Cross-talk between the Aryl Hydrocarbon Receptor and Hypoxia
Inducible Factor Signaling Pathways
DEMONSTRATION OF COMPETITION AND COMPENSATION*
William K.
Chan
,
Guang
Yao§,
Yi-Zhong
Gu§, and
Christopher A.
Bradfield§¶
From the
Department of Pharmaceutics and Medicinal
Chemistry, School of Pharmacy and Health Sciences, University of the
Pacific, Stockton, California 95211 and the § McArdle
Laboratory for Cancer Research, University of Wisconsin School of
Medicine, Madison, Wisconsin 53706
 |
ABSTRACT |
The aryl hydrocarbon receptor (AHR) and the
-class hypoxia inducible factors (HIF1
, HIF2
, and HIF3
) are
basic helix-loop-helix PAS (bHLH-PAS) proteins that heterodimerize with
ARNT. In response to 2,3,7,8-tetrachlorodibenzo-p-dioxin,
the AHR·ARNT complex binds to "dioxin responsive enhancers"
(DREs) and activates genes involved in the metabolism of xenobiotics,
e.g. cytochrome P4501A1 (Cyp1a1). The
HIF1
·ARNT complex binds to "hypoxia responsive enhancers" and
activates the transcription of genes that regulate adaptation to low
oxygen, e.g. erythropoietin (Epo). We
postulated that activation of one pathway would inhibit the other due
to competition for ARNT or other limiting cellular factors. Using
pathway specific reporters in transient transfection assays, we
observed that DRE driven transcription was markedly inhibited by
hypoxia and that hypoxia responsive enhancer driven transcription was
inhibited by AHR agonists. When we attempted to support this cross-talk model using endogenous loci, we observed that activation of the hypoxia
pathway inhibited Cyp1a1 up-regulation, but that activation of the AHR actually enhanced the induction of Epo by
hypoxia. To explain this unexpected additivity, we examined the
Epo gene and found that its promoter harbors DREs
immediately upstream of its transcriptional start site. These
experiments outline conditions where inhibitory and additive cross-talk
occur between the hypoxia and dioxin signal transduction pathways and
identify Epo as an AHR-regulated gene.
 |
INTRODUCTION |
The AHR1 regulates a
variety of biological responses to environmentally ubiquitous
polycyclic aromatic hydrocarbons and dioxins (1, 2). In what can be
defined as an adaptive pathway, the AHR up-regulates a battery of XMEs
that often metabolize many of these agonists to more soluble and
excretable products. A classic example of this pathway is observed upon
exposure to benzo[a]pyrene. This chemical binds to the AHR
leading to the up-regulation of a battery of genes including
Cyp1a1, Cyp1a2, and Cyp1b1 (3). The enzymes
encoded by these loci have metabolic activity toward benzo[a]pyrene and thus play an important role in its
elimination (4). At present, we understand many of the molecular events in what appears to be an adaptive response to polycyclic aromatic hydrocarbon exposure. In brief, the up-regulation of genes like Cyp1a1 are regulated by an agonist-induced
heterodimerization between two bHLH-PAS proteins, the AHR and ARNT (5,
6). This heterodimeric pair interacts with DREs upstream of the
regulated promoters leading to an increase in their transcription rate
and a resultant increase in XME activity (7).
Although we have developed models to describe how the AHR regulates the
expression of XMEs, we still have very little knowledge about how this
protein mediates the toxicity of potent agonists like dioxin. The
molecular mechanisms of dioxin-induced effects like lymphoid
involution, epithelial hyperplasia, tumor promotion, teratogenesis, or
even death remain unclear. Moreover, although genetic studies indicate
the involvement of the AHR in these toxic end points, we have
essentially no information that can allow us to conclude that the
AHR-mediated mechanisms underlying these effects are similar to the
regulation of XMEs. In fact, pharmacological evidence suggests that the
mechanisms may be unique (1, 8). Taken in sum, these observations have
led us to postulate the existence of a toxic response pathway that may
be mechanistically distinct from the adaptive one.
In an effort to provide evidence for the existence of alternative
methods of dioxin signaling, we have explored the possibility that
activation of the AHR may inhibit homologous pathways by sequestering
limiting cellular factors (2). This idea has its roots in the
observation that most bHLH proteins function in highly complex
signaling networks that involve multiple combinations of bHLH partners,
with each pair having a unique effect on gene expression and the
cellular environment (9-11). In its simplest form, this model predicts
that the activation of the AHR may sequester ARNT, preventing this
protein from fully participating in other ARNT-dependent
pathways. The recent discovery that HIF1
·ARNT complexes bind to
HREs and activate the transcription of a battery of hypoxia responsive
genes provided a system to test the model of dioxin toxicity described
above (12). Therefore, we examined the cross-talk between the dioxin
and hypoxia signal transduction pathways in both an in vitro
and cell culture model system. Our experiments outline conditions where
reciprocal inhibitory cross-talk between the hypoxia and dioxin signal
transduction pathways occurs and describe a compensatory mechanism that
explains the unexpected effects of dioxin on Epo expression in cell culture.
