Involvement of the Xenobiotic Response Element …dihydrodiol (B[a]P-trans-4,5diol) was obtained...
Transcript of Involvement of the Xenobiotic Response Element …dihydrodiol (B[a]P-trans-4,5diol) was obtained...
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Involvement of the Xenobiotic Response Element (XRE) in Ah-Receptor Mediated
Induction of Human UDP-glucuronosyltransferase 1A1.
Mei-Fei Yueh, Yue-Hua Huang, Shujuan Chen, Nghia Nguyen and Robert H. Tukey.
Department of Pharmacology, Chemistry & Biochemistry
Laboratory of Environmental Toxicology
University of California, San Diego
La Jolla, California, 92093
Running Title: UGT1A1 induction by the Ah-receptor.
Keywords: Glucuronidation, UGT1A1, dioxin receptor, transcription,
Correspondence:
Robert H. Tukey, Ph.D.
Department of Pharmacology
UCSD
La Jolla, CA 92093-0636
Phone: 858-822-0288
FAX: 858-822-0363
Email: [email protected]
Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on February 3, 2003 as Manuscript M300645200 by guest on February 27, 2020
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UGT1A1 induction by the Ah-receptor
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UDP-glucuronosyltransferase 1A1 (UGT1A1) plays an important physiological role by
contributing to the metabolism of endogenous substances such as bilirubin in addition to
xenobiotics and drugs. The UGT1A1 gene has been shown to be inducible by nuclear
receptors PXR and CAR. In this report, we show that in human hepatoma HepG2 cells
the UGT1A1 gene is also inducible with aryl hydrocarbon receptor (Ah-receptor) ligands
such as 2,3,7,8-tetrachlodibenzo-p-dioxin (TCDD), β-naphthoflavone (BNF) and
benzo[a]pyrene (B[a]P) metabolites. Induction was monitored by increases in protein,
catalytic activity, as well as UGT1A1 mRNA. To examine the molecular interactions that
control UGT1A1 expression, the gene was characterized and induction by Ah-receptor
ligands regionalized to bases -3529 to –3143. Nucleotide sequence analysis of this
UGT1A1 enhancer region revealed a xenobiotic response element (XRE) at –3319/-
3300. The dependence of the XRE on UGT1A1-luciferase activity was demonstrated by
a loss of Ah-receptor ligand inducibility when the XRE core region (CACGCA) was
deleted or mutated. Gel mobility shift analysis confirmed that TCDD induction of nuclear
proteins specifically bound to the UGT1A1-XRE, and competition experiments with Ah-
receptor and Arnt antibodies confirmed that the nuclear protein was the Ah-receptor.
These observations reveal that the Ah-receptor is involved in human UGT1A1 induction.
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Introduction
Glucuronidation has evolved in vertebrates to catalyze the transfer of glucuronic
acid from uridine 5’-diphosphoglucuronic acid (UDPGlcA) to soluble non-lipid dependent
substances generated as by-products of dietary and cellular metabolism (1). Some of
the endogenous agents that are targets for glucuronidation are bilirubin, many of the
steroids as well as several phenolic neurotransmitters. In addition, hundreds of drugs
and xenobiotics are subject to glucuronidation (2;3). The vast numbers of endogenous
and exogenous substances that are susceptible to glucuronidation in humans are
catalyzed by the family of UDP-glucuronosyltransferases (UGTs). A comparison of the
deduced amino acid sequence of the UGTs in mammalian species has helped in
classifying these proteins as members of the UGT1 or UGT2 gene family (4). In
humans, 16 cDNAs have been identified and shown through expression experiments in
tissue culture to encode proteins that display functional glucuronidation activity (3). It is
generally felt that evolutionary constraints associated with the UGT1 family of proteins
leads to more efficient glucuronidation of drugs and xenobiotics, while the UGT2 family
of proteins displays far greater catalytic diversity toward endogenous agents such as
steroids.
Regulation of the UGTs in human tissues is tightly controlled. Analysis of RNA
expression patterns has demonstrated that no two tissues display the same pattern of
UGT gene expression, indicating that regulatory control is occurring in a tissue specific
manner (5). In addition, environmental influences on gene control clearly indicate that
the UGTs are capable of undergoing differential regulation resulting in enhanced
glucuronidation capacity. The treatment of Caco-2 cells with the antioxidant tert-
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butylhydroquinone (tBHQ) leads to induction of UGT1A6, UGT1A9 and UGT2B7 (6;7).
Transcriptional regulation of UGT1A6 and UGT1A9 occurs following exposure to Ah
receptor ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (6;8). Human UGT1A1 has
recently been shown to be under control by agents that induce gene expression in
concordance with the constitutive active receptor (CAR) (9) and the steroid xenobiotic
receptor (SXR). The treatment of HepG2 and Caco-2 cells with the flavonoid chrysin
leads to the induction of UGT1A1 (10-12). Interestingly, flavonoids have also been
shown to induce CYP1A1 (13) in a CYP1A1-luciferase reporter HepG2 cell line (14)
implicating a potential role for the induction of UGT1A1 through a similar mechanism.
