The expression of pregnane X receptor and its target gene, cytochrome P450 3A1, in perinatal mouse

Molecular and Cellular Endocrinology 172 (2001) 47 – 56 www.elsevier.com/locate/mce

The expression of pregnane X receptor and its target gene, cytochrome P450 3A1, in perinatal mouse H. Masuyama *, Y. Hiramatsu, Y. Mizutani, H. Inoshita, T. Kudo Department of Obstetrics and Gynecology, Okayama Uni6ersity Medical School, 2 -5 -1, Shikata, Okayama 700 -8558, Japan Received 12 May 2000; accepted 29 August 2000

Abstract Recently, pregnane X receptor (PXR) has been described to mediate the genomic effects of several steroid hormones, such as progesterone (P), glucocorticoid (Dex), pregnenolone (Preg), and xenobiotics through the cytochrome P-450 3A gene family (CYP3A), which are monooxygenases, responsible for the oxidative metabolism of some endogenous substrates and xenobiotics. In the present study, we used a transient transfection reporter gene expression assay of COS-7 cells to demonstrate that P, Dex and Preg significantly stimulate PXR-mediated transcription at relatively high concentration comparable with that of progesterone near term pregnancy. In yeast two-hybrid protein interaction assay, PXR interacted with nuclear receptor coactivator proteins, SRC1, RIP140, and SUG1 in a ligand-dependent manner. The expression of PXR mRNA was observed in the liver, intestine, uterus, ovary and placenta. The expressions of PXR mRNA in the liver and ovary increased towards term about fifty-fold compared with that of non-pregnancy and decreased postpartum. Its expression in the placenta was not drastically changed towards term. CYP3A, a target gene of PXR, was also expressed in the liver, ovary, and placenta. The expressions of CYP3A mRNA as well as PXR in the liver and ovary increased about 20-fold during prenatal period. These data suggest that PXR may play certain roles in perinatal period, possibly in the protection of the feto-maternal system from the toxic effect of endogenous steroids and foreign substrates. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Pregnane X receptor; Steroidogenesis; Cytochrome P450; Fetus; Placenta

1. Introduction The endocrine alterations that accompany pregnancy are among the most remarkable in mammalian physiology or pathophysiology (Casey et al., 1992; Liu and Rebar, 1999). Estrogen and progestins play several important roles in pregnancy. Progestins appear to be important in implantation, maintaining uterine quiescence and delaying cervical ripening by the inhibition of prostaglandin production, and in the protection of tissue rejection mediated by T lymphocyte (Casey et al., 1992; Liu and Rebar, 1999). Estrogens are also important for parturition as well as implantation. The amounts of hormone produced by the ovary and placenta during pregnancy is about 100 or greater than the concentration of the hormone in non-pregnant women * Corresponding author. Tel.: +81-86-2357320; fax: + 81-862259570. E-mail address: [email protected] (H. Masuyama).

(Casey et al., 1992; Liu and Rebar, 1999). Thus, a remarkable aspect of pregnancy is the establishment of mechanisms, whereby the gravid woman and her fetus are able to adapt to this unusual endocrine milieu. However, the regulatory mechanism for hypersteroidemia in feto-maternal system has not been investigated in detail. Steroid hormones, including estrogen and progesterone, and non-steroid hormones, vitamin D, retinoids, thyroid hormone, and prostanoids regulate their specific genes through the binding to their specific receptors, comprising the nuclear receptor superfamily. These receptors form homodimer or heterodimer with retinoid X receptor (RXR) and directly associate with specific DNA sequences, known as hormone responsive elements (HRE), at the upstream region of specific genes (Evans, 1988; Tsai and O’Malley, 1994; Mangelsdorf and Evans, 1995). The DNA-receptor complex interacts with basal transcriptional machinery and nuclear receptor coactivator proteins, resulting in the lig-

