by hCAR Fig. 6. Dominance of mCAR over other NRs on mP-
BREM. Activation of mPBREM- or mNR1-driven re-
porter 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 trans-
fected cells were treated with vehicle (䡺), mCAR-
specific deactivator ANDR (f), competitor NR-specific
activating ligand (u), or their combination (o). Activ-
ity with empty vector plus vehicle only was set to 100.
Columns and bars denote mean and S.D., respec-
tively, from three independent experiments.
Ma¨kinen et al.
(6-fold) and hPXR (4-fold) (data not shown). Transfection of that (rER6) -tk-luc and CYP3A23-luc reporters were induced hVDR and VD3 treatment increased XREM-driven activity 5- and 20-fold, respectively, by ligand-activated hVDR (data very strongly (Fig. 8, middle). Transfection of equal amounts not shown). Figure 8, bottom, shows that mCAR and mPXR of hPXR and hVDR resulted in more than 60 and 40% sup- activated the CYP3A23-luc reporter over 4- and 3-fold, re- pression of hVDR- and hPXR-dependent activities, respec- spectively. Mouse CAR has substantial activity over mPXR, tively. This suggested similar competition between hVDR because the combined RU486⫹ANDR treatment began to and hPXR for the XREM binding sites but higher activation increase reporter activity above ANDR levels only at 25:25 potential by hVDR. This notion was supported by the finding ratio of mPXR to mCAR. Similar mCAR dominance over 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
(䡺), hCAR-specific partial deactivator EE2 (f),
competitor NR-specific activating ligand (u) or
their combination (o).
Nuclear Receptor Cross-Talk on PB-Responsive CYP2B Enhancers
mPXR were seen with (rER6) -tk-luc reporter (data not of CYP2B gene regulation, mechanisms, and species differ- shown). Our results indicated that, in contrast to PBREM ences therein. Our studies were aimed at resolving the func- elements more selective for CAR, the CYP3A enhancers are tional interplay between several NRs expressed in the liver very responsive to hVDR, CAR, and PXR isoforms.
and the mouse and human PBREM elements. To help in thistask, CAR ligand binding specificities had to be defined in more detail as well.
The preference of PBREM for various NRs is not known Ligand Specificities of mCAR and hCAR. The known
although many NRs can bind to DR4-type motifs contained in ligand profiles of mCAR and PXR isoforms (Forman et al., PBREM. Thus, assessment of NRs with respect to their 1998; Lehmann et al., 1998; Sueyoshi et al., 1999; Moore et PBREM-modulating activity is important for understanding al., 2000) were well reproduced in our GAL4 fusion protein 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 (䡺), hPXR-specific RIF or mPXR-specific RU486
(f), competitor NR-specific ligand (u), or their combina-
Ma¨kinen et al.
assays. With respect to hCAR, we confirmed that 5␤-preg- ple DR4 motifs from the repressive effects, as shown by nanedione was a modest activator, TCPOBOP had no ef- diminished suppression by, for example, cTR␣, hVDR, PXR, fect, and ANDR was a weak deactivator, as shown earlier and PPAR␣ isoforms. Only COUP-TFI, a well-known sup- by Moore et al. (2000). Intriguingly, EE2 proved to be an pressor (Cooney et al., 1993), could bring the CAR-dependent activator of mCAR but a partial deactivator of hCAR.
PBREM activity below 50%. We observed that hPBREM is Because HEK293 cells do not express estrogen receptors notably less selective for CAR and more prone to NR-medi- (Kahlert et al., 2000), EE2 cannot inhibit hCAR activity ated suppression than mPBREM. In hPBREM, the NFI site via estrogen receptor-dependent squelching. EE2 was seems to be mutated (Sueyoshi et al., 1999); therefore, NFI found to strongly promote the interaction between hCAR might play a role in the high selectivity of mPBREM. Kim et LBD and a NR corepressor, lending strong support for al. (2001) have recently shown that NFI and CAR can bind direct inhibitory action of EE2 on hCAR. In contrast to the simultaneously to rat PBREM in vitro and that NFI coex- report by Moore et al. (2000), we did not detect any sup- pression may enhance trans-activation by CAR. This attrac- pression by CLOTR of hCAR activity. Instead, CLOTR tive mechanism cannot yet explain the selectivity of PBREM activated GAL4-hCAR on its own and could also overcome for CAR because CAR and NFI bound independently of each the inhibition by EE2. This finding was also supported by other, at least in vitro, and other NRs could potentially sub- our yeast two-hybrid experiments. Moore et al. (2000) re- stitute for CAR. It may be possible that specific cofactors, ported decreases by CLOTR in hCAR activity in CV-1 cells lacking from in vitro studies, mediate the interaction be- and in in vitro association between hCAR LBD and coac- tween NFI and CAR. Other possibilities include co-operation tivator SRC-1 with a FRET-based assay. Perhaps cell- and between CAR-bound NR1 and NR2 sites that cannot be re- assay-specific differences may explain these differences.
