Gene-Specific TCDD Suppression of RAR{alpha}- and RXR-Mediated Induction of Tissue Transglutaminase

Sheryl R. Krig*, Roshantha A. S. Chandraratna{dagger}, Mary M. J. Chang{ddagger}, Reen Wu{ddagger} and Robert H. Rice*,1

* Department of Environmental Toxicology, University of California, Davis, 1 Shields Avenue, California 95616–8588; {dagger} Retinoid Research, Departments of Chemistry and Biology, Allergan, Inc., Irvine, California 92623–9534; and {ddagger} Department of Internal Medicine, University of California, Davis, California 95616–8685;

Received December 18, 2001; accepted March 12, 2002


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The malignant human keratinocyte line SCC4 provides a model system to study the mechanism by which 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) suppresses retinoid induction of the tissue transglutaminase gene (TGM2). The current work explores the nature of TCDD suppression of retinoid action to determine whether it is gene specific, whether it is retinoid receptor isoform-dependent, and whether it requires close proximity of retinoid and TCDD response elements. First, two other retinoid-inducible genes were identified in SCC4 by microarray screening whose induction was unaffected by TCDD, clearly demonstrating the gene specificity of TCDD suppression. Second, the receptor isoform dependence of retinoid responsiveness in SCC4 was tested. TGM2 was found to be inducible by an RAR{alpha}-specific but not by an RAR{gamma}-selective agonist. A lack of responsiveness to RAR{gamma} agonists was found to be characteristic of SCC4, however, inasmuch as transcription driven by a retinoid response element in transfections was also stimulated only by the {alpha}-specific agonist in these cells. Because SCC4 lacks expression of RARß, the gene specificity evidently was not attributable to differential TCDD targeting of retinoid receptor isoforms. Finally, the proximal 5 kb of the TGM2 promoter was found to be retinoid responsive in stable transfections, but the induction was not suppressed by TCDD. These results indicate that the suppressive action of TCDD occurs indirectly and through a separate DNA site likely located outside the 5-kb region, not by direct interference with retinoid action or at retinoid response elements.

Key Words: dioxin; keratinocytes; retinoids; TGM2.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Exposure to the persistent and extremely potent environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can result in a multitude of chronic toxic effects in a variety of animal tissues and species (Poland and Knutson, 1982Go). Among these effects are altered development of hormonally responsive tissues in utero (cf. Lewis et al., 2001Go) and interference with the action of steroid superfamily and other hormonal effectors in cell culture (Wilson and Safe, 1998Go). The tremendous potency with which TCDD acts has raised concern that it could have adverse consequences by this mode of action at currently encountered concentrations, but developmental effects for its potent endocrine disruptor action may not become evident until later in life (Dienhart et al., 2000Go). Thus, model systems are sought in which the mechanism by which it acts can be elucidated.

TCDD is well known to serve as a ligand for the Ah receptor and thus to induce alterations in gene expression (Denison et al., 1998Go; Whitlock, 1999Go). The best studied alteration is induction of transcription at dioxin-responsive elements (DREs) in the promoter regions of responsive genes. Negative regulation, which appears to result from preventing the positive effects of agents such as hormones, is not well understood. One proposed mechanism involves overlapping or closely spaced response elements, in which Ah receptor binding to a poorly active DRE could interfere with the binding of a positive acting factor at, for example, an Sp1 (Krishnan et al., 1995Go) or an AP-1 (Gillesby et al., 1997Go) site. Interactions of the Ah receptor with steroid hormone superfamily receptors or even their transcriptional cofactors at the protein level in principle might also occur.