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and-dependent induction of transcription (Mangelsdorf and Evans, 1995; Horwitz et al., 1996; Masuyama et al., 1997a). Recently, pregnane X receptor (PXR), a new member of the nuclear receptor superfamily, has been shown to mediate the genomic effects of several steroid hormones such as progesterone (P), pregnenolone(Preg), glucocorticoid, synthetic glucocorticoids anti-glucocorticoids, and xenobiotics in the mouse, rat and human (Bertilsson et al., 1998; Blumberg et al., 1998; Kliewer et al., 1998; Lehmann et al., 1998; Schuetz et al., 1998; Pascussi et al., 1999; Zhang et al., 1999). Like non-steroid hormone receptor, it binds as a heterodimer with RXR to specific DNA sequences including the upstream of the cytochrome P-450 3A (CYP3A) gene family (Bertilsson et al., 1998; Kliewer et al., 1998; Lehmann et al., 1998; Pascussi et al., 1999), which are monooxygenases responsible for the oxidative metabolism of certain endogenous substrates and xenobiotics (Nebert and Gonzalez, 1987; Juchau, 1990). In our experiments, we analyzed the tissue distribution of PXR mRNA in the mouse using reverse transcriptase-polymerase chain reaction (RT-PCR). The PXR mRNA was also detected in the ovary and uterus as well as liver and intestine, as described earlier by Kliewer et al. (Kliewer et al., 1998). Since PXR was expressed in reproductive organs including ovary and uterus, we performed several experiments to examine the role of PXR in the perinatal period, which is an unusual hypersteroidemia. First, we examined which steroid hormones at what concentrations affected the PXR-mediated transcription through the CYP3A motif in the transient reporter assay. We also checked whether PXR interacted with nuclear receptor coactivators, steroid hormone receptor coactivator-1 (SRC1) (Onate et al., 1995), receptor interacting protein 140 (RIP140) (Cavailles et al., 1995), and suppressor for gal 1 (SUG1) (vom Bauer et al., 1996; Rubin et al., 1996) in the presence of certain steroids, which enhanced PXR-mediated transcription. Then, the expression of PXR and its target gene, CYP3A1 mRNA in the liver, ovary and placenta were analyzed during the perinatal period using semi-competitive RT-PCR. The results of these experiments show some evidences that PXR may regulate its specific gene expression via a similar mechanism to that of other nuclear receptors and play particular roles in perinatal period, possibly in the protection of the feto-maternal system from the toxic effects of endogenous steroids and foreign substrates.

2. Materials and methods

2.1. Materials 5-Pregneno-3b-ol-20-one

(pregnenolone),

proges-

terone (P), and estradiol (E2) were purchased from Sigma Chemical Co., Ltd. (St. Louis, MO). 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) was kindly provided by Dr M.R. Uskokovic. BALB/cA mice, bred at our medical school, were used in these studies. Food and water were available ad libitum. Gestational age was calculated based on the estimated time of insemination with the day of mating being considered as gestation day 0. All the materials for RT-PCR were purchased from TAKARA Co., Ltd. (Kyoto, Japan).

2.2. Tissue and blood collection and hormone measurement The animals were killed under ether anesthesia and the target tissues were removed, immediately frozen and stored at − 70°C. The frozen tissue was homogenized in a Polytron homogenizer and total RNA was extracted using the guanidine idiothiocyanate method (Trizol; GibcoBRL, Grand Island, NY) according to the manufacturer’s instructions. Maternal blood was also collected from these examined mice and the levels of serum progesterone and estradiol were measured using ELISA assay (Immuno Biological Laboratories, Hamburg, Germany).