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EXPERIMENTAL PROCEDURES |
Oligonucleotides--
The underlined nucleotides define the core
sequences of the DRE or HRE: OL73:
5'-TCGAGTAGATCACGCAATGGGCCCAGC; OL74:
5'-TCGAGCTGGGCCCATTGCGTGATCTAC; OL116:
5'-GGCCACATCCGGGACATCACAGA; OL117: 5'-TGGGGATGGTGAAGGGGACGAA; OL135:
5'-GAAGATCTTCCAGTGGTCCCAGCCTACACC; OL200:
5'-GAAGATCTTCTTAGTGATGGTGATGGTGATGGAAGTCTAGTTTGTGTTTGGTTC; OL497:
5'-CGCCCCACCACGCCTCAT; OL498: 5'-AGCCCCCATCCTGTCTTCAT; OL523:
5'-GCCCTACGTGCTGTCTCA; OL524:
5'-TGAGACAGCACGTAGGGC; OL1204: 5'-GGAGATCTGGTACCGGTGGCCCAGGGACTCTGCG; OL1205:
5'-GGAGATCTGATGCCCCCCAGGGGAGGTG.
Materials--
The Cyp1a1 enhancer-luciferase
reporter plasmid, PL1A1N, and the HepG2-101L cells that stably express
this reporter plasmid were a gift from Dr. Robert Tukey (13). The Hep3B
cells were obtained from American Type Culture Collection (Mannassas,
VA). The plasmid pbEP-luciferase, containing the HRE and
promoter from the Epo gene driving luciferase expression,
was a gift of Jaime Caro (14). The plasmid PL412, containing the HRE
from the Epo gene fused to the SV40 promoter driving
luciferase expression, was described previously (15). The plasmid
PL1018, containing the Epo promoter driving luciferase
expression was constructed by amplifying the 327-bp Epo
promoter from the pbEP-luciferase plasmid using OL1204 and 1205 as
primers. The amplification product was cloned into the BglII
site of the promoterless luciferase reporter plasmid pGL2-Basic
(Promega, Madison, WI). The plasmid pCH110 containing the
-galactosidase gene, driven by the SV40 early promoter was obtained
from Pharmacia Biotech Inc. (Piscataway, NJ). The plasmid pGemEpo2
harbors a 998-bp fragment of the human EPO cDNA. This cDNA was
amplified using OL497 and OL498 as primers. The template was a reverse
transcription reaction generated from CoCl2 induced
mRNA from Hep3B cells. The fragment of the human EPO cDNA was
cloned into the pGEM-T vector in the SP6 orientation (Promega, Madison,
WI). The plasmid PL449 harbors a 340-bp fragment of the human CYP1A1
cDNA. This cDNA was amplified from a HepG2 cDNA library
using OL116 and OL117 as primers. The cDNA fragment was cloned into
the PGEM-T vector in the SP6 orientation. The plasmid phuAHR1267
harbors the first 1267 nucleotides of the human AHR. This cDNA was
amplified from phuAHR using OL135 and OL200 as primers (16). The
cDNA fragment was cloned into PGEM-T in the SP6 orientation.
DNA Gel Shift Assay--
Oligonucleotides containing the DRE
(OL73/74) and the HRE (OL523/524) were synthesized by Life
Technologies, Inc. (Grand Island, NY) (17). The double-stranded
32P-labeled oligonucleotide probes were generated by
kinasing a single-stranded oligonucleotide with
[
-32P]ATP (10 Ci/mmol, NEN Life Science Products Inc,,
Boston, MA) and then allowing it to anneal with a 10-fold excess of its
complementary strand. The human HIF-1
(PL611) and AHR (phuAHR)
proteins were expressed in reticulocyte lysate as described previously
(15, 18). The human ARNT protein was generated using a baculovirus expression system (19). Based upon our previous estimates,
approximately 1-2 fmol of each protein was used in a gel shift assay.
The AHR/ARNT and HIF-1
/ARNT samples were "activated" by
incubation at 30 °C for 10 and 30 min, respectively, followed by the
addition of 200 ng of poly(dI-dC) and 500,000 cpm of the
32P-labeled oligo. In experiments where ligand activation
of the AHR was required, the agonist
NF was added at the beginning
of the activation step. Following addition of oligo, samples were incubated for 10 min at room temperature prior to loading. Samples were
loaded onto a 4% nondenaturing polyacrylamide gel buffered with
0.5 × TBE and subjected to electrophoresis at constant voltage of
135 volts for approximately 3 h at 4 °C. Migration of DNA bound complexes were analyzed by autoradiography of the dried gels.
Nuclear Extract and Microsome Preparation--
Nuclear extracts
and microsomes were prepared from Hep3B cells as follows. Cells were
harvested, washed with phosphate-buffered saline, and subjected to
centrifugation at 100 × g for 5 min at 4 °C. The
washed cells were resuspended in "lysis buffer" (20 mM
HEPES-KOH, pH 7.8, containing 1.5 mM MgCl2, 10 mM KCl, 0.5 mM phenylmethylsulfonyl fluoride,
and 2 mg/ml leupeptin) and subjected to three cycles of freeze-thaw,
followed by centrifugation at 1,000 × g for 10 min at
4 °C. The supernatant was subjected to centrifugation at
100,000 × g for 1 h. The microsomal pellet was resuspended in 15 mM, Tris-HCl, pH 8, containing 250 mM sucrose. Nuclear extracts were prepared by resuspending
the nuclei-enriched fraction (1,000 × g supernatant
from above) in 50 µl of "nuclear extract buffer" (20 mM HEPES-KOH, pH 7.8, containing 0.42 M KCl, 1.5 mM MgCl2, 20% glycerol, 0.5 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, and 2 mg/ml leupeptin). After incubation on ice for 30 min, the sample was
subjected to centrifugation at 16,000 × g for 30 min
at 4 °C. The supernatant was used directly and defined as nuclear extract.
