From the Departments of Pharmacology, Chemistry & Biochemistry, Laboratory of Environmental Toxicology, University of California San Diego, La Jolla, California 92093-0636
Received for publication, January 21, 2003, and in revised form, February 3, 2003
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ABSTRACT |
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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 steroid xenobiotic receptor (SXR) and the
constitutive active receptor, 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),
Glucuronidation has evolved in vertebrates to catalyze the
transfer of glucuronic acid from uridine 5'-diphosphoglucuronic acid to
soluble non-lipid dependent substances generated as byproducts of
dietary and cellular metabolism (1). Some of the endogenous agents that
are targets for glucuronidation are bilirubin and 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).1 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, whereas 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 occurs 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-butylhydroquinone leads to induction of
UGT1A6, UGT1A9, and UGT2B7 (6, 7). Transcriptional regulation of
UGT1A6 and UGT1A9 occurs after 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) (33). 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. Although the mechanisms surrounding expression of
CYP1A1 after 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.
Materials--
1-Naphthol, 17 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
37 °C in 95% air and 5% CO2 in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum. Cells were
trypsinized 24 h before chemical treatment, and 106
cells were seeded in P100 plates. Cells were treated for 24-72 h with
either TCDD (10 nM) or BNF (20 µM). For
transient transfection experiments, 105 cells were split
into 12-well plates ~24 h before transfection followed by chemical
treatment for 48 h. Chemicals were first dissolved in
Me2SO, and Me2SO concentration in media never
exceeded 0.1% (v/v). Fresh media and chemical treatment were changed
every 24 h.
Enzyme Analysis--
UDP-glucuronosyltransferase analysis was
determined using 1-naphthol and 17 Western Blot Analysis--
HepG2 cells were collected and washed
in cold phosphate-buffered saline and resuspended in ~5 volumes of
phosphate-buffered saline. The cells were sonicated on a 10-s pulse
cycle for 2 min at 6 watts with a Sonic Dismembrator (Fisher). Each
extract was centrifuged first for 5 min at 1000 × g in
a refrigerated Eppendorf centrifuge followed by centrifugation for 10 min at 9000 × g. This supernatant was then centrifuged
at 100,000 × g in a Beckman TL-100 tabletop
ultracentrifuge, and the microsomes were resuspended in
phosphate-buffered saline. Western blots were carried out on Nupage
Bis-Tris 10% polyacrylamide gels as outlined by the manufacturer (Invitrogen). Protein (10 µg) was run at 200 V for 50 min and transferred at 30 V for 1 h to nitrocellulose membranes. The
membrane was blocked with 5% nonfat dry milk in Tris-buffered saline
for 1 h 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 h at room temperature.
Detection of the proteins was conducted by chemiluminescence.
Northern Blot Analysis--
HepG2 cells were treated for 24-72
h with 10 nM TCDD or 20 µM BNF, and total RNA
was prepared using TRIZOL Reagent (Invitrogen). For Northern blots, 15 µg of total RNA was separated through 1% formaldehyde agarose gels.
RNA was subsequently blotted onto GeneScreen membrane (PerkinElmer Life
Sciences) 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 (Invitrogen) and purified using a nucleotide removal kit
(Qiagen). After boiling, the probe was added to hybridization solution
(Stratagene) and incubated with the filters at 68 °C for 6 h
followed by washing in 0.1× SSC (1× SSC = 0.15 M
NaCl and 0.015 M sodium citrate) and 0.1% SDS at 60 °C for 30 min. Visualization was performed using a Storm 860 PhosphorImager (Molecular Dynamics).
UGT1A1 Promoter Cloning--
Genomic clones containing the
UGT1 locus were characterized by screening a human bacterial
artificial chromosome (BAC) library. Enhancer DNA fragments as well as
a
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 internal primers
that carried the mutations were
5'-CTTGGTAAGACCGCAATGAAC-3 and
5-GTTCATTGCGGTCTTACCAAG-3'. 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 Fig. 4A). After digestion of the amplified sequence with
SacI and XhoI, 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 h using LipofectAMINE Plus reagent as described by the
manufacture's protocol (Invitrogen). In general, transfection mixtures
contained 500 ng of UGT1A1-reporter plasmid and 300 ng of
Generation of G418-resistant UGT1A1-luciferase MH1A1L
Cells--
Using HepG2 cells that were seeded at ~106
cells/100-mm tissue culture dish, the pLUGT1A1N plasmid was transfected
as outlined above. After 48 h of growth, the cells were
trypsinized, 1/10 volume of the collected cells was plated into
a 100-mm tissue culture dish, and the cells were 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 was expanded and treated with 5 µM TCDD
for 24 h 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 (22), with all of
the procedures performed at 4 °C. After 48 h of treatment with
10 nM TCDD, 20 µM BNF, or Me2SO, HepG2 cells were washed twice with 10 mM HEPES buffer, pH
7.5, collected by scraping into MDH buffer (3 mM
MgCl2, 1 mM DTT, 25 mM HEPES, pH
7.5), and homogenized with a Potter-Elvehjem tissue grinder driven by
an electric motor. The homogenate was centrifuged at 1000 × 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) 3 times. The
pellet was then lysed in HDK buffer (25 mM HEPES, pH 7.5, 1 mM DTT, 0.4 M KCl) and centrifuged at
105,000 × g for 60 min, and the supernatant was
designated as nuclear extract.
