Effects of ß-Estradiol and Bisphenol A on Heat Shock Protein Levels and Localization in the Mouse Uterus Are Antagonized by the Antiestrogen ICI 182,780

Andriana D. Papaconstantinou*, Benjamin R. Fisher{dagger},1, Thomas H. Umbreit{dagger}, Peter L. Goering{dagger}, Nicholas T. Lappas{ddagger} and Ken M. Brown*,2

* Department of Biological Sciences, George Washington University, Washington, D.C. 20052; {dagger} Center for Devices and Radiological Health, Food and Drug Administration, Rockville, Maryland 20857; and {ddagger} Forensic Sciences Department, George Washington University, Washington, D.C. 20052

Received March 16, 2001; accepted June 26, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bisphenol A (BPA) exhibits many estrogen-like effects in the rodent uterus, but not all of these can be attenuated by antiestrogens. This suggests the involvement of alternate pathways of BPA action that do not involve the estrogen receptor (ER). An examination of the in vivo effects of BPA on uterine gene expression and protein levels should contribute to an understanding of its mechanism of action. In this study we examined the dose-related effects of BPA on levels of a suite of heat shock proteins (hsps) and on the localization of hsp90{alpha}, a chaperone of the ER, in uteri of ovariectomized B6C3F1 mice and compared these effects with those of ß-estradiol (E2). The antiestrogen ICI 182,780 (ICI) was co-administered with BPA or E2 in order to examine the potential role of the ER. BPA, although less potent than E2, increased hsp90{alpha} and grp94 to similar levels, but was much less effective than E2 in increasing levels of hsp72. Treatment with 100 mg BPA/kg/day or 2 µg E2/kg/day increased hsp90{alpha} to 300% of control levels and altered its tissue expression pattern. In uteri of corn oil (control)-treated mice, hsp90{alpha} predominantly localized in the cytoplasm and nuclei of epithelial cells. Upon treatment with BPA or E2 there was increased intensity of staining in the stroma and myometrium, and in the epithelium hsp90{alpha} was localized almost exclusively in the cytoplasm. The effects of BPA or E2 on hsp levels and hsp90{alpha} localization were attenuated by ICI. These results suggest an involvement of the ER in BPA- and E2-induced increases in uterine levels of hsp90{alpha}, grp94, and hsp72, and localization of hsp90{alpha}.

Key Words: bisphenol A (BPA); ICI 182,780 (ICI); ß-estradiol (E2); estrogen receptor (ER); uterus; heat shock proteins; hsp90{alpha} localization; B6C3F1 mouse.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Endocrine disruptors are chemicals found in the environment that are able to act as estrogens, androgens, thyroid, or other hormones and therefore upset normal endocrine function. Bisphenol A (BPA), a chemical used in the manufacture of several types of plastics including polycarbonates, epoxy resins, and polyvinyl chloride (Staples et al., 1998Go), exhibits estrogenic properties. Concern about potential exposure of humans to BPA is based on reports indicating leaching of BPA from plastics (Krishnan et al., 1993Go; Mountfort et al., 1997Go; Yamamoto and Yasuhara, 1999Go), food cans lined with epoxy resins (Brotons et al., 1995Go), and certain dental sealants (Olea et al., 1996Go). Low levels of BPA have also been detected in serum samples of adult Japanese men and women, and higher levels of BPA have been detected in animal plasma or serum samples purchased from Japanese pharmaceutical companies (Sajiki et al., 1999Go). Low concentrations of BPA were found in water samples collected near manufacturing plants in the U.S. (Staples et al., 2000Go) and Canada (Lee and Peart, 2000Go), whereas higher concentrations were found in municipal wastewater samples collected in Germany (Körner et al., 2000Go).

