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Regular Article |
a Departments of Medical Nutrition and Bioscience, Karolinska Institute, Novum, Huddinge, Stockholm S-141 86, Sweden
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(ER) is involved in proliferation and progesterone receptor in differentiation, the role of the two other gonadal-hormone receptors expressed in the uterus, androgen receptor (AR) and estrogen receptor ß (ERß), remains undefined. In this study, the involvement of AR in 17ß-estradiol (E2)-induced cellular proliferation in the immature rat uterus was investigated. AR levels were low in the untreated immature uterus, but 24 h after treatment of rats with E2, there was an increase in the levels of AR and of two androgen-regulated genes, IGF-I and Crisp (cysteine-rich secretory protein). As expected, E2 induced proliferation of luminal epithelial cells. These actions of E2 were all blocked by both the antiestrogen tamoxifen and the antiandrogen flutamide. The E2-induced AR was found by immunohistochemistry to be localized exclusively in the stroma, mainly in the myometrium, where it colocalized with ER but not with ERß. ERß, detected with two different ERß-specific antibodies, was expressed in both stromal and epithelial cells either alone or together with ER. Treatment with E2 caused down-regulation of ER and ERß in the epithelium. The data suggest that, in E2-induced epithelial cell proliferation, ER induces stromal AR and AR amplifies the ER signal by induction of IGF-I. Because AR is never expressed in cells with ERß, it is unlikely that ERß signaling is involved in this pathway. These results indicate an important role for AR in proliferation of the uterus, where estrogen and androgen do not represent separate pathways but are sequential steps in one pathway.
androgen receptor, estradiol receptor, female reproductive tract
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(ER) mutation [1, 2] and of male mice in which ER was inactivated [3] revealed an important role for E2 in the skeleton, the cardiovascular system, and the reproductive tract of males. On the other hand, the parturition defects of 5-reductase-deficient mice [4, 5] and impaired reproductive performance and accelerated ovarian aging phenotypes of testicular feminized female (Tfm/Tfm) mice [6] revealed that androgens are important for females.
The uterus is a classical 17ß-estradiol (E2) target tissue, which has been used extensively to study gonadal-hormone-regulated cell proliferation. It is composed of a variety of epithelial, stromal, and smooth muscle cells, the function and hormone responsiveness of which are influenced by their position within the organ. All of the gonadal steroid hormone receptors, ER/estrogen receptor ß (ERß), progesterone receptor (PR), and androgen receptor (AR), are expressed in this organ [7, 8]. E2 induces epithelial cell proliferation in the uterus, and this induction is known to be indirect, i.e., the presence of ER in the epithelial cells is not required. Evidence suggests that epithelial cell proliferation is mediated via stromal ER, which stimulates secretion of growth factors [9]. Although ER is the predominant ER in the adult rodent uterus, we have previously shown that ERß also has a role. In the uteri of ERß knockout (ERß-/-) mice, there is hyperresponsiveness to E2 [8]. Progesterone is antiproliferative in the uterus, an action mediated by PR, which itself is an E2-inducible gene. Recent studies have shown that the induction of PR by E2 is complex, with both ER and/or ERß pathways involved [8, 10].
ER and ERß can have opposite effects on transcription of certain genes. Whether an ER will act as a transcriptional activator or repressor is influenced both by the ligand and response element on DNA with which the ER interacts. E2 in the presence of ER can activate transcription from AP-1 (activator protein-1) sites, while tamoxifen can activate AP-1 sites via ERß [11]. E2 is known to induce AR in the uterus, but the roles of the two ERs in this induction remain to be defined. In the promoter region of AR, there are several AP-1 response elements [12]. Tamoxifen could be expected to decrease AR in ER-containing cells and increase it in ERß-containing cells. AR is abundant in the uterus [13, 14], and androgens have uterotrophic effects [15]. Expression of the growth factor IGF-1 as well as the multifunctional protein thioredoxin is regulated by both estrogen and androgen in the uterus [16–18]. When genes are regulated by both estrogen and androgen, it is usually assumed that the ER- and AR-dependent pathways are separate, not sequential [14, 19–22].