produced on multimeric NR1 sites. This option is consistent For instance, the decrease by ANDR of mCAR–SRC1 in- with the earlier report that mutation of any NR half-site in teraction that was seen in a GST pulldown assay (Forman mPBREM reduced the PB inducibility to a similar extent et al., 1998) could not be reproduced by Moore et al. (2000).
(Honkakoski et al., 1998). Further studies into these hypoth- In our view, deactivators may be studied best with core- eses are warranted.
pressor association assays.
Preference of PBREM and XREM for NRs. The NR
NR1 Binding and Activation Specificity. Although
preference studies indicated that mCAR predominates on NR1 sites alone confer CAR responsiveness (Sueyoshi et al., mPBREM over weaker effectors such as mPXR. Therefore, 1999), the presence of both NR1 and NR2 sites in the natural only weak activation by pure NR ligands of Cyp2b10 gene PBREM enhancer seems crucial for optimal activation might be expected in vivo. Indeed, hepatocytes transfected (Honkakoski et al., 1998; Goodwin et al., 2001). Despite pre- with a PBREM construct showed only 2-fold activation after vious observations that mouse CAR/RXR␣ heterodimer binds PCN treatment (Xie et al., 2000b; Smirlis et al., 2001); he- to NR1 and NR2 sites with equal efficiency in vitro (Tzameli patic CYP2B10 was induced 37-fold by PB but only 7-fold by et al., 2000), the present functional studies indicated that PCN (Pellinen et al., 1994); CYP2B10 mRNA induction was NR1 site is the stronger of these DR4 motifs. Paquet et al.
not affected either by thyroid hormone or by retinoic acid in (2000) have also suggested that NR1 and NR2 in rat CYP2B2 mouse hepatocytes (Honkakoski and Negishi, 1998); T3 does gene are not identical. Among many NRs capable of DR4 not seem to affect PBREM or its associated factors in rats binding, only hPXR and mPXR have been reported to bind to (Ganem et al., 1999). The identity of 5⬘-flanking nucleotides the NR1 site with affinity similar to CAR (Xie et al., 2000b; in DR4 motifs is important for TR-mediated activation (Har- Goodwin et al., 2001; Smirlis et al., 2001). Here, many other bers et al., 1996; Zhang and Lazar, 2000) and this property NRs were assessed through in vitro translation and NR1 may explain the discrepancy between the strong binding and probe binding under optimized conditions. Human VDR and inefficient function by TR␣ on PBREM. Although TR iso- cTR␣ bound to NR1 with greater efficiency than CAR, which forms are expressed in liver (Zhang and Lazar, 2000), and TR in turn displayed better binding than PXR isoforms. If the can inhibit CAR LBD, the levels of TR relative to CAR may be binding efficiency to NR1 were the sole determinant of too low for significant suppression via competition for NR PBREM activation, then one would predict that cTR␣ and coregulators. On the other hand, hPBREM seems to allow hVDR would be strong activators of PBREM. Clearly, this some hVDR and especially hPXR interactions. This probably was not the case. On simple NR1 sites, activation by CAR explains why RIF, a specific hPXR ligand, can efficiently greatly surpassed that of cTR␣, VDR, or PXR, which showed induce CYP2B6 mRNA in human hepatocytes (e.g., Goodwin a maximal 2- to 3.5-fold activation. On natural PBREM ele- et al., 2001). To our knowledge, there are no data available on ments, these three receptors were even less efficient. This is the response of CYP2B genes to VDR ligands or VDR status.
in contrast with the results of Smirlis et al. (2001) and Xie et The CYP3A4 and CYP3A23 enhancers, in contrast, re- al. (2000b), who found similar or 40% smaller activation of spond not only to PXR but also to CAR and VDR. These rodent PBREMs by mPXR or hPXR than by mCAR, respec- experiments now give, for the first time, a mechanistic ex- tively. However, they found ⬃2-fold activation by PCN of planation of the strong inducibility of Cyp3a genes by the PBREM in hepatocytes, which is similar to the 2- to 2.5-fold mCAR ligand TCPOBOP (e.g., Smith et al., 1993), to the activation by RU486 seen in HEK293 cells.
induction of CYP3A4 mRNA by VD3 in Caco-2 cells NR Cross-Talk Is Attenuated on PBREM Elements.
