Dioxin Inhibition of Estrogen-Induced Mouse Uterine Epithelial Mitogenesis Involves Changes in Cyclin and Transforming Growth Factor-ß Expression

David L. Buchanan*,{dagger},1, Seiichiro Ohsako{dagger},{ddagger}, Chiharu Tohyama{dagger},{ddagger}, Paul S. Cooke§ and Taisen Iguchi*

* Center for Integrative Bioscience, Okazaki National Research Institutes, Okazaki, Aichi 444-8585, Japan; {dagger} CREST, Japan Science and Technology Corporation, Kawaguchi, Saitama 332-0012, Japan; {ddagger} National Institute for Environmental Studies, Tsukuba, 305-8506, Japan; and § Department of Veterinary Biosciences and Division of Nutritional Sciences, University of Illinois, Urbana 61802, Illinois

Received June 5, 2001; accepted November 19, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A single dose of dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin or TCDD; 5 µg/kg, ip) inhibits 17ß-estradiol (E2)-induced uterine epithelial mitogenesis, apparently through disruption of stromal-epithelial interactions. To understand if TCDD alters early uterine (Ut) responses to E2, young adult C57BL/6J mice were ovariectomized and given (ip) either oil or 5 µg/kg TCDD. After 24 h, TCDD-treated mice received E2, and oil-treated mice were given E2 or oil. Body and Ut weights were collected 6 and 18 h later. Ut were flash-frozen at 6 h. E2 increased Ut weight (p < 0.0001) and Ut/body weight ratio (p < 0.0001), compared to mice given oil alone. Ut cyclin expression was assessed by an RNase protection assay. E2 increased mRNA expression for cyclin A2 and B1 (p < 0.05), in addition to D1, D2, and D3 (p < 0.001), while cyclin C was unchanged from oil controls and cyclins A1 and B2 were undetectable. In contrast, TCDD completely abolished E2-induced cyclin A2, which has been associated with S phase initiation, and reduced B1 and D2 (p < 0.05). Interestingly, TCDD did not alter E2-induced Ut weight increases at 6 h, but inhibited E2-induced Ut weight gain at 18 h. A 10-µg/kg TCDD dose was necessary for attenuation of the early E2-induced Ut weight increases (p < 0.01). Since TGF-ß regulates cyclins, Ut TGF-ß was also assessed in TCDD + E2-treated and control mice. TGF-ß mRNA levels were increased after TCDD compared to E2 alone (p < 0.01), suggesting a possible mechanism for TCDD inhibition of Ut cyclin A2. Thus, TCDD alters specific E2-regulated Ut G1 phase activities and may inhibit E2-induced Ut epithelial mitogenesis by disrupting specific cell signaling mechanisms necessary for S phase initiation in vivo.

Key Words: cytokine; uterus; estrogen; proliferation; antiestrogen; aryl hydrocarbon receptor; ovariectomy.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
2,3,7,8-Tetrachlorodibenzo-p-dioxin (dioxin; TCDD) is the most potent congener within a large class of chlorinated hydrocarbon environmental contaminants known as dibenzo-p-dioxins. TCDD elicits a variety of biochemical and toxicological outcomes, including immunotoxicity, carcinogenicity and alterations in endocrine responses (Barsotti et al., 1979Go; Geyer et al., 2000Go; Huff et al., 1991Go). Most TCDD effects have been attributed to its ability to bind the cytosolic aryl hydrocarbon receptor (AhR), a member of the basic helix-loop-helix/PAS superfamily of DNA binding proteins (Swanson and Bradfield, 1993Go). The AhR is a promiscuous receptor in that its activity is modulated by not just one but numerous compounds, and dioxins compose just one class in a large family of manmade polyhalogenated chemicals that interact with it; polychlorinated dibenzofurans and coplanar polychlorinated biphenyls are examples of other types of compounds that also have strong affinity for AhR (Geyer et al., 2000Go).

