Endocrine Disrupting Chemicals, Phthalic Acid and Nonylphenol, Activate Pregnane X Receptor-Mediated Transcription

Hisashi Masuyama, Yuji Hiramatsu, Mamoru Kunitomi, Takafumi Kudo and Paul N. MacDonald

Department of Obstetrics and Gynecology (H.M., Y.H., M.K., T.K.) Okayama University Medical School Okayama, 700-8558, Japan
Department of Pharmacology (P.N.M.) Case Western Reserve University Cleveland, Ohio 44106


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Recently, Pregnane X receptor (PXR), a new member of the nuclear receptor superfamily, was shown to mediate the effects of several steroid hormones, such as progesterone, glucocorticoid, pregnenolone, and xenobiotics on cytochrome P450 3A genes (CYP3A) through the specific DNA sequence for CYP3A, suggesting that PXR may play a role in steroid hormone metabolism. In this paper, we demonstrated that phthalic acid and nonylphenol, endocrine-disrupting chemicals (EDCs), stimulated PXR-mediated transcription at concentrations comparable to those at which they activate estrogen receptor-mediated transcription using a transient reporter gene expression assay in COS-7 cells. However, bisphenol A, another EDC, had no effect on PXR-mediated transcription, although this chemical significantly enhanced ER-mediated transcription. In the yeast two-hybrid protein interaction assay, PXR interacted with two nuclear receptor coactivator proteins, steroid hormone receptor coactivator-1 and receptor interacting protein 140, in the presence of phthalic acid or nonylphenol. Thus, EDC-occupied PXR may regulate its specific gene expression through the receptor-coactivator interaction. In contrast, these EDCs had no effect on the interaction between PXR and suppressor for gal 1, a component of proteasome. Finally, the expression of CYP3A1 mRNA in the liver of rats exposed to phthalic acid or nonylphenol markedly increased compared with that in rats treated with estradiol, bisphenol A, or ethanol as assessed by competitive RT-PCR. These data suggest that EDCs may affect endocrine functions by altering steroid hormone metabolism through PXR.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The nuclear receptor superfamily consists of more than 150 different proteins that have evolved to mediate a complex array of extracellular signals into transcriptional responses. There are many ligands for these nuclear receptors, including steroid hormones, such as estradiol (E2), progesterone (P), and glucocorticoid, and nonsteroid hormones, such as vitamin D, retinoids, thyroid hormone, and prostanoids. These receptors form homodimers or heterodimers with RXR and directly associate with specific DNA sequences known as hormone-responsive elements located in the promoters of specific genes (1, 2, 3). The DNA-receptor complex interacts with basal transcriptional machinery and nuclear receptor coactivator proteins, resulting in ligand-dependent induction of transcription (3, 4, 5). Recently, pregnane X receptor (PXR), a new member of the nuclear receptor superfamily, has been shown to mediate the effects of several steroid hormones, such as P, pregnenolone, glucocorticoid, synthetic glucocorticoids, antiglucocorticoids, and xenobiotics on the cytochrome P450 3A genes (CYP3A) in the mouse, rat, and man (6, 7, 8, 9, 10, 11, 12). Like nonsteroid hormone receptors, PXR binds as a heterodimer with retinoid X receptor to specific DNA sequences, including those upstream of CYP3A, and regulates expression of target genes (6, 7, 8, 11).

The cytochrome P450 family consists of heme-containing monooxygenases that function in the oxidative metabolism of a wide variety of endogenous substances and xenobiotics. Specifically, the CYP3A subfamily is involved in the metabolism of endogenous substrates such as steroids, bile acids, and retinoic acid. In addition, this subfamily also plays important roles in the metabolism of procarcinogen and pharmaceutical agents, including innumerable drugs, chemical carcinogens, mutagens, and other environmental contaminants (13, 14). The fact that PXR has been shown to be activated by several steroids and other exogenous compounds that are known to induce the CYP3A genes (6, 7, 8) suggests a novel endocrine signaling pathway that regulates the metabolism of steroids and xenobiotics through PXR.

