Estrogen Activation of the Nuclear Orphan Receptor CAR (Constitutive Active Receptor) in Induction of the Mouse Cyp2b10 Gene
Takeshi Kawamoto,
Satoru Kakizaki,
Kouich Yoshinari and
Masahiko Negishi
Pharmacogenetics Section Laboratory of Reproductive and
Developmental Toxicology National Institute of Environmental Health
Sciences National Institutes of Health Research Triangle Park,
North Carolina 27709
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ABSTRACT
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The nuclear orphan receptor CAR (constitutively
active receptor or constitutive androstane receptor) can be activated
in response to xenochemical exposure, such as activation by
phenobarbital of a response element called NR1 found in the
CYP2B gene. Here various steroids were screened for
potential endogenous chemicals that may activate CAR, using the NR1
enhancer and Cyp2b10 induction in transfected HepG2 cell
and/or in mouse primary hepatocytes as the experimental criteria.
17ß-Estradiol and estrone activated NR1, whereas estriol,
estetrol, estradiol sulfate, and the synthetic estrogen
diethylstilbestrol did not. On the other hand, progesterone and
androgens repressed NR1 activity in HepG2 cells, and the repressed NR1
activity was fully restored by estradiol. Moreover, estrogen treatment
elicited nuclear accumulation of CAR in the mouse livers, as well as
primary hepatocytes, and induced the endogenous Cyp2b10
gene. Ovariectomy did not affect either the basal or induced level of
CAR in the nucleus of the female livers, while castration slightly
increased the basal and greatly increased the induced levels in the
liver nucleus of male mice. Thus, endogenous estrogen appears not to
regulate CAR in female mice, whereas endogenous androgen may be the
repressive factor in male mice. Estrogen at pharmacological levels is
an effective activator of CAR in both female and male mice, suggesting
a biological and/or toxicological role of this receptor in estrogen
metabolism. In addition to mouse CAR, estrogens activated rat CAR,
whereas human CAR did not respond well to the estrogens under the
experimental conditions.
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INTRODUCTION
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The nuclear orphan receptor CAR (constitutive active receptor or
constitutive androstane receptor) was originally characterized as a
receptor that activates an empirical set of retinoic acid response
elements without the presence of ligands such as retinoic acid (1, 2).
The first gene identified as a direct target of CAR in vivo
is the hepatic CYP2B in the mouse, rat, and human (3, 4).
Treatment with phenobarbital (PB) translocates cytoplasmic CAR into the
nucleus of liver or primary hepatocytes. Forming a heterodimer with
retinoid X receptor, the CAR binds to and activates NR1 enhancer within
the conserved 51-bp PB response element called PBREM
(phenobarbital-responsive enhancer module) found in the mouse and human
CYP2B genes (3, 4, 5, 6). The corresponding enhancer sequence has
also been defined in the rat CYP2B genes (7, 8, 9). Although
CAR can respond to structurally diverse xenochemicals, its response is
relatively limited to those of so-called PB-type inducers including
pesticides (e.g. methoxychlor and
1,1,1-trichloro-1,2-bis(o.p-chlorophenyl)ethane),
chlorpromazine, polychlorinated biphenyls, and organic solvents
(e.g. acetone and pyridine) (4, 5). Thus, CAR can be
characterized as a xenochemical receptor that is activated in response
to environmental insults. Yet, an endogenous chemical that could
activate CAR remains of major interest to the current
investigations.
16-Androstenes are primarily produced in the testis and are the odorous
compounds secreted into apocrine glands and urine (10). These steroids,
such as androstenol, are known to inhibit a mouse CAR (mCAR)-mediated
transactivation of retinoic acid response element in HepG2 cells (11).
In fact, treatment with androstenol represses the endogenous
CYP2B6 gene in HepG2 cells transfected transiently or
permanently with an mCAR-expression plasmid (4). Moreover, the
repressed gene can be reactivated (i.e. induced) by
treatment with PB and other PB-type inducers. These studies have led us
to envision additional steroid molecules that repress or even activate
the receptor CAR, regulating CYP2B and other potentially
CAR-targeted genes. In fact, the induction of the Cyp2b10
gene by estrogens was previously reported in mouse primary hepatocytes
as well as in mice (12, 13). Liver is the major organ that metabolizes
steroids, and hepatic CYP enzymes are the key metabolizing enzymes.
