The Pregnane X Receptor Regulates Gene Expression in a Ligand- and Promoter- Selective Fashion

Hisashi Masuyama, Naoko Suwaki, Yoko Tateishi, Hideki Nakatsukasa, Tomonori Segawa and Yuji Hiramatsu

Department of Obstetrics and Gynecology, Okayama University Medical School, Okayama 700-8558, Japan

Address all correspondence and requests for reprints to: Hisashi Masuyama, M.D., Ph.D., 2-5-1, Shikata, Okayama 700-8558, Japan. E-mail: masuyama{at}cc.okayama-u.ac.jp.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Recent studies have revealed that pregnane X receptor (PXR) can function as a master regulator to control the expression of phase I and phase II drug-metabolizing enzymes, as well as members of the drug transporter family, including multiple drug resistance (MDR) 1, which has a major role in multidrug resistance. Previously, we have demonstrated that steroid/xenobiotics metabolism by tumor tissue through the PXR-cytochrome P-450 3A (CYP3A) pathway might play an important role in endometrial cancer. In this study, we examined which endocrine-disrupting chemicals (EDCs) and anticancer agents might be ligands for PXR and whether these chemicals enhanced PXR-mediated transcription through two different PXR-responsive elements (PXREs), CYP3A4 and MDR1, in endometrial cancer cell lines. Some steroids/EDCs strongly activated PXR-mediated transcription through the CYP3A4-responsive element compared with the MDR1-responsive element, whereas these steroids/EDCs also enhanced the CYP3A4 expression compared with the MDR1 expression. In contrast, the anticancer agents, cisplatin and paclitaxel, strongly activated PXR-mediated transcription through the MDR1-responsive element compared with the CYP3A4-responsive element, whereas these drugs also enhanced the MDR1 expression compared with the CYP3A4 expression. We also analyzed how these ligands regulated PXR-mediated transcription through two different PXREs. In the presence of PXR ligands, there was no difference in the DNA binding affinity of the PXR/retinoid X recptor heterodimer to each PXRE, but there were different interactions of the coactivator to each PXR/PXRE complex. These data suggested that PXR ligands enhanced PXR-mediated transcription in a ligand- and promoter-dependent fashion, which in turn differentially regulated the expression of individual PXR targets, especially CYP3A4 and MDR1.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
PREGNANE X RECEPTOR (PXR), a new member of the steroid receptor superfamily, has been shown to mediate the genomic effects of several steroid hormones, including progesterone, pregnenolone, and estrogen, and of xenobiotics in the mouse, rat, and human (1, 2, 3, 4, 5, 6, 7). Like nonsteroid hormone receptors, it binds as a heterodimer with retinoid X receptor (RXR) to specific DNA sequences, PXR-responsive element (PXRE), including the upstream region of the cytochrome P-450 3A (CYP3A) gene family (1, 2, 3, 6), which are monooxygenases responsible for the oxidative metabolism of certain endogenous substrates and xenobiotics (8, 9), and multiple drug resistance 1 (MDR1) (10, 11), which encodes the P-glycoprotein, a multidrug transporter and which has a major role in multidrug resistance (12). Because the PXR pathway is activated by a large number of prescription drugs designed to treat infection, cancer, convulsion, and hypertension (13), PXR is thought to play roles in drug metabolism/efflux and drug-drug interaction. Recent research demonstrated that PXR regulates the metabolism of bile acid, which is essential for the elimination of excess cholesterol from the body and the transport of dietary lipids in the intestine (14). These data suggest that PXR regulates an entire program of genes in the liver and intestine that are involved in the metabolism and elimination of potentially toxic substrates from the body (13). Previously, we demonstrated the expression of PXR in mouse reproductive tissues, uterine and ovarian, as well as the liver and intestine, and we showed that the expressions of PXR and CYP3A1 in the liver and ovary significantly increased with the progression of hypersteroidemia evaluated toward term during pregnancy, suggesting that PXR may play a role in the regulation of steroid hormone metabolism during reproduction (15). In addition, our recent data suggested that steroid/xenobiotics metabolism by tumor tissue through the PXR-CYP3A pathway might play an important role in endometrial cancer, especially as an alternative pathway for gonadal hormone and EDC effects on endometrial cancer expressing low estrogen receptor-{alpha} (16).

