From the Department of Molecular Pharmacology, Stanford University
School of Medicine, Stanford, California 94305-5332
We used differential display to discover a new
gene that the environmental contaminant
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) regulates in
mouse hepatoma cells. Its predicted amino acid sequence suggests that
the gene encodes an ecto-ATPase that contains multiple glycosylation
sites, conserved cysteine residues, and apyrase conserved regions.
cDNA expression experiments in mouse hepatoma cells confirm that
the new gene encodes an ecto-ATPase. Wild-type mouse hepatoma cells
contain both constitutive and TCDD-inducible ecto-ATPase activity.
Induction of ecto-ATPase gene expression by TCDD is direct and
occurs at the transcriptional level. Studies in mutant hepatoma cells
indicate that induction requires both the aromatic hydrocarbon
receptor (AhR) and the AhR nuclear translocator (Arnt). Furthermore,
induction requires AhR's transactivation domain, but not that of
Arnt. Our findings reveal new aspects of dioxin's biological effects
and TCDD-dependent gene regulation.
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INTRODUCTION |
2,3,7,8-Tetrachlorodibenzo-p-dioxin
(TCDD)1 is the prototype for
a class of halogenated aromatic environmental contaminants that have
generated concern because of their persistence and potential toxicity.
TCDD elicits numerous adverse responses in animals including neoplasia,
immunosuppression, hepatotoxicity, epithelial dysplasia, reproductive
toxicity, and teratogenesis (1, 2). However, the risk that dioxin poses
to human health is uncertain; it is a suspected human carcinogen (3,
4). Dioxin also elicits adaptive responses, including the induction of
xenobiotic-metabolizing enzymes. One such enzyme is cytochrome P4501A1,
which is encoded by the CYP1A1 gene (5, 6). Cytochrome
P4501A1 induction has been a useful model response for analyzing the
mechanism of TCDD action; mouse hepatoma cells constitute a
particularly powerful experimental system, because the existence of
induction-defective mutants permits genetic analyses of the mechanism
(7-9). Studies of CYP1A1 gene regulation have revealed an
interesting transcriptional control system by which mammalian cells
respond to certain environmental chemicals (10). The response involves
two transcription factors, the aromatic hydrocarbon receptor (AhR) and
the AhR nuclear translocator (Arnt), which heterodimerize and bind to
an enhancer upstream of the target CYP1A1 gene (9-11). AhR
and Arnt are members of a novel class of basic helix-loop-helix PAS
proteins, which also mediate responses to other environmental stimuli,
such as hypoxia or light (12-15). Activation of CYP1A1
transcription by the AhR/Arnt heterodimer is associated with
alterations in the chromatin structure and the pattern of protein
binding at the CYP1A1 enhancer and promoter (16-19). AhR's
transactivation domain communicates the induction signal from enhancer
to promoter and facilitates the binding of general transcription
factors to the promoter (20, 21).
Although studies on CYP1A1 have revealed important aspects
of dioxin action, additional mechanistic issues remain to be analyzed. For example, we and others have shown that dioxin can down-regulate the
expression of certain genes (22-24). Furthermore, unlike the CYP1A1 gene, some dioxin-inducible genes are constitutively
expressed in the absence of TCDD (25-28). Such observations indicate
that the dioxin-responsive gene regulatory system can function in
different contexts; the mechanisms responsible for this versatility are not well understood. To begin to address this issue, we have identified a new TCDD-inducible gene in mouse hepatoma cells, and we have analyzed
basic aspects of the mechanism by which it responds to TCDD.
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EXPERIMENTAL PROCEDURES |
Materials--
Kits for total RNA isolation (RNeasy) and DNA
purification were from QIAGEN (Chatsworth, CA). RNAimage kit for
differential display was from GenHunter (Nashville, TN). Taq
polymerase was from Perkin-Elmer (Fremont, CA). Random primer labeling
kit (rediprime), [
-32P]dCTP,
[
-33P]dATP, [
-35S]dATP, and Hyperfilm
MP were from Amersham (Arlington Heights, IL). Radioactive probe
purification columns (NucTrap) were from Stratagene (La Jolla, CA).
