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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go). 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{alpha}-hydroxyprogesterone did not affect activity. Pregnenolone and its 17{alpha}-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%.

 
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. 2Go). 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. 1Go. 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%.

 
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. 3Go). 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. 4Go). Both estradiol and estrone fully restored the KN-62-repressed NR1 activity at concentrations of 3–10 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.

 
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. 5Go). 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. 5Go). 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.

 
Repression of NR1
Since the initial screening of various steroids suggested that progesterone and androgens repressed NR1 activity (Figs. 1Go and 2Go), 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. 6Go). 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. 7Go). 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.

 
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. 8AGo). 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. 8Go, 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. 8BGo). 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.

 
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. 9AGo). 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. 9BGo). In addition to estradiol, estrone translocated mCAR into the hepatic nucleus, whereas estriol and DES did not (Fig. 9CGo). 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.

 
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. 10AGo). 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. 10BGo). 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. 9Go. 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.

 
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{alpha}-hydroxypregnenolone, 17{alpha}-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{alpha}-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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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).


    FOOTNOTES
 
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.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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