Effects of exogenous estrogens in the rodent uterus include hypertrophy (Branham et al., 1993Go), water imbibition (Astwood, 1938Go), and secretory protein production (Buchanan et al., 1999Go). Uterine genes regulated by ß-estradiol (E2) and thought to have a role in the uterotrophic response include the estrogen receptor (ER; Ing and Ott, 1999Go; Katsuda et al., 1999Go), progesterone receptor (PR; Ing and Ott, 1999Go), the protooncogenes c-fos and c-jun (Yamashita et al., 1996Go), insulin-like growth factor-I (IGF-I; Ghahary and Murphy, 1989Go), and epidermal growth factor (DiAugustine et al., 1988Go). Several members of the heat shock protein (hsp) 90 and 70 families of genes, including the hsp homologous glucose regulated proteins (grps), are estrogen-regulated and may be indirectly involved in the uterotrophic response. The products of these hsp genes are chaperone proteins involved in the correct folding and processing of proteins (Welch, 1987Go) and in the signal transduction of various steroid receptors, including the ER, and also of growth factor receptors (Pratt, 1998Go) and other tyrosine kinases (Buchner, 1999Go). Members of the hsp90 and hsp70 families have been shown to associate with the ER (Chambraud et al., 1990Go; Landel et al., 1995Go; Sabbah et al., 1996Go). Estrogen has been shown to regulate uterine mRNA levels of hsp90 (Shyamala et al., 1989Go; Tang et al., 1995Go; Wu et al., 1996Go), grp94 (Shyamala et al., 1989Go), hsp70 (Rivera-Gonzalez et al., 1998Go; Tang et al., 1995Go; Wu et al., 1996Go), and the heat shock transcription factor, HSF-1 (Yang et al., 1995Go), which is involved in the transcriptional regulation of hsp70 and hsp90 gene expression (Morimoto et al., 1992Go). Estrogen has also been shown to alter levels of the hsp90 isoforms, hsp90{alpha} and hsp90ß, in the murine uterus (Shyamala et al., 1989Go).

BPA competes with E2 for binding to the ER in human breast cancer MCF-7 cells (Krishnan et al., 1993Go; Nagel et al., 1997Go) or to the purified ER{alpha} or ERß (Kuiper et al., 1998Go). In in vivo studies in rodents, BPA, analogous to other estrogens, increases uterine weight (Ashby and Tinwell, 1998Go; Cook et al., 1997Go; Laws et al., 2000Go; Papaconstantinou et al., 2000Go), uterine vascular permeability (Milligan et al., 1998Go), and BrdU labeling in cells of all the uterine layers (Tinwell et al., 2000Go), and results in hypertrophy of the luminal epithelium (Papaconstantinou et al., 2000Go; Steinmetz et al., 1998Go; Tinwell et al., 2000Go), stroma, and myometrium (Papaconstantinou et al., 2000Go).

The precise mechanism of BPA action in the rodent uterus is unknown. We have previously shown (Papaconstantinou et al., 2000Go) that the BPA-induced growth of the uterus and increase in luminal epithelium height can be reversed by the antiestrogen ICI 182,780 (ICI), suggesting a role for the ER. However, the maximal effect of BPA was much lower than that of E2, and a higher dose of ICI was needed to reverse the effect of BPA on the uterine epithelium when compared to E2. These observations suggest potential alternate mechanisms for BPA-stimulated uterotrophism. A comparative assessment of the in vivo effects of BPA and E2 on uterine gene expression and protein levels will aid in the eventual understanding of the mechanism of BPA action. In the present study, we compared the effects of BPA on protein levels of uterine hsp90{alpha}, the hsp90 endoplasmic reticulum homologue, grp94, and the inducible and constitutive members of the hsp70 family, hsp72 and hsc73, respectively, to those of E2, and also examined the ability of ICI to antagonize these effects. Since E2 often regulates the expression of uterine proteins in a cell-type specific manner, it was of interest to compare the E2- and BPA-mediated localization of hsp90{alpha}, a protein that was shown to be up-regulated by the 2 estrogens to similar levels.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals and treatment protocols.
All procedures requiring the use of animals were conducted according to the National Institutes of Health's Using Animals in Intramural Research: Guidelines (1998) as previously described (Papaconstantinou et al., 2000Go). Ovariectomized female B6C3F1 mice supplied from Charles River Laboratories Inc. (Wilmington, MA) were housed in groups of 4 to 6 in polypropylene cages with stainless steel wire lids and heat treated chips (Cellu-Dri; Shepherd Specialty Papers, Kalamazoo, MI), with access to food (Lab Rodent Diet 5001; PMI Nutrition International Inc., St. Louis, MO) and water ad libitum. The animal rooms were maintained on a 12-h light/dark cycle (0600 to 1800 h) at 23 ± 1°C with 30–50% relative humidity.