Thus, we screened various steroids with respect to their ability to
modulate CAR function using the enhancer activity of NR1 and induction
of CYP2B gene as the experimental criteria. Estrogens have
appeared as CAR activators, whereas androgens and progesterone seem to
be CAR repressors. The activation by estrogens suggests that CAR may
play a biological and/or toxicological role in estrogen metabolism.
Species differences in the function of CAR are also investigated.
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RESULTS AND DISCUSSION
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Effects of Various Steroids on NR1 Activity in g2car-3 and HepG2
Cells
The nuclear receptor CAR is spontaneously localized in the nucleus
of the transfected HepG2 cells and is constitutively activated (6),
meaning that the receptor does not require ligand binding for its
activation. We have previously constructed a stable HepG2 cell line
(called g2car-3) transfected permanently with a mCAR expression plasmid
(4). The constitutive expression of mCAR resulted in activation of the
enhancer element NR1 to high levels in g2car-3 cells. Using this
g2car-3 system, various steroids were tested to determine whether they
altered NR1 activity (Fig. 1
). Both
testosterone and androstenedione decreased the NR1 activity to one
third of that observed in control cells. Progesterone abrogated NR1
activity almost completely, while 17
-hydroxyprogesterone did not
affect activity. Pregnenolone and its 17
-hydroxy product,
glucocorticoids, dehydroepiandrosterone (DHEA), and
cholesterol exhibited no effect on NR1 activity in the g2car-3 cells.
Only estradiol and estrone, on the other hand, increased the activity
significantly over control levels. In addition, as shown previously,
the most potent PB-type inducer 1,4-bis-[2-(3,
5-dichloropyridyloxy)]benzene (TCPOBOP) also activated NR1. It appears
that estrogens activate mCAR, whereas androgens and progesterone
repress the receptor.

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Figure 1. Effects of Steroid Hormones on NR1 Activity in
g2car-3 Cells
(NR1)5-tk-luciferase plasmid was cotransfected with
pRL-SV40 into g2car-3 cells. The transfected cells were incubated with
various steroids (10 µM) or TCPOBOP (250 nM),
harvested, and assayed for luciferase activity. Relative activity
levels are expressed by taking the control values obtained from the
nontreated cells as 100%.
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This activation and repression, in fact, depended on the presence of
the receptor CAR in the cells. While treatment with various steroids
modulated the NR-1 activity in the cotransfected HepG2 cells in the
ways reminiscent of those in the g2car-3 cells, steroids exhibited no
effect on the activity in normal HepG2 cells (Fig. 2
). Since CAR is already active in the
cells unexposed to steroids, the in vitro transfection
assays may have accurately determined the levels of repression by
progesterone or androgens. However, for the same reason, the activation
capability of estrogens may have been underestimated.

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Figure 2. Transient Transfection Assay of Steroid-Dependent
NR1 Activity in HepG2 Cells
(NR1)5-tk-luciferase plasmid was cotransfected with (A) or
without (B) an mCAR expression plasmid in HepG2 cells. All other
experimental conditions were the same as described in the legend for
Fig. 1 . The steroid-dependent NR1 activity is first calculated and
expressed by taking that value from the nontreated cells transfected
with the expression plasmid as 100%.
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NR1 Activation by Estrogens
In light of the finding that estrogens were possibly mCAR
activators, we examined the activation by estrogen in further detail.
Recently, the Ca2+/calmodulin-dependent kinase (CaMK) inhibitor KN-62
has been shown to repress mCAR-mediated activation of NR1 in HepG2 as
well as g2car-3 cells and treatment with TCPOBOP reactivated
(induced) the repressed NR1 (our unpublished observation).
