* Department of Biological Sciences, George Washington University, Washington, D.C. 20052;
Center for Devices and Radiological Health, Food and Drug Administration, Rockville, Maryland 20857; and
Forensic Sciences Department, George Washington University, Washington, D.C. 20052
Received March 16, 2001; accepted June 26, 2001
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ABSTRACT |
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Key Words: bisphenol A (BPA); ICI 182,780 (ICI); ß-estradiol (E2); estrogen receptor (ER); uterus; heat shock proteins; hsp90 localization; B6C3F1 mouse.
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INTRODUCTION |
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Effects of exogenous estrogens in the rodent uterus include hypertrophy (Branham et al., 1993), water imbibition (Astwood, 1938
), and secretory protein production (Buchanan et al., 1999
). 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, 1999
; Katsuda et al., 1999
), progesterone receptor (PR; Ing and Ott, 1999
), the protooncogenes c-fos and c-jun (Yamashita et al., 1996
), insulin-like growth factor-I (IGF-I; Ghahary and Murphy, 1989
), and epidermal growth factor (DiAugustine et al., 1988
). 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, 1987
) and in the signal transduction of various steroid receptors, including the ER, and also of growth factor receptors (Pratt, 1998
) and other tyrosine kinases (Buchner, 1999
). Members of the hsp90 and hsp70 families have been shown to associate with the ER (Chambraud et al., 1990
; Landel et al., 1995
; Sabbah et al., 1996
). Estrogen has been shown to regulate uterine mRNA levels of hsp90 (Shyamala et al., 1989
; Tang et al., 1995
; Wu et al., 1996
), grp94 (Shyamala et al., 1989
), hsp70 (Rivera-Gonzalez et al., 1998
; Tang et al., 1995
; Wu et al., 1996
), and the heat shock transcription factor, HSF-1 (Yang et al., 1995
), which is involved in the transcriptional regulation of hsp70 and hsp90 gene expression (Morimoto et al., 1992
). Estrogen has also been shown to alter levels of the hsp90 isoforms, hsp90
and hsp90ß, in the murine uterus (Shyamala et al., 1989
).
BPA competes with E2 for binding to the ER in human breast cancer MCF-7 cells (Krishnan et al., 1993; Nagel et al., 1997
) or to the purified ER
or ERß (Kuiper et al., 1998
). In in vivo studies in rodents, BPA, analogous to other estrogens, increases uterine weight (Ashby and Tinwell, 1998
; Cook et al., 1997
; Laws et al., 2000
; Papaconstantinou et al., 2000
), uterine vascular permeability (Milligan et al., 1998
), and BrdU labeling in cells of all the uterine layers (Tinwell et al., 2000
), and results in hypertrophy of the luminal epithelium (Papaconstantinou et al., 2000
; Steinmetz et al., 1998
; Tinwell et al., 2000
), stroma, and myometrium (Papaconstantinou et al., 2000
).
The precise mechanism of BPA action in the rodent uterus is unknown. We have previously shown (Papaconstantinou et al., 2000) 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
, 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
, a protein that was shown to be up-regulated by the 2 estrogens to similar levels.
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MATERIALS AND METHODS |
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After an acclimation period of 12 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., 2000) 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, 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
, 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.
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, 1999). 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
(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.
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RESULTS |
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The E2-induced effects on hsp levels were attenuated by ICI as shown in the representative blots in Figure 2A. 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. 2B
). The ICI antagonism of the E2-induced levels of hsp90
and grp94 was also significant (p < 0.05), but much less pronounced than for hsp72.
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DISCUSSION |
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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, hsp72, hsc73, and grp94. BPA was as efficacious but less potent than E2 in increasing levels of hsp90
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
or grp94, appear to correlate with the respective curves for increases in uterine weight seen in our previous study (Papaconstantinou et al., 2000
). 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, 1987
) 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., 1995
; Nanbu et al., 1996
).
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., 2000). 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
and grp94 is at the level of transcription (Shyamala et al., 1989
). In this case, ICI may be acting by increasing the degradation of ER
, as shown in in vitro studies (Van Den Bemd et al., 1999
), and/or by preventing ER nuclear localization (Dauvois et al., 1993
). 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., 1997
) but not in MDA-MB-231 ER negative breast cancer cells (Kiang et al., 2000
). Furthermore, HSF-1, the transcriptional factor involved in the regulation of hsp70 and hsp90 genes (Morimoto et al., 1992
) has been shown to be under the control of estrogen in the uterus (Yang et al., 1995
).
Since hsp90 is involved in the signal transduction of several factors including the steroid receptors, kinases, growth factors, and HSF (Buchner, 1999
), some of which are localized mainly in the nucleus and others in the cytoplasm, it was of interest to examine the localization of hsp90
upon treatment with E2 or BPA, alone or in combination with ICI. The localization of hsp90
under different treatment conditions should assist in determining whether these 2 estrogens elicit similar functions for this hsp in the uterus. Localization of hsp90
in uteri of control mice in the present study correlates with the localization of the ER
in rat uteri (Saunders et al., 1997
). The decrease in nuclear epithelial hsp90
immunostaining with E2 or BPA treatment may be attributed to a dissociation of hsp90
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., 1998
). In addition, uterine HSF-1 is a cytoplasmic protein mainly localized in the epithelium upon E2 treatment (Yang et al., 1995
). The cytoplasmic presence of this factor that associates with hsp90 potentially increases the requirement for hsp90
in the cytoplasm. Increase in total hsp90
immunostaining in the stroma and myometrium after E2 or BPA treatment may be explained by increased levels of proteins chaperoned by hsp90
in these uterine layers. Such proteins whose levels are known to increase in response to estrogen treatment include stromal ER
(Cooke et al., 1997
), and stromal and myometrium PR (Kurita et al., 2000
). The antiestrogen ICI 182,780 antagonized the E2- and BPA-induced effects on hsp90
localization. Decreased hsp90
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
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., 1991
) potentially decreasing the total population of proteins chaperoned by hsp90
.
In conclusion, BPA, although less potent than E2, induced comparable increases in hsp90 and grp94 levels in the mouse uterus, and this induction was mediated through the ER. The intracellular localization of hsp90
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
, this suggests that hsp90
localization is regulated by the ER or other ER-regulated uterine gene products.
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NOTES |
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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.
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