From the Environmental Toxicology Center and
School of Pharmacy, University of Wisconsin, Madison, Wisconsin
53706 and the § Department of Biology, Woods Hole
Oceanographic Institution, Woods Hole, Massachusetts 02543
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ABSTRACT |
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Two aryl hydrocarbon receptors
(AhRs), rtAhR2 Trout and other salmonids are especially sensitive to
polychlorinated dibenzo-p-dioxins, dibenzofurans, and
biphenyls (1-3). The toxicity of planar polychlorinated
dibenzo-p-dioxins, dibenzofuran, and biphenyl congeners is
mediated by the aryl hydrocarbon receptor (AhR).1 While this is well
studied in mammals (4), the AhR pathway is less well characterized in
fish (5, 6). Experiments with a photoaffinity ligand have identified a
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-binding protein
in trout with a molecular mass of 145 kDa, somewhat similar to the
95-110-kDa AhR proteins found in mammals (7, 8). In addition, the AhR
dimerization partner, ARNT, has been cloned from rainbow trout (9) and
more recently, AhR homologs have been cloned in other fish species
(10-12).
The AhR is a ligand-activated transcription factor in the PAS family of
proteins. Transcriptional activity requires dimerization with another
PAS protein, ARNT. The unliganded AhR resides in the cytosol as a
multiprotein complex that includes hsp90 and AhR interacting protein
(13). Ligand binding causes the AhR to move to the nucleus. During
transport to the nucleus, AhR dissociates from hsp90 dimer and AhR
interacting protein and subsequently forms a dimer with ARNT. This new
complex binds to specific double-stranded DNA sequences, generally
referred to as dioxin response elements (DREs), to alter the expression
of genes (14, 15). The DRE consensus sequence has been defined as
TnGCGTG for the mouse and human AhR (14). This sequence has been
identified and shown to be transcriptionally active in the 5'-flanking
region of genes such as CYP1A1 in mammals. This sequence has
also been identified in the 5'-flanking region of the rainbow trout and
Atlantic tomcod CYP1A genes, but has not been directly
tested for functionality (16, 17).
The structure-activity relationship for both early life stage mortality
and induction of CYP1A by polychlorinated dibenzo-p-dioxins and dibenzofuran congeners in rainbow trout is generally similar to
that for mammals, but for planar polychlorinated biphenyls the
structure activity relationship in fish does not correspond to that
observed in mammals (3, 18, 19). The lower potency of the
mono-ortho-polychlorinated biphenyls in fish may be
attributable to differences between mammalian and fish AhRs, but proof
of this hypothesis requires direct comparison of the receptor proteins from each species. As a step toward this analysis, we set out to clone
AhR cDNAs from a rainbow trout gonad cell line (RTG-2). In this
report, we describe the cloning of two distinct rainbow trout AhR
cDNAs. The proteins encoded by the two clones are very similar in
sequence. However, they show surprising differences in tissue
distribution, regulation of expression, and transcriptional activity.
These results suggest that the two types of receptors may mediate
distinct responses in different tissues.
Rainbow Trout--
Six female juvenile rainbow trout were
obtained from the Aquaculture Program at the University of Wisconsin
and held in flowing water at 8 °C for 3 days to acclimate. Trout
were injected intraperitoneally with TCDD (10 µg/kg) or the same
volume (2 ml/kg) of vehicle (5% acetone, 95% corn oil) as control.
After 3 days tissues were harvested and immediately frozen in liquid
nitrogen and stored at Cell Culture--
RTG-2 cells were obtained from ATCC (Manassas,
VA) and cultured in modified Eagle's medium supplemented with 10%
fetal bovine serum at 21 °C in an atmosphere of normal air. Cells
were split and final media changes were completed at least 24 h
prior to dosing. Cells were exposed to graded concentrations of TCDD
(0.0005-1.0 nM) dissolved in Me2SO with a
final Me2SO concentration in the culture media of 0.1%.
TCDD exposure continued for 6 h before cells were harvested for
RNA. COS-7 (monkey kidney epithelial cells) were obtained from ATCC and
were cultured in Dulbecco's modified eagle's medium supplemented with
10% fetal bovine serum in an atmosphere of 5% CO2. Cells
were split the day prior to transfection.
Library Construction and Screening--
Two cDNA libraries
were constructed using RNA isolated from RTG-2 cells. A random primed
Construction of Full-length AhR Clones--
Two independent
clones were isolated from library 1 which encompass the 5' UTR and part
of the coding sequence of each rtAhR2 and two more independent clones
were isolated from library 2. After complete sequencing, overlapping
sequences were found and a single full-length cDNA was constructed
corresponding to each mRNA. The nucleotide sequence for the rainbow
trout AhR2 Sequencing--
Sequencing of isolated clones was completed by
manual sequencing using a Sequenase kit (U. S. Biochemical Corp.,
Cleveland, OH), and by automated sequencing using ABI ready reaction
mixture and an ABI 377 automated sequencer (Perkin Elmer, Foster City, CA). Sequences were determined for each strand and each strand was
sequenced at least 3 times.
In Vitro Transcription/Translation--
In vitro
transcription/translation reactions were completed using a TNT kit from
Promega essentially as directed by the manufacturer. For each reaction
1 µg of plasmid DNA was used in a 50-µl total reaction volume.
Amino acid mixtures lacking methionine were added to each reaction and
reactions were supplemented with translational grade
[35S]methionine (NEN Life Science Products Inc., Boston,
MA). 10 µl of each reaction was diluted with an equal volume of
2 × loading dye and separated on 10% SDS-polyacrylamide gel
electrophoresis. Gels were fixed, dried onto filter paper, and exposed
to PhosphorImager screens overnight.