Restriction endonucleases were from New England Biolabs (Beverly, MA).
TA cloning kit was from Invitrogen (San Diego, CA). DNA sequencing kit
(Sequenase Version 2.0) was from U. S. Biochemical Corp.
(Cleveland, OH). SequiTherm EXCEL DNA sequencing kit was from Epicentre
Technologies (Madison, WI). Digoxigenin (DIG) RNA/DNA labeling kit and
chemiluminescent detection substrate (CDPstar) were from
Boehringer Mannheim (Indianapolis, IN). Reagents for ecto-ATPase assays
were from Sigma. SuperFect Transfection reagent was from QIAGEN.
Cell Culture--
Wild-type Hepa1c1c7, AhR-defective, and
Arnt-defective mouse hepatoma cells were maintained in
-minimal
essential medium containing 10% fetal bovine serum as described
previously (8). Phoenix cells (29) were grown in Dulbecco's modified
Eagle's medium containing 10% fetal bovine serum.
Differential Display--
We used an mRNA differential
display system (30) according to the manufacturer's instructions
(GenHunter). Briefly, wild-type hepatoma cells were exposed to 1 nM TCDD or dimethyl sulfoxide (as a control) for 16 h.
Total RNA was isolated using an RNeasy kit and reverse transcribed with
oligo(dT) primers (H-T11M, where M is A, C, or G) annealed
to the poly(A) tail. Polymerase chain reactions were performed in the
presence of [
-33P]dATP, using 30 arbitrary 5' 13-mer
and three 3' oligo(dT)M primers. Amplified cDNA fragments were
resolved on a 6% DNA sequencing gel and visualized by autoradiography.
A differentially expressed cDNA fragment (designated as C9) was
eluted from the gel and reamplified using the same set of polymerase
chain reaction primers. Amplified cDNA was subcloned into a TA
cloning vector, pCRII (Invitrogen), followed by DNA sequencing using a
Sequenase kit.
cDNA Library Screening--
A cDNA library was
constructed from Hepa1c1c7 cells (31). A 171-base pair fragment of C9
cDNA in the pCRII vector was used as a template for synthesis of
digoxigenin-labeled DNA probe according to the manufacturer's
instructions (Boehringer Mannheim). 106 plaques were
screened using this probe. Positive clones, contained within the
pBK-CMV phagemid, were excised in vivo from the ZAP Express
vector using an ExAssist-SOLR system, following the manufacturer's protocol (Stratagene). A Sequenase kit and a SequiTherm EXCEL DNA
sequencing kit were used to sequence cDNA clones.
Northern Blot Analyses--
Total RNA was isolated from
untreated or TCDD-treated cells using an RNeasy kit. Ten µg of total
RNA was separated on a 1% formaldehyde-agarose gel and transferred
onto a nylon membrane (Schleicher & Schuell) by capillary blotting,
followed by UV cross-linking. A 1.3-kilobase cDNA fragment of the
ecto-ATPase was used as a template to generate a
32P-labeled hybridization probe using the random primer
labeling kit. Membranes were prehybridized and hybridized in the
presence of 32P-labeled probe as described previously (21).
Signals were detected by autoradiography with Hyperfilm MP.