In this study, we examined which EDCs and anticancer agents might be ligands for PXR and whether these chemicals enhanced PXR-mediated transcription through two different PXREs, CYP3A4 and MDR1. We also analyzed how these ligands regulated PXR-mediated transcription through these PXREs. The data suggested that PXR might regulate gene expression, especially CYP3A4 and MDR1, in a ligand- and promoter-selective fashion in endometrial cancer.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The Effect of PXR Ligands on PXR-Mediated Transcription through Different PXREs
Because we have demonstrated that PXR was abundant in HEC-1 cells, but was weakly detected in Ishikawa cells (16), we used these cell lines derived from endometrial cancer to examine the effect of steroids, EDCs, and anticancer agents on the PXR-mediated transcription through different PXREs. We used two endocrine-disrupting chemicals, phthalate and bisphenol A, as well as the steroids estradiol, pregnenolone, and progesterone, as PXR ligands because these chemicals have been demonstrated to activate PXR-mediated transcription (16, 17, 18). In addition, we tested whether the anticancer agents, cisplatin, carboplatin, paclitaxel, and docetaxel, and anticancer hormone medroxyprogesterone acetate (MPA) enhanced PXR-mediated transcription, because paclitaxel, which is a commonly used chemotherapeutic agent, has been demonstrated to activate PXR-mediated transcription (10). Two different reporter gene constructs, (CYP3A4)3-tk (thymidine kinase)-CAT (chloramphenicol acetyl transferase) and (MDR1)3-tk-CAT, were introduced into Ishikawa cells and HEC-1 cells. In HEC-1 cells using (CYP3A4)3-tk-CAT, the steroids 17ß-estradiol, pregnenolone, progesterone, and MPA and the EDCs bisphenol A and phthalate significantly activated native PXR-mediated transcription (P < 0.01). The antitumor agents cisplatin and paclitaxel also significantly activated the transcription (P < 0.01), but the fold increases (2.9- to 3.1-fold) were lower compared with those in the presence of steroids or EDCs (3.9- to 6.5-fold) in HEC-1 cells (Fig. 1AGo). In contrast, these cisplatin and paclitaxel had a stronger effect on this transcription through MDR1-responsive element compared with steroids or EDCs (4.8- to 5.4-fold vs. 1.8- to 2.6-fold). The other drugs we tested, carboplatin and docetaxel, had no effect on this transcription. We observed a similar result in Ishikawa cells (Fig. 1BGo). In this transient transfection assay, the ranges of fold increase in HEC-1 cells, in which PXR is abundant (1.8- to 6.5-fold; Fig. 1AGo) were much higher than those in Ishikawa cells, in which PXR is weakly expressed (1.6- to 4.0-fold; Fig. 1BGo). In HEC-1 cells, these effects through CYP3A4-responsive element were dependent on the ligand concentration and significantly increased at 10 nM phthalate or estradiol and 1 µM cisplatin or paclitaxel compared with ethanol treatment (Fig. 1CGo). In contrast, the effects caused by MDR1-responsive element were significantly increased at 10 nM cisplatin or paclitaxel and 1 µM phthalate or estradiol compared with ethanol treatment.



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Fig. 1. Effect of PXR Ligands on PXR-Mediated Transcription through Different PXREs

A, HEC-1 cells were transfected with 1 µg of the (CYP3A4)3-tk-CAT, (MDR1)3-tk-CAT or tk-CAT vector. The cells were treated with ethanol vehicle or 10–6 M steroids, EDCs, or anticancer agents for 24 h. The amount of CAT was determined with a CAT ELISA kit (Roche Diagnostics Co., Tokyo, Japan) according to the manufacturer’s instruction. The results represent the mean ± SD of triplicate determinations (*, P < 0.01 compared with ethanol-treated control). The number above each bar represents fold increase relative to the ethanol-treated control. B, Ishikawa cells were transfected with 1 µg of the (CYP3A4)3-tk-CAT, (MDR1)3-tk-CAT or tk-CAT vector. The cells were treated with ethanol vehicle or 10–6 M steroids, EDCs, or anticancer agents for 24 h. The amount of CAT was determined with a CAT ELISA kit. The results represent the mean ± SD of triplicate determinations (*, P < 0.01 compared with ethanol-treated control). The number above each bar represents fold increase relative to the ethanol-treated control. C, HEC-1 cells were transfected with 1 µg of the (CYP3A4)3-tk-CAT, (MDR1)3-tk-CAT or tk-CAT vector. The cells were treated with ethanol vehicle or increasing concentrations of steroids, EDCs, or anticancer agents for 24 h. The amount of CAT was determined with a CAT ELISA kit (Roche Diagnostics Co., Tokyo, Japan) according to the manufacturer’s instruction. The results represent the mean ± SD of triplicate determinations (*, P < 0.01 compared with ethanol-treated control).