(Schmiedlin-Ren et al., 1997), and suggest why CYP3A and The activation potential of NRs was significantly weaker on CYP2B genes tend to be coregulated in humans. Our results PBREM enhancers compared with NR1 sites. This suggests are in contrast with Moore et al. (2000), who found that hPXR that NR interaction with DR4 motifs imbedded in PBREM is predominated hCAR on the XREM enhancer. Their conclu- restricted, resulting in increased specificity for CAR. Fur- sion was based on the repressive effect of CLOTR on hCAR, thermore, PBREM enhancers are more ‘insulated' than sim- a finding that we could not reproduce with either full-length Nuclear Receptor Cross-Talk on PB-Responsive CYP2B Enhancers
TABLE 2Summary of NR effects on CAR signaling Activation of NR1/ Suppression of NR1/ Predicted Relevance of CAR Cross-Talk in Vivo ⫹⫹/⫹⫹ b ⫹⫹⫹⫹ Human, ⫹⫹ Mouse ⫹⫹⫹/⫹ b ⫹⫹ Human, 0 Mouse ⫹ Human, 0 Mouse ⫹ Human, ⫹⫹ Mouse ⫹⫹⫹⫹, Strong effect; ⫹⫹⫹, significant effect; ⫹⫹, moderate effect; ⫹, weak effect; 0, no effect; N.A., not applicable.
a COUP-TFI represses NR1 and PBREM.
b PXR, VDR, and COUP-TFI suppress hPBREM more than mPBREM.
c Hepatic expression of hPPAR␣ is significantly lower than that of mPPAR␣.
hCAR, GAL4 fusion plasmids, or with yeast two-hybrid as- T3 suppresses CYP2B mRNA expression in rats but does not affect PBREM (Ganem et al., 1999). Moreover, these experi- Interference of CAR Signaling without Significant
ments would require truly monospecific NR ligands to avoid, NR1 Binding. PPAR␣ and FXR that bind poorly if at all to
for example, interference with glucocorticoid signaling that is NR1 sites can still significantly inhibit CAR-mediated sig- essential for CYP2B regulation (Honkakoski and Negishi, naling. This suppression seems to be caused by reversible 2000) that plagues the mPXR ligands PCN and RU486, and competition for coactivators. Recent experiments with NR the cross-activation of NRs such as FXR and PXR by bile null mice suggest that the interference of CAR function, acids. Given the lower selectivity of human PBREM and detected here by cotransfection assays, may have physiolog- XREM for NRs, human hepatocytes would probably be the ical relevance in the liver where all these NRs are predomi- best system in which to run the experiments. Most impor- nantly expressed. For example, the lack of PPAR␣ greatly tantly, tissue or hepatocytes from NR null mice would be enhances the mitogenic effects of TCPOBOP (Columbano et most valuable in confirming the observed NR interferences.
al., 2001) that are mediated by CAR (Wei et al., 2000). Be- In conclusion, our results indicate that binding of an NR to cause CAR/RXR␣ heterodimer binding is important for both NR1 sites does not correlate with its functional effects in the basal and inducible CYP2B gene expression (Wan et al., context of PBREM. The use of simple NR motifs for binding 2000; Wei et al., 2000), it is possible that PPAR␣ suppresses and trans-activation assays may not reveal actual function of CAR and exerts an effect on PBREM. The activation of an NR on natural DNA elements. Mouse PBREM was found CYP3A and CYP2B gene expression in the absence of FXR to be more selective for CAR than human PBREM, which is (Schuetz et al., 2001) is difficult to interpret similarly be- also activated by PXR, VDR, and TR␣. In contrast to PBREM cause bile acids that accumulate in FXR null mice are also elements, CYP3A enhancers were highly responsive to VDR, activators for PXR (e.g., Schuetz et al., 2001) and possibly CAR, and PXR. PPAR␣ and FXR may use mechanisms de- weak ligands for mCAR as well (see Table 1). An FXR-specific pendent on coactivators to interfere with CAR signaling.