Nuclear Transcription Studies--
Nuclear run-on experiments
were performed as described by Ausubel et al. (32) with
modifications. 2 × 107 uninduced or TCDD-induced
cells were lysed in 4 ml of Nonidet P-40 lysis buffer (10 mM Tris-Cl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, and 0.5% Nonidet P-40). Nuclei were
collected by centrifugation at 500 × g and stored in
100 µl of glycerol storage buffer (50 mM Tris-Cl, pH 8.3, 40% glycerol, 5 mM MgCl2, 0.1 mM
EDTA). Ten µl of 10 × DIG mixture (10 mM ATP, CTP,
GTP, 6.5 mM UTP, and 3.5 mM DIG-UTP, Boehringer
Mannheim) was added to 100 µl of 2 × transcription buffer (10 mM Tris-Cl, pH 8.0, 5 mM MgCl2, 0.3 M KCl, 0.2 mM EDTA, 1 mM
dithiothreitol). The mixture was added to the nuclei and in
vitro transcription was carried out at 30 °C for 30 min with shaking. DIG-labeled nuclear RNA was isolated using an RNeasy kit. Ten
µg of cDNA were denatured and immobilized onto a nylon membrane
and hybridized with isolated nuclear RNA at 42 °C overnight in
standard buffer (Boehringer Mannheim) + 50% formamide. The membrane
was washed and signals were detected by chemiluminescence according to
the manufacturer's instructions (Boehringer-Mannheim).
Ecto-ATPase Assay--
The colorimetric assay measures inorganic
phosphate released during ATP hydrolysis (33). Uninduced and
TCDD-induced cells were detached from tissue culture dishes by adding
10 mM EDTA and incubating at 37 °C for 20 min. Cells
were collected by centrifugation at 500 × g for 5 min
at 4 °C and washed twice with assay buffer (20 mM HEPES,
pH 7.4, 120 mM NaCl, 5 mM KCl, 1 mM
EGTA, 0.5 mM Na3VO4, and 1 mM NaN3). 1.5 × 105 intact
cells in 300 µl of assay buffer were used for each reaction. ATP and
MgCl2 were added to a final concentration of 2 mM, followed by incubation for 15 min at 37 °C. ATP
hydrolysis was linear for at least 20 min under these experimental
conditions. Seven hundred µl of ice-cold ascorbic/molybdate mixture
(1 part of 10% ascorbic acid and 6 parts of 0.42% ammonium
molybdate·4 H2O in 1 N
H2SO4) was added to stop the reaction and the
incubation was continued for 20 min to allow color development. Cells
were pelleted and the absorbance of the supernatant at 820 nm was
measured. Ecto-ATPase activity was defined as the difference between
reactions without cation (Mg2+) and those with cation.
Expression of Ecto-ATPase cDNA--
The full-length
ecto-ATPase cDNA plus 81 base pairs of 3'-untranslated region was
cloned into the pMFG vector (20). The recombinant DNA was introduced
into Phoenix cells using a SuperFect Transfection reagent according to
the manufacturer's instructions (QIAGEN). Culture medium containing
recombinant virus was used to infect Arnt-defective mouse hepatoma
cells as described (20). Infected Arnt-defective cells were assayed for
ecto-ATPase activity.
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RESULTS |
Cloning of a Novel Dioxin-regulated Gene--
We utilized a
differential display technique (30) to identify new dioxin-regulated
genes in mouse hepatoma cells. We isolated a dioxin-inducible cDNA
fragment, which we designated as C9 (Fig. 1). Sequencing revealed approximately 170 nucleotides including the 3' poly(A) tail. A GenBankTM
search did not show significant homology to any known gene.

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Fig. 1.
Differential display. Uninduced ( ) and
TCDD-induced (+, 1 nM, 16 h) wild-type mouse hepatoma
cells were analyzed by differential display, as described under
"Experimental Procedures." The arrow indicates the
TCDD-inducible cDNA fragment, C9.
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We screened a mouse cDNA library (31) using C9 as a hybridization
probe; we isolated four overlapping cDNA clones whose sizes ranged
from 0.9 to 1.3 kilobases. Sequence analyses indicated that each clone
contained C9; however, none of the clones was full-length, which we
estimated to be 1.9 kilobases based upon Northern analyses. Therefore,
we rescreened the library using the 1.3-kilobase fragment as a
hybridization probe and isolated a 1872-base pair cDNA clone, whose
nucleotide sequence is shown in Fig. 2.