 
The Effect of PXR Ligands on the Expression of PXR, MDR1, and CYP3A4
To investigate the effect of PXR ligands on the expression of CYP3A4, MDR1, and PXR in vitro, we examined the protein levels of CYP3A4, MDR1, and PXR qualitatively in HEC-1 cells and Ishikawa cells that had been exposed to steroids, EDCs, and anticancer drugs. In HEC-1 cells, the CYP3A4 protein level was significantly increased in the presence of PXR ligands, which activated PXR-mediated transcription in the transient transfection assay (Fig. 2AGo). The steroids/EDCs had a significant and stronger positive effect on CYP3A4 expression compared with the anticancer agents paclitaxel and cisplatin. In contrast, the MDR1 level was significantly increased only in the presence of cisplatin and paclitaxel. The CYP3A4 level did not change in response to docetaxel or carboplatin, which did not enhance PXR-mediated transcription. In addition, PXR ligands had different effects on the PXR level itself. PXR was down-regulated in the presence of cisplatin but was up-regulated in the presence of bisphenol A. The mRNA levels of CYP3A4, MDR1, and PXR were also examined by semiquantitative RT-PCR. The effect of PXR ligands on mRNA level of these PXR target genes and PXR itself were compatible with these effects on protein levels (Fig. 2BGo).



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Fig. 2. Effect of PXR Ligands on the Expression of PXR, MDR1, and CYP3A4 in HEC-1 Cells

A, HEC-1 cells were treated with dimethylsulfoxide (DMSO), various steroids, EDCs or anticancer agents for 36 h and nuclear extracts were prepared as described in Materials and Methods. Equivalent amounts of each extract (25 µg/sample) were resolved by 10% SDS-PAGE, and CYP3A4, MDR1, and PXR protein levels were determined by Western blotting using anti-CYP3A4, -MDR1, and -PXR antibody. As a loading control, ß-actin protein levels were also examined using anti-ß-actin antibody. Each bar represents the mean ± SD from three independent experiments (*, P < 0.01 compared with DMSO-treated control). B, HEC-1 cells were treated with DMSO or 10–6 M various steroids, EDCs or anticancer agents for 36 h and the total RNA was obtained from HEC-1 cells and analyzed for the expression of CYP3A4, MDR1, PXR, and GAPDH mRNA using semicompetitive 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 results represent the mean ± SD of triplicate determinations (*, P < 0.01 compared with DMSO-treated control).

 
PXR/RXR Heterodimer Interacted with PXREs, CYP3A4 and MDR1 in Gel Shift Assay
To determine whether the PXR/RXR heterodimer alters the DNA binding properties in the presence of PXR ligands, we performed EMSAs using overexpressed pSG5-PXR and pSG5-RXR{alpha} proteins in HEC-1 cells and a biotin-labeled PXRE. There were no differences in DNA binding of PXR/RXR heterodimer to each PXRE in the presence of PXR ligands, estradiol, paclitaxel, or phthalate. However, the interaction of PXR/RXR with the CYP3A4-responsive element was slightly stronger than that with the MDR1-responsive element (Fig. 3Go). Western analysis showed that expressions of PXR and RXR{alpha} proteins were at similar levels (data not shown).



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Fig. 3. Interaction of PXR/RXR Heterodimer with PXREs of both CYP3A4 and MDR1

Cellular extracts of HEC-1 cells expressing PXR and/or RXR{alpha} were incubated with a biotin-labeled PXRE and electrophoresed on a 4% nondenaturing polyacrylamide gel. The biotin-labeled DNA/protein complexes were detected using chemiluminescent solution according to the manufacturer’s protocol.

