Antiestrogenic Effects of 2,3,7,8-Tetrachlorodibenzo-p-dioxin in Mouse Uterus: Critical Role of the Aryl Hydrocarbon Receptor in Stromal Tissue

David L. Buchanan*,1, Tomomi Sato*, Richard E. Peterson{dagger} and Paul S. Cooke*,2

* Department of Veterinary Biosciences, University of Illinois, Urbana, Illinois 61802; and {dagger} School of Pharmacy and Environmental Toxicology Center, University of Wisconsin, Madison, Wisconsin 53706

Received March 13, 2000; accepted July 10, 2000


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and the role of aryl hydrocarbon receptor (AhR) in estradiol (E2)-induced uterine epithelial mitogenic activity and secretory protein mRNA expression were determined. Ovariectomized wild-type (wt) and AhR-knockout (AhRKO) mice received oil, E2, or 5 µg/kg TCDD+E2. E2 stimulated similar large increases in the uterine epithelial labeling index (LI) and mRNA abundance for the E2-dependent epithelial secretory protein, lactoferrin (LF), in both wt and AhRKO mice. However, uterine epithelial LI and LF mRNA were significantly reduced by TCDD+E2 in wt but not AhRKO mice. To determine the roles of stromal and epithelial AhR in the TCDD effect, uterine stroma and epithelium from AhRKO and wt mice were enzymatically separated and recombined into four types of tissue recombinants that either contained or lacked AhR in one or more tissue compartments. Tissue recombinants were grafted into nude mice, which were later ovariectomized and given oil, E2, or TCDD+E2. Epithelial LI was significantly reduced by TCDD in grafts containing stromal AhR, regardless of epithelial AhR status. However, LI was unaffected by TCDD in grafts lacking stromal AhR, even when epithelial AhR was present. Thus, TCDD inhibits E2-induced uterine epithelial mitogenic and secretory activity, and this requires AhR. Anti-proliferative effects of TCDD on uterine epithelia appear to be mediated indirectly through stromal AhR, suggesting that liganded AhR alters epithelial function by disrupting normal E2-induced stromal activity. This is the first demonstration that TCDD impairs uterine epithelial function by altering normal stromal-epithelial interactions in vivo.

Key Words: mouse; estrogen receptor-{alpha}; mitogenesis; epithelium; stroma; antiestrogen; lactoferrin; TCDD; dioxin; uterus; aryl hydrocarbon receptor.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A number of xenobiotic compounds commonly found in terrestrial and aquatic environments, including certain polychlorinated dibenzo-p-dioxin congeners, have been identified as endocrine disrupters. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), the most potent congener in this class of endocrine disrupters, has antiestrogenic activity both in vitro and in vivo (Gallo et al., 1986Go; Gierthy et al., 1987Go). Estrogen normally stimulates increases in progesterone and epidermal growth factor receptor levels in rodent uteri, but TCDD inhibits these responses in vitro (Safe, 1995Go). In addition, TCDD has negative effects on ovarian function and fertility (Li et al., 1995Go; Moore et al., 1989Go). Of particular interest, TCDD has also been shown to decrease uterine wet weight in intact mice and alter endometrial histology (Gallo et al., 1986Go; Umbreit et al., 1988Go), thereby raising questions as to the site and mechanism of action of TCDD in affecting uterine growth and function.

Many effects of TCDD and other known hormone-modulating chemicals, such as some of the coplanar polychlorinated biphenyls and polychlorinated dibenzofurans, appear to be mediated through the cytosolic aryl hydrocarbon receptor (AhR; Fernandez-Salguero et al., 1995; Hansen, 1998; Poland et al., 1976; Safe et al., 1991). This receptor is a ligand-activated transcription factor and a member of the basic helix–loop–helix/PAS (Per ARNT Sim) superfamily of DNA binding proteins (Swanson and Bradfield, 1993Go). However, because AhR-independent effects exist (Fernandez-Salguero et al., 1996Go; Hoffer et al., 1996Go; Lin et al., 1991Go; Puga et al., 1997Go), it is unclear if the antiestrogenic effects of chemicals such as TCDD are mediated through AhR in the uterus.