As an endocrine disruptor, TCDD has antiestrogenic activity both in vitro and in vivo (Gallo et al., 1986Go; Safe, 2001Go). TCDD decreases mouse uterine wet weight (Gallo et al., 1986Go), suggesting inhibitory effects on estrogen-controlled cellular processes contributing to water imbibition. Whereas estrogen induces proliferation and secretory protein productions in uterine epithelia, these responses are inhibited by TCDD in ovariectomized wild-type but not AhR gene knockout mice (Buchanan et al., 2000Go). Both TCDD and unbound AhR downregulate cell cycle progression in absence of hormone administration in vitro, albeit through different mechanisms (Kolluri et al., 1999Go; Puga et al., 2000Go). Mechanistic influences of TCDD and AhR on stimulation and inhibition of cell cycle progression in nonreproductive tissues in vitro and in vivo have been described (Abbott et al., 1987Go; Lucier et al., 1991Go; Stohs et al., 1990Go). Yet, TCDD effects are cell type- and species-specific (DeVito and Birnbaum, 1994Go), and the cellular and molecular mechanisms underlying antiestrogenic TCDD effects on processes such as uterine epithelial proliferation have not been reported.

Within the first 24 h, a single injection of 17ß-estradiol (E2), the most potent endogenous estrogen, stimulates multiple responses in the rodent uterus. For example, uterine epithelial proliferation and differentiation depend on stromal cell responses to E2 (Buchanan et al., 1999Go; Cooke et al., 1986Go, 1997Go). Shortly after E2 treatment, stromal cells undergo differentiative changes associated with dramatic increases in uterine fluid uptake. Subsequently, endothelial, along with luminal and glandular epithelial, cells enter G1 in preparation for DNA synthesis before proceeding to mitosis. The greatest proportion of maximal responses to E2 in mouse uterus correspond to G1 since they occur within 6 h after exposure (Buchanan, unpublished report) and include hyperemia, water imbibition, and variations in expression of delayed early genes (e.g., cytokines and cyclins; Geum et al., 1997Go; Martin et al., 1976Go; Takahashi et al., 1994Go). These early preparatory events are critical for onset and normal progression of S phase, which is confined to epithelia in the uterus and begins about 8.5 h after E2 treatment (Martin et al., 1973Go). As an antiestrogen, TCDD may alter events necessary for these early uterine responses.

Uterine epithelial S phase peaks between 13 and 25 h after E2 treatment in ovariectomized mice (Martin et al., 1973Go), and E2 acts through uterine stromal estrogen receptor-{alpha} (ER{alpha}) to induce epithelial mitogenesis in vivo (Cooke et al., 1997Go). We recently determined that a single TCDD exposure of 5.0 µg/kg (ip) inhibits E2-induced uterine epithelial mitogenic activity in ovariectomized mice and that this inhibition requires stromal AhR, while epithelial AhR is not involved (Buchanan et al., 2000Go). Thus, TCDD inhibits epithelial proliferation prior to S phase in vivo, and liganded AhR alters stromal-epithelial interactions by disrupting stromal responses to E2. While TCDD inhibition of uterine epithelial proliferation may be initiated through alterations in E2-induced stromal responses, factors under E2 control that regulate epithelial proliferation and differentiation have not yet been identified either in vitro or in vivo.

The use of AhR ligands to understand E2/TCDD interactions and control E2 signaling has been suggested (Safe and Krishnan, 1995Go). To better understand the mechanisms responsible for the TCDD effect and the E2 signaling pathway in vivo, TCDD influence on uterine responses during the early E2-induced preparatory period was examined in the absence of endogenous hormonal influences. We determined whether TCDD alters early E2 regulation of uterine events such as wet weight, in addition to G1 phase activity, as indicated by cyclin and cytokine gene expressions. Our data show that TCDD alters early E2-regulated uterine processes critical for cell cycle progression in vivo and emphasize the use of TCDD as a tool for understanding steroid regulatory pathways.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals.
Adult C57BL/6J mice (40- to 50-day-old; Chu-bu Kaggaku Shizai, Nagoya, Japan) were given CE-2 rodent chow (Clea Japan, Tokyo) and tap water ad libitum. Animals within similar treatment groups were housed 2 to 4 per cage with controlled lighting (12L:12D), temperature (22–24°C), and humidity (50 ± 5%) in clear plastic cages with hardwood bedding and were maintained in accordance with the Guiding Principles in the Use of Animals in Toxicology. The Animal Research Committees at Okazaki National Research Institutes and National Institute for Environmental Studies, Japan, approved all experiments.