 
The Different Interaction of Coactivators with PXR/PXRE Complexes
Because we didn’t observe a different DNA binding property of the PXR/RXR heterodimer in the presence of PXR ligands, we next used the DNA affinity immunoblotting assay to examine which coactivator was recruited by the PXR/PXRE complex. Because steroid receptor coactivator-1 (SRC-1) and amplified in breast cancer 1 (AIB1), members of the p160 nuclear receptor coactivator family, were expressed and up-regulated in endometrial cancer (19), and we have previously demonstrated that PXR interacted with SRC-1 (17), we examined whether SRC-1 and AIB1 associated on PXR/PXRE complex in the presence of PXR ligands. In addition, thyroid receptor-associated protein 220 (TRAP220), a member of another nuclear receptor coactivator family called the vitamin D receptor (VDR)-interacting protein/TRAP complex, was also examined because TRAP220 played important roles in nuclear receptor-mediated transcription as coactivators (20). On CYP3A4-responsive element, SRC-1 was strongly detected in the presence of estradiol, phthalate, bisphenol A, or pregnenolone, much less than paclitaxel or cisplatin (Fig. 4Go). There was no detection in the presence of a control vehicle. In contrast, we observed a strong association of AIB1 on MDR1-responsive element in the presence of paclitaxel or cisplatin compared with that in the presence of estradiol, phthalate, bisphenol A, or pregnenolone. Moreover, there was no difference in the interaction between TRAP220 and PXR/PXRE complex in the presence of any PXR ligand, whereas PXR ligands didn’t affect the binding affinity of PXR to PXRE. There was no detection of other nuclear receptors including constitutive androstane receptor (CAR), VDR and thyroid hormone receptor (TR), which could interact with the direct repeat (DR)-3 or DR-4 hormone-responsive element, in this assay.



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Fig. 4. Different Interactions of Coactivators with the PXR/PXRE Complex

Two hundred micrograms of nuclear extracts obtained from HEC-1 cells were incubated with DNA binding reaction mixture including biotin-labeled PXRE. DNA/protein complexes were captured with 0.1 mg of magnetic streptavidin beads at 4 C for 30 min. The beads were pelleted using a magnet and washed three times with DNA binding buffer. The bound proteins were eluted from the magnetic beads by heating and analyzed by Western blot analysis using goat polyclonal antibody for PXR or CAR, rabbit polyclonal antibody for TRAP220, SRC-1, AIB1, VDR, or TR.

 
The Effect of PXR Ligands on the Interaction between PXR and Coactivator AIB-1, SRC-1, or TRAP220 in the Yeast Two-Hybrid Assay
We used the two-hybrid protein interaction assay to examine whether PXR interacts with the coactivators SRC-1, AIB1, or TRAP220 in the presence of PXR ligands. AIB1 strongly interacted with PXR in the presence of paclitaxel or cisplatin, which stimulated PXR-mediated transcription especially through the MDR1-responsive element, compared with estradiol or phthalate (Fig. 5Go). In contrast, SRC-1 preferably interacted with PXR in the presence of estradiol or phthalate, which stimulated PXR-mediated transcription especially through the CYP3A4-responsive element. However, there was no difference in the interaction between PXR and TRAP220 in the presence of any PXR ligand.



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Fig. 5. Effect of PXR Ligands on the Interaction between PXR and Coactivator AIB1, SRC-1, or TRAP220

Yeast expressing the pAS1-PXR and pAD-Gal4-AIB1, -SRC-1, -TRAP220 or pAD-Gal4 empty vector were grown for 24 h at 30 C in the presence of ethanol vehicle or 10–6 M phthalate, estradiol, paclitaxel, or cisplatin. PXR-AIB1, -SRC-1, or -TRAP220 interaction was assessed in ß-galactosidase assay. Results are presented as the mean ± SD of triplicate independent cultures.

 
The Effect of Overexpressed Coactivators on PXR-Mediated Transcription through the PXREs CYP3A4 or MDR1
We used the transient reporter expression assay in HEC-1 cells to examine whether coactivators affect PXR-mediated transcription through two different promoters, CYP3A4 and MDR1. As described in Fig. 6Go, the coactivators tested here enhanced PXR-mediated transcription in the presence of PXR ligands through both PXREs. The effect of SRC-1 on PXR-mediated transcription in the presence of phthalate or estradiol was more efficient than that in the presence of paclitaxel or cisplatin through the CYP3A4-responsive element (3.5- to 4.3-fold vs. 2.0- to 2.1-fold). In contrast, the effect of AIB1 on PXR-mediated transcription in the presence of paclitaxel or cisplatin was significantly increased compared with that in the presence of estradiol or phthalate through the MDR1-responsive element (3.7- to 3.8-fold vs. 1.6- to 1.8-fold). TRAP 220 moderately enhanced PXR-mediated transcription in the presence of any ligand, and there was no different effect of TRAP220 on PXR-mediated transcription between CYP3A4- and MDR1-responsive elements (1.5- to 2.5-fold vs. 1.7- to 2.4-fold).