Velocity Sedimentation
Analysis--
2,3,7,8-Tetrachloro[1,6-3H]dibenzo-p-dioxin
(35 Ci/mmol) was obtained from Chemsyn Science Laboratories (Lenexa,
KS) and purified to In Vitro Electrophoretic Mobility Shift Assay--
All sense
strand oligonucleotides are listed 5' to 3', complementary strands are
not shown. The consensus DRE core is underlined and mutated bases are
in bold. Oligonucleotide wt rtDRE1 (ACCTTTGCACGCTATCGAAAT) was 5'-end labeled with 32P using T4 polynucleotide kinase
and annealed to a 3-fold molar excess of the complementary
oligonucleotide followed by probe purification. Unlabeled competitor
DNAs were similarly produced by annealing unlabeled wt rtDRE1 with its
complementary oligonucleotide, mut rtDRE1
(ACCTTTGCGCGCTATCGAAAT and its complementary oligonucleotide. For in vitro DNA binding assays,
approximately equal amounts of in vitro produced rtAhR2 Genomic Southern Blots--
Genomic DNA was isolated from
rainbow trout livers and Southern blots were prepared using standard
techniques (27). Membranes were hybridized overnight at 42 °C in
50% formamide hybridization solution with random primed
32P-labeled DNA probes. Membranes were washed with high
stringency (0.1 × SSC, 0.1% SDS). Probe 1 was made by digesting
rtAhR2 Total RNA and Poly(A+) RNA Isolation--
Total RNA
was isolated using two methods. Plates of cells exposed to TCDD in
Me2SO or Me2SO alone were rinsed with culture media to remove dosing solution, lysed, and scraped into Qiashredder homogenizers (Qiagen, Chatsworth, CA). RNA was isolated from the lysates using Qiagen RNeasy kits. RNA was isolated from cells and organ
tissues using TRI reagent (Molecular Research Laboratories, Cincinnati,
OH) as directed by the manufacturer. Poly(A+) RNA was
isolated from total RNA using PolyATtractTM mRNA
isolation kits (Promega, Madison, WI).
Northern Blots--
RNA was separated in 1.2% denaturing
formaldehyde agarose gels, blotted to nylon with 20 × SSC, and
hybridized overnight at 42 °C with random primed
32P-labeled DNA probes in 50% formamide hybridization
solution (19). Relative message levels were determined using ImageQuant
software (Molecular Dynamics, Sunnyvale, CA).
Reporter Vectors--
prt1Aluc was constructed using
PCR amplification of rainbow trout genomic DNA (forward primer
5'-AGGTTGGTTGAGTGAGATG-3'; reverse primer 5'-TGCAGGGAGATCGAAGAAG-3') to
amplify a 1540-bp portion of the 5'-flanking region of the rainbow
trout CYP1A gene promoter from base pair 139-1678 (GenBank
accession number S69277). This includes 2 DREs and the transcriptional
start site (position 1594). The PCR product was TA cloned into
pGemT-Easy. The resulting plasmid was digested in its multiple cloning
region with SacI and NcoI and ligated into
pGL3-Basic. This plasmid was named prt1Aluc and provided a
TCDD responsive firefly luciferase reporter vector under control of the
rainbow trout CYP1A gene promoter.
The pGudluc 1.1 reporter vector (28) was obtained from Dr. Michael
Denison (University of California, Davis, CA). This reporter vector is
based on pGL2-Basic and has the firefly luciferase gene under control
of a 484-bp fragment of the mouse CYP1A1 enhancer, that
contains 4 DREs and the murine mammary tumor virus promoter. The pRL-TK
vector (Promega) was used in all experiments as a control for
transfection efficiency. This vector contains the Renilla reniformis luciferase gene under the control of a herpes simplex virus thymidine kinase promoter.
Reporter Gene Assays--
Assays were performed 24 h after
exposure to TCDD. A Dual Luciferase Assay (Promega) was used to
determine firefly (AhR agonist-dependent) and
Renilla (transfection control) luciferase activity for each well. Media was removed by vacuum aspiration, each well was washed with
1 × PBS and 100 µl of passive lysis buffer was added. Plates were incubated 20 min at room temperature on an orbital shaker. Cell
lysis was confirmed microscopically and a 10-µl aliquot was transferred to a 96-well luminometer plate. Luminescence assays were
completed using a Dynatech Laboratories ML-2250 luminometer (Chantilly,
VA). Assays for luciferase activity were conducted as follows: 50 µl
of luciferase assay buffer II was injected into each well, incubated
2 s, and the resulting luminescence integrated over the next
10 s. After reading each plate the assay buffer was changed to
Stop & Glo and identical assay conditions were used to measure
Renilla activity in the same wells. Because the Renilla luciferase control vector is susceptible to
induction by trans effects when a second reporter construct
with a strong promoter is activated, the amount of transfected control
plasmid was reduced to 3 ng of pRL-TK/µg DNA in each well in order to avoid this problem.
