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TOXICOLOGICAL SCIENCES 89(1), 75–82 (2006)
doi:10.1093/toxsci/kfi344
Advance Access publication September 28, 2005
The Aryl Hydrocarbon Receptor Directly Regulates Expression
of the Potent Mitogen Epiregulin
Rushang D. Patel, Dae Joon Kim, Jeffrey M. Peters, and Gary H. Perdew1
Graduate Programs in Molecular Medicine and Molecular Toxicology, The Huck Institutes of the Life Sciences, Department of Veterinary Science and
The Center for Molecular Toxicology and Carcinogenesis, The Pennsylvania State University, University Park, Pennsylvania 16802
Received June 27, 2005; accepted September 20, 2005
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to cause
a large number of adverse effects, mediated largely by its binding to
the aryl-hydrocarbon receptor (AhR) and subsequent modulation
of gene expression. It is thought that AhR mediates these effects
through the untimely and disproportionate expression of specific
genes. However, the exact mechanism, or the genes involved,
through which TCDD leads to these effects is still unknown. This
study reports the discovery of a novel target gene, epiregulin, which
is regulated by TCDD-activated AhR. Epiregulin is a growth
regulator which belongs to the epidermal growth factor (EGF)
family. Using real time quantitative PCR (qPCR), it was established that TCDD upregulates epiregulin gene expression. The
promoter region of epiregulin has a dioxin responsive element
(DRE) 56 nucleotides upstream of the transcription start site, along
with three potential Sp1 binding sites. Chromatin immunoprecipitation (ChIP) assays with an anti-AhR antibody showed promoter
occupancy upon TCDD treatment. Luciferase reporter assays
using a vector harboring the first 125 base pairs of the epiregulin
rat promoter revealed an increase in signal on TCDD treatment,
which was lost upon mutation of the DRE. Epiregulin and TCDD
treatment mediated a dose-dependent increase in primary mouse
keratinocyte growth. These results demonstrate that AhR directly
increases epiregulin expression, which could play an important role
in TCDD mediated tumor promotion observed in rodent models.
Key Words: AhR; epiregulin; transcription; growth factor;
TCDD.
The aryl hydrocarbon receptor (AhR) is a member of the
basic helix-loop-helix (bHLH) PER-ARNT-SIM (PAS) family
of transcription factors. AhR exists in the cytoplasm complexed
with the chaperone HSP90 together with co-chaperones Xassociated protein 2 (also referred to as AIP or ARA9) and p23
(reviewed in Petrulis and Perdew, 2002). The AhR translocates
to the nucleus after ligand binding and heterodimerizes with
AhR nuclear translocator (ARNT). AhR mediates its effects in
1
To whom correspondence should be addressed at Center for Molecular
Toxicology and Carcinogenesis, 309A Life Sciences Building, The Pennsylvania
State University, University Park, PA 16802. Fax: (814) 863-1696. E-mail:
[email protected].
a fashion similar to nuclear hormone receptors. The AhR/
ARNT heterodimer binds to dioxin response elements (DRE)
and modulates transcription of target genes. The consensus
core binding site for the AhR-ARNT complex is TNGCGTG.
ARNT binds the GTG half-site while AhR binds the other half.
The majority of the genes known to be regulated by AhR are
involved in xenobiotic metabolism; the well-characterized
genes include CYP1A1, CYP1A2, CYP1B1, glutathione Stransferase Ya, and aldehyde dehydrogenase 3A1 (reviewed in
Nebert et al., 1993, 2000).
Aside from its role in xenobiotic metabolism, there is an
increasing body of evidence implicating AhR in a diverse range
of patho-physiological processes involving the immune system,
liver, and the cardiovascular system (Heid et al., 2001) as well
as in cell cycle control (Elizondo et al., 2000). AhR activation
in response to xenobiotic exposure leads to a variety of adverse
effects, including tumor promotion. Studies using mice deficient in AhR have provided clues to its role in growth and
development. AhR null mice exhibit increased hepatic fibrosis,
decreased liver weight, abnormalities of the immune system in
the form of a reduced number of peripheral lymphocytes
(Fernandez-Salguero et al., 1995; Schmidt et al., 1996),
decreased fertility, vascular abnormalities in heart, liver, and
uterus, delayed wound healing, impaired skin homeostasis, and
an overall slower growth (Fernandez-Salguero et al., 1997). In
addition, studies using null mice have also shown that AhR
might have a role in the formation of primordial follicles and in
regulating the number of antral follicles, thus affecting development of the mouse ovary (Benedict et al., 2000). Also, the
incidence of cardiac hypertrophy in AhR null mice suggests
a role for AhR in normal cardiovascular development and
angiogenesis (Lund et al., 2003). However, it has not been
clearly defined how AhR mediates these functions. Differential
regulation of genes, other than those involved in xenobiotic
metabolism, could possibly account for these AhR mediated
effects. Alternate mechanisms have also been suggested in the
recent years to explain the different functions of AhR. For
example, it has been shown to interact with retinoblastoma (Rb)
(Ge and Elferink, 1998) and nuclear factor j-B (Koo and Kim,
2003) transcription factors and modulate cell proliferation.