Some environmental agents have been shown to disrupt the endocrine functions in many species through a variety of pathways including the change of steroidogenesis (15). Nonylphenol, one of these environmental agents, has been demonstrated to induce CYP3A expression (16). Thus, we examined whether some endocrine-disrupting chemicals (EDCs) including nonylphenol activate the PXR-mediated transcription through the CYP3A1 motif in the transient reporter assay. We also checked whether PXR interacts with two nuclear receptor coactivators, steroid hormone receptor coactivator-1 (SRC-1) (17) and receptor interacting protein 140 (RIP140) (18), in the presence of EDCs. Finally, the effect of EDCs on CYP3A1 mRNA expression in the liver of EDC-treated rats was analyzed using competitive RT-PCR. The results of these experiments provide some evidence that EDCs stimulate PXR-mediated transcription through the interaction between PXR and nuclear receptor coactivator proteins, suggesting that EDCs may affect the endocrine functions by altering the steroid hormone metabolism through PXR.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
EDCs Stimulate PXR-Mediated Transcription
A transient reporter expression assay was performed in COS-7 cells to examine whether EDCs enhance PXR-mediated transcription. Interestingly, phthalic acid and nonylphenol enhanced PXR-mediated transcription, and this effect was almost equal to the transcriptional level of the cells treated with P, one of the natural steroids that activate PXR (6). In contrast, E2, dithiothreitol (DDT), and bisphenol A had no effects on the transcription (Fig. 1AGo). These effects were dependent on the concentration of ligands and significantly increased at 10 nM, but bisphenol A had no effect on PXR-mediated transcription at any of the concentrations tested (Fig. 1BGo). These data suggest that phthalic acid and nonylphenol are exogenous ligands for PXR. We also determined the concentration of EDCs that enhanced ER-mediated transcription in this assay. Bisphenol A, phthalic acid, and nonylphenol had similar positive effects comparable to those of E2 on ER-mediated transcription (Fig. 1CGo). However, these EDCs had significant effects only at high concentrations, while E2 significantly stimulated the transcription at 10 pM (Fig. 1DGo).



View larger version (26K):
[in this window]
[in a new window]
 
Figure 1. EDCs Enhanced PXR-Mediated Transcription

A, COS-7 cells were transfected with 1 µg of (CYP3A1)2-tk-CAT reporter gene construct together with 0.5 µg of the PXR expression plasmid or empty vector (pSG5). The cells were treated with 10-6 M of estradiol, progesterone, DDT, bisphenol A, phthalic acid, nonylphenol, or ethanol vehicle for 36 h. CAT activity was quantified using an ELISA kit. The results represent the mean ± SD of triplicate determinations. B, COS-7 cells were transfected with 1 µg of (CYP3A1)2-tk-CAT reporter gene construct together with 0.5 µg of the PXR expression plasmid. The cells were treated with increasing concentrations of progesterone, phthalic acid, nonylphenol, or bisphenol A for 36 h. CAT activity was quantified using an ELISA kit. The results represent the mean ± SD of triplicate determinations. Student’s t test was used to determine whether treated values were significantly different from the control with P < 0.05 as the limit of significance (*, P < 0.01, **, P < 0.05). C, COS-7 cells were transfected with 1 µg of (ERE)2-G-CAT reporter gene construct together with 0.5 µg of the ER{alpha} expression plasmid or empty vector (pSG5). The cells were treated with 10-6 M of estradiol, DDT, bisphenol A, phthalic acid, nonylphenol, or ethanol vehicle for 36 h. CAT activity was quantified using ELISA kit. The results represent the mean ± SD of triplicate determinations. D, COS-7 cells were transfected with 1 µg of (ERE)2-G-CAT reporter gene construct together with 0.5 µg of the ER{alpha} expression plasmid. The cells were treated with increasing concentrations of estradiol, phthalic acid, or nonylphenol for 36 h. CAT activity was quantified using an ELISA kit. The results represent the mean ± SD of triplicate determinations. Student’s t test was used to determine whether treated values were significantly different from the control with P < 0.05 as the limit of significance (*, P < 0.01, **, P < 0.05).