2.3. Transient transfection studies COS-7 cells were cultured in D-MEM medium without phenol red supplemented with 10% charcoal-striped calf serum. The (CYP3A1)2-tk-CAT containing two copies of the CYP3A1 motif, which is a direct repeat of the non-steroid nuclear receptor half-site sequence AGTTCA separated by a three-nucleotide spacer (Quattrochi et al., 1995; Huss et al., 1996), and pSG5PXR expression plasmid containing full-length mouse PXR cDNA were obtained from S.A. Kliewer (Kliewer et al., 1998). COS-7 cells were cotransfected with 1 mg of reporter gene construct (CYP3A1)2-tk-CAT) and 0.5 mg of receptor expression vector (pSG5-PXR) or empty vector (pSG5). In all transfections, liposome-mediated transfections were accomplished with lipofectamine (Life Technologies Inc., Gaithersburg, MD) according to the manufacturer’s protocol. Transfected cells were treated for 36 h with either vehicle alone or the indicated concentrations of steroid hormones. Cell extracts were prepared and assayed for CAT activity. The amount of CAT was determined using a CAT ELISA kit (5%Prime-3%Prime, Inc., Boulder, CO) according to the manufacturer’s protocol. In this transient expression assay, western analysis using these cell extracts revealed equivalent levels of PXR in COS-7 cells transfected with pSG5-PXR (data not shown).

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2.4. Preparation of two-hybrid expression 6ectors and b-galactosidase assays All two-hybrid plasmid constructs used the pAS1 (Durfee et al., 1993) and pAD-GAL4 (Stratagene, La Jolla, CA) yeast expression vectors. The pAD-GAL4SUG1, -SRC1,and -RIP140 and pAS1-PXR were described earlier (Masuyama et al., 1997b, 2000). The pAS1-PXR, or empty vector (pAS1) was cotransformed with pAD-GAL4-SUG1, -SRC1, or -RIP140 into the yeast strain Hf7c, which was made competent with lithium acetate, in order to examine the interaction with coactivator proteins in the two-hybrid assay. Transformants were plated on media lacking leucine and tryptophan (SC-leu-trp) and were grown for 4 days at 30°C to select for yeast that had acquired both plasmids. Triplicate independent colonies from each plate were grown overnight in 2 ml of SC-leu-trp with or without the indicated concentrations of steroids. The cells were harvested and assayed for b-galactosidase activity as described earlier (Fagan et al., 1994).

2.5. Re6erse transcription-polymerase chain reaction (RT-PCR) Each sample was treated with DNase I to remove genomic DNA contamination. To confirm the absence of genomic DNA in the RNA samples, PCR was performed directly on each RNA sample using primers for PXR, CYP3A1, b-actin and cyclophilin with no PCR products detected under this condition. According to the protocol of the RNA PCR kit, 0.1 mg of total RNA was reverse transcribed at 42°C for 20 min in 20 ml of reaction solution containing 1× PCR buffer, 5 mM MgCl2, 1 mM dNTPs, 2.5 mM random 9 mers primer, 10 U RNase inhibitor and 5 U AMV reverse transcriptase. Amplification for mPXR and b-actin was carried out as described earlier (Masuyama et al., 2000). The primer for cyclophilin was as follows: sense: 5%-GCACAGGAGGAAAGAGCATC-3%, antisense: 5%TGACATCCTTCAGTGGCTTG-3% and the primer for CYP3A1 was as follows: sense: 5%-CATGCGGAGGCTACAGGTAT-3%, antisense: 5%-AGGAAGGGAAAAGCCCTTG-3%. Each PCR sample contained 1xPCR buffer, 2.5 mM MgCl2, 0.1 mM primers, and 2.5U TAKARA LA Taq. Amplification for PXR and b-actin was carried out on a TAKARA PCR thermocycler with initial denaturation at 94°C for 5 min, followed by 30 cycles of 94, 60 and 72°C, each for 1 min, and a final extension at 72°C for 15 min. Amplification for CYP3A and cyclophilin was carried out on a TAKARA PCR thermocycler with initial denaturation at 94°C for 2 min, followed by 24 cycles of 94°C, 56 and 72°C, each for 30 s, and a final extension at 72°C for 2 min. The number of PCR cycles resulting in PCR products in the linear logarithmic phase of the amplification curve was

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determined. PCR samples were electrophoresed on 3% Nu-Sieve agarose gel and visualized by ethidium bromide. The amount of each electrophoretically-separated cDNA was quantitated densitometrically using an Image Scanner GT-9500 (Epson, Suwa, Japan) and Bio Image BQ 2.0 software (Bio Image, Ann Arbor, MI).

