ORIGINAL

RESEARCH

Neonatal estrogen receptor ␤ is important in the permanent inhibition of epithelial cell proliferation in the mouse uterus Tadaaki Nakajima1),2), Yuki Tanimoto1), Masami Tanaka3), Pierre Chambon4), Hajime Watanabe5), Taisen Iguchi6), and Tomomi Sato1) 1)

Graduate School of Nanobioscience, Yokohama City University, Yokohama 236 – 0027, 2) Department of Biological Science and Technology, Tokyo University of Science, Tokyo 125– 8585, 3) Department of Food and Nutrition, Junior College of Aizu, Aizu 965– 8570, 4) Centre National de la Recherche Scientifique/Institut National de la Santé et de la Recherche Médicale/Université Louis Pasteur, Collège de France, Illkirch, France, 5) Graduate School of Engineering, Osaka University, Suita 565– 0871, 6) Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, Okazaki 444 – 8787, Japan

Estrogen receptor ␣ (ER␣) plays a pivotal role in the mouse uterine and vaginal epithelial cell proliferation stimulated by estrogen, whereas ER␤ inhibits cell proliferation. ER␤ mRNA is expressed in neonatal uteri and vaginae, however, its functions in neonatal tissues were not ascertained. In this study, we investigated the ontogenic mRNA expression and localization of ER␤, and roles in cell proliferation in neonatal uteri and vaginae of ER␤ knockout (␤ERKO) mice. ER␤ mRNA and protein were abundant in the uterine and vaginal epithelia of 2-day-old mice, and decreased with age. In uterine and vaginal epithelia of 2-day-old ␤ERKO mice, cell proliferation was greater than that in wild-type animals, as well as uterine epithelia of 90- and 365-day-old ␤ERKO mice. In addition, p27 protein known as a cyclin-dependent kinase inhibitor was decreased in the uteri of 90- and 365-day-old ␤ERKO mice. Inhibition of neonatal ERs by ICI (ER antagonist) treatment stimulated cell proliferation and decreased p27 protein in the uterine luminal epithelium of 90day-old mice, but not in the vaginal epithelium. These results suggest that neonatal ER␤ is important in the persistent inhibition of epithelial cell proliferation with accumulation of p27 protein in the mouse uterus. Thus, suppression of ER␤ function in the uterine epithelium during the neonatal period may be responsible for a risk for proliferative disease in adults.

E

strogen stimulates epithelial cell proliferation in the female reproductive tracts of adult mice (1). Mouse estrogen receptor (ER) is distinguished as two subtypes, ER␣ (2) and ER␤ (3, 4). ER␣ is a major receptor mediating most of the estrogen actions in the female mouse reproductive tracts (5). In adult mice, ER␣ in the stroma activated by estrogen stimulates epithelial cell proliferation both in the uteri and vaginae, whereas ER␣ both in the epithelium and stroma is essential for the estrogen-induced expression of uterine epithelial secretory proteins and vaginal epithelial cornification (6). ER␤ is expressed in the uterine epithelia and stroma, and prostatic epithelia

of adult mice (7, 8). Epithelial cell proliferation in the uteri and prostates of adult ER␤ knockout (␤ERKO) mice is higher than that in wild-type (WT) mice, and epithelial cell apoptosis is reduced in the prostatic epithelia of adult ␤ERKO mice (7–10). Therefore, ER␤ is involved with antiproliferative and apoptotic actions in the epithelium of adult uterus and prostate. ER␤ mRNA is found in the rat Müllerian ducts from fetal through postnatal period, but ER␤ protein is not detected in the uteri (11–13). Thus, ER␤ mRNA is expressed in the neonatal uterus and vagina, however, its roles in cell proliferation of the neonatal mouse uterus and vagina have not been investigated yet.

ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in USA Copyright © 2015 by the Endocrine Society Received January 7, 2015. Accepted May 21, 2015.