TCDD and E2 treatments.
TCDD (>99.5% pure; Cambridge Isotope Laboratory, Andover, MA) was dissolved in n-Nonane (Nacalai Tesque, Kyoto, Japan) and diluted in sesame oil to the appropriate concentration. The final formulation for each TCDD dose included 10% n-Nonane. To determine the influence of TCDD on E2-stimulated increases in uterine (Ut) weight and regulation of early gene expression, mice were ovariectomized and one week later given 1 of 4 TCDD doses (0.2, 1.0, 5.0, or 10.0 µg TCDD + 10% nonane/kg body weight) or 10% nonane + oil vehicle by ip injection. The next day, 10% nonane + oil-treated animals received pure oil vehicle or E2 (30 ng/mouse in 0.05 ml corn oil, which is equivalent to 1.5 µg/kg); all TCDD-treated animals received E2 (30 ng). Body, liver, and Ut weights were collected 6 or 18 h later. After weighing, Ut were flash frozen in liquid N2.

Probe synthesis, RNA isolation.
For RNase protection assay, antisense biotin-labeled probe syntheses was carried out using PharMingen multiprobe template sets for mouse cyclin and cytokine mRNAs (mCYC and mCK-3b; BD-PharMingen International, San Diego, CA) with Biotin RNA Labeling Mix (10x; Roche Diagnostics GmbH, Mannheim, Germany) by in vitro transcription at 37°C according to standard protocols. Labeled probe was extracted by phenol-chloroform and then precipitated with 4 M LiCl and 100% EtOH to remove free nucleotides. Total RNA was isolated from whole frozen Ut using TRIzol reagent (Gibco BRL, Rockville, MD) according to the manufacturer's protocol. To remove residual contaminants, RNA was further purified using the Qiagen RNeasy total RNA kit (Qiagen K.K., Tokyo, Japan).

RNase protection assay.
Ut mRNA expression levels for cyclins (A2, B1, C, D1, D2, D3, A1, and B2) and cytokines (TGF-ß1, TGF-ß2, TGF-ß3, MIF, TNF-{alpha}, IL-6, and IFN-{gamma}) were assessed in 5.0 µg TCDD-exposed and control Ut after 6 h E2 or oil by RNase protection assay. Yeast control and target RNAs (12 µg) were incubated with 20 ng labeled probe at 90°C for 10 min, then 56°C for 15 h and 37°C for a final 30 min. Unhybridized probe and unprotected RNA were digested in a 1:100 dilution of RNase A + T1 mix in hybridization buffer (Ambion, Inc., Austin, TX) for 30 min at 37°C. RNases were inactivated with RNase Inactivation/Precipitation III Solution (Ambion, Inc.). Precipitation of yeast control RNA that had been incubated with or without RNase and protected probe samples was achieved at –20°C. Samples were then resolved on a denaturing 5% polyacrylamide gel and blotted by semidry electro-transfer to a nylon (+) membrane (Hybond-N+, Amersham Pharmacia Biotech, Buckinghamshire, UK). Biotin signals from blot membranes were captured on X-ray film by chemiluminescence (BrightStar BioDetect, Amersham) and converted to electronic form, using an Epson ES2000 scanner interfaced with a Macintosh G4 computer utilizing Adobe Photoshop software. Autoradiograms for cyclins and cytokines were quantitated using Image Gauge software (Fuji Photo Film Co., Ltd., Tokyo, Japan). Relative mRNA transcript levels were normalized based on densitometric analysis of hybridization signals for mRNA of the ribosomal protein L32 to compensate for loading differences between gel lanes. For all treatment groups, RNase Protection assays were replicated at least 3 times in duplicate for cyclins and 2 times in triplicate for cytokines.