In this study, using immunohistochemical, Western blot, and reverse transcription-polymerase chain reaction (RT-PCR) techniques and combined treatments of E2 with or without the antiestrogen tamoxifen and with or without the antiandrogen flutamide, we have explored the relations between ER/ß and AR and their roles in controlling cell proliferation in the uterus.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Female Sprague-Dawley rats, 21 days old, were used. E2, tamoxifen, and flutamide were dissolved in Intralipid (Pharmacia & Upjohn AB, Stockholm, Sweden). There were six animals in each group, and all of them received their treatment either s.c. (for estradiol and tamoxifen) or via i.p. injections (for flutamide). The control group received vehicle. All other groups were given 10 µg/kg body weight E2. The E2 plus tamoxifen group was, in addition, given 60 µg/kg body weight tamoxifen and the E2 plus flutamide group 1 mg/kg body weight flutamide. Four hours after drug treatment, 100 mg/kg body weight of BrdU (5-bromo-2'-deoxyuridine dissolved in PBS buffer) was given to all the animals via s.c. injection. Animals were asphyxiated by CO2 at 6, 12, or 24 h after initial treatment, and the reproductive tracts were collected. The uteri were either frozen in liquid nitrogen for RNA and protein preparation or fixed in 4% PFA (paraformaldehyde) for immunohistochemical staining.
Chemicals and Antibodies
17ß-Estradiol, tamoxifen, and flutamide were from Sigma (St. Louis, MO). 5-Bromo-2'-deoxyuridine (BrdU) was from Roche (Mannheim, Germany). Polyclonal antibodies against AR (N-20, rabbit) for immunohistochemical staining and cyclin A (H-432, rabbit) were from Santa Cruz (Santa Cruz, CA). AR (PA1-111A, rabbit) antibody for Western blotting was from Affinity Bioreagents (Golden, CO). Monoclonal antibody against ER (6F-11) was from NovoCastra (Newcastle, U.K.). Chicken anti-human ERß antibody 503 was produced in this laboratory [8] and ERß protein 503 was provided by KaroBio (Stockholm, Sweden). Rabbit anti-rodent ERß (cat. no. 51-7900) was from Zymed (South San Francisco, CA). Peroxidase-conjugated anti-rabbit IgG was from Sigma. Biotin-conjugated goat anti-rabbit secondary antibody and VECTASTAIN ABC kits were from Vector Laboratories (Burlingame, CA). DAB (3,3'-diaminobenzidine) solution was from DAKO Corporation (Carpinteria, CA). Cy3-conjugated anti-chicken and anti-rabbit IgG and fluorescein isothiocyanate (FITC)-conjugated anti-rabbit and anti-mouse IgG raised in the donkey were obtained from Jackson ImmunoResearch Products (Cambridge, MA).
Detection of AR Expression by Western Blotting
With a Polytron PT3100 (Kinematica AG, Littau, Switzerland), frozen tissues were homogenized for a few seconds in a high-salt buffer (600 mM Tris-HCl, 1 mM EDTA, pH 7.4, with 1/10 w/v of homogenate). Two tablets of cocktail protease inhibitors (Boehringer Mannheim, GmbH, Germany) were added per 50 ml buffer before use. The homogenates were centrifuged at 105 000 x g for 1 h at 4°C. Supernatants (whole cell extracts) were aliquoted and kept at -80°C until use. Before Western blotting, protein contents were measured by the Bio-Rad protein assay with BSA as the standard. Equal amounts of protein were loaded onto each lane of a 9% polyacrylamide gel. Western blotting was done according to the protocol described previously [23]. Antibody dilutions were 1:1000 for the anti-AR antibody (PA1-111A) and 1:3000 for the peroxidase conjugated goat anti-rabbit IgG. Signals were detected with ECL (Amersham Life Science, Buckinghamshire, U.K.).
Reverse Transcription-PCR
Tissue from each treatment group was pooled, and total RNA was isolated with a RNAWIZ kit (Ambion, Austin, TX) according the protocol provided by the company. For the RT reaction, 5 µg of RNA was used from the pooled RNA of individuals of each group. The RT-PCR reactions were carried out with One-Step RT-PCR kits from Gibco BRL (cat. no. 10928-042) in a Gene Amp PCR system 2400 (Perkin-Elmer, Norwalk, CT). Primers, together with their annealing temperatures and the number of cycles, were IGF-1: forward, 5'-CACATCTCTTCTACCTGGCA-3', reverse, 5'-TGAGTCTTGGGCATGTCAGT (56°C, 35 cycles) [24]; Crisp: forward, 5'-TGTTCCTGGCTGCTGTATTG-3', reverse, 5'-AAGACCACGATGCAGGGTAA-3' (58°C, 35 cycles); ß-actin: forward, 5'-GGGCACAGTGTGGGTGAC-3', reverse, 5'-CTGGCACCACACCTTCTAC-3' (56°C, 15 cycles). PCR products were resolved in a 1.5% agarose gel and stained with ethidium bromide, and DNA bands from triplicate reactions were quantified using Fluor-S MultiImager (Bio-Rad). The values of IGF-I and Crisp were normalized against ß-actin. Results were statistically analyzed with a Student t-test, and P < 0.05 was considered as statistically significant.