Uterine epithelium plays a critical role in uterine function and in reproduction and fertility in general. 17ß-Estradiol (E2), the most potent natural form of estrogen, acts through estrogen receptor-alpha (ER{alpha}) to induce uterine epithelial mitogenesis and secretory protein production in vivo. These critical processes are potentially important targets of an antiestrogenic effect. However, because uterine luminal epithelium comprises only about 5–10% of total uterine wet weight (Bigsby et al., 1986Go), it has been unclear if reported TCDD-induced decreases in uterine wet weight were accompanied by concomitant inhibitions of critical E2-induced epithelial responses. Thus, the use of wet weight as an endpoint for monitoring the antiestrogenic effects of TCDD is limited since it is not apparent if this decrease involves changes in cell proliferation, cell death, and/or other estrogen-regulated processes such as water imbibition. Similarly, it has not been definitively established if the antiestrogenic effects of TCDD in vivo are mediated through AhR. Normal E2-induced uterine epithelial proliferation and secretory protein production are dependent on ER{alpha} in the stroma (Buchanan et al., 1999Go; Cooke et al., 1997Go). However, it is entirely unknown whether the antiestrogenic uterine effects of TCDD are due to actions on epithelial and/or stromal tissue. To better understand the antiestrogenic actions of TCDD and the role of AhR at the cellular and molecular levels, it is essential to determine if TCDD can interfere with critical E2-regulated epithelial events in vivo.

In the present study, we used ovariectomized wild-type (wt) and AhR-knockout (AhRKO) mice to determine the role of AhR and the effect of TCDD exposure on E2-induced uterine epithelial mitogenic activity and secretory protein mRNA expression. We then used tissue recombination methodology in conjunction with AhRKO mice to determine if TCDD effects on E2-induced epithelial mitogenic activity are mediated through AhR in the epithelium, stroma, or both of these tissue compartments. Our results show that antiestrogenic TCDD effects on epithelial mitogenic activity are mediated through stromal AhR and this finding provides important insight into the mechanism of the antiestrogenic effects of TCDD and liganded AhR action on uterine epithelial function.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
All experiments described here were approved by the Laboratory Animal Care Advisory Committee of the University of Illinois. Mice heterozygous for the AhR gene deletions were mated and their offspring were genotyped as described previously (Abbott et al., 1999Go). Neonatal and young adult (40- to 60-day-old) offspring homozygous for the AhR gene disruption (AhRKO) and homozygous wt animals of the AhRKO background strain (C57BL/6N) were used for these studies. Mice were given TekLad rodent chow (Madison, WI) and tap water ad libitum. All animals were housed individually with controlled lighting (14L:10D) and temperature (21–22°C) in clear plastic cages with hardwood bedding and maintained in accordance with the Guiding Principles in the Use of Animals in Toxicology.

Western blot.
To establish the pattern of AhR and aryl hydrocarbon receptor nuclear translocator (ARNT) protein expression, uteri from young adult C57BL/6N mice were placed in calcium- and magnesium-free Hank's Balanced Salt Solution (Sigma, St. Louis, MO) containing 1% trypsin (Life Technologies, Grand Island, NY) for 90 min at 4°C. Uterine epithelia and stroma were then separated as described previously (Buchanan et al., 1998Go; Cooke et al., 1997Go). Whole uteri and epithelial and stromal tissue fractions were then subjected to Western blot analysis (Sommer et al., 1999Go). Briefly, frozen samples were placed in ice-cold 1X lysis buffer (25 mM HEPES, pH 7.4, 20 mM sodium molybdate, 5 mM EGTA, 3 mM MgCL, 10% glycerol, 100 U/ml aprotinin, and 50 µM leuprotin) containing 1% Nonidet P-40 at a concentration of 100 mg tissue per ml buffer. Samples were sonicated, phenylmethylsulfonyl fluoride was added to a concentration of 100 µM, and samples were sonicated again. A 10 µl aliquot was used for protein concentration assay (Coomassie Blue Plus Protein Assay, Pierce, Rockford, IL) with BSA as the standard, and a 2X gel sample buffer (25 mM Tris, pH 6.8, 4% SDS, 25% glycerol, 4mM EDTA, 20 mM DTT, 0.005% bromophenol blue) was added to the remainder. Tissue lysates were heated at 95°C for 5 min and stored at –20°C. Lysates were diluted to 1 µg/µl, resolved by SDS–PAGE and electrophoretically transferred to nitrocellulose; mouse liver was used as a positive control for both AhR and ARNT antibodies. Blots were incubated with affinity-purified polyclonal antibodies against mouse ARNT or mouse AhR (Pollenz et al., 1994Go, 1998Go) at 1 µg/ml in BLOTTO (5% Carnation Instant Milk in Tris-buffered Saline containing 0.2% Tween-20 and 20 mM histidine [TTBS+]) for 1 h at 25°C and washed in TTBS+. Blots were next incubated for 1 h at 25°C in BLOTTO buffer containing goat anti-rabbit-horseradish peroxidase diluted 1:10,000 and then washed in TTBS+. After a final wash in TBS, bands were visualized with the Enhanced Chemiluminescence kit (Amersham, Arlington Heights, IL). Western blot for AhR and ARNT was repeated twice on whole and separated uterine tissue fractions from 3 wt mice.

