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J Biol Chem, Vol. 273, Issue 25, 15358-15365, June 19, 1998


A Novel Response to Dioxin
INDUCTION OF ECTO-ATPase GENE EXPRESSION*

Lin Gao, Liqun Dong, and James P. Whitlock Jr.Dagger

From the Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California 94305-5332

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

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), [alpha -32P]dCTP, [alpha -33P]dATP, [alpha -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 alpha -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 [alpha -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.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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.

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 (up-arrow ) indicates the potential tyrosine kinase phosphorylation site. Triangles (black-triangle) indicate shared cysteine residues. Four putative apyrase conserved regions (ACRs) are boxed.

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 beta - and gamma -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).

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. open circle  represents the average of three independent experiments. Brackets indicate standard deviation.

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.

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.

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.

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.

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.

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.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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.

    ACKNOWLEDGEMENTS

We thank Margaret Tuggle for secretarial assistance and Vatis A. Jeen for comments on the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant ES03719 (to J. P. W.) and a Pharmaceutical Research and Manufacturers of America Foundation (PhRMA) Fellowship for Advanced Predoctoral Training in Pharmacology/Toxicology (to L. G.).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) AF042811.

Dagger To whom correspondence should be addressed. Tel.: 650-723-8233; Fax: 650-725-2952.

1 The abbreviations used are: TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; ACR, apyrase conserved regions; AhR, aromatic hydrocarbon receptor; Arnt, Ah receptor nuclear translocator; DIG, digoxigenin; PAS, a homologous region shared by Per, Arnt, and Sim.

2 L. Gao and J. P. Whitlock, Jr., unpublished results.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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