Endocrine Disrupting Chemicals,
Phthalic Acid and Nonylphenol,
Activate Pregnane X ReceptorMediated 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
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 ERmediated 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 metab-
olism through PXR. (Molecular Endocrinology 14:
421–428, 2000)
INTRODUCTION
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 (1–3). The DNA-receptor complex interacts with basal transcriptional
machinery and nuclear receptor coactivator proteins,
resulting in ligand-dependent induction of transcription (3–5). 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 (6–12). 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 (6–8, 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
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Molecular Endocrinology
Copyright © 2000 by The Endocrine Society
421
MOL ENDO · 2000
422
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 (6–8) 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
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. 1A). 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. 1B). 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 ERmediated transcription (Fig. 1C). However, these EDCs
had significant effects only at high concentrations,
while E2 significantly stimulated the transcription at 10
pM (Fig. 1D).
Vol 14 No. 3
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 (3–5), in the presence of
EDCs. As illustrated in Fig. 2, 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. 1A). However, bisphenol A and dichlorodiphenyltrichloroethane (DDT), which had no effect on PXRmediated 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. 2C). We also
examined the effects of these EDCs on the interaction
of another steroid hormone receptor with SRC-1. As
shown in Fig. 2D, 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. 2D). 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. 2E).
Effect of Phthalic Acid on the Expression of
CYP 3A1 in the Rat Liver
The expression of CYP3A1 and ␤-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. 3, 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, ␤-actin, was used to determine the constitutive
level of gene transcription and to control for variations
in RNA recoveries from each liver specimen.
DISCUSSION
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 synthe-
EDCs Activate PXR-Mediated Transcription
423
Fig. 1. EDCs Enhanced PXR-Mediated Transcription
A, COS-7 cells were transfected with 1 ␮g of (CYP3A1)2-tk-CAT reporter gene construct together with 0.5 ␮g 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 ␮g of (CYP3A1)2-tk-CAT reporter gene construct
together with 0.5 ␮g 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 ␮g
of (ERE)2-G-CAT reporter gene construct together with 0.5 ␮g of the ER␣ 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 ␮g of (ERE)2-G-CAT reporter gene construct together with 0.5 ␮g of the ER␣ 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).
sis 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 PXRmediated transcription at concentrations comparable to
those at which phthalic acid and nonylphenol activated
PXR-mediated transcription in the present experiments
MOL ENDO · 2000
424
Vol 14 No. 3
Fig. 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 ␤-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 ␤-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 ␤-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 ␤-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 ␤-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).
EDCs Activate PXR-Mediated Transcription
425
Fig. 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 ␤-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).
(12). Since the CYP3A family, which has been shown to
be one of the specific genes regulated by PXR (6–8),
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 PXRmediated 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
MOL ENDO · 2000
426
(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–
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 EDCoccupied 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.
Vol 14 No. 3
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 pSG5PXR expression plasmid containing full-length mouse PXR
cDNA were obtained from Dr. S. A. Kliewer (6). COS-7 cells
were cotransfected with 1 ␮g of reporter gene construct
[CYP3A1)2-tk-CAT or (ERE)2-G-CAT] and 0.5 ␮g of receptor expression vector (pSG5-PXR or pSG5-ER␣) 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 Prime33 Prime, Inc., Boulder, CO) according to the
manufacturer’s protocol.
Preparation of Two-Hybrid Expression Vectors and ␤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-GAL4SUG1, -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 (SCleu-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 SCleu-trp with or without the indicated concentrations of steroids or EDCs. The cells were harvested and assayed for
␤-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.
MATERIALS AND METHODS
RT-PCR
Materials
Isopropylidenediphenol (bisphenol A), phthalic acid bis (2ethylhexel 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).
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 ␤-actin,
and no PCR products were detected under this condition.
According to the protocol of the RNA PCR kit, 0.1 ␮g of
total RNA was reverse transcribed at 42 C for 20 min in 20
␮l of reaction solution containing 1⫻PCR buffer, 5 mM
MgCl2, 1 mM deoxynucleoside triphosphates, 2.5 ␮M random 9 mers primer, 10 U ribonuclease inhibitor, and 5 U
AMV reverse transcriptase. The primers for CYP3A1 (36)
and ␤-actin were as follows: CYP3A1 sense: 5⬘-ATCCGATATGGAGATCAC-3⬘,3⬘; antisense: 5⬘-GAAGAAGTC-
EDCs Activate PXR-Mediated Transcription
CTTGTCTGC-3⬘, ␤-actin sense: 5⬘-GTTTGAGACCTTCAACACCC-3⬘; 3⬘ antisense: 5⬘-CTTGATCTTCATGGTGCTAG3⬘. Each PCR sample contained 1⫻PCR buffer, 2 mM
MgCl2, 10 pmol of primer mix for CYP3A1 or ␤-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).
427
11.
12.
13.
14.
Acknowledgments
The authors thank Dr. Steven A Kliewer for providing mouse
PXR.1 expression vector and CYP3A1 reporter vector.
Received June 15, 1999. Re-revision received November
23, 1999. Accepted December 1, 1999.
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, 7008558, Japan. E-mail: [email protected]
15.
16.
17.
18.
19.
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