3. Results

3.1. The expression of mouse PXR mRNA in non-pregnant mice The tissue distribution of mouse PXR in non-pregnant mice was examined by RT-PCR. PXR mRNA was expressed abundantly in the ovary and, to a lesser extent, the uterus of non-pregnant mice as well as the liver and intestine as described by Kliewer et al (Kliewer et al., 1998) using northern blotting (Fig. 1). This distribution of mouse PXR in the ovary and placenta differs from that of human PXR (Lehmann et al., 1998). Such differences may result from the detection method for PXR mRNA, i.e. RT-PCR and northern blotting because RT-PCR is generally more sensitive to mRNA expression than northern analysis. However, it may be due to the interspecies difference because mouse and human PXR have been demonstrated to display some differences in their activation profile (Blumberg et al., 1998; Lehmann et al., 1998).

3.2. Steroid hormones stimulate PXR-mediated transcription at the normal concentration near term Transient reporter expression assay was analyzed in COS-7 cells to examine which steroid hormones enhanced PXR-mediated transcription. Preg, P, and Dex enhanced PXR-mediated transcription (Fig. 2A). In contrast, E2 and 1,25(OH)2D3 had no effects on transcription. These effects were dependent on the ligand

Fig. 1. The expression of PXR mRNA in non-pregnant mouse. The total RNA was isolated from several tissues of non-pregnant mouse and analyzed for the expression of PXR mRNA using RT-PCR. The PCR products were separated on 3% Nu-Sieve agarose gels and visualized by ethidium bromide.

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Fig. 2. Steroid hormones enhanced PXR-mediated transcription. (A) COS-7 cells were transfected with 1 mg of (CYP3A1)2-tk-CAT reporter gene construct together with 0.5 mg of the PXR expression plasmid or empty vector (pSG5). The cells were treated with 10 − 6 M of progesterone, dexamethasone, pregnenolone, estradiol or 1,25-dihydroxyvitamin D3, or ethanol vehicle for 36 h. CAT activity was quantified using ELISA kit. The results represent the mean9S.D. of triplicate determinations. (B) COS-7 cells were transfected with 1 mg of (CYP3A1)2-tk-CAT reporter gene construct together with 0.5 mg of the PXR expression plasmid. The cells were treated with increasing concentrations of progesterone, dexamethasone, pregnenolone, or estradiol for 36 h. CAT activity was quantified using ELISA kit. The results represent the mean 9 S.D. of triplicate determinations. The student’s t-test was used to determine whether treated values were significantly different from the control with P B0.05 as the limit of significance. (*; P B0.01, **; PB 0.05)

concentration and significantly increased at 10 – 100 nM (Fig. 2B). Serum progesterone and estradiol levels of non-pregnant and pregnant mice were measured using ELISA assay. On day 19, progesterone level of pregnant mouse was about 100 ng/ml, and estradiol concentrations reached 80 pg/ml prior to parturition (Table 1), suggesting that progesterone level near term might activate PXR-mediated transcription in vivo.

3.3. Effect of steroid hormones on the interaction between PXR and coacti6ator proteins The two-hybrid protein interaction assay was used to examine whether PXR interacts with coactivator proteins, which are very important for nuclear receptormediated transcription (Mangelsdorf and Evans, 1995; Horwitz et al., 1996; Masuyama et al., 1997b) in the presence of steroid hormones. PXR interacted with nuclear receptor coactivators, SRC1, RIP140, and SUG1 in the presence of Preg, P, and Dex, which stimulated PXR-mediated transcription. However, E2 and 1,25(OH)2D3, which had no effect on PXR-mediated transcription, did not affect this interaction. (Fig. 3A – C). The effect on the interaction between PXR and SRC1 was dependent on the concentration of the ligands and significantly increased at 10 – 100 nM, which is compatible with the transcriptional activity (Fig. 3D).