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PATEL ET AL.
Co-expression of AhR and BRG-1 restored Rb-sensitivity to
a tumor cell line, C33A, which could otherwise progress
through the cell cycle even in the presence of active Rb protein
(Strobeck et al., 2000).
A number of exogenous and endogenous ligands are capable
of activating the AhR (Denison et al., 2002). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to be the most
potent exogenous AhR ligand. While there is evidence suggesting that TCDD exerts almost all its toxic effects through the
AhR, the exact mechanisms underlying these effects are not
clear. TCDD has been shown to be more effective as a tumor
promoter than as a tumor initiator, due to the fact that TCDD is
essentially not metabolized (reviewed in Dunson et al., 2000).
TCDD has been shown to promote skin tumor formation in
hairless mice (Poland et al., 1982) as well as promote liver
tumor formation (reviewed in Dragan and Schrenk, 2000). The
importance of AhR in tumor promotion is also highlighted by
the results obtained in a recent study using transgenic mice
expressing a constitutively active AhR where a significant
increase in hepatocarcinogenesis was noted compared to wildtype mice (Moennikes et al., 2004). In addition, studies using
constitutively active AhR have demonstrated that the receptor
can induce tumors in the stomach (Andersson et al., 2002). The
fact that benzo[a]pyrene, a known AhR ligand, is unable to
exhibit its carcinogenic effects in the skin of AhR null mice,
provides further evidence supporting a role for AhR in
tumorigenesis (Shimizu et al., 2000).
Epidermal growth factor (EGF) and other members of its
family are potent peptide growth factors and are involved in
a plethora of physiological and pathological processes through
their signaling properties (Harris et al., 2003). Mutations and/or
over-expression of EGF-family members cause cells to acquire
an oncogenic phenotype. Epiregulin is a relatively newly
identified member of the EGF family, isolated from the
conditioned medium of NIH/3T3/clone 7 cells (Toyoda et al.,
1995). It is secreted as a 46-amino acid single chain polypeptide
with contrasting growth regulatory properties, inhibiting the
growth of several epithelial cell lines while promoting growth
of other cell types, like vascular smooth muscle cells (Koo and
Kim, 2003), hepatocytes (Komurasaki et al., 2002; Toyoda
et al., 1995), and keratinocytes (Shirakata et al., 2000).
Epiregulin is expressed during development as well as later
on in the genitourinary tract (Sekiguchi et al., 2002, 2004),
gastrointestinal tract, vascular smooth muscle cells, and skin. In
the latter two tissues, it has been shown to function in a
paracrine as well as autocrine fashion (Takahashi et al., 2003).
This study demonstrates, for the first time, that ligand
activated AhR binds to its cognate element in the epiregulin
promoter and mutation of this binding element results in loss of
AhR-driven reporter activity. AhR increases epiregulin transcription in cultured primary mouse keratinocytes, immortalized hepatocytes, and mouse hepatoma derived cell line. It is
also shown that epiregulin is capable of significantly enhancing
the proliferation of cultured primary mouse keratinocytes. This
could possibly account for one of the mechanisms by which
TCDD exerts its tumor promotional effects in rodent skin.