Statistical Analysis--
TCDD dose-response experiments in
RTG-2 cells were completed in triplicate. AhR or CYP1A mRNA
PhosphorImager signals were normalized by dividing by the corresponding
Cloning of Two Rainbow Trout AhR cDNAs--
A random primed
rainbow trout cDNA library was screened using a previously cloned
F. heteroclitus AhR cDNA fragment as a probe (20). A
total of 8.0 × 106 plaques were screened, and 16 clones were isolated. These clones fell into 2 groups, termed Comparison of Rainbow Trout AhRs to AhRs and PAS
Proteins from Other Species--
The full-length sequences
for rtAhR2
Fig. 1 presents an alignment of the
rtAhR2 In Vitro Translation and Functional Characterization of rtAhR2
The in vitro translated proteins were tested for ability to
bind TCDD (Fig. 3). Lysates containing
unlabeled in vitro translated proteins were incubated with
[3H]TCDD and analyzed by velocity sedimentation on
sucrose density gradients by the method of Tsui and Okey (23). Both
trout AhR2s exhibited a peak of [3H]TCDD specific binding
(i.e. binding that was abolished by a 200-fold excess of
2,3,7,8-TCDF) with a sedimentation coefficient of 10.6 S.
This peak was not seen when unprogrammed lysate was used in place of
lysate containing the AhR translation products. This experiment shows
that rtAhR2
To further confirm that the clones encode functional AhR proteins, gel
shift experiments were used to demonstrate specific binding to
double-stranded DNA fragments containing a DRE from the rainbow trout
CYP1A promoter. In vitro translated proteins were
preincubated with in vitro translated rainbow trout ARNT along with 10 nM TCDD in order to form an active, DNA
binding complex. After activation, a 32P-labeled
double-stranded oligonucleotide containing the DRE (core TAGCGTG) was
added, and the bound probe was separated from free oligonucleotide
probe by native gel electrophoresis (Fig.
4). This experiment shows that both the
Evidence for Distinct Genes--
Several pieces of evidence
suggest that the TCDD Induction of rtAhR2 mRNA Abundance--
RTG-2 cells were
treated with graded concentrations of TCDD in Me2SO, and
total RNA was isolated 6 h later for Northern blotting (Fig.
6). Using a probe that hybridizes with
both the
TCDD treatment also induced both rtAhR2 Differences in rtAhR2 Comparison of Transcriptional Activity of rtAhR2
In these experiments, we compared the activity of rtAhR2
When the reporter construct contained the rainbow trout
CYP1A enhancer, along with huAhR, zfAhR2, or rtAhR2 Rainbow Trout Express Two Distinct AhR Transcripts--
We have
isolated two different AhR2 clones from rainbow trout cDNA
libraries. These two cDNAs encode proteins of 1058 and 1059 amino
acids that are 95% identical at the amino acid level. The rainbow
trout AhRs are substantially longer than mammalian AhR proteins, which
range from 805 amino acids for mouse, to 853 amino acids for the rat
AhR. For comparison, the Atlantic tomcod (Microgadus tomcod)
receptor contains 823 amino acids (10), and the zebrafish AhR is 1027 amino acids long (11). The rtAhR2s show strong similarities to
mammalian receptors over the first 400 amino acids, but the carboxyl
termini are much less conserved. Notably, the rainbow trout AhRs do not
contain the Gln-rich domain made up of repeated glutamines that are
thought to play an important role in mediating transactivation (34).
The zebrafish and Atlantic tomcod sequences also lack Gln-rich domains
(10, 11). While the stretches of polyglutamine are absent in the fish
receptors, there appear to be corresponding segments of polyasparagine
in approximately the same locations. In addition to the repeated glutamines, several other regions in the carboxyl-terminal half of the
mammalian AhR are also important in transactivation (35). Three motifs
have been found to generally mediate transactivation: acidic amino
acid-rich, glutamine-rich, or areas rich in a mixture of
proline/serine/threonine (36). Examining the distal end of the rainbow
trout AhRs we find several regions that match these specifications. The
most striking of these in rtAhR2
For both the
There are several possible explanations for our finding two distinct
AhR clones: they could represent products from two different genes,
they might be splice variants from a single gene, or they might
represent allelic variants of a single gene. The wide distribution of
substitutions between the two clones is difficult to account for by
splicing, requiring either a very large number of splices, or almost
total gene duplication to produce the two different messages. In
addition, when a genomic Southern blot was probed with a labeled
fragment that was expected to cross-hybridize with both sequences, a
consistent pattern of at least two bands was observed. Because the
probe was relatively short (598 bp) it is unlikely that in all cases
multiple bands were produced by cleavage of a single gene within the
region corresponding to the probe. Indeed, when the blot was reprobed
with a similar sized fragment corresponding to the 3'-UTR of rtAhR2 Multiple AhRs in Fish--
Other fish species also appear to have
more than a single AhR gene. The cloning of multiple AhR types from
several fish species (rainbow trout, zebrafish, killifish, and smooth
dogfish) raises questions regarding the function of these multiple
genes (12). It is hypothesized that vertebrates underwent an ancient
genome duplication event and that some fish, including salmonids and catastomids, underwent a second such duplication more recently (38-40). Comparison of rtAhR2 Differences between rtAhR2
We have found that TCDD induces rtAhR2
Our finding that AhR isoforms from the same species have different
specificities for enhancer sequences indicates that the rtAhR2 and rtAhR2
, were cloned from rainbow trout (rt)
cDNA libraries. The distribution of sequence differences, genomic
Southern blot analysis, and the presence of both transcripts in all
individual rainbow trout examined suggest that the two forms of rtAhR2
are derived from separate genes. The two rtAhR2s have significant
sequence similarity with AhRs cloned from mammalian species, especially
in the basic helix-loop-helix and PAS functional domains located in the
amino-terminal 400 amino acids of the protein. In contrast, the
Gln-rich transactivation domain found in the carboxyl-terminal half of
mammalian AhRs is absent from both rtAhR2s. Both clones were expressed
by in vitro transcription/translation and proteins of
approximately 125 kDa were produced. These proteins bind
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and are able
to bind dioxin response elements in gel shift assays. rtAhR2
and
rtAhR2
are expressed in a tissue-specific manner with the highest
expression of rtAhR2
in the heart. Expression of rtAhR2
and
rtAhR2
mRNAs is positively regulated by TCDD. Both rtAhR2
and
rtAhR2
produced TCDD-dependent activation of a reporter
gene driven by dioxin response elements. Surprisingly, the two
receptors showed distinct preferences for different enhancer sequences.