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Fig. 6. Effect of Coactivators on PXR-Mediated Transcription through the PXREs, CYP3A4 or MDR1

HEC-1 cells were cotransfected with 1 µg of the (CYP3A4)3-tk-CAT or (MDR1)3-tk-CAT reporter gene constructs with 1 µg of pcDNA3-AIB1, -SRC-1, or -TRAP220 or pcDNA3 expression vector. The cells were treated with ethanol vehicle or 10–6 M phthalate, estradiol paclitaxel, cisplatin or ethanol for 24 h. The amount of CAT was determined with a CAT ELISA kit. The results represent the mean ± SD of triplicate determinations.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have demonstrated the expression of PXR and its target gene, CYP3A4, in endometrial cancer tissues (16). There was a significant inverse correlation between PXR and estrogen receptor and the strong transcriptional response of the PXR-CYP3A pathway to the PXR ligands in endometrial cancer cells, suggesting that PXR-mediated pathways might be more active in endometrial tumors, which are less likely to respond to estrogen, or that the up-regulation of PXR by a local hyperestrogenic state might introduce a poor response to estrogen (16). The CYP3A subfamily is involved in the metabolism of endogenous substrates such as steroid hormones and bile acids (8, 9). And this subfamily also plays important roles in the metabolism of procarcinogens and pharmaceutical agents, including innumerable drugs, chemical carcinogens, mutagens, and other environmental contaminants (8, 9, 21). In humans, CYP3A4 mRNA is revealed in uterine endometrium by RT-PCR (22, 23), and we found that the expression of CYP3A4 mRNA in endometrial cancer tissue was significantly higher than that in normal endometrium and that CYP3A4 expression positively correlated with PXR expression (16). On the other hand, MDR1 was originally identified because of its overexpression in cultured cancer cells associated with an acquired cross-resistance to multiple anticancer drugs and has been demonstrated to be an ATP-dependent efflux pump of hydrophobic anticancer drugs (12). Recently, it has been reported that MDR1 was also regulated by PXR (10, 11). Some endometrial carcinomas as well as the normal endometrial controls from both the proliferative and the secretory phase of the menstrual cycle overexpressed P-glycoprotein, which was encoded by MDR1 (24). In this study, PXR ligands, especially the steroids estradiol, MPA, and pregnenolone, and the EDCs bisphenol A and phthalate, strongly activated PXR-mediated transcription through the CYP3A4-responsive element compared with the MDR1-responsive element, whereas these steroids/EDCS also enhanced the CYP3A4 expression compared with the MDR1 expression in endometrial cancer cell lines. In contrast, the anticancer agents paclitaxel and cisplatin strongly activated PXR-mediated transcription through the MDR1-responsive element compared with the CYP3A4-responsive element, whereas these drugs also enhanced the MDR1 expression compared with the CYP3A4 expression. These data suggested that PXR might regulate its target genes, CYP3A4 and MDR1, in a ligand- and promoter-selective fashion in endometrial cancer.

There are several different PXREs, DR-4 and everted repeat-6 (ER-6) at the upstream portion of the MDR1 gene and DR-3 and ER-6 at the upstream portion of the CYP3A gene (11, 25). In this study, we examined the DR-4 and DR-3 as PXRE for MDR1 and CYP3A4, respectively, because previous reports demonstrated that both PXREs play important roles for the PXR-mediated transactivation of MDR1 and CYP3A4 compared with the ER-6 element (11, 25). We showed that steroids/EDCs preferred DR-3/CYP3A4 promoter in PXR-mediated transcription of endometrial cancer cells, but that anticancer drugs mainly affected the transcription through DR-4/MDR promoter. To investigate the mechanism of this difference by PXR ligands, we have performed additional experiments including a gel shift assay and a DNA affinity immunoblotting assay. These experiments showed that there was no difference in DNA binding affinity of the PXR/RXR heterodimer to either of the PXREs in the presence of any PXR ligand tested here, but there were different interactions of coactivator to PXR/PXRE complex in the presence of PXR ligands. CAR and PXR have been demonstrated to share some xenobiotics and steroids as ligands, although CAR was much less promiscuous in its interaction with chemicals than PXR (26). Thus, we confirmed that there was no detection of CAR and other nuclear receptors, VDR and TR, that could interact with the DR-3 or DR-4 hormone-responsive element, on two PXREs, DR-3/CYP3A4 and DR-4/MDR1, in the presence of various PXR ligands. The data of the yeast two-hybrid assay showed that PXR preferably interacted with SRC-1 in the presence of estradiol and phthalate, but with AIB1 in the presence of cisplatin and paclitaxel. Ligand-occupied nuclear receptors showed different conformational changes in the presence of each ligand (17, 20, 27, 28), suggesting that nuclear receptors including PXR have ligand-specific interactions with coactivators. Finally, in the transient transfection assay, SRC-1 strongly enhanced the PXR-mediated transcription in the presence of bisphenol A or estradiol through the CYP3A4-responsive element compared with paclitaxel or cisplatin, but the effect of AIB1 on PXR-mediated transcription in the presence of paclitaxel or cisplatin was significantly increased compared with that in the presence of estradiol or phthalate. These data suggested that the binding of PXR ligands results in a different conformation on each PXRE, which in turn differentially regulates the expression of each PXR target gene. Other nuclear receptors, including estrogen receptor and farnesoid X receptor, have also been demonstrated to interact with ligands for each receptor in a unique fashion that leads to a ligand and promoter selectivity for estrogen receptor- or FXR-mediated gene transcription (29, 30, 31). In our study, although PXR strongly interacted with SRC-1 in the presence of steroids/EDCs in the two-hybrid assay, we observed that there were different accumulations of SRC-1 on CYP3A4-responsive element compared with MDR1-responsive element in the presence of the same ligand. These findings suggested that the interaction of coactivators with PXR/PXRE complex might be dependent on the promoter. Further analysis will be required to elucidate the mechanism of promoter-selective fashion and the potential role of the ER-6 element in PXR-mediated transcription.