MATERIALS AND METHODS
Cell culture. Primary mouse keratinocytes were obtained from two-day
old neonatal C57BL/6 mice and cultured using a previously described method
(Dlugosz et al., 1995). NIH/3T3 cells and Hepa1c1c7 were grown in a-minimal
essential medium (a-MEM) supplemented with 10% FBS (HyClone Laboratories, Logan, UT), 100 IU/ml penicillin, and 0.1 mg/ml streptomycin (Sigma)
at 37C in 5% CO2 atmosphere. Hepatocytes from two-week old C57BL/6N
mice were infected with SV40 virus to prepare temperature sensitive SV40
immortalized mouse hepatocytes in our laboratory (Murray et al., in press)
using a previously described protocol with modifications (Chou, 1985;
Lorenzen et al., 1997). Three cell lines of immortalized hepatocytes were
established. Cells from line 2 were used for experiments. These cells were
maintained in 4% FBS and 0.1 lM dexamethasone at 34C.
Real time quantitative PCR (qPCR). Real time qPCR was performed on
the DNA Engine Opticon (MJ Research, Inc.) using DyNAmo Hot Start SYBR
Green qPCR kit purchased from MJ Research, Inc. cDNA synthesis was carried
out using High Capacity cDNA Archive Kit from Applied Biosystems. The
reverse transcription reactions were set up according to the manufacturer’s
instructions. cDNA synthesized from 50 ng of total RNA was used per qPCR
reaction. Epiregulin mRNA was detected using 5#-TGGGTCTTGACGCTGCTTTGTCTA-3# and 5#-AAGCAGTAGCCGTCCATGTCAGAA-3# primers.
Epiregulin promoter in ChIP assays was detected using 5#-TTCCTGAGAGGGAGGATGACAT-3# and 5#-CCCACCAAGTCGCTGTGACT-3# primers.
Thermal cycling conditions were setup according to the manufacturer’s protocol.
Plasmids. The following plasmids were used for reporter assays:
pEpi125 – derived by inserting rat epiregulin promoter region 125/þ12
(genomic contig: NW_047424.1) in pGL3-Basic luciferase vector, pmutABC –
pEpi125 with all three GT and CT boxes mutated (Sato et al., 2003),
pmutARNT – pEpi125 with ARNT half-site of DRE mutated and pmutAhR –
pEpi125 with AhR half-site of DRE mutated. pEpi125 and pmutABC were a
kind gift of Dr. Kaoru Miyamoto (Fukui Medical University, Japan). pmutARNT
and pmutAhR were modified forms of pEpi125, generated by site-directed
mutagenesis using the QuikChange mutagenesis kit (Stratagene). Forward and
reverse primer pairs used to mutate the ARNT and AhR half-sites, respectively
were (5#-GTAAGTCCTCGCTGGCCTAAGCACC-3# and 5#-GGTGCTTAGGCCAGCGAGGACTTAC-3#) and (5#-GTAAGTCCTCGAGTGCCTAAGCACC-3# and 5#-GGTGCTTAGGCACTCGAGGACTTAC-3#).
Transient transfections and luciferase reporter assays. These experiments were carried out using NIH/3T3 cells, obtained from the American Type
Culture Collection (ATCC). Transfections were performed using Lipofectamine
Plus (Invitrogen). Cells at 70% confluency in 6-well plates were washed with
PBS and 1.5 ml Optimem (Life Technologies) containing 5 ll of Lipofectamine,
1 ll of Plus, and 1.5 lg DNA were added per well. The DNA was comprised of
500 ng of a luciferase reporter vector harboring wild-type or mutated versions of
the rat epiregulin promoter, 100 ng of pDJM/b-gal to control for transfection
efficiency and empty expression vector. After 4 h cells were washed with PBS
and a-MEM containing 10% FBS and antibiotics was added. Cells were allowed
to grow overnight and treated with 10 nM TCDD or DMSO the next day for 7 h
and lysed with 1X cell lysis buffer (25 mM Tris buffer pH 7.8, 2 mM DTT, 2 mM
EDTA, 10% glycerol, and 1% Triton X-100). Luciferase activity from cell
lysates was measured using a Turner TD-20e luminometer (Turrner Designs,
Sunnyvale, CA) the and values were normalized to b-gal values.
