Ah Receptor and NF-
B Interactions, a Potential Mechanism for
Dioxin Toxicity*
Yanan
Tian
,
Sui
Ke
,
Michael. S.
Denison§,
Arnold B.
Rabson¶
, and
Michael A.
Gallo
**
From the
Department of Environmental and Community
Medicine, Environmental & Occupational Health Sciences Institute,
University of Medicine and Dentistry of New Jersey-Robert Wood Johnson
Medical School, Piscataway, New Jersey 08854, the
§ Department of Environmental Toxicology, University of
California, Davis, California 95616, the ¶ Center for Advanced
Biotechnology and Medicine, Department of Molecular Genetics and
Microbiology, University of Medicine and Dentistry of New Jersey-Robert
Wood Johnson Medical School, Piscataway, New Jersey 08854, and the
Cancer Institute of New Jersey, New Brunswick, New Jersey
08901
 |
ABSTRACT |
The Ah receptor (AhR) mediates many of the toxic
responses induced by polyhalogenated and polycyclic hydrocarbons (PAHs)
which are ubiquitous environmental contaminants causing toxic responses in human and wildlife. NF-
B is a pleiotropic transcription factor controlling many physiological functions adversely affected by PAHs,
including immune suppression, thymus involution, hyperkeratosis, and
carcinogenesis. Here, we show physical interaction and mutual functional repression between AhR and NF-
B. This mutual repression may provide an underlying mechanism for many hitherto poorly understood PAH-induced toxic responses, and may also provide a mechanistic explanation for alteration of xenobiotic metabolism by cytokines and
compounds that regulate NF-
B.
 |
INTRODUCTION |
The aryl hydrocarbon receptor
(AhR)1 is a ligand-activated
basic helix-loop-helix transcription factor (bHLH) (1, 2) that plays a
pivotal role in mediating a broad range of distinct toxic responses
induced by polyhalogenated and polycyclic aromatic hydrocarbons,
such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and related compounds (3). These responses include immune
suppression, thymic involution, endocrine disruption, wasting syndrome,
chloracne (keratinocyte proliferation), birth defects, and
carcinogenesis (4). The mechanism for these AhR-mediated
pathophysiological responses is not well understood.
Unliganded AhR is located in the cytoplasm associated with heat shock
protein 90 (hsp90) (5-7) and a 38-kDa, immunophilin-related protein
(8-10). Upon ligand binding, hsp90 is released from the complex, and
the receptor translocates into the nucleus and dimerizes with the aryl
hydrocarbon receptor nucleus translocator (ARNT) protein (11). The
heterodimer binds to the xenobiotic response element (XRE) (12) and
alters expression of genes controlled by enhancer XREs. XREs, with the
conserved core sequences "GCGTG", are found in the promoter regions
of several genes involved in the metabolism of xenobiotics, including
cytochromes P-450 (CYP1A1, CYP1A2, and CYP1B1) (13-16), and
NAD(P)H-quinone oxidoreductase (17). Studies of the regulation of these
genes, especially the regulation of CYP1A1, have provided a basis for
understanding the mode of action of dioxin and related compounds (18,
19).