TCDD and E2 treatments.
To determine the effect of TCDD and involvement of AhR in E2-induced uterine epithelial proliferation and LF mRNA expression, young adult AhRKO and wt mice were ovariectomized and injected ip one week later with either oil, E2 (30 ng/mouse in 0.05 ml corn oil, which is equivalent to 1.5 µg/kg), or a combination of 5 µg TCDD/kg (in 0.05 ml corn oil; 98% purity, Cambridge Isotope Laboratories, Woburn, MA), followed 24 h later by 30 ng E2. To conserve animals, whole uteri were removed 24 h after the final injection and divided into 2 portions: one horn was used to assess epithelial mitogenic activity while the other was analyzed by Northern blot for LF mRNA expression as an indicator of E2-induced secretory protein production. The TCDD dose used in this study, 5 µg/kg, is too low and the time after treatment, 2 days, too short to cause overt toxicity in young adult C57BL/6 mice (DeVito and Birnbaum, 1994Go). A recent study using C57BL/6J mice also reported no significant increase in liver-to-body weight ratio following 5 µg/kg TCDD exposure (Tian et al., 1998Go).

Tissue recombination.
The tissue separation and recombination procedure for uterine epithelium (E) and stroma (S) was carried out as described previously (Cooke et al., 1997Go). Briefly, uteri were removed from neonatal (0–3 day) AhRKO (knockout [ko]) and wt mice immediately following sacrifice and subjected to trypsin digestion as described above. Tissue recombinants composed of wtS+wtE, wtS+koE, koS+wtE, and koS+koE were then prepared, allowed to adhere overnight, and grown as subrenal grafts in intact, adult female athymic (nude) mice (Harlan, Indianapolis, IN) for 4 weeks, at which time all hosts were ovariectomized. To investigate the specific uterine tissue compartment through which the liganded AhR mediates antiestrogenic effects on uterine epithelium, host animals were injected ip with either oil, 30 ng E2, or a combination of 5 µg/kg TCDD followed 24 h later by 30 ng E2, beginning one week after ovariectomy. Uterine tissue recombinants were harvested 18 h after the final injection, then formalin-fixed and paraffin-embedded for autoradiographic analysis.

RNA isolation and Northern blot analysis.
To assess mRNA expression levels for the E2-inducible uterine epithelial secretory protein lactoferrin (LF; Teng et al., 1986), AhRKO and wt uteri were immediately flash frozen in liquid nitrogen and stored at –80°C for subsequent RNA isolation. Total RNA was extracted from frozen uteri and used for Northern analysis as described by Buchanan et al. (1999). For normalization of total RNA loading between lanes, all membranes were stripped of the LF cDNA probe by incubation in 50% formamide at 65°C for 1 h and rehybridized with labeled cDNA probe for 28S rRNA ([32P]deoxy-CTP, using the Multiprime DNA Labeling System, Amersham Pharmacia Biotech, Buckinghamshire, UK). X-ray film images developed against Northern blot membranes were captured, using a Leaf Lumina scanner interfaced to a Macintosh computer utilizing Adobe Photoshop software. Autoradiograms for LF and 28S rRNA were scanned and quantitated, using a computer-linked scanning laser densitometer and RFL-Print software (pdi, Huntington Station, NY). Relative mRNA transcript levels were normalized based on densitometric analysis of 28S rRNA hybridization signals to compensate for loading differences between gel lanes (Bunick et al., 1994Go). In all cases, Northern blots were replicated at least 4 times for each treatment group.

[3H]-Thymidine autoradiography.
[3H] Autoradiography was performed as described previously (Cooke et al., 1997Go). [3H]-Thymidine injections were given 2 h before tissue collection. Epithelial mitogenic activity in terms of DNA synthesis in whole uteri from C57BL/6N, AhRKO, and host mice was expressed as LI or [3H]-thymidine-labeled cells per total cells, and each point is based on data from 5–6 C57BL/6N or AhRKO mice and 8 host mice for each treatment group. As an internal control, uterine luminal epithelia in host uteri were also evaluated for mitogenic activity. The extent of uterine epithelial mitogenic activity in the various tissue recombinants was evaluated as described above, and is based on autoradiographic analysis of 4–6 separate specimens for each tissue recombinant type.

Statistical analysis.
Data on epithelial proliferation and LF mRNA expression were evaluated by ANOVA using the General Linear Models Procedure (GLM) from Statistical Analyses Software (SAS; SAS Institute, Inc., Cary, NC). Comparison between means were based on the least significant difference (LSD) test. In all cases, differences between means were considered significantly different at p < 0.05.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
E2 stimulated a marked increase in epithelial labeling index (LI) and strong LF mRNA expression in uteri from both wt and AhRKO mice compared to oil-treated controls (Figs. 1–4GoGoGoGo), indicating that the presence or absence of AhR does not affect normal estrogen responsivity. However, when wt mice were given 5 µg/kg TCDD 24 h before E2 treatment, uterine epithelial LI was reduced by 42% (p < 0.05) compared to wt mice given E2 alone (Fig. 1Go). Normalized densitometric analysis of Northern blots indicated LF mRNA expression was reduced by 38% (p < 0.05) compared to wt mice that received E2 alone (Fig. 2Go). Conversely, TCDD treatment did not alter uterine epithelial proliferation or LF mRNA expression in AhRKO mice compared to AhRKOs that received E2 alone (Figs. 3 and 4GoGo), showing that AhR is necessary for the antiestrogenic effects of TCDD.