Chromatin Immunoprecipitation assay and PCR. Chromatin Immunoprecipitation (ChIP) assays were performed as previously described (Spencer
et al., 2003) with some modifications. Briefly, 80% confluent cells were treated
with 10 nM TCDD or DMSO for 100 min and crosslinked for 8 min at room
EPIREGULIN, A DIRECT AhR TARGET
temperature with 0.33% formaldehyde (SIGMA, F-8775). Cells were harvested
and lysed with 750 ll/flask lysis buffer (1% SDS, 10 mM EDTA, 50 mM TrisHCl pH 8). Lysates were sonicated with Branson Sonifier 250 using 40% duty
cycle and output set at 4 for 8 cycles of 12 pulses each. Protein-DNA complexes
were immunoprecipitated from 200 ll lysate using either 6 ll of rabbit polyclonal anti-AhR antibody (Biomol, SA-210), control IgG or no antibody and
50 ll Goat Anti-Rabbit IgG-Agarose resin (SIGMA, A1027). Resin was washed
once each with low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA,
20 mM Tris-HCl pH 8 and 150 mM NaCl), 1X RIPA buffer (0.1% SDS, 0.1%
sodium deoxycholate, 1% Triton X-100, 1 mM EDTA, 0.5 mM EGTA, 140 mM
NaCl, 10 mM Tris-HCl pH 8) and 13MENG buffer (25 mM MOPS, 2 mM
EDTA, 0.02% sodium azide, 10% glycerol pH 7.5) in that order. Immunoprecipitated protein-DNA complexes were incubated in 300 ll digesting buffer (50
mM Tris-HCl pH 8, 1 mM EDTA, 100 mM NaCl, 0.5% SDS, 100 lg/ml
Proteinase K). DNA was isolated by phenol-chloroform extraction and
concentrated using ethanol precipitation. PCR was performed using following
primers: Epiregulin (5#-TTCCTGAGAGGGAGGATGACAT-3# and 5#CCCACCAAGTCGCTGTGACT-3#; located 107 bases upstream and 75 bases
downstream relative to TATA box), Cyp1A1 (5#-GCCGAGCATCGCACGCAAACC-3# and 5#-GGATCCACGCGAGACAGCAGG-3#; located 1168 and
784 bases upstream relative to TATA box) and GAPDH (5#CATGGCCTTCCGTGTTCCTA-3# and 5#-GCGGCACGTCAGATCCA-3#).
Ten percent DMSO was added to the reactions.
Keratinocyte proliferation studies. Equivalent number of keratinocytes
were seeded in 12 well plates and cultured in low calcium medium (0.05 mM)
containing 8% chelexed FBS for 24 h before treatment. To test the effect of
epiregulin or TCDD on keratinocyte growth, cells were cultured in low calcium
medium containing epiregulin (R&D Systems, 1068-EP), at a concentration
from 1– 20 ng/ml, or TCDD at a concentration from 0.1– 1.0 nM, continuously
with medium change after 24 and 72 h. After 24, 72, or 120 h incubation, cell
number was measured using a Z1 coulter particle counter (Beckman Counter,
Inc., Hialeah, FL).
RESULTS
77
FIG. 1. TCDD increases Epiregulin mRNA. Primary mouse keratinocytes,
SV-40 immortalized hepatocytes and Hepa1c1c7 cells were treated with 10 nM
TCDD and total RNA was extracted at 90 min. Real time qPCR was performed
using cDNA synthesized from 50 ng RNA. Experiments were performed four
times with primary keratinocytes and twice each with immortalized hepatocytes and Hepa1c1c7 cells. qPCR was performed in duplicate for each
biological replicate. Data from representative experiments are presented as
fold increase in relative fluorescence units upon TCDD treatment compared to
untreated samples. Data is normalized to GAPDH mRNA levels within each
cell type. *p < 0.05 as determined by Student’s t-test.
10 nM TCDD for 90 min and a change in the level of epiregulin
mRNA was compared by real time qPCR. TCDD increased
epiregulin mRNA expression by 3.3-fold (Fig. 1) in primary
mouse keratinocytes also. Thus, AhR activation resulted in
epiregulin upregulation in primary cells as well as immortalized
cell lines.
