Antiestrogenic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin are mediated by direct transcriptional interference with the liganded estrogen receptor. Cross-talk between aryl hydrocarbon- and estrogen-mediated signaling.

Aryl hydrocarbon receptor (AhR) ligands have diverse biological effects including striking antiestrogenic activity. We have investigated at the molecular level the antiestrogenic activity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). We show that the previously documented TCDD-mediated decrease in estradiol-inducible gene products such as cathepsin D (cat D) is due to a sharp decline in mRNA accumulation despite any change in estrogen receptor (ER) mRNA levels. The decline in cat D mRNA level is most likely due to a decrease in transcription of the cat D gene since TCDD blocks the ability of ER to transactivate from an estrogen response element. AhR is required for this activity as TCDD is no longer antiestrogenic in a mutant cell line that is deficient in functional AhR. We provide evidence that the loss of transactivation potential by ER in the presence of TCDD is due to a sharp decrease in its ability to bind to an estrogen response element. Reciprocally, estradiol treatment blocked TCDD-induced accumulation of CYP1A1 mRNA and AhR-mediated activation of the CYP1A1 promoter. This is due to the ability of liganded ER to interfere with the binding of AhR to the xenobiotic response element. These results provide a molecular mechanism for the antiestrogenic effects of TCDD and demonstrate the presence of a two-way crosstalk between the intracellular signaling pathways involving estrogens and aryl hydrocarbons.

Aryl hydrocarbon receptor (AhR) ligands have diverse biological effects including striking antiestrogenic activity. We have investigated at the molecular level the antiestrogenic activity of 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD). We show that the previously documented TCDD-mediated decrease in estradiol-inducible gene products such as cathepsin D (cat D) is due to a sharp decline in mRNA accumulation despite any change in estrogen receptor (ER) mRNA levels. The decline in cat D mRNA level is most likely due to a decrease in transcription of the cat D gene since TCDD blocks the ability of ER to transactivate from an estrogen response element. AhR is required for this activity as TCDD is no longer antiestrogenic in a mutant cell line that is deficient in functional AhR. We provide evidence that the loss of transactivation potential by ER in the presence of TCDD is due to a sharp decrease in its ability to bind to an estrogen response element. Reciprocally, estradiol treatment blocked TCDD-induced accumulation of CYP1A1 mRNA and AhR-mediated activation of the CYP1A1 promoter. This is due to the ability of liganded ER to interfere with the binding of AhR to the xenobiotic response element. These results provide a molecular mechanism for the antiestrogenic effects of TCDD and demonstrate the presence of a two-way crosstalk between the intracellular signaling pathways involving estrogens and aryl hydrocarbons.
The potent environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) 1 has been used as a model compound for investigating the cellular and molecular mechanisms of aryl hydrocarbon (Ah) action such as those found in drugs, carcinogens, mutagens, dietary components, and other environmental pollutants (1,2). The mechanism of action of these compounds is to activate the Ah receptor (AhR) to a form that binds to specific gene regulatory sequence elements, called xenobiotic response elements (XREs), through heterodimerization with the AhR nuclear translocator protein (Arnt) (3,4). AhR and Arnt have a similar overall structure and belong to the basic helix-loop-helix class of transcription factors (5)(6)(7). Upon binding XREs, the AhR⅐Arnt complex activates transcription of adjacent structural genes which encode enzymes that are involved in the oxidative metabolism of these compounds (for a review, see Refs. 8 and 9).
TCDD elicits a broad spectrum of biochemical responses in laboratory animals and mammalian cells in culture (1, 2), including striking antiestrogenic activity (10 -14). TCDD displays significant activity as a hepatocellular and squamous carcinogen in chronic bioassays, yet in other tissues it can be protective: long term feeding of TCDD results in significant decreases in incidence of spontaneous mammary, uterine, adrenal, and pituitary tumors in female rats (15,16). This tissuespecific action may extend to other AhR ligands, such as those from cigarette smoke (17), since tobacco smoke has antiestrogenic effects on a variety of biological parameters such as early onset of menopause, increased risk of postmenopausal osteoporosis and decreased levels of circulating urinary estrogens during the menstrual cycle (for a review, see Ref. 18). In addition, smoking is inversely correlated with endometrial cancer risk (19). AhR ligands derived from the diet may also be significant contributors in this area (20,21).