PXR is an orphan receptor originally identified as a xenobiotics receptor that regulates CYP gene expression (13). Subsequent studies have revealed much more complex regulatory pathways governed by this receptor. PXR can function as a master regulator to control the expression of phase I and phase II drug-metabolizing enzymes, as well as members of the drug transporter family (13). In this study, we have demonstrated that, in endometrial cancer, both a metabolizing enzyme and transporter were expressed and these expressions were regulated by PXR. However, there was a different property of PXR ligands between steroids/EDCs and anticancer drugs. Moreover, in PXR ligands, steroids/EDCs significantly increased PXR protein and anticancer drugs decreased this protein, suggesting the existence of different autoregulatory loops in the PXR pathway. Because cisplatin and paclitaxel are used for the systemic chemotherapy for advanced endometrial cancer patients (32), further analysis will be required to determine the biological roles of the PXR pathway in endometrial cancer tissues including drug resistance.

In summary, we examined PXR-mediated transcription in endometrial cancer cells. Some steroids/EDCs strongly activated PXR-mediated transcription through the CYP3A4-responsive element compared with the MDR1-responsive element. These steroids also enhanced the CYP3A4 expression compared with the MDR1 expression. In contrast, some anticancer agents strongly activated PXR-mediated transcription through the MDR1-responsive element. These drugs also enhanced the MDR1 expression compared with the CYP3A4 expression. Moreover, in PXR ligands, steroids/EDCs significantly increased PXR protein, but anticancer drugs decreased this protein, suggesting the existence of different autoregulatory loops in the PXR pathway. Finally, we investigated how these ligands regulated PXR-mediated transcription through hormone-responsive elements. There was no difference in DNA binding affinity of the PXR/RXR heterodimer to the tested PXREs in the presence of PXR ligands, but there were different interactions of coactivators to the PXR/PXRE complex in the presence of each PXR ligand. These data suggested that PXR ligands enhanced PXR-mediated transcription in a ligand- and promoter-dependent fashion, which in turn differentially regulates the expression of individual PXR targets.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Isopropylidenediphenol (bisphenol A), phthalic acid bis (2-ethylhexel ester) (phthalic acid), 5-Pregneno-3ß-ol-20-one (pregnenolone), progesterone, MPA, and 17ß-estradiol (estradiol) were purchased from Sigma Co., Ltd. (St. Louis, MO). Carboplatin, cisplatin, docetaxel, and paclitaxel were purchased from Calbiochem-Novabiochem Corp. (San Diego, CA). Ishikawa cells were kindly provided by Dr. M. Nishida (Tsukuba University, Tochigi, Japan), and HEC-1 cells were obtained from the Health Science Research Resources Bank (Osaka, Japan).