TCDD Treatment Upregulates Epiregulin Transcription
AhR Binds the DRE in the Epiregulin Promoter
The principal mode of action of AhR is through binding its
cognate cis-regulatory element, the DRE, in the promoter
region of its target genes. AhR heterodimerizes with ARNT
and then recruits coregulators to enhance transcription (reviewed in Swanson et al., 2002). Despite the fact that AhR is
implicated in a diverse range of physiological processes, as
discussed in the introduction, few genes outside the gamut of
xenobiotic metabolism have been reported to be directly
regulated by the AhR pathway. In an attempt to identify novel
target genes, whose expression is under the regulation of AhR,
microarray experiments (unpublished data) were performed
using SV-40 immortalized mouse hepatocytes. These experiments revealed epiregulin as one of the target genes whose
mRNA levels increased on TCDD treatment. Microarray results
were confirmed with real-time qPCR (Fig. 1). Effect of TCDD
on epiregulin mRNA levels was also tested in Hepa1c1c7,
a mouse hepatoma cell line. Epiregulin was upregulated by 2.6fold, 90 min after TCDD exposure. As TCDD exerts significant
adverse effects on skin, including tumor promotion, upregulation of epiregulin in response to TCDD was assessed in primary
keratinocytes. Primary mouse keratinocytes were treated with
In accordance with the currently accepted theory, AhR must
bind its response element in the promoter of a gene to directly
regulate its expression. A search for binding sites for AhRARNT heterodimer by sequence analysis identified a consensus
DRE (TCGCGTG) 56 nucleotides upstream of the transcription
start site in the epiregulin promoter in mouse and rat genomic
DNA. To assess if AhR binds the DRE in the epiregulin
promoter sequence, ChIP assays were performed with SV40
virus immortalized mouse hepatocytes treated with TCDD.
DNA fragments isolated from ChIP assays were analyzed using
PCR (Fig. 2A). CYP1A1 was used as a positive control.
GAPDH and immunoprecipitations with no antibody or with
control IgG were used as negative controls. CYP1A1 showed
a strong signal in the TCDD treated sample, compared to
a carrier solvent treated sample, while there was no difference
in the case of GAPDH (data not shown). Immunoprecipitations
with control IgG or no antibody showed minimal background
signals. While the epiregulin signal was more intense in TCDD
samples compared to carrier solvent treated samples, the
difference was less than that of CYP1A1. This difference could
be explained by the fact that epiregulin has only one DRE in its
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PATEL ET AL.
FIG. 2. Epiregulin promoter occupancy by the ligand-activated AhR. (A)
SV-40 immortalized mouse hepatocytes were treated with TCDD or carrier
solvent (DMSO), crosslinked with formaldehyde, lysed and sonicated. ProteinDNA complexes were immunoprecipitated with anti-AhR antibody, control
IgG or no antibody as outlined in materials and methods. The crosslinked DNA
was resolved by heating, PCR amplified using appropriate primers and
visualized on agarose gel. Immunoprecipitations using anti-AhR antibody
were done in duplicate. Experiment was repeated three times and similar results
were obtained. (B) Real time qPCR revealed an increase in AhR binding
to the DRE in epiregulin promoter. Reverse crosslinked DNA from ChIP assay
was analyzed by real time qPCR using primers surrounding the DRE in
Epiregulin promoter. Data is represented as average relative fluorescence units
in TCDD and carrier solvent treated samples across two ChIP assays, each
measured in duplicate. (*p-value ¼ 0.014 on Student’s t-test) AhR IP ¼
immunoprecipitation using anti-AhR antibody; IgG IP ¼ control IgG antibody;
No Ab IP ¼ no antibody; RFU ¼ relative fluorescence units; Input ¼ 10% of
lysate used for immunoprecipitation with different antibodies.
promoter while the CYP1A1 promoter has multiple DREs, and
that AhR upregulates CYP1A1 expression by a greater magnitude than epiregulin. These results were confirmed by realtime
qPCR (Fig. 2B) performed on DNA isolated from ChIP assays.
The relative signal for epiregulin in TCDD treated samples was
1.7-fold greater than carrier solvent treated samples. Combined,
these results demonstrate an increase in epiregulin promoter
occupancy by AhR in response to TCDD treatment.
AhR Driven Promoter Activity Is Dependent on the DRE
To further confirm the role of the DRE in modulating
epiregulin gene expression, a luciferase reporter assay was
performed using a construct containing the first 125 bases of the
rat epiregulin promoter cloned into pGL3-Basic vector
(pEpi125). There is a single nucleotide difference between the
first 125 bases of the mouse (genomic contig: NT_039308.3)
and the rat (genomic contig: NW_047424.1) epiregulin promoters, which is not a part of the DRE or the Sp1 binding sites.
This construct has been previously used by Sekiguchi et al. to
study transcriptional regulation of the epiregulin gene in the rat
ovary (Sekiguchi et al., 2002). They showed by deletion and
mutation analyses that the region encompassing 125 bp
upstream of the transcription start site was essential for
controlling transcription of the epiregulin gene. According to
their results, Sp1/Sp3 proteins bind to one or more of the two CT
boxes and one GT box within this 125 bp region and are
involved in regulating epiregulin gene expression.