One potential mechanism that may link the expression of UGT1A1 and CYP1A1 by
flavonoids is the ability of these agents to activate the Ah receptor. While the
mechanisms surrounding expression of CYP1A1 following activation of the Ah receptor
are well documented (15-17), there is little information linking expression of the human
UGT1A1 gene through an Ah receptor dependent mechanism. Experiments were
undertaken in this study to examine the actions of several Ah receptor ligands to
modulate expression of the UGT1A1 gene.
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Experimental Procedures
Materials. 1-Naphthol, 17α-ethynylestradiol, o-nitrophenyl-β-D-
galactopyranoside (ONPG) and ß-naphtholflavone (BNF) were purchased from Sigma
(St. Louis, MO). 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 1-hydroxybenzo[a]pyrene
(1B[a]P), 2-hydroxybenzo[a]pyrene (2B[a]P), 3-hydroxybenzo[a]pyrene (3B[a]P, 4-
hydroxybenzo[a]pyrene (4B[a]P), 6-hydroxybenzo[a]pyrene (6B[a]P), 7-
hydroxybenzo[a]pyrene (7B[a]P), 8-hydroxybenzo[a]pyrene (8B[a]P), 9-
hydroxybenzo[a]pyrene (9B[a]P), 10-hydroxybenzo[a]pyrene (10B[a]P),
benzo[a]pyrene-cis-4,5-dihydrodiol (B[a]P-cis-4,5diol), benzo[a]pyrene-trans-4,5-
dihydrodiol (B[a]P-trans-4,5diol) was obtained from the National Cancer Institute
Chemical Carcinogen Reference Standard Repository (Kansas City, MO). Bio-Rad
protein assay for protein concentration analysis was purchased from Bio-Rad (Hercules,
CA). Restriction enzymes and T4 ligase were from New England Biolabs (Beverly, MA).
Taq polymerase and the reporter plasmids PGL3-basic vector and PGL3-promoter
vector were from Promega (Madison, WI). Custom oligonucleotides used in PCR
cloning, DNA sequencing and electrophoretic mobility shift assay (EMSA) were
purchased from Genbase (San Diego, CA). The β-galactosidase expression vector
PCMVβGal was purchased from CLONTECH (Palo Alto, CA). Thin-layer
chromatography plates for enzyme analysis were from Whatman (Clifton, NJ)
Cell culture: The human cell lines used in this study are the hepatoma derived
HepG2 and the human CYP1A1-luciferase reporter gene TV101 cell line (14). Both cell
lines were maintained at 37oC in 95% air and 5% CO2 in Dulbecco’s Modified Eagle’s
Medium supplemented with 10% fetal bovine serum. Cells were trypsinized 24 hours
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before chemical treatment and 106 cells were seeded in P100 plates. Cells were treated
for 24 to 72 hours with either TCDD (10 nM) or BNF (20 µM). For transient transfection
experiments, 105 cells were split into 12-well plates approximately 24 hours before
transfection followed by chemical treatment for 48 hours. Chemicals were first dissolved
in DMSO, and DMSO concentration in media never exceeded 0.1% (v/v). Fresh media
and chemical treatment were changed every 24 hours.
Enzyme analysis: UDP-glucuronosyltransferase analysis were determined using
1-naphthol and 17α-ethynylestradiol as substrates (18). HepG2 cells were treated with
either 10 nM TCDD or 20 µM β-napthoflavone (BNF) and the cells collected after 24-72
hours of treatment. Whole cell lysates were prepared. Each UGT assay was conducted
in a total volume of 100 µl containing 50 mM Tris-HCl (pH7.6), 10 mM MgCl2, 500 µM
UDPGlcA, 0.04 µCi [14C]UDPGlcA (0.14 nmol), 8.5 mM sacchrolactone, 100 µM
substrate and 100 µg of protein. Each reaction was incubated at 37oC for 90 min. TLC
plates were visualized with a Molecular Dynamics Storm 820 phosphorimager. Resident
glucuronides were then removed and quantitated by liquid scintillation counting.
Western blot analysis: HepG2 cells were collected and washed in cold
phoshate buffered saline (PBS) and resuspended in approximately 5 volumes of PBS.
The cells were sonicated on a 10 second pulsatable cycle for 2 minutes at 6 watts with
a Sonic Dismembrator (Fisher Scientific). Each extract was centrifuged first for 5
minutes at 1000 x g in a refrigerated Eppendorf centrifuge followed by centrifugation for
10 minute at 9000 x g. This supernatant was then centrifuged at 100,000 x g in a
Beckman TL-100 tabletop ultracentrifuge and the microsomes resuspended in PBS.