Perturbation of vitamin A homeostasis by TCDD is a consistent effect of exposure across species, appears to include downregulation of retinoid action, and could contribute to the wasting syndrome (Fletcher et al., 2001Go). The tissue transglutaminase gene (TGM2) is retinoid inducible in a variety of cell types and cell lines. The finding that TCDD prevents the retinoid induction in SCC4 cells (Rubin and Rice, 1988Go) suggests that this line could serve as a good culture model for understanding the nature of the negative regulation. Krig and Rice (2000) have shown that TCDD suppression occurs at the level of transcription and cannot be attributed to depletion of active retinoid in the cell culture medium. However, an important remaining uncertainty was whether TCDD suppression affected retinoid-induced genes in general or only certain retinoid-induced genes. The latter would leave open the possibility of differential TCDD perturbation of the action of RAR isoforms acting on different genes. However, this work also raised the prospect that retinoid induction might occur by an indirect pathway and that TCDD suppression could, therefore, also be indirect. Current efforts were aimed at helping resolve these uncertainties.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture.
SCC4 and SCC12B2 keratinocytes, derived from squamous carcinomas of human tongue and epidermis, respectively (Rheinwald and Beckett, 1981Go), were cultivated with a feeder layer of lethally irradiated 3T3 cells in Dulbecco modified Eagle/Ham's F-12 media (2:1) supplemented with fetal bovine serum (5%), hydrocortisone (0.4 µg/ml), insulin (5 µg/ml), T3 (20 pM), and adenine (0.18 mM) as described by Allen-Hoffmann and Rheinwald (1984). HeLa cells were grown in the same media without 3T3 support and supplemented with 10% serum. Cultures were treated with retinoids and TCDD at confluence and harvested after 48 or 72 h. TCDD (>= 98% pure; AcuStandard, New Haven, CT) was used at 10 nM, sufficient to produce maximal suppressive effects (Rubin and Rice, 1988Go). All-trans retinoic acid (ATRA), used at 3-µM concentration for maximal effect, was purchased from Sigma (St. Louis, MO). Retinoids and TCDD were dissolved in dimethylsulfoxide (DMSO) for addition to culture medium.

Synthetic retinoids.
The RXR panagonist AGN 194204, used at 30 nM, has a median effective concentration (EC50) of < 1 nM for transactivation of RXR{alpha}, ß, or {gamma} and does not activate RARs (Vuligonda et al., 2001Go). The RAR{alpha} antagonist AGN 194301, used in the range of 0.1–100 nM, is completely inactive in transactivation assays but displays dissociation constants (Kds) of 2.8, 320, and 7258 nM for binding to RAR{alpha}, ß, and {gamma}, respectively (Teng et al., 1997Go). Transactivating activities of the RAR{alpha}- and {gamma}-selective agonists (AGN 194078 and 194433, respectively), used in the range of 0.1 nM–3 µM, were assayed in quadruplicate in CV1 cells (which lack retinoid receptors) transfected with an expression vector for an individual retinoid receptor and a luciferase reporter plasmid as previously described (Klein et al., 1996Go).

Northern blotting.
After treatment, cultures were rinsed with isotonic phosphate buffer and dissolved in Trizol (GibcoBRL) for RNA isolation (Krig and Rice, 2000Go). Total cellular RNA (20 µg) was electrophoresed in a 1% agarose gel containing 5% formalin and transferred to a Nytran membrane (Schleicher and Schuell, Keene, NH). The RNA was cross-linked to the membrane by ultraviolet irradiation (Stratalinker, Stratagene, La Jolla, CA) and baked 1 h at 80°C in a vacuum oven. The blot was prehybridized for a minimum of 1 h in 7% SDS-0.5 M sodium phosphate, pH 7.2. Membranes were probed with cDNA as previously described, labeled with [32P]-{alpha}-deoxycytidine triphosphate by random labeling (Ambion, Austin, TX), and hybridized overnight. Before autoradiography, membranes were washed 3 times in 2X SSC-0.1% SDS for 5 min and once in 0.5X SSC-0.1% SDS for 30 min. Quantitation was performed using a Molecular Dynamics SI phosphorimager (Amersham, Piscataway, NJ).