Immunofluorescence Labeling of ER, ERß, and AR
ER, ERß, and AR were detected in tissue sections by standard immunofluorescence. Six-micrometer frozen sections were air dried for 30 min. After washing with ice-cold methanol and acetone, each for 3 min, sections were fixed for 10 min at room temperature in 4% paraformaldehyde. Antigens were retrieved by boiling in 10 mM citrate buffer in a microwave oven for 15 min. Sections were then incubated first with PBS containing 0.5% Triton X-100 for 1 h, then with 5% normal serum of the host of secondary antibodies for 30 min. For double stainings of ER/ERß and ER/AR, sections were incubated sequentially with each of the two primary antibodies. Preadsorbed ERß antibody was prepared by incubating ER 503 IgY for 12 h at 4°C with ERß protein coupled to activated Sepharose. The ERß protein used was either the ERß 503 that was used as antigen or recombinant human ERß obtained from Panvera (Madison, WI).
All incubations with primary antibodies were done overnight at 4°C in PBS buffer containing 3% BSA and 0.3% Triton X-100. All incubations with secondary antibodies were done for 1 h at room temperature in PBS buffer containing 2% normal rat serum. After washing, the sections were mounted in Vectashield antifading medium (Vector Laboratories). The sections were examined under a Zeiss fluorescence microscope by using suitable filters for selectively detecting the fluorescence of FITC (green) and Cy3 (red). Colocalization was indicated by yellow or orange color in cells in which both FITC- and Cy3-conjugated secondary antibodies are sequestered.
For confirmation of the validity of the signals obtained with ERß 503 IgY, a second antibody, rabbit anti-rodent ERß from Zymed, was also used to detect ERß in the paraffin-embedded uterus. Two modes of antigen retrieval were tested with this commercial antibody. One was the citrate buffer described above and the other was the use of 0.8 M urea in place of citrate, with boiling in a microwave oven for 20 min. The avidin-biotin-peroxidase method was used for staining as described previously [8].
Immunohistochemical Evaluation of Proliferation
Six-micrometer paraffin sections were used. After antigen retrieval in citrate buffer, sections were incubated with mouse anti-BrdU IgG (1:100; PharMingen, San Diego, CA) in 3% BSA for 3 h at room temperature. For negative controls, primary antibody was replaced by 3% BSA. Slides were washed with PBS and incubated with peroxidase-conjugated secondary rabbit anti-mouse antibody (Sigma) for 1 h at room temperature. After thorough washing in PBS, sections were developed with DAB substrate (DAKO), slightly counterstained with Mayer hematoxylin, dehydrated, and mounted. Three randomly selected areas in each sample were counted for BrdU-positive cells and total cells in the luminal epithelium.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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With cytosols prepared from the uteri of immature rats, an AR band at 110 kDa was detected by Western blotting. When 50 µg of protein was loaded in each lane, the AR signal was weak in samples from untreated mice, but after E2 treatment, there was increased intensity of this AR band. Administration of tamoxifen alone also resulted in a small increase in AR over the level seen in untreated mice, which supports the partial agonist activity of tamoxifen. The E2-induced increase in AR was almost completely abolished by coadministration of tamoxifen together with E2. The 66-kDa bands on the blot were unspecific and served as a loading control (Fig. 1).
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Cellular Localization and Regulation of ER and AR
To delineate which of the two ERs is involved in the induction of AR, immunocolocalizations of ER, ERß, and AR were done. In the untreated immature uterus, there was no detectable expression of AR. Upon treatment of mice with E2, AR was induced in stromal cells close to myometrium and in the myometrium, where it colocalized with ER. There was no AR expression in the epithelium.
In untreated mice, ER was expressed in the luminal epithelium, in the periluminal stroma, and in the myometrium. E2 treatment caused a decrease in the level of ER in the luminal epithelium, but stromal and myometrial ER remained unchanged (Fig. 2).
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In untreated mice, ERß was detected in both the luminal epithelium and the periluminal stroma. The ERß signals were completely extinguished by preadsorption of the ERß503 antibody with ERß protein (Fig. 3). Upon E2 treatment, ERß signals were reduced in the epithelium but expression remained in the stromal cells surrounding the luminal epithelium. No ERß was detected in stroma close to myometrium and in the myometrium, regions where AR is located. Some ERß-containing cells in the periluminal stroma also expressed ER and some did not.