After an acclimation period of 1–2 weeks, animals were dosed sc with solutions of bisphenol A (Sigma Chemical Co., St. Louis, MO), ß-estradiol (Sigma), ICI 182,780 (Tocris, Ballwin, MO), BPA + ICI, E2 + ICI, or with corn oil (vehicle control; Sigma) once a day between 0830 and 1100 h for 4 consecutive days. In a first set of experiments to assess dose-related effects of E2 and BPA on hsp induction, animals were treated with 0.02, 0.2, 2, or 20 µg E2/kg/day or 1, 10, 40, 100, or 400 mg BPA/kg/day. In separate experiments to study the antagonism of the E2- and BPA-induced effects by ICI, doses of the estrogens were selected based on the observations of the dose response experiments, and doses of ICI were selected based on our previous observation (Papaconstantinou et al., 2000Go) of doses needed to reverse the effects of E2 and BPA on uterine weights. Animals were euthanized 24 h after the last treatment by carbon dioxide inhalation followed by cervical dislocation.

SDS-polyacrylamide gel electrophoresis.
After euthanasia, uteri were removed, blotted, and weighed to the nearest 0.1 mg. A 5-mm posteriormost segment of 1 uterine horn was fixed overnight in 10% neutral buffered formalin for immunohistochemical analysis. Each remaining uterus was pooled with the other 3 to 5 uteri of the same treatment group. There were 2 groups per treatment. The pooled uteri were homogenized with 0.5 µl of 10 mM Tris buffer, pH 7.4, per mg of tissue. The homogenates were centrifuged at 15,000 x g for 10 min, and the total protein concentrations of the supernatants were determined by the method of Lowry et al. (1951) with bovine serum albumen (Sigma) as the standard.

Uterine homogenates were diluted to 3.5 mg protein/ml, and 3 µl of sample (i.e., 10.5 µg/lane) were separated by 12.5% homogeneous SDS-polyacrylamide gel electrophoresis using the Pharmacia Phast System (Pharmacia Biotechnologies, Piscataway, NJ) as described by Goering et al. (1992). Equal loading was verified by coomassie staining (Pharmacia) of proteins in representative gels.

Immunologic detection and densitometric and statistical analysis of heat shock proteins.
Electrophoretic transfer of proteins onto nitrocellulose membranes and immunochemical detections of hsp72, hsp90{alpha}, hsc73, and grp94 were performed as described by Goering et al. (1992). Briefly, blots were blocked in Tris-buffered saline (TBS; 20 mM Tris, 500 mM NaCl, pH 7.5) with 4% milk for a minimum of 4 h. Solutions of stress protein antibodies (Stress-Gen Biotechnologies, Inc., Vancouver, BC, Canada) at concentrations of 1:500 for hsp72, hsp90{alpha}, and grp94 or 1:1200 for hsc73 were prepared in TTBS (TBS containing 0.05% Tween 20) with 4% milk. Blots were incubated with hsp antibodies overnight followed by a 3-h incubation with alkaline phosphatase conjugated goat-anti-mouse (Bio-Rad Laboratories, Hercules, CA), goat-anti-rabbit (Sigma), or rabbit-anti-rat (Sigma) secondary antibodies. Blots were scanned with an ARCUS II AGFA scanner, saved as TIFF images, and imported into the Kodak Digital ScienceTM 1D Image Analysis Software package (Version 3.0; Eastman Kodak Company, Rochester, NY) for densitometric analysis. The average percent control band intensity of each protein from each of 2 separate groups/treatment was calculated and standard deviations determined. One-way analysis of variance (ANOVA) was used to assess the variation of the means among the treatments. If the variation was greater than expected by chance alone, the Dunnett's test was performed for a comparison of the means with that of control. Significance was established when the p value was less than 5%.