These results suggest that the two receptor forms may regulate
different sets of genes, and may play different roles in the toxic
responses produced by AhR agonists such as TCDD.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C until RNA was isolated.
ZAP cDNA library (library 1) was constructed by Stratagene (La
Jolla, CA). cDNAs were created using a random hexamer and a library
which contained 5.8 × 106 primary clones was
produced. A second library (library 2) was constructed in our
laboratory using a
ZAP express kit from Stratagene and a poly(dT)
primer. Libraries were screened as described by Stratagene. Briefly,
50,000 plaque forming units were diluted in fresh XL1-Blue MRF
bacteria, mixed with 0.7% agarose, and plated on 150-mm LB plates.
After overnight incubation, nylon membranes were used to lift plaques.
The membranes were hybridized with probe in a 50% formamide
hybridization solution at 42 °C. Initial screens of library 1 were
made using a portion of the Fundulus heteroclitus AhR2 (20)
as a probe; 8.0 × 106 plaques were screened.
Subsequent screens of library 2 used portions of the rainbow trout AhR
as a probe; 6 × 106 plaques were screened. Probes
were labeled with [32P]dCTP by random priming. Phagemids
(pBluescript II SK and pBK-CMV) were excised from purified plaques with
helper phage as described by the manufacturer. For use as a loading
control, we cloned rainbow trout glyceraldehyde-3-phosphate
dehydrogenase from library 2 using a probe generated by RT-PCR
amplification of a conserved portion of glyceraldehyde-3-phosphate
dehydrogenase (GenBank accession number AF027130).
cDNA has been submitted to GenBank with accession
number AF065137. The nucleotide sequence for the rainbow trout AhR2
cDNA has been submitted to GenBank with accession number AF065138.
For expression studies these fragments were ligated into pBK-CMV that
had been digested with XbaI and NotI.
95% by high performance liquid chromatography
according to the method of Gasiewicz and Neal (21). 2,3,7,8-TCDF was
obtained from Ultra Scientific (Hope, RI).
Methylated-[methyl-14C]ovalbumin was from NEN
Life Science Products Inc. (Boston, MA). Methylated-[methyl-14C]catalase was
synthesized as described previously (22). AhR proteins were expressed
by in vitro transcription and translation (TnT) and analyzed
by velocity sedimentation on sucrose gradients in a vertical tube rotor
by the method of Tsui and Okey (23). For each AhR, two identical TnT
reactions (100 µl total) were combined, diluted 1:1 with MEEDMG
buffer (8) (25 mM MOPS, pH 7.5, 20 °C, containing 1 mM dithiothreitol, 1 mM EDTA, 5 mM
EGTA, 0.02% NaN3, 20 mM
Na2MoO4, 10% (v:v) glycerol), split into two 100-µl aliquots, and incubated with [3H]TCDD (2 nM) ± TCDF (400 nM) for 1-2 h at 4 °C.
[3H]TCDD concentration was verified by sampling each tube
for total counts. No charcoal-dextran treatment was used to remove
unbound [3H]TCDD, as trout AhR has been shown to be
sensitive to small amounts of charcoal (24). After incubation, 90 µl
of each incubation was applied to 10-30% sucrose gradients prepared
using the method of Coombs and Watts (25); tubes were then spun for 140 min at 60,000 rpm at 4 °C in a VTi 65.2 rotor. Gradients were
fractionated (150 µl per fraction) and counted using a Beckman
LS5000TD scintillation counter. Specific binding is defined as the
difference between total binding (incubations containing
[3H]TCDD) and nonspecific binding (incubations containing
[3H]TCDD plus a 200-fold excess of TCDF).
[14C]Catalase (11.3 S) and
[14C]ovalbumin (3.6 S) were added as internal
sedimentation markers; they eluted in fractions ~15-16 and ~4,
respectively, as indicated. Sedimentation coefficients were determined
by the method of Martin and Ames (26).
or rtAhR2
and rtARNTb proteins were incubated in the presence of 10 nM TCDD in 0.2% Me2SO or Me2SO
alone for 90 min at 22 °C. Following incubation, 1.5 µg of
poly(dI-dC) and binding buffer (20 mM Hepes, pH 7.9, 100 mM NaCl, 1 mM dithiothreitol, 6% (v:v)
glycerol) were added and the incubation continued for an additional 20 min at 22 °C before the addition of 100,000 cpm of the wt rtDRE
probe and 10-fold molar excess of unlabeled wild type rtDRE, or mutated
rtDRE competitor DNAs. After 20 min incubation at 22 °C, complexes
were resolved on a 0.5 × TBE (90 mM Tris, 64.6 mM boric acid, and 2.5 mM EDTA, pH 8.3) 4.5%
acrylamide gel at 4 °C. The dried gels were exposed to a phosphor
screen overnight before analysis.
with SpeI and HindIII to release a
520-bp fragment of the 3'-UTR which specifically bound to rtAhR2
sequences. Probe 2 was made by digesting rtAhR2
with
PvuII and gel isolating a 598-bp fragment from the 5' end of
the coding sequence. Probe 2 cross-hybridizes with both rtAhR2
and
rtAhR2
sequences.