The relative contributions of the AhR and Sp1/Sp3 were
studied using plasmids derived from pEpi125 by mutating their
binding sites, the DRE and the CT and GT boxes, respectively
(Fig. 3A). Luciferase reporter plasmid pEpi125 was transiently
transfected in NIH/3T3 cells, while pDJM/b-gal vector was
cotransfected to control for transfection efficiency. Reporter
assays were also carried out with pmutABC plasmid, derived
from pEpi125 by mutating both the CT boxes as well as the GT
box, pmutARNT plasmid, derived from pEpi125 by mutating
the ARNT half-site of DRE and pmutAhR plasmid, derived
from pEpi125 by mutating the AhR half-site of DRE (Fig. 3C).
There was a two-fold increase (2.3-fold) in luciferase reporter
activity observed upon TCDD treatment (Fig. 3B) with the
pEpi125 transfections. Surprisingly, a similar increase in
luciferase activity (2.2-fold) was observed with the pmutABC
transfections compared to vehicle treated control samples.
There was no increase in luciferase activity on TCDD
treatment when the AhR and ARNT binding half-sites were
mutated in pmutARNT and pmutAhR, respectively (~1.2-fold
in pmutARNT and pmutAhR each). Interestingly, the reporter
gene activity in pmutABC transfections was 3.2 times lower
compared to pEpi125 transfections, both in TCDD and carrier
solvent treated samples. This decrease in reporter activity was
not observed when the DRE was mutated (pmutAhR and
pmutARNT transfections). These results, along with ChIP
assays, demonstrate that the activated AhR binds to the DRE
within the epiregulin promoter and is responsible for the
observed increase in transcriptional activity.
Effect of Epiregulin and TCDD on Mouse Keratinocyte
Proliferation
To characterize the functional role of increased epiregulin
expression on cell growth, the effect of epiregulin treatment
was determined using primary mouse keratinocytes. It has
previously been shown that epiregulin stimulates proliferation
of cultured human keratinocytes in a dose-dependent manner
and acts as an autocrine growth factor (Shirakata et al., 2000).
Recombinant human epiregulin at a concentration of 1 ng/ml
resulted in a three-fold increase in cell growth. In the present
EPIREGULIN, A DIRECT AhR TARGET
79
study, primary mouse keratinocytes showed a dose-dependent
increase in cell number when treated with recombinant mouse
epiregulin, even in the presence of other growth factors found
in fetal bovine serum (Fig. 4A). Epiregulin stimulated a statistically significant proliferation of primary mouse keratinocytes
across a dose range of 1–20 ng/ml at 24, 72, and 120 h of
treatment, as determined by ANOVA (p < 0.05). Significant
increase in proliferation was observed at all three time points
with the higher doses (10 and 20 ng/ml), whereas 1 ng/ml
epiregulin induced proliferation only at 72 and 120 h.
Similar cell proliferation studies were performed using
TCDD as a stimulant (Fig. 4B). Primary mouse keratinocytes
were treated with 0.1 nM, 0.5 nM, and 1 nM TCDD, or vehicle
FIG. 3. AhR binds DRE in rat Epiregulin promoter. (A) NIH/3T3 cells were
transfected in 6-well dishes with pEpi125, pmutABC, pmutARNT or pmutAhR
plasmids using Lipofectamine Plus protocol as described in materials and
methods. Cells were subjected to no treatment, carrier solvent (DMSO), or
10 nM TCDD. Transcriptional activity was assessed by measuring luciferase
assay signals. Data is shown as mean and standard deviation of triplicate
transfections. Experiment was repeated four times with similar results.
Statistical analysis is performed using Student’s t-test; *p < 0.001 in both cases
(significant); #p > 0.05 in both cases (insignificant). (B) Graphic representation
of the fold-increase in luciferase assay data from panel A. (C) Schematic
diagram of first 125 bases of rat epiregulin promoter present within the plasmid
pEpi125. Transcription start site is indicated by an arrow. DRE is shown as two
half-sites (AhR and ARNT) represented by ovals. Box A and C are the two CT
boxes and Box B is the GT box. In pmutABC, pmutARNT, and pmutAhR,
mutation of respective elements are shown as blacked out boxes/ovals.