Immunohistochemical analysis of hsp90{alpha}.
Uterine tissues fixed in 10% neutral buffered formalin overnight were dehydrated in a graded ethanol series, cleared in xylene, and embedded in paraffin. Sections (5 µm) were mounted on poly-lysine coated slides, deparaffinized in xylene, rehydrated, and refixed in 10% neutral buffered formalin for 10 min. Sections were immersed in antigen unmasking solution (Vector Laboratories, Burlingame, CA) in a pyrex container and boiled in a microwave set on high for 3 min followed by 15 min cooling. This process was repeated 4 times (Tang, 1999Go). Sections were then incubated in 0.3% H2O2 for 30 min to quench endogenous peroxidase, and blocked in normal goat serum (Vector) for 30 min. The excess of serum was blotted and the sections were incubated in a 1:20,000 solution of rabbit anti-hsp90{alpha} (ABR, Golden, CO) or rabbit IgG (1:20,000; Vector) in phosphate-buffered saline (PBS; 10 mM sodium phosphate, 0.9% NaCl, pH 7.5) for 2 h at room temperature. Slides were washed in buffer twice for 20 min each followed by incubation in secondary antibody solution (biotinylated goat anti-rabbit IgG; Vector) for 30 min at room temperature. After washing twice for 20 min each in PBS buffer, slides were incubated with avidin-biotin-peroxidase complex in PBS (Vector) for 20 min at room temperature, washed twice for 20 min each in PBS buffer and developed with 3,3'-diaminobenzidine substrate (Vector), counterstained with hematoxylin (Gill's formula, Vector) for 3 min, dehydrated, and mounted with Permount. Slides were observed with an Olympus BH microscope and photographs were taken with an Olympus C-35A camera containing Kodak Ektachrome 160T 35 mm film.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Uterine Heat Shock Protein Levels
Both E2 and BPA treatments resulted in dose-dependent increases in levels of uterine hsp72, hsp90{alpha}, and grp94 (Fig. 1Go). Of the stress proteins examined, hsp72 synthesis appeared to be more sensitive to E2 induction followed by hsp90{alpha} and grp94. The lowest dose of E2 administered, 0.02 µg/kg/day, induced an approximate 3-fold increase in hsp72 levels compared to control (p < 0.05). The induction of hsp72 levels appeared to plateau at 2 µg E2/kg/day with 2 or 20 µg E2/kg/day each increasing hsp72 levels to approximately 600% of control values. Significant increases in hsp90{alpha} and grp94 levels were first observed at 0.02 and 0.2 µg E2/kg/day, respectively. Induction of hsp90{alpha} and grp94 levels peaked at 2 µg E2/kg/day. Levels of the constitutive form of hsp70, hsc73, were basically unaltered by E2.



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FIG. 1. Effects of ß-estradiol (E2) or bisphenol A (BPA) on the induction of the heat shock proteins hsp72, hsp90{alpha}, grp94, and hsc73 in uteri of B6C3F1 mice. Animals were treated sc with corn oil (control), 0.02, 0.2, 2, or 20 µg E2/kg/day or with 1, 10, 40, 100, or 400 mg BPA/kg/day for 4 days. Uteri were removed on the fifth day, homogenized and centrifuged at 15,000 x g. Heat shock proteins present in the supernatant were electrophoretically separated on SDS-PAGE gels and detected by Western analysis. Graphs represent the average of 2 values obtained by densitometric analysis of blots of 2 different samples per treatment and are expressed as % of control levels. Standard deviations did not exceed 28% of the value for any protein at any treatment. a,b,c,dSignificant differences from control for hsp72, hsp90{alpha}, grp94, and hsc73, respectively, as determined by one-way ANOVA and the Dunnett's post-hoc test (p < 0.05).

 
In contrast to E2, BPA was less effective in increasing levels of hsp72 than hsp90{alpha} and grp94. The 2 proteins of the hsp90 family followed a similar dose-response curve with maximum levels induced by 100 mg BPA/kg/day. The lowest dose of BPA examined was able to significantly increase levels of grp94 (p < 0.05 vs. control), but not of hsp90{alpha} or hsp72. Induction of hsp72 did not plateau by the highest dose of BPA administered. The maximal effect of BPA on grp94 levels (264% control) was slightly higher than the maximal effect of E2 on this protein (182% control). In contrast, the maximal BPA-induced increase in hsp72 (296%) was lower than that of E2 (620%).