The transcription of CYP1A1 and CYP1A2 is also regulated by cytokines
(20-24). It has been reported that TNF-
suppresses CYP1A1 and
CYP1A2 levels in human primary hepatocytes (22), and IL-1
has been
shown to suppress TCDD-mediated induction of P-4501A1 and P-4501A2 in
hepatocytes (23). The mechanism(s) of the observed suppression is
unknown. It is known, however, that these cytokines induce NF-
B
(25). NF-
B is a pleiotropic transcription factor that participates
in many of the physiological responses that have been shown to be
affected by xenobiotics, especially TCDD. The classic inducible NF-
B
heterodimer typically consists of a p65(RelA) and a p50(NF-
B-1)
subunit, with RelA being the subunit conferring strong transcriptional
activation (26). We hypothesized that NF-
B may mediate interactions
between cytokines and the AhR signaling pathway. In this report, we
demonstrate that these two pathways interact by physical association of
their respective critical components, i.e. RelA and the AhR,
and that this interaction is associated with mutual functional
modulation of gene expression controlled by the AhR and NF-
B.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture and Transient Transfection--
Cells were
maintained in
MEM supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 250 ng/ml
amphotericin B (Life Technologies, Inc.), 5% CO2, and 37 °C. The plasmid constructs used in this study were as follows: human AhR expression plasmid (phuAhR), pNF
B-Luc (Stratagene), human
NF-
B RelA expression plasmid (pCMV65) (27) and pGudLuc6.1, a
reporter plasmid containing the firefly luciferase gene under control
of four XRE segments derived from the upstream region of the murine
CYP1A1 gene and the mouse mammary tumor virus promoter. This plasmid
was generated as described previously (28), except that the Promega
vector pGL3 was used instead of pGL2. Plasmid DNAs used were purified
using the Qiagen Maxi-Prep DNA Isolation system. For transient
transfection, Hepa1c1c7 and COS-7 cells were seeded in 6-cm dishes on
day 1, and transfection was performed using LipofectAMINE (Life
Technologies, Inc.) when cell density reached 80% confluence.
pSV-
-galactosidase Control Vector (Promega) was used for
normalization of transfection efficiency. Twelve h after transfection,
cells were treated with AhR ligands (TCDD or BNF) or Me2SO
(solvent control) for 18 h before harvest for determination of
luciferase activity or testing in EMSA for DNA-protein binding.
Co-immunoprecipitation/Western Blot Analysis--
Mouse hepatoma
cells (Hepa1c1c7) were treated with TCDD (10 nM) or
Me2SO (vehicle control) for 2 h. Before harvest, the
cells were washed twice with ice-cold phosphate-buffered saline,
harvested by scraping, and collected by centrifugation at 1500 × g. The cells were then lysed in lysis buffer (20 mM Hepes, pH 7.4, 125 mM NaCl, 1% Triton
X-100, 10 mM EDTA, 2 mM EGTA, 2 mM
Na3VO4, 50 mM sodium fluoride, 20 mM ZnCl2, 10 mM sodium
pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin) and centrifuged for 15 min at 12,000 × g, and supernatant fractions were collected. The indicated
antisera were added to the lysate, and the binding reactions were
performed at 4 °C for 2 h on a rotary shaker, following which
30 µl of GammaBind Plus Sepharose beads (Amersham Pharmacia Biotech)
were added to precipitate the antibody-antigen complexes. The beads
were washed three times in lysis buffer and boiled in 2 × SDS
sample buffer containing dithiothreitol. The proteins were separated by
SDS, 8% polyacrylamide gels. Proteins on the gels were transferred to
nitrocellulose membranes (Bio-Rad), and the membranes were blocked with
5% bovine serum albumin in TBST buffer (20 mM Tris-HCL, pH
7.6, 137 mM NaCl, 0.5% Tween 20) and incubated with
appropriate primary antibodies at 37 °C for 2 h. Blots were
then incubated with a 1:2000 dilution of immunoaffinity purified goat
anti-rabbit IgG linked to alkaline phosphatase. Blots were washed three
times with TBST with subsequent color development using nitro blue
tetrazolium/BCIP (Sigma) as the substrate.
Luciferase Reporter Gene Activity Assay--
Luciferase assays
were performed using the Luciferase Assay System (Promega). Briefly,
the transfected cells were lysed in the culture dishes with reporter
lysis buffer, and the lysates were centrifuged at maximum speed for 10 min in an Eppendorf microfuge. Ten µl of the supernatant fraction was
incubated with 100 µl of luciferase substrate, and relative
luciferase activity was determined with a luminometer (Turner Designs).