The ability of TCDD and other AhR ligands to reduce cancer incidence in hormonally responsive tissues can be correlated with the antiestrogenic action of these compounds which has become apparent in recent years (10 -14, 22-26). In mice and rats, TCDD exposure counteracts the effects of estrogens, such as 17␤-estradiol, on uterine hypertrophy, peroxidase activity, ER binding activity, progesterone receptor binding activity, and epidermal growth factor receptor binding activity (10 -14). In human mammary cell lines, TCDD exposure results in decreased secretion of tissue plasminogen activator (24) and decreased secretion of estrogen-induced proteins, such as cathepsin D (cat D) (23). TCDD exposure also blocks the estradioldependent proliferation response (23,27), and the occurrence of multicellular foci in postconfluent cultures of human mammary cell line MCF-7 (27,28).
Estrogens influence cellular function by binding to intranuclear receptor molecules (ERs) which are ligand-activated transcription factors belonging to the nuclear receptor superfamily (for a review, see Ref. 29). Upon ligand binding, ER interacts with cis-acting DNA elements, called estrogen response elements (EREs), in the vicinity of estrogen responsive genes and activates transcription.
Understanding the mechanism by which AhR agonists exert their antiestrogenic effects may be useful in evaluating their potential role as therapeutic and/or protective agents in breast and endometrial carcinogenesis. To that end, we have investigated at the molecular level antiestrogenic activities of TCDD. We present evidence that the TCDD-mediated decrease in estradiol induction of the cat D gene is due to a sharp drop in mRNA accumulation. This is most likely due to interference with the ER transactivation potential caused by abrogation of its ability to interact with an ERE. Interestingly, we find reciprocal, similar effects of estradiol on TCDD-mediated mRNA accumulation and AhR-dependent transactivation. These results demonstrate an extensive cross-talk between two signal transduction pathways, one used by estrogens and the other by AhR ligands.

MATERIALS AND METHODS
RNA Isolation and Analysis-Total RNA was isolated by the acid guanidine thiocyanate-phenol-chloroform technique. For Northern analysis, RNA was electrophoresed through formaldehyde-agarose gels (10 g/lane) and transferred to nitrocellulose in 20 ϫ SSC, and the filters were air-dried and baked in vacuo for 2 h at 80°C (30). Prehybridization, hybridization, and washing were performed as described elsewhere (30).
Cell Culture and DNA Transfections-Hepa1c1c7 (Hepa-1) cells and the mutant derivative B13NBii1 (a nuclear translocation defective, or C Ϫ mutant, a generous gift from O. Hankinson, University of California, Los Angeles) were maintained at 37°C in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F12 medium supplemented with 5% fetal bovine serum that had been charcoal-treated. In all experiments phenol red-free medium was used to minimize basal estrogenic activity. MCF-7 cells were maintained in a 1:1 mixture of Dulbecco's modified Eagle's medium/F12 supplemented with 10% fetal bovine serum. All cultures were maintained in a humidified atmosphere containing 5% CO 2 and 95% air. Transient transfections and assay of chloramphenicol acetyltransferase (CAT) activity were carried out as described previously (35).
Mobility Shift Assay-The mobility shift assay, preparation of 32 Plabeled oligonucleotides, and preparation of nuclear extracts were described previously (35). The XRE probe is the XRE1-40 oligonucleotide which spans the core region of the Ah response element in the CYP1A1 gene promoter, bases Ϫ1007 to Ϫ1021 (35). Vitellogenin ERE oligonucleotide was described previously (32).

Reciprocal Inhibitory Effects of TCDD and Estradiol on the Level of mRNA Accumulation of TCDD or Estradiol Responsive
Genes-A mechanism by which the liganded AhR may decrease ER activity is interference with the ability of the activated ER to stimulate gene expression. This possibility was examined in the experiment shown in Fig. 1. One of the estradiol responsive genes is cat D (31). Northern blot analysis indicated that treatment of MCF-7 cells with estradiol strongly activated the accumulation of cat D mRNA, whereas TCDD treatment did not have an effect. However, after simultaneous incubation of cells with estradiol and TCDD, a significant decrease in the cat D mRNA levels, relative to the cells treated with estradiol alone, was observed. Thus, these data suggest that TCDD interferes with the ability of estradiol to activate the cat D gene.