-actin mRNA signals. This normalized value obtained for each
TCDD dose was divided by the mean value for the Me2SO
control to determine the magnitude of induction by TCDD. For
transactivation experiments in transiently transfected COS-7 cells,
graded concentrations of TCDD were used to produce dose-response curves
with each AhR/reporter gene pair and each experiment was repeated 3-5
times. A normalized luciferase activity number was determined for each
assay well by dividing the firefly luciferase activity by
Renilla luciferase activity. EC50 values were
determined using a nonlinear estimation process for determination of
half-maximal response in the Statistica software package (StatSoft,
Tulsa, OK). Level of significance for all analyses was
p < 0.05.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
, by restriction digest and sequence. Sequencing demonstrated that
both classes of clones were homologous to mammalian AhRs; however,
these sequences were incomplete: each clone contained a section of
5'-untranslated region (UTR), an initiation ATG codon and a single open
reading frame which continued to the end of the insert without a stop codon. A second cDNA library was constructed using a poly(dT) primer so that cDNAs encompassing the 3' end of mRNAs would be favored. Screening this library yielded clones corresponding to the
and
clones. These had regions overlapping the previously isolated
clones at their 5' ends, and as expected, these clones contained
complete 3' ends. From this it was deduced that two distinct AhR
mRNAs are expressed in RTG-2 cells.
and rtAhR2
were obtained by sequencing each strand at
least 3 times. These sequences were each found to have a single open
reading frame, encoding proteins of 1059 and 1058 amino acids,
respectively. This is approximately 200 amino acids longer than most
other AhR sequences previously reported. The predicted molecular mass
of unmodified rtAhR2
and rtAhR2
is 115 kDa, somewhat smaller than
the 145-kDa mass estimated for AhR in RTG-2 cells and trout liver by
photoaffinity labeling (7, 8). The two predicted amino acid sequences
show 95% identity with each other. Most of the differences between the two amino acid sequences occur in the first 345 amino acids, however, occasional differences appear throughout the length of the protein sequences.
and rtAhR2
predicted amino acid sequences with the human
AhR sequence (29). The greatest similarity between the sequences is
found within the conserved basic helix-loop-helix and the PAS A and B
functional domains. The basic helix-loop-helix domain has been shown to
mediate DNA binding and play a secondary role in AhR/ARNT dimerization in mammalian AhRs. The PAS region is important in ligand binding and
AhR/ARNT dimerization (30, 31). Notably, the rainbow trout AhRs do not
contain a Gln-rich region that is thought to play an important role in
mediating transactivation by the AhR. The zebrafish AhR and the
Atlantic tomcod AhR sequences also lack a Gln-rich domain (10, 11).
BLASTP comparison of the rainbow trout AhRs to the GenBank data base
showed that the rtAhR2 sequences are most similar to existing AhR
sequences, with significant but lower similarity to other PAS family
proteins. The amino-terminal halves of the protein sequences (including
the basic helix-loop-helix and PAS domains) have the greatest
similarity. Both sequences resemble the fish AhR2 sequences more
closely than the AhR1 sequences reported by Hahn et al. (11)
and are therefore designated AhR2s.
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Fig. 1.
Comparison of
rtAhR2 , rtAhR2
, and
human AhR deduced amino acid sequences. Periods
indicate identities and dashes indicate gaps. Functional
domains defined for the human AhR (29) are indicated (DNA-binding
domains, dark box; ARNT dimerization, underlined;
ligand binding, open box; transactivation, vertical
box). Sequence alignment was completed using ClustalW (51).
GenBank accession numbers for the sequences are as follows: rtAhR2
(AF065137), rtAhR2
(AF065138), and human AhR (S41124).
and rtAhR2
--
To demonstrate the ability of the rtAhR2
and
rtAhR2
cDNAs to encode proteins, we used a coupled in
vitro transcription/translation system to make
[35S]methionine-labeled translation products. Fig.
2 shows the phosphorimage of the SDS-PAGE
separated in vitro translation products. A single band of
approximately 125 kDa was produced with each cDNA. This is
intermediate between the 115-kDa size predicted from the coding sequence and the estimated 145-kDa size observed in the photoaffinity ligand binding experiments.
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Fig. 2.
In vitro translation of
rtAhR2 and
rtAhR2
.
[35S]Methionine-labeled in vitro translated
rtAhR2
, rtAhR2
, and rtARNTb proteins were resolved on an 8%
SDS-polyacrylamide gel. A phosphorimage of the dried gel is shown.
Arrows indicate position of the full-length proteins.
and rtAhR2
both encode proteins capable of specific,
high-affinity binding of TCDD.
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Fig. 3.
Specific binding of [3H]TCDD to
rtAhR2 and rtAhR2
as
analyzed by velocity sedimentation on sucrose gradients. AhR
proteins were expressed by in vitro transcription and
translation (TnT), diluted 1:1 with MEEDMG buffer containing molybdate
(see "Experimental Procedures"), and incubated with
[3H]TCDD (2 nM) ± TCDF (400 nM)
for 1-2 h at 4 °C. After each incubation, 90 µl was applied to
10-30% sucrose gradients and spun for 140 min at 60,000 rpm in a VTi
65.2 rotor. Gradients were fractionated (150 µl per fraction) and
counted. Specific binding is the difference between total binding
(
TCDF, solid circles) and nonspecific binding (+TCDF,
open circles). [3H]TCDD concentration was
verified by sampling each tube for total counts.