The first ATG codon is in-frame with the longest open reading frame,
which encodes a polypeptide of 495 amino acids.

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Fig. 2.
Nucleotide and predicted amino acid sequence
of the cDNA clone. The initiation codon is in
boldface. The stop codon is indicated by a star
(*). The 171-base pair cDNA fragment identified by differential
display (C9) is in italics. The polyadenylation signaling
site (aataaa) is in boldface and
italics. The predicted amino acid sequence is compared with
that of the rat brain ecto-ATPase. Identical amino acids are indicated
by dots (·). Potential N-glycosylation sites
are underlined. Arrow ( ) indicates the
potential tyrosine kinase phosphorylation site. Triangles
( ) indicate shared cysteine residues. Four putative apyrase
conserved regions (ACRs) are boxed.
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The cDNA encodes a protein whose predicted molecular mass is 54,307 Da and whose pI is 9.08. GenBankTM searches at the
nucleotide and the predicted amino acid sequence levels reveal
homologies between the cDNA and a family of
ecto-ATPase/ecto-apyrase (ATP diphosphohydrolase) enzymes. The highest
homology is with a rat brain ecto-ATPase (34), where there is 94%
identity and 98% similarity at the amino acid level (Fig. 2). The
predicted amino acid sequence for the cloned cDNA reveals six
potential N-glycosylation sites, a potential tyrosine kinase
phosphorylation site, and four putative apyrase conserved regions
(ACRs). ACR1 and ACR4 are similar to the
- and
-phosphate binding
motifs that are present in actin, hsp70, and hexokinase (35). In
addition, there are 11 conserved cysteine residues between the cloned
cDNA and the rat brain ecto-ATPase; these may form intramolecular
disulfide bonds, as hypothesized for the chicken muscle ecto-ATPase
(36). Together, these structural data imply that the cloned cDNA
encodes an ecto-ATPase; expression studies, described later, confirm
this hypothesis.
Regulation of Ecto-ATPase Gene Expression--
Northern blot
analyses using the cloned cDNA as a probe reveal the presence of
ecto-ATPase mRNA in uninduced wild-type mouse hepatoma cells; this
finding implies that the gene is constitutively expressed in the
absence of TCDD. After exposure of cells to 1 nM TCDD,
ecto-ATPase mRNA increases about 15-fold over the course of 16 h (Fig. 3).

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Fig. 3.
Time course of ecto-ATPase induction by
TCDD. Mouse hepatoma cells were treated with 1 nM TCDD
for the indicated times, and total RNA was analyzed by Northern
analyses, using ecto-ATPase cDNA as a probe (upper
panel). An identical blot was stained with 0.04% methylene blue
to verify equal RNA loading (lower panel).
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Dose-response experiments indicate that the induction of ecto-ATPase
mRNA by TCDD is dose-dependent; the estimated
EC50 is between 10 and 100 pM (Fig.
4). This dose range is similar to that of
other TCDD-inducible responses that are mediated by the Ah receptor
(37).

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Fig. 4.
Concentration dependence of ecto-ATPase
induction by TCDD. Panel A, mouse hepatoma cells were
exposed for 16 h to the indicated concentrations of TCDD. Total
RNA was analyzed by Northern analyses, using ecto-ATPase cDNA as a
probe. Equal RNA loading was verified as in Fig. 3. Panel B,
Northern blots were quantitated by densitometry using the NIHimage
program. represents the average of three independent experiments.
Brackets indicate standard deviation.
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Nuclear run-on experiments show that TCDD induces the rate of
ecto-ATPase gene transcription by about 10-fold in wild-type mouse
hepatoma cells (Fig. 5). The increase in
transcription rate is similar in magnitude to the TCDD-induced increase
in ecto-ATPase mRNA accumulation (Fig. 3). Therefore, we conclude
that TCDD acts at the transcriptional level to induce ecto-ATPase gene
expression.