The antiestrogenic action of TCDD and the results presented above would be consistent with a mechanism in which the TCDD⅐AhR complex down-regulates transcription of the ER gene itself, resulting in decreased levels of ER and thus decreased transcriptional activation in response to estradiol. We therefore examined whether the decrease in cat D mRNA levels in the presence of TCDD was due to a decrease in ER mRNA levels. MCF-7 cells were either left untreated or treated with estradiol and/or TCDD, and ER mRNA levels were determined by Northern analysis. As shown in Fig. 1, none of the treatments significantly altered ER mRNA levels. These data suggest that changes in ER mRNA levels cannot account for the antiestrogenic effect of TCDD.
To assess the possibility of any reciprocal effect, that is, whether estradiol might influence AhR-mediated gene expression, we determined the mRNA levels of the CYP1A1 gene which is regulated by TCDD on the transcriptional level (for a review, see Refs. 36 and 37). MCF-7 cells were either left untreated or treated with estradiol and/or TCDD, and CYP1A1 mRNA levels were determined by Northern analysis. As shown in Fig. 1, CYP1A1 mRNA was increased following TCDD treatment, whereas estradiol did not have an effect. However, when estradiol was added together with TCDD, there was a marked reduction in CYP1A1 mRNA levels compared with TCDD alone. These data suggest that estradiol inhibits AhR-mediated gene activation.
TCDD Interferes with Estradiol-dependent Transactivation by the ER-Above findings are compatible with two possibilities: first, the TCDD⅐AhR complex has a direct effect on the transactivation potential of the estradiol-ER complex, and second, the TCDD⅐AhR complex affects the mRNA stability of estradiol-responsive genes. To determine whether the liganded AhR is involved in the repression of ER transactivation potential, the reporter plasmid 2xERE-CAT, in which the ERE from the vitellogenin A2 gene is fused to the CAT reporter gene (32), was transfected into MCF-7 cells. Cells were grown in estrogenfree medium and supplemented with estradiol, TCDD, or both. As shown in Fig. 2, CAT activity was induced approximately 15-fold in cells treated with estradiol but was not affected by TCDD treatment. However, simultaneous treatment of estradiol and TCDD resulted in abrogation of the estradiol induction of CAT activity. Addition of TCDD 6 or 12 h before estradiol and at levels 100-fold less (0.1 nM) than what is commonly used in transient transfection assays yielded similar results (data not shown). These data suggest that the antiestrogenic action of TCDD is directed against the transcriptional activation potential of ER.
The data presented in Fig. 1 showed that ER gene transcription is not a target for the antiestrogenic action of TCDD. To further study whether changes in ER levels in TCDD-treated cells may be involved in the antiestrogenic action of TCDD, we performed transient transfection experiments under conditions where the ER is maintained at moderate constant levels by using an ER expression vector. Hepa-1 cells were cotransfected with 2xERE-CAT and the ER expression vector HEO (32) and the cells were either left untreated or treated with TCDD FIG. 1. Reciprocal inhibitory effects of TCDD and estradiol on the level of mRNA accumulation. MCF-7 cells were left untreated (C) or were treated with estradiol (E, 1 ϫ 10 9 M) and/or TCDD (T, 1 ϫ 10 9 M). Following a 12-h treatment period, total cellular RNA was isolated, electrophoresed (10 g/lane) through a formaldehyde agarose gel, transferred to nitrocellulose, and hybridized sequentially to 32 Plabeled cDNAs specific for cathepsin D (CatD), CYP1A1, estrogen receptor (ER), and glyceraldeyde-3-phosphate dehydrogenase (GADPH). Results shown are from a representative experiment repeated at least three times. and/or estradiol. As shown in Fig. 2B, treatment with estradiol alone, but not TCDD, gave rise to a 10-fold increase in 2xERE-CAT expression which was significantly reduced when cells were simultaneously treated with TCDD and estradiol. Qualitatively similar results were obtained without ectopic expression of ER except that the activation of 2xERE-CAT was substantially lower in response to estradiol treatment alone (data not shown). These data are consistent with ER mRNA analysis presented in Fig. 1 and further indicates that a decrease in ER levels is not an obligatory step in the antiestrogenic action of TCDD.