Using the repressive activity to fullest advantage, g2car-3
cells were first incubated with KN-62, and then treated with estrogens
or other steroids to examine the activation of CAR activity (Fig. 3
). Estradiol and estrone effectively
reactivated NR1 activity 15- to 20-fold, as observed with TCPOBOP. No
other steroids were capable of reactivating NR1 in KN-62-treated
g2car-3 cells. The activation by estrogens of NR1 occurred in a
dose-dependent fashion (Fig. 4
). Both
estradiol and estrone fully restored the KN-62-repressed NR1 activity
at concentrations of 310 µM. On the other
hand, estriol and estetrol were totally ineffective in reactivating NR1
in the KN-62-treated g2car-3 cells. Unexpectedly, the hormonally
inactive estradiol sulfate slightly increased NR1 activity only at a
high concentration (10 µM), although this
increase could have been due to the desulfation in the cells. In
contrast, the potent synthetic estrogen diethylstilbestrol (DES)
displayed absolutely no effect on NR1 activity in the KN-62-treated
g2car-3 cells. Thus, only endogenous estrogens have appeared to be mCAR
activators.

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Figure 3. Activation of NR1 by Estrogens in KN-62-Treated
g2-car3 Cells
Cells cotransfected with (NR1)5-tk-luciferase and
pRL-SV40 plasmids were incubated with KN-62 (10 µM) for
1 h and subsequently treated with various steroids (10
µM) or TCPOBOP (250 nM) for 24 h. These
cells were harvested and assayed for luciferase activity. Relative
activity levels are expressed as induction fold by taking the control
values in the KN-treated cells as 1.
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Figure 4. Dose-Dependent Activation of NR1 by Estrogen in
KN-62-Treated g2car-3 Cells
Estrone (E1), estradiol (E2), estriol (E3), estetrol (E4), estradiol
sulfate (E2SO3), or DES was added to the KN-62-treated and
(NR1)5-tk-luciferase plasmid-transfected g2car-3 cells at
the indicated concentrations. The fold activation was calculated using
the activity in g2car-3 cells treated with KN-62 alone as 1.
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Species Differences in CAR to Respond to Estrogens
Having established the activation by estrogens of mCAR in g2car-3
cells, we investigated whether estrogens were also able to activate
CARs from other species. For this, HepG2 cells were transiently
transfected with an expression vector containing mouse, rat, or human
CAR (rCAR, hCAR) (Fig. 5
). As observed in
the g2car-3 cells, mCAR enhanced NR1 activity that was repressed by
KN-62 and reactivated by either estradiol or estrone in the HepG2
cells. Similar to what happened with mCAR, rCAR effectively activated
the NR1 activity in the transfected HepG2 cells. The rCAR-mediated NR1
activity was repressed by KN-62 treatment and was reactivated after
treatment with estrogens. Human hCAR enhanced NR1 activity only 8-fold
compared with the 25-fold activation by mCAR or rCAR (Fig. 5
). This
hCAR-mediated activity, however, was insensitive to KN-62 inhibition
and did not respond to estrogens. Androstenol also did not repress NR1
activity in hCAR-transfected HepG2 cells (our unpublished
observation). Function of a given nuclear receptor can be activated or
repressed differently depending on the type of target enhancer
sequences and chemicals. hCAR was capable of activating NR1 in HepG2
cells, but KN-62 failed to repress the hCAR activity. As a result, hCAR
appeared not to be activated by estrogens under the present
experimental conditions. Thus, these results do not mean that hCAR can
not be activated by estrogens under any conditions, and findings of
estrogen-dependent regulation of hCAR remains of interest for future
investigations. An alternative is to search for a second hCAR
resembling its activity with the rodent counterparts if it
exists. Expression of pregnane X receptor (PXR) did not modulate
NR1 in the transfected HepG2 cells at all.

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Figure 5. Species Differences in CAR to Response to Estrogens
Each of the mouse, rat, and human CAR expression plasmids or the human
PXR expression vector was co-transfected with
(NR1)5-tk-luciferase and pRL-SV40 plasmids into HepG2
cells. The transfected cells were incubated for 24 h with KN-62
(10 µM), estrone (10 µM), and estradiol (10
µM) in the combinations indicated, harvested, and
subjected to luciferase assay.