Preparation of Nuclear Extract and EMS--
Nuclear extracts
were prepared using a small scale procedure as described (29). The
cytosolic fractions of the extraction were used for the Western blot
analysis of the I
B
. Oligonucleotides used for EMSA were
commercially synthesized (Life Technologies, Inc.) and included
B(WT): GGCAGGGGAATTCCCC and
B (MT): GGCAGCTCAATTGAGCT corresponding to a consensus NF-
B binding site (used as the probe for EMSA) and a mutant (used for competition assay in EMSA). These oligonucleotides can self-anneal, and
B(WT) was labeled with [
-32P]dCTP by using Klenow enzyme (Amersham Pharmacia
Biotech). For EMSA assay, 3 µg of nuclear extract protein was
incubated in a reaction mixture containing 40 mM KCl, 1 nM MgCl2, 0.1 mM EGTA, 0.5 mM
dithiothreitol, 20 mM Hepes, pH 7.9, and 4% Ficoll (400 K)
and approximately 30000 cpm of radiolabeled double-stranded oligonucleotide probe. After incubation for 30 min, the reaction mixture was then separated by electrophoresis through a 4.5%
nondenaturing polyacrylamide gel.
 |
RESULTS |
To test the effects of NF-
B on gene expression of CYP1A1, we
first investigated the effects of TNF-
treatment on the CYP1A1 promoter activity. For this, we transiently transfected Hepa1c1c7 mouse
hepatoma cells, which express wild-type AhR, with a XRE-driven luciferase reporter gene (pGudLuc6.1). Treatment with TNF-
, which is
a strong inducer of NF-
B, markedly suppressed TCDD-induced promoter
activity as determined by the luciferase reporter gene assay,
suggesting that cytokine-induced NF-
B could suppress the activity of
AhR (Fig. 1A). To further
define the role of NF-
B in the observed suppression, we co-expressed
pCMV65, which expresses RelA, with the pGudLuc6.1 reporter gene.
Transfection of increasing amounts of the RelA expression plasmid
effectively suppressed TCDD-induced XRE-dependent promoter
activity (Fig. 1B). These results demonstrated that RelA can
suppress AhR-mediated induction of gene expression, suggesting that an
underlying mechanism for the observed suppressive effects of cytokines
on the CYP1A1 and CYP1A2 expression (20-24) is through the induction
of NF-
B.

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Fig. 1.
NF- B suppresses the TCDD-inducible
promoter. A, Hepa1c1c7 cells were transiently transfected
with pGudLuc6.1 (1 µg), a luciferase plasmid containing the firefly
luciferase gene under control of four XRE segments derived from the
upstream region of the murine CYP1A1 gene and the mouse mammary tumor
virus promoter. This plasmid was generated as described previously (14)
except that the Promega vector pGL3 was used instead of pGL2. After
12 h, the cells were treated with TCDD (10 nM) and
TNF- for 18 h, and the activity of the reporter gene was
determined with a luminometer. B, Hepa1c1c7 cells were
co-transfected with pGudLuc6.1 (1 µg), and pCMV65, which expresses
RelA protein (10). The transfected genes were allowed to express for
12 h, and then cells were treated with TCDD (10 nM,
18 h). The cells were then harvested and luciferase activity was
determined as above.
|
|
The NF-
B proteins have been previously shown to interact with
several other transcriptional regulators, including several nuclear
hormone receptors (30-34), Sp1 (35), and p300/CBP (36, 37), resulting
in modulation of different transcriptional regulatory pathways. In
several of these cases, direct physical interactions between RelA and
other transcription factors have been demonstrated (30-37). We
therefore performed co-immunoprecipitation assays to detect possible
complex formation between AhR and RelA in vivo (Fig.
