From the McArdle Laboratory for Cancer Research and
the § Training Program in Environmental Toxicology,
University of Wisconsin Medical School, Madison, Wisconsin 53706
Received for publication, September 18, 2002, and in revised form, February 27, 2003
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ABSTRACT |
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The Ah receptor (AHR) mediates the
metabolic adaptation to a number of planar aromatic chemicals.
Essential steps in this adaptive mechanism include AHR binding of
ligand in the cytosol, translocation of the receptor to the nucleus,
dimerization with the Ah receptor nuclear translocator, and
binding of this heterodimeric transcription factor to dioxin-responsive
elements (DREs) upstream of promoters that regulate the
expression of genes involved in xenobiotic metabolism. The AHR is also
involved in other aspects of mammalian biology, such as the toxicity of
molecules like 2,3,7,8-tetrachlorodibenzo-p-dioxin as well
as regulation of normal liver development. In an effort to test whether
these additional AHR-mediated processes require a nuclear event, such
as DRE binding, we used homologous recombination to generate mice
with a mutation in the AHR nuclear localization/DRE binding domain.
These Ahrnls mice were found to be resistant to all
2,3,7,8-tetrachlorodibenzo-p-dioxin-induced toxic responses
that we examined, including hepatomegaly, thymic involution, and cleft
palate formation. Moreover, aberrations in liver development observed
in these mice were identical to that observed in mice harboring a null
allele at the Ahr locus. Taken in sum, these data support a
model where most, if not all, of AHR-regulated biology requires nuclear localization.
The aryl hydrocarbon receptor
(AHR)1 regulates an adaptive
metabolic response to a variety of planar aromatic chemicals that are
widely dispersed in the environment. Over the last 20 years, the
mechanistic details of this adaptive signaling pathway have been well
characterized (1-4). The AHR is a basic helix-loop-helix-PAS (bHLH-PAS) transcription factor. Upon binding agonists, the AHR translocates from the cytoplasm to the nucleus, where it forms a
heterodimer with another bHLH-PAS protein known as the aryl hydrocarbon
nuclear translocator (ARNT). This heterodimeric complex interacts
with dioxin-responsive elements (DREs) within the genome and
up-regulates the transcription of a battery of xenobiotic metabolizing
enzymes (XMEs). These regulated XMEs include the cytochrome P450s
Cyp1a1, Cyp1b1, and Cyp1a2 and the
phase II enzymes Gst-a1 and Ugt1-06 (reviewed in
Refs. 2 and 3).
In addition to regulating an adaptive metabolic response, the AHR also
mediates toxic responses to
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and plays an
important role in normal development. Early genetic and pharmacological
experiments provided evidence that the AHR mediates toxic responses to
TCDD and related pollutants (5). Highly reproducible toxic endpoints in
rodent species include thymic involution, hepatomegaly, epithelial
hyperplasia, and teratogenesis. More recently, generation of null
alleles at the Ahr locus in mice revealed that the AHR also
plays an important role in normal mammalian development (6-9). Across
laboratories, the most reproducible phenotype associated with the
homozygous null allele is a smaller liver. We have proposed that
smaller liver size is the result of the persistence of a fetal vascular structure known as the ductus venosus (DV) (10). Our hypothesis is that
smaller liver size is due to hepatocyte atrophy, resulting from partial
shunting of portal blood flow directly to the vena cava. Importantly,
the mechanistic role of AHR in the persistence of the DV as well as the
appearance of other vascular aberrations is unknown.
Our laboratory is interested in determining if the adaptive, toxic, and
developmental pathways of AHR differ in any mechanistic aspect. In
particular, we are interested in understanding whether cytosolic
events, nuclear events, or DRE-mediated transcription play a role in
TCDD-induced toxicity or in the establishment of a normal liver size.
Although it may be assumed that all actions of AHR are dependent upon
DRE-mediated transcription, a considerable body of evidence has been
reported to the contrary. In this regard, signal transduction through
cellular factors such as the cSrc kinase, the retinoblastoma protein
(Rb), ceramide, steroid receptors, NF- In an effort to formally examine the possibility that aspects of AHR
biology are mediated through cytosolic events that do not require the
AHR to translocate to the nuclear compartment, we undertook the
approach of using gene targeting in mouse embryonic stem (ES) cells to
generate a novel allele at Ahr that harbors a mutation that
blocks both nuclear localization and DRE binding (designated as
Ahrnls). By examining the responses of these mutant
animals to TCDD and through a comparison with other Ahr
alleles, we are able to define the relative importance of cytosolic
versus nuclear events in various aspects of AHR biology.
Strategy--
The design of the AHRnls allele (and
therefore the Ahrnls protein) is based on published
results from several laboratories as well as practical considerations.
Foremost is the observation that the residues required for DRE binding
overlap with those required for nuclear localization. Previous
mutational analysis reveals that the AHR nuclear localization sequence
(NLS) is bipartite and resides within the basic region residues 12-17
and 37-42 (18-20). These regions significantly overlap with those
shown to be critical for DRE binding, including residues 9-14 and
36-39 (21-23) (Fig. 1A). Because of this domain overlap,
we chose to introduce a series of nonconservative mutations to change
Arg37, His38, and Arg39 to
Ala37, Gly38, and Ser39,
respectively. Our prediction was that this would generate an AHR
protein that is deficient in both nuclear localization and DRE binding.
Based upon the work cited above and our previous domain mapping work,
we predicted that these mutations would not influence ligand binding,
chaperone binding, or dimerization with ARNT (24).