Statistical analysis.
Data on Ut and liver weights were evaluated by ANOVA (StatView, version 5.0; SAS Institute, Cary, NC) followed by Dunnett's test for pairwise comparisons. Ut mRNA expressions were evaluated by Student's t-test using StatView. All data are reported as means ± SEM. In all cases, means were considered significantly different when p < 0.05. For the various endpoints, data are from 6 to 12 mice per treatment group.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Body, Liver, and Uterine Weights
Eighteen h of E2 exposure induced a marked hyperemia and increased Ut weight by 42% (p < 0.0001) and Ut-to-body weight ratio by 41% (p < 0.0001), compared to oil-treated mice. In contrast, when 5.0 µg/kg TCDD was given prior to the 18 h E2 exposure, Ut weight increased by only 10% and Ut-to-body weight ratio increased by only 5% compared to oil-treated mice (Table 1Go). Thus, both absolute and relative Ut weights achieved 18 h after E2 were dramatically decreased by TCDD (p < 0.01). The shorter 6-h E2 exposure elicited responses similar to those seen 18 h after E2. Specifically, 6 h of E2 exposure induced Ut hyperemia and increased Ut weight by 45% (p < 0.0001) and Ut-to-body weight by 42% (p < 0.0001), compared to oil. Importantly, 0.2, 1.0, or 5.0 µg/kg TCDD did not alter E2-induced increases in hyperemia, Ut weight, or Ut-to-body weight ratio 6 h after E2. However, although 10.0 µg/kg TCDD had no effect on relative uterine weight, 10.0 µg/kg TCDD abrogated the early (6 h) E2-induced hyperemic response and diminished the early E2-induced Ut weight increase (p < 0.01; Table 1Go). Thus, at least 10.0 µg/kg TCDD was required to achieve inhibition of the early E2-stimulated gain in Ut weight and hyperemia while only 5.0 µg/kg TCDD was sufficient to inhibit Ut weight gain and hyperemia 18 h after E2 treatment.


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TABLE 1 TCDD Effect on Uterine Weight Increases after 6 or 18 Hours Estradiol
 
Absolute and relative liver weights were unchanged by E2 given alone or by 0.2 µg/kg TCDD compared to oil controls. Liver-to-body weight ratios were slightly increased (9%; p < 0.05) in the 1.0, 5.0, and 10.0 µg/kg TCDD groups 6 h after E2, but absolute liver weights in these groups were not altered by TCDD when compared to mice given E2 alone (Table 1Go). Relevant to previous findings that antiestrogenic TCDD effects may not be related to changes in liver enzyme activity (DeVito et al., 1992Go), the lack of effect on absolute liver weight by TCDD minimizes the probability of a general toxicological response in this study.

Uterine Cyclin Gene Expression
Uterine epithelial cells are in G1 phase through 6 h of E2 treatment. Cyclin mRNA expression levels were determined and compared between uteri from mice given 5.0 µg/kg TCDD + 6 h E2, oil vehicle, or 6 h E2 alone. In the absence of TCDD, 6 h of E2 exposure significantly induced Ut mRNA expression for cyclins A2 and B1 (p < 0.01 and p < 0.05, respectively), and also cyclins D1, D2, and D3 (p < 0.001) relative to oil controls. Cyclin C expression was unchanged compared to oil control levels, and cyclins A1 and B2 were undetectable. In contrast, 5.0 µg/kg TCDD given 24 h before E2 completely abolished E2-induced cyclin A2 expression (p < 0.05), and cyclin B1 and D2 expression levels were significantly reduced compared to those of E2 alone (p < 0.05 and p < 0.01, respectively; Fig. 1Go). Expression levels for cyclins C, D1, and D3 did not change after TCDD compared to E2 levels, suggesting that the inhibitory effect of TCDD on the E2-induced increases in cyclins A2, B1, and D2 was specific.



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FIG. 1. RNase protection assay for cyclin expression in mouse uteri. Representative blot with normalized densitometric data shown graphically. Mice were given oil or TCDD (5 µg/kg, ip) 24 h before estrogen treatment (E2). Uteri were collected 6 h post-E2 or oil. Uterine mRNA levels from oil-treated mice were considered baseline as indicated by a line through the data. The E2 dose increased cyclin A2 and B1 (p < 0.01 and p < 0.05, respectively) and cyclins D1, D2, and D3 (p < 0.001), while cyclin C was unchanged compared to oil. TCDD dosing completely abolished E2-induced cyclin A2 expression (p < 0.05) and reduced B1 and D2 levels (p < 0.05 and p < 0.01, respectively), compared to E2 alone (asterisks). RNA was pooled from 8 to 12 mice for each treatment. Densities were normalized to that of ribosomal protein L32, which served as a loading control. Inset: Y–, yeast RNA minus RNase; Y+, yeast RNA plus RNase.