To investigate whether the TCDD⅐AhR complex is directly involved in the antiestrogenic action of TCDD, we used a derivative of Hepa-1 cells, Hepa-C Ϫ (38), that lacks a functional AhR due to mutations in the Arnt subunit. Hepa-C Ϫ cells were grown in estrogen-free medium, transfected with the 2xERE-CAT reporter and ER expression vector HEO. Cells were treated with TCDD for 12 h before addition of estradiol and further incubated for 24 h. As shown in Fig. 2C, estradiol increased 2xERE-CAT expression approximately 7-fold, but TCDD treatment did not have an effect. In contrast to MCF-7 cells ( Fig. 2A) and Hepa-1 cells (Fig. 2B), 2xERE-CAT activity was not diminished in extracts prepared from Hepa-C Ϫ cells treated with estradiol and TCDD. These results suggest that a functional AhR complex that can translocate to the nucleus is required for the antiestrogenic action of TCDD.
TCDD Treatment Blocks Binding of ER to the ERE-DNA binding by ER is necessary for its ability to activate transcription. Therefore, a possible mechanism for transcriptional interference of liganded ER by the TCDD⅐AhR complex is interference with the ability of ER to bind to DNA. We tested this possibility by using the mobility shift assay. Nuclear extracts were prepared from MCF-7 cells which were either left untreated or treated with estradiol and/or TCDD, and DNA binding activities present in the extracts were detected using a 32 P-labeled vitellogenin A2 ERE (32) oligonucleotide as probe. As shown in Fig. 3A, estradiol treatment of cells increased the formation of two protein-DNA complexes. When a 25-fold excess of unlabeled A2 ERE was included in the binding reaction, all binding was lost indicating that the two complexes represent specific binding to the A2 ERE. Similar patterns of DNA binding activity in MCF-7 cell extracts treated with estradiol have been observed by others (for example, see Ref. 32). When an ER-specific antiserum (39), but not nonimmune serum, was included in the binding reaction, the intensity of the upper band decreased sharply and a slower migrating complex appeared, whereas the intensity of the faster migrating complex did not change. These results indicate the presence of ER in the upper complex. Although we do not know at present the nature of the species in the faster moving complex, it could represent a proteolytic fragment of the full-length ER in which the epitope that was recognized by the anti-ER antiserum was lost.
As shown in Fig. 3B, estradiol treatment increased ER⅐ERE complex formation, whereas TCDD treatment did not have an effect. In contrast, the binding activity was completely lost in extracts prepared from cells that were simultaneously treated with both estradiol and TCDD. These results suggest that the TCDD⅐Ah receptor complex interferes with the transactivation potential of ER by inhibiting the ERE binding activity of ER.
Liganded ER Inhibits AhR Transactivation Function-As shown in Fig. 1, estradiol treatment inhibited the TCDD induction of CYP1A1 mRNA accumulation in MCF-7 cells. To assess whether this inhibition was directed at the level of CYP1A1 transcription, Hepa-1 cells were transfected with a reporter construct in which the 3.1-kb upstream sequences of the

FIG. 2. TCDD blocks transactivation by ER.