chemical probe should help resolve this question and furtherelucidate interactions between NRs and P450 gene expres- We thank Drs. Pierre Chambon (IGBMC, Illkirch, France), Ronald Finally, we have attempted to classify NRs based on the Evans, Frank Gonzalez (NCI, Bethesda, MD), Hinrich Gronemeyer, observed DNA binding and PBREM-suppressive effects (Ta- Steven Kliewer, David Mangelsdorf, (University of Texas Southwest- ble 2). This data combined with approximate hepatic levels of ern Medical Center, Dallas, TX), Masahiko Negishi and Cary Wein- mouse NRs reported in the literature (e.g., Wan et al., 2000; berger (NIEHS, Research Triangle Park, NC), Ming-Jer Tsai (Baylor Xie et al., 2000a; Zhang and Lazar, 2000) may allow us to College of Medicine, Houston, TX), Bjo¨rn Vennstro¨m (Karolinska predict the relevance of NR cross-talk with CAR signaling. As Institute, Stockholm, Sweden), Steven Kliewer, Chris Liddle, David positive signs of this predictability, the moderate or weak Moore, for plasmids, and Kaarina Pitka¨nen for technical assistance.
efficiency of mPXR, TR␣, and RAR␣ for PBREM activation is indeed reflected in some in vivo studies (Pellinen et al., 1994; Choi HS, Chung M, Tzameli I, Simha D, Lee YK, Seol W, and Moore DD (1997) Honkakoski and Negishi, 1998; Ganem et al., 1999). Simi- Differential trans-activation by two isoforms of the orphan nuclear hormone re- larly, PPAR␣ seems to suppress CAR activity in vivo (Colum- ceptor CAR. J Biol Chem 272:23565–23571.
Columbano A, Ledda-Columbano GM, Pibiri M, Concas D, Reddy JK, and Rao MS bano et al., 2001).
(2001) Peroxisome proliferator-activated receptor-␣(⫺/⫺) mice show enhanced he- Collectively, our studies generate hypotheses for further in patocyte proliferation in response to the hepatomitogen 1,4-bis[2-(3,5-dichloro-
pyridyloxy)]benzene, a ligand of constitutive androstane receptor. Hepatology 34:
vivo experiments that must, however, be carefully controlled to rule out any nonspecific effects occurring outside PBREM.
Cooney AJ, Tsai SY, O'Malley BW, and Tsai M-J (1993) Chicken ovalbumin up- stream promoter transcription factor (COUP-TF) dimers bind to different GGTCA As examples of the associated problems, CYP3A and CYP2B response elements, allowing COUP-TF to repress hormonal induction of the vita- genes contain NR binding sites also outside of XREM and min D3, thyroid hormone and retinoic acid receptors. Mol Cell Biol 12:4153– 4163.
PBREM. For examples, the CYP3A promoter contains DR1 Forman BM, Goode E, Chen J, Oro AE, Bradley DJ, Perlmann T, Noonan DJ, Burka LT, McMorris T, Kozak CA, et al. (1995) Identification of a nuclear receptor that sites that are crucial for CYP3A basal activity (Quattrochi is activated by farnesol metabolites. Cell 81:687– 693.
and Guzelian, 2001) but are also targets for COUP-TFI, Forman BM, Tzameli I, Choi HS, Chen J, Simha D, Seol W, Evans RM, and Moore DD (1998) Androstane metabolites bind to and deactivate the nuclear receptor hepatocyte nuclear factor-4, and perhaps other NRs as well.
CAR-␤. Nature (Lond) 395:612– 615.
Ma¨kinen et al.
Ganem LG, Trottier E, Anderson A, and Jefcoate CR (1999) Phenobarbital induction Quack M and Carlberg C (2000) Ligand-triggered stabilization of vitamin D receptor/ of CYP2B1/2 in primary hepatocytes: endocrine regulation and evidence for a retinoid X receptor heterodimer conformations on DR4-type response elements.
single pathway for multiple inducers. Toxicol Appl Pharmacol 155:32– 42.
J Mol Biol 296:743–756.
Goodwin B, Hodgson E, and Liddle C (1999) The orphan human pregnane X receptor Quattrochi LC and Guzelian PS (2001) CYP3A regulation: from pharmacology to mediates the transcriptional activation of CYP3A4 by rifampicin through a distal nuclear receptors. Drug Metab Dispos 29:615– 622.
enhancer module. Mol Pharmacol 56:1329 –1339.