Oligonucleotides--
Oligonucleotides (Invitrogen) are
designated as follows: OL72, 5' -GGTTCGAATTTCCAGGATG-3'; OL73,
5'-TCGAGTAGATCACGCAATGGGCCCAGC-3'; OL74,
5'-TCGAGCTGGGCCCATTGCGTGATCTAC-3'; OL659,
5'-ATCCAGAAGAGCTTATCAGTGGTTCTGC-3'; OL941, 5'-CTGAGGGGACGTTTTAATG-3';
OL942, 5'-AACATTTGCACTCATGGATAG-3'; OL975, 5'-
GCGTCGACCCACCATGAGCAGCGGCGCCAACATC-3'; OL1352,
5'-GGTACCTCTGAGTTCAAGTCTAGTCTG-3'; OL1353,
5'-GGTACCGCATGCTTACTAGTAGTTTTTCTAG-3'; OL1503,
5'-GCCACCATGAGCAGCGGCGCCAACATCACCTATGCCAGCCGCAAGCGGCGCAA GCCGGTGCAGAAAACAGTAAAGCCCGGGCCCGCTGAA-3', OL1500,
5'-GTAAAGCCCGGGCCCGCTGAAGGAATTAAGTCAAATCCTTCTAA GGCAGGATCCGACCGGCTGAACACAGAGTTAGA-3'; OL2639,
5'-ACTAGTCGACCTAACCCATTTGCTGTCCACCAGTCATGCTAGCCATACTCTGCACCTTGCTTAG-3'.
Expression Constructs--
The construct pSV-AHR (PL 65) is
previously described (25, 26). To construct pTgTAHRT7 (PL1550), PL65
was used as a template for 22 rounds of PCR amplification using OL1503
(forward) and OL2639 (reverse) as primers. The resulting amplicon was
cloned into the pTargetTM mammalian expression vector
(Promega, Madison, WI). OL1503 contains a consensus Kozak start codon
and a conservative mutation creating an SrfI site to match
the targeting construct sequence (see below). The oligonucleotide
OL2639 contains the region of AHR cDNA preceding the stop codon,
followed by sequences for the T7 epitope and for a translational stop
codon. The plasmid pSVAHRnls (PL1108) was made by
three-step PCR using PL65 as template. In the first step, the forward
oligonucleotide, OL1500, containing a 9-base substitution that changed
codons Arg37 to Ala37, His38 to
Gly38, and Arg39 to Ser39,
respectively, was used in a PCR-amplification with the reverse oligonucleotide OL72. The product was then used as template for 20 rounds of amplification using OL1503 and OL72 to add the 5' amino
acids. This product was then amplified using OL975 and OL72 to add a
consensus Kozak start codon for increased expression. An
SpeI/NaeI fragment was excised and used to
replace the SpeI/NaeI fragment from PL65 to
create pSVAHRnls (PL1108). Sequencing of the full-length
constructs ensured that no other mutations were present.
Protein Analysis--
Gel shifts and coimmunoprecipitations were
performed as described previously (16, 27, 28). In vitro
protein expression was carried out using the TNT® coupled
transcription/translation reticulocyte lysate system (Promega). For gel
shift assays, in vitro expressed proteins were incubated
with a 32P-labeled DRE fragment derived from annealing OL73
and OL74 in the presence or absence of 10 µM
Western blots, EROD assays, and photoaffinity labeling of the AHR were
performed using methods described previously (7, 29, 31). Microsomal
and cytosolic fractions were isolated from ~0.5 g of mouse liver that
was homogenized in ice-cold MENG buffer followed by two high speed
centrifugations at 10,000 × g and 100,000 × g. The microsomal pellet was resuspended in 1 ml of
resuspension buffer (15 mM Tris-Cl, pH 8.0, 250 mM sucrose). The supernatant, containing cytosolic protein,
was saved for later AHR analysis. Western blot analysis was performed
as described using the BEAR-3 antibody and a secondary antibody labeled
with alkaline phosphatase (29). The EROD assays were performed in a
96-well format. In each well, one-one thousandth of the total microsomal preparation was diluted into 200 µl in MENG buffer. To
start the reaction, 3 µl of 0.1 mM 7-ethoxyresorufin and
20 µl of 5 mM NADPH were added. The production of
7-hydroxyresorufin was measured by fluorometry (fMax;
Molecular Devices) at excitation of 510 nm, emission of 590 nm
every 30 s over 10 min at 25 °C. Total protein concentration
was determined using the BCA assay (Pierce). The results are expressed
as relative fluorescence units/min as calculated using SoftMaxPro
software (Molecular Devices) and normalized to total protein levels.
Photoaffinity labeling of the AHR protein with
2-azido-3-iodo[125I]iodo-7,8-dibromodibenzo-p-dioxin
([125I]Br2DpD) was performed essentially as described
(31). Briefly, for labeling of the cytosolic AHR, liver cytosolic
protein (300 µg/ml) was incubated with [125I]Br2DpD at
the indicated concentrations for 2 h at 4 °C. This was followed
by the addition of one-tenth volume of 1%/0.1% activated charcoal/gelatin for 30 min at 4 °C to remove unbound ligand. Following centrifugation, supernatants were exposed to UV light at 310 nm (80 watts, 4 cm) for 30 s. Protein was precipitated in acetone
and separated by 7.5% SDS-PAGE. The location of labeled receptor was
determined by autoradiography, and the band was excised and counted by
Cell Culture Conditions--
ES cells, designated GS-1, were
purchased from Genome Systems (St. Louis, MO). The ES cells were
cultured on a confluent layer of Mitomycin-c-treated mouse embryonic
fibroblasts derived from PGK-NeoR transgenic mice (formal designation
C57BL/6-TgN(pPGKneobpA)3Ems; Jackson Laboratory, Bar Harbor, ME) in
Dulbecco's modified Eagle's medium-high glucose supplemented with
20% fetal bovine serum (Hyclone, Logan, UT), 0.1 mM
nonessential amino acids, 2 mM L-glutamine, 10 mM HEPES, 100 units/ml penicillin, 100 µg/ml
streptomycin, and 1000 units/ml ESGROTM (Invitrogen).