Electrophoretic Mobility Shift Assay--
A complementary pair
of synthetic oligonucleotides,
5'-GCTAGGCACTTGGTAAGCACGCAATGAACAGTCA-3' and
5'-GCTATGACTGTTCATTGCGTGCTTACCAAGTGCC-3', encoding
the 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 [ 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 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 (Fig. 1A).
Treatment of HepG2 cells with 10 nM TCDD for 72 h 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
17 Characterization of the UGT1A1 Promoter and Ah Receptor Binding
Site--
To examine the mechanism of UGT1A1 induction, an
11-kilobase region of the UGT1A1 promoter was cloned from a
human BAC containing the entire UGT1A1 locus.
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
Induction of the
To localize the region on the UGT1A1 gene that controls
induction, further mutational analysis on the E4 clone demonstrated that a sharp drop in induction was observed between bases
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 (Fig. 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 was 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 whether 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 toward 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 anti-mouse AhR and
anti-mouse Arnt antibodies. No inhibition was observed when the binding
reactions were incubated with a mouse anti-human 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 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 controlled in part
by binding of the activated Ah receptor-Arnt complex to the
UGT1A1-XRE sequence.
The human UGT1A1 gene plays an important role in normal
physiology by serving as the only source for the glucuronidation of bilirubin (28), the byproduct 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, oltipraz, 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 HepG2 cells exposed to TCDD and BNF induces UGT1A1, as shown by Western
blot analysis and indirectly by an increase in 17 The identification of the UGT1A1-XRE suggests that Ah
receptor ligands may regulate UGT1A1 in a fashion comparable
with 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.2 Studies in rats show 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). Although not
directly demonstrating induction of UGT1A1, omeprazole therapy has been shown to increase duodenal 3-hydroxybenzo[a]pyrene UGT activity greater than 5-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 UGT1A1 (32),
has very limited capacity to induce CYP1A1, as measured by
induction of CYP1A1-luciferase in TV101 cells (13). Apigenin
may regulate 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, SXR, and the Ah
receptor. These cis-acting regulatory elements are
positioned within a 125-base pair region on the UGT1A1 gene
between bases -naphthoflavone, and benzo[a]pyrene metabolites. Induction was
monitored by increases in protein and 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 was regionalized to bases
3338 to
3287. Nucleotide sequence analysis of this UGT1A1 enhancer region
revealed a xenobiotic response element (XRE) at
3381/
3299. 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 demonstrated that the nuclear protein
was the Ah receptor. These observations reveal that the Ah receptor is involved in human UGT1A1 induction.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-ethynylestradiol,
o-nitrophenyl-
-D-galactopyranoside and
-naphthoflavone (BNF) were purchased from Sigma. TCDD,
1-hydroxybenzo[a]pyrene (1B[a]P), 2B[a]P, 3B[a]P, 4B[a]P, 6B[a]P, 7B[a]P, 8B[a]P, 9-9B[a]P, 10B[a]P,
benzo[a]pyrene-cis-4,5-dihydrodiol, and
benzo[a]pyrene-trans-4,5-dihydrodiol were obtained from
the National Cancer Institute, National Institutes of Health, Chemical Carcinogen Reference Standard Repository (Kansas City, MO). The Bio-Rad
protein assay for protein concentration analysis was purchased from
Bio-Rad. 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 were purchased
from Genbase (San Diego, CA). The
-galactosidase expression vector
PCMV
Gal was purchased from Clontech (Palo Alto,
CA). Thin-layer chromatography plates for enzyme analysis were from Whatman (Clifton, NJ).
-ethynylestradiol as substrates
(18). HepG2 cells were treated with either 10 nM TCDD or 20 µM BNF, and the cells were collected after 24-72 h of
treatment. Whole cell lysates were prepared. Each UGT assay was
conducted in a total volume of 100 µl containing 50 mM
Tris-HCl, pH 7.6, 10 mM MgCl2, 500 µM uridine 5'-diphosphoglucuronic acid, 0.04 µCi of
[14C]uridine 5'-diphosphoglucuronic acid (0.14 nmol), 8.5 mM saccharolactone, 100 µM substrate, and 100 µg of protein. Each reaction was incubated at 37 °C 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.