 
Effect of EDCs on the Interaction between PXR and Coactivator Proteins
A two-hybrid protein interaction assay was used to examine whether PXR interacted with coactivator proteins, which are very important for the nuclear receptor-mediated transcription (3, 4, 5), in the presence of EDCs. As illustrated in Fig. 2Go, A and B, PXR interacted with two nuclear receptor coactivators, SRC-1 and RIP140, in the presence of P, phthalic acid or nonylphenol, which stimulated PXR-mediated transcription (Fig. 1AGo). However, bisphenol A and dichlorodiphenyltrichloroethane (DDT), which had no effect on PXR-mediated transcription, did not affect this interaction. In contrast, EDCs had no effect on the interaction between PXR and suppressor for gal-1 (SUG1), a component of proteasome (19, 20) (Fig. 2CGo). We also examined the effects of these EDCs on the interaction of another steroid hormone receptor with SRC-1. As shown in Fig. 2DGo, none of the EDCs tested here affected the interaction between vitamin D receptor (VDR) and SRC-1, suggesting that the effects of EDCs are specific for the interactions between PXR and coactivators (Fig. 2DGo). The effect on the PXR interaction with SRC-1 was dependent on the concentration of ligands and significantly increased at 10 nM, a concentration comparable to those that activated PXR-mediated transcription (Fig. 2EGo).



View larger version (19K):
[in this window]
[in a new window]
 
Figure 2. The Effect of EDCs 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, DDT, bisphenol A, phthalic acid, nonylphenol, or ethanol vehicle. PXR-SRC-1 interaction was assessed in a ß-galactosidase assay. The results represent the mean ± SD 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, DDT, bisphenol A, phthalic acid, or nonylphenol, or ethanol vehicle. PXR-RIP140 interaction was assessed in a ß-galactosidase assay. The results represent the mean ± SD 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, DDT, bisphenol A, phthalic acid, or nonylphenol, or ethanol vehicle. PXR-SUG1 interaction was assessed in a ß-galactosidase assay. The results represent the mean ± SD of triplicate independent cultures. D, Yeast expressing the pAS1-VDR 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, DDT, bisphenol A, phthalic acid, nonylphenol, 1,25-dihydroxyvitamin D3, or ethanol vehicle. VDR-SRC-1 interaction was assessed in a ß-galactosidase assay. The results represent the mean ± SD of triplicate independent cultures. E, 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, phthalic acid, or nonylphenol. PXR-SRC-1 interaction was assessed in a ß-galactosidase assay. The results represent the mean ± SD of triplicate independent cultures. Student’s t test was used to determine whether treated values were significantly different from the control with P < 0.05 as the limit of significance (*, P < 0.01).

 
Effect of Phthalic Acid on the Expression of CYP 3A1 in the Rat Liver
The expression of CYP3A1 and ß-actin mRNA in the livers of rats after 24 h exposure to several chemicals was analyzed using the competitive RT-PCR method. The expression of CYP3A1 mRNA increased markedly in rats exposed to phthalic acid, nonylphenol, or P, relative to the expression in rats treated with E2, bisphenol A, or ethanol (Fig. 3Go, A and B). Treatment with E2 moderately enhanced the expression of CYP3A1 mRNA, and bisphenol A had a weak effect on the expression of CYP3A1 mRNA. The housekeeping gene, ß-actin, was used to determine the constitutive level of gene transcription and to control for variations in RNA recoveries from each liver specimen.



View larger version (35K):
[in this window]
[in a new window]
 