The E2-induced effects on hsp levels were attenuated by ICI as shown in the representative blots in Figure 2AGo. Treatment of mice with 1 mg ICI/kg/day alone did not affect the levels of any of the hsps examined in the uterus. When 1 mg ICI/kg/day was co-administered with 2 µg E2/kg/day, levels of hsp72 dropped from 694% to 292% of control (Fig. 2BGo). The ICI antagonism of the E2-induced levels of hsp90{alpha} and grp94 was also significant (p < 0.05), but much less pronounced than for hsp72.



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FIG. 2. (A) Western analysis of hsp90{alpha}, hsp72, hsc73, and grp94 protein levels in 15,000 x g supernatants from uterine tissue homogenates of B6C3F1 mice. Animals were treated with corn oil (CO), 2 µg ß-estradiol (E2)/kg/day, or 1 mg ICI 182,780 (ICI)/kg/day alone or in combination with E2. (B) Densitometric analysis of Western blots of heat shock proteins. Averages of 2 Western blots for each protein for each treatment were calculated, and results are expressed as % of control. *,aStatistically significant difference from control or E2, respectively, as determined by one-way ANOVA and the Dunnett's post-hoc test (p < 0.05).

 
ICI also antagonized the BPA-induced increases in hsps (Fig. 3AGo). A BPA dose of 100 mg/kg/day significantly increased the levels of uterine hsp90{alpha} and grp94 (p < 0.05), and caused an increase in hsp72 that was not statistically significant (Fig. 3BGo). Administration of 10 mg ICI/kg/day with 100 mg BPA/kg/day completely blocked the BPA-induced effect on levels of hsp90{alpha}, and attenuated its effect on grp94. ICI alone at this concentration had no significant effect on the levels of any of the stress proteins examined.



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FIG. 3. (A) Western analysis of hsp90{alpha}, hsp72, hsc73, and grp94 protein levels in 15,000 x g supernatants from uterine tissue homogenates of B6C3F1 mice. Animals were treated with corn oil (CO), 100 mg bisphenol A (BPA)/kg/day, or 10 mg ICI 182,780 (ICI)/kg/day alone or in combination with BPA. (B) Densitometric analysis of Western blots of heat shock proteins. Averages of 2 Western blots for each protein and for each treatment group were calculated, and results are expressed as % of control. *,aStatistically significant difference from control or BPA, respectively, as determined by one-way ANOVA and the Dunnett's post-hoc test (p < 0.05).

 
Immunolocalization of Hsp90{alpha}
In uteri of corn oil-treated mice hsp90{alpha} was predominantly localized in luminal and glandular epithelial cells. Immunostaining of hsp90{alpha} was seen in the nuclei and cytoplasm of epithelial cells but also in occasional nuclei of the stroma and myometrium (Fig. 4AGo). This immunostaining is due to specific antibody binding of hsp90{alpha} since incubation of uterine tissue with rabbit IgG did not result in staining in any of the uterine tissue layers (Fig. 4BGo). In the uterine epithelium of mice treated with 2 µg E2/kg/day (Fig. 4CGo) hsp90{alpha} localized almost exclusively in the cytoplasm. Treatment with E2 resulted in intense cytoplasmic immunostaining of stromal and myometrial cells and approximately half the nuclei of these cells were positively stained. The antiestrogen ICI antagonized the effects of E2 on hsp90{alpha} immunolocalization. Uteri of mice co-treated with 2 µg E2/kg/day and 1 mg ICI/kg/day (Fig. 4DGo) had an appearance between that of E2 and corn oil-treated uteri. Specifically, in cells of the epithelium, most nuclei were positively stained, similarly to control. In the stroma and myometrium, staining of the cytoplasm was less intense and the number of stained nuclei was lower than in uteri of E2-treated mice.



