Departments of Pharmaceutics (J.K., J.J., P.H.), Biochemistry
(C.F., C.C.), and Pharmaceutical Chemistry (J.G.), University of
Kuopio, Kuopio, Finland
 |
Introduction |
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 |
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-mCAR
8 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.
|
|
Human and mouse CAR LBDs were inserted between EcoRI and
BamHI sites in pGBKT7 plasmid. The NR interaction domains
from mouse (residues 1988-2304) and human (residues 1972-2290)
corepressor NCoR (Hu and Lazar, 1999
) were cloned from liver RNAs and
inserted between EcoRI and BamHI sites in pGADT7
plasmid (Matchmaker GAL4 System 3, BD Clontech). All the manipulations
were done essentially according to manufacturer's instructions. Random
yeast colonies selected on SD/Leu
/Trp
plates were picked,
amplified, and aliquots of cells were then treated with vehicle or test
chemicals for 3.5 h before measurement of
-galactosidase
activities and cell densities according to Nishikawa et al. (1999)
.
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.
After a 4-h transfection period, the medium was changed. The fresh
medium additionally contained an established NR ligand/activator at
concentration sufficient for maximal or near-maximal NR response: 20 µM WY-14643 for h/mPPAR
, 0.1 µM 1
,25-dihydroxycholecalciferol (VD3) for hVDR, 10 µM rifampicin (RIF) for hPXR, 10 µM mifepristone (RU486) for mPXR, 10 µM 3
-androstenol (ANDR) or 0.5 µM TCPOBOP for mCAR, 10 µM 5
-pregnanedione, 2 µM clotrimazole (CLOTR), or 10 µM 17
-ethynyl-3,17
-estradiol (EE2) for hCAR (see Table 1), 10 µM arotinoid acid for h/mRAR
, 50 µM chenodeoxycholic acid for
hFXR, 10 µM 25OH-cholesterol for hLXR
, and 0.1 µM
tri-iodothyronine (T3) for cTR
.
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 |
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.
Yeast two-hybrid assays supported the finding that ANDR and EE2 can
deactivate mCAR and hCAR, respectively, because they could dose-dependently increase association between the CAR LBD and the NR
interaction domain of the corepressor NCoR by 25- to 50-fold (Fig.
1, left,
,
). This association
could be reversed by TCPOBOP and CLOTR (Fig. 1, right,
), whereas
these activators themselves had little if any effect on the CAR
LBD-NCoR interaction. These results obtained from two independent
systems strongly suggest that the EE2 and CLOTR are true, reciprocally
acting hCAR ligands.

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.
|
|
Gel shift assays were performed to compare the ability of NR/RXR
heterodimers to form complexes with NR1 sites (Fig.
3). The human and mouse NR1 sites were
similar in their binding patterns. In the absence of specific
activating ligands, the ranking of complex formation was found to be
cTR
mCAR > hCAR
hVDR > hPXR > mPXR
hCOUP-TFI > hRAR
> hLXR
. Heterodimers
of hFXR and hPPAR
showed no binding to NR1 sites but were
demonstrated to bind to consensus DR1-type motifs (data not shown).
Addition of specific ligands enhanced complex formation only for mCAR, hCAR, and hVDR. However, the VD3-induced complex formation of hVDR was
so strong that it practically equalled that of cTR
. Thus, cTR
and
hVDR surpass both CAR and PXR isoforms in their ability to bind to
mouse and human NR1 sites.

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.
First, the maximal effect of NRs on simple DR4 motifs was tested (Fig.
4, top,
,
). Mouse
(NR1)3-tk-luc was activated, in descending
order, by mCAR (11.2-fold)
hVDR (3.4-fold) > cTR
(2.6-fold) > mPXR (2.0-fold)
mRAR
(1.9-fold).