 
Uterine Cytokine Gene Expression
Total RNA collected from Ut of mice given 5.0 µg/kg TCDD + 6 h E2, oil vehicle, or 6 h E2 alone was also used to determine cytokine mRNA expression levels. Ut mRNA expression levels for TGF-ß1 were not altered after 6 h of E2 compared to oil control animals, and TGF-ß3 mRNA levels were suppressed (p < 0.01) below control levels after 6 h of E2. After TCDD, TGF-ß1 expression was increased (p < 0.0001) compared to mice that received E2 alone, and TGF-ß3 suppression by E2 was reversed and appeared similar to the level of oil controls (p < 0.01; Figure 2Go). Uterine mRNA expression levels for the cytokines macrophage migration inhibiting factor (MIF), tumor necrosis factor-{alpha} (TNF-{alpha}), interleukin-6 (IL-6), and interferon-{gamma} (IFN-{gamma}) were also evaluated. MIF increases in response to E2 (Suzuki et al., 1996Go). The E2-induced increase in MIF mRNA over oil-treated mice (p < 0.01) was further increased by 5.0 µg/kg TCDD (p < 0.0001; Figure 2Go). Neither E2 nor 5.0 µg/kg TCDD + E2 altered uterine mRNA levels for TNF-{alpha}, IL-6, or IFN-{gamma} compared to uteri from oil control mice (data not shown), confirming specificity of the E2 and TCDD effects.



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FIG. 2. TCDD effect on estrogen (E2)-induced uterine TGF-ß mRNA expression by RNase protection assay. Representative blot with normalized densitometric data shown graphically. Animals received oil or TCDD (5 µg/kg, ip) 24 h before E2. Uteri were collected 6 h post-E2 or oil; mRNA levels from oil controls were considered baseline. TGF-ß1 was unchanged and TGF-ß3 was suppressed by E2 (p < 0.01) compared to baseline. However, TCDD dosing completely abolished E2 suppression of TGF-ß3 (p < 0.01) and increased TGF-ß1 by 28% (p < 0.0001), compared to E2 (asterisks). E2 induction of migration inhibiting factor (MIF) was augmented by TCDD (p < 0.0001). Total RNA was pooled from 8 to 12 mice for each treatment. Densities were normalized to mRNA for the ribosomal protein L32. Inset: Y–, yeast RNA minus RNase; Y+, yeast RNA plus RNase.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
To better understand the mechanisms underlying the antiestrogenic TCDD effect and the E2 signaling pathway in vivo, the influence of TCDD on early E2-induced uterine fluid uptake and G1 phase activity was examined. As seen in the present study, uterine fluid uptake approaches maximum by 6 h after E2 treatment, and the resulting increase in wet weight is maintained at a similar level by 18 h (Martin et al., 1976Go). DeVito et al. (1992) found no change in mouse serum E2 levels after TCDD and proposed that observed decreases in uterine ER levels after TCDD may be responsible for the antiestrogenic TCDD effect on uterine weight seen in nonovariectomized (intact) mice (Gallo et al., 1986Go). However, uterine ER levels were high and were not altered by TCDD in ovariectomzed mice (DeVito et al., 1992Go). Taken together, these findings suggested ovariectomy renders mice insensitive to the TCDD effect on uterine weight. Nonetheless, TCDD completely reduced uterine weight gain and hyperemia 18 h after E2 in ovariectomized mice to near control levels in the present study. This indicates ER levels may not contribute to TCDD effects on uterine weight in intact mice.

TCDD (5 µg/kg) did not alter E2-induced increases in uterine weight at 6 h but did alter G1 phase gene transcription for factors associated with epithelial cell cycle progression. When considered with findings by Buchanan et al. (2000) that TCDD inhibits E2-induced uterine epithelial proliferation, the inhibition of E2-induced uterine weight gain by 5 µg/kg TCDD at 18 h but not at 6 h suggests normal epithelial proliferative functioning may be critical for maintenance of uterine fluid uptake and weight at 18 h. Further, 10.0 µg/kg TCDD reduced uterine weight after 6 h E2, even though 5 µg/kg TCDD was insufficient, demonstrating a lower sensitivity to TCDD at this earlier time point and suggesting the antiestrogenic TCDD effects on early weight increases and G1 phase activity may be elicited through different mechanisms.