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Fig. 5.
Nuclear transcription experiments.
Nuclei were isolated from uninduced ( ) and TCDD-induced (+, 1 nM, 16 h) cells and used in nuclear run-on studies.
Ecto-ATPase cDNA, glyceraldehyde 3'-phosphate dehydrogenase
cDNA (GAPDH), and CYP1A1 cDNA were
immobilized on a nylon membrane and hybridized to digoxigenin-labeled
RNA as described under "Experimental Procedures." Signals were
visualized by chemiluminescence.
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Cycloheximide, at a concentration that inhibits protein synthesis by
>95% (38), does not block the induction of ecto-ATPase mRNA by
TCDD (Fig. 6). This observation implies
that the proteins necessary for induction pre-exist within the cell and
that induction is a direct effect of TCDD. In the presence of TCDD,
cycloheximide "superinduces" ecto-ATPase mRNA accumulation
about 1.5-fold (Fig. 6); previously, we reported that cycloheximide
superinduces the accumulation of cytochrome P4501A1 mRNA to a
substantially greater extent (10-15-fold) (38, 39). This difference in
superinducibility might reflect a subtle difference in the regulatory
mechanisms for the two genes.

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Fig. 6.
Effect of cycloheximide on ecto-ATPase
induction by TCDD. Mouse hepatoma cells were exposed to 10 µg/ml
cycloheximide (CHX) for 30 min and/or to 1 nM
TCDD for an additional 4 h, as indicated. Total RNA was analyzed
by Northern analyses, using ecto-ATPase cDNA as a probe. Equal RNA
loading was verified as in Fig. 3.
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We utilized AhR-defective and Arnt-defective cells to determine whether
the induction of ecto-ATPase gene expression requires AhR and/or Arnt.
Northern analyses reveal that TCDD induces a slight accumulation of
ecto-ATPase mRNA in AhR-defective cells; reconstitution of
AhR-defective cells with AhR restores the responsiveness of the
ecto-ATPase gene to TCDD (Fig.
7A). These findings implicate AhR in the ecto-ATPase induction mechanism. Analogous findings in
Arnt-defective cells also implicate Arnt in the induction process (Fig.
7B).

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Fig. 7.
AhR and Arnt dependence of ecto-ATPase
induction by TCDD. Wild-type, AhR-defective, Arnt-defective, and
reconstituted mouse hepatoma cells were exposed to TCDD (1 nM, 16 h), as indicated. Total RNA was analyzed by
Northern analyses, using ecto-ATPase or CYP1A1 cDNA as
probes. WT, wild-type cells; AhR-def.,
AhR-defective cells; Arnt-def., Arnt-defective cells. Cells
were reconstituted with cDNA, as indicated. Equal RNA loading was
verified as in Fig. 3.
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Our observations indicate that ecto-ATPase mRNA is present in
uninduced Arnt-defective cells (Fig. 7B). Thus, although
induction of ecto-ATPase mRNA by TCDD requires Arnt, constitutive
expression is independent of Arnt. This pattern differs from that for
the CYP1A1 gene, whose expression requires Arnt even in the
absence of TCDD (Fig. 7B). We infer, therefore, that
regulation of basal expression differs for these two TCDD-inducible
genes, and we envision that studies of their promoters will reveal
interesting differences in function and chromatin structure.