Generation of Mouse Embryonic Fibroblasts--
We have
previously generated Ahr null mice that will be hereafter
referred to as Ahr Transfection of Mouse Embryonic Fibroblasts--
Transient
transfections of mouse embryonic fibroblasts were performed using
Fugene6 reagent (Roche Applied Sciences) with 1 µg of total DNA and a
3:1 Fugene6/DNA ratio. When TCDD was used, cells were allowed to
recover for 1 day after transfection. Following this period, 1 nM TCDD was added directly to the medium in
Me2SO (final Me2SO concentration 0.1%)
and cells were harvested after 2 h. Immunofluorescence was
performed as described (26) using BEAR-3 and a fluorescein
isothiocyanate-conjugated goat anti-rabbit secondary antibody (Jackson
Immunoresearch, West Grove, PA) for fluorescence detection.
Generation of Ahrnls/nls Mice--
A
15-kb region of homology surrounding exon 2 of AHR was isolated from a
129SvJ genomic library (Genome Systems) as described (7, 33). To make
the Ahrnls allele, a 9-nucleotide replacement was
introduced by using the PCR product of OL1500 and OL942 as megaprimer
with 22 rounds of amplification with OL941 (23). The SphI
fragment from the mutant PCR product was used to replace exon 2 in an
8-kb BamHI genomic fragment. A 5.5-kb region containing the
mutated exon 2 was amplified with OL1352 and OL1353 and cloned into the
KpnI site of ploxPNT (34). A 7-kb SphI fragment
from the 5' region of exon 2 was cloned into the
NotI/XhoI site of this construct to make the
final targeting construct that was designated pNTAHRnls
(PL1075). For homologous recombination in ES cells, ~10 µg of
targeting construct was electroporated into GS1 cells (Genome Systems),
and selection was performed using 200 µg/ml G418 and 1 mM
Ganciclovir. Clones were screened by Southern blot of
BamHI-digested genomic DNA using a probe 3' to the end of
the targeting construct (PL311). Correctly targeted clones were
injected into 3.5-day postcoital C57BL/6 blastocysts, and resulting
chimeras were backcrossed to C57BL/6 to determine contribution of the
ES clones to the germ line. For experimental analysis, animals were
backcrossed to C57BL/6 mice that are congenic for the
Ahrd allele (35). Mice were genotyped using the PCR
primers OL941 and OL942. The PCR was carried out for 35 cycles
(95 °C, 1 min; 63 °C, 1 min; 72 °C, 1 min) in buffer
containing 4 mM MgCl2; a BamHI
digest cuts the 380-bp PCR product from the Ahrnls
allele into two small fragments of 240 and 140 bp that were detected on
a 2% agarose gel.
Animals--
Animals were housed in a selective pathogen-free
facility on corncob bedding with food and water ad libitum
according to the rules and guidelines set by the University of
Wisconsin. When TCDD was used, 5-week-old animals were injected once
intraperitoneally with 100 µg/kg TCDD in p-dioxane or with
p-dioxane alone. After 6 days, animals were weighed and
sacrificed, and organs were immediately removed and weighed. For
angiography, ~1 ml of Omnipaque 300 (Nycomed, Inc., Princeton, NJ)
was injected into the hepatic portal vein postmortem. Continuous x-ray
images were obtained over ~10 s using an OEC 9800 Portable Vascular
C-ARM (Medical Systems, Inc., Salt Lake City, UT).
In order to directly compare the null, wild type, and
Ahrnls/nls alleles, genetic
background had to be taken into consideration. Mice used in these
experiments that are defined as harboring the Ahr Palate Cultures--
Suspended palate organ cultures were
performed essentially as described (36, 37). Palatal shelves were
dissected from embryonic day 12.5 embryos of the
Ahr+/+, Ahr+/ Statistics--
In the situation where multiple comparisons
could be made, an analysis of variance was performed, and Tukey's test
was used to determine differences with a p Preliminary Characterization of the Ahrnls Mutation in
Vitro--
In order to generate an AHR protein deficient in nuclear
localization, we replaced amino acids Arg37,
His38, and Arg39 in the mouse AHR cDNA
(pSV-AHR) with nonconservative amino acids Ala, Gly, and Ser,
respectively, to generate the plasmid pSV-AHRnls (Fig.
1A). Given the overlap between
the NLS and the DRE recognition sequences, this mutant was also
predicted to have deficiencies in DRE binding. As a preliminary
examination of the characteristics of the mutant AHRnls
protein, we translated the cDNA in a reticulocyte lysate system. To
demonstrate that the mutant receptor bound ligand normally, we
performed labeling experiments with the photoaffinity ligand 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin.
We observed that the mutant receptor bound this ligand with a capacity
that was similar to the wild-type protein (Fig. 1B). To
determine the DNA binding capacity of the AHRnls protein,
we performed gel shift analysis with a consensus DRE oligonucleotide
(Fig. 1C). In the absence of agonists and in the absence or
presence of ARNT, neither the AHR nor the AHRnls proteins
interacted significantly with a 32P-labeled DRE
oligonucleotide. The addition of the agonist
We used indirect immunofluorescence to determine the influence of the
NLS mutation on the subcellular localization of the AHR in mammalian
cells. In order to perform this experiment in the absence of endogenous
wild-type AHR, we generated "immortalized" fibroblasts from
Ahr
To demonstrate the capacity of the AHRnls protein to form
normal complexes with its cognate cytosolic chaperones, we performed coimmunoprecipitation experiments with HSP90 and ARA9. For these experiments, proteins were expressed in vitro in
reticulocyte lysate. For interaction analysis with HSP90,
35S-labeled AHR or AHRnls were
immunoprecipitated with antibodies to HSP90 (Fig.
2A). For interaction analysis
with ARA9, 35S-labeled ARA9 was incubated in the presence
of unlabeled AHR and AHRnls and precipitated with anti-AHR
antibody (Fig. 2B). Results from three independent
experiments indicated that both the AHR and AHRnls proteins
formed complexes with the chaperones ARA9 and HSP90 with similar
capacity. The results from representative experiments are shown
in Fig. 2. Together these results indicate that AHRnls
is capable of interacting with both HSP90 and ARA9 in a manner similar
to the wild type AHR yet is no longer capable of translocating to the
nucleus and interacting with DNA at DRE elements (Figs. 1 and 2).