Western Blot Analysis--
Anti-AHR monoclonal antibody (VG9)
was raised in mice against the C-terminal half of the human AHR (20).
Anti-ARNT polyclonal antibodies were a gift from Dr. Alan Poland (21).
Anti-HIF-1
polyclonal antibodies, R35732B, were raised in rabbits
against a protein fragment corresponding to amino acids 328-527 of the human HIF-1
(15). Rabbit anti-CYP1A1 polyclonal antibodies were
purchased from Human Biologics, Inc. (Phoenix, AZ). Poly(dI-dC) was
purchased from Pharmacia Biotech Inc. Western blot analyses of ARNT,
AHR, and CYP1A1 were performed using alkaline phosphatase-coupled secondary antibodies and developed by a colorimetric assay, as described previously (19). The HIF-1
Western blot was performed using horseradish peroxidase-coupled secondary antibodies and developed with the chemiluminescent "SuperSignal" substrate
(Pierce, Rockford, IL).
Ribonuclease Protection Assays--
The effects of 75 µM CoCl2 and/or 10 nM dioxin on
the mRNA levels from the Epo and Cyp1a1 genes
were monitored by an RPA assay using Hep3B cell lysates as the target
(Direct Protect Lysate RPA kit, Ambion, Austin, TX) (22). A
350-nucleotide EPO riboprobe was generated with T7 RNA polymerase using
the template pGemEpo2 that had been linearized with BglII. A
390-nucleotide CYP1A1 riboprobe was generated with T7 polymerase using
PL449 that had been linearized with SalI. As a loading
control in EPO measurements, a 265-nucleotide AHR riboprobe was
generated with T7 polymerase using phuAHR1267 as template that had been
linearized with NdeI. Similarly, as a loading control in
CYP1A1 measurements, a 580-nucleotide AHR riboprobe was generated with
T7 polymerase using phuAHR1267 that had been linearized with
EcoRI. Analysis was performed on lysates from cells grown in
75-cm2 flasks at 37 °C at 5% CO2. Upon
harvest, cells were washed once with 5 ml of cold phosphate-buffered
saline and scraped into 5 ml of cold phosphate-buffered saline,
followed by centrifugation at 100 × g for 5 min at
4 °C. The pellet was resuspended into 1 ml of "Direct Protect
Lysis Solution" and stored at
20 °C until use. The CYP1A1 and
EPO riboprobes generated protected fragments of 340 and 310 bp,
respectively. The 265-nucleotide AHR riboprobe generated a 225-bp
protected fragment. The 580-nucleotide AHR riboprobe generated a 539-bp
protected fragment. Riboprobes were synthesized using Maxiscript
in vitro translation kit (Ambion), with a specific activity
of at least 1 × 108 cpm/mg RNA and a 60-90%
incorporation. The riboprobes (1 × 105 cpm each) were
added to the lysates and incubated at 37 °C overnight. Following
this incubation, 0.5 ml of an RNase A/T1 mixture (Ambion's Direct
Protect Digestion Buffer) was added and incubated at 37 °C for 40 min. Sodium Sarcosyl (10%, 20 µl) and proteinase K (20 µg/ml, 10 µl) were then added, followed by another 37 °C incubation for 30 min. Samples were precipitated by the addition of 0.5 ml of isopropyl
alcohol, stored at
20 °C for 30 min, and subjected to
centrifugation at 16,000 × g for 15 min at 4 °C.
The pellets were dried and resuspended into 10 µl of buffer, before
being loaded onto a 4% acrylamide gel containing 8 M urea.
After electrophoresis (50 constant watts for 90 min), the gel was dried
and the protected fragments were quantitated using a Fuji BAS2000 PhosphorImager.
Choice of Model Cell Lines and Pharmacology--
To induce AHR
activation, we chose dioxin and/or
NF. Dioxin was chosen for most
in vivo experiments because it is one of the most potent AHR
agonists ever described and it is not known to activate other cellular
signal transduction pathways. The agonist
NF was used in some
experiments because this molecule is known to have an affinity for the
AHR that is approximately 3 orders of magnitude lower than dioxin (23).
Thus, its relative potency can be used as pharmacological proof that
the AHR and DREs are involved in the observed phenomenon. To
up-regulate HIF1
in vivo, we chose to expose cells to
75-100 µM CoCl2 (12). Although we have
reproduced most of our observations using hypoxic conditions (i.e. 1% O2, data not shown), we have found the
addition of the CoCl2 yields identical results, is less
labor intensive, and allows more experimental flexibility. Our choice
of Hep3B cells was based upon the observation that this hepatoma cell
line displayed both the AHR and HIF1
signal transduction pathways.
The use of the HepG2 derived CYP1A1 reporter line, 101L cells, was a
matter of convenience and was used sparingly, primarily as a
confirmation of our data generated in Hep3B cells (13).