Schmiedlin-Ren P, Thummel KE, Fisher JM, Paine MF, Lown KS, and Watkins PB Goodwin B, Moore LB, Stoltz CM, McKee DD, and Kliewer SA (2001) Regulation of (1997) Expression of enzymatically active CYP3A4 by Caco-2 cells grown on the human CYP2B6 gene by the nuclear pregnane X receptor. Mol Pharmacol extracellular matrix-coated permeable supports in the presence of 1␣,25- dihydroxyvitamin D3. Mol Pharmacol 51:741–754.
Harbers M, Wahlstro¨m GM, and Vennstro¨m B (1996) Transactivation by the thyroid Schuetz EG, Strom S, Yasuda K, Lecureur V, Assem M, Brimer C, Lamba J, Kim RB, hormone receptor is dependent on the spacer sequence in hormone response Ramachandran V, Komoroski BJ, et al. (2001) Disrupted bile acid homeostasis elements containing directly repeated half-sites. Nucl Acids Res 24:2252–2259.
reveals an unexpected interaction among nuclear hormone receptors, transporters Honkakoski P, Jaaskelainen I, Kortelahti M, and Urtti A (2001) A novel drug- and cytochrome P450. J Biol Chem 276:39411–39418.
regulated gene expression system based on the nuclear receptor constitutive Sher T, Yi HF, McBride OW, and Gonzalez FJ (1993) cDNA cloning, chromosomal androstane receptor (CAR). Pharm Res 18:146 –150.
mapping and functional characterization of the human peroxisome proliferator- Honkakoski P, Moore R, Gynther J, and Negishi M (1996) Characterization of activated receptor. Biochemistry 32:5598 –5604.
phenobarbital-inducible Cyp2b10 gene transcription in primary hepatocytes.
Smirlis D, Muangmoonchai R, Edwards M, Phillips IR, and Shephard EA (2001) J Biol Chem 271:9746 –9753.
Orphan receptor promiscuity in the induction of cytochromes P450 by xenobiotics.
Honkakoski P and Negishi M (1998) Protein serine/threonine phosphatase inhibitors J Biol Chem 276:12822–12826.
suppress phenobarbital-induced Cyp2b10 gene transcription in mouse primary Smith G, Henderson CJ, Parker MG, White R, Bars RG, and Wolf CR (1993) hepatocytes. Biochem J 330:889 – 895.
Honkakoski P and Negishi M (2000) Regulation of cytochrome P450 (CYP) genes by 1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene, an extremely potent modulator of nuclear receptors. Biochem J 347:321–337.
mouse hepatic cytochrome P-450 gene expression. Biochem J 289:807– 813.
Honkakoski P, Zelko I, Sueyoshi T, and Negishi M (1998) The orphan nuclear Sueyoshi T, Kawamoto T, Zelko I, Honkakoski P, and Negishi M (1999) The re- receptor CAR-retinoid X receptor heterodimer activates the phenobarbital- pressed nuclear receptor CAR responds to phenobarbital in activating the human responsive enhancer module of the CYP2B gene. Mol Cell Biol 18:5652–5658.
CYP2B6 gene. J Biol Chem 274:6043– 6046.
Hu X and Lazar MA (1999) The CoRNR motif controls the recruitment of corepres- Teboul M, Enmark E, Li Q, Wikstrom AC, Pelto-Huikko M and Gustafsson J-Å sors by nuclear hormone receptors. Nature (Lond) 402:93–96.
(1995) OR-1, member of the nuclear receptor superfamily that interacts with the Janowski BA, Willy PJ, Devi TR, Falck JR, and Mangelsdorf DJ (1996) An oxysterol 9-cis-retinoic acid receptor. Proc Natl Acad Sci USA 92:2096 –2100.
signalling pathway mediated by the nuclear receptor LXR␣. Nature (Lond) 383:
Tzameli I, Pissios P, Schuetz EG, and Moore DD (2000) The xenobiotic compound 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene is an agonist ligand for the nuclear re- Kahlert S, Nuedling S, van Eickels M, Vetter H, Meyer R, and Grohe C (2000) ceptor CAR. Mol Cell Biol 20:2951–2958.
Estrogen receptor alpha rapidly activates the IGF-1 receptor pathway. J Biol Voegel JJ, Heine MJ, Zechel C, Chambon P, and Gronemeyer H (1996) TIF2, a 160 kDa transcriptional mediator for the ligand-dependent activation function AF-2 of Kim J, Min G, and Kemper B (2001) Chromatin assembly enhances binding to the nuclear receptors. EMBO (Eur Mol Biol Organ) J 15:3667–3675.