We have previously shown that TCDD-induced CYP1A1 gene
expression requires AhR's transactivation domain but not that of Arnt (20). To determine whether the same situation exists for the ecto-ATPase gene, we reconstituted AhR- or Arnt-defective cells with
mutant AhR or Arnt cDNA that lack transactivation capability, and
we measured the TCDD-inducible accumulation of ecto-ATPase mRNA in
the reconstituted cells. Our findings reveal that an AhR mutant lacking
its transactivation domain (designated as AhR515 (20)) cannot restore
TCDD responsiveness to the ecto-ATPase gene in AhR-defective cells
(Fig. 8A). Thus, we conclude
that induction of ecto-ATPase gene expression by TCDD requires AhR's transactivation domain. In contrast, analogous experiments with a
mutant Arnt (designated as Arnt574 (20)) reveal that Arnt's transactivation domain is not required for the response of the ecto-ATPase gene to TCDD in vivo (Fig. 8B).

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Fig. 8.
Dependence of ecto-ATPase induction on AhR's
transactivation domain. Wild-type, AhR-defective, Arnt-defective,
and reconstituted mouse hepatoma cells were exposed to TCDD (1 nM, 16 h), as indicated. Total RNA was analyzed by
Northern analyses, using ecto-ATPase or CYP1A1 cDNA as
probes. Exposure time was 72 h for ecto-ATPase and 24 h for
CYP1A1. WT, wild-type cells; AhR-def.,
AhR-defective cells; Arnt-def., Arnt-defective cells.
cDNA used for reconstitution: AhR515, AhR lacking its
transactivation domain (20); AhR, full-length AhR; Arnt574, Arnt
lacking its transactivation domain (20), Arnt, full-length Arnt. Equal
RNA loading was verified as in Fig. 3.
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Ecto-ATPase Enzyme Activity in Mouse Hepatoma Cells--
The
preceding findings predict that mouse hepatoma cells will express
ecto-ATPase activity constitutively and that TCDD will induce enzyme
activity to higher levels. In confirmation of this hypothesis, we find
that wild-type cells exhibit both constitutive and TCDD-inducible
ecto-ATPase activity (Fig. 9). These
enzyme activity measurements are consistent with our measurements of mRNA accumulation and document that TCDD induces ecto-ATPase
activity in this cell system.

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Fig. 9.
TCDD-inducible ecto-ATPase activity in
wild-type hepatoma cells. Ecto-ATPase activity was measured in
uninduced ( ) or TCDD-induced (+, 1 nM, 24 h) cells.
Data represent the average of four experiments. Brackets
indicate standard deviation.
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To show more definitively that the cDNA we isolated encodes an
ecto-ATPase, we expressed the cDNA in Arnt-defective cells, which
exhibit low constitutive enzyme activity. Our findings reveal that
expression of the cDNA in these cells increases their ecto-ATPase activity by about 30-fold (Fig. 10).
These observations document that the cDNA encodes an ecto-ATPase.

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Fig. 10.
Cloned cDNA encodes ecto-ATPase
activity. The indicated cDNA (LacZ, pMFG/LacZ vector;
ecto-ATPase, pMFG/ecto-ATPase) were introduced into Arnt-defective
cells by retroviral infection and ecto-ATPase activity was measured as
described under "Experimental Procedures." Data represent the
average of four experiments. Brackets indicate standard
deviation.
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DISCUSSION |
Our understanding of the mechanism of dioxin action is based
largely upon studies of CYP1A1 induction; such analyses have revealed several important components of an interesting gene regulatory system and have generated major insights into AhR/Arnt function (9, 10,
40, 41). However, because of the focus on CYP1A1 regulation,
our mechanistic knowledge of TCDD action is probably incomplete, in
view of the range of responses that TCDD elicits. We envision that
studies in other regulatory contexts will add perspective to the
CYP1A1 model and reveal new features of TCDD-inducible gene
expression. Such studies also have the potential to uncover new aspects
of dioxin biology and toxicology. Therefore, we have used differential
display to identify new dioxin-responsive genes in mouse hepatoma
cells. This cell system is especially useful because AhR- and
Arnt-defective strains permit both genetic and biochemical analyses of
TCDD-dependent gene regulation.