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To test the validity of ERß staining in paraffin-embedded uterus, we compared a commercial antibody from Zymed, which is raised against a C-terminal peptide of ERß, with ERß 503 IgY antibody that was raised against the whole ERß molecule. Ovaries of normal and ERß-/- mice were used as positive and negative controls, respectively. When citrate buffer was used for antigen retrieval, ERß was easily detected with the Zymed antibody in granulosa cells of the ovary in wild type mice (Fig. 4a) and rats (Fig. 4c) but not in ERß-/- mice (Fig. 4b). With this antigen retrieval method, no ERß could be detected in the uterus (Fig. 4d). When 0.8 M urea was used instead of citrate buffer as the antigen retrieval buffer, ERß was detected in most of the epithelial cells and stromal cells of the uterus of control animals (Fig. 4f). Ovaries of ERß-/- mice were used as negative controls. No ERß signals were detected in these ovaries with either citrate (Fig. 4b) or urea as retrieval buffer (data not shown). In the positive control tissue, normal mouse ovaries, ERß was detected in the granulosa cells and the detection was not influenced by the retrieval procedure (Fig. 4, e and g). With the urea antigen retrieval, it was possible to confirm, with the paraffin-embedded samples, that E2 treatment down-regulated ERß in the uterine epithelium but not in the periluminal stromal cells (Fig. 4h).
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IGF-1 and Crisp in the Uterus
As judged from semiquantitative RT-PCR analysis, E2 treatment of immature rats resulted in an increase in mRNA of two well-characterized androgen-regulated genes, IGF-1 and cysteine-rich secretory protein (Crisp) [24], in the uterus. The E2 induction of both genes was inhibited by tamoxifen and by flutamide (Fig. 5, A and B).
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Effects of Flutamide and Tamoxifen on the E2 Induction of the Cell Proliferation
Animals received BrdU 4 h after hormone treatment. Uterine tissues were collected at 6, 12, and 24 h after hormone treatment. The effect of E2 treatment on proliferation of luminal epithelial cells was evaluated immunohistochemically with specific BrdU antibody. The total BrdU-positive luminal epithelial cells were counted at each time point. As expected, in the uterus of untreated immature rats, very few luminal epithelial cells were positive. After E2 treatment, the number of BrdU-positive cells was increased in the luminal epithelium. When administered together with E2, tamoxifen and flutamide each significantly inhibited E2-induced proliferation of the luminal epithelium (Fig. 6, A and B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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followed by elevation of the levels of AR, increased IGF-1 secretion, and epithelial cellular proliferation. The immunohistochemical data strongly indicate that ER and not ERß is the estrogen receptor involved in the induction of AR. AR is not detected in the epithelial cells but is present in stromal cells, mainly myometrium, where it colocalizes with ER but not with ERß. There are conflicting reports about whether there is ERß protein expression in the rodent uterus. Some laboratories have detected it and some have reported absence of immunohistochemical detection. These different experimental results may be due to differences in antibodies used and in antigen retrieval techniques employed in different laboratories [7, 25–28]. In this study, we showed that the method used for antigen retrieval is a key determinant of whether or not ERß is detected. With a commercial antibody available from Zymed, ERß was detected in both the luminal epithelium and the periluminal stroma when the antigen retrieval buffer was 0.8 M urea but not when citrate was used. With the 503 IgY antibody in frozen sections, the pattern of distribution of ERß was similar to what is seen in urea retrieved sections with the Zymed antibody, but the 503 IgY did not give satisfactory results with paraffin-embedded sections whether citrate or urea was used for retrieval. In the granulosa cells of the ovary, ERß could be detected with either retrieval method. The need for a harsher retrieval method in sections from the uterus could be due to the lower levels of ERß in the uterus or might indicate that ERß in the uterus exists in a receptor complex that is different from that in the granulosa cells.
Since the antiandrogen flutamide can block E2 induction of IGF-1 as well as Crisp and luminal epithelial proliferation as measured by BrdU labeling, AR most likely participates in E2-induced luminal epithelial proliferation in the rat uterus. It has been shown that the E2-induced proliferation of uterine epithelial cells is controlled by growth factors from the stroma and that it is the ER in the stroma but not in the epithelium that is responsible [9]. IGF-1 is one of the major growth factors involved in stimulation of cellular proliferation in the uterus [29] and has been shown to be regulated by both E2 and androgen in this tissue [30, 31]. The major IGF-1-producing compartment of the uterus is the myometrium [32], where both ER and AR are induced by estradiol. We found in this study that most of the induction of IGF-1 by E2 is mediated by AR because the E2-induced expression of IGF-1 mRNA was reduced from 3-fold to 1.3-fold by flutamide. The residual induction of IGF-1 mRNA expression in the presence of flutamide may reflect the involvement of PR in mediating estradiol-induced IGF-1 expression [33].