Generation of Ahrnls/nls Mice and Their
Controls--
To generate mice with mutant AHRnls protein,
we used PCR to alter the above mentioned sequences of the basic region
of AHR (exon 2) in a 15-kb region of homologous genomic DNA from the
Ahr locus (Fig.
3A). The targeting construct
was designed to insert the neomycin cassette into a similar region of
Ahr as was done previously to generate the null mutation
(Ahr Characterization of Ahrnls Mutation in
Vivo--
Expression levels of each mutant AHR protein were determined
by Western blot and photoaffinity labeling of receptor from liver protein extracts. Visual inspection of the Western blots suggested that
the expression of receptor from the
Ahrnls/nls mouse liver was
approximately equivalent to that observed in the
Ahr+/
To determine whether the mutant AHR protein signals in vivo,
Ahrnls/nls,
Ahr+/+, and Ahr+/ Ahrnls/nls Mice Are Resistant to TCDD
Toxicity--
To determine whether the NLS mutation influenced the
role of the AHR in the toxicological response to TCDD, we exposed
male Ahrnls/nls,
Ahr
We also examined the closure of palatal shelves in an organ culture
model system, where we can directly expose the tissue to TCDD (36, 37).
Palates were excised from embryonic day 12.5 Ahr+/+, Ahr+/ Ahrnls/nls Mice Show Developmental Defects
Similar to Ahr
In Ahr Previous cell culture and in vitro analyses of AHR
signaling have provided a detailed picture of the mechanism by which
the ligand-activated AHR up-regulates the expression of certain XMEs through receptor-DRE interactions within the nucleus (44). However, this understanding of the DRE-linked transcriptional targets of AHR has
not yet explained the variety of toxic events that occur in response to
potent agonists like TCDD; nor have they been used to explain the
aberrant liver/vascular development observed in Ahr In an effort to better understand the importance of DRE-independent
signaling, we are constructing mutant mouse models that directly test
the roles of receptor functional domains on various aspects of AHR
biology in vivo. The AHRnls mouse was designed
specifically to examine the role that cytosolic events play in
TCDD-induced toxicity and AHR-dependent liver development. In this endeavor, we chose to first generate animals with an AHR protein that can be activated by ligand but that is not capable of
translocating to the nucleus or binding to DREs. The rationale for such
a model is that it can be used to identify AHR-mediated end points that
do not require nuclear localization of the AHR. Put another way, with
this model we may be able to identify cytosolic signaling pathways
related to AHR biology, or we can provide evidence to refute their existence.
Generation of this mouse model required consideration of the
overlapping domain structure of the basic There are two major points regarding controls that arose during the
course of these experiments. The first relates to genetic background.
Because embryonic stem cells are derived from 129Sv mice that carry the
d allele of Ahr, we utilized the
C57BL/6-Ahrd animals for backcrossing and to
generate control animals for these experiments. Mice harboring the
wild-type d allele are not used as frequently as those strains
harboring the more common b alleles due to the fact that the d allele
encodes a receptor with a lower binding affinity for TCDD (49).
Importantly, this lower binding affinity exhibited by the d allele has
been shown to be similar to the human AHR and thus may serve as a more
relevant model for human toxicity (47). The major influence this
parental allele had on our experimental design was that it necessitated the development of dosing protocols that would induce toxic end points
in mice harboring this lower affinity receptor. Thus, it will be noted
that the doses of TCDD used in these studies are about 5-10-fold
higher than are commonly used in most mouse studies.
The second point relevant to control animals is that although the AHR
from the d allele and the nls allele display similar affinities for
ligand, the nls allele is expressed at a lower level (Fig. 4). Our
photoaffinity labeling of the AHR found in the livers of
Ahrnls/nls mice indicated that the
mutant protein was expressed at a level that was approximately one-half
of that observed in Ahr+/+ mice. Fortunately,
AHR expression in wild-type animals appears to be a direct function of
gene copy number.2 Therefore,
mice that were heterozygous for the d allele and the null allele served
as controls (i.e. Ahr+/ To determine whether nuclear events were required for the role of AHR
in TCDD toxicity or normal liver development, we performed a number of
toxicology experiments with the
Ahrnls/nls mice. First, we
examined the toxic response of the
Ahrnls/nls mice by injecting the
animals with TCDD and assaying for liver hypertrophy and thymic
involution. We found that Ahrnls/nls
mice failed to exhibit these obvious liver and thymic end points, whereas the corresponding Ahr+/+ and
Ahr+/ We also assessed the role of cytosolic events in the developmental
aspects of AHR biology. In this regard, all of the known developmental
defects we have previously observed in Ahr The Ahrnls/nls mice provided us with
a model system to test the importance of cytosolic interactions in
certain aspects of AHR biology. Given that it would be impossible to
test each proposed model in our system (i.e. cSrc, ceramide,
etc.), our strategy was to carry out experiments that would examine the
relative importance of cytosolic versus nuclear signaling
with regard to a few classic receptor-mediated end points
(i.e. liver hypertrophy, thymic involution, cleft palate
formation, and persistence of the DV). Although our results are not
supportive of a role for cytosolic events for these end points, it is
still possible that cytosolic signaling by the AHR is important in end
points not examined here (e.g. chloracne, altered T-cell
responses, etc.). It is also important to point out that we cannot rule
out the possibility that cytosolic signaling does occur and that it is
dependent upon the exact identity of residues Arg37,
His38, and Arg39. In an effort to address such
possibilities, we plan to make this mutant available to any laboratory
interested in formally examining the influence of the nls allele on any
proposed cytosolic mechanism. Finally, given that the NLS mutation
eliminates nuclear localization, it remains possible that nuclear, yet
non-DRE binding events are important in AHR biology. Nuclear events
such as cross-talk with the hypoxia-inducible factor and interactions
with steroid receptors are still potentially important mechanisms of
TCDD action or AHR-mediated developmental signaling.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B, and HIF1
has been
reported to be modulated by AHR agonists in a manner that is
independent of AHR DRE binding and transcriptional activation roles
(11-17).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-naphthoflavone (
NF). The presence of the AHR in the DRE binding
complex was confirmed using a high affinity rabbit polyclonal antibody
raised against recombinant AHR (BEAR-3). Co-immunoprecipitation
experiments were performed by co-incubating ~10 fmol of reticulocyte
lysate-expressed proteins with 2 µg of antibody in 500 µl of cold
MENG buffer (25 mM MOPS, pH 7.5, 0.025% sodium azide, 1 mM EGTA, 10% glycerol, pH 7.5), supplemented with 15 mM NaCl, 0.1 mM dithiothreitol, and 0.1% Nonidet P-40. Bound protein-antibody complexes were precipitated with
protein A-Sepharose (Sigma) for 1.5 h at 4 °C, washed four times with cold MENG buffer, eluted in 2× SDS sample buffer, and analyzed by SDS-PAGE.