2). Hepa1c1c7 cells were lysed with
buffer containing Triton X-100. The presence of AhR·RelA complexes
was detected by sequential immunoprecipitation and Western blot
analysis, initially using an antibody against AhR to immunoprecipitate
the complex, and the presence of RelA in the complex was detected by
Western blot analysis (Fig. 2A). RelA was detected by
Western blot following immunoprecipitation of the Hepa1c1c7 cell
lysates with either AhR- or RelA-specific antibodies, but not by
immunoprecipitation with control antisera. The association of AhR and
RelA was further confirmed with reciprocal immunoprecipitation with an
antibody against RelA as the immunoprecipitating antibody, followed by Western blotting using the antibody directed against the AhR (Fig. 2B). This reciprocal co-immunoprecipitation confirmed the
physical association of AhR and RelA. Because the activated AhR is
known to associate with the ARNT protein in the nucleus, we tested the possible association of the ARNT with the RelA protein. No significant association between the ARNT and RelA was detectable by
co-immunoprecipitation (Fig. 2C). Thus, AhR·RelA complexes
appear to be distinct from the AhR·ARNT complexes associated with
TCDD-induced transcriptional activation. No association between AhR and
the NF-
B p50 protein, which forms heterodimers with RelA, was
detected by co-immunoprecipitation (data not shown), suggesting that
RelA·AhR complexes are also distinct from the classic p50/p65 NF-
B
heterodimers. The results of reciprocal coimmunoprecipitation also
demonstrated that AhR·RelA association can occur in the absence of
exogenous ligand. The same type of ligand-independent association has
also been observed in the interaction between RelA and the progesterone
receptor (34).

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Fig. 2.
Physical association of AhR and RelA.
Hepa1c1c7 cells were treated with TCDD (10 nM, 2 h)
and lysed with buffer containing Triton X-100.
Co-immunoprecipitation/Western blot analysis was performed to detect
specific association of AhR and RelA (15). A,
immunoprecipitation of RelA with antibody against AhR. Samples of total
cell lysates were incubated with rabbit lgG (Sigma) as the negative
control, anti-RelA antibody (positive control), and antibody against
AhR. The complex was precipitated with protein G coupled to Sepharose
beads (GammaBind G Sepharose, Amersham Pharmacia Biotech), and after
Western transfer, the blot was probed with antibody against RelA
protein (Santa Cruz Biotechnology). IP Abs,
immunoprecipitation antibodies. B, immunoprecipitation of
AhR with antibody against RelA. The cell lysates were incubated with
rabbit IgG (negative control), antibody against AhR (positive control),
and antibody against RelA. After Western transfer, the blot was stained
with antibody against AhR. C, ARNT is not associated with
RelA. The cell lysates were incubated with antibody against RelA, ARNT
(positive control), and rabbit IgG (negative control). The Western blot
was stained with an antibody against the ARNT protein.
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|
Because we had previously shown that RelA was capable of inhibiting
ligand-induced AhR transcriptional activation, we were interested in
assessing the reciprocal effects of AhR activation on NF-
B function.
To test the potential suppressive effects of AhR ligands on endogenous
NF-
B activity, we treated Hepa1c1c7 cells with BNF and TCDD. BNF,
which is an AhR ligand (38-42), is known to cause immune suppression
(43). Twelve h later, the cells were treated with TNF-
for induction
of NF-
B, and activity of NF-
B was determined by EMSA.
Pretreatment with BNF and TCDD markedly reduced the induction of
NF-
B by TNF-
(Fig. 3, A
and B). These data suggest that activation of AhR by ligand
treatment can inhibit NF-
B binding activity. In further experiments,
we tested the effects of AhR activation on NF-
B binding to the
B site DNA motifs using transiently transfected AhR and RelA expression plasmids (Fig. 3C). COS-7 cells were utilized for these
experiments, as they exhibited a lower basal NF-
B binding activity
than the Hepa1c1c7 cells, allowing more efficient detection of changes in NF-
B binding induced by the transfected RelA expression plasmid (data not shown). Twelve h after transfection, cells were treated with
TCDD for 18 h, and nuclear extracts were prepared and were assayed
by EMSA for
B site binding activity. As expected, transfection of
pCMVRelA led to readily detectable NF-
B activation (Fig.