A, MCF-7 cells were grown in estrogen-free medium and transfected with the 2xERE-CAT reporter construct (7.0 g). After transfection, the cells were either left untreated (C) or incubated with estradiol (E, 1 ϫ 10 9 M) and/or TCDD (T, 1 ϫ 10 9 M) for 24 h as indicated. Cells were harvested and CAT activities were determined. Results shown represent at least three independent experiments. B, TCDD is antiestrogenic even in the presence of constant levels of ER. Hepa-1 cells were grown in estrogen free medium and transfected with the 2 ϫ ERE-CAT reporter construct (6.9 g) and the ER expression vector HEO (0.1 g). After transfection, the cells were either left untreated (C) or incubated with estradiol (E, 1 ϫ 10 9 M) and/or TCDD (T, 1 ϫ 10 9 M) for 24 h as indicated. Cells were harvested, extracts prepared, and CAT activities were determined. Results shown represent at least three independent experiments. C, a functional AhR is required for the antiestrogenic action of TCDD. Hepa-C Ϫ cells which are defective in a functional AhR were grown in estrogen-free medium and transfected with the 2xERE-CAT reporter (6.9 g) and the ER expression vector HEO (0.1 g). After transfection, the cells were either left untreated (C) or incubated with TCDD (T, 1 ϫ 10 9 M) for 12 h. Estradiol (E, 1 ϫ 10 9 M) was then added and incubation continued for an additional 24 h. Cells were then harvested, extracts prepared, and CAT activities were determined. Results shown represent at least three independent experiments. CYP1A1 gene drives expression of the CAT gene, 3.1CYP1A1-CAT (34). Cells were either left untreated or treated with TCDD and/or estradiol and CAT activites were determined. Fig. 4A shows that TCDD treatment induced 3.1CYP1A1-CAT activity consistent with previous findings (8), whereas estradiol did not have any effect. However, when the cells were treated simultaneously with both TCDD and estradiol, the 3.1CYP1A1-CAT activity was significantly reduced relative to the cells treated with TCDD alone. Similar results were obtained in MCF-7 cells (data not shown). These results suggest that similar to the antiestrogenic action of TCDD, estradiol exerts its anti-AhR effects, at least in part, by inhibiting the ability of TCDD⅐AhR complex to activate transcription.
A possible mechanism of estradiol-induced repression of AhR activity is interference with DNA binding by the AhR. As shown in Fig. 4B, we tested this possibility in a mobility shift assay by using nuclear extracts prepared from Hepa-1 cells which were either left untreated or treated with TCDD and/or estradiol. 32 P-Labeled XRE oligonucleotide (35) was incubated with the nuclear extracts, and the resulting complexes were resolved by polyacrylamide gel electrophoresis. No significant binding was observed in extracts prepared from untreated cells or cells treated with estradiol, whereas TCDD treatment induced the formation of a strong shifted band previously shown to contain the Ah receptor (8, 35, 40). Intriguingly, the TCDD-induced AhR binding activity was almost completely lost when extracts from cells treated with both TCDD and estradiol were used. Similar results were obtained using MCF-7 cells (data not shown). These results suggest that the inhibitory effect of estradiol on AhR transactivation function is mediated by blocking of AhR binding to the XRE. DISCUSSION It has been known for some time that TCDD and other AhR ligands reduce cancer incidence in estrogen-responsive tissues. Since TCDD does not bind ER and estradiol does not bind AhR (10,11), the basis of the antiestrogenic activity of TCDD is thought to be indirect. Several mechanisms may account for the antiestrogenic action of TCDD. First, it has been proposed that the antiestrogenic effect of TCDD in MCF-7 cells and in other systems is due to the ability of this compound to increase the expression of P4501A1 and P4501A2 enzymatic activity, which hydroxylates estrogens to less active forms and facilitates their further metabolism and clearance from the system (24,27,28,41). The rapid onset of the antiestrogenic action of TCDD on DNA binding of ER presented here argues against this mechanism. In addition recent evidence which shows that an analog of TCDD acts antiestrogenically in both MCF-7 and Hepa-1 cells, yet does not induce P4501A1 (42), suggests that new gene induction by AhR may not be required for the antiestrogenic action of TCDD.
A second possible mechanism for the antiestrogenic action of TCDD has been proposed based on the observation that TCDD causes the levels of nuclear ER to decrease (43) and the observation that sequences similar to XREs through which the AhR acts are present upstream of the ER gene (44). Thus, it is possible that the primary antiestrogenic action of TCDD is to down-regulate expression of the ER gene, thereby reducing cellular ER levels. In contrast, we find that TCDD is strongly antiestrogenic in cells in which the levels of ER cannot be down regulated due to constitutive expression of this receptor from an ER expression vector (Fig. 2B). In addition, we do not find any changes in ER mRNA levels upon treatment with TCDD, regardless of the presence of estradiol (Fig. 1), consistent with earlier findings (45). Together, these observations suggest that TCDD may exert its antiestrogenic effects independent of changes in ER levels.
