Department of Environmental Toxicology, One Shields Avenue, University of California, Davis, California 95616-8588
Received January 20, 2000; accepted April 10, 2000
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ABSTRACT |
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Key Words: Ah receptor; butyrate; dioxin; tissue transglutaminase (TGM2); (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthylenyl)-1-propenyl] benzoic acid (TTNPB).
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INTRODUCTION |
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While the molecular basis of TCDD toxicity beyond the role of the AhR remains mysterious, numerous studies have suggested that this agent interferes with normal hormonal regulatory mechanisms. Early investigations in animals noted a correlation between toxic effects from TCDD exposure and reductions in hepatic vitamin A or altered retinoid processing (Hakansson, 1997). More recent work shows that TCDD prevents the retinoid induction of the retinoic acid receptor-ß (RARß) and the cellular retinoic acid binding protein type II in mice (Weston et al., 1995
). In culture, TCDD prevents the retinoid induction of TGFß activity in a human keratinocyte line derived from a squamous cell carcinoma (Lorick et al., 1998
). In humans, as well as in higher primates and several domestic animal species, TCDD produces hyperkeratosis of the skin, and characteristic symptoms of exposure (in humans, chloracne) resemble those produced by vitamin A deficiency (Kimbrough, 1984
). TCDD exposure in animals has also been correlated with reduced thyroid hormone levels and goiter (Bastomsky, 1977
). These and other studies demonstrate the ability of TCDD and related chemicals to adversely affect the normal functioning of hormones and vitamins that act through the steroid, thyroid hormone, and retinoic-acid receptor superfamily.
The present work was prompted by the observation that TCDD largely prevents the retinoid stimulation of tissue transglutaminase (TGM2) in the squamous carcinoma line SCC-4 in an AhR-mediated process (Rubin and Rice, 1988). This transglutaminase is expressed in many cell types, where it is commonly stimulated by retinoids. It came to prominence as one of the earliest and most easily assayed enzyme markers for retinoid action in culture. A number of functions have since been ascribed to it, particularly stabilization of tissues by cross-linking of extracellular matrix (Greenberg et al., 1991
) in wound healing (Upchurch et al., 1991
), fibrosis (Kuncio et al., 1998
), and developing organs such as lung (Schittny et al., 1997
). For some time it has been proposed as serving an important role in apoptosis (Fesus and Thomazy, 1988
), and more recently has been found to function as a G-protein (Feng et al., 1996
). Although of uncertain importance in the physiology of keratinocytes, TGM2 in the present work serves as a valuable indicator of retinoid action.
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MATERIALS AND METHODS |
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Northern blotting.
After treatment cultures were rinsed with isotonic phosphate buffer, dissolved in 1 ml of Trizol (BRLGibco) and stored at 80°C until extraction. Following isopropanol precipitation, RNA was isolated by a second phenol extraction and ethanol precipitation. Total cellular RNA (15 µg) was electrophoresed at 70 v in a 1% agarose gel containing 5% formalin, and was transferred in 10X SSC to a Nytran membrane (Schleicher and Schuell, Keene, HN). The RNA was cross-linked to the membrane by UV 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% SDS0.5 M sodium phosphate. Membranes were probed with 1 x 106 cpm/ml cDNA labeled with [32P]--dCTP by random labeling (Ambion, Austin, TX) and hybridized for 18 h. Prior to autoradiography membranes were washed 3 times in 2X SSC0.1% SDS for 5 min and once in 0.5X SSC for 30 min. Quantitation was carried out using a Molecular Dynamics SI phosphorimager. The TGM2 cDNA probe (0.58 kb) was prepared by polymerase chain reaction from the 3'-untranslated region of the mRNA. A glyceraldehyde phosphate dehydrogenase (GAPDH) probe was employed as a loading control.
mRNA stability.
Cultures were treated at confluence with 3 µM ATRA plus or minus 10 nM TCDD. After 48 h, the medium was supplemented with 5,6-dichlorobenzimidazole riboside (30 µg/ml) and RNA isolated after additional time periods of incubation. Northern blotting was performed and the blots probed with the TGM2 cDNA and an 18S ribosomal cDNA as a loading control.
