* Department of Environmental Toxicology, University of California, Davis, 1 Shields Avenue, California 956168588;
Retinoid Research, Departments of Chemistry and Biology, Allergan, Inc., Irvine, California 926239534; and
Department of Internal Medicine, University of California, Davis, California 956168685;
Received December 18, 2001; accepted March 12, 2002
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ABSTRACT |
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Key Words: dioxin; keratinocytes; retinoids; TGM2.
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INTRODUCTION |
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TCDD is well known to serve as a ligand for the Ah receptor and thus to induce alterations in gene expression (Denison et al., 1998; Whitlock, 1999
). 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., 1995
) or an AP-1 (Gillesby et al., 1997
) 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., 2001). 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, 1988
) 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.
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MATERIALS AND METHODS |
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Synthetic retinoids.
The RXR panagonist AGN 194204, used at 30 nM, has a median effective concentration (EC50) of < 1 nM for transactivation of RXR, ß, or
and does not activate RARs (Vuligonda et al., 2001
). The RAR
antagonist AGN 194301, used in the range of 0.1100 nM, is completely inactive in transactivation assays but displays dissociation constants (Kds) of 2.8, 320, and 7258 nM for binding to RAR
, ß, and
, respectively (Teng et al., 1997
). Transactivating activities of the RAR
- and
-selective agonists (AGN 194078 and 194433, respectively), used in the range of 0.1 nM3 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., 1996
).
Northern blotting.
After treatment, cultures were rinsed with isotonic phosphate buffer and dissolved in Trizol (GibcoBRL) for RNA isolation (Krig and Rice, 2000). 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]-
-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, 2000). 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 12 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.
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RESULTS |
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DISCUSSION |
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In a human myeloma cell line expressing RAR, ß, and
, 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., 1998
). By contrast, in a human neuroblastoma cell line, RAR
- or
-selective retinoids alone induce TGM2 (Melino et al., 1997
). As an example of further variation, SCC4 cells express both RAR
and RAR
, but TGM2 induction could be demonstrated only with a ligand selective for RAR
. 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
-selective agonist (data not shown). TGM2 induction specifically by RAR
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., 1995
). In SCC4 cells, RAR
specificity reflects a general inactivity of RAR
as evidenced in the transfection studies. Attempts to confer sensitivity to the RAR
-selective ligand by cotransfecting human RAR
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 isoform, they were suitable to test the dependence of the degree of TCDD suppression on target genes of the RAR
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., 1995
), 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., 1996
; Yan et al., 1996
), 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, 2000
), which alters the acetylation state of the chromatin and thus the promoter microenvironment.
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ACKNOWLEDGMENTS |
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NOTES |
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