Using differential display, we identified a TCDD-inducible cDNA
that is related to a family of ecto-ATPases. Structurally, the deduced
amino acid sequence reveals four apyrase conserved regions; such
domains may contribute to nucleotide binding and catalytic activity
(35). The sequence also reveals multiple conserved cysteine
residues and several potential glycosylation sites; intramolecular
disulfide bonds, together with glycosylation, may contribute to the
protease resistance that is typical of ecto-ATPases (36).
Functionally, our expression experiments confirm that the cDNA
encodes ecto-ATPase activity.
Using a commercially available multiple-tissue Northern blot, we find
constitutive ecto-ATPase mRNA expression in many mouse tissues,
such as heart, brain, liver, kidney, and skeletal
muscle.2 Therefore, we
envision that TCDD will induce ecto-ATPase gene expression in numerous
tissues; however, this hypothesis remains to be tested in intact
animals and/or other cell types.
Ecto-ATPases may influence several physiological processes. For
example, in hydrolyzing extracellular ATP and other nucleotides, ecto-ATPases have the potential to modulate the ligand concentration at
P2 purinergic receptors, which bind ATP (42, 43).
Ecto-ATPases may also act in conjunction with ecto-5'-nucleotidases to
convert extracellular AMP to adenosine, which is a ligand for
P1 purinergic receptors (44). Ecto-ATPase/ecto-apyrase
enzymes probably also participate in the recycling of nucleosides for
purine/pyrimidine biosynthesis and general cell metabolism (45).
Therefore, induction of ecto-ATPase activity by TCDD may perturb
purinergic signaling and cellular metabolic pathways. In addition,
CD39, which is an ecto-apyrase that contributes to homotypic cell
adhesion in activated lymphocytes (46), shares substantial homology
with the mouse hepatoma ecto-ATPase. This finding suggests that the
ecto-ATPase could play a role in cell adhesion; if so, induction by
TCDD might affect the adhesion process.
Our findings indicate that TCDD induces ecto-ATPase gene expression at
the transcriptional level; furthermore, the response is direct and
requires both AhR and Arnt. In these respects, the induction mechanism
resembles that for the CYP1A1 gene (10). In contrast to
CYP1A1, the ecto-ATPase gene exhibits constitutive expression, which is Arnt-independent. This difference suggests to us
that the chromatin structures for the ecto-ATPase and CYP1A1 promoters are likely to differ. The uninduced CYP1A1
promoter assumes a nucleosomal configuration (17, 19); this chromatin structure can account for the lack of constitutive CYP1A1
expression, because nucleosomes repress transcription (47-49). Its
constitutive expression leads us to envision that, in uninduced cells,
the ecto-ATPase promoter is maintained in a relatively accessible (i.e. non-nucleosomal) chromatin configuration, which allows
binding of general transcription factors in the absence of TCDD. Thus, induction of ecto-ATPase gene expression provides an opportunity to
analyze AhR/Arnt function in a new structural and functional context.
The induction of ecto-ATPase gene expression represents an unexplored
aspect of dioxin biology. We assume that TCDD mimics a naturally
occurring, perhaps endogenous, substance that can induce the gene. We
also envision that induction evolved as an adaptive response and that
the effect of TCDD could be either advantageous or adverse. For
example, induction could be beneficial if increased ecto-ATPase
activity provides protection against high levels of extracellular ATP
associated with increased cell lysis during trauma or inflammation
(50). On the other hand, responses to TCDD are sustained, because
dioxin is resistant to metabolism and accumulates in the cell (1, 51,
52). Persistent induction of ecto-ATPase activity could have adverse
consequences, by causing long-term changes in purinergic signaling
and/or nucleotide metabolism. Our findings support the general concept
that TCDD produces (some of) its adverse biological effects through
sustained disruptions of cellular signaling and metabolic pathways that are critical for maintaining homeostasis.
We thank Margaret Tuggle for secretarial
assistance and Vatis A. Jeen for comments on the manuscript.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF042811.