emissions. The quantity of bound ligand is expressed as dpm/lane.
The in vitro translated proteins were labeled by diluting 10 µl of the translation reaction in 0.5 ml of MENG. We ensured that the
10 µl of translation reaction contained equal amounts of translated
receptor by 35S calibration. The receptor was then labeled
with 1 nM [125I]Br2DpD for 30 min at 20 °C
followed by 4 °C for 5 min. Unbound ligand was removed by
charcoal/gelatin, and the quantity of bound ligand was determined in
the same manner as above. The specific activity of the radioligand is
2176 Ci/mmol, and 1 pmol = 4,830,700 dpm. For Scatchard analysis,
it was assumed that free probe was equal to total counts added and that
total bound was equal to specific bound (this assumes that nonspecific
binding is negligible following SDS-PAGE and band excision) (30).
/
(formal genetic
designation is AhrtmBra1). To generate matched mouse
embryonic fibroblasts, Ahr+/
mice that have
been backcrossed to C57BL/6 for 16 generations were intercrossed to
generate littermate +/+, +/
, and
/
embryos. Embryos were isolated
from their yolk sacs, and heads and livers were removed by dissection.
DNA was isolated from the heads of individual embryos and was used for
genotyping as described (10). At passage 2 (P2),
Ahr+/+ and Ahr
/
fibroblasts were maintained on a "3T3 protocol" until passage 25 and then passaged regularly at subconfluence as described (32). Briefly, in the 3T3 protocol, cells were grown in 6-cm dishes in
Dulbecco's modified Eagle's medium-high glucose, supplemented with
10% fetal bovine serum, 0.1 mM nonessential amino acids, 2 mM L-glutamine, 10 mM HEPES, 100 units/ml penicillin, and 100 µg/ml streptomycin. Every 3rd day, cells
were trypsinized and replated at a density of 3 × 105. At passage 28, individual clones were isolated from
one line of each genotype and passaged regularly at subconfluence. The Ahr
/
subcloned cell line used for these
experiments was designated B4.4.
allele have been previously backcrossed to
C57BL/6 for 16 generations. Because C57BL/6 carries the high affinity
Ahrb allele and the Ahrnls allele
was created in 129 ES cells carrying the lower affinity Ahrd allele, we used a C57BL/6 strain congenic for
DBA2-derived Ahrd allele to make subsequent
backcrosses of both the Ahr
and
Ahrnls alleles (generous gift from Alan Poland).
This breeding generated wild type Ahrd allele
controls on the C57BL/6 background (35). For clearer presentation, the
wild type Ahrd allele is referred to as
Ahr+ ("wild type") throughout this paper.
Mice harboring the Ahrnls allele were backcrossed
for three generations to C57BL/6. To further minimize the influence of
genetic background, all experiments were repeated from multiple
independently derived sublines. Only a single representative experiment
is presented.
, and
Ahrnls/nls genotypes. The palates
were placed into cold phosphate-buffered saline and then transferred to
prewarmed 1:1 Dulbecco's modified Eagle's medium/F-12 medium
supplemented with 1% L-glutamine, 1% ascorbate, and 1%
penicillin/streptomycin and immediately treated with either 0.03%
Me2SO or 3.3 nM TCDD in 0.033%
Me2SO (v/v). After exposure for 4 days, the palates were
fixed in 10% formalin and stained with hematoxilin for visualization.
Palates were scored as not fused or fused. Ambiguous partially fused
palates accounted for only a small percentage and were not included in
the data set.
0.05 (38). When only two groups were being compared, a two-tailed
t test was performed with the level of significance set at
p
0.05 (Fig. 5) (38).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
NF induced formation of
the wild-type recombinant AHR·ARNT·DRE complex that could be
blocked by the addition of the anti-AHR antibody. However, the addition
of
NF failed to induce formation of a recombinant AHRnls/ARNT/DRE complex, indicating that DNA binding is
disrupted by the amino acid replacements (Fig. 1C).
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Fig. 1.
In vitro characterization of
AHRnls recombinant protein. A, schematic of
AHR protein domain structure. Amino acids 1-43 are depicted.
Bars above and below show locations of
basic residues involved in nuclear localization (NLS) and
DRE binding (DRE). The mutated amino acids in the
AHRnls recombinant protein (Arg37,
His38, and Arg39) are marked with
asterisks. B, photoaffinity labeling of wild-type
and Ahrnls recombinant proteins. Recombinant proteins were
labeled with [125I]Br2DpD and activated at
310-nm light (described under "Experimental Procedures"). Labeled
proteins were separated by PAGE and visualized by autoradiography. A
representative experiment with duplicate samples is shown.