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FIG. 1. Labeling index (LI) of wild-type (wt) uterine luminal epithelium, expressed as mean percent labeled cells ± SEM. Wild-type C57BL/6N mice were ovariectomized and then injected with oil, E2, or 5 µg/kg TCDD 24 h before E2. Different letters above columns indicate significant differences between groups (p < 0.05). Results are based on an n of 5–6 mice for each treatment group.

 


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FIG. 2. Northern blot analysis of uterine lactoferrin (LF) mRNA expression in wt C57BL/6N mice. Normalized densitometric data, based on a reference 28S rRNA signal, is shown graphically as a percentage of the E2-treated positive control value. Asterisks indicate significant differences compared to E2 control. Ovariectomized wt mice treated with E2 expressed high levels of a 2.6 kb LF transcript compared to oil-treated mice, while mice that received 5 µg/kg TCDD injected 24 h before E2 had a significant decrease (p < 0.05) in uterine LF mRNA relative to E2-treated controls. Values are means ± SEM, based on an n of 5–6 mice for each treatment group.

 


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FIG. 3. Labeling index (LI) of AhRKO uterine luminal epithelium, expressed as mean percent labeled cells ± SEM. AhRKO mice were ovariectomized and then injected with E2. Although some animals were injected with 5 µg/kg TCDD 24 h before E2 exposure, LI was not significantly different between E2- and TCDD+E2-treated groups. Results are based on an n of 5 mice for each treatment group.

 


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FIG. 4. Northern blot analysis of uterine lactoferrin (LF) mRNA expression in AhRKO mice. Normalized densitometric data, based on the 28S rRNA signal, is shown graphically as a percentage of the positive E2-treated control value. Ovariectomized AhRKO mice treated with either E2 or 5 µg/kg TCDD injected 24 h before E2 expressed LF mRNA at similar levels and were not significantly different, while LF mRNA was low in the oil-treated group. Values are means ± SEM, based on an n of 5 mice for each treatment group.

 
Using Western blot analysis, AhR and ARNT proteins were detected in whole uteri from wt mice (Fig. 5Go). Similarly, AhR and ARNT were each expressed in isolated uterine stroma and epithelium, suggesting that both of these tissue types may be directly responsive to TCDD.



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FIG. 5. Expression pattern of uterine AhR and ARNT proteins in C57BL/6N mice. Western blot analysis indicated that the AhR, as well as its transcriptional heterodimerization partner, ARNT, are expressed in whole uteri. Importantly, stromal and epithelial uterine tissue also express both AhR and ARNT proteins. Bands for AhR and ARNT were compared to those for a molecular weight standard and those for AhR and ARNT in positive control mouse liver.

 
For all tissue recombinants, epithelial LI was low and similar in oil-treated hosts (data not shown), and strongly increased with E2. The magnitude of the LI in E2-treated groups was similar among the various tissue recombinant types (Figs. 6 and 7GoGo). Epithelial LI in tissue recombinants composed of wtS+wtE from host animals exposed to 5 µg/kg TCDD 24 h before E2 treatment was reduced by about 34% (p < 0.05) compared to the same type of tissue recombinants exposed to E2 alone (Fig. 6Go). Thus, the inhibitory effects of TCDD on epithelial proliferation in the tissue recombinants closely paralleled that observed in whole uteri in vivo.



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FIG. 6. Epithelial labeling index (LI) in uterine tissue recombinants containing wtS, expressed as mean percent labeled cells ± SEM. Tissue recombinants with wtS and either wt or ko epithelium were grafted into female nude host mice which were later ovariectomized and injected with either oil, E2 or 5 µg/kg TCDD+E2. LI in tissue recombinants from oil-treated hosts was consistently low and similar to oil treated controls in Figures 1 and 3GoGo (not shown). Asterisks indicate significant differences (p < 0.05) between treatment groups (n = 4–6) within each tissue recombinant type.

 


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FIG. 7. Epithelial labeling index (LI) in uterine tissue recombinants containing koS, expressed as mean percent labeled cells ± SEM. In contrast to tissue recombinants with wtS, LI in tissue recombinants containing ko stroma was similar between E2- and TCDD+E2-treated groups, even when epithelial AhR was present as in koS+wtE. LI in tissue recombinants from oil-treated hosts were consistently low and similar to oil-treated controls in Figures 1 and 3GoGo. There were no significant differences (p < 0.05) between treatment groups (n = 4–6).