Western blots were carried out on Nupage Bis-Tris 10% polyacrylamide gels as outlined
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UGT1A1 induction by the Ah-receptor
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by the manufacturer (Invitrogen). Protein (10 µg) was run at 200 V for 50 min and
transferred at 30V for 1 hour to nitrocellulose membranes. The membrane was blocked
with 5% nonfat dry milk in Tris-buffered saline for 1 hour at room temperature followed
by incubation with anti-human UGT1A1 (19) (1:1000) or antihuman CYP1A1 (20)
(1:5000) in Tris-buffered saline overnight at 4°C. The membranes were washed and
then treated with horseradish peroxidase conjugated anti-mouse (for UGT1A1) or anti-
rabbit (for CYP1A1) antibody for 1 hour at RT. Detection of the proteins was conducted
by chemiluminescence.
Northern blot analysis: HepG2 cells were treated for 24-72 hours with 10 nM
TCDD or 20 µM BNF and total RNA prepared using TRIZOL Reagent (Life
Technologies Gaithersburg, MD). For Northern blots, 15 µg of total RNA was separated
through 1% formaldehyde agarose gels. RNA was subsequently blotted onto
GeneScreen membrane (NEN, Boston MA) by capillary transfer. After transfer, the blot
was stained with methylene blue to visualize RNA for loading efficiency. A 423-bp
fragment recovered by digesting the UGT1A1 cDNA with AvaI/ExoRI was 32P-labeled by
random priming (Life Technologies) and purified using a Nucleotide Removal Kit
(Qiagen). After boiling, the probe was added to hybridization solution (Stratagene) and
incubated with the filters at 68oC for 6 hours followed by washing in 0.1 X SSC and
0.1% SDS at 60oC for 30 min. Visualization was performed using a Storm 860
Phosphorimager (Molecular Dynamics).
UGT1A1 promoter cloning: Genomic clones containing the UGT1 locus were
previously characterized in this lab by screening a human bacterial artificial
chromosome (BAC) library 1. Enhancer DNA fragments as well as a –3716/-5 UGT1A1
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UGT1A1 induction by the Ah-receptor
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promoter fragment containing the TATA box were amplified by PCR using primers
corresponding to sites on the promoter sequence, as published in NCBI GenBank
accession number AF297093 (21). The restriction enzyme sites SacI and XhoI were
incorporated at the 5’ end of the sense and antisense primers, respectively. The PCR
product for the –3715/-5 UGT1A1 promoter was generated with oligonucleotides 5’-
tttaggagctcTCAGACAAAAGGAA-3’ and 5-tcctgctcgagGTTCGCCCTCTCCT-3’,
digested with SacI/XhoI (the sites are in lower case and underlined) and subcloned into
SacI/XhoI digested PGL3-basic vector. This plasmid was identified as pLUGT1A1.
Using the pL1A1Neo plasmid originally cloned in the laboratory (14), the neomycin gene
was removed and cloned into the SalI site of pLUGT1A1, generating the pLUGT1A1N
plasmid.
The sequences of the primers used for the enhancers are as follows. E1, 5’-
atatggagctcAAAGAAGAGAACT-3’ and 5’-atctactcgaGGGAATGATCCTTT-3’. E2, 5’-
atattgagctcTTGCTTGCTGC-3’ and 5’-aatttctcgagACCATGGCTGGTT-3’. E3, 5’-
tttaggagctcTCAGACAAAAGGAA-3’ and 5’-ttacactcgagAACCACTACTAAGC-3’. E4, 5’-
tccttgagctcTTTTTGACACTGGA-3’ and 5’-aaattctcgagCTCATTCCTCCTCT-3’. E5, 5’-
aaagggagctcTAACGGTTCATAAA-3’ and 5’-aaattctcgagCTTACTATGACTG-3’. E6, 5’-
aaagggagctcTAACGGTTCATAAA-3’ and 5’-aatggctcgagGTTATGTAACTAGA-3’. Each
of these amplified inserts were digested with SacI and XhoI site and subcloned into the
SacI/XhoI digested PGL3-promoter vector.
For construction of the mutant UGT1A1-XRE enhancer plasmid, E4 was used as
template. The primers used for amplification of the insert were 5’-
tccttgagctcTTTTTGACACTGGA-3’ and 5’-aaattctcgagCTCATTCCTCCTCT-3’. The two
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UGT1A1 induction by the Ah-receptor
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internal primers that carried the mutations were 5’-CTTGGTAAGACCGCAATGAAC-3
and 5-GTTCATTGCGGTCTTACCAAG-3’ and’. The underlined region represents the
area of the Ah receptor core binding region and the bold and italicized bases are those
that were changed form the normal XRE sequence to disrupt the Ah-receptor binding
region (see Figure 4A). Following digestion of the amplified sequence with Sacl and
Xhol, the insert was cloned into these same sites in the PGL3-promoter vector.
Transfection Assays: HepG2 cells were plated in 12-well tissue culture plates at
30-40 % confluence and transfected after 24 hours using lipofectamine plus reagent as
described by the manufacture’s protocol (Life Technologies). In general, transfection
mixtures contained 500 ng of UGT1A1-reporter plasmid and 300 ng of ß-galactosidase
expression vector (PCMVß) as an internal control to monitor for transfection efficiency.