Transfections.
Retinoic acid response element (RARE) constructs, based on the sequence from the RARß promoter (Umesono et al., 1991), contained two half sites (underlined) with a 5 bp spacer. The 33 bp monomer oligonucleotide (5'-TCGAGAAGCCGAACTCGCATC-3') included XhoI site overhangs (in boldface) to facilitate generation of contcatamers and ligation into the pGL3 promoter vector (Promega, Madison, WI). The final construct contained two monomers ligated in the 5'
3' direction and two in the 3'
5' direction. The day prior to 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 3 µM ATRA or TTNPB ((E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthylenyl)-1-propenyl] benzoic acid, Ro 137410) with or without 10 nM TCDD. After 48 h of treatment, the cultures were lysed and the luciferase activities measured using the Dual-Luciferase Reporter system (Promega).
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-RARE construct and 5 µg of pPUR (Clontech, Palo Alto, CA) for puromycin selection. After one 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. The macroscopic clones were then pooled. Retinoid and TCDD treatments were as described above. Firefly luciferase reporter activity was assayed using the Promega reporter system.
Replication.
Each experiment illustrated is representative of 3 or more trials. Error bars show the mean and range of duplicate or triplicate samples from representative experiments. Statistical significance of the results was calculated by a 2-tailed t-test.
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RESULTS |
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Interactions of butyrate with ATRA and TCDD were then investigated. As shown in Figure 8, butyrate alone at 1 mM had little effect on TGM2 stimulation by ATRA, although mRNA levels in cultures treated with butyrate, ATRA, and TCDD approached those treated with butyrate and ATRA alone. At 3 mM, butyrate alone clearly induced TGM2, and the effect was nearly additive with that of ATRA. Moreover, TCDD no longer showed a suppressive effect in the presence of ATRA and even increased the TGM2 induction by butyrate alone. Cultures treated with both TCDD and ATRA had essentially the same levels as those treated with either TCDD or ATRA. A similar result was obtained using 5-mM butyrate (not shown).
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DISCUSSION |
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One plausible explanation for the TCDD action was that it could induce cytochrome P450 and perhaps other enzyme activities that inactivate the retinoid. Support for this notion could be inferred from findings in AhR homologous knockout mice, where liver retinoid levels are substantially higher, that ATRA catabolism is markedly reduced and hepatic expression of TGM2 is nearly 80% higher than in normal control mice (Andreola et al., 1997). However, several lines of evidence indicate this is not a major factor in SCC-4 cultures. First, if TCDD-induced metabolic inactivation were occurring, then adding the TCDD before the retinoid could substantially increase the observed TGM2 suppression. But adding TCDD 6 or 24 h in advance had the same effect as adding it 6 h afterward. Second, TTNPB resists metabolic inactivation, but its stimulation of TGM2 was suppressed essentially to the same extent as ATRA. Along these lines, a metabolic inactivation process might be saturable, leading to alteration of the concentration dependence of TGM2 induction, but this effect was not seen. Finally, and most conclusively, neither ATRA nor TTNPB was at all inactivated by TCDD addition in the transfection experiments, judging by the magnitude of the effect or its concentration dependence. In what is likely an analogous phenomenon, TCDD suppresses the hydrocortisone-stimulated accumulation of keratins and keratinocyte transglutaminase (TGM1) in human squamous carcinoma cells without affecting hydrocortisone levels in the medium (Rice and Cline, 1984
).
Negative interactions among a variety of transcription factors, including those of the steroid superfamily, have been documented since the original observation of interference with AP-1 protein action by the glucocorticoid receptor (Karin et al., 1997). Thus, another plausible explanation for the TCDD action was that it prevents proper RAR function, perhaps by direct protein-protein binding. However, no evidence for this type of interaction (squelching) was evident in the transfection experiments, where RAR function was fully maintained. This result contrasts with loss of the ligand-binding function of the RAR that reportedly contributes to the anti-retinoid action of TCDD in human SCC-12F cells (Lorick et al., 1998
). In that study, however, significant loss of specific binding was not observed below 100 nM TCDD, far above the Kd of ligand binding and thus arguing against a role for the AhR.