FIG. 4. Epiregulin and TCDD increase primary mouse keratinocyte
proliferation in a dose-dependent manner. 105 cells were seeded per well in
12-well plates. After 24 h, fresh medium containing either recombinant mouse
epiregulin (A) or TCDD (B) was added at indicated doses. Cells were counted
24, 72, and 120 h after adding epiregulin or TCDD. Data is presented as the
mean and standard error of cells measured in triplicate wells. Data presented
was checked for statistical significance by ANOVA and Tukey HSD test (see
text). Observed increase in proliferation was significant for both epiregulin and
TCDD (p < 0.05).
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PATEL ET AL.
solvent, and cells counted at 24, 72, and 120 h after addition of
TCDD. TCDD-induced cell proliferation was statistically
significant, as determined by ANOVA (p < 0.05). Individually,
all three doses induced statistically significant increase in cell
proliferation when compared to vehicle treated cells. However,
a statistically significant difference was not observed amongst
the different doses, as determined by Tukey’s HSD test. When
the effects of different doses were compared at individual timepoints, an increase in the number of cells was observed at all
three time-points with 0.1 nM and 0.5 nM TCDD treatments.
Primary mouse keratinocytes treated with 1 nM TCDD showed
a slight reduction in cell numbers at the 120 h time point. This
could be due to increasing toxicity owing to prolonged TCDD
exposure at this latest time point.
DISCUSSION
TCDD causes a wide range of toxic effects including tumor
promotion, but the exact mechanisms responsible for these
effects are not known. However, most of the efforts investigating genes regulated by AhR have been focused on enzymes
involved in xenobiotic metabolism. Though there are indications for involvement of AhR in a number of cellular processes
(Carlson and Perdew, 2002), there is no well-described role for
the AhR in normal cellular function or tumor promotion that
has been delineated at the gene expression level. In this report
we have examined the ability of the AhR to regulate expression
of the potent mitogen epiregulin.
Skin and liver have been observed to be two of the most
common sites of tumor development in TCDD tumor promotion studies. AhR-dependent upregulation of genes involved
in growth promotion could be an important mechanism for
TCDD mediated carcinogenesis. One of the functional classes
of genes that could mediate these effects would be growth
factors. The ability of TCDD to increase epiregulin mRNA in
cell types known to exhibit TCDD-mediated toxicity, as
revealed by the microarrays and real time qPCR experiments
in hepatocytes and keratinocytes, support the hypothesis that
TCDD exposure could stimulate the growth potential of
precancerous cells through the enhanced expression of potent
mitogens like epiregulin. Further, results from the ChIP assay
and luciferase reporter assay, reveal that an increase in
epiregulin mRNA levels is brought about by ligand-activated
AhR binding to the DRE in the epiregulin promoter and thus,
epiregulin is directly regulated by the AhR in response to
TCDD. Earlier studies have demonstrated that two of the three
Sp1 binding elements, in the epiregulin promoter, are actively
involved in regulating epiregulin mRNA levels. A cooperative
interaction between AhR-ARNT and Sp1 in the CYP1A1 gene
promoter has been reported previously (Kobayashi et al., 1996).
According to their results, binding of either AhR-ARNT or Sp1
to its element in the CYP1A1 promoter facilitated binding of
the other factor and increased reporter activity in a synergistic
fashion. The DRE and GC/CT boxes are found in close
proximity in a number of other AhR regulated gene promoters
like UDP-glucuronosyl transferase, aldehyde dehydrogenase-3,
and quinine reductase. An interaction between the AhR/ARNT
complex and Sp1 has been shown to exist even in the absence of
exogenous ligand, for the Cathepsin D gene promoter (Shelly
et al., 1998). Since the epiregulin promoter has both the AhR
and Sp1 binding elements, it was interesting to determine
whether this kind of cooperative interaction existed at the
epiregulin promoter. Notably, as revealed in the reporter assays,
even when all three Sp1 binding sites were mutated within the
epiregulin promoter (pmutABC transfections), the fold-increase
in luciferase activity on TCDD induction was similar to that
observed in the wild-type promoter, 2.2- and 2.3-fold respectively. However, the overall amount of luciferase signal
decreased by approximately two-thirds in pmutABC transfections. This shows that Sp1 is not necessary for AhRmediated induction of epiregulin gene expression, but enhances
the overall transcription rate of the epiregulin promoter. After
demonstrating that TCDD-activated AhR increases epiregulin
transcription, the next logical step would be to show a corresponding increase in epiregulin secreted in conditioned media.