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FIG. 4. Immunohistochemical localization of hsp90{alpha} in uteri of B6C3F1 mice. Animals were treated with corn oil (A and B); 2 µg ß-estradiol (E2)/kg/day (C); 2 µg E2/kg/day + 1 mg ICI 182,780 (ICI)/ kg/day (D); 100 mg bisphenol A (BPA)/kg/day (E); 100 mg BPA/kg/day + 10 mg ICI/kg/day (F); 1 mg ICI/kg/day (G); or 10 mg ICI/kg/day (H). Shown are representative sections incubated with 1:20,000 rabbit anti-hsp90{alpha} (A, C–H) or rabbit IgG (B). Cells immunostained for hsp90{alpha} are brown. Epi, stro, and myo in photomicrograph A designate uterine epithelium, stroma and myometrium, respectively. Bar = 40µ.

 
Treatment with 100 mg BPA/kg/day (Fig. 4EGo) showed a similar pattern with that of E2 even though there were fewer stained nuclei and a lower intensity of cytoplasmic staining in the stroma or myometrium when compared to E2-treated animals. In uteri of mice co-treated with 100 mg BPA/kg/day and 10 mg ICI/kg/day (Fig. 4FGo), the pattern of hsp90{alpha} immunolocalization closely resembled that of control uteri (Fig. 4AGo). In epithelial cells, most nuclei were positive and in the stroma and myometrium there was no detectable cytoplasmic staining but there were occasional stained nuclei whose number appeared to be greater than that in control. Treatment of mice with either dose of the antiestrogen alone did not result in any significant change in the hsp90{alpha} localization or staining intensity (Figs. 4G and 4HGo).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Many of the effects of BPA on the rodent uterus have been shown to mimic those of E2, but its mechanism of action remains mostly unknown. A requirement for ER{alpha} in the BPA-induced increases of uterine IGF-I mRNA, proliferating cell nuclear antigen (PCNA) expression, and phosphorylation of IGF-I receptor has been shown (Klotz et al., 2000Go). Previous studies of ours have suggested an involvement of the ER in the BPA-induced increases in uterine weight and luminal epithelial height since the antiestrogen, ICI, reversed these BPA-induced effects (Papaconstantinou et al., 2000Go). In other studies, BPA was unable to antagonize E2-induced increases in uterine weight suggesting that BPA acts through a mechanism different from E2, but was able to antagonize E2-induced effects on PR levels and peroxidase activity (Gould et al., 1998Go). Furthermore, BPA has a differential effect from that of ethinyl estradiol on the expression of uterine PR, clusterine and glyceraldehyde phosphate dehydrogenase mRNAs (Diel et al., 2000Go). In addition, whereas ER{alpha} and ERß equally recruit the coactivators SRC-1 and TIF-2 in the presence of E2, these coactivators are preferentially bound to ERß in the presence of BPA (Routledge et al., 2000Go). These observations demonstrate the need to study the in vivo effects of BPA on all factors potentially involved in estrogenic action in the uterus before we can understand its mechanism of action.

In the present study, we chose to examine the in vivo effects of a weak estrogen, BPA, as compared to E2 on uterine levels of hsp90{alpha}, hsp72, hsc73, and grp94. BPA was as efficacious but less potent than E2 in increasing levels of hsp90{alpha} and grp94. BPA was much less effective than E2 in increasing levels of the inducible hsp70 protein, hsp72. The E2 and BPA dose-response curves for uterine hsp72, but not for hsp90{alpha} or grp94, appear to correlate with the respective curves for increases in uterine weight seen in our previous study (Papaconstantinou et al., 2000Go). Both hsp72 levels and uterine weights increased with E2 dose and plateaued at 2 µg E2/kg/day. With respect to BPA, 40 mg/kg/day was the lowest dose able to significantly increase both hsp72 levels and uterine weights. These results are in agreement with a role of hsp72 in the correct folding and functioning of proteins (Welch, 1987Go) necessary for uterine growth and synthesized in response to E2 or BPA treatment. In addition, hsp72 shows an inverse correlation with the ER in both normal human endometrium and in endometrial carcinomas, and is thus thought to have a role in down-regulation of the ER (Koshiyama et al., 1995Go; Nanbu et al., 1996Go).