3C, lanes 3 and 4) as compared with
untransfected cells (lanes 1 and 2). Transfection
of increasing amounts of AhR led to suppression of
B binding
activity even in the absence of ligand (lanes 5, 7, and 9). At each level of transfected AhR
plasmid, treatment with TCDD reduced the levels of NF-
B binding as
compared with untreated cells (compare lanes 5,
7, and 9 with 6, 8, and
10, respectively). TCDD treatment coupled with
cotransfection of 2 µg of the AhR expression vector (lane
10) resulted in complete repression of NF-
B activity as
compared with RelA-transfected cells (lanes 3 and
4). These results demonstrated that activation of the AhR by
ligand treatment suppressed NF-
B activity, either because of TNF-
treatment (Fig. 3, A and B) or transfection of RelA (Fig. 3C). It is interesting to note that transfection
of low levels of AhR actually increased NF-
B binding in the absence of ligand (lanes 5-8). In an analogous case, Palvimo
et al. showed that cotransfection of the androgen receptor
increased NF-
B activity in whole cell extracts (31). The reason for
the increased NF-
B activity in the presence of low levels of AhR is
not known; however, what is clearly demonstrated by these experiments
(Fig. 3) is that activation of AhR reduced the binding of NF-
B to
its cognate enhancer sequence.

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Fig. 3.
AhR suppresses the NF- B binding to the
B site. A, suppression of TNF- -induced NF- B
binding activity by BNF. Hepa1c1c7 cells were treated with BNF (0-10
µM) for 12 h and then NF- B activity was induced
by TNF- (1 ng) treatment for 1 h. The NF- B activity was
determined by EMSA. The radiolabeled B(WT) oligonucleotide was
competed with the unlabeled B(WT) and B(MT) to demonstrate
protein binding specificity. B, suppression of
TNF- -induced NF- B binding activity by TCDD. Hepa1c1c7 cells were
treated with TCDD (20 nM) for 18 h, and NF- B
activity was induced and measured as described in panel A.
C, AhR-mediated suppression of NF- B binding by
TCDD. COS-7 cells were transfected with pCMV65 and phuAhR. After
12 h, the transfected cells were treated with 10 nM
TCDD. The cells were harvested 18 h later, and the nuclear
proteins were extracted and assayed for binding activity to a
radiolabeled B-containing oligonucleotide by EMSA (16). *, indicates
lanes (11 and 12) in which unlabeled
oligonucleotide B(WT) was included in the reaction as a specific
competitor of binding.
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|
The role of AhR in the suppression of NF-
B-mediated transcriptional
activation was demonstrated in a transient transfection study in which
transfection of AhR caused suppression of
B-directed promoter
activity. Treatment with AhR ligand (BNF) markedly accentuated the
suppressive effects (Fig. 4A),
consistent with the results from EMSA (Fig. 3C). This
transient transfection study (Fig. 4A) also demonstrated
that treatment with BNF alone, without exogenously transfected AhR
caused suppression of NF-
B activity, suggesting that the COS-7 cells
express AhR. We noticed that Hepa1c1c7 cells express high background
levels of NF-
B activity. These cells are also known to express
wild-type AhR. AhR ligands, TCDD and BNF, both suppressed the
endogenous NF-
B activity in the Hepa1c1c7 cells (Fig.
4B). The effects of AhR activation on NF-
B
transcriptional activity were further tested in an additional transient
transfection assay (Fig. 4C). BNF, as well as
-naphthoflavone (ANF), an isomer and a competitive antagonist of BNF
(38-41), were used as the AhR agonist and antagonist, respectively.
BNF suppressed the RelA-induced activity of a reporter gene driven by
B enhancer sequences, whereas treatment with the AhR antagonist ANF
completely reversed the BNF-induced suppression (Fig. 4C),
strongly suggesting that the repression is through an AhR-mediated
mechanism. Thus, ligand activation of AhR repressed transcriptional
activation by NF-
B as well as binding to the
B motif.

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Fig. 4.