Transient Transfection Experiments--
Hep3B cells were
transiently transfected using a LipofectAMINE reagent (Life
Technologies, Inc.). Cells were grown to 50% confluence in 6-well
plates before transfection. A transfection solution of 1.0 ml of
serum-free minimal essential medium containing plasmid (0.5 µg of
PL1A1N, 1.0 µg of PL412, 1.0 µg of PL1018 or 1.0 µg of
pbEP-luciferase) and 5 µl of LipofectAMINE reagent was added to each
well. Each plasmid transfection was accompanied by 0.2 µg of the LacZ
reporter pCH110, which acted as an efficiency control. Transfection was
allowed to occur by incubation at 37 °C for 5 h. After this
time, minimal essential medium plus 10% fetal bovine serum was added
prior to addition of dioxin or CoCl2. Dioxin and/or
CoCl2 treatments were started at this time, or after 20 additional hours of incubation. Cells were then washed with 2.0 ml of
phosphate-buffered saline per well, followed by addition of 150 µl of
cell lysis buffer (Promega, Madison, WI). Following 5 min at room
temperature, the lysed cells were collected and cleared by
centrifugation at 12,000 × g for 1 min. The
supernatants were assayed for luciferase activity (20 µl/reaction)
and
-galactosidase activity (6-10 µl/reaction) as described
previously (15).
Statistics--
Differences between treatment groups were
identified by the Bonferrani Multiple Comparison Test (24). Statistical
significance was set at p < 0.05.
 |
RESULTS |
Gel Shift Assays--
In the early stages of this work, we asked
whether the activation of either the dioxin or hypoxia pathway would
inhibit signaling by the other. As an initial test of this idea, we
employed gel shift assays where the readout of pathway activity was the
interaction of each heterodimer with their corresponding response
element. In gel shift assays with fixed amounts of radiolabeled DREs,
AHR, and ARNT, we first identified the specific complex using the well characterized inducibility of AHR agonist
NF. Using this system, we
observed that the addition of increasing amounts of HIF-1
protein
inhibited the amount of AHR·ARNT·DRE complex in a
dose-dependent fashion (Fig.
1, A and C). In gel
shift assays using fixed amounts of radiolabeled HREs, HIF1
, and
ARNT, we first identified the specific complex by demonstrating its
formation only in the presence of both bHLH-PAS proteins. In a manner
similar to that described above, we observed that increasing the amount
of AHR in the system inhibited the formation of the
HIF-1
·ARNT·HRE complex (Fig. 1, B and D).
Interestingly, this inhibition of HIF1
·ARNT·HRE binding by AHR
was observed in both the presence and absence of the AHR ligand
NF.
Although, in the presence of the ligand, the competition was increased
compared with lack of agonist (compare 4 × plus or minus
NF,
Fig. 1, B and D). Analysis from three separate
experiments revealed strong reproducibility of these conclusions (Fig.
1, C and D).

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Fig. 1.
Gel shift assays to examine in
vitro competition between HIF-1 and
AHR. A, inhibition of the AHR·ARNT·DRE complex by
the addition of HIF1 . The arrow indicates the
AHR·ARNT·DRE complex formed in the presence or absence of 10 µM NF. Increasing amounts of HIF-1 (1, 2, and 4 times in excess relative to the amount of AHR) were added as competitor
to the incubation mixture as illustrated. AHR or ARNT alone was
included as negative controls. B, inhibition of the
HIF1 ·ARNT·HRE complex by the addition of AHR. The
arrow indicates the HIF1 ·ARNT·HRE complex. Increasing
amounts of AHR (2 and 4 times in excess relative to the amount of
HIF1 ) were added as competitor to the incubation mixture in the
presence or absence of 10 µM NF as illustrated.
HIF1 or ARNT alone was included as negative controls. C,
histogram of the relative radioactive counts of 32P-labeled
AHR·ARNT·DRE complexes from A. The gels as illustrated
in A were put on a PhosphorImager overnight and the
radioactive counts from the bands containing the AHR·ARNT·DRE
complexes were measured. All the counts are normalized with the control
(AHR + ARNT in the presence of NF). The data shows the mean from
three separate experiments and the error bars show the
standard error of the mean. D, histogram of relative
radioactive counts from the 32P-labeled
HIF1 ·ARNT·HRE complex. The gels as illustrated in B
were put on a PhosphorImager overnight and the radioactive counts from
the bands containing the HIF1 ·ARNT·HRE complexes were measured.
All the counts are normalized with the control (HIF1 + ARNT in the
absence of NF). The data shows the mean from three separate
experiments and the error bars show the standard error of
the mean.
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Cross-talk with Synthetic Reporters--
We also examined the
cross-talk phenomenon in Hep3B cells using transient transfection of
reporter constructs that were specific for activation of either the
dioxin or hypoxia pathway. To characterize the AHR/ARNT mediated
response, a time course was performed in cells transiently transfected
with the dioxin inducible reporter PL1A1N (13). In keeping with the
expected pharmacology of this reporter, luciferase activity was
markedly up-regulated after 5, 10, and 23 h of dioxin exposure
(14-, 16-, and 27-fold, respectively), but was unaffected by exposure
to CoCl2 (Fig. 2).
Importantly, co-exposure to both dioxin and CoCl2 led to a
decrease in the luciferase response at all time points, as compared
with dioxin alone (Fig. 2). To characterize the HIF1
/ARNT response,
a similar experiment was performed using a reporter plasmid, PL412,
with a single HRE element derived from the Epo gene upstream
of an SV40 promoter driving luciferase expression (Fig.
3). In keeping with the expected
pharmacology of this reporter we observed induction of luciferase
activity at all time points (4-, 9-, and 10-fold for 5, 10, and 23 h, respectively). Importantly, co-exposure to both CoCl2
and dioxin led to a decrease in the luciferase response at all time
points, as compared with CoCl2 alone (Fig. 3).

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Fig. 2.