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Repression of NR1
Since the initial screening of various steroids suggested
that progesterone and androgens repressed NR1 activity (Figs. 1
and 2
),
we examined the dose-dependent repression in g2car-3 cells.
Progesterone completely repressed NR1 activity at 10 µM,
which was reminiscent of the repression observed with androstenol (Fig. 6
). Androgens (testosterone and
androstenedione) also repressed NR1 activity in a dose-dependent
fashion, although they decreased the activity only to 40% of control
levels at 10 µM. The best-known PXR activator
5ß-pregnane-3,20-dione was not effective in modulating NR1 activity.
Next, NR1 activity was first repressed by progesterone (10
µM) and then challenged by estrogens to see whether the
steroid hormone could restore it (Fig. 7
). Treatments with estradiol and estrone
reactivated completely the repressed NR1 activity to the control levels
at 1 µM and increased the activity another 2-fold over
the control at 10 µM. Thus, these results clearly showed
that progesterone and androgens antagonize the estrogen activation of
the receptor mCAR.

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Figure 6. Dose-Dependent Repression of NR1 Activity by
Progesterone and Androgens in g2car-3 Cells
g2car-3 cells transfected with (NR1)5-tk-luciferase and
pRL-SV40 plasmids were treated with different steroids at various
concentrations for 24 h. These cells were harvested and subjected
to luciferase assay. The NR1 activity is indicated as a percent of the
corresponding activity in transfected g2car-3 cells without the
steroids.
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Figure 7. Activation by Estrogens of the
Progesterone-Repressed NR1 Activity
The g2car-3 cells transfected with (NR1)5-tk-luciferase and
pRL-SV40 plasmids were treated with progesterone (10 µM)
for 1 h, followed by incubation with or without estradiol or
estrone for 24 h. Then, the cells were harvested and subjected to
luciferase assay. The Arabic numbers in parentheses
indicate the steroid concentrations in µM. The levels of NR1 activity
are expressed as percent by taking the activity in the nontreated
g2car-3 cells (without progesterone or estrogens) as 100.
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Estrogen Induction in Primary Hepatocytes
The activation by estrogens of NR1 in g2car-3 and HepG2 cells
suggested that these steroids may induce the Cyp2b10 gene.
To examine this induction, mouse primary hepatocytes were prepared and
treated with estrogens or other steroid hormones. Then, hepatic RNAs
were isolated and subjected to quantitative PCR analysis (Fig. 8A
). Estradiol and estrone consistently
induced CYP2B10 mRNA a maximum of 3-fold, although the induction by the
estrogens was less effective compared with the 9-fold induction by
TCPOBOP. Consistent with the induction of CYP2B10 mRNA, NR1 activity
was also enhanced by treatment with estradiol approximately 4-fold
(Fig. 8
, A and B). On the other hand, the action of androgens or
progesterone was found to be repressive in the primary hepatocytes,
except that progesterone was much less effective in decreasing the NR1
activity (Fig. 8B
). Thus, estrogen has appeared to be a natural inducer
of the Cyp2b10 gene in mouse primary hepatocytes.

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Figure 8. Effect of Estrogens in Mouse Primary Hepatocytes
A, Cells were treated with 10 µM of indicated steroid or
50 nM TCPOBOP for 9 h. Total cellular RNAs were
prepared from the treated cells and subjected to quantitative real time
RT-PCR of CYP2B10 mRNA. The levels of CYP2B10 mRNA were normalized by
the ß-actin mRNA levels and are expressed as fold induction taking
the control value as 1. B, For NR1 activity,
(NR1)5-tk-luciferase plasmid was cotransfected with
pRL-SV40 into primary hepatocytes. The transfected cells were incubated
with 10 µM of the indicated steroids or 50 nM
of TCPOBOP for 24 h, harvested, and assayed for luciferase
activity. Activity levels are expressed as fold induction by taking the
control value obtained from the nontreated hepatocytes as 1.