Figure 3. The Effect of EDCs on the Expression of CYP3A1 mRNA

A, The total RNA was isolated from livers of rat exposed to phthalic acid, nonylphenol, bisphenol A, estradiol, progesterone, or ethanol and analyzed for the mRNA expression of CYP3A1 gene and ß-actin using competitive RT-PCR. The PCR products were separated on 3% Nu-Sieve agarose gels and visualized by ethidium bromide. B, The band intensities were densitometrically measured and quantified using Image Scanner T-9500 and Bio Image software. The data are averages of two determinations of mRNA from two rats. Student’s t test was used to determine whether treated values were significantly different from the control with P < 0.05 as the limit of significance (*, P < 0.01).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The endocrine disrupting chemical (EDC) has been defined as an exogenous agent that interferes with the synthesis, secretion, transport, binding, action, or elimination of natural hormones in the body, which are responsible for the maintenance of homeostasis, reproduction, development, and/or behavior (21). These chemicals can alter endocrine functions through a variety of mechanisms, including steroid hormone receptor-mediated changes in protein synthesis, interference with membrane receptor binding, steroidogenesis, or synthesis of other hormones (15). Major chemicals, such as phthalates, alkylphenols, bisphenol A, and DDT, have been shown to disrupt estrogenic actions mainly through the binding to estrogen or androgen receptors (15). However, other potential cellular mechanisms have not been vigorously explored. Here, we demonstrate that a kind of EDCs enhanced PXR-mediated transcription at concentrations comparable to those at which they activate ER-mediated transcription and that the expression of endogenous CYP3A1 mRNA increased by treatment with these EDCs. Other EDCs, nonplanar polychlorinated biphenyls, are known to up-regulate CYP3A expressions, and these compounds have been shown to activate PXR-mediated transcription at concentrations comparable to those at which phthalic acid and nonylphenol activated PXR-mediated transcription in the present experiments (12). Since the CYP3A family, which has been shown to be one of the specific genes regulated by PXR (6, 7, 8), hydroxylates endogenous steroids, including cortisol, progesterone, and testosterone (13, 14), it is possible that these EDCs might have some effects on endocrine function by altering steroid hormone metabolism through PXR as well as by the binding to estrogen receptor. This speculation is supported by the fact that rifampicin, a PXR ligand in humans (7), has been shown to affect plasma levels of estradiol and the pharmacokinetics of oral contraception (22, 23). We here demonstrated that, although E2 and bisphenol A had no effect on PXR-mediated transcription in our experiments, both induced moderate or weak CYP3A gene expression in the treated rats. In addition, E2 has been shown to up-regulate the expression of other CYP3A family members (24). Further experiments will be needed to determine the mechanisms by which E2 induces CYP3A expression.

It is becoming increasingly clear that protein-protein contacts between the receptor and the basal transcriptional machinery are important for ligand-mediated transactivation or repression by nuclear receptors. Nuclear receptors directly contact several general transcription factors (GTFs) in the preinitiation complex (PIC). The interaction of receptors with these GTFs is thought to either recruit these limiting factors to PIC assembly or to stabilize the PIC itself (2). Moreover, coactivator proteins, including SRC-1 (17), estrogen receptor-associated protein (ERAP 160) (25), and RIP140 (18), interact in a ligand-dependent manner with several members of the nuclear receptor superfamily to enhance ligand-induced transactivation (3, 4, 26). In this paper, we showed that PXR interacted with the coactivator proteins SRC-1 and RIP140 in a ligand-dependent manner similar to that of other nuclear receptors. In addition, we demonstrated that phthalic acid and nonylphenol enhanced the interaction between PXR and these coactivator proteins. These data suggest that phthalic acid and nonylphenol enhanced PXR-mediated transcription through the interaction of PXR with coactivators. Importantly, these compounds did not affect the interaction between PXR and SUG1. SUG1 has been described as a component of proteasome (20), which is an enzyme complex responsible for major protein degradation (27, 28, 29). We have also reported that SUG1 plays some roles in nuclear receptor degradation by proteasome (30). PXR does not interact with SUG1 in the presence of EDCs, although progesterone enhanced the interaction between PXR and SUG1, suggesting that EDC-occupied PXR might have a conformational change distinct from that of the natural steroid-occupied receptor. The crystallization of the ligand-binding domain of the PXR with progesterone and EDCs should provide important insights into the conformational changes that occur in the liganded receptor. Since PXR interacts with SUG1 in the presence of natural steroids, but does not interact in the presence of phthalic acid or nonylphenol, the EDCs-occupied PXR may be more slowly degraded than the steroid hormone-occupied receptor, which might result in the difference of the PXR-mediated gene expression. Additional studies are ongoing to investigate this question.

In summary, we demonstrated that some EDCs enhanced PXR-mediated transcription through the PXR interaction with coactivator proteins. Also, these EDCs had more positive effects on the expression of CYP3A1 mRNA, which is a target gene through PXR and plays important roles in the metabolism of steroid hormones and exogenous substrates, than did vehicle, other EDCs, or estradiol. These data suggest that EDCs may affect the endocrine functions by altering the steroid hormone metabolism through PXR.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Isopropylidenediphenol (bisphenol A), phthalic acid bis (2-ethylhexel ester) (phthalic acid), P, and E2 were purchased from Sigma (St. Louis, MO). 1,25-Dihydroxyvitamin D3 was kindly provided by Dr. M. R. Uskokovic. 4-Nonylphenol (nonylphenol), which is a mixture of compounds with branched side chains, and DDT were obtained from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan). Male Wistar rats, 200–300 g, bred in our laboratory, were used in these studies. Food and water were available ad libitum. All materials for competitive RT-PCR were purchased from TAKARA Co., Ltd. (Kyoto, Japan).