Microarray.
Nylon membrane arrays of 1.8 x 2.7 cm were prepared using at least 10 ng of DNA per spot. For spotting, cDNA was prepared by polymerase chain reaction (PCR) from human EST clones obtained from the IMAGE consortium. Product quality was verified by gel electrophopresis, and only single-band cDNAs were spotted. Messenger RNA was isolated using a Poly-A-Pure mRNA kit (Ambion) from SCC4 cultures treated with ATRA, TCDD, ATRA plus TCDD, or DMSO (solvent control). Biotin-16-deoxyuridine triphosphate-labeled cDNA was prepared from 1 µg of mRNA using Superscript II (Gibco) with random hexamer primers. Each 2000-cDNA membrane was prehybridized in a Seal-a-Meal bag for 1 h at 65°C with 1% salmon sperm DNA in 1.5 ml of 1X hybridization solution: 20X SSC, 1% N-laurylsarcosine, 10% SDS, and 1% blocking reagent (Roche). Hybridization was continued overnight in a fresh Seal-a-Meal bag in 150 µl of 2X hybridization solution, including poly A (10 µg/µl), Cot-1 DNA (10 µg/µl), and the biotin-labeled cDNA probe. The next day the membrane was washed in 2X SSC, 0.1% SDS at room temperature a minimum of 2 times for 5 min followed by a second wash series 3 times with 0.1X SSC, 0.1% SDS at 65°C for 15 min. Membranes were incubated in blocking solution for 1 h at room temperature in 4 ml of 80% blocking dilution buffer (1 M maleic acid, 0.15 M sodium chloride, 7.5% solid sodium hydroxide), 0.5 ml of 20% dextran sulfate solution, and 0.5 ml of 10% blocking reagent followed by 1 h in a solution containing 4.1 ml of 1X tris-buffered saline (TBS)–0.3% bovine serum albumin (pH 7.4), 0.5 ml of 10% blocking reagent, 0.4 ml of water, and 8.4 µl of streptavidin-galactosidase 1:700 (GibcoBRL). Next, the membrane was washed 3 times in 1X TBS at room temperature for 10 min. Color development required 3 h to overnight incubation at 37°C in 5 ml of X-gal substrate buffer (10X TBS/pH 7.4, 3 mM potassium ferrocyanide, 3 mM potassium ferricyanide, 1 mM magnesium chloride) with 120 mM X-gal. After color development, membranes were rinsed in water, dried at room temperature, scanned on a UMAX Powerlook 3000 flatbed scanner at 3000 dpi, and analyzed with the Gene Pix program. Clones selected for further study were sequence verified.

Transfections.
The retinoic acid response element (RARE) construct based on the sequence from the RARß promoter was described previously (Krig and Rice, 2000Go). The day before transient transfection, 5 x 105 cells per well were inoculated into 6-well culture plates. The medium was changed the next day, and each well was transfected with a calcium phosphate coprecipitate containing 3.8 µg of the pGL3-RARE construct, 3.8 µg of pGL3 Basic, and 0.8 µg of pRL-null renilla luciferase vector. After 16 h, the cultures were rinsed with isotonic phosphate buffer and treated with the indicated concentration of retinoids with or without 10 nM TCDD, all dissolved in DMSO. After 48 or 72 h of treatment, the cultures were lysed and the luciferase activities measured using the Dual-Luciferase Reporter system (Promega, Madison, WI). The 5-kb human TGM2 promoter sequence was obtained by PCR from a 140-kb region of chromosome 20 (PAC clone containing the human TGM2 gene, accession no. AL031651) used as template and cloned into the pGL3 (Basic) luciferase reporter vector. For stable transfections, 3 x 106 cells were plated in a 10-cm dish and the next day treated with a calcium phosphate coprecipitate of 40 µg of the pGL3 construct and 5 µg of pPUR (Clontech, Palo Alto, CA) for puromycin selection. After 1 day the medium was changed, and the next day the transfected cells were passaged 1:10 onto a puromycin-resistant 3T3 feeder layer. Selection with 0.3 µg/ml puromycin was continued for 2 weeks, at which time a new, nonresistant 3T3 feeder layer was added and culture continued without puromycin for 1–2 weeks. Macroscopic clones were then pooled. Retinoid and TCDD treatments were as described previously. Firefly luciferase reporter activity was assayed using the Promega reporter system.

Replication.
Each experiment illustrated is representative of 3 or more experiments. Values represent the mean ± SD of duplicate or triplicate samples from representative experiments. Statistical significance of the results was calculated using a 2-tailed t-test; a Bonferroni correction was included for multiple comparisons.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To investigate the generality of TCDD suppression of retinoid action in SCC4 cells, a cDNA microarray study was conducted similar to those previously described (Chen et al., 2001Go). Because an exhaustive analysis was not required, limited arrays of 2000 genes encoding enzymes of metabolism, cell cycle proteins, transcription factors, and other signal transduction components were screened initially for those induced by ATRA. A number of genes responsive to TCDD alone were detected but not investigated further. In addition to TGM2, 3 retinoid-induced genes were identified: isocitrate dehydrogenase (IDH), interleukin enhancer binding factor (ILF1), and an ETS domain transcription factor (E7-4). In the microarrays and in subsequent Northern blots, the retinoid induction of the first 2 genes was largely unaffected by TCDD. Figure 1Go shows the induction of IDH and ILF1 by the RAR{alpha}-specific ligand AGN 194078 and their lack of TCDD suppression as demonstrated by Northern blotting. The third gene was substantially induced by TCDD alone in arrays and Northern blots, preventing clear interpretation.