 
In tissue recombinants that contained only stromal AhR (wtS+koE), treatment with 5 µg/kg TCDD, at 24 h before E2, reduced epithelial LI by about 37% (p < 0.05) compared to the same tissue recombinants in E2-treated hosts, indicating normal TCDD responsiveness despite the absence of epithelial AhR (Fig. 6Go). Importantly, epithelial LI in tissue recombinants containing koS (koS+koE and koS+wtE) was not significantly different between E2- and TCDD+E2-exposed groups (Fig. 7Go). Thus, tissue recombinants show normal decreases in epithelial LI in response to TCDD when stromal AhR is present, whereas epithelial AhR status has no effect.

As an additional control, we also monitored luminal epithelial DNA synthesis in uteri of the ovariectomized, nude host animals following recovery of tissue recombinants. In all cases, LI was low in oil-treated hosts and increased dramatically in E2-treated hosts. TCDD treatment produced consistent and significant decreases in the LI of host uterine epithelia (Fig. 8Go). These clear antiestrogenic effects in host animals indicated that the lack of a TCDD effect on epithelial mitogenic activity in tissue recombinants prepared with koS was strongly correlated with the genotypic composition of the tissue recombinant grafts.



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FIG. 8. Luminal epithelial labeling Index (LI) in uteri of nude mouse hosts, expressed as mean percent labeled cells ± SEM. After recovery of tissue recombinants, uteri were collected from ovariectomized host animals. Hosts injected with E2 showed high epithelial labeling, as expected. However, labeling was reduced by 26% in hosts injected with TCDD+E2, in contrast to normal labeling in koS+wtE tissue recombinants grown in the same host animals. Different letters above columns indicate significant differences between groups (p < 0.05). Results are based on an n of 8 mice for each treatment group.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
E2 normally regulates a wide variety of processes within the uterus, including water imbibition, proliferation, and functional activity of the epithelial, stromal, myometrial, and vascular cells that comprise the uterus. Previous studies (Gallo et al., 1986Go; Safe, 1995Go) strongly suggested that TCDD had antiestrogenic effects on mouse uterine cellular function in vivo. These and other studies raised questions regarding the role of factors such as the AhR that may participate in the TCDD effect. Lin et al. (1991) used an AhR nonresponsive mouse strain to demonstrate that TCDD-induced decreases in hepatic glucocorticoid receptor binding and tyrosine aminotransferase activity may not be mediated by the AhR in C57BL/6J mice. Findings such as these by others (Fernandez-Salguero et al., 1996Go; Hoffer et al., 1996Go; Puga et al., 1997Go) suggest that AhR-independent TCDD effects in uterus cannot be ruled out. Thus the present study sought to determine if the antiestrogenic effects of TCDD in the uterus involved inhibition of critical E2-induced epithelial processes and to establish whether or not the AhR was necessary for any such TCDD effect. Finally, the specific role of both stromal and epithelial AhR in TCDD disruption of uterine epithelial function was determined. These findings may have relevance for understanding how E2/ER{alpha} signaling may be influenced by putative endocrine disrupters.

Stimulation of uterine epithelial mitogenesis and secretory protein production are important and well studied E2 actions. The inhibitory effects of TCDD on proliferation of cell lines (Safe et al., 1998Go), most of which are epithelial in derivation, suggested TCDD might also inhibit E2 actions on epithelia in vivo, though the effect of TCDD on uterine epithelia and uterine stromal-epithelial interactions has not been previously described. Our results show for the first time that TCDD inhibits specific uterine epithelial responses to E2 by acting through the stromal AhR to perturb uterine function in mice. The inhibitory effect of TCDD on uterine epithelial mitogenic and secretory protein activity in C57BL/6N mice most likely leads to a decrease in overall cell number and therefore contributes to the reduction in uterine weight (Gallo et al., 1986Go; Umbreit et al., 1988Go). This weight reduction was observed following exposure to higher doses of TCDD than that used in the present study.

Uterine epithelial proliferation and LF mRNA expression occurred normally in ovariectomized AhRKO mice given E2. Though AhRKOs are sub-fertile (Abbott et al., 1999; D. L. Buchanan, unpublished data), their normal estrogen responsiveness in terms of uterine epithelial DNA synthesis and LF mRNA production is apparently not affected by the absence of AhR. Importantly, epithelial labeling and LF mRNA expression in ovariectomized AhRKO mice exposed to TCDD plus E2 were not altered compared to the AhRKOs that had received E2 alone, and were similar to wt controls exposed to E2 alone, indicating that E2-induced uterine epithelial activity in the AhRKO mouse is not altered by TCDD. These findings are consistent with previous reports that AhRKO mice do not show uterine histopathological changes following TCDD exposure (Fernandez-Salguero et al., 1996Go). However, epithelial proliferative activity and LF mRNA expression were substantially reduced in TCDD+E2-treated wt animals compared to E2-treated wt controls. Altogether, these results indicate that AhR expression is not necessary for normal E2-induced uterine epithelial function, and that TCDD requires AhR expression to inhibit E2-induced uterine epithelial mitogenic activity and secretory protein production in C57BL/6N mice.