AhR-mediated suppression NF- B
transcriptional activation. A, suppression of NF- B
transcriptional activation by BNF. COS-7 cells were transiently
transfected with pSVsporthuAhR, RelA, and pNF B-Luc (Stratagene)
luciferase reporter gene at the indicated amounts. 12 h after
transfection, the cells were treated with BNF (200 nM), and
18 h later, the cells were processed for determination of luciferase activity. B, suppression of NF- B
activity by activating the endogenous AhR in Hepa1c1c7 cells with BNF
and TCDD. Hepa1c1c7 cells were transiently transfected with pNF B-Luc
luciferase reporter gene, and 12 h after transfection, the cells
were treated with BNF or TCDD for 18 h and luciferase activity was
determined. C, AhR-mediated suppression of NF- B
functional activity by BNF. COS-7 cells were transiently transfected
with pCMV65 and pNF B-Luc, a luciferase reporter gene driven by B
enhancer sequences. Twelve h after transfection, the cells were treated
with BNF (200 nM), ANF (0.5-2 µM), or
Me2SO (1 µl/ml, solvent control), and luciferase activity
was determined as above.
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 |
DISCUSSION |
AhR-mediated gene regulation has been defined by the induction of
CYP1A1 in which the ligand-activated AhR translocates into the nucleus
and binds to a promoter containing the XRE sequence, thereby altering
gene expression controlled by that promoter. This mode of action limits
the regulation of genes by the ligands of the AhR to those containing
XRE enhancer sequences or "negative regulatory" sequences
(silencers) (44). However, a rich body of literature indicates that
ligands of AhR including polyhalogenated and polycyclic aromatic
hydrocarbons cause a wide spectrum of toxic responses ranging from
apoptosis to cell proliferation, suggesting that AhR is likely to
interact with other signaling pathways to cause the observed toxic
responses without acting through the XREs. It is now recognized that
protein-protein interaction is an important mode of action for
transcription factors to increase their regulatory repertoire. For
example, it has recently been shown that a DNA-binding deficient
glucocorticoid receptor mutant retains its transcriptional
transrepression activity without binding to the glucocorticoid response
elements (45).
In the present study, we demonstrated that the AhR and NF-
B
signaling pathways interact by physical association and functional modulation. Although addition of TCDD did not significantly alter the
apparent association between AhR and NF-
B in the in vitro co-immunoprecipitation assay (Fig. 2, A and B), the
functional interactions between the AhR and NF-
B are clearly
ligand-dependent as shown in the transient transfection
assay (Figs. 3 and 4). Our data show that the AhR and RelA proteins
have a cognate ability to associate. One model would suggest that
unactivated AhR and NF-
B are sequestered in the cytoplasm and kept
apart by their respective regulatory mechanisms, i.e. hsp90
associates with AhR and I
Bs associate with RelA. This
compartmentalization would serve as a regulatory mechanism to keep
cellular signaling in order. The AhR and NF-
B subunits would then
interact with each other upon provision of their respective activating
signals (TCDD and BNF for AhR, and TNF for NF-
B). These signals
would cause the dissociation of the hsp90 and I
Bs from the AhR and
RelA, respectively, allowing the association of AhR and the RelA
in vivo. The data presented in Fig. 2, A and
B, revealed that AhR and RelA are physically capable of
associating with each other within total cell lysates, in which the
entire cellular pools of AhR and RelA are exposed to each other,
thereby potentially obscuring the effects of ligands. We believe that
this specific association of these two proteins underlies the potential
functional modulations that were shown in the subsequent transfection
assays and EMSA (Figs. 3 and 4).
The mechanism for the observed functional mutual repression is not
clear. One possibility is that AhR and NF-
B RelA form an inactive
complex, thereby causing mutual repression. Another scenario is that
the mutual modulation between the AhR and NF-
B is mediated by a
transcription coactivator, such as the p300/CBP. It has been found
recently that the transcription coactivator p300/CBP serves as
integrator for many signaling pathways (46), ultimately leading to
histone acetylation and activation of gene expression. Intriguingly,
p300/CBP was found to associate with both RelA (36, 37) and ARNT (47).