The day after transfection, the cells were treated with 20 µM BNF, 10nM TCDD or
DMSO for 48 hours. The cells were harvested, lysed and analyzed for luciferase and ß-
galactosidase activity. Luciferase activities were assessed by the methods described
previously (22) using a Monolight 2001 luminometer (Analytical Luminescence
Laboratory, Ann Arbor, MI). Briefly, HepG2 cells were harvested in lysis buffer (1%
Triton, 25 mM Tricine, 15 mM MgSO4, 4 mM EDTA, and 1 mM DTT). Cell lysates were
centrifuged, and 10 µl of the supernatant was mixed with 300 µl of reaction mixture (15
mM potassium phosphate, pH 7.8, 15 mM MgSO4, 2 mM ATP, 4 mM EDTA, 25 mM
Tricine and 1 mM DTT). Reactions were started by adding 100 µl of luciferin (0.3 mg/ml)
dissolved in 0.1 M potassium phosphate, pH 7.4; light output was measured for 10
seconds, and the luciferase activity is expressed as relative light units. β-galactosidase
activities were determined using a standard ONPG colorimetric assay (Promega’s
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UGT1A1 induction by the Ah-receptor
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instructions). Data represent the mean ± SD of experiments performed in duplicate or
triplicate.
Generation of G418-resistant UGT1A1-luciferase MH1A1L cells. Using
HepG2 cells that were seeded at approximately 106 cells per 100-mm tissue culture
dish, the pLUGT1A1N plasmid was transfected as outlined above. After 48 hours of
growth, the cells were trypsinized and 1/10 volume of the collected cells plated into a
100-mm tissue culture dish and the cells exposed to media containing 0.8 mg/ml G418.
After 2-3 weeks, individual colonies of selected cells were removed and re-cultured in
60-mm plates with continued G418 selection. A final round of clonal selection was made
and each clone expanded and treated with 5 µM TCDD for 24 hours followed by
analysis of induced luciferase activity. For these studies, the cell line selected is referred
to as MH1A1L cells.
Preparation of Nuclear Proteins: Nuclear extracts from HepG2 cells were
isolated as described previously (Miller et al., 1983), with all of the procedures
performed at 4oC. After 48hours of treatment with 10 nM TCDD, 20 µM BNF or DMSO,
HepG2 cells were washed twice with 10 mM HEPES buffer (pH 7.5), collected by
scraping into MDH buffer (3 mM MgCl 2 1mM DTT, 25mM HEPES, pH 7.5) and
homogenized with a Potter-Elvehjem tissue grinder driven by an electric motor. The
homogenate was centrifuged at 1000 x g for 5 min, and the pellet was washed with
MDHK buffer (3 mM MgCl2, 1 mM DTT, 25 mM HEPES, pH 7.5, 0.1 M KCl) three times.
The pellet was then lysed in HDK buffer (25 mM HEPES, pH 7.5, 1 mM DTT, 0.4 M
KCl), centrifuged at 105,000 x g for 60 min, and the supernatant was designated as
nuclear extract.
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UGT1A1 induction by the Ah-receptor
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Electrophoretic mobility shift assay: A complementary pair of synthetic
oligonucleotides, 5’-GCTAGGCACTTGGTAAGCACGCAATGAACAGTCA-3’ and 5’ –
GCTATGACTGTTCATTGCGTGCTTACCAAGTGCC-3’, encoding consensus core
sequence (underlined) of the UGT1A1 XRE element were synthesized. For analysis of
Ah-receptor activation, the human CYP1A1 DRE3 oligonucleotides (5’-
GATCCGGCTCTTGTCACGCAACTCCGAGCTCA-3’ and 5’-
GATCTGAGCTCGGAGTTGCGTGAGAAGAGCCG-3’) were used as previously
described (22). Double-stranded oligonucleotides were assembled by annealing equal
concentrations of either the XRE or DRE and then labeled with [α-32P]-CTP in the
presence of Klenow and 25 µM dATP, dGTP and dTTP. Binding assays were carried
out on ice containing 3 x 104 cpm of labeled oligonucleotide, 10 µg of nuclear extract, 2
µg of poly (dI/dC) and 1 µg of salmon sperm DNA in a final reaction volume of 30 µl
containing 25 mM HEPES, Ph 7.5; 1.5 mM EDTA, 1 mM DTT, 10 % glycerol (22). To
examine the specificity of Ah-receptor binding, 100 µg of anti-Ah receptor or anti-Arnt
antibody (generous gift from Christopher Bradfield) was included in the binding reaction.
Protein-DNA complex was then separated on a 6% nondenaturing polyacrylamide gel
using 1 x TBE as a running buffer. Competition assays were performed by adding 50-
fold excess of unlabeled CYP1A1 DRE or UGT1A1 XRE oligonucleotide. The gels were
then dried, and protein-DNA complexes were visualized by phosphorimager.
Induction of CYP1A1 in TV101 cells. Human TV101 cells were derived from
the human hepatoma cell line HepG2 but carry the human CYP1A1 promoter fused to
the firefly luciferase gene (14). The -1600-bp of the CYP1A1 promoter contains three Ah
receptor specific XRE sites. Luciferase activity results from Ah receptor activation
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following treatment with Ah receptor ligands. The TV101 cells were grown under the
same conditions as HepG2 cells but supplemented with 0.8 mg/ml G418. The TV101
cells were treated with TCDD, BNF or DMSO at different time points to evaluate their
ability to activate CYP1A1 gene transcription. Luciferase activity was measured and
normalized for protein concentration.