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In contrast to the nuclear localization of CAR in the transfected HepG2
cells, the receptor is present in the cytoplasm and translocates to the
nucleus after induction by a PB-type inducer in mouse liver as well as
primary hepatocytes (6). Thus, the nuclear translocation is the first
step occurring to CAR during the induction. Knowing that estrogens
regulated the mCAR-mediated transactivation of NR1 in mouse primary
hepatocytes, we performed Western blot analysis to determine whether
steroids also elicited nuclear translocation of mCAR. The nuclear
extracts were prepared from mouse primary hepatocytes treated with
various steroids at 1 µM and were subjected to Western
blot analysis using anti-CAR antibody (Fig. 9A
). Treatment with estradiol resulted in
the nuclear accumulation of CAR, as did TCPOBOP. The dose-dependent
experiments demonstrated that estradiol was able to elicit the nuclear
translocation of mCAR at 1 µM (Fig. 9B
). In addition to
estradiol, estrone translocated mCAR into the hepatic nucleus, whereas
estriol and DES did not (Fig. 9C
). All other steroids (testosterone,
progesterone, pregnenolone, and corticosterone) were incapable of
translocating CAR into the nucleus, except that testosterone treatment
might have accumulated a subtle amount of CAR in the nucleus.
Nevertheless, our present results clearly show that estrogen elicits
the nuclear translocation of CAR, activates the NR1, and induces the
Cyp2b10 gene in mouse primary hepatocytes.

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Figure 9. Nuclear Accumulation of CAR in Estrogen-Treated
Hepatocytes
A, Mouse primary hepatocytes were treated with 1 µM of
the indicated steroids or 50 nM of TCPOBOP for 1 h.
Nuclear extracts were prepared from these cells and subjected to
Western blot analysis using anti-CAR antibody. Prestained Protein
Marker Broad Range (New England Biolabs, Inc., Boston, MA)
was used as the molecular marker. B, Dose dependency of the
estradiol-elicited nuclear translocation. Mouse primary hepatocytes
were treated with 1, 3, or 10 µM of estradiol or 50
nM of TCPOBPOP for 1 h, and then nuclear extracts were
prepared from the treated cells and subjected to Western blot analysis
using anti-CAR-antibody. C, Association of the nuclear accumulation
with natural estrogenic steroids. Primary hepatocytes were treated with
10 µM of the indicated steroids, DES, or TCPOBOP (50
nM) for 1 h. The nuclear extracts from these cells
were used for Western blot analysis using anti-CAR antibody.
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Estrogen Induction in Mice
Basal levels of hepatic nuclear CAR were extremely low in
unexposed male mice (6), although the receptor levels were not
investigated in female mice at that time. To examine an initial event
of estrogen action on the receptor, we performed Western blot analysis
on the nuclear extracts prepared from female as well as male mice. The
basal nuclear level of CAR was much higher in the control females
compared with that in the control males. When female mice were treated
with various doses of estradiol, CAR began to accumulate in the nucleus
at the dose of 0.1 mg E2/kg of body weight. The
male mice, on the other hand, exhibited an increase of the receptor at
the higher dose, although the levels were significantly lower than that
in the female, even at 1.0 mg/kg body weight (Fig. 10A
). Presuming that endogenous levels
of sex hormones may have affected the basal and inducible accumulation
of CAR in the nucleus, we then examined the receptor in castrated and
ovariectomized male and female mice, respectively (Fig. 10B
). The
levels of CAR in the sham-operated mice were essentially correlated
with those in the control mice. In contrast to what happened in the
sham-operated males, treatment with estradiol for 3 h dramatically
increased CAR in the liver nucleus of the castrated males to the same
level observed in the estrogen-treated females. These results suggest
that estrogen at endogenous concentration may not regulate CAR in the
female mice, although the subtle decrease of the basal level of nuclear
CAR in the ovariectomized females remains an interesting question for
future investigation. On the other hand, estrogen at pharmacological
levels induces the nuclear accumulation of CAR in both female and male
mice. Androgen at physiological concentration appeared to repress the
estrogen-induced nuclear accumulation of CAR in male mice. This
antagonistic nature of androgen in the liver may not be consistent with
the subtle increase of CAR in the testosterone-treated primary
hepatocytes shown in Fig. 9
. Thus, it remains to be established in
future investigations whether the repression may or may not be a direct
action of androgen to the liver of mice in vivo.