C, gel shift analysis of AHR, ARNT, and AHRnls
recombinant proteins. Recombinant proteins were expressed alone or in
the presence of ARNT or ARNT + NF and analyzed for binding to a
32P-labeled oligonucleotide with a single DRE
element by nondenaturing PAGE. The anti-AHR antibody
(AHR-Ab) was added to indicated reaction to block formation
of the complex. D-G, subcellular localization
of recombinant AHR and AHRnls proteins.
Ahr
/
cells were transiently transfected with
either AHR (D and E) or AHRnls
(F and G) cDNA (pSV-AHR and
pSV-AHRnls, respectively). Cells were treated with
either 1% Me2SO (C and E) or 1 nM TCDD plus 1% Me2SO (D and
F), fixed in cold methanol, and analyzed for localization of
AHR using anti-AHR antibody and detected with fluorescein
isothiocyanate-conjugated secondary antibody. Digital images were
collected by standard fluorescence microscopy.
/
embryos using a 3T3 protocol
(32). The Ahr
/
fibroblasts were transiently
transfected with AHR or AHRnls cDNAs (pTgTAHRT7 or
pSV-AHRnls, respectively). Following treatment with either
vehicle or 1 nM TCDD, cells were fixed, and the
localization of the AHR or AHRnls protein was visualized
using anti-AHR antibody and a fluorescein isothiocyanate-conjugated
secondary antibody. We observed that wild-type AHR localizes to the
cytosol in untreated fibroblasts but moves to the nucleus within 2 h after treatment of cells with 1 nM TCDD, (Fig. 1,
D and E). As predicted, the AHRnls
protein was constitutively cytoplasmic, and its localization was not
affected by TCDD (Fig. 1, F and G). This block in
nuclear localization was also observed in COS7 and murine hepatoma
cells (data not shown).
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Fig. 2.
Capacity of AHRnls to form
complexes with HSP90 and ARA9. A, co-immunoprecipitation of
AHRnls with HSP90. Five microliters of in vitro
translated [35S]methionine-labeled wild type AHR or
AHRnls was incubated with 2 µg of anti-HSP90 antibody and
precipitated with Protein A-Sepharose. Reticulocyte lysates contain
large amounts of HSP90, so no additional protein is added.
B, co-immunoprecipitation of ARA9 with AHRnls.
Five microliters of in vitro translated
[35S]methionine-labeled full-length ARA9 was incubated
with either wild type AHR or AHRnls. Protein complexes were
incubated with 2 µg of the AHR-specific antibody and precipitated
using Protein A-Sepharose. All proteins in A and
B were eluted from Protein A-Sepharose and separated by
SDS-PAGE gels and visualized by autoradiography. Input, a
loading control representing 100% of the radiolabeled ARA9 used in the
assay. PreI, the preimmune control.
/
) (7). After selection in both G418 and
Ganciclovir, surviving clones were screened by Southern blot, and
correctly targeted clones were used to generate chimeras and ultimately
germ line transmission of the AHRnls mutation to mice. A
representative map of the resulting Ahrnls allele is
depicted in Fig. 3A beside the
Ahr
/
allele and wild type alleles. The
resulting Ahrnls/nls mice (formal
genetic designation AhrtmBra2) are born at a
frequency consistent with a nonlethal allele, and the animals are
fertile (Fig. 3B and data not shown).
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Fig. 3.
Gene targeting of pNTAHRnls to
the germ line in mice. A, schematic of targeting
construct and resulting alleles. The pNTAHRnls targeting construct is
depicted at the top, indicating positions of exon 2, the
neomycin resistance gene cassette (Neo), the herpes simplex
virus thymidine kinase cassette (TK), and informative
restriction sites (Mlu (M), BamHI
(B), BglII (Bg), SrfI
(Sf)). Amino acid replacements are depicted
above. Below the targeting construct are wild
type allele (Ahr+) and the resulting mutant
allele (Ahrnls). The Ahr-null allele
(Ahr ) is included for comparison.
Below each are lengths of diagnostic BamHI
fragments for Southern genotyping. Locations of probe and primers used
for genotyping in ES cells and mice are shown below the
Ahrnls allele. B, representative PCR
genotyping results. Primers amplify the 380-bp wild type
Ahr+ allele. Product is cut into 140- and 240-bp
fragments by BamHI after amplification of the
Ahrnls allele. Genotype is listed above
each representative lane, and the percentage of animals of
each genotype recorded at weaning is noted below each
lane (n = 86). MW, 100-bp ladder
molecular weight marker.
liver. Both of these samples expressed
the receptor at a level that was approximately one-half of what we
observed from the Ahr+/+ mouse liver (Fig.
4A). Since Western blots are a
semiquantitative method, we provided an independent and more
quantitative assessment of AHR expression levels by photoaffinity
labeling the receptors with increasing amounts of the ligand
2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin
(Fig. 4B). To quantitate bound photoaffinity ligand in the
104-kDa AHR bands, excised radioactive gel slices were quantified in a
counter. In keeping with what we observed based upon Western blots,
we found that the level of functional binding sites for this ligand in
liver extracts of Ahrnls/nls mice
approximated the levels found in Ahr+/
animals
and was half of the level found in Ahr+/+ mice
(Fig. 4B). In an effort to determine whether the ligand binding affinity was the same for receptor found in
Ahr+/
and
Ahrnls/nls mice, we generated a
saturation binding isotherm with increasing amounts of radioligand. A
modified Scatchard analysis of the binding data revealed similar
affinities between wild-type AHR and AHRnls proteins
derived from liver cytosols (Fig. 4, C and D). As
a result of these findings, we included heterozygous
Ahr+/
mice as controls in most
experiments.
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Fig. 4.
The AHRnls protein binds ligand
but does not activate DRE-mediated transcription in
vivo. A, Western blot analyses showing
protein levels in liver cytosolic extracts from three individual
animals from each genotype Ahr+/+,
Ahr+/ , and
Ahrnls/nls. Cytosolic protein (150 µg) is loaded in each well of a 7.5% polyacrylamide gel.