Cyclin activity has been well characterized in vitro, yet cyclin regulation and function in the uterus and in vivo are not well understood. Consistent with our data, mRNA for cyclins D1 and D3 are maximally increased 6 h after E2 in mouse uterus (Geum et al., 1997Go). D-type cyclins regulate passage through G1, and B cyclins are initially active during late S phase (Hunter and Pines, 1994Go). In MCF-7 cells, TCDD inhibits E2-induced cyclin D1 (Wang et al., 1998Go) but had no effect on 6-h E2-induced uterine D1 mRNA levels in vivo. Transcription of cyclin A2, which is required for initiation and progression of S phase, is confined to proliferating cells, and deregulation of cyclin A2 expression can disturb normal G1-S transition (Huet et al., 1996Go; Resnitzky et al., 1995Go). In mice, uterine cyclin A2 increases by 6 h after E2 and is predominately epithelial (Tong and Pollard, 1999Go). Of note, cyclin E regulates G1-S transition in vitro (Hunter and Pines, 1994Go). In mouse uterus, E2-induced cyclin E protein levels are 2- to 5-fold lower than those of cyclin A2 throughout G1 and S (Tong and Pollard, 1999Go), pointing to cyclin A2 as a substantial E2-induced cyclin for controlling the initiation of DNA synthesis. When mice were given TCDD in the present study, cyclin A2 mRNA induction by E2 was specifically abolished, while cyclin D2 and B1 expressions were decreased compared to animals given E2 alone. This antimitogenic effect during G1 by TCDD has never before been described in the uterus, nor in opposition to hormone treatment in vivo. Although it is not clear how E2 regulates cyclin A2 or how TCDD disrupts E2 signaling necessary for cyclin expression, the TCDD effect on cyclin A2 is maximal and may be useful for understanding E2 signaling. To further investigate this, the TCDD antiestrogenic effect on uterine cytokine gene expression was examined.

TGF-ß promotes stromal growth and extracellular matrix deposition (Sporn and Roberts, 1990Go). In epithelial cell cultures, TGF-ß is a potent antimitogen and inhibits several G1 phase cyclins (Geng and Weinberg, 1993Go). TGF-ß blocks proliferation early by preventing hyperphosphorylation of retinoblastoma (Rb) protein, but it loses this effect by late G1 (Geng and Weinberg, 1993Go). Importantly, TGF-ß specifically suppresses cyclin A2 gene expression in vitro (Feng et al., 1995Go; Yoshizumi et al., 1997Go). The present results show in vivo that low uterine TGF-ß levels are associated with increased cyclin expression. These events are inversely correlated as indicated by TCDD treatment. TCDD results in the opposite response to E2 so that increased TGF-ß1 and ß3 levels are accompanied by decreases in mRNAs for specific E2-induced cyclins that are important for normal G1 phase progression and S phase initiation. This correlation is indicative of alterations in normal uterine E2 signaling and further underscores the use of TCDD to understand E2 signaling in vivo.

Uterine TGF-ß1, ß2, and ß3 mRNAs are maximally increased by 3 h after E2, but ß1 and ß3 are suppressed below oil control levels and ß2 is undetectable at 6 h after injection of an E2 analogue in weanling mice (Takahashi et al., 1994Go). Consistent with this, TGF-ß1 and ß3 were similar to or suppressed below oil control levels, respectively, while ß2 was undetectable after 6 h of E2 exposure in the present study. Importantly, TCDD increased E2-regulated TGF-ß1 and ß3 levels, suggesting a possible mechanism for TCDD suppression of E2-induced uterine cyclins and consequently epithelial mitogenesis. Unexpectedly, E2 induction of MIF was augmented by TCDD. MIF expression is increased by TGF-ß in murine colon carcinoma cells (Takahashi et al., 1998Go). Thus, the additional increase in uterine MIF after TCDD likely tracks the TGF-ß increase and evidently results from increased TGF-ß protein signaling. These findings, are the first indication that TCDD alters E2 regulation of uterine TGF-ß.