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Results
Induction of UGT1A1 by TCDD and BNF
The small phenolic compound, 1-naphthol, was used as a substrate to examine
UGT activity in HepG2 cells (Figure 1A). Treatment of HepG2 cells with 10 nM TCDD
for 72 hours led to a time-dependent increase in 1-naphthol UGT activity that
consistently was determined to be 3 fold over untreated cells. Similar treatment of cells
with 20 µM BNF resulted in a 4-5-fold increase in 1-naphthol UGT activity. Simple
phenols have been shown to be glucuronidated by most of the UGT1A proteins (3), with
a preference for UGT1A1, UGT1A6, UGT1A8 and UGT1A9. Glucuronidation of 17a-
ethynylestradiol, a substrate that is preferentially glucuronidated by UGT1A1, was
increased 2.5 to 5-fold in TCDD or BNF treated cells (Figure 1A). Quantitation of
UGT1A1 RNA transcripts by Northern blot analysis demonstrated that both TCDD and
BNF induced UGT1A1 (Figure 1B) in a time dependent fashion. Slightly greater
increases in RNA were observed with BNF treated cells, a pattern that was also
reflected in catalytic activity. It was also observed that induction of UGT1A1 RNA and
17a-ethynylestradiol glucuronidation by TCDD and BNF correlated with increased levels
of UGT1A1 protein (Figure 1C), with BNF generating slightly greater levels of induced
UGT1A1 in microsomes. In HepG2 cells, TCDD and BNF are capable of inducing
CYP1A1, as shown by induction of CYP1A1 (Figure 1C) and activation of the human
CYP1A1-luciferase gene in TV101 cells (Figure 2). Induction of CYP1A1-luciferase in
TV101 cells has been linked to activation of the Ah-receptor (22;23). Although maximal
CYP1A1-luciferase activity is achieved between 8-24 hours in TV101 cells with TCDD
and BNF, maximal levels of UGT1A1 RNA and protein are evident at around 48 hours
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(Figure 1C), indicating that slightly different regulatory events may be controlling the
CYP1A1 and UGT1A1 genes. Combined, these results indicate that induction of
UGT1A1 may be occurring through an Ah-receptor dependent mechanism.
Characterization of the UGT1A1 promoter and Ah-receptor binding site.
To examine the mechanism of UGT1A1 induction, an 11 kb region of the
UGT1A1 promoter was cloned from a human BAC containing the entire UGT1A1 locus1.
UGT1A1 promoter and enhancer regions, cloned by PCR, were subcloned into the
pGL3 basic or pGL3 promoter vectors, respectively. Portions of the regulatory region
including the promoter constituted a fragment from –3716 to -5, while the individual
enhancer sequences contained bases from -11015/-8174 (Enhancer 3, E3), -8551/-
4650 (Enhancer 2, E2), and –3714/-2068 (Enhancer 1, E1). Each plasmid was
transfected transiently into HepG2 cells and expression of luciferase activity determined
following treatment of cells for 48 hours with TCDD or BNF (Figure 3). Our selection of
48 hours for the treatment time was selected since we had observed adequate
accumulation of both RNA and protein in TCDD/BNF treated HepG2 cells. The UGT1A1
–3716/-5 luciferase promoter fragment was induced following treatment with TCDD and
BNF. An enhancer sequence from –3714 to –2068 (E1) relative to the transcriptional
start site was also responsive. Enhancer sequences E2 and E3, which covered a region
from –11001 to –4636, were refractory to both TCDD and BNF.
Induction of the –3716/-5 promoter-luciferase construct with TCDD indicates that
the transcriptional activation may be occurring through an Ah-receptor dependent
mechanism. Compounds that have been shown to be ligands for the Ah-receptor are
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UGT1A1 induction by the Ah-receptor
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classically polycylic aromatic hydrocarbons. To examine this possibility further, we
developed MH1A1L cells carrying the UGT1A1-luciferase plasmid and demonstrated
that classical polycyclic aromatic hydrocarbons composed of hydroxylated
benzo[a]pyrene were capable of inducing UGT1A1 driven luciferase. We examined 1-,2-
,3-,4-,6-,8-,9- and 10-hydroxylated isomers of benzo[a]pyrene in addition to cis- and
trans-4,5-dihydrodiol benzo[a]pyrene (Figure 4). Along with TCDD induction, we
observed a 2-5 fold induction of luciferase activity with the 3- and 9-hydroxy
benzo[a]pyrene and the trans-4,5-dihydrodiol serving as the most efficient inducers.