Transient Transfection Studies
COS-7 cells were cultured in DMEM medium without phenol red supplemented with 10% charcoal-stripped calf serum. The (CYP3A1)2-tk-CAT containing two copies of the CYP3A1 motif, which is a direct repeat of the nonsteroid nuclear receptor half-site sequence AGTTCA separated by a three-nucleotide spacer (31, 32), and pSG5-PXR expression plasmid containing full-length mouse PXR cDNA were obtained from Dr. S. A. Kliewer (6). COS-7 cells were cotransfected with 1 µg of reporter gene construct [CYP3A1)2-tk-CAT or (ERE)2-G-CAT] and 0.5 µg of receptor expression vector (pSG5-PXR or pSG5-ER{alpha}) 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 with either vehicle alone or the indicated concentrations of steroid hormones or EDCs for 36 h. Cell extracts were prepared and assayed for CAT (chloramphenicol acetyltransferase) activity. The amount of CAT was determined with a CAT enzyme-linked immunosorbent assay (ELISA) kit (5 Prime->3 Prime, Inc., Boulder, CO) according to the manufacturer’s protocol.

Preparation of Two-Hybrid Expression Vectors and ß-Galactosidase Assays
All two-hybrid plasmid constructs used the pAS1 (33) and pAD-GAL4 (Stratagene, La Jolla, CA) yeast expression vectors. The pAD-GAL4-SUG1, -SRC-1, and -RIP140 were previously described (26). The full-length PXR was subcloned into the pAS1 to examine the interaction with coactivator proteins in the two-hybrid assay. The pAS1-PXR, -VDR (34), or empty vector (pAS1) was cotransformed with pAD-GAL4-SUG1, -SRC-1, or -RIP140 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 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 or EDCs. The cells were harvested and assayed for ß-galactosidase activity as described previously (35).

Administration of Chemicals and Tissue Collection
The animals were administered phthalic acid, nonylphenol, bisphenol A, E2, P (0.3 mg/kg), or ethanol via intraperitoneal injection. Twenty-four hours after the injection, the animals were killed under ether anesthesia, and the livers 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 iodothiocyanate method (Trizol; Life Technologies, Inc.) according to the manufacturer’s instructions.

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 CYP3A1 and ß-actin, and no PCR products were detected under this condition. According to the protocol of the RNA PCR kit, 0.1 µg of total RNA was reverse transcribed at 42 C for 20 min in 20 µl of reaction solution containing 1xPCR buffer, 5 mM MgCl2, 1 mM deoxynucleoside triphosphates, 2.5 µM random 9 mers primer, 10 U ribonuclease inhibitor, and 5 U AMV reverse transcriptase. The primers for CYP3A1 (36) and ß-actin were as follows: CYP3A1 sense: 5'-ATCCGATATGGAGATCAC-3',3'; antisense: 5'-GAAGAAGTCCTTGTCTGC-3', ß-actin sense: 5'-GTTTGAGACCTTCAACACCC-3'; 3' antisense: 5'-CTTGATCTTCATGGTGCTAG-3'. Each PCR sample contained 1xPCR buffer, 2 mM MgCl2, 10 pmol of primer mix for CYP3A1 or ß-actin, and 1.25 U TAKARA LA Taq. Amplification was carried out on a TAKARA PCR thermocycler with initial denaturation at 94 C for 2 min, followed by 24 cycles of 94 C for 30 sec, 56 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 (Bio Image, Ann Arbor, MI).


    ACKNOWLEDGMENTS
 
The authors thank Dr. Steven A Kliewer for providing mouse PXR.1 expression vector and CYP3A1 reporter vector.


    FOOTNOTES
 
Address requests for reprints to: Hisashi Masuyama, M.D., Ph.D., Department of Obstetrics and Gynecology, Okayama University Medical School, 2–5-1, Shikata, Okayama, 700-8558, Japan.