Our results provide a novel mechanism distinct from those proposed earlier for the antiestrogenic activities of TCDD, the AhR⅐TCDD complex interferes with the ability of the liganded ER to activate transcription. Several lines of evidence support this conclusion. The most significant of these are, first, in the presence of TCDD and estradiol, ER-dependent activation of cat D mRNA accumulation is abrogated. Second, in the presence of AhR, TCDD inhibits ER-mediated ligand-dependent transcriptional activation of a reporter construct in a transient transfection assay.
Interestingly, in reciprocal fashion, estradiol-ER complex interferes with AhR transactivation potential. TCDD-dependent increase in CYP1A1 mRNA accumulation is blocked by estradiol treatment and TCDD-dependent activation of the CYP1A1 promoter is diminished in the presence of estradiol and ER in a transient transfection assay. Thus ER and AhR mutually inhibit each other's transactivation potential depending on the availability of their ligands.
Similar cases of transcriptional interference have been observed between other transcription factors. For example, nuclear receptors can interfere with transcriptional activation by several transcription factors including CREB (46,47), Oct1 (48), basic helix-loop-helix proteins (49), and the AP-1 oncoprotein complex (for a review, see Ref. 50). Repression by transcriptional interference results when a transcripton factor is FIG. 4. Estradiol blocks transactivation by AhR. A, Hepa-1 cells were grown in estrogen free medium and transfected with the 3.1CYP1A1-CAT reporter construct (7 g). After transfection, the cells were either left untreated (C) or incubated with estradiol (E, 1 ϫ 10 9 M) and/or TCDD (T, 1 ϫ 10 9 M) for 24 h as indicated. Cells were harvested, and CAT activities were determined. Results shown are from a representative experiment. B, Hepa-1 cells were either left untreated (Ϫ) or were treated with TCDD (T, 1 ϫ 10 9 M), and estradiol (E, 1 ϫ 10 9 M) alone or in combination for 1 h and nuclear extracts were prepared. These extracts (6 g/reaction) were used in a mobility shift assay with 32 P-labeled XRE1 oligonucleotide as probe. AhR-specific shifted band (B) and free probe (F) are indicated. occluded from fruitfully interacting with the transcription initiation complex through direct or indirect interactions with another factor. This may involve competition for binding to a common or overlapping cis element, competition for a common mediator, a phenomenon also known as sequelching, or formation of inactive complexes (for a review, see Refs. 51 and 52).
DNA binding experiments indicate that ER does not bind directly to the XRE and that the AhR does not bind to the ERE, as would be expected from the dissimilar sequences of the response elements (Figs. 3 and 4B; data not shown). On the other hand, cotreatment of cells, such as MCF-7, which contain both receptors, with estradiol and TCDD completely blocks ERE-specific DNA binding activity of ER. Similarly, cotreatment of cells with estradiol and TCDD completely blocks XREspecific DNA binding activity of AhR. Remarkably, in both cases the block in DNA binding is complete within 1 h. The mutual inhibition of DNA binding activities of ER and AhR may be mediated by either direct interactions between the receptor molecules, or stabilized or catalyzed by an accessory cofactor(s). There is no evidence for the formation of a ternary DNA-protein complex by the liganded ER and AhR on the XRE or ERE or inhibition of each other's DNA binding by mixing extracts that contain activated ER and AhR, 2 which suggests that the interaction may be indirect involving a bridging factor which is lost or inactivated during the preparation of the extracts. Additional studies are needed to answer these questions. Furthermore, in vivo footprinting studies of cellular promoters bearing EREs or XREs and their occupancy in the presence and absence of the appropriate ligand will have to be performed to determine the significance of these in vitro findings in living cells.
Whereas the physiological effects of the antiestrogenic activity of TCDD and AhR ligands have been studied extensively, that of the anti-AhR activity of estradiol is not clear at present. It would be interesting to determine whether the increased risk of cancer associated with length of time that a woman is subject to estrogenic stimulation correlates with a decrease in the levels of gene products induced by AhR ligands, such as P4501A1, which are involved in detoxification processes. Such a decrease may compromise the ability of the physiology to eliminate mutagens and carcinogens ingested in the diet or contracted from the environment leading to their accumulation in the body and thereby giving rise to increased incidence of cancer. Further studies are needed to assess this possibility.