TGF-ß1 and ß3 upregulation following TCDD has been reported in MCF-7 cell and thymus organ cultures (Lai et al., 1997Go; Vogel and Abel, 1995Go), but the mechanism leading to the increase is not known. Mouse TGF-ß1 contains dioxin response elements (DREs) in its gene promoter (Lai et al., 1996Go), but no DREs have been found in the ß3 promoter. Similar to the results of Kover et al. (1995), E2 did not alter uterine expression of cytokines TNF-{alpha}, IL-6, or IFN-{gamma}, and their levels were unaffected by TCDD under the present conditions, even when their promoters contained a DRE (IL-6 and IFN-{gamma}; Lai et al., 1996Go). This lack of effect by TCDD is consistent with the theory that increased TGF-ß levels after TCDD may not be due to TGF-ß gene induction, but could result from stabilization of TGF-ß mRNA levels reported to be maximum at 3 h post-E2 (Takahashi et al., 1994Go) and/or inhibition of E2-induced suppression at 6 h. Alternatively, since the TGF-ß1 promoter has AP-1 elements, it may be upregulated by that mechanism (Fos-Jun; Kim et al., 1990Go) since TCDD upregulates both c-fos and c-jun and increases AP-1 DNA binding (Hoffer et al., 1996Go). TCDD may also behave as an AhR antagonist for TGF-ß regulation. Liver abnormalities in AhR-null mice are attributed to TGF-ß production by hepatocytes, resulting in apoptosis (Zaher et al., 1998Go). Thus, possible AhR suppressive effects may be antagonized by TCDD in vivo to allow TGF-ß upregulation. Studies are now under way to better understand the TCDD effect and role of AhR on uterine TGF-ß gene expression and protein signaling.

Several explanations for the TCDD inhibitory effect on cyclins are possible. In vitro, the Rb protein is required for cyclin A2 and cyclin D activity during G1 (Feng et al., 1995Go; Hunter and Pines, 1994Go). Puga and colleagues (2000) found that AhR interaction appears to maintain Rb in the hypophosphorylated state and contributes to cell cycle arrest in cultures. AhR may exhibit such effects in the presence of hormone in vivo, but this remains to be investigated. Another possibility is that liganded AhR can interfere with liganded ER binding at the gene promoter (Krishnan et al., 1995Go). However, a DRE has not been described in either mouse or human cyclin A2 genes.

The changes we observed in cyclin and MIF levels, which correlated with changes in TGF-ß following E2 and TCDD treatments in the present study, are consistent with TGF-ß protein signaling (Slingerland et al., 1994Go). Moreover, the TCDD effect on TGF-ß and cyclin mRNAs are actual fundamental changes that likely upset transcript level balance and cellular signaling, and as such, could alter cellular functioning as demonstrated by TCDD inhibition of E2-induced uterine epithelial proliferation and secretory protein activity (Buchanan et al., 2000Go).

The findings herein are the first to show that TCDD may inhibit uterine epithelial mitogenesis by altering E2-regulated cellular signaling necessary for cyclin production and S phase initiation. We have demonstrated that a TCDD dose capable of inhibiting uterine epithelial proliferation 18–24 h after E2 also increases TGF-ß mRNAs and abolishes cyclin A2 mRNA at 6 h after E2. Thus, TCDD alters E2-regulated transcription for cell signaling factors associated with normal cyclin gene expression. Further, TCDD-induced decreases in mRNAs of cyclins critical for G1 phase progression and S phase entry suggest increased TGF-ß protein activity and provide a possible explanation for TCDD inhibition of E2-induced uterine epithelial proliferation in vivo. Additional research on the antiestrogenic TCDD effect in vivo should continue to provide valuable clues for understanding steroid signaling pathways.


    ACKNOWLEDGMENTS
 
This work was supported by CREST, Japan Science and Technology Corporation. The authors are grateful to Yoshinao Katsu, Okazaki National Research Institutes, for expert technical advice, and Byung-Woun Seo, currently at SankyoPharma, for generous assistance during preliminary TCDD treatments.


    NOTES
 
Presented in part at the 40th Annual Meeting of the Society of Toxicology, San Francisco, CA, March 2001.

1 To whom correspondence should be directed at National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709. Fax: (919) 316-4626. E-mail: buchana1{at}niehs.nih.gov. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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