It is conceivable that the mutual antagonism found between the AhR and
NF-
B signaling pathways also converges upon this central
transcriptional coactivator. Competition between ligand-AhR/ARNT
complexes and RelA for p300/CBP binding could affect the levels of
transcriptional activation seen in these two pathways. Some nuclear
hormone receptors have also been reported to repress NF-
B activity
(30-34), and the effects of the glucocorticoid receptor in this regard
have been shown to be mediated at least in part through induction of
I
B
(48, 49). In transient transfection, we were unable to observe
any alteration of I
B
level by transfected AhR (data not shown), thus at this point, we do not believe that the repressive effects of
AhR on NF-
B are mediated through induction of I
B
.
In this study, both TCDD and BNF were used as the ligands for the
activation of the AhR. These two ligands have been used interchangeably
for binding to the AhR and induction of CYP1A1. Both ligands suppressed
NF-
B activity in gel-shift assay (Fig. 3, A and
B) as well as transient transfection assays in Hepa1c1c7 cells (Fig. 4B). In a transient transfection assay with COS
cells, BNF effectively suppressed the RelA-induced
B enhancer-driven luciferase reporter gene, whereas co-treatment with
-naphthoflavone reversed the suppressive effects of BNF, strongly suggesting that the
suppression is mediated by the AhR (Fig. 4C).
In conclusion, we have shown in this study a direct interaction between
the AhR and NF-
B signaling pathways. The association between the AhR
and RelA provides a physical basis for the functional antagonism, which
in turn provides a possible mechanistic explanation for the toxicity of
the TCDD. For example, TCDD-induced immune suppression could be a
result of AhR-induced suppression of NF-
B activity in a manner
somewhat analogous to the immune suppressive effects of glucocorticoid.
Recent studies using AhR
/
mice have shown that the AhR is required
for thymic toxicity and that cell-autonomous function of the AhR in
thymocytes is required for the TCDD toxic effects (3, 50). These
results, coupled with our data and the emerging role of NF-
B in
inhibition of apoptosis (51-54) suggest a model whereby repression of
NF-
B by ligand-AhR interactions could result in enhanced
susceptibility of lymphoid cells to apoptotic stimuli, which could
contribute to TCDD-mediated immune suppression. Similarly, TCDD-induced
skin proliferation could be mediated through inhibition of NF-
B in
epidermal cells. A recent report has demonstrated enhanced epidermal
hyperplasia in transgenic mice expressing a constitutive I
B
molecule (55). Conversely, the transrepression of AhR activity by
NF-
B may be the underlying mechanism for the suppression of CYP1A1
and CYP1A2 by cytokines or other substances, which are capable of
inducing NF-
B. Thus, AhR-NF-
B interactions may underlie important
aspects of the pathophysiological responses to polyhalogenated and
polycyclic aromatic hydrocarbons.
 |
ACKNOWLEDGEMENTS |
We thank C. A. Bradfield for phuAhR
plasmid, J. P. Whitlock, Jr. for Hepa1c1c7 cell line, A. Poland
for antibodies against AhR and ARNT, S. Safe for TCDD, and C. Gu and J. Suh (CABM, UMDNJ) for assistance with NF-
B functional assays.
 |
FOOTNOTES |
*
The research was funded in part by National Institutes of
Health, NIEHS Center Grant ES05022 (to M. A. G.), National Institutes of Health Grant CA69281 (to A. B. R.), the New Jersey Commission on
Science and Technology (to A. B. R.), and National Institutes of
Health Grant ES07072 (to M. S. D).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.: 732-445-0175;
Fax: 732-445-4161; E-mail: gallo{at}umdnj.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
AhR, aryl
hydrocarbon receptor;
ARNT, aryl hydrocarbon receptor nucleus
translocator;
XRE, xenobiotic response element;
TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin;
CYP1A1, cytochrome
P-4501A1;
CYP1B1, cytochrome P-4501B1;
EMSA, electrophoretic mobility
shift assay;
TNF-
, tumor necrosis factor;
IL, interleukin;
BSA, bovine serum albumin;
ANF,
-naphthoflavone;
BNF,
-naphthoflavone;
BCIP, 5-bromo-4-chloro-3-indolyl phosphate;
WT, wild type;
MT, mutant type..
 |
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