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FIG. 1. Retinoid-induced genes in SCC4 cells not suppressed by TCDD. Results of a 2000-clone cDNA microarray identified 2 ATRA-induced genes: isocitrate dehydrogenase (IDH) and interleukin enhancer binding factor (ILF1). Cultures were treated with 3 µM RAR{alpha}-specific agonist AGN 194078 (RAR{alpha}) with and without TCDD for 72 h followed by Northern analysis. Values represent the mean (n = 2) ± SD; *p < 0.05 for retinoid induction over DMSO solvent control.

 
The RAR isoform dependence of TGM2 induction was investigated in SCC4 cells using isoform-selective agonists. As shown in Figure 2Go, the RAR{alpha}-selective ligand AGN 194078 produced a 3-fold induction at 3 nM and a 4-fold increase over background at 300 nM (Fig. 2AGo). By contrast, the RAR{gamma}-selective ligand AGN 194433 was inactive (Fig. 2CGo). For comparison, dose–response relations in CV1 cells transfected with individual RAR isoforms are shown in Fig. 2B and 2DGo, demonstrating the selectivities of each synthetic retinoid in standard functional tests. As shown in Figure 3Go, these agents were then tested for activity in transient transfections of SCC4 using the retinoid response element from the RARß gene promoter to drive a luciferase reporter as previously described (Krig and Rice, 2000Go). In this assay, the RAR{alpha}-selective agonist was highly active at 3 nM and above, but the RAR{gamma}-selective agonist was inactive below 300 nM despite its EC50 of {approx}30 nM in RARE-reporter transactivation systems (see Fig. 2DGo). These results indicated that SCC4 cells were generally unresponsive to the RAR{gamma}-selective ligand. Previous work of others using SCC4, unlike normal human epidermal cells, has revealed the inability of RAR{gamma}1 in cooperation with the vitamin D receptor to stimulate transcription of the phospholipase C-{gamma}1 gene (Xie and Bikle, 1998Go).



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FIG. 2. RAR-selective agonist concentration dependence of TGM2 or reporter transactivation. (A, C) SCC4 cultures were harvested after 72-h exposure to the indicated concentrations of retinoid and analyzed for TGM2 mRNA by Northern blotting. Values represent the mean (n = 2) ± SD. (B, D) Retinoid transactivation assay in CV1 cells transiently cotransfected with a RARE-driven luciferase reporter and with the indicated RAR construct. Values represent the mean ± SD of quadruplicate transfections. *Significant retinoid induction (p < 0.03).

 


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FIG. 3. Concentration dependence of RAR-selective agonists on RARE luciferase-reporter activity. SCC4 cells were transiently transfected and the cultures treated for 72 h with the indicated concentration of RAR{gamma}-specific agonist AGN 194433 (left panel) or RAR{alpha}-specific agonist AGN 194078 (right panel) and then assayed for luciferase activities. Values represent the mean (n = 2) ± SD. *Significant retinoid induction (p < 0.03).

 
When SCC4 cells were treated with the RAR{alpha}-selective ligand at 300 nM, the RAR{alpha}-selective antagonist AGN 194301 suppressed TGM2 induction with an IC50 of {approx}3 nM (Fig. 4Go), in agreement with its Kd of 3 nM for binding to RAR{alpha} (Teng et al., 1997Go). This antagonist similarly suppressed the small TGM2 induction and retinoid response element transcriptional activity of the RAR{gamma}-selective ligand evident at higher concentrations (data not shown), indicating that only RAR{alpha} was mediating transcriptional responses in these cells.



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FIG. 4. Suppression of RAR{alpha}-specific agonist induction of TGM2 by RAR{alpha}-selective antagonist. Cultures were harvested after 72-h exposure to the indicated concentrations of agonist AGN 194078 and antagonist AGN 194301 and analyzed by Northern blotting. A representative experiment of 3 trials is illustrated.