3.4. The expression of PXR and CYP3A1 mRNA in perinatal period The house keeping genes, b-actin and cyclophilin were used to determine the relative level of PXR and

CYP3A1 gene transcription and to control for variations in RNA recoveries from each specimen. Normalization of the data was accomplished by quantifying the amount of amplified cDNA products by calculating the ratio of the amount of PXR cDNA relative to the amount of b-actin or cyclophilin cDNA. This ratio was used to compare the relative amounts of PXR and CYP3A1 mRNA in each tissue. PXR mRNA in the liver and ovary gradually increased and reached a peak on the gestation day 19 and then quickly decreased after birth (Fig. 4A and B). PXR mRNA was also expressed in the placenta and showed no remarkable changes during the perinatal period compared with that in the liver and ovary (Fig. 4C). Simultaneously, the expressions of CYP3A1 mRNA in the liver and ovary increased towards term (Fig. 4D and E), but in placenta, it was not drastically changed compared with those in the liver and ovary (Fig. 4F). The expressions of PXR and CYP3A1 mRNA in uterus and intestine could not be detected with sufficient accuracy for quantifications under these conditions. Table 1 Serum progesterone and estradiol level of perinatal mousea

Non-pregnancy Pregnancy

Postpartum a

Day Day Day Day

Mean9 S.D., n =3

12 15 19 3

Progesterone (ng/ml)

Estradiol (pg/ml)

4.1 91.3 73.6 98.4 92.4 97.9 99.4 9 9.6 3.9 90.9

11.0 94.7 37.4 93.4 69.2 99.8 81.0 9 8.9 8.0 93.3

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Fig. 3. The effect of steroids on the interaction between PXR and coactivator proteins. (A) Yeast expressing the pAS1-PXR or empty vector (pAS1) and pAD-SRC-1 two hybrid plasmids were grown for 24 h at 30°C in the presence of 10 − 6 M progesterone, dexamethasone, pregnenolone, estradiol or 1,25-dihydroxyvitamin D3, or ethanol vehicle. PXR-SRC-1 interaction was assessed in a b-galactosidase assay. The results represent the mean 9 S.D. of triplicate independent cultures. (B) Yeast expressing the pAS1-PXR or pAS1 and pAD-RIP140 two hybrid plasmids were grown for 24 h at 30°C in the presence of 10 − 6 M of progesterone, dexamethasone, pregnenolone, estradiol or 1,25-dihydroxyvitamin D3, or ethanol vehicle. PXR-RIP140 interaction was assessed in a b-galactosidase assay. The results represent the mean 9S.D. of triplicate independent cultures. (C) Yeast expressing the pAS1-PXR or pAS1 and pAD-SUG1 two hybrid plasmids were grown for 24 h at 30°C in the presence of 10 − 6 M of progesterone, dexamethasone, pregnenolone, estradiol or 1,25-dihydroxyvitamin D3, or ethanol vehicle. PXR-SUG1 interaction was assessed in a b-galactosidase assay. The results represent the mean 9 S.D. of triplicate independent cultures. (D) Yeast expressing the pAS1-PXR and pAD-SRC-1 two hybrid plasmids were grown for 24 h at 30°C with increasing concentrations of progesterone, dexamethasone, pregnenolone or estradiol. PXR-SRC-1 interaction was assessed in a b-galactosidase assay. The results represent the mean 9 S.D. of triplicate independent cultures. The student’s t-test was used to determine whether treated values were significantly different from the control with P B0.05 as the limit of significance. (*; PB 0.01)

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activity of catabolic p450 enzyme in response to the presence of their substrates (Giguere, 1999). The expression of PXR mRNA has been observed in reproductive organs including the ovary, which is steroid-producing and metabolizing tissue and uterus, which is a target of steroid hormones, as well as in the liver and intestine as shown by Kliewer et al. (Kliewer et al., 1998), suggesting that PXR may play some roles in reproduction. In general, the production of steroids, especially estrogen and progesterone, during pregnancy is much greater than that in non-pregnancy (Pasqualini and Kincl, 1985). We have also shown that the serum

4. Discussion Several ligands for PXR including Dex, P, Preg, and xenobiotics have been demonstrated, and relatively high concentration of these ligands were required for the activation of PXR-mediated transcription (Bertilsson et al., 1998; Blumberg et al., 1998; Kliewer et al., 1998; Lehmann et al., 1998; Schuetz et al., 1998; Pascussi et al., 1999; Zhang et al., 1999; Masuyama et al., 2000). The broader activation profile and low affinities for PXR suggest that PXR could function as a steroid and xenobiotic sensor that directly regulates the

Fig. 4.