[14C]Catalase (11.3 S) and
[14C]ovalbumin (3.6 S) were added as
sedimentation markers; their elution positions are indicated.
A, rtAhR2
; B, rtAhR2
; and C,
unprogrammed lysate.
and
forms of the receptor produced a shifted band (solid
arrow) that could be specifically competed by a 10-fold excess of
unlabeled oligonucleotide. This competition was substantially reduced
when the unlabeled oligonucleotide was mutated at a single position
(core TAGCGCG; mutated base
underlined). Addition of either ARNT alone or the AhRs
without ARNT, failed to produce specific bands, although nonspecific
bands (open arrows) were observed in these lanes. Although
both forms of the receptor produced a readily visible shifted band, the
form of the receptor consistently produced a stronger band with this probe than the
form. These results also demonstrate that in vitro rtAhR2/rtARNTb DRE binding is independent of added
ligand. Ligand-independent DNA binding has also been reported for the mammalian AhR (32).
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Fig. 4.
Gel-shift analysis of rtAhR2s and rtARNTb
interactions in vitro. Equal amounts of in
vitro translated rtAhR2 or rtAhR2
proteins were incubated
with equal molar amounts of rtARNTb with, or without, 10 nM
TCDD. The samples were then incubated with a 32P-labeled
oligonucleotide probe derived from a DRE in the rainbow trout
CYP1A enhancer. In some lanes a 10-fold molar excess of
unlabeled competitor oligonucleotides was also added as indicated. The
bound and free oligonucleotides were separated on a native acrylamide
gel, and a phosphorimage of the dried gel is shown. The solid
arrow indicates the rtAhR2·rtARNTb·DRE complexes. Open
arrows indicate positions of nonspecific complexes.
and
clones are encoded by distinct genes.
First, the differences between the two sequences are widely scattered
throughout the sequence, making differential splicing an unlikely
mechanism for producing the two messages. Second, probing a genomic
Southern blot with a cDNA expected to hybridize with both sequences
consistently produced at least two bands (Fig.
5A). In contrast, a probe
expected to hybridize with only rtAhR2
sequences consistently
produced a more simple pattern (Fig. 5B). Finally, RT-PCR
analysis of mRNA from RTG-2 cells, 6 individual juvenile rainbow
trout, and 24 individual rainbow trout fry demonstrated that the two
different transcripts were present in all samples (data not shown). If
these two transcripts were allelic variants, it would be predicted that some of the 30 individuals tested would be homozygous for one of the
alleles. Taken together, our results suggest that these transcripts are
likely to be the products of different genes.
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Fig. 5.
Southern blot with rainbow trout genomic
DNA. Genomic DNA, 25 µg/lane, was digested with the indicated
enzyme, separated on a 0.8% agarose gel, and blotted. A,
the blot was probed with probe 2 which cross-reacts with both rtAhR2
and rtAhR2
sequences. B, the blot was stripped and probed
with probe 1, which hybridizes specifically with rtAhR2
.
and
sequences (probe 2), we observed two bands on the
RTG-2 total RNA blot. These bands correspond to rtAhR2
and rtAhR2
mRNA in mobility. The blot was also probed with a fragment
complimentary to 3'-untranslated portions of the rtAhR2
message
(probe 1), sequences that are not found in the rtAhR2
mRNA. With
this probe, we observed only a single band, consistent with the
expected position of rtAhR2
. We therefore conclude that the upper
band represents rtAhR2
mRNA, and that TCDD produces a
dose-related increase in both rtAhR2
and rtAhR2
mRNA
abundance. The blot was also probed with a fragment complimentary to
the rainbow trout CYP1A mRNA (19), demonstrating that the TCDD
dose-responsive induction of rtAhR2
, rtAhR2
, and CYP1A mRNAs
occurs over a similar range of TCDD concentrations.
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Fig. 6.
Induction of rtAhR2
and rtAhR2
mRNA by TCDD in a rainbow
trout gonadal cell line (RTG-2). RTG-2 cells were treated with
graded concentrations of TCDD as indicated, and RNA was isolated after
6 h. Phosphorimage of a representative Northern blot, with 20 µg
of total RNA, hybridized with probe 1, probe 2,
-actin, and CYP1A
probes is indicated.
and rtAhR2
mRNAs in
kidney of juvenile rainbow trout. Trout were injected intraperitoneally with TCDD, (10 µg/kg) or vehicle as a control, and tissues were collected for Northern blotting 3 days after injection. As shown in
Fig. 7, TCDD induced both rtAhR2
and
rtAhR2
mRNAs in kidney, but failed to change these message
levels in spleen and liver. This result confirms that the TCDD
induction of rtAhR2
and rtAhR2
seen in RTG-2 cells also occurs in
a least some cells in the whole organism.
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Fig. 7.