The use of cell lines deficient in Ah-receptor function have shown that polycyclic
aromatic hydrocarbons induce gene expression in an Ah-receptor dependent fashion
(24). It has also been demonstrated through the use of reporter gene assays that are
controlled by the Ah-receptor enhancer sequence that polycyclic aromatic hydrocarbons
induce transcription through activation of the Ah-receptor (14;25;26). Combined, the
results of TCDD, BNF and B[a]P induction of the UGT1A1 promoter constructs strongly
indicates that these agents are eliciting transcriptional activation through and Ah-
receptor dependent pathway.
To localize the region on the UGT1A1 gene that controls induction, further
mutational analysis on the E1 clone demonstrated that a sharp drop in induction was
observed between bases –3285 and –3337 (Figure 5A). Sequence analysis in this
region revealed the presence of a single copy of the Ah-receptor XRE motif (CACGCA)
starting at position –3311 (Figure 5B). Using DNA fragments spanning –3529 to –3142,
site-directed mutagenesis was carried out on the conserved UGT1A1 XRE sequence
altering the CACGCA to ACCGCA. Transient transfection of this plasmid demonstrated
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UGT1A1 induction by the Ah-receptor
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that the mutated UGT1A1-XRE resulted in a loss of inducibility (Figure 5C) by TCDD
and BNF.
Regulation of UGT1A1 by the XRE core sequence indicates that the CACGCA
motif may be a binding site for the Ah-receptor. Binding of Ah-receptor complex to the
XRE response element in the UGT1A1 promoter region was examined by gel mobility
shift analysis (Figure 6). When nuclear extract prepared from TCDD-treated HepG2
cells was incubated with a 32P-labeled UGT1A1-XRE probe, an induced DNA-protein
complex was detected. Competition for the labeled XRE was evident when excess
unlabeled UGT1A1-XRE as well as CYP1A1-DRE were included in the reaction. A
similar series of experiments were conducted using the CYP1A1 DRE as probe. Binding
of a TCDD inducible nuclear protein to the CYP1A1 DRE could be blocked when the
binding reactions were conducted in the presence of unlabeled UGT1A1-XRE and
CYP1A1-DRE.
To determine if the TCDD activated nuclear protein that associates with the
UGT1A1 XRE is the Ah-receptor, gel mobility shift analysis experiments were carried
out in the presence of antibodies directed towards the Ah-receptor and its dimerization
partner Arnt. Binding of the TCDD induced nuclear protein to the UGT1A1 XRE
sequence was blocked by the IgG purified rabbit antimouse-AhR and antimouse-Arnt
antibodies. No inhibition was observed when the binding reactions were incubated with
a mouse antihuman monoclonal UGT antibody (27), demonstrating that the inhibition of
UGT1A1 XRE binding by the Ah-receptor and Arnt antibodies was specific. As a control
experiment to assure the specificity of the antibodies in blocking the functional Ah-
receptor, a similar experiment was carried out using CYP1A1 DRE as probe. The Ah-
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UGT1A1 induction by the Ah-receptor
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receptor and Arnt antibodies inhibited binding of the TCDD induced nuclear protein to
the labeled CYP1A1 DRE. Combined, these experiments demonstrate that the induction
of UGT1A1 by TCDD is being controlled in part by binding of the activated Ah-
receptor/Arnt complex to the UGT1A1-XRE sequence.
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UGT1A1 induction by the Ah-receptor
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Discussion
The human UGT1A1 gene plays an important role in normal physiology by
serving as the only source for the glucuronidation of bilirubin (28), the by-product of
heme degradation. The gene is expressed differentially in a tissue specific fashion in
humans (29-31) indicating that multiple regulatory factors are involved in UGT1A1
expression. Several recent findings confirm that UGT1A1 can also be regulated by
environmental exposure. Exposure of HepG2 (10) and Caco-2 Cells (11) by specific
bioflavonoids (10;32) induces UGT1A1. In primary human hepatocytes, treatment with
phenobarbital, oltiprz and 3-methylcholanthrene led to the induction of UGT1A1 mRNA
and protein (19). The phenobarbital-type inducer, TCPOBOP, activates the human
UGT1A1 gene through CAR at a nuclear receptor sequence (NR1) between bases –
3483/-3194. Work in our laboratory has recently identified a human steroid and
xenobiotic-receptor (SXR) binding site in this same region (33). These results
demonstrate that the UGT1A1 gene undergoes differential regulation because of tissue
specific expression and inducibility with drugs and xenobiotics. In addition to these
responses, we have demonstrated that the UGT1A1 gene is also regulated by the
human Ah-receptor in response to TCDD, BNF and B[a]P metabolites.
HepG2 cells exposed to TCDD and BNF induces UGT1A1, as shown by Western
blot analysis and indirectly by an increase in 17α-ethynylestradiol UGT activity. The Ah
receptor is functional in these cells as evident from the induction of CYP1A1 protein as
well as regulation of a CYP1A1-luciferase promoter. We have mapped a regulatory
sequence on the UGT1A1 gene that contains an XRE core sequence, which is
positioned in close proximity to the NR1 (9) and SXR binding sites (Figure 5B). An
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UGT1A1 induction by the Ah-receptor
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oligonucleotide encoding bases –3318/-3294 containing the Ah receptor binding
sequence CACGCA associates with the activated nuclear Ah receptor in HepG2 cells.