 
Consistent with previous observations that TCDD suppresses TGM2 stimulation by ATRA (Krig and Rice, 2000Go), TCDD also suppressed the stimulatory action of the RAR{alpha}-selective agonist AGN 194078 in the current work. During the course of these experiments, the RXR panagonist AGN 194204 was found to stimulate substantially TGM2 expression in cooperation with the RAR{alpha}-selective agonist but not by itself (Fig. 5Go). As illustrated, TCDD suppressed TGM2 stimulation to the same low level regardless of whether the RXR panagonist was included.



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FIG. 5. TCDD suppresses TGM2 stimulation by RAR{alpha}-specific agonist with or without RXR panagonist. Cultures were treated with 3 µM RAR{alpha}-specific agonist AGN 194078, 30 nM RXR panagonist AGN 194204, or 10 nM TCDD where indicated. After 72 h, treatment cultures were harvested and analyzed by Northern blotting. Values represent the mean (n = 3) ± SD. *Significant retinoid induction; **Significant TCDD suppression of the retinoid induction (p < 0.04).

 
The ability of TCDD to suppress retinoid action was examined in several other cell lines. ATRA is known to stimulate TGM2 in the malignant epidermal keratinocyte line SCC12B2 (Rubin and Rice, 1986Go). Current work now shows this stimulation to be greatly suppressed by TCDD in these cells (Fig. 6Go), similar to SCC4. In contrast, ATRA stimulates TGM2 in HeLa cells, but this action is unaffected by TCDD (see Fig. 6Go). Resistance rather than sensitivity to TCDD suppression was more commonly observed in this work, a result obtained as well with human HL60 leukemia cells, mouse Hepa-1 hepatoma cells, and continuous lines derived from rat prostate, endometrium, and bladder (Phillips and Rice, 1983Go), known to display retinoid-inducible TGM2 (Rice et al., 1988Go).



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FIG. 6. Differing ability of TCDD to suppress retinoid induction of TGM2 in representative cell lines. ATRA-induced TGM2 in HeLa cells is unaffected by TCDD. TCDD suppression of ATRA-induced TGM2 in SCC12B2 malignant epidermal keratinocytes. Cultures were treated 72 h with 3 µM ATRA ± 10 nM TCDD followed by Northern blotting analysis. Above each graph, phosphorimages are shown of the duplicate lanes for TGM2 (T) and GAPDH (G, used for normalization) from which the graphs were generated. Values represent the mean (n = 2) ± SD; p < 0.05 for ATRA induction over DMSO control.

 
Because the TCDD effect on retinoid induction appeared insignificant in most other cell lines and genes, an analysis of the TGM2 promoter in SCC4 cells was undertaken. Initial experiments using 5 kb of 5`-flanking DNA proximal to the transcription start site to drive a luciferase reporter exhibited little retinoid inducibility in transient transfections. However, stable transfections were clearly retinoid (and TCDD) responsive, as shown in Figure 7Go. This result raised the possibility of localizing a retinoid responsive element, but the ultimate goal of elucidating the action of TCDD appeared elusive inasmuch as the combination of ATRA and TCDD was stimulatory in pools of stable transfectants, giving much higher transcriptional activity than with either agent alone.



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FIG. 7. Stable transfection of TGM2 5-kb promoter luciferase-reporter construct is ATRA responsive. Pooled transfectants were cultured 72 h with 3 µM ATRA ± 10 nM TCDD followed by firefly luciferase assay of cell lysates. Values represent the mean (n = 6) ± SD. *Significant induction above DMSO control (p < 0.03).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous work suggested that TCDD acts transcriptionally but indirectly to suppress retinoid induction of TGM2 in SCC4 cells (Krig and Rice, 2000Go). That the action was indirect was inferred from the observation that TCDD did not interfere with transcription driven by a retinoid response element in transfections. It is also consistent with the finding that TGM2 is induced with a considerable lag time, suggesting that the retinoid action itself has an indirect component. However, it is also possible that TCDD interferes selectively with different retinoid receptor isoforms. Thus, retinoid action at a given gene may be suppressed, whereas that at another gene or at a model response element is affected differently as a result of alternate isoforms involved. Although a number of explanations must be considered, this latter possibility seems plausible in view of the lack of TCDD suppression of TGM2 induction in a variety of other cell lines that may differ in RAR complement. SCC4 is not uniquely sensitive to TCDD suppression of retinoid action because the independently derived human skin carcinoma line SCC12B2 (Rheinwald and Beckett, 1981Go) showed similar sensitivity. However, these keratinocyte lines are known to lack RARß (Hu et al., 1991Go), raising the possibility that this property could contribute to their sensitivity.