However, attempts to detect epiregulin using Western blot were
unsuccessful (data not shown).
Increased epiregulin mRNA expression has been observed in
pancreatic cancer tissue samples (Zhu et al., 2000) as well as in
prostate and pancreatic cell lines (Torring et al., 2000; Zhu
et al., 2000). It has been proposed that over-expression of
epiregulin is an important factor in growth regulation of
malignant fibrous histiocytoma (Yamamoto et al., 2004), colon
cancer cells (Baba et al., 2000), and various gynecological
malignancies (Freimann et al., 2004). These data, along with
the fact that ligands and receptors of the EGF-family have been
implicated in a number of tumors, suggest that epiregulin might
be an important factor in mediating TCDD dependent skin
tumor promotion in mice. To show that modulation of
epiregulin levels has a functionally significant role in primary
mouse keratinocytes, cells were treated with increasing concentrations of epiregulin. Epiregulin treatment caused an
increase in proliferation of primary mouse keratinocytes as
observed at three time points (24, 72, and 120 h), even at
a concentration as low as 1 ng/ml. It is worth noting that this
increase occurred in the presence of 8% fetal bovine serum.
Different concentrations of TCDD also increased cell proliferation, albeit at a lower rate than epiregulin. Statistical
analysis revealed this effect of TCDD to be significant, even
though apparently modest. Although it is not possible to rule out
the contribution of other growth factors, it is possible that this
effect is due, at least in part, to an increase in epiregulin level.
Results from these studies also suggest that caution should be
taken when extrapolating results across different species. The
International Agency for Research on Cancer (IARC) updated
its classification of TCDD as a group 1 carcinogen in 1997 from
its previous evaluation that classified it as a group 2B (possible)
EPIREGULIN, A DIRECT AhR TARGET
81
Involvement of deregulated epiregulin expression in tumorigenesis in vivo
through activated Ki-Ras signaling pathway in human colon cancer cells.
Cancer Res. 60, 6886–6889.
Benedict, J. C., Lin, T. M., Loeffler, I. K., Peterson, R. E., and Flaws, J. A.
(2000). Physiological role of the aryl hydrocarbon receptor in mouse ovary
development. Toxicol. Sci. 56, 382–388.
FIG. 5. The DRE is absent in the human epiregulin promoter. First 125
bases of epiregulin promoter, in mouse, rat and human, were aligned.
Nucleotides representing Box A, B, and C and DRE are enclosed. The DRE
sequence is conserved in rodents but lost in human epiregulin promoter. Box A
and C are conserved in all three species while Box B is lost in human promoter.
human carcinogen (IARC, 1997). This upgrade has been
debated in the literature (Cole et al., 2003; Steenland et al.,
2004). However, it is clear from these discussions that not all
data obtained from animal studies can be applied directly to
humans. The toxic effects of TCDD appear to be less pronounced in humans than in rodents. A number of factors could
be responsible for this, including differences in AhR, its
cytosolic or nuclear binding partners, its affinity for ligands,
endogenous ligands, transcriptional coregulators or the battery
of genes whose transcription is modulated by AhR. If a gene
involved in a cellular process is regulated differently in humans
and mice, it could be an important reason for the possible differences in response to TCDD. We compared the first 125 bases
of epiregulin promoter sequence from human, mouse, and rat
and found that the DRE is absent in humans (Fig. 5). Thus,
TCDD-activated AhR may not regulate expression of epiregulin
in humans. However, further studies in human tissue samples or
cell lines will be needed to clarify this issue. In conclusion, we
have shown for the first time that epiregulin is a direct target
gene for the AhR pathway in rodents. Considering the mitogenic potential of epiregulin and the possibility that it might not
be regulated through the AhR pathway in humans, these results point to a potential inter-species difference in regulation
of a gene that could participate in TCDD-mediated tumor
promotion.
ACKNOWLEDGMENTS
We thank Dr. Kaoru Miyamoto at the Fukui Medical University, Japan, for
kindly providing pEpi125 and pmutABC plasmids. We also thank Marcia
Perdew and Dr. Iain Murray for editorial review of the manuscript. This study
was funded by NIH grants ES04869 and ES011834.
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