AhR expression in mouse uterus has been reported (Nesaretnam et al., 1996Go), but it was not known if AhR was present in both epithelium and connective tissue of the uterus, as is typical in other organs (Abbott et al., 1994Go). Our results showing that AhR protein, as well as its dimerization partner, the ARNT protein, are present in both uterine epithelium and stroma, indicate that both of these tissues have the intracellular machinery to respond directly to TCDD. Thus, TCDD or other AhR ligands could exert antiestrogenic effects on uterine epithelium, either directly through the epithelia, indirectly via the stroma, or through both the epithelia and stroma.

Initially, it would appear likely that the inhibitory effects of TCDD on uterine epithelial mitogenic activity and secretory protein mRNA expression induced by E2 would involve direct actions on the epithelium. However, E2-induced uterine and vaginal epithelial proliferation is mediated indirectly through stromal ER{alpha}, while epithelial ER{alpha} is not involved (Bigsby and Cunha, 1986Go; Buchanan et al., 1998Go; Cooke et al., 1997Go). Additionally, stromal ER{alpha} is essential for epithelial production of the secretory protein LF in response to E2 (Buchanan et al., 1999Go). These previous findings along with indications that TCDD antagonizes normal estrogen responsiveness suggest that inhibition of these uterine E2 effects by TCDD acting on AhR in the stroma may involve interference with the E2 response of the stromal cells themselves and/or the stromal-epithelial communication that is induced by E2. Consistent with this hypothesis, Near et al. (1999) has shown that an AhR agonist induces apoptosis in bone marrow-derived preB cells cultured on AhR-positive, but not AhR-negative, primary bone marrow stromal cell monolayers. This indirect mediation of preB cell apoptosis by an AhR agonist through stromal AhR suggests TCDD could antagonize E2 effects in uterine epithelium by a similar indirect mechanism.

The tissue recombination methodology, in conjunction with the AhRKO mouse, allows construction of uterine tissue recombinants that express AhR in both stroma and epithelium, or lack AhR in one or both tissue compartments. By examining the effects of TCDD on these various types of tissue recombinants, the role of both stromal and epithelial AhR on processes such as E2-induced epithelial mitogenesis and secretory protein production may be established.

Thymidine labeling in the tissue recombinations indicated significant differences in epithelial DNA synthesis between E2- and TCDD+E2-treated groups as long as stromal AhR was present. Reduction of epithelial LI in wtS+wtE but not koS+koE tissue recombinants from TCDD+E2-treated nude host animals validated the results we observed in whole uteri and established TCDD as having antiestrogenic activity in tissues grafted into the host mouse strain. However, TCDD also inhibited the epithelial mitogenic response in tissue recombinants composed of wtS+koE, despite the lack of epithelial AhR. This demonstrated that stromal AhR is sufficient to mediate the normal TCDD antiestrogenic effect in the absence of epithelial AhR. In contrast, tissue recombinants containing epithelial but not stromal AhR were unaffected by TCDD exposure, confirming that the TCDD antiestrogenic effect observed in wtS+wtE tissue recombinants does not involve epithelial AhR. This is the first indication that antiestrogenic epithelial effects of TCDD occur indirectly through stromal AhR and may involve disruption of normal E2-induced stromal-epithelial communication.

Stromal-epithelial interactions in mouse uterus are believed to involve paracrine factors produced by an ER{alpha}-expressing stroma (Cooke et al., 1998bGo). Estrogen stimulates the production of a number of growth factors in uterus, and paracrine regulation of epithelial function by stromal growth factors has been proposed (Cooke et al., 1998aGo). It is unclear which growth factors are involved, but presumably TCDD is altering the production and/or response to one or more of these factors to exert its antiestrogenic effect. Experiments are currently underway to investigate the antiestrogenic influence of TCDD on possible stromal paracrine factors.

Compared to other mouse strains, C57BL/6 mice are typically regarded as one of the most sensitive to TCDD (Safe, 1995Go), whereas the nude mice used as host animals in this study are derived from the less sensitive Balb/c strain (Nemoto et al., 1990Go; Smith et al., 1998Go). The attenuated decreases in epithelial LI induced by TCDD in host uteri supported previous evidence that Balb/c mice are less sensitive to the antiestrogenic effects of TCDD. The results in host uteri also confirmed that the reduction in epithelial LI observed in tissue recombinants in response to TCDD was due to treatment conditions and stromal AhR, and not some deficiency in the tissue recombination/grafting system.