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progesterone level of the prenatal mouse rose to about 500 nM, which was 100-fold greater than that in the non-pregnant mouse, while the estradiol level of the prenatal rat was about 500 pM near term. Moreover, the ovary produces large amounts of progesterone and probably has a progesterone concentration exceeding that in the peripheral blood. Such progesterone levels might enhance the PXR-mediated regulation of CYP3A in vivo because transient transfection assay showed that progesterone could significantly activate PXR-mediated transcription at 10– 100 nM. In addition, we demonstrated that the mRNA expression of PXR and its target gene, CYP3A1, in the liver and ovary gradually increased as pregnancy progressed and quickly decreased to the level of non-pregnancy after birth. These expressions in the placenta were not drastically changed

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towards term compared with those in ovary and liver. In the rat, the ovary is thought to be the primary source of steroid hormones, while the placental contribution is of secondary importance during pregnancy, which is completely different from humans, in which the placenta primarily contributes to steroid production during the second and third trimesters (Pasqualini and Kincl, 1985). These data suggest that PXR may play the role of steroid sensor in pregnancy-induced increase in plasma steroid concentrations, and regulate perinatal steroidogenesis through cytochrome p450 enzymes in steroid-producing and -metabolizing tissues. Moreover, since CYP3A is involved in the hydroxylation of steroid hormones as well as various toxic xenobiotics (Nebert and Gonzalez, 1987; Juchau, 1990) and the induction of CYP3A family is believed to confer protection against

Fig. 4. (Continued)

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drugs and toxic xenobiotics by increasing their catabolism (Kourounakis et al., 1977), PXR play some roles for protection of feto-maternal system from the toxic effect of endogenous steroids and foreign substrates during the perinatal period. Coactivator proteins, including SRC-1 (Onate et al., 1995), estrogen receptor-associated protein (ERAP 160) (Halachmi et al., 1994), and RIP140 (Cavailles et al., 1995), interact in a ligand-dependent manner with several members of the nuclear receptor superfamily to enhance ligand-induced transactivation (Mangelsdorf and Evans, 1995; Horwitz et al., 1996; Masuyama et al., 1997b). Here, we demonstrate that PXR interacted with coactivator proteins, SRC1 and RIP140 in a ligand-dependent manner, suggesting that ligands might enhance PXR-mediated transcription through the interaction of PXR with coactivators such as other nuclear receptors (Mangelsdorf and Evans, 1995; Horwitz et al., 1996; Masuyama et al., 1997a,b). The activation function-2 (AF-2) domains of these nuclear receptors are essential for interaction with coactivator proteins and ligand-induced transactivation (Mangelsdorf and Evans, 1995; Horwitz et al., 1996; Masuyama et al., 1997b). The amino acid sequences of PXRs showed that PXRs also have the conserved AF-2 domain (Giguere, 1999), suggesting that PXR might activate transcription through interaction between the AF-2 domain and coactivator proteins. Moreover, we showed that ligand-occupied

PXR interacted with SUG1. SUG1 has been described as a component of proteasome (Rubin et al., 1996), which is an enzyme complex responsible for major protein degradation (Rock et al., 1994; Tanaka, 1995; Coux et al., 1996), and we have earlier reported that SUG1 might play certain roles in the degradation of vitamin D receptor by proteasome (Masuyama and MacDonald, 1998). Thus, SUG1 might play some role in the degradation of this receptor. However, our recent data (Masuyama et al., 2000) shows that some endocrine disrupting chemicals that activate PXR-mediated transcription also enhanced the interaction with SRC-1 and RIP140, but did not enhance the interaction with SUG1 suggesting a different conformational change of ligand-occupied PXR in the presence of progesterone or endocrine disrupting chemicals. Further analysis is required to clarify this issue. In summary, we demonstrated that several steroids enhanced PXR-mediated transcription probably through interaction with coactivator proteins at concentrations compatible to those near term in pregnancy. Also, the expressions of PXR and CYP3A in the liver and ovary significantly increased with the progression of hypersteroidemia evaluated towards term, however, those in placenta were not drastically changed. These data suggest that PXR may play roles in the regulation of steroid hormones in pregnancy.