Cross-talk between dioxin and
CoCl2 characterized using a DRE driven reporter.
Above, schematic of the PL1A1N plasmid.
CYP1A1-DRE is the 1612-bp region from the Cyp1A1 gene that
harbors three DREs. SV40 is the minimal early promoter from the SV40
genome. Arrow indicates the predicted transcriptional start
site. LUC is the luciferase open reading frame. Below,
results from transient transfection assays with PL1A1N under various
chemical treatments at different time points. RLU, relative
light units. All transfections were normalized to a -galactosidase
internal control (see "Experimental Procedures"). The four
treatments are dimethyl sulfoxide (DMSO) control,
CoCl2 (100 µM), dioxin (10 nM),
or dioxin (10 nM) plus CoCl2 (100 µM). Each time point represents the mean of triplicates
and the error bars represent the standard error of the mean.
Statistical analysis is described in text. For each time point, those
determinations not sharing a superscript (a, b, or
c) are significantly different (p < 0.05).
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Fig. 3.
Cross-talk between dioxin and
CoCl2 characterized using an HRE driven reporter.
Above, schematic of the PL412 plasmid. EPO-HRE is the 193-bp
region derived from the Epo gene that harbors a single HRE.
SV40 is the minimal early promoter from the SV40 genome.
Arrow indicates the predicted transcriptional start site.
LUC is the luciferase open reading frame. Below,
results from transient transfection assays with PL412 under various
chemical treatments at different time points. RLU, relative
light units. All transfections were normalized to a -galactosidase
internal control (see "Experimental Procedures"). The four
treatments are dimethyl sulfoxide (DMSO) control,
CoCl2 (100 µM), dioxin (10 nM),
or dioxin (10 nM) plus CoCl2 (100 µM). Each time point represents the mean of triplicates
and the error bars represent the standard error of the mean.
Statistical analysis is described in text. For each time point, those
determinations not sharing a superscript (a, b, or
c) are significantly different (p < 0.05).
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CoCl2 and Dioxin Effects on Endogenous CYP1A1
Expression in Hepatoma Cells--
To minimize the possibility that
cross-talk was a phenomenon specific to cellular conditions observed
only in vitro or in transient transfection assays, we
performed additional experiments to demonstrate that cross-talk between
hypoxia and dioxin signaling could occur using the endogenous
Cyp1a1 and Epo loci as reporters. Using Hep3B
cells, we first analyzed the amount of HIF1
, ARNT, AHR, and CYP1A1
protein that was expressed under the various treatment conditions (Fig.
4A). As expected, the amount
of HIF1
protein was markedly up-regulated by exposure to
CoCl2 and CoCl2 plus dioxin. Although
expression of the AHR protein was not affected by any of the
treatments, we did observe a small but reproducible CoCl2
related up-regulation of the ARNT protein over basal expression levels.
Finally, the microsomal CYP1A1 protein was up-regulated by dioxin
exposure and this up-regulation was markedly inhibited by co-exposure
to CoCl2.

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Fig. 4.
A, Western blot analysis of HIF-1 ,
ARNT, AHR, and CYP1A1 protein levels in Hep3B cells after various
treatments. Cells were treated for 23 h at 37 °C with dimethyl
sulfoxide (DMSO), 75 µM CoCl2, 10 nM dioxin, or 75 µM CoCl2 plus 10 nM dioxin. Nuclear extracts containing 40 µg of proteins were used to detect the protein
levels of HIF-1 , AHR, and ARNT, whereas microsomes containing 20 µg of proteins were used to detect CYP1A1 protein levels. Antibodies
are described under "Experimental Procedures." The migration of
each band corresponds to molecular masses of 120, 86, 104, and 58 kDa,
respectively, for HIF-1 , ARNT, AHR, and CYP1A1. B,
quantitation of the CYP1A1 mRNA levels by RPA upon various
treatments. Above, an example of the RPA result from Hep3B
cells exposed to dimethyl sulfoxide (DMSO), 75 µM CoCl2, 10 nM dioxin, or 75 µM CoCl2 plus 10 nM dioxin for
4 h. An AHR probe was used as the internal standard. The size of
the protected fragments of CYP1A1 and AHR were 340 and 539 bp,
respectively. Below, data from five replicates were
quantitated on a Fuji PhosphorImager and the mRNA levels were
presented as fold induction compared with the dimethyl sulfoxide
control. C, characterization of cross-talk using a stable
cell line (HepG2-101L) expressing a DRE driven luciferase reporter.
Above, schematic of the reporter. CYP1A1-DREs is the 1612 bp
of the 5'-flanking region derived from the human Cyp1a1
locus that harbors three bona fide DREs. SV40 is the minimal promoter
regions the SV40 early promoter. LUC defines the luciferase
reporter open reading frame. Below, cells were
exposed to dimethyl sulfoxide, 75 µM CoCl2,
10 nM dioxin, or 75 µM CoCl2 plus
10 nM dioxin for 23 h. The luciferase activity was
then assayed as described under "Experimental Procedures." The data
are normalized to total protein in the cellular extracts.