3712/
7 UGT1A1 promoter fragment containing the TATA
box were amplified by PCR using primers corresponding to sites on the
promoter sequence, as published in NCBI GenBankTM accession
number AF297093 (21). The cap site in AF297093 is at base pair 175,027, as characterized previously (38). 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
3712/
7 UGT1A1 promoter was generated with oligonucleotides 5'-tttaggagctcTCAGACAAAAGGAA-3' and
5-tcctgctcgagGTTCGCCCTCTCCT-3', digested with
SacI/XhoI (the sites are in lowercase 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.
-galactosidase expression vector (PCMV
) as an internal control to
monitor for transfection efficiency. The day after transfection, the
cells were treated with 20 µM BNF, 10 nM
TCDD, or Me2SO for 48 h. The cells were harvested, lysed, and analyzed for luciferase and
-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 s, and the
luciferase activity is expressed as relative light units.
-Galactosidase activities were determined using a standard
o-nitrophenyl-
-D-galactopyranoside
colorimetric assay (with instructions from Promega). Data represent the
mean ± S.D. of experiments performed in duplicate or triplicate.
-32P]CTP in the presence of
Klenow and 25 µM dATP, dGTP, and dTTP. Binding assays
were carried out on ice containing 3 × 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
g of anti-Ah
receptor or anti-Arnt antibody (a generous gift from Christopher
Bradfield) was included in the binding reaction. Protein-DNA complex
was then separated on a 6% nondenaturing polyacrylamide gel using 45 mM Tris-borate, 10 mM EDTA as a running buffer.
Competition assays were performed by adding a 50-fold excess of
unlabeled CYP1A1 DRE or UGT1A1 XRE oligonucleotide. The gels were then
dried, and protein-DNA complexes were visualized by a PhosphorImager.
1600 bp of the CYP1A1 promoter contains 3 Ah
receptor-specific XRE sites. Luciferase activity results from Ah
receptor activation after 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 Me2SO at different time points to evaluate their ability to activate CYP1A1 gene transcription.
Luciferase activity was measured and normalized for protein concentration.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-ethynylestradiol, a substrate that is preferentially
glucuronidated by UGT1A1, was increased 2.5-5-fold in TCDD- or
BNF-treated cells (Fig. 1A). Quantitation of
UGT1A1 RNA transcripts by Northern blot analysis demonstrated that both TCDD and BNF induced UGT1A1 (Fig.
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 17
-ethynylestradiol
glucuronidation by TCDD and BNF correlated with increased levels of
UGT1A1 protein (Fig. 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
(Fig. 1C) and activation of the human
CYP1A1-luciferase gene in TV101 cells (Fig.
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 h in
TV101 cells with TCDD and BNF, maximal levels of UGT1A1 RNA and protein
are evident at around 48 h (Fig. 1C), indicating that
slightly different regulatory events may control the CYP1A1
and UGT1A1 genes. Combined, these results indicate that
induction of UGT1A1 may occur through an Ah
receptor-dependent mechanism.
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Fig. 1.
Induction of UGT1A1 in HepG2
cells. A, 1-naphthol and 17 -ethynylestradiol UGT
activity after treatment with 10 nM TCDD (blue
bars) or 20 µM BNF (brown bars) for 24, 48, and 72 h. B, Northern blot of UGT1A1 RNA in HepG2
cells after treatment with 10 nM TCDD and 20 µM BNF from 8 to 72 h. C, Western blot
analysis of UGT1A1 and CYP1A1 protein in HepG2 cells after treatment
with Me2SO4 (D), 10 nM TCDD or 20 µM BNF for 48 and 72 h. DMSO,
Me2SO.
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Fig. 2.
Induction of CYP1A1-luciferase. TCDD and
BNF were evaluated for their ability to induce luciferase activity in
HepG2 TV101 cells at various times after treatment. Luciferase activity
was expressed as relative light unitsµg of protein. The results
reported at each time represent the average of two separate
determinations. DMSO, dimethyl sulfoxide.
3712 to
7, whereas the individual enhancer sequences
contained bases from
10998/
8134 (Enhancer 1, E1),
8533/
4738
(Enhancer 2, E2), and
3712/
2081 (Enhancer 3, E3). Each plasmid was
transfected transiently into HepG2 cells, and expression of luciferase
activity was determined after treatment of cells for 48 h with
TCDD or BNF (Fig. 3). Our selection of
48 h for the treatment time was selected because we had observed
adequate accumulation of both RNA and protein in TCDD/BNF-treated HepG2
cells. The UGT1A1
3712/
7 luciferase promoter fragment
was induced after treatment with TCDD and BNF. An enhancer sequence
from
3712 to
2081 (E3) relative to the transcriptional start site
was also responsive. Enhancer sequences E2 and E1, which covered a
region from
10998 to
4738, were refractory to both TCDD and
BNF.