Fig. 4. The expression of PXR and CYP3A1 mRNA in perinatal period. (A) The total RNA was isolated from liver of non-pregnant, pregnant (day 12, 15 and 19) and postpartum (3 days after birth) mouse, and analyzed for the expression of PXR and b-actin mRNA using semi-competitive RT-PCR. The PCR products were separated on 3% Nu-Sieve agarose gels and visualized by ethidium bromide. The band intensities were densitometrically measured and quantified using Image Scanner T-9500 and Bio Image software. The data are averages of three determinations of mRNA from three mice. The student’s t-test was used to determine whether treated values were significantly different from the control with PB 0.05 as the limit of significance. (*; PB 0.01) (B) The total RNA was isolated from ovary of non-pregnant, pregnant (Day 12, 15 and 19) and postpartum (3 days after birth) mouse and analyzed for the mRNA expression of PXR and b-actin using semi-competitive RT-PCR. The band intensities were densitometrically measured and quantified using Image Scanner T-9500 and Bio Image software. The data are averages of three determinations of mRNA from three mice. The student’s t-test was used to determine whether treated values were significantly different from the control with PB 0.05 as the limit of significance. (*; P B0.01) (C) The total RNA was isolated from placenta of pregnant mouse (day 12, 15 and 19) and analyzed for the mRNA expression of PXR and b-actin using semi-competitive RT-PCR. The band intensities were densitometrically measured and quantified using Image Scanner T-9500 and Bio Image software. The data are averages of three determinations of mRNA from three mice. The student’s t-test was used to determine whether treated values were significantly different from the control with P B 0.05 as the limit of significance. (*; PB 0.01) (D) The total RNA was isolated from liver of non-pregnant, pregnant (day 12, 15 and 19) and postpartum (3 days after birth) mice, and analyzed for the expression of CYP3A1 and cyclophilin mRNA using semi-competitive RT-PCR. The PCR products were separated on 3% Nu-Sieve agarose gels and visualized by ethidium bromide. The band intensities were densitometrically measured and quantified using Image Scanner T-9500 and Bio Image software. The data are averages of three determinations of mRNA from three mice. The student’s t-test was used to determine whether treated values were significantly different from the control with P B0.05 as the limit of significance. (*; PB 0.01) (E) The total RNA was isolated from ovary of non-pregnant, pregnant (day 12, 15 and 19) and postpartum (day 3 after birth) mice, and analyzed for the expression of CYP3A1 and cyclophilin mRNA using semi-competitive RT-PCR. The PCR products were separated on 3% Nu-Sieve agarose gels and visualized by ethidium bromide. The band intensities were densitometrically measured and quantified using Image Scanner T-9500 and Bio Image software. The data are averages of three determinations of mRNA from three mice. The student’s t-test was used to determine whether treated values were significantly different from the control with P B 0.05 as the limit of significance. (*; P B0.01) (F) The total RNA was isolated from placenta of pregnant (day 12, 15 and 19) mice, and analyzed for the expression of CYP3A1 and cyclophilin mRNA using semi-competitive RT-PCR. The PCR products were separated on 3% Nu-Sieve agarose gels and visualized by ethidium bromide. The band intensities were densitometrically measured and quantified using Image Scanner T-9500 and Bio Image software. The data are averages of three determinations of mRNA from three mice. The student’s t-test was used to determine whether treated values were significantly different from the control with PB 0.05 as the limit of significance. (*; P B0.01)

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Acknowledgements The authors gratefully thank Steven A. Kliewer for providing mouse PXR.1 expression vector and CYP3A1 reporter vector and Paul N. MacDonald for his continuing kind support. This work was supported in part by research grants from the Ministry of Education, Science and Culture of Japan.

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