The inhibition of E2-induced uterine epithelial mitogenic activity and secretory protein mRNA production by a TCDD dose that is lower than that used in previous studies on mouse uterine responses suggests that these endpoints may be relatively sensitive to antiestrogen exposure. Previous studies with mice used TCDD doses equal to or greater than 30 µg/kg in non-ovariectomized mice to demonstrate antiestrogenic effects of TCDD on uterine weight (DeVito et al., 1992Go; Gallo et al., 1986Go; Umbreit et al., 1988Go). However, increases in hepatic CYP1A1 mRNA levels were detected after exposure to 10 and 25 ng/kg of TCDD in the mouse and hepatic ethoxyresorufin-O-deethylase (EROD) activity was induced by 100 ng/kg TCDD (Narasimhan et al., 1994Go). These results, along with the present findings demonstrating uterine epithelial effects at 5 µg TCDD/kg in 2 mouse strains, suggest that sublethal TCDD exposure exerts important biological effects in the mouse, a species typically regarded as being less sensitive to TCDD. Furthermore, the AhR-mediated inhibitory effect of TCDD on E2-induced uterine epithelial responses in the mouse also may be a relevant endpoint in a more TCDD-sensitive species such as the rat.

Normal uterine epithelial function is highly dependent on hormonal regulation, making it potentially vulnerable to endocrine-disrupting chemicals. However, the molecular mechanism by which the liganded AhR exerts antiestrogenic effects is not known. The inhibition of epithelial estrogenic responses by TCDD has been attributed to a reduction in ER levels, suppression of ER transactivation and/or limited transcriptional cofactor availability (Kumar et al., 1999Go; Romkes and Safe, 1988Go; Safe and Krishnan, 1995Go). Reduction in ER levels by TCDD accompany a decrease in uterine weight (Romkes and Safe, 1988Go), but regulation of uterine ER concentrations by TCDD is dependent on physiologically functioning ovaries, in that a TCDD-induced decrease in uterine ER below normal levels is not seen in ovariectomized mice (DeVito et al., 1992Go). The fact that the ovariectomized animals used in our studies were susceptible to the antiestrogenic effects of TCDD suggests that the ER level may not be a determinant for TCDD effects in uterine epithelia, and shows TCDD may induce its antiestrogenic effects on uterine epithelia, and possibly uterine weight in general, by an alternative mechanism.

Suppression of ER transactivation by liganded AhR has also been proposed (Duan et al., 1999Go; Kharat and Saatcioglu, 1996Go; Safe and Krishnan, 1995Go). AhR ligands induce the production of enzymes, such as CYP1A1, 1A2, and 1B1, which are encoded by genes containing dioxin response elements (DREs; Safe, 1995; Whitlock, 1993). In addition, DREs have been identified in several E2-responsive genes (Safe and Krishnan, 1995Go), and the ability of the liganded AhR to interfere with liganded ER action and inhibit E2-induced gene expression by binding an overlapping DRE in vitro has been demonstrated (Krishnan et al., 1995Go). Although the molecular intricacies of the uterine epithelial mitogenic response to estrogen have not been fully elucidated, we have shown that E2-induction of uterine epithelial proliferation is mediated entirely through the stroma (Cooke et al., 1997Go). Therefore, if an interaction between liganded AhR and liganded ER{alpha} or its ERE disrupts epithelial mitogenesis at the level of ER{alpha} transactivation, such inhibitory AhR-ER cross-talk would likely occur in the stroma and could apply only to estrogenic responses involving genes that contain a DRE.

Although the mouse and human LF genes do not contain a consensus DRE sequence within 2 kb upstream of the promoter, they do contain a number of other potential DRE elements, yet none of these overlap or juxtapose the ERE (Christina Teng, NIEHS, personal communication). Therefore, the hypothesis that ER transactivation is inhibited by the liganded AhR occupying an adjacent DRE (Duan et al., 1999Go; Krishnan et al., 1995Go) may explain our results showing TCDD inhibition of LF mRNA if the 3-dimensional structure of the LF gene is considered. Alternatively, LF mRNA decreases by TCDD could involve other inhibitory mechanisms. The uterine LF gene contains several response elements that may be either directly or indirectly influenced by an AhR ligand. For example, the LF gene contains a proximal epidermal growth-factor (EGF) response element and has been shown to be upregulated by EGF and EGF response element (EGFRE) binding in vitro (Teng et al., 1999). EGF is an example of a stromal regulatory factor that is bioactivated by E2 (Mukku and Stancel, 1985Go) and may be involved in stimulating mitogenic or secretory protein responses in uterine epithelia following E2 treatment (Nelson et al., 1991Go). Cross-talk between the E2 and EGF signaling pathways involving phosphorylation of ER and affecting interactions between the ER protein and cofactors or DNA has been suggested (Ignar-Trowbridge et al., 1992Go). However, because TCDD exposure inhibits E2-induced expression of EGF receptor in mouse uterus (Astroff et al., 1990Go), uterine epithelial LF expression in vivo may also be altered at the EGFRE. While such a mode of action by TCDD may reduce LF expression, factors such as transcriptional coactivators involved in ER-mediated LF induction may also be influenced by TCDD.