In a human myeloma cell line expressing RAR{alpha}, ß, and {gamma}, TGM2 is inducible by agonists selective for any of these receptor isoforms but only if the cells are also treated with an RXR ligand (Joseph et al., 1998Go). By contrast, in a human neuroblastoma cell line, RAR{alpha}- or {gamma}-selective retinoids alone induce TGM2 (Melino et al., 1997Go). As an example of further variation, SCC4 cells express both RAR{alpha} and RAR{gamma}, but TGM2 induction could be demonstrated only with a ligand selective for RAR{alpha}. An RXR agonist, although inactive alone and not required for the induction, substantially augmented it. However, it did not augment induction in SCC4 of 2 other identified retinoid-inducible genes (IDH and ILF1) in the presence of the RAR{alpha}-selective agonist (data not shown). TGM2 induction specifically by RAR{alpha} agonists has also been reported for rat tracheobronchial cells, in which RXR-specific ligands did not augment the induction in that case (Zhang et al., 1995Go). In SCC4 cells, RAR{alpha} specificity reflects a general inactivity of RAR{gamma} as evidenced in the transfection studies. Attempts to confer sensitivity to the RAR{gamma}-selective ligand by cotransfecting human RAR{gamma} were unsuccessful, suggesting that this anaplastic cell line lacks a specific coactivator for this receptor isoform.

Because SCC4 cells were responsive to RAR agonists only of the {alpha} isoform, they were suitable to test the dependence of the degree of TCDD suppression on target genes of the RAR{alpha} signaling pathway. The finding of additional genes, in contrast to TGM2, in which retinoid induction was minimally if at all affected by TCDD indicates that the repressive interaction is gene specific. This finding confirms that the suppression does not reflect general perturbation of the retinoid signaling pathway itself, including differential RAR isoform effects. It does leave open the possibility that DNA binding of the AhR could interfere with retinoid-responsive element function as a result of the proximity of a DRE-like element, because this presumably would be highly gene dependent. However, the proximity scenario is not supported in the case of TGM2. Stable transfections of the 5-kb proximal promoter sequence suggest that the elements mediating retinoid stimulation and TCDD suppression are not in close proximity because of a lack of TCDD suppression of the ATRA-induced reporter activity through the 5-kb promoter region. Because previous promoter analysis studies have not identified a retinoid-responsive element within 1.7 kb upstream of the transcription start site (Lu et al., 1995Go), such an element appears likely to be found in the region between 1.7 and 5 kb upstream. Whether it may constitute a site for RAR/RXR binding, such as a traditional DR5 or a multicomponent element as found for mouse TGM2 (Nagy et al., 1996Go; Yan et al., 1996Go), or for a different type of transcription factor resulting from action of retinoids at an RARE elsewhere remains to be seen. Regardless, the suppressive TCDD interaction is likely a secondary effect arising from AhR induction of other genes negatively regulating the TGM2 promoter. In any case, because the responsive site for TCDD suppression appears to be separate from that for retinoids, its localization presents a considerable challenge for identification, permitting more direct analysis of the underlying mechanism by which TCDD suppresses retinoid induction of the TGM2 gene. The unexpected stimulation of TGM2 in the stable transfectants may reflect the presence of 1 or more cryptic Ah receptor response elements in the promoter that become active in new chromosomal locations. Consistent with this speculation, previous work showed that transcription of the endogenous gene is stimulated by TCDD in the presence of 3 mM butyrate (Krig and Rice, 2000Go), which alters the acetylation state of the chromatin and thus the promoter microenvironment.


    ACKNOWLEDGMENTS
 
This work was supported by U.S. Public Health Service Grants AR27130, ES07059, ES04699 and ES05707. We thank Drs. P. J. A. Davies and M. A. Phillips for valuable experimental advice and Dr. Donald D. Phillips for valuable statistical advice.


    NOTES
 
1 To whom correspondence should be addressed. Fax: (530) 752–3394. E-mail: rhrice{at}ucdavis.edu. Back


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 DISCUSSION
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