TCDD induction of rtAhR2
and rtAhR2
mRNA in kidneys of
juvenile rainbow trout. Rainbow trout were treated with vehicle or
10 µg/kg TCDD and kidneys, livers, and spleens were isolated for
Northern blotting as described under "Experimental Procedures." The
blot was probed with probe 2, which hybridizes with both rtAhR2
and
rtAhR2
as indicated by the arrows. The blot was stripped
and a probe for glyceraldehyde-3-phosphate dehydrogenase was used to
control for variations in loading and transfer.
and rtAhR2
mRNA Abundance in
Rainbow Trout Tissues--
A Northern blot using RNA samples isolated
from different rainbow trout organs showed tissue-specific differences
in the expression of rtAhR2
and rtAhR2
. A Northern blot using
poly(A+) RNA samples from the indicated rainbow trout
organs was probed with a fragment that hybridizes with both of the AhR
transcripts (Fig. 8A). The
blot shows that rtAhR2
mRNA is consistently expressed at lower
levels than the rtAhR2
message. However, the ratio of AhR2
to
AhR2
message varied between tissues (Fig. 8B). The rank order for organs with the highest AhR2
message abundance relative to
rtAhR2
was heart > liver= brain > kidney = blood = intestine = spleen. Expression of both messages was low and similar in the ovary.
This tissue-specific expression suggests that the two forms of AhR may
play distinct roles in different tissues.
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Fig. 8.
Organ-specific expression of
rtAhR2 and rtAhR2
in
juvenile rainbow trout. Poly(A+) RNA (3 µg/lane)
from the indicated organs of juvenile rainbow trout was separated in
1.2% denaturing agarose, blotted to nylon, and hybridized with a probe
that recognizes both rtAhR2
and rtAhR2
transcripts.
and rtAhR2
with Different Enhancers--
To further demonstrate functional
activity of rtAhR2
and rtAhR2
, we used a transfection assay to
measure transactivation. This assay system uses transfected COS-7
monkey kidney cells to express the different AhR/ARNT proteins, and a
luciferase reporter vector driven by promoter fragments from either the
rainbow trout CYP1A or the mouse CYP1A1 genes to
measure transactivation activity. This allowed us to compare the
activity of different AhR/ARNT proteins from 3 different species,
rainbow trout, zebrafish, and human, in a setting where the cellular
background remained constant, and only the receptor molecules and the
enhancer elements were varied.
and
rtAhR2
from rainbow trout to that of human AhR (huAhR) and a
zebrafish AhR (zfAhR2). The AhR cDNAs were transfected together with ARNT cDNAs from the same species. In the case of rainbow trout, the same form of ARNT (rtARNTb) was paired with both the
and
forms of the AhR. Transcription of the AhR and ARNT constructs was
verified by Northern blot (data not shown). COS cells do not express
endogenous AhR, but do express a small amount of endogenous ARNT
protein (33). The COS cells were transfected with the constructs, exposed to graded concentrations of TCDD, and assayed for luciferase activity as a measure of transactivation. The transfection efficiency in each assay well was monitored by inclusion of a control construct, pRL-TK, expressing Renilla luciferase activity in all transfections.
,
addition of TCDD gave a dose-dependent increase in
luciferase expression (Fig. 9,
top). In contrast, cells transfected with rtAhR2
showed
little if any transcriptional response to TCDD. However, rtAhR2
is
not transcriptionally inactive in all settings. When cells containing the reporter driven by the mouse enhancer elements were transfected with rtAhR2
, there was a TCDD dose-related increase in luciferase expression (Fig. 9, bottom). On the other hand, zfAhR2 was
less active with the mouse enhancer fragment. Thus, in addition to tissue differences in message expression pattern, the two forms of
rainbow trout AhR differ in their preference for enhancers. This
suggests that the two receptors may regulate distinct sets of genes.
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Fig. 9.
Transactivation activity of
rtAhR2 , rtAhR2
,
zfAhR2, and huAhR with two different AhR-responsive luciferase reporter
vectors. COS-7 cells were transfected with the indicated AhR and
the species-specific ARNT expression construct, along with pRL-TK and
one of the reporter vectors, either prt1Aluc or pGudluc 1.1. Points represent fold induction of luciferase activity relative to
Me2SO control (mean ± S.E., n = 3).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
runs from amino acid 985 to 994 as
... STSTSPPSRP ... with 9 of 10 residues falling into this
category. Serine/threonine-specific phosphatases have been found to
alter the rate of AhR transcriptional enhancement at a step subsequent
to AhR/ARNT DNA binding (37). Confirmation of transactivation activity
in these sequences will require further study.
and
clones, open reading frames were found out of
frame and just upstream of the coding sequence. Multiple resequencing
experiments confirm that these are truly out of frame with the AhR
ORFs, do not encode part of the receptor, and are highly similar to
each other. The strong conservation between these two upstream
sequences suggests that they serve an important function. One
possibility is that they encode a functional product. If so, this would
be a novel protein, in that the predicted amino acid sequences encoded
by these regions failed to match any known protein sequences in the
data bases. Perhaps a more likely possibility is that these sequences
are involved in some form of regulation of AhR gene expression, either
controlling transcription or translation.
,
with no similarities to the rtAhR2
clone, a much less complex
pattern was consistently observed. In addition to the genomic Southern
blot, an experiment using reverse transcriptase/PCR amplification of
sequences from the mRNA of 30 individual fish demonstrated the
presence of both mRNAs in all of the samples. If the two sequences
are allelic variants, this would indicate that 30 out of 30 fish are
heterozygotes, with 0 out of 30 fish homozygous for either allele.
Since with two alleles, an individual fish has a 50% chance of being
homozygous for one allele or the other, the odds of obtaining no
homozygotes in 30 fish are very small. Our results are most consistent
with at least two distinct AhR genes in rainbow trout.
and rtAhR2
to the partial
sequences of AhR1 and AhR2 from killifish and smooth dogfish show that
both rtAhR2s are more similar to the AhR2 forms than either is to AhR1. Therefore, an AhR1 gene may exist in rainbow trout. A portion of a
third AhR-like transcript in rainbow trout has been isolated but not
fully characterized (41). It has been suggested that complete
redundancy of function after gene duplication is unstable, and over
time one of the duplicated genes will become inactive, or will diverge
in function. We have demonstrated that the two rtAhR2s are
differentially expressed between organs, suggesting divergence in function.
and rtAhR2
--
The two forms of
AhR from rainbow trout show remarkable similarity in size and sequence.