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Figure 10. Nuclear Accumulation of CAR in Estrogen-Treated
Mice
Each group of four mice was treated by ip injection of E2
at the doses indicated. Western blot was performed on the liver nuclear
extracts as described in Materials and Methods. A, For
dose-dependent experiments, the mice were treated with 0.1, 0.5, or 1.0
mg/kg body weight of E2 or DMSO (100 µl) for 3 h. B,
To examine the estrogen effect in the castrated males (Cast) and the
ovariectomized females (Ovex), these and sham-operated mice (Sham) were
treated with 1.0 mg/kg body weight of E2 or 100 µl of
DMSO (C) for 3 h.
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General Discussion
CAR now appears to be a nuclear receptor that can be activated or
repressed in response to endogenous steroid hormones, at least in the
mouse and rat. Estrogen is the first endogenous chemical found to
activate CAR in HepG2. Estrogen also elicits the nuclear accumulation
of the receptor in mouse primary hepatocytes and mice, leading to the
induction of the Cyp2b10 gene. Since estrogen and androgen
are metabolized by CYP2B enzyme, its induction would result in
decreasing precursors and increasing estrogen metabolism, resulting in
lowering estrogen level. Thus, the estrogen responsiveness of CAR in
activating the CYP2B gene implies that this receptor may be
involved in the regulation of active estrogens. Consistently, the
nonestrogenic metabolites (estriol, estetrol, and estradiol sulfate)
did not activate CAR. Progesterone and androgens repress the
estrogen-activated CAR, suggesting that these steroid hormones may act
as antagonists to counterbalance the decreased levels of estrogen.
It is logical to find that glucocorticoids do not affect the function
of CAR, since these hormones are not directly in the metabolic pathways
leading to estrogens. However, it is surprising that the
intermediate steroid metabolites, pregnenolone,
17
-hydroxypregnenolone, 17
-hydroxyprogesterone, and
DHEA exert neither activation nor repression of CAR. PXR
and CAR belong to the same nuclear receptor subfamily 1I (15), and
exhibit some degree of overlapping properties. In the case of PXR,
however, the intermediate metabolites 5ß-pregnane-3,20-dione and
17
-hydroxyprogesterone are far better activators compared with their
parent steroids (16, 17). PXR activates the CYP3A genes and
CYP3A enzymes also metabolize estrogens and glucocorticoids. As yet,
the activation of PXR by these steroid hormones was found to be weak
(16, 17). It appears that the steroid hormones may regulate CAR,
whereas the intermediate steroid metabolites are the modulators of PXR
function. Although the biological implication in these differences
remains elusive, the distinct nature of CAR leads us to think that CAR
may, in fact, be involved in estrogen metabolism.
The basal levels of nuclear CAR in the castrated or ovariectomized mice
have suggested that the physiological concentration of estrogen may be
a CAR activator, whereas that of androgen can be a CAR repressor.
Presuming that CAR may play a role in steroid metabolism, the efficacy
of exogenous estrogen (a low µM) to activate CAR does not appear to
be physiological in mice. One possibility is that CAR plays a role in a
defense mechanism against possible adversity caused by estrogen at high
levels. Alternatively, the receptor may be present in a developmental
period of the organs such as ovary, uterus, testis, brain, and placenta
in which estrogen level may become high enough to activate or repress
CAR. Yet another possibility is that the apparent activation by
estrogen of CAR is an indirect reflection of the more sensitive
hormonal effect. It is not likely, however, that the estrogen
activation of CAR is mediated by the estrogen receptor, since the
potent synthetic estrogen DES did not activate CAR at all. A previous
report has already shown that, unlike natural estrogen, DES does not
induce the Cyp2b10 gene in mouse primary hepatocytes (12).
The receptor CAR seems to regulate not only the CYP2B but
also many other genes. Those genes include human bilirubin
UDP-glucuronosyl transferase (UGT1A1) (18), acetyl-CoA oxidase, and
enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (19), and genes
potentially regulated by retinoic acid response element (1).