Received for publication June 15, 1999. Revision received November 23, 1999. Accepted for publication December 1, 1999.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Evans RM 1988 The steroid and thyroid hormone receptor superfamily. Science 240:889–895[Medline]
  2. Tsai MJ, O’Malley BW 1994 Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu Rev Biochem 63:451–486[CrossRef][Medline]
  3. Mangelsdorf DJ, Evans RM 1995 The RXR heterodimers and orphan receptors. Cell 83:841–850[Medline]
  4. Horwitz KB, Jackson TA, Bain DL, Richer JK, Takimoto GS, Tung L 1996 Nuclear receptor coactivators and corepressors. Mol Endocrinol 10:1167–1177[Abstract]
  5. Masuyama H, Jefcoat SC, MacDonald PN 1997 The N-terminal domain of transcription factor IIB is required for direct interaction with vitamin D receptor and participates in vitamin D-mediated transcription. Mol Endocrinol 11:218–228[Abstract/Free Full Text]
  6. Kliewer SA, Moore JT, Wade L, Staudinger JL, Watson MA, Jones SA, Mckee DD, Oliver BB, Willson TM, Zetterstrom RH, Perlmann T, Lehmann JM 1998 An orphan nuclear receptor activated by pregnanes defines a novel steroid signaling pathway. Cell 92:73–82[Medline]
  7. Lehmann JM, Mckee DD, Watson MA, Willson TM, Moore JT, Lehmann JM 1998 The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions. J Clin Invest 102:1016–1023[Abstract/Free Full Text]
  8. Bertilsson G, Heidrich J, Svensson K, Asman M, Jendeberg L, Sydow-Backman M, Ohlsson R, Postlind H, Blomquist P, Berkenstam A 1998 Identification of a human nuclear receptor defines a new signaling pathway for CYP3A induction. Proc Natl Acad Sci USA 95:12208–12213[Abstract/Free Full Text]
  9. Zhang H, LeCulyse E, Liu L, Hu M, Matoney L, Zhu W, Yan B 1999 Rat pregnane x receptor: molecular cloning, tissue distribution, and xenobiotic regulation. Arch Biochem Biophys 368:14–22[CrossRef][Medline]
  10. Blumberg B, Sabbagh Jr W, Juguilon H, Bolado Jr J, van Meter CM, Ong ES, Evans RM 1998 SXR, a novel steroid and xenobiotic-sensing nuclear receptor. Genes Dev 12:3195–3205[Abstract/Free Full Text]
  11. Pascussi JM, Jounaidi Y, Drocourt L, Domergue J, Balabaud C, Maurel P, Vilarem MJ 1999 Evidence for the presence of a functional pregnane X receptor response element in the CYP3A7 promoter gene. Biochem Biophys Res Commun 260:377–381[CrossRef][Medline]
  12. Schuetz EG, Brimer C, Schuetz JD 1998 Environmental xenobiotics and the antihormones cyproterone acetate and spironolactone use the nuclear hormone pregnenolone x receptor to activate the CYP3A23 hormone response element. Mol Pharmacol 54:1113–1117[Abstract/Free Full Text]
  13. Nebert DW, Gonzalez FJ 1987 P450 genes; structure, evolution and regulation. Ann Rev Biochem 56:945–993[CrossRef][Medline]
  14. Juchau MR 1990 Substrate specificities and functions of the P450 cytochromes. Life Sci 47:2385–2394[CrossRef][Medline]
  15. Cooper RL, Kavlock RJ 1997 Endocrine disrupters and reproductive development: a weight-of-evidence overview. J Endocrinol 152:159–166[Abstract/Free Full Text]
  16. Lee PC, Patra SC, Struve M 1996 Modulation of rat hepatic CYP3A by nonylphenol. Xenobiotica 26:831–838.[Medline]
  17. Onate SA, Tsai SY, Tsai M-J, O’Malley BW 1995 Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science 270:1354–1357[Abstract]
  18. Cavailles V, Dauvois S, L’Horset F, Lopez G, Hoare S, Kushner PJ, Parker MG 1995 Nuclear factor RIP140 modulates transcriptional activation by the estrogen receptor. EMBO J 14:3741–3751[Abstract]
  19. vom Bauer E, Zechel C, Heery D, Heine MJS, Garnier JM, Vivat V, Le Douarin B, Gronemeyer H, Chambon P, Losson R 1996 Differential ligand-dependent interactions between the AF-2 activating domain of nuclear receptors and the putative transcriptional intermediary factors mSUG1 and TIF1. EMBO J 15:110–124[Abstract]