The effect of flutamide on estrogen-induced rat uterine growth was studied decades ago [34, 35]. In these studies, no effects of flutamide were found on the estradiol-induced uterine weight change. It is known that the uterine weight gain induced by estradiol is mostly due to the increased water imbibition and protein secretion [36], which do not necessarily indicate proliferation. With BrdU labeling in this study, we found that the estradiol-induced uterine luminal epithelial cell proliferation was inhibited by both tamoxifen and flutamide at three time points, 6, 12, and 24 h.
Crisp is transcriptionally regulated by androgens in several tissues in the male urogenital tract [37], in the lacrimal gland of both male and female mice [38], and in human and mouse glandular tissues [39]. The presence of Crisp in the uterus is a novel finding. It was found, by protein sequencing, to be one of several proteins that were overexpressed in the uterine secretion fluid of E2-treated ERß-/- mice (unpublished data). Its regulation in the uterus by E2, and inhibition of this E2 induction by an antiestrogen and an antiandrogen provides further support for the existence of an ER-AR sequential pathway.
Before puberty, the ovary secrets androgens. Just before first ovulation, the ovary turns from an androgen-producing tissue to an estrogen-producing tissue [40]. The ovarian androgens produced before puberty are most likely the source of the ligands for AR in the E2-treated uterus shown in this study.
ERß is mostly expressed in the stromal cells surrounding the lumen, a population of cells lacking AR expression. Some of these ERß-containing cells do not express ER. In a previous study, we found that, in ERß-/- mice, there is increased IGF-I expression and exaggerated cellular proliferation in response to E2 in the immature mouse uterus [8]. ER and ERß are known to have opposite effects on expression of genes that are regulated by the AP-1 site [11]. AR is a candidate gene in this category since there are several AP-1 sites in the promoter region of the AR gene [12]. In the presence of tamoxifen, transcription of genes, which is regulated by ERß via AP-1 sites, should be increased. We speculate that AR is such a gene and that tamoxifen-induced proliferation in the uterus is a result of this ERß-AP-1 pathway. This interpretation remains speculative because tamoxifen can also act as a partial agonist with ER.
Upon E2 treatment, both ER and ERß were down-regulated in the luminal epithelial cells, but in the periluminal stromal cells, ERß was not down-regulated. Because these are the cells that undergo decidualization during implantation of the embryo and because E2 is essential for this process, ERß may play a role in implantation [27].
In many systems where E2 and testosterone have similar effects [41, 42], the reason for the apparent dual regulation is the conversion of testosterone to estrogen by local aromatase. In such systems, antiandrogens do not block the effects of androgen since the response is mediated by ER. In other systems, nonaromatizable androgens can mimic the effects of E2 [43–45]. In these systems, both ER and AR participate. In such situations, the E2 response is blocked by antiestrogens and the androgen response by antiandrogens. In the present study, we have shown that the proliferative response of the uterus to E2 can be blocked by both an antiestrogen and an antiandrogen. This suggests that ER and AR constitute two sequential steps in a single pathway, where ER regulates AR expression and AR amplifies the E2 signaling. Because ER but not ERß colocalizes with AR, this pathway is composed of ER and AR. There is much evidence that E2-induced proliferation in the epithelium is indirect and requires ER in the stroma, not in the epithelium. We suggest that proliferation is due to IGF-1 released from the stroma through the action of AR. In those regions where ER, AR, and IGF-1 are colocalized, ER induces AR and AR increases IGF-1 levels.
There is much discussion today about whether or not androgens should be included along with estrogen and progesterone in hormone replacement therapy (HRT) of postmenopausal women [46]. There appear to be beneficial effects of androgen on maintenance of bone, improvement of mood, and reversal of sexual dysfunction [47]. The present data suggests that androgens can also stimulate proliferation of the uterine epithelium. Whether or not inclusion of androgen in HRT increases the risk for uterine proliferation cannot be evaluated from the present studies. However, it is clear that, for optimal HRT, the roles of all four gonadal hormone receptors should be considered.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by the Swedish Cancer Fund and by KaroBio AB. S.J. has a scholarship from Wenner Gren Foundation and Scandinavian-Japan Osakawa Foundation. 
2 Correspondence. FAX: 46 8 7116659; zhang.weihua{at}mednut.ki.se
Accepted: March 18, 2002.
Received: January 10, 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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