Differential expression in g2car-3 over HepG2 cells has already
suggested that many other genes can be under the control of CAR
(our unpublished data). Thus, there appear to be ample places in
which CAR may play roles in the regulation of genes in response to
steroid hormones. Our present findings may provide some insights for
future investigations of these roles.
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MATERIALS AND METHODS
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Animals
Cr1:CD-1(ICR)BR mice were castrated or sham operated at
the age of 6 weeks and housed for another 4 weeks before experiments.
Each group of mice was treated by intraperitoneal injection of
17ß-estradiol [in 100 µl of dimethylsulfoxide (DMSO)] at
various doses for 3 h. Then, the livers from all of the mice were
pooled and the liver nuclear extracts were prepared for Western blot
analysis as described previously (3).
Materials
Androstenol and estetrol were purchased from Steraloids
(Newport, RI), whereas all other steroids were obtained from
Sigma (St. Louis, MO). DES was kindly provided by Drs. Kun
Chae and Lisa Newbold. Drs. David Moore and Steve Kliewer kindly
provided the human CAR- and PXR-expression plasmids, respectively. For
the rat CAR expression vector, the EcoRI fragment that
encodes the entire coding sequence was generated from the original rat
CAR cDNA (our unpublished data) and cloned at the EcoRI site
of pCR3 plasmid. All other cell lines and recombinant plasmids were
previously produced (4, 6).
Cells and Transfection Assay
HepG2 and g2car-3 cells were cultured in MEM supplemented with
10% FBS. (NR1)5-tk-luciferase plasmid (0.1 µg)
was cotransfected with pRL-SV40 (0.1 µg) into g2car-3 cells (17-mm
well) using a calcium phosphate coprecipitation method, or it was
cotransfected with pRL-SV40 (0.1 µg) as well as CAR- or PXR
expression plasmid (0.2 µg) into HepG2 cells. Mouse primary
hepatocytes were prepared from males of 2-month-old Cr1:CD-1(ICR)BR by
a two-step collagenase perfusion method and cultured as previously
described (21). Electroporation was employed to cotransfect
(NR1)5-tk-luciferase plasmid (15 µg) with
pRL-SV40 (5 µg) into mouse primary hepatocytes. These cells were
treated with various chemicals, and luciferase activity was measured
using the Dual-Luciferase reporter assay system (Promega Corp., Madison, WI).
RT-PCR
To quantify CYP2B10 mRNA, cDNA prepared from total cellular RNA
of mouse primary hepatocytes was subjected to quantitative real time
PCR using ABI Prism 7700 (PE Applied Biosystems, Foster
City, CA). CYP2B10 cDNA was amplified using 5'-AAAGTCCCGTGGCAACTTCC-3'
and 5'-TCCCAGGTGCACTGTGAACA-3' for 5'- and 3'-primers, respectively.
Amplified cDNA was measured using 6FAM-ACCCCGTCCCCTGCCCCTCTT-TAMRA as a
CYP2B10 probe. For an internal control, ß-actin mRNA level was also
measured with a VIC-TAGCCATCCAGGCTGTGCTGT-TAMRA probe using
5'-TTCAACACCCCAGCCATGTA-3' and 5'-TGTGGTACGACCAGAGGCATAC-3' as 5'- and
3'-primers, respectively.
Western Blots
Nuclear extracts were prepared from mouse primary hepatocytes,
resolved on a SDS-10% polyacrylamide gel, transferred to a
polyvinylidene difluoride membrane, and incubated with anti-hCAR
antibody. After being incubated with an antirabbit IgG-horseradish
peroxidase conjugate, the polypeptide band on the membrane was
visualized with an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Arlington Heights, IL).
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FOOTNOTES
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Address requests for reprints to: Dr. Masahiko Negishi, Pharmacogenetics Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institute of Health, Research Triangle Park, North Carolina 27709. E-mail: negishi{at}niehs.nih.gov
Received for publication March 27, 2000.
Revision received July 27, 2000.
Accepted for publication August 4, 2000.
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