Visualization is performed with anti-AHR antibody. B, ligand
binding capacity of cytosolic AHR proteins using 1 nM
photoaffinity ligand. 150 µg of the same protein extracts in
A were incubated with [125I]Br2DpD
and activated at 310-nm light. Samples were washed to remove unbound
ligand and separated by SDS-PAGE. Ligand binding was analyzed by
autoradiography. C and D, ligand binding of the
AHR from Ahr+/
and
Ahrnls/nls animals. Cytosolic
extracts from Ahr+/
(C) and
Ahrnls/nls (D) animals
were incubated with increasing amounts of
[125I]Br2DpD ligand. Saturation binding
isotherms are plotted as a function of total ligand versus
bound (dpm counted from excised gel pieces). Each point on the plot
represents the average of two experiments. Insets, Scatchard
analysis of binding data calculated according to "Experimental
Procedures." Bmax values are expressed as
relative dpm: Ahr+/
Bmax = 1290;
Ahrnls/nls
Bmax = 1110. Because of significant overlap
between curves and for clarity, results from
Ahr+/
(C) and
Ahrnls/nls (D) are plotted
separately. E, EROD activity analyses from microsomes in
Ahr+/+, Ahr+/
,
Ahr
/
, and
Ahrnls/nls animals. Microsomal
isolations from liver extracts were incubated with ethoxyresorufin in
the presence of NADPH. EROD activity was measured from
p-dioxane-treated (
) and p-dioxane plus
TCDD-treated (+) animals at excitation of 510 nm and emission of 590 nm. Fluorescent values were normalized to total protein levels.
Error bars, S.D. values from three individual
animals.
animals were injected intraperitoneally with 100 µg/kg TCDD. After 6 days, liver microsomes were isolated and analyzed for EROD
activity. Microsomes from wild type and Ahr+/
mice show a similar basal EROD activity that was induced nearly 1000-fold by TCDD (Fig. 4E). In contrast, microsomes from
Ahrnls/nls mice showed only basal
EROD activity that was not altered by TCDD treatment. This result
indicates that the AHRnls protein cannot activate gene
transcription from DRE elements in vivo.
/
, Ahr+/
, and
Ahr+/+ mice to TCDD and measured liver
hypertrophy and thymic involution, two highly reproducible end points
associated with TCDD toxicity in animals. Male, 5-6-week-old animals
were treated with 100 µg/kg TCDD in p-dioxane or an
equivalent volume of p-dioxane alone. Mice were sacrificed 6 days later, and liver and thymus wet weights were recorded. Liver
weights in Ahr+/+ and
Ahr+/
animals increased significantly in
response to TCDD, yet neither the Ahr
/
and
Ahrnls/nls animals showed any
TCDD-induced increase in liver weights in response to TCDD (Fig.
5A, p
0.05). In addition, we observed that the relative liver weights of both
the Ahrnls/nls and the
Ahr
/
mice were reduced ~28% as compared
with the Ahr+/+ or
Ahr+/
controls (p
0.05;
Fig. 5A). Thymus weights in Ahr+/+
and Ahr+/
animals decreased significantly in
response to TCDD, yet Ahr
/
and
Ahrnls/nls, animals did not show any
significant TCDD-induced decreases in thymus weights (p
0.05; Fig. 5B). In this experiment, there was a trend
suggesting that TCDD might have a subtle influence on relative thymus
weights of Ahr
/
and
Ahrnls/nls mice, but independent
experiments did not support this relationship (data not shown).
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Fig. 5.
Comparison of toxicity of TCDD measured in
liver and thymus in Ahr+/+, Ahr+/ ,
Ahr
/
, and
Ahrnls/nls animals. Animals 5 weeks old were exposed
by intraperitoneal injection to a single 100 µg/kg dose TCDD
dissolved in p-dioxane (vehicle) or equivalent volume of
p-dioxane alone. After 6 days, liver (A) and
thymus (B) were excised and weighed and normalized to total
body weight. Gray bars, vehicle-treated animals;
white bars, TCDD-treated animals.
Error bars, S.D. The number of animals used in
each group is noted within each bar. Those groups not
sharing a superscript letter differ significantly
at p
0.05.
, and
Ahrnls/nls embryos, and the tissue
was directly exposed to TCDD in culture for 4 days. In response to
0.03% (v/v) Me2SO alone, genotype did not affect palatal
closure. However, when exposed to 3 nM TCDD in
Me2SO, no palates from Ahr+/+ or
Ahr+/
mice fused, whereas the TCDD-exposed
palates from Ahrnls/nls animals all
fused (Table I). This result was not
different from Me2SO-treated controls. Therefore,
AHRnls protein fails to activate those events, leading to
cleft palate formation in response to TCDD.
Closure in wild type and mutant palates following direct exposure
to TCDD in organ culture
/
Mice--
Wild type and
Ahrnls/nls animals were examined for
developmental phenotypes that have been previously reported in
Ahr
/
mice (6-8, 10, 39-43). Tissue wet
weights were measured on major organs of 8-week-old male mice. We
observed that relative liver weights in
Ahrnls/nls animals were 27% smaller
when compared with Ahr+/+ controls (Fig.
6A; p
0.001). The reduced Ahrnls/nls liver
weights were identical to those reported previously in Ahr
/
animals (7). Also similar to what has
been reported for Ahr
/
animals, normalized
spleen and heart weights were moderately increased in
Ahrnls/nls compared with
Ahr+/+ (Fig. 6, B and C;
p
0.001). Importantly, relative weights of thymus,
kidney, lung, testes, and brain remained unaffected by genotype (thymus
weight is shown as an example) (Fig. 6D).
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Fig. 6.
Developmental phenotype of the
Ahrnls/nls mouse. Weights of excised organs from
8-week-old animals were normalized to body weight (b.w.).