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Fig. 3.
Activation of the UGT1A1 promoter in response
to TCDD and BNF. The UGT1A1 promoter and ~11
kilobases of flanking DNA was cloned and characterized from a human BAC
clone. Three fragments of DNA, from 10998 to
8134 (E1),
8533 to
4738 (E2), and
3712 to
2081 (E3) were subcloned into the
pGL3-promoter luciferase plasmid. A heterologous SV40 promoter drove
transcription. A fourth fragment spanning
3712 to
7 (promoter) was
subcloned into the pGL3-basic vector. The UGT1A1 promoter
drove transcription from this plasmid. Each of these plasmids were
transfected into HepG2 cells followed by a 48-h treatment with TCDD or
BNF. Transient transfection experiments were carried out using
luciferase reporter plasmids cotransfected with
-galactosidase to
normalize transfection efficiency. Luciferase activity was measured in
the cytosolic fraction and normalized by
-galactosidase activity.
The fold induction was calculated from those values of the treated
cells compared with Me2SO-treated transfected cells.
3712/
7 promoter-luciferase construct with TCDD
indicates that the transcriptional activation may occur through an Ah
receptor-dependent mechanism. Compounds that have been
shown to be ligands for the Ah receptor are classically polycyclic 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 (Fig. 4). Along with
TCDD induction, we observed a 2-5-fold induction of luciferase activity with the 3- and 9-hydroxybenzo[a]pyrene and the
trans-4,5-dihydrodiol serving as the most efficient
inducers. The use of cell lines deficient in Ah receptor function show
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 elicit transcriptional activation through
and Ah receptor-dependent pathway.
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Fig. 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),
B[a]P metabolites were examined for their ability to induce
UGT1A1 promoter driven luciferase activity. Treatment of
cells was carried out for 48 h with 5 µM samples of
each B[a]P metabolite. B[a]P-cis-4,5-diol,
B[a]P-cis-4,5-dihydrodiol;
B[a]P-trans-4,5-diol,
B[a]P-trans-4,5-dihydrodiol. Activity is expressed as
relative light units (RLU)/µg of protein. Each assay was
conducted in triplicate. DMSO, dimethyl sulfoxide.
3338 and
3287 (Fig. 5A). Sequence
analysis in this region revealed the presence of a single copy of the
Ah receptor XRE motif (CACGCA) starting at position
3309 (Fig.
5B). Using DNA fragments spanning
3525 to
3144,
site-directed mutagenesis was carried out on the conserved
UGT1A1 XRE sequence, altering CACGCA to ACCGCA. Transient transfection of this plasmid demonstrated that the mutated
UGT1A1-XRE resulted in a loss of inducibility (Fig.
5C) by TCDD and BNF.
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Fig. 5.
Functional characterization of the UGT1A1-XRE
sequence. A, an additional series of expression
plasmids were generated from E1 (Fig. 3) to identify the
TCDD-responsive region. A region of ~200 bases (E5) was identified
that supports enhancer activity after treatment with TCDD (blue
bars) and BNF (brown bars). B, nucleotide
sequence of a 130 base pair region spanning from 3425 to
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 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.
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Fig. 6.
Ah receptor binding to UGT1A1-XRE. HepG2
cells were treated with Me2SO or 10 nM TCDD for
48 h. As outlined under "Experimental Procedures," nuclear
extract was isolated from Me2SO-treated (DMSO-E)
or TCDD-treated (TCDD-E) HepG2 cells, and 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 unlabeled UGT1A1-XRE (XRE ×50) or
CYP1A1-DRE (DRE ×50) To determine whether 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.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3483/
3194. Work in our laboratory has recently identified a human 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.
-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 (Fig. 5B). An 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,
whereas 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
after exposure to TCDD and BNF.
3424 and
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. Although 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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ACKNOWLEDGEMENTS |
---|
We 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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FOOTNOTES |
---|
* This work was supported in part by United States Public Health Service Grants GM49135 and ES10337.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 858-822-0288;
Fax: 858-822-0363; E-mail: rtukey@ucsd.edu.
Published, JBC Papers in Press, February 3, 2003, DOI 10.1074/jbc.M300645200
2 A. Galijatovic and R. H. Tukey, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
UGT, glucuronosyltransferase;
TCDD, 2,3,7,8-tetrachlodibenzo-p-dioxin;
CAR, constitutive active
receptor;
SXR, steroid xenobiotic receptor;
BNF, -naphthoflavone;
B[a]P, benzo[a]pyrene;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
DTT, dithiothreitol;
XRE, xenobiotic response element;
DRE, drug response
element.
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