Transient Transfection Studies
The pSG5-PXR expression plasmid containing full-length human PXR cDNA were kindly provided by Dr. S. A. Kliewer (5). The (CYP3A4)3-tk-CAT was generated by insertion of three copies of a double-strand oligonucleotides containing the DR-3/CYP3A4 (5'-gggtcagcaagttca-3'), and the (MDR1)3-tk-CAT was generated by insertion of three copies of a double-strand oligonucleotides containing the DR-4/MDR1 (5'-aggtcaagttagttca-3') as described before (11, 25). Ishikawa cells and HEC-1 cells were cotransfected with 1 µg of a reporter gene construct [(CYP3A4)3-tk-CAT or (MDR1)3-tk-CAT] or tk-CAT vector. For coactivator expression, 1 µg of pcDNA3-SRC-1, -AIB1, -TRAP220, or pcDNA3 alone was also transfected into the cells. In all transfections, liposome-mediated transfections were accomplished by using lipofectamine (Invitrogen Corp., Carlsbad, CA) according to the manufacturer’s instructions. The AIB1 cDNA, which was generated by RT-PCR and the SRC-1 cDNA, which was a gift from Dr. M. J. Tsai (Baylor College, Houston, TX), were subcloned into pcDNA3 expression vectors (Invitrogen). The pcDNA-TRAP220 has been described previously (33). Transfected cells were treated either with vehicle alone or with the indicated concentrations of steroid hormones, EDCs, or anticancer agents for 24 h. The cell extracts were prepared and assayed for CAT activity. The amount of CAT was determined using a CAT ELISA kit (Roche Diagnostics Co., Tokyo, Japan) according to the manufacturer’s instructions.

Cell Culture and Western Blot Analysis
HEC-1 cells and Ishikawa cells were cultured in DMEM without phenol red supplemented with 10% charcoal-striped fetal bovine serum. The medium and serum were purchased from Invitrogen. Nuclear extracts were obtained from both cell types using NE-PER nuclear and cytoplasmic Extraction Reagents (Pierce Chemical Co., Rockford, IL) according to the manufacturer’s protocol and stored at –80 C until analysis. Equivalent amounts of nuclear protein (25 µg/sample) from each extract were determined by bicinchoninic acid protein assay (Pierce Chemical Co.), solubilized in sodium dodecyl sulfate (SDS) buffer [0.05 M Tris-HCl, 2% SDS, 6% mercaptoethanol, 10% glycerol (pH 6.8)] and analyzed by Western blot analysis as previously described (34). We employed goat polyclonal antibody for PXR (A-20; 1:1000 dilution), rabbit polyclonal antibody for TRAP220 (M-255; 1:1000 dilution), SRC-1 (M-341; 1:1000 dilution), AIB1 (C-20; 1:1000 dilution) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and CYP3A4 (1:1000) (PanVera Corp., Madison, WI). The amount of each band was quantitated densitometrically using Image Scanner GT-9500 (Epson, Suwa, Japan) and Bio Image BQ 2.0 software (Bio Image, Ann Arbor, MI).

RT-PCR
Total RNA was also extracted from endometrial cancer cells using Trizol reagent (Invitrogen). Each sample was treated with deoxyribonuclease I to remove genomic DNA contamination. According to the protocol of the RNA PCR kit (TAKARA Co., Ltd., Kyoto, Japan), 0.1 µg of total RNA was reverse transcribed at 42 C for 20 min in 20 µl of reaction solution containing 1x PCR buffer, 5 mM MgCl2, 1 mM deoxynucleotide triphosphate, 2.5 µM random 9 mers primer, 10 U RNase inhibitor, and 5 U avian myeloblastosis virus reverse transcriptase. The primers for human CYP3A4, PXR and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were described before (16) and those for MDR1 were as follows: MDR1 sense: 5'-gctcctgactatgccaaagc-3', 3' antisense: 5'-tcttcacctccaggctcagt-3'. Amplification for CYP3A4, PXR, MDR1 and GAPDH was carried out on a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA) with initial denaturation at 94 C for 2 min, followed by 25 cycles of 94 C for 30 sec, 60 C for 30 sec, 72 C for 30 sec, 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 determined. PCR samples were electrophoresed on 3% Nu-Sieve agarose gel and visualized by ethidium bromide. The amount of each electrophorectically separated cDNA was quantitated densitometrically using an Image Scanner GT-9500 (Epson, Suwa, Japan) and Bio Image BQ 2.0 software. The housekeeping gene, GAPDH, was used to determine the relative levels of CYP3A4, MDR1, and PXR 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 CYP3A4, MDR1, and PXR cDNA relative to the amount of GAPDH cDNA. This ratio was used to compare the relative amounts of CYP3A4, MDR1 and PXR mRNA in each sample.