As with ER, AhR/ARNT also recruit basal transcription factors that require cofactor binding to bridge the transcriptional apparatus to DNA (Rowlands et al., 1996Go; Swanson and Yang, 1998Go). The ER coactivator, RIP140, enhanced DRE-driven luciferase reporter activity by liganded AhR in 3 different cell lines (Kumar et al., 1999Go). Since RIP140 also binds AhR (Kumar et al., 1999Go), competition for transcriptional coactivators following TCDD exposure may diminish the ER-mediated uterine epithelial response. Further, the RIP140 domain that binds AhR is distinct from that involved in ER binding (Kumar et al., 1999Go), creating the possibility of a bridging effect by RIP140 to form a nonfunctional AhR-RIP140-ER complex. Thus, cofactor availability following TCDD treatment may limit ER mediation of the uterine epithelial response. Clearly, further studies are necessary to determine the specific molecular mechanism by which liganded AhR exerts antiestrogenic effects on uterine epithelial proliferation and LF mRNA expression.

Previously, we have demonstrated the efficacy of the tissue separation and recombination technique in uterine tissue recombinants constructed from ER{alpha} knockout and wt mice by ER{alpha} immunohistochemistry. Although antibodies for AhR exist and show staining where AhR is known to be abundant, we have found positive staining in AhRKO tissues with available AhR antibodies; these antibodies may recognize proteins other than AhR (Roman et al., 1998Go). Thus, genotypes of the various tissues in the tissue recombinants used here were not directly monitored. However, our ability to reliably and consistently construct heterogeneous tissue recombinations devoid of contaminating epithelial or stromal tissue has been repeatedly demonstrated to be extremely efficient, with a >95% success rate (Buchanan et al., 1998Go; 1999Go; Cooke et al., 1986Go; 1997Go).

In conclusion, we have shown that TCDD inhibits E2-induced uterine epithelial mitogenic activity and secretory protein mRNA production, and have definitively established that both effects require AhR. We have previously demonstrated that the E2-induced uterine epithelial proliferative response is mediated by stromal ER{alpha}, and our present results are the first evidence that TCDD inhibits uterine epithelial mitogenic activity through AhR in the stroma, while epithelial AhR is not involved in this effect. Thus, the antiestrogenic effect of TCDD on uterine epithelium that is possibly due to suppression of ER{alpha} transactivation by liganded AhR and/or limited transcriptional co-activator availability may result from TCDD actions in the stroma. This finding could have important clinical benefits in other female reproductive tract tissues affected by TCDD. For example, since liganded AhR inhibits growth of a breast cancer cell line (Wang et al., 1997Go); understanding the tissue sites and mechanisms of AhR action in the mammary gland may be useful in developing new therapeutic approaches to controlling mammary gland tumor growth (Safe et al., 1998Go; Weiss et al., 1996Go). The examination of tissue compartment-specific effects of the liganded AhR on E2-induced uterine epithelial function may be used as a model system for future research into the mechanism of action of AhR agonists in vivo, AhR-ER{alpha} cross-talk, and the risk that AhR ligand exposure poses to growth, development and function of the female reproductive tract.


    ACKNOWLEDGMENTS
 
This work was supported by an Interdisciplinary Environmental Toxicology Scholarship from the University of Illinois (to DLB) and NIH Grants AG15500 (to PSC) and ES01332 (to REP). Initial breeding pairs of mice used to obtain AhRKO animals were a generous gift from Dr. Barbara Abbott (Environmental Protection Agency, Research Triangle Park, NC) and Dr. Frank Gonzalez (National Cancer Institute). We also thank Dr. Richard S. Pollenz (Department of Biochemistry, University of South Florida, Tampa, FL) for performing Western blot analysis and Dr. Christina Teng (NIEHS, Research Triangle Park, NC) for providing LF cDNA.


    NOTES
 
1 Present address: The National Institute for Basic Biology, Nishigonaka 38, Myodaiji, Okazaki 444-8585, Aichi, Japan. Back

2 To whom correspondence should be addressed at the Department of Veterinary Biosciences, 2001 S. Lincoln Ave., University of Illinois, Urbana, IL 61802. Fax: (217) 244–1652. E-mail: p-cooke{at}uiuc.edu. Back

Presented in part at the 32nd Annual Meeting of the Society for the Study of Reproduction, Pullman, WA, August 1999 and the 39th Annual Meeting of the Society of Toxicology, Philadelphia, PA, March 2000.


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 MATERIALS AND METHODS
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 DISCUSSION
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