Left bar, Ahr+/+;
right bar,
Ahrnls/nls. A, liver;
B, spleen; C, heart; D, thymus.
Error bars, S.D. of at least four animals.
Statistical differences are noted by a star
(p 0.001). E-H, radiographs of portal
vein-injected contrast agent in animals. E,
Ahr+/+; F,
Ahr+/
; G,
Ahr
/
; H,
Ahrnls/nls. The arrows in
G indicate key features. PV, portal vein;
shIVC, suprahepatic inferior vena cava; ihIVC,
infraheptaic inferior vena cava. Representative images were taken ~10
s after injection of contrast agent. G and H are
enlarged ~2× the size of E and F for labeling
clarity.
/
mice, the DV remains open throughout
adulthood. To determine whether a similar phenotype exists in
Ahrnls/nls animals, we performed time
lapse angiography to observe the flow of contrast medium through the
perfused liver. In the control Ahr+/+ and
Ahr+/
animals, contrast medium flowed into the
portal vein and immediately into the portal branches of the liver
vasculature (Fig. 6, E and F, respectively).
After filling the major branching veins of the liver, contrast entered
the suprahepatic IVC and then filled the infrahepatic IVC. However,
contrast medium in Ahrnls/nls mice
flowed directly from the portal vein to the IVC. The shunt between the
portal vein and the IVC was clearly visible as a short segment that
runs perpendicular to both the portal vein and IVC within the liver
(Fig. 6H). This vascular pattern is consistent with patent
DV in Ahr
/
mice (Fig. 6G and Ref.
10)
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
mice. Although it may be simple to
assume that these receptor dependent processes are related to
DRE-driven transcription, several reports have suggested that the AHR
may signal through DRE-independent pathways (11, 13-16, 17, 45, 46,
48). Thus, we are still in need of a formal proof to demonstrate that
the mechanism by which XMEs are up-regulated is similar to the
mechanism by which the AHR plays a role in TCDD toxicity or regulates
aspects of mammalian development.
-helix of AHR
within the bHLH domain. This basic region encodes the nuclear
localization motif that is revealed upon ligand binding, and it harbors
the DRE contact residues by which the AHR interacts with its DNA
half-site within these enhancers (20, 28). In early characterization studies, we demonstrated that the NLS mutation resulted in an AHR
protein that interacts normally with chaperones but that could not
translocate to the nucleus or bind to DREs upon exposure to agonists.
To this end, we used immunocytochemistry to demonstrate that the mutant
protein remained cytosolic in the presence of an agonist. We then also
used a battery of in vitro assays to demonstrate that the
mutant protein was capable of interactions with its chaperones HSP90
and ARA9 (Figs. 1 and 2). Once the predicted consequences of our
mutations were confirmed, we used homologous recombination in ES cells
to replace the endogenous bHLH region of the Ahr locus (exon
2) with a mutated exon carrying the NLS amino acid substitutions.
).
controls were quite sensitive (Fig. 5,
A and B). We also examined a TCDD-induced
teratogenic response, namely the formation of cleft palate. To this
end, we exposed embryonic day 12.5 palatal shelves directly to TCDD in
culture. This protocol directly exposes palate tissue in culture to
TCDD and eliminates issues related to maternal influence. Just like the
hepatic and thymic responses, we observed that the palates from
Ahrnls/nls animals were completely
resistant to TCDD exposure, whereas the corresponding controls were
sensitive (Table I). From these results, we conclude that nuclear
localization is required for TCDD-induced liver hypertrophy, thymic
atrophy, and cleft palate formation.
/
mice are also observed in the
Ahrnls/nls mice. To this point, the
Ahrnls/nls mice display a similar
reduction in relative liver weight and moderate increases in heart and
spleen weights. In more recent experiments, we have also observed that
the livers of Ahrnls/nls display
microvesicular fatty changes around day 6 after birth (7). Just as in
the null allele these fatty changes resolve by adulthood (data not
shown). Finally, the presence of a patent DV was observed in both the
Ahrnls/nls mice and in the
Ahr
/
mice (Fig. 6). Just as in the
toxicology experiments described above, we are led to conclude that the
nuclear localization of the AHR is an essential step in the receptor
pathways that regulate liver development/DV closure.
![]() |
ACKNOWLEDGEMENTS |
---|
We acknowledge members of the University of Wisconsin-Madison Transgenic Animal Facility for expert technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants P01-CA22484, P30-CA07175-CA14520, T32-CA09135, T32-ES07015, and F32-ES05877 and a fellowship from the Mary Engsberg 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@oncology.wisc.edu.
Published, JBC Papers in Press, March 5, 2003, DOI 10.1074/jbc.M209594200
2 M. K. Bunger, S. M. Moran, E. Glover, T. L. Thomae, G. P. Lahvis, B. C. Lin, and C. A. Bradfield, unpublished observation.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
AHR, aryl
hydrocarbon receptor;
Ahrnls, the Ahr nls
allele generated;
AHRnls, the AHRnls protein
that arises from the mutant allele;
ARNT, aryl hydrocarbon receptor
nuclear translocator;
BEAR, bacterially expressed aryl hydrocarbon
receptor;
bHLH, basic helix-loop-helix;
NF,
-naphthoflavone;
dpm, disintegrations/min;
DRE, dioxin response element;
DV, ductous venosus;
EROD, ethoxyresorufin-o-de-ethylase;
ES, embryonic stem;
HSP90, 90-kDa heat shock protein;
NLS, nuclear localization signal;
PAS, Period-ARNT-Singleminded;
Rb, retinoblastoma;
TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin;
XME, xenobiotic
metabolizing enzyme;
MOPS, 4-morpholinepropanesulfonic acid;
[125I]Br2DpD, 2-azido-3-iodo[125I]iodo-7,8-dibromodibenzo-p-dioxin;
IVC, inferior vena cava.
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