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To control for the possibility that CoCl2 might be acting
by influencing the stability of CYP1A1 protein, we monitored the response by directly measuring the levels of CYP1A1 mRNA (Fig. 4B). Using an RPA assay at a 5-h time point, we observed
that the level of CYP1A1 transcript was up-regulated by dioxin,
~4-fold, and unaffected by CoCl2 exposure. Importantly,
co-administration of CoCl2 inhibited the dioxin induced
levels of the CYP1A1 mRNA by ~40%. As a loading control, we
analyzed the levels of the AHR mRNA, which was unaffected by any
treatment. To demonstrate that CoCl2 inhibition of CYP1A1
induction was mediated by genomic regulatory elements, we employed the
HepG2-101L cell line with an integrated reporter constructed from a
fusion of the DREs from the CYP1A1 gene to an SV40 promoter driven
luciferase reporter (Fig. 4C). Using this reporter cell
line, we observed pharmacological results that were essentially
identical to those observed using the RPA. That is, after 23 h of
exposure, 10 nM dioxin induced luciferase 73-fold and this
induction was reduced approximately 70% in the presence of 75 µM CoCl2. CoCl2 alone did not
have any effect on the luciferase activity.
CoCl2 and Dioxin Effects on Endogenous EPO Expression
in Hepatoma Cells--
Using Hep3B cells, we used an RPA assay to
examine the effects of CoCl2 and/or dioxin exposure on
expression of the endogenous Epo gene (Fig.
5). In agreement with the known
regulation of this gene, we observed that Epo mRNA was
up-regulated more than 5-fold over basal levels by exposure to
CoCl2. To our surprise, we also found that dioxin
up-regulated the Epo mRNA expression greater than
3-fold. Co-exposure to both CoCl2 and dioxin led to an
additive effect, with an 8-fold Epo mRNA induction over
basal levels.

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Fig. 5.
Quantitation of EPO mRNA levels by RPA
following various treatments. Above, an
example of an RPA result from Hep3B cells exposed to dimethyl sulfoxide
(DMSO), 75 µM CoCl2, 10 nM dioxin, and 75 µm CoCl2 plus 10 nM dioxin after 18 h. EPO mRNA level was
normalized to AHR mRNA as the internal standard. The sizes of the
protected EPO and AHR fragments were 312 and 225 bp, respectively.
Below, data from triplicate samples were quantitated on a
Fuji PhosphorImager and the mRNA levels were presented as fold
induction compared with the dimethyl sulfoxide control. Statistical
analysis is described in text. For each time point, those
determinations not sharing a superscript (a, b, or
c) are significantly different (p < 0.05).
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The Epo Promoter Contains a Functional Dioxin Responsive
Enhancer--
We performed a number of experiments to test the idea
that additive induction by dioxin and CoCl2 at the
endogenous Epo locus was due to the presence of dioxin
responsive elements within the Epo gene. First, we examined
the available DNA sequence of the human Epo gene (GenBank
accession number M11319) for sequences that conformed to the core
sequence of the DRE, i.e. 5'-CACGC-3' (25). We found five
such sequences within 600 nucleotides upstream of the Epo
translational start site (Fig.
6A). All five sequences within
the upstream promoter region were in the same orientation and had a
consensus of CACGCNC. To determine if this region could act
as a functional dioxin responsive enhancer, we assayed luciferase reporter constructs that were regulated by the 330-bp promoter derived
from the Epo gene, which harbored the putative DREs. We observed that this reporter was unaffected by exposure to
CoCl2, but was up-regulated by exposure to dioxin and that
the dioxin effects were inhibited by co-exposure to CoCl2
(Fig. 6B).

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Fig. 6.
Analysis of the consensus DREs within the
Epo gene promoter. A, sequence of the
Epo gene promoter region. The sequence was obtained from
GenBank data base (accession number M11319). The five consensus DREs
are underlined and in bold. The nucleotide
"A" in the translational start site (ATG) is defined as position
number +1. The arrows indicate the boundaries of the element
used in the construction of PL1018. B, above, schematic of the
Epo promoter reporter plasmid PL1018. EPO-PROM represents
the 327-bp Epo promoter region that harbors five putative
DREs. The arrow represents the predicted transcriptional
start site. LUC is the luciferase open reading frame.
Below, the response of the Epo
promoter to CoCl2 and/or dioxin. Results from transient
transfection assays with PL1018 under various chemical treatments for
18 h. RLU, relative light units. All transfections were
normalized to a -galactosidase internal control (see "Experimental
Procedures"). The four treatments are dimethyl sulfoxide control,
CoCl2 (100 µM), dioxin (10 nM),
or dioxin (10 nM) plus CoCl2 (100 µM). Each time point represents the mean of triplicates
and the error bars represent the standard error of the mean.
Statistical analysis is described in the text. For each time point,
those determinations not sharing a superscript (a, b, or
c) are significantly different (p < 0.05).
|
|
In an effort to demonstrate that we could recapitulate the additive
cross-talk observed using the endogenous Epo locus, we repeated these experiments using the pbEP-luciferase reporter which was
regulated by both the Epo HRE and the Epo
promoter (Fig. 7). We observed that this
construct displayed pharmacology identical to the endogenous
Epo reporter and that additive cross-talk could be observed
with two distinct AHR agonists,
NF and dioxin (compare Fig. 5 to
Fig. 7, A and B). As an additional proof that
region
499 to
275 of the Epo promoter is a functional
dioxin responsive enhancer, we compared the structure-activity
relationship of this response to that observed using the CYP1A1 dioxin
responsive enhancer. In this experiment, we examined the dose-response
curves for the two AHR agonists
NF and dioxin. We observed that the
relative potencies and efficacies of these two agonists were
essentially identical when using a reporter plasmid driven by the
CYP1A1 dioxin responsive enhancers (PL1A1N) or driven by an
Epo promoter harboring the putative dioxin responsive
enhancers (PL1018) (Fig. 8).