CYP2B1 pheno- barbital-responsive unit (PBRU) of nuclear factor-1, which binds Wan YY, An D, Cai Y, Repa JJ, Chen TH, Flores M, Postic C, Magnuson MA, Chen simultaneously with constitutive androstane receptor (CAR)/retinoid X receptor J, Chien KR, et al. (2000) Hepatocyte-specific mutation establishes retinoid X (RXR) and enhances CAR/RXR-mediated activation of the PBRU. J Biol Chem receptor ␣ as a heterodimeric integrator of multiple physiological processes in the liver. Mol Cell Biol 20:4436 – 4444.
Laffitte BA, Kast HR, Nguyen CM, Zavacki AM, Moore DD, and Edwards PA (2000) Waxman DJ (1999) P450 gene induction by structurally diverse xenochemicals: Identification of the DNA binding specificity and potential target genes for the central role of nuclear receptors CAR, PXR and PPAR. Arch Biochem Biophys farnesoid X-activated receptor. J Biol Chem 275:10638 –10647.
Lehmann JM, McKee DD, Watson MA, Willson TM, Moore T, and Kliewer SA (1998) Wei P, Zhang J, Egan-Hafley M, Liang S, and Moore DD (2000) The nuclear receptor The human orphan nuclear receptor PXR is activated by compounds that regulate CAR mediates specific xenobiotic induction of drug metabolism. Nature (Lond) CYP3A4 gene expression and cause drug interactions. J Clin Invest 102:1016 –
Xie W, Barwick JL, Downes M, Blumberg B, Simon CM, Nelson MC, Neuschwander- Mangelsdorf DJ and Evans RM (1995) The RXR heterodimers and orphan receptors.
Tetri BA, Brunt EM, Guzelian PS, and Evans RM (2000a) Humanized xenobiotic Cell 83:841– 850.
response in mice expressing nuclear receptor SXR. Nature (Lond) 406:435– 438.
Moore LB, Parks DJ, Jones SA, Bledsoe RK, Consler TG, Stimmel J, Goodwin B, Xie W, Barwick JL, Simon CM, Pierce AM, Safe S, Blumberg B, Guzelian PS, and Liddle C, Blanchard SG, Willson TM, et al. (2000) Orphan nuclear receptors Evans RM (2000b) Reciprocal activation of xenobiotic response genes by nuclear constitutive androstane receptor and pregnane X receptor share xenobiotic and receptors SXR/PXR and CAR. Genes Dev 14:3014 –3023.
steroid ligands. J Biol Chem 275:15122–15127.
Zelent A, Krust A, Petkovich M, Kastner P, and Chambon P (1989) Cloning of murine Nishikawa J, Saito K, Goto J, Dakeyama F, Matsuo M, and Nishihara T (1999) New alpha and beta retinoic acid receptors and a novel receptor gamma predominantly screening methods for chemicals with hormonal activities using interaction of expressed in skin. Nature (Lond) 339:714 –717.
nuclear hormone receptor with coactivator. Toxicol Appl Pharmacol 154:76 – 83.
Paquet Y, Trottier E, Beaudet MJ, and Anderson A (2000) Mutational analysis of the Zhang J and Lazar MA (2000) The mechanism of action of thyroid hormones. Annu CYP2B2 pheno- barbital response unit and inhibitory effect of the constitutive Rev Physiol 62:439 – 466.
androstane receptor on phenobarbital responsiveness. J Biol Chem 275:38427–
Pellinen P, Honkakoski P, Stenback F, Niemitz M, Alhava E, Pelkonen O, Lang MA, Address correspondence to: Dr. Paavo Honkakoski, Department of Phar-
and Pasanen M (1994) Cocaine N-demethylation and the metabolism-related hep- maceutics, University of Kuopio, P.O.Box 1627, FIN-70211 Kuopio, Finland.
atotoxicity can be prevented by cytochrome P450 3A inhibitors. Eur J Pharmacol
William E. Seidelman MDScience and Inhumanity: The Kaiser-Wilhelm/Max Planck Society First Published in: If Not Now an e-journal Volume 2, Winter 2000 Revised February 18, 2001. One hundred years ago this past December a German scientist by the name of Max Karl Ernst Ludwig Planck gave a lecture in Berlin to the German Physical