Addition of hLXR
resulted in a slight 30% increase in activity,
whereas COUP-TFI suppressed it by 25%. Human
(NR1)3-tk-luc was activated by hCAR (8.9-fold)
hPXR (3.5-fold) > hVDR (2.2-fold)
cTR
(2.1-fold). Human LXR
and mRAR
increased and hFXR and COUP-TFI decreased the
human NR1-driven activity slightly. Control experiments with tk-luc
plasmid lacking any enhancers established the specificity of NR effects
(data not shown). In addition, control experiments with activating NR
ligands (Fig. 4, top,
,
) showed that NR1-elements were not
activated in the absence of NR expression vectors. In summary, almost
all NRs capable of NR1 binding in vitro were able to activate
NR1-driven gene transcription to varying degrees, whereas COUP-TFI
inhibited it.

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.
|
|
When the same experiment was done with PBREM-driven reporters (Fig. 4,
top,
,
), a
more restricted and attenuated response to NRs was noted. Mouse CAR was
by far the strongest activator of the mPBREM (10.1-fold), followed by
2-fold activation by cTR
, hVDR, and mPXR. The human PBREM was
activated, in descending order, by hCAR (7.9-fold) and
2.1-fold by
hPXR, hVDR, and cTR
. Human RAR
did not affect human PBREM, even
though the NR1 element was modestly but reproducibly activated. In
addition, the extent of activation by other NRs was always less on
PBREM than on NR1 sites. For instance, the activation of NR1 by hVDR or
PXR reached 28 and 40% of that by CAR, respectively. On PBREM, hVDR
and PXR reached only 18 and 27% of CAR-dependent activity. PBREM seems
to be activated preferentially by CAR and then by similar efficiency
(
2-fold) by cTR
, hVDR, and PXR. Mouse PXR was a poorer activator
of both NR1 and PBREM elements than hPXR.
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.
Figure 4, bottom, indicates that mCAR-activated NR1-driven activity
(Fig. 4,
) was suppressed most efficiently by COUP-TFI (to 11% of
control activity), followed by cTR
(32%) and hVDR (44%). Mouse
PPAR
, hFXR, and mPXR displayed a comparable 50 to 60% decrease,
followed by mRAR
and hLXR
. Human CAR (Fig. 4,
) was
suppressed, in descending order, by COUP-TFI (to 16% of control
activity) > cTR
hVDR (about 30%) > hPXR
hPPAR
(about 50%), followed by hFXR (65%). Again, hRAR
and
hLXR
had little or no effect. A less prominent suppression by the
NRs was found on PBREM-driven reporters (Fig. 4, bottom,
,
). Instead
of the 50 to 90% decrease in activity that was observed on NR1 sites with COUP-TFI, cTR
, hVDR, PXR, or PPAR
isoforms, PBREM was
inhibited by only 20 to 60% by the same receptors. There was also a
tendency for human NR1 and PBREM to be inhibited more than
corresponding mouse elements by NR1-binding PXR isoforms, hVDR, and
cTR
. In line with activation results, mPXR was a weaker suppressor
than hPXR. In the context of PBREM, hFXR, and PPAR
isoforms
suppressed CAR as efficiently as PXR isoforms hVDR and cTR
, which
bind to and inhibit NR1 more avidly.
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-mCAR
8 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.
It should be noted that in preference studies, much lower total amounts
of NRs than in suppression assays (Fig. 4, bottom) were used (50 versus
250 ng), and therefore unspecific squelching effects are not likely. In
activation experiment titrations (data not shown), we did not see any
significant self-suppression by increasing amounts of CAR, PXR, or any
other NR plasmid. This would happen if NR coregulators were a limiting
factor and result in squelching. This did not seem to be the case.
Figure 6, top, shows that in the
presence of saturating amounts of mPXR only, mPBREM was activated
2-fold by RU486, regardless of the presence of ANDR, as expected from
data in Table 1. In the presence of mCAR only, mPBREM was activated
7-fold, which was completely abolished by ANDR. Already at 1:25 ratio
of mCAR to mPXR expression vectors, the combined RU486+ANDR treatment suppressed the mPBREM-driven activity to control levels, suggesting that mCAR clearly dominates mPXR on PBREM. Mouse PBREM was activated 2-fold by 0.1 µM VD3 in the presence of hVDR (Fig. 6, middle). When
the ratio of mCAR to hVDR was increased stepwise, the combined VD3+ANDR
treatment gave slightly higher LUC activities than ANDR alone up to
1:25 ratio, suggesting that hVDR binds to mPBREM slightly more avidly
than mPXR. However, the hVDR-mediated suppression at 5:25 ratio (about
15%) was less than for mPXR. Both these results are well in line with
DNA binding and GAL4-mCAR suppression studies (Figs. 3 and 4, bottom).