They both bind TCDD and interact with DRE containing sequences. Despite
these similarities, the two receptor forms show surprising differences
in expression pattern and transactivation properties. The two rtAhR2s
are expressed in different tissues and at different levels. rtAhR2
mRNA was identified in all organs examined, albeit at very low
levels in the ovary, and at strikingly high levels in the heart. The
high level of rtAhR2
mRNA in the heart is of interest
toxicologically because the heart has been identified as a key target
organ for TCDD developmental toxicity in early life stages of rainbow
trout (42-44).
and rtAhR2
mRNA levels
in RTG-2 cells as well as in kidney cells in juvenile trout. The
elevation in rtAhR2
and rtAhR2
mRNA in RTG-2 cells mirrors the induction of CYP1A mRNA, which is believed to be the result of
AhR activation. The TCDD EC50 values for induction of the
three transcripts in RTG-2 cells are about 0.025 nM TCDD
and are maximally elevated at 6 h. These findings suggest that
rtAhR2
and rtAhR2
expression is directly induced by ligand
activation of the rainbow trout AhR. This appears to be distinct from
the regulation of AhR expression observed in mammals. In mammalian cell
lines and organs, AhR protein is rapidly depleted after acute exposure
to TCDD (45-48). Exposure to TCDD during mouse embryonic development caused a decrease in the amount of AhR mRNA and AhR protein in the
palate of embryonic mice (49). The difference between our results with
rtAhR2
, rtAhR2
, and the previous results might be ascribed to a
fundamental difference between fish and mammalian systems, as the
zebrafish AhR2 mRNA is also induced by TCDD (11). However, the
Atlantic tomcod AhR mRNA in liver does not appear to be regulated
by aryl hydrocarbons (10).
and
rtAhR2
isoforms may regulate distinct sets of genes. This is
surprising given the high degree of sequence conservation between the
two receptor forms. The zebrafish receptor also showed a distinct
preference for one reporter construct over another. In contrast to
rtAhR2
, the zfAhR2 recognizes the trout enhancer, but is only weakly
active with the mouse reporter. We have previously shown that the
zebrafish AhR2/ARNT dimer only weakly binds to a DRE based on the mouse
CYP1A1 gene enhancer, although it does bind a similar
oligonucleotide based on the rainbow trout CYP1A gene
enhancer (11). This is despite the fact that both enhancers contain
nearly identical core consensus DRE sequences. Other researchers have
reported reduced activity of the rainbow trout CYP1A
promoter in a mammalian cell line when compared with mammalian
CYP1A1 promoter sequences (50). The possibility that the two
trout AhRs induce distinct sets of genes, taken with the observed
variations in tissue distribution, suggests that the two isoforms may
mediate different biological responses. Whether one form or the other contributes more to mediating TCDD toxicity in rainbow trout remains to
be seen. These results also suggest that there may be more than one
form of the AhR in other vertebrate classes and should serve as an
impetus for their discovery and functional characterization.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Sibel Karchner for assistance in the cloning of the partial F. heteroclitus AhR, Eric Andreasen for help with RTG-2 experiments, and Zhengjin Cao and Dr. Judd Aiken for assistance with in vivo rtAhR2 expression experiments.
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FOOTNOTES |
---|
* This work was supported in part by the University of Wisconsin Sea Grant Institute under the National Sea Grant College Program, National Oceanic and Atmospheric Administration, United States Department of Commerce, and the State of Wisconsin, Federal Grant NA46RG0481 and Sea Grant project number R/MW-58 (to W. H. and R. E. P.), National Institutes of Health NIEHS Grant R29 ES06272 and Sea Grant College Program Office Grant NA46RG0470, Sea Grant project number R/B-124, and the Woods Hole Oceanographic Institute (to M. E. H.). This is contribution 9742 from the Woods Hole Oceanographic Institution and contribution 324 of the University of Wisconsin, Environmental Toxicology Center.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF027130, AF065137, and AF065138.
¶ Supported by a National Institutes of Health NIEHS Training Grant, awarded to the University of Wisconsin, Environmental Toxicology Center, and National Institutes of Health NIEHS Post-doctoral Fellowship F32 ES05786-01.
** To whom correspondence and reprint requests should be addressed: School of Pharmacy, 425 N. Charter St., University of Wisconsin, Madison, WI 53706. Tel.: 608-263-5453; Fax: 608-265-3316; E-mail: repeterson{at}pharmacy.wisc.edu.
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ABBREVIATIONS |
---|
The abbreviations used are: AhR, aryl hydrocarbon receptor; rt, rainbow trout; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; ARNT, aryl hydrocarbon receptor nuclear translocator; P450lA, cytochrome P4501A protein; cyp1A cytochrome P4501A, DRE, dioxin response element; RTG-2, rainbow trout gonadal cells; UTR, untranslated region; GCG, genetics computer group; EC50, half-maximal response; Me2SO, dimethyl sulfoxide; RT-PCR, reverse transcriptase-polymerase chain reaction; bp, base pair(s); MOPS, 4-morpholinepropanesulfonic acid; TCDF, 2,3,7,8-tetrachlorodibenzofuran.
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REFERENCES |
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