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Fig. 7.
A synthetic reporter construct mimics the
cross-talk and pharmacology of the endogenous Epo
gene. A, response of the Epo enhancer and
promoter to CoCl2 and/or NF. Above, schematic
of the luciferase reporter. EPO-PROM represents the 327-bp
Epo promoter region that harbors five putative DREs. EPO-HRE
is a 193-bp region derived from the Epo gene enhancer that
harbors a single HRE. LUC is the luciferase open reading frame.
Below, results from transient transfection assays with
pbEP-luciferase under various chemical treatments for 18 h.
RLU, relative light units. All transfections were normalized
to a -galactosidase internal control (see "Experimental
Procedures"). The four treatments are dimethyl sulfoxide
(DMSO) control, CoCl2 (100 µM),
NF (1 µM), and NF (1 µM) plus
CoCl2 (100 µM). Each time point represents
the average of duplicates. B, response of the Epo
enhancer and promoter to CoCl2 and/or dioxin.
Above, schematic of the luciferase reporter, same as in
A. Below, same as in A except that the
four treatments are dimethyl sulfoxide control, CoCl2 (100 µM), dioxin (10 nM), and dioxin (10 nM) plus CoCl2 (100 µM). Each
time point represents the mean of triplicates and the error
bars represent the standard error of the mean. Statistical
analysis is described in text. For each time point, those
determinations not sharing a superscript (a, b, or
c) are significantly different (p < 0.05).
|
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Fig. 8.
Pharmacological comparison of a bona fide DRE
reporter (PL1A1N) and a putative one (PL1018). Above,
schematics, see figure legends 2 and 6. Below, dose-response
curves for dioxin or NF using PL1A1N or PL1018 as reporters. The
ordinate is "% Maximal response" with responses for each reporter
normalized to the greatest response achieved. RLU, relative
light units. All transfections were normalized to a -galactosidase
internal control (see "Experimental Procedures"). Each point
represents the mean of triplicates and the error bars
represent the standard error of the mean.
|
|
 |
DISCUSSION |
Ah receptor agonists can initiate pathways that mediate metabolic
adaptation to environmental pollutants in addition to a number of toxic
responses (1-3). In the well characterized "adaptive pathway," the
ligand-induced AHR-ARNT heterodimers bind to DREs resulting in the
transcriptional activation of genes encoding a variety of XMEs.
Unfortunately, an understanding of the molecular details that underlie
dioxin's "toxic pathway" have yet to be developed. We postulate
that the high binding affinity of dioxins may translate into a high
fractional activation of the receptor, and a corresponding depletion of
limiting factors within a cell that are required not only for AHR
signaling, but also for parallel signal transduction pathways. The
discovery that ARNT was also a heterodimeric partner of HIF1
and
that this complex bound to HREs to activate transcriptional responses
to hypoxia, CoCl2, and certain iron chelators provided a
system to test this idea. In its simplest form, this model predicts
that a reciprocal relationship would exist between the dioxin and
hypoxia signaling pathways. For example, if ARNT was the putative
limiting factor, agonists like dioxin would up-regulate DRE driven
genes through the formation of AHR-ARNT heterodimers, while at the same
time inhibiting the hypoxia response due to the decreased availability
of ARNT for HIF1
dimerization. In a similar manner, this model also
predicts that activation of HIF1
would up-regulate the levels of
hypoxia responsive genes, while at the same time decreasing the
availability of ARNT and inhibiting the dioxin response.
In an effort to test this model, we first examined a number of simple
systems to see if we could find evidence that the AHR and HIF1
were
in competition for limiting cellular factors in vitro. In
gel shift experiments we observed that the AHR inhibited HIF1
-ARNT
interactions with an HRE and that HIF1
inhibited AHR-ARNT interactions with a DRE (Fig. 1). These experiments were performed in a
preliminary phase of our investigation and led us to perform additional
tests of the cross-talk model. The observation that the AHR was capable
of inhibiting HIF1
-ARNT interactions in the absence of agonist is
consistent with a high level of constitutive activity of the human AHR
in vitro.2 The
human AHR was chosen for these studies so that only human proteins
would be used. The high level of constitutive human AHR would also
explain why the addition of
NF to these reactions only modestly
increased the inhibition of the HIF1
·ARNT complex. Given that the
in vitro constitutive activity of the human AHR is a
peripheral issue to this report, it has not been pursued further by
using a more tightly controlled AHR form (e.g. murine b-1
allele) or by optimizing incubation conditions. We can only note that
the reciprocal nature of the inhibition is reproducible, and that these
observations led us to test this model in vivo, where
compelling evidence was also obtained.
Transient transfection assays using a luciferase reporter that was
linked to either the HRE derived from the Epo gene or the multiple DREs derived from the Cyp1A1 gene also supported
this model. That is, dioxin inhibited the CoCl2 induction
of an HRE driven promoter and CoCl2 inhibited the
dioxin-induction of a DRE driven promoter (Figs. 2 and 3). Finally,
experiments where we monitored the impact of CoCl2 on
dioxin's induction of the endogenous CYP1A1 gene provided
additional evidence that hypoxia signaling inhibited the dioxin pathway
(Fig. 4). These experiments also indicated that this effect was
occurring at the level of Cyp1A1 transcription and via DRE
enhancer elements.