Because of very modest activation of mPBREM by cTR
(see Fig. 3), the
experiment was done with mNR1 reporter (Fig. 6, bottom). Already at
1:25 and greater ratios of mCAR to cTR
, the combined T3+ANDR
treatment decreased activities to control levels, suggesting a strong
mCAR dominance over cTR
.

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.
|
|
Human PBREM was activated 1.8-fold by EE2 and 2.5-fold by RIF in the
presence of hPXR only. As predicted, hPBREM activity was decreased to
40% by EE2 but not affected by RIF in the presence of hCAR only (Fig.
7, top). Compared with results with
mCAR-to-mPXR titration on mPBREM, hPXR predominated strikingly over
hCAR at a 1:25 ratio, showed substantial activity at a 5:25 ratio; hCAR predominated only at a 25:25 ratio. Cotransfection of hVDR (Fig. 7,
middle) that combined VD3+EE2 treatment reduced the activity below
those elicited by VD3 alone beginning at hCAR-to-hVDR ratio of 5:25. In
contrast to mPBREM, hPBREM activity was substantially reduced (to 60%)
by VD3, matching the similar difference seen in suppression assay in
Fig. 4. On human NR1 sites, cTR
was the dominant receptor up to 5:25
ratio of hCAR to cTR
, above which the combined T3+EE2 treatment
began to decrease the activity (Fig. 7, lower). These results show that
human PBREM was less selective for CAR than mouse PBREM.

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 ( ).
|
|
For comparison, PXR-responsive XREM-3A4-luc and CYP3A23-luc reporters
were used in similar preference studies with hCAR, mCAR, and hVDR.
Figure 8, top, shows that the human XREM
enhancer was activated 2- to 3-fold by EE2, RIF, and their combination
when hPXR only was present. XREM activity driven by hCAR only was again decreased 50% by EE2 but not affected by RIF. Titration with
increasing amounts of hPXR vector indicated that the decreasing effect
of EE2 on XREM-driven activity was lost only at 25:25 ratio of hPXR to
hCAR. This suggests that hCAR has considerable affinity to XREM motifs
ER6 and/or DR3. Experiments with (rER6)3-tk-luc
reporter proved that at least ER6-driven activity could be enhanced
comparably by hCAR (6-fold) and hPXR (4-fold) (data not shown).
Transfection of hVDR and VD3 treatment increased XREM-driven activity
very strongly (Fig. 8, middle). Transfection of equal amounts of hPXR and hVDR resulted in more than 60 and 40% suppression of hVDR- and
hPXR-dependent activities, respectively. This suggested similar competition between hVDR and hPXR for the XREM binding sites but higher
activation potential by hVDR. This notion was supported by the finding
that (rER6)3-tk-luc and CYP3A23-luc reporters
were induced 5- and 20-fold, respectively, by ligand-activated hVDR (data not shown). Figure 8, bottom, shows that mCAR and mPXR activated the CYP3A23-luc reporter over 4- and 3-fold, respectively. Mouse CAR
has substantial activity over mPXR, because the combined RU486+ANDR treatment began to increase reporter activity above ANDR levels only at
25:25 ratio of mPXR to mCAR. Similar mCAR dominance over mPXR were seen
with (rER6)3-tk-luc reporter (data not shown). Our results indicated that, in contrast to PBREM elements more selective for CAR, the CYP3A enhancers are very responsive to hVDR,
CAR, and PXR isoforms.

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 |
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.