Gel Mobility Shift Analysis
The PXRE oligonucleotides (CYP3A4 and MDR1) were synthesized from the sequences in the upstream portion of human CYP3A4 or MDR1 gene as previously described (11) and 5' end-labeled with biotin. Double-stranded forms were prepared by incubating at 100 C and cooling to room temperature. Using a LightShift chemiluminescent EMSA kit (Pierce Chemical Co., Rockford, IL), we incubated 10 µg of the lysates obtained from HEC-1 cells, to which were introduced overexpressed PXR and/or RXR{alpha} by liposome-mediated transfection, with biotin-labeled PXRE probe for 20 min at room temperature, and then unbound probe and protein-DNA complexes were separated by nondenaturing electrophoresis on a 4% polyacrylamide gel in 0.25x Tris-borate-EDTA. Binding reactions were electrophoretically transferred to nylon membrane, and the DNAs were cross-link transfected to the membrane using a UV-light cross-linker instrument according to the manufacturer’s protocol. The biotin-labeled DNA/protein complexes were detected using chemiluminescent solution according to the manufacturer’s protocol.

DNA Affinity Immunoblotting
Two hundred micrograms of nuclear extracts obtained from HEC-1 cells were incubated at 4 C for 30 min with DNA binding reaction mixture containing 25 nM biotin-labeled PXRE, 2 µg of poly (deoxyinosine/deoxycytosine), 5 mM dithiothreitol, and 40 µl of 10x DNA binding buffer [200 mM Tris-HCl (pH 7.2), 10 mM EDTA, 1% Triton X-100, and 40% glycerol]. DNA/protein complexes were captured with 0.1 mg of magnetic streptavidin beads (Dynabeads, Dynal Biotech, Oslo, Norway) at 4 C for 30 min. The beads were pelleted using a magnet and washed three times with DNA binding buffer. The bound proteins were eluted from the magnetic beads by heating at 70 C in 20 µl of SDS buffer [0.05 M Tris-HCl, 2% SDS, 6% mercaptoethanol, 10% glycerol (pH 6.8)] and analyzed by Western blot analysis using goat polyclonal antibody for PXR or CAR, rabbit polyclonal antibody for TRAP220, SRC-1, AIB1, VDR or TR (Santa Cruz Biotechnology Inc.).

Preparation of Two-Hybrid Expression Vectors and ß-Galactosidase Assays
All two-hybrid plasmid constructs used the pAS1 (35) and pAD-GAL4 (Stratagene, La Jolla, CA) yeast expression vectors. The pAD-GAL4-SRC-1, -TRAP220, and pAS1-PXR were previously described (17, 33, 36). AIB1 cDNA was subcloned into the pAD-GAL4 expression vector. To examine the interaction with coactivator proteins in the two-hybrid assay, we cotransformed the pAS1-PXR with pAD-GAL4-SRC-1, -AIB1 or -TRAP220 into the yeast strain Hf7c, which was made competent with lithium acetate. Transformants were plated on media lacking leucine and tryptophan (SC-leu-trp) and were grown for 4 d 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 in the presence of ethanol vehicle or 10–6 M phthalate, estradiol, paclitaxel, or cisplatin. The cells were harvested and assayed for ß-galactosidase activity as described previously (37).

Statistical Analysis
Statistical analysis was evaluated by one-way ANOVA followed by Dunnett’s test, as shown in Figs. 1Go and 2Go. Data are the mean ± SD. P < 0.05 denoted the presence of a statistically significant difference.


    ACKNOWLEDGMENTS
 
The authors gratefully thank Dr. Steven A. Kliewer for providing CYP3A4 reporter vector and Dr. M. Nishida for providing Ishikawa cells.


    FOOTNOTES
 
This work was supported in part by research grants (14042236, 14571562) from the Ministry of Education, Science and Culture of Japan and the Okayama Medical Foundation (to H.M.).

First Published Online January 13, 2005

Abbreviations: AIB1, Amplified in breast cancer 1; CAR, constitutive androstane receptor; CAT, chloramphenicol acetyl transferase; CYP3A, cytochrome P-450 3A; DR, direct repeat; EDC, endocrine-disrupting chemical; ER, everted repeat; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MDR, multiple drug resistance; MPA, medroxyprogesterone acetate; PXR, pregnane X receptor; PXRE, PXR-responsive element; RXR, retinoid X receptor; SDS, sodium dodecyl sulfate; SRC-1, steroid receptor coactivator-1; tk, thymidine kinase; TR, thyroid hormone receptor; TRAP, thyroid receptor-associated protein; VDR, vitamin D receptor.

Received for publication October 25, 2004. Accepted for publication January 7, 2005.


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