In order to investigate the role of the ER in the E2- and BPA-induced increases in hsp levels, 1 or 10 mg/kg/day of the antiestrogen ICI was co-administered with 2 µg E2/kg/day or 100 mg BPA/kg/day, respectively. The selection of these doses of ICI was based on our previous observations of the doses needed to attenuate the E2 and BPA effects on uterine weights (Papaconstantinou et al., 2000Go). ICI attenuated all the E2- and BPA-induced effects on uterine hsp levels, suggesting an involvement of the ER in the regulation of hsp proteins. Previous reports have indicated that estrogenic regulation of uterine hsp90{alpha} and grp94 is at the level of transcription (Shyamala et al., 1989Go). In this case, ICI may be acting by increasing the degradation of ER{alpha}, as shown in in vitro studies (Van Den Bemd et al., 1999Go), and/or by preventing ER nuclear localization (Dauvois et al., 1993Go). Involvement of the ER in grp94 regulation can be indirectly inferred by studies showing that upon heat shock, levels of grp94 increase in T47D ER positive (Kiang et al., 1997Go) but not in MDA-MB-231 ER negative breast cancer cells (Kiang et al., 2000Go). Furthermore, HSF-1, the transcriptional factor involved in the regulation of hsp70 and hsp90 genes (Morimoto et al., 1992Go) has been shown to be under the control of estrogen in the uterus (Yang et al., 1995Go).

Since hsp90{alpha} is involved in the signal transduction of several factors including the steroid receptors, kinases, growth factors, and HSF (Buchner, 1999Go), some of which are localized mainly in the nucleus and others in the cytoplasm, it was of interest to examine the localization of hsp90{alpha} upon treatment with E2 or BPA, alone or in combination with ICI. The localization of hsp90{alpha} under different treatment conditions should assist in determining whether these 2 estrogens elicit similar functions for this hsp in the uterus. Localization of hsp90{alpha} in uteri of control mice in the present study correlates with the localization of the ER{alpha} in rat uteri (Saunders et al., 1997Go). The decrease in nuclear epithelial hsp90{alpha} immunostaining with E2 or BPA treatment may be attributed to a dissociation of hsp90{alpha} from the nuclear localized ER and the translocation of the protein to the cytoplasm and is supported by similar observations in E2-treated COS-7 cells transfected with hsp90 (Devin-Leclerc et al., 1998Go). In addition, uterine HSF-1 is a cytoplasmic protein mainly localized in the epithelium upon E2 treatment (Yang et al., 1995Go). The cytoplasmic presence of this factor that associates with hsp90 potentially increases the requirement for hsp90{alpha} in the cytoplasm. Increase in total hsp90{alpha} immunostaining in the stroma and myometrium after E2 or BPA treatment may be explained by increased levels of proteins chaperoned by hsp90{alpha} in these uterine layers. Such proteins whose levels are known to increase in response to estrogen treatment include stromal ER{alpha} (Cooke et al., 1997Go), and stromal and myometrium PR (Kurita et al., 2000Go). The antiestrogen ICI 182,780 antagonized the E2- and BPA-induced effects on hsp90{alpha} localization. Decreased hsp90{alpha} immunostaining in the stroma and myometrium of E2- and BPA-treated mice by ICI can be accounted for by the decrease in the levels of hsp90{alpha} shown by Western analysis. Furthermore, ICI 164,384, a congener of ICI, has been shown to downregulate the ER and to antagonize E2-induced increases in uterine PR levels (Gibson et al., 1991Go) potentially decreasing the total population of proteins chaperoned by hsp90{alpha}.

In conclusion, BPA, although less potent than E2, induced comparable increases in hsp90{alpha} and grp94 levels in the mouse uterus, and this induction was mediated through the ER. The intracellular localization of hsp90{alpha} in the different uterine tissue layers upon treatment with BPA resembles that of E2, suggesting that this hsp associates with similar proteins and performs similar functions under these 2 conditions. Since ICI antagonized the E2 and BPA effects on the localization of hsp90{alpha}, this suggests that hsp90{alpha} localization is regulated by the ER or other ER-regulated uterine gene products.


    NOTES
 
1 Present address: Covance Laboratories, 9200 Leesburg Pike, Vienna, VA 22182. Back

2 To whom all correspondence should be addressed at 332 Lisner Hall, Department of Biological Sciences, George Washington University, 2023 G. St. N.W., Washington, D.C. 20052. Fax: (202) 994-6100. E-mail: kmb{at}gwu.edu. Back


    REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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