Mutation of this sequence eliminates binding of the Ah receptor while the generation of
enhancer constructs containing the same mutation leads to a loss of TCDD and BNF
induction of transfected reporter gene activity. It would appear that this single
responsive element plays an important role in regulation of UGT1A1 following exposure
to TCDD and BNF.
The identification of the UGT1A1-XRE suggests that Ah-receptor ligands may
regulate UGT1A1 in a fashion comparable to CYP1A1. Along with results that we have
presented for TCDD and BNF, other polycyclic aromatic hydrocarbons such as
metabolites of B[a]P are capable of inducing UGT1A1. In addition, there is building
evidence that some of the flavonoids modulate gene regulation in part through the Ah
receptor. Chrysin is a potent inducer of UGT1A1 (10) and is able to induce the
expression of CYP1A1, as demonstrated through induction of CYP1A1-luciferase in
TV101 cells (unpublished results). Studies in rats have shown that Ah receptor ligands
such as 3-methylcholanthrene are capable of inducing intestinal Ugt1a1 (34), and it is
well known that 3-methylcholanthene is a potent Ah receptor ligand. Omeprazole, a
benzimidazole used in the treatment of peptic ulcer disease, activates the Ah receptor
and induces CYP1A1 (23). While not directly demonstrating induction of UGT1A1,
omeprazole therapy has been shown to increase duodenal 3-hydroxybenzo[a]pyrene
UGT activity greater than five fold (35). UGT1A1 is abundantly expressed in the small
intestine (31). However, it is important to appreciate that dual regulation of UGT1A1 and
CYP1A1 may not always occur. Apigenin, a flavonoid that is a potent inducer of human
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UGT1A1 induction by the Ah-receptor
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UGT1A1 (32), has very limited capacity to induce CYP1A1, as measured by induction of
CYP1A1-luciferase in TV101 cells (13). Apigenin may be regulating UGT1A1 in a
manner that is independent of the Ah receptor.
As described by Sugatani et al. (9) and expanded by these studies and others
(33), the UGT1A1 gene can be regulated by ligands that activate nuclear receptors
CAR, PXR (SXR) and the Ah-receptor. These cis-acting regulatory elements are
positioned within a 125 base pair region on the UGT1A1 gene between bases –3424/-
3299. The location of these xenobiotic receptors in close proximity to each other may
serve an important biological role in maintaining adequate expression levels UGT1A1.
SXR and CAR are part of the orphan nuclear receptors that are structurally related to
nuclear hormone receptors. It has been proposed that the xenobiotic nuclear receptors
compose a family of regulatory proteins that are involved in steroid and xenobiotic
sensing leading to altered gene expression patterns essential for normal homeostasis
(36;37). Originally postulated to regulate CYP3A genes, these nuclear receptors are
now known to regulate a number of phase I and phase II xenobiotic enzymes. While not
part of the nuclear receptor family, the Ah receptor also serves to modulate phase I and
phase II enzymes in response to environmental stimuli. Thus, regulation of UGT1A1 can
be controlled by numerous endogenous agents that are ligands for SXR and CAR, as
well as xenobiotics that are ligands for SXR, CAR and the Ah receptor.
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UGT1A1 induction by the Ah-receptor
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Acknowledgements: This work was supported in part by United States Public Health
Service Grant GM49135 and ES10337. The authors thank Dr. Joe Ritter, Department of
Pharmacology and Toxicology, Virginia Commonwealth University for a sample of the
anti-UGT1A1 antibody and Dr. Fred Guengerich, Department of Biochemistry,
Vanderbilt University, for a sample of the anti-CYP1A1 antibody. Dr. Christopher
Bradfield, McArdle Laboratory for Cancer Research, University of Wisconsin provided
aliquots of the anti-Ah-receptor and anti-Arnt antibodies, and Dr. Wilbert H. Peters,
Department of Gastroenterology, St Radbound University Hospital, Njimegen, The
Netherlands, provided a sample of the anti-UGT antibody.
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30. Strassburg, C. P., Strassburg, A., Nguyen, N., Li, Q., Manns, M. P., and Tukey, R.
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31. Strassburg, C. P., Kneip, S., Topp, J., Obermayer-Straub, P., Barut, A., Tukey, R.
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B. S., Cabrera, G. Y., Tukey, R. H., and Evan, R. M. (2003) PNAS
34. Grams, B., Harms, A., Braun, S., Strassburg, C. P., Manns, M. P., and Obermayer-
Straub, P. (2000) Arch.Biochem.Biophys. 377, 255-265
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36. Blumberg, B., Sabbagh, W., Jr., Juguilon, H., Bolado, J., Jr., van Meter, C. M.,
Ong, E. S., and Evans, R. M. (1998) Genes Dev. 12, 3195-3205
37. Jones, S. A., Moore, L. B., Shenk, J. L., Wisely, G. B., Hamilton, G. A., McKee, D.
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A., and Moore, J. T. (2000) Mol.Endocrinol. 14, 27-39
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Footnotes
1. Personal communication. Strassburg, C.P., Hiller, A., Geske, D., Karin, M. and Tukey, R.H. Submitted for publication.
LEGENDS Figure 1. Induction of UGT1A1 in HepG2 cells.