The CYP3A4 and CYP3A23 enhancers, in contrast, respond not only to PXR
but also to CAR and VDR. These experiments now give, for the first
time, a mechanistic explanation of the strong inducibility of
Cyp3a genes by the mCAR ligand TCPOBOP (e.g., Smith et al., 1993
), to the induction of CYP3A4 mRNA by VD3 in Caco-2 cells (Schmiedlin-Ren et al., 1997
), and suggest why CYP3A and
CYP2B genes tend to be coregulated in humans. Our results
are in contrast with Moore et al. (2000)
, who found that hPXR
predominated hCAR on the XREM enhancer. Their conclusion was based on
the repressive effect of CLOTR on hCAR, a finding that we could not
reproduce with either full-length hCAR, GAL4 fusion plasmids, or with
yeast two-hybrid assays.
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.
Finally, we have attempted to classify NRs based on the observed DNA
binding and PBREM-suppressive effects (Table
2). This data combined with approximate
hepatic levels of mouse NRs reported in the literature (e.g., Wan et
al., 2000
; Xie et al., 2000a
; Zhang and Lazar, 2000
) may allow us to
predict the relevance of NR cross-talk with CAR signaling. As positive
signs of this predictability, the moderate or weak efficiency of mPXR,
TR
, and RAR
for PBREM activation is indeed reflected in some in
vivo studies (Pellinen et al., 1994
; Honkakoski and Negishi, 1998
;
Ganem et al., 1999
). Similarly, PPAR
seems to suppress CAR activity
in vivo (Columbano et al., 2001
).
Collectively, our studies generate hypotheses for further in vivo
experiments that must, however, be carefully controlled to rule out any
nonspecific effects occurring outside PBREM. As examples of the
associated problems, CYP3A and CYP2B genes
contain NR binding sites also outside of XREM and PBREM. For examples, the CYP3A promoter contains DR1 sites that are crucial for CYP3A basal
activity (Quattrochi and Guzelian, 2001
) but are also targets for
COUP-TFI, hepatocyte nuclear factor-4, and perhaps other NRs as well.
T3 suppresses CYP2B mRNA expression in rats but does not affect PBREM
(Ganem et al., 1999
). Moreover, these experiments would require truly
monospecific NR ligands to avoid, for example, interference with
glucocorticoid signaling that is essential for CYP2B
regulation (Honkakoski and Negishi, 2000
) that plagues the mPXR ligands
PCN and RU486, and the cross-activation of NRs such as FXR and PXR by
bile acids. Given the lower selectivity of human PBREM and XREM for
NRs, human hepatocytes would probably be the best system in which to
run the experiments. Most importantly, tissue or hepatocytes from NR
null mice would be most valuable in confirming the observed NR interferences.
In conclusion, our results indicate that binding of an NR to NR1 sites
does not correlate with its functional effects in the context of PBREM.
The use of simple NR motifs for binding and trans-activation
assays may not reveal actual function of an NR on natural DNA elements.
Mouse PBREM was found to be more selective for CAR than human PBREM,
which is also activated by PXR, VDR, and TR
. In contrast to PBREM
elements, CYP3A enhancers were highly responsive to VDR,
CAR, and PXR. PPAR
and FXR may use mechanisms dependent on
coactivators to interfere with CAR signaling.
We thank Drs. Pierre Chambon (IGBMC, Illkirch, France), Ronald
Evans, Frank Gonzalez (NCI, Bethesda, MD), Hinrich Gronemeyer, Steven
Kliewer, David Mangelsdorf, (University of Texas Southwestern Medical
Center, Dallas, TX), Masahiko Negishi and Cary Weinberger (NIEHS,
Research Triangle Park, NC), Ming-Jer Tsai (Baylor College of Medicine,
Houston, TX), Björn Vennström (Karolinska Institute, Stockholm, Sweden), Steven Kliewer, Chris Liddle, David Moore, for plasmids, and Kaarina Pitkänen for technical assistance.
This study was supported by Academy of Finland grants 44040 and
51610 (to P.H.) and 50331 (to C.C.).
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 domain;
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.