The possible action of the AhR as a gene repressor must be considered. By binding to dioxin-response elements in the upstream promoter, the liganded AhR complex normally stimulates gene transcription. However, AhR-dependent repression of expression by TCDD has been observed, as for the major histocompatibility complex Q1b gene (Dong et al., 1997). TCDD suppression of estrogen induction of cathepsin D has been attributed to occupation by the liganded AhR of a site that overlaps an important estrogen responsive Sp1 site (Krishnan et al., 1995
). Similarly the liganded AhR reportedly suppresses expression of the human breast cancer marker pS2 by binding to its response element abutting a critical AP-1-like site (Gillesby et al., 1997
). Whether this type of interaction occurs in the present case of TGM2 may become evident from promoter analysis. In any case, finding whether the TCDD suppression of retinoid action occurs generally, or is limited to a very few genes, would attest to the plausibility of this scenario. To this end, several candidate retinoid-inducible genes have been surveyed, including keratin 19, thioredoxin, transforming growth factor-ß, RARß and TIG-1. However, either these genes are not expressed in SCC-4 cells or they are expressed constitutively (not retinoid-inducible).
The gene specificity of TCDD suppression of retinoid action could be tested in cell lines where the above genes are known to be retinoid-inducible. However, a preliminary survey of several lines indicates that this suppression phenomenon is not found generally in cultured cells. On the one hand, ATRA induced TGM2 in the human keratinocyte line SCC-12B2 (Rheinwald and Beckett, 1981), and the induction was suppressed similarly to that in SCC-4. In preliminary experiments, on the other hand, TGM2 was inducible in HeLa and HL-60, but TCDD produced little if any suppression. Similarly, present work confirmed that TGM2 is inducible in epithelial lines derived from normal rat tissues (Rice et al., 1988
), but no TCDD suppression was evident in the lines examined (bladder, endometrial, prostate). We did not investigate whether relative levels of AhR in these lines varied substantially.
Sodium butyrate has long been known to have a variety of effects on cells in culture, most of which could result from its inhibition of histone deacetylase activity (Kruh, 1982). In present work, the effect of butyrate was striking, not only in reversing TCDD suppression of retinoid action but also in permitting TCDD to act as an inducer of TGM2. This observation is consistent with a recent report that the AhR can interact with transcriptional coactivators and corepressors (Nguyen et al., 1999
). One hypothesis to explain TCDD suppression is that the AhR complex, in the absence of butyrate, interacts with histone deacetylase in chromatin at the TGM2 promoter, preventing its release by retinoids and in this way suppressing induction. In the presence of butyrate, where deacetylation is inhibited, the interaction with coactivators dominates, yielding a stimulation. This scenario is also consistent with the observation that, in the presence of butyrate, retinoid and TCDD addition individually give the same stimulation as they do together. However, no indication was obtained that TCDD interfered with the retinoid stimulation of RARE-driven reporter activity in stable transfections, where interaction of the AhR complex with corepressors could have inhibited the response.
The peculiar selectivity of tissues and species to TCDD toxicity in vivo suggests that important phenomena may not be generalizable in culture but become revealed only in special circumstances. One special circumstance may be reflected in the evident delay between retinoid treatment and TGM2 expression in the SCC-4 cultures. This response resembles the retinoid induction of laminin B1 in F9 teratocarcinoma cells, where little transcription is evident for a day and maximal accumulation of the mRNA is seen after 72 h. In that instance, retinoids are proposed to act directly through a RARE, but the delay in transcription is attributed to an indirect effect through another important upstream regulatory site (Li and Gudas, 1996). If a similar phenomenon pertains to TGM2 in SCC-4, TCDD might not interfere with retinoid action directly but rather through the indirect pathway. Since the kinetics of TGM2 induction by retinoids differ substantially among cell types, the relative importance of the indirect route, and thus the efficacy of TCDD treatment, might differ accordingly. Elucidating TCDD action then could depend upon understanding the nature of the delayed retinoid response. Further work will be required to distinguish among the possible mechanisms by which TCDD suppresses retinoid action.
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ACKNOWLEDGMENTS |
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NOTES |
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