A). 1-naphthol and 17α-ethynylestradiol UGT activity following treatment with 10 nM
TCDD or 20 µM BNF for 24, 48 and 72 hours.
B). Northern blot of UGT1A1 RNA in HepG2 cells following treatment with 10 nM TCDD
and 20 µM BNF from 8 to 72 hours.
C). Western blot analysis of UGT1A1 and CYP1A1 protein in HepG2 cells following
treatment with 10 nM TCDD or 20 µM BNF for 48 and 72 hours.
Figure 2. Induction of CYP1A1-luciferase. TCDD and BNF were evaluated for their
ability to induce luciferase activity in HepG2 TV101 cells at various times following
treatment. Luciferase activity was expressed as RLU/µg of protein. The results reported
at each time represent the average of two separate determinations.
Figure 3. Activation of the UGT1A1 promoter in response to TCDD and BNF. The
UGT1A1 promoter and approximately 11 kb of flanking DNA was cloned and
characterized from a human BAC clone. Three fragments of DNA, from –11001 to –
8311 (E3), -8537 to –4636 (E2), and -3714 to –2568 (E1) were subcloned into the
pGL3-promoter luciferase plasmid. A heterologous SV40 promoter drives transcription.
A fourth fragment spanning –3716 to –5 (promoter) was subcloned into the pGL3-basic
vector. The UGT1A1 promoter drives transcription from this plasmid. Each of these
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plasmids were transfected into HepG2 cells followed by a 48-hour treatment with TCDD
or BNF. Transient transfection experiments were carried out using luciferase reporter
plasmids cotransfected with ß-galactosidase to normalize transfection efficiency.
Luciferase activity was measured in the cytosolic fraction and normalized by ß-
galactosidase activity. The fold induction was calculated from those values of the
treated cells compared to DMSO-treated transfected cells.
Figure 4. Induction of UGT1A1-luciferase activity in MH1A1L cells by B[a]P
metabolites. Using plasmid pLUGT1A1N to establish the MH1A1L cells from HepG2
cells (Experimental Procedures), benzo[a]pyrene (B[a]P) metabolites were examined for
their ability to induce UGT1A1 promoter driven luciferase activity. Treatment of cells
was carried out for 48 hours with 5 µM of each B[a]P metabolite. Abbreviations for each
of the metabolites can be found in the Materials section of Experimental Procedures.
Activity is expressed as Relative Light Units (RLU)/µg protein. Each assay was
conducted in triplicate.
Figure 5. Functional characterization of the UGT1A1-XRE sequence.
A). An additional series of expression plasmids were generated from E1 (Figure 3) to
identify the TCDD responsive region. A region of approximately 200 bases (E5) was
identified that supports enhancer activity following treatment with TCDD (solid bars) and
BNF (open bars). B). Nucleotide sequence of a 131 base pair region spanning from –
3426/-3295. Shown in bold are binding regions for SXR, CAR (NR1) and the Ah
receptor (XRE). C). Activity of an enhancer region that contains a mutation in the XRE
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sequence. The reporter plasmid containing either wild type or mutated UGT1A1-XRE
(see Experimental Procedures) was inserted into the PGL3-promoter vector and then
used in transient transfections. The core binding sequence of CACGCA was changed to
ACCGCA. This mutation resulted in a lack of TCDD-dependent induction of
transcriptional activity.
Figure 6 Ah receptor binding to UGT1A1-XRE. HepG2 cells were treated with DMSO
or 10 nM TCDD for 48 hrs. As outlined under “Experimental Procedures”, nuclear
extract was isolated from DMSO treated (DMSO-E) or TCDD treated (TCDD-E) HepG2
cells, 10 µg of protein from each extract was incubated with labeled UGT1A1-XRE or
CYP1A1-DRE probe (indicated at the bottom of the autoradiographs) and subjected to
6% non-denaturing acrylamide gel electrophoresis. Competition was performed in the
presence of a 50-fold excess of unlabelled UGT1A1-XRE (XRE x 50) or CYP1A1-DRE
(DRE x 50) To determine if the induced nuclear protein represented the Ah-
receptor/Arnt complex, binding reactions were also carried out in the presence of
antibody generated toward the mouse Ah-receptor (Anti-AhR) or mouse Arnt (anti-Arnt).
Control experiments were also conducted with an antibody generated toward the UDP-
glucuronosyltransferases (Anti-UGT). The arrow indicates the TCDD inducible protein-
DNA complex.
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Mei-Fei Yueh, Yue-Hua Huang, Shujuan Chen, Nghia Nguyen and Robert H. Tukeyinduction of human UDP-glucuronosyltransferase 1A1
Involvement of the xenobiotic response element (XRE) in Ah-receptor mediated
published online February 3, 2003J. Biol. Chem.
10.1074/jbc.M300645200Access the most updated version of this article at doi:
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