Given the supportive data generated in early experiments, we were
surprised when we observed that dioxin exposure did not inhibit the
induction of EPO mRNA by CoCl2 in Hep3B cells (Fig. 5).
Fortunately a clue was found in the observations that dioxin alone
up-regulated EPO mRNA and that co-administration of
CoCl2 and dioxin displayed an additive effect in this
system. This led us to investigate the possibility that the
Epo gene may be a dioxin inducible gene and harbor DREs
within its regulatory regions. The identification of five sequences
within the proximal Epo promoter that closely matched the
functional consensus sequence found in bona fide DREs, supported this
idea (Fig. 6A) (26). To support the functional identity of
this region as a composite dioxin response enhancer, we performed a
pharmacological comparison with dioxin and
NF. We found that the
Epo promoter and the CYP1A1 enhancer region displayed the
same relative transcriptional responses to
NF and dioxin (Fig. 8).
Two important conclusions arise from these observations. First, that
Epo is a dioxin responsive gene. Second, that the DREs
within the promoter region of the Epo gene can compensate
for the inhibitory effects that dioxin has on HRE mediated
transcription at this locus. The net result of these multiple elements
is a dampening of the inhibitory/reciprocal cross-talk between these
two pathways. At the present time, it is not known whether this
phenomenon extends to other HRE driven genes like Vegf (27),
Pgk (28), or if this scenario is specific to Epo expression.
Our experiments also support the idea that the hypoxia and dioxin
response pathways can compete for limiting cellular factor(s) and that
reciprocal cross-talk is probably occurring at certain loci within
mammalian cells. Although we have used ARNT as an example of this
limiting factor in model development, it is important to point out that
we have not proven this to be the case. Although our attempts have been
limited, our preliminary experiments to reduce this cross-talk by
transfection of ARNT, yielded ambiguous results. Although we observed
some degree of reduction, we also observed that the basal activities of
each promoter increased, thus clouding any potential conclusions. An
argument for the importance of limiting factors other than ARNT can be
found in the recent observation that demonstrates that dioxin can also
inhibit progesterone receptor signaling (29). This may be an indication
that the limiting factor(s) may also be shared with nuclear receptor
signal transduction pathways. Although the candidates are many, it is tempting to speculate that competition for shared coactivators like
SRC-1 may be important (30). Finally, this "limiting cellular factor" may not be a single protein, but rather may be a composite of
limiting levels of multiple factors.
Interestingly, data from other investigators has demonstrated that
dioxin signaling can be inhibited by activation of HIF1
even though
inhibition of hypoxia signaling was not observed by dioxin (31,
32). Data from these earlier papers suggested that this
unidirectional cross-talk might be related to the fact that HIF1
has
a greater affinity for ARNT than the AHR. At first inspection, our gel
shift experiments are in agreement with the idea that HIF1
has a
greater affinity for ARNT than does the AHR. Nevertheless, it may be
premature to compare the relative affinities of these two proteins
since we do not know the fraction of the AHR that is activated in gel
shift assays (or in any in vitro assays), nor do we know how
long this species remains active. Thus, the increased binding of
HIF1
for ARNT may be simply due to the fact that more HIF1
per
unit time is in a receptive form to bind to ARNT, as compared with AHR
in in vitro assays. Moreover, these earlier studies did not
reveal that dioxin up-regulated EPO mRNA. This may be due to the
use of a weaker agonist than dioxin, or to the fact that readily
metabolized agonists do not exhibit their activity for the prolonged
time periods as compared with dioxin (33, 34). Thus the differences in
EPO response may simply be due to the different time points examined in
these studies.
In summary, we have shown that reciprocal cross-talk between the AHR
and HIF-1
signaling pathways can occur in vitro and in vivo and that this cross-talk occurs at their cognate
response elements. These experiments also support the idea that a
limiting cellular factor is shared by these two pathways, the obvious
candidate being ARNT. Our data also point to the complexity of
cross-talk. Although the activation of the hypoxia pathway inhibited
the up-regulation of the Cyp1a1 gene, the effect of dioxin
on the up-regulation of the Epo gene is more complex. Most
importantly, we examined the Epo gene and found that its
promoter functions as a dioxin responsive enhancer and thus,
Epo transcription can be up-regulated by both hypoxia and
dioxin. Studies are now in progress to determine the importance of
these novel responses and whether they are essential steps in the toxic
pathway of dioxins.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grants R01-ES05703, T32-CA09681, and P30-CA07175 and a fellowship from
the Burroughs Wellcome Fund.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: McArdle Laboratory
for Cancer Research, 1400 University Ave., Madison, WI 53706. Tel.:
608-262-2024; Fax: 608-262-2824; E-mail:
bradfield{at}oncology.wisc.edu.
2
W. K. Chan, G. Yao, Y-Z. Gu, and C. A. Bradfield, unpublished observations.
 |
ABBREVIATIONS |
The abbreviations used are:
AHR, Ah receptor;
dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin;
NF,
-napthoflavone;
bHLH, basic helix-loop-helix;
PAS, Per, ARNT, AHR,
SIM homology domain;
ARNT, Ah-receptor nuclear translocator;
HIF1
, hypoxia inducible factor 1
;
XME, xenobiotic metabolizing enzyme,
RPA, RNase protection assay;
EPO, erythropoietin;
CYP1A1, the 1A1
isoform of cytochrome P450;
bp, base pair;
DRE, dioxin responsive
enhancer;
HRE, hypoxia responsive enhancer.
 |
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