  20. Rubin DM, Coux O, Wefes I, Hengartner C, Young RA, Goldberg AL, Finley, D 1996 Identification of the gal4 suppressor Sug1 as a subunit of the yeast 26 S proteasome. Nature 379:655–657[CrossRef][Medline]
  21. Kavlock RJ, Daston GP, DeRosa C, Fenner-Crisp P, Gray LE, Moore J, Rolland R, Scott G, Sheehan DM, Sinks T, Tilson HA 1996 Research needs for the risk assessment of health and environmental effects of endocrine disruptors. A report of the US EPA-sponsored workshop. Environ Health Perspect 104:715–740[Medline]
  22. Lonning PE, Bakke P, Thorsen T, Olsen B, Gulsvik A 1989 Plasma levels of estradiol, estrone, estrone sulfate and sex hormone binding globulin in patients receiving rifampicin. J Steroid Biochem 33:631–635[CrossRef][Medline]
  23. Lebel M, Masson E, Guilbert E, Colborn D, Paquet F, Allard S, Vallee F, Narang PK 1998 Effects of rifabutin and rifampicin on the pharmacokinetics of ethinylestradiol and norethindrone. J Clin Pharmacol 38:1042–1050[Abstract/Free Full Text]
  24. Wang H, Stribel HW 1997 Regulation of CYP3A9 gene expression by estrogen and catalytic studies using cytochrome P450 3A9 expressed in Escherichia coli. Arch Biochem Biophys 344:365–372[CrossRef][Medline]
  25. Halachmi S, Marden E, Martin G, MacKay H, Abbondanza C, Brown M 1994 Estrogen receptor-associated proteins: possible mediators of hormone-induced transcription. Science 264:1455–1458[Medline]
  26. Masuyama H, Brownfield C, St-Arnaud R, MacDonald P 1997 Evidence for ligand-dependent intramolecular folding of the AF-2 domain in vitamin D receptor-activated transcription and coactivator interaction. Mol Endocrinol 11:1507–1517[Abstract/Free Full Text]
  27. Rock KL, Gramm C, Rothstein L, Clark K, Stein R, Dick L, Hwang D, Goldberg AL 1994 Inhibitors of the proteasome block degradation of most cell proteins and the generation of peptides presented on MHC class I molecules. Cell 78:761–771[Medline]
  28. Tanaka K 1995 Molecular biology of proteasomes. Mol Biol Rep 21:21–26[Medline]
  29. Coux O, Tanaka K, Goldberg AL 1996 Structure and functions of the 20S and 26S proteasome. Annu Rev Biochem 65:801–847[CrossRef][Medline]
  30. Masuyama H, MacDonald PN 1998 Proteasome-mediated degradation of the vitamin D receptor (VDR) and a putative role for SUG1 interaction with the AF-2 domain of VDR. J Cell Biochem 71:429–440[CrossRef][Medline]
  31. Huss JM,Wang SI, Astrom A, McQuiddy P, Kasper CB 1996 Dexamethasone responsiveness of a major glucocorticoid-inducible CYP3A gene is mediated by elements unrelated to a glucocorticoid receptor binding motif. J Biol Chem 93:4666–4670
  32. Quattrochi LC, Mills AS, Barwick JL, Yockey CB, Guzelian PS 1995 A novel cis-acting element in a liver cytochrome P450 3A gene confers synergistic induction by glucocorticoids plus antiglucocorticoids. J Biol Chem 270:28917–28923[Abstract/Free Full Text]
  33. Durfee T, Becherer K, Chen P-L, Yeh S-H, Yang Y, Kilburn AE, Lee W-H, Elledge SJ 1993 The retinoblastoma protein associates with the protein phosphatase type I catalytic subunit. Genes Dev 7:555–569[Abstract]
  34. MacDonald PN, Sherman DR, Dowd DR, Jefcoat SC, DeLisle RK 1995 The vitamin D receptor interacts with general transcription factor IIB. J Biol Chem 270:4748–4752[Abstract/Free Full Text]
  35. Fagan R, Flint KJ, Jones N 1994 Phosphorylation of E2F-1 modulates its interaction with the retinoblastoma gene product and the adenoviral E4 19 kDa protein. Cell 78:799–811[Medline]
  36. Morris DL, Davila JC 1996 Analysis of rat cytochrome P450 Isoenzyme expression using semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Biochem Pharmacol 52:781–792[CrossRef][Medline]