Abbreviations:

doi: 10.1210/en.2015-1012

Endocrinology

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Neonatal ER␤ persistently inhibits cell proliferation

Epithelial cell proliferation is high in the uteri and vaginae of intact neonatal mice, and neonatal 17␤-estradiol (E2) treatment stimulates cell proliferation in the uterine epithelia at day 1 but inhibits at day 2, and inhibits cell proliferation in the vaginal epithelium (14). Thus, the regulation of epithelial cell proliferation in the uteri and vaginae of neonatal mice may be different from that in adult mice regardless of E2 treatment. In addition, exposure to natural or synthetic estrogens during perinatal period results in the persistent cell proliferation, stratification and cornification in the mouse vagina, and uterine hypoplasia and epithelial metaplasia (15, 16). This fact suggests that alteration of cell proliferation by changes of estrogen signaling during neonatal period leads to the abnormalities in adult tissues. A single injection of E2 into ovariectomized mice shortens G1 phase in the uterine epithelial cells (17). The epithelial cells passing G1 phase proceed into S phase, peaking at 13 to 16 hours after E2 treatment, followed by a cell division (18). At 21 hours after E2 treatment, cell-death in uterine epithelial cells is minimal (18). A G1/S progression is stimulated by complexes of cyclin with cyclin-dependent kinases (CDKs) (19, 20). Cyclin/CDK complexes are inhibited by CDK inhibitors, such as p21 (21), p27 (22, 23) and p57 (24). In the uterine luminal epithelia of adult mice, E2 stimulates the protein expression of cyclin A and cyclin E, and induces a nuclear translocation of cyclin D1 from cytoplasm in addition to a decrease of p27 protein (25). In the uteri of adult mice, E2 induces nuclear translocation of cyclin D1 and epithelial cell proliferation through ER␣ (26). In contrast, the overexpression of ER␤ in human breast cancer cell lines inhibits cell proliferation, accompanied with inhibition of cyclin A and cyclin D1 and stimulation of p21 and p27 protein expression (27). ER␤ knockdown stimulates cell proliferation with a decrease of p21 mRNA expression in human breast cancer cell lines (28). In human prostate cancer cell lines, ER␤ also stimulates of p21 and p27 protein expression (29). In HeLa cells, ER␤ completely inhibits activation of cyclin D1 promoter by estrogen-ER␣ complex (30). Also, extreme endometrial hyperplasia is reported in aged ␤ERKO mice (31). These results suggest that ER␤ inhibits cell proliferation through the regulation of cyclin A, cyclin D1, p21 and p27 expression in the various epithelial cancer cells. In this study, the roles of ER␤ in epithelial cell proliferation of the uteri and vaginae of neonatal mice were examined. First, the ontogenic mRNA and protein expression and mRNA localization of ER␤ in the uteri and vaginae were determined. Cell proliferation, apoptosis and gene expression involved in the cell cycle and cell proliferation in uteri and vaginae of ␤ERKO mice from days 2 to 365 were examined. In order to investigate the involve-

Endocrinology

ment of neonatal ER with epithelial cell proliferation of the uteri and vaginae in adulthood, effects of neonatal treatment with ICI 182 780 (ICI), an antagonist of both activation function 1 and 2 of ER␣ and ER␤ (4, 32, 33), on cell proliferation of uteri and vaginae at day 90 were examined.

Materials and Methods Animals ␤ERKO mice were obtained by mating mice of mixed C57BL6/129sv background that were heterozygous for the ER␤ gene disruption, as described previously (34). Pup genotypes were determined by multiplex PCR. Female C57BL/6J (CLEA, Tokyo, Japan), WT and ␤ERKO mice were given a commercial diet (MF, Oriental Yeast Co., Tokyo, Japan) and tap water ad libitum, and kept at 22 ⫾ 1.0°C under 12 hours light/12 hours darkness by artificial illumination (lights on 0800 –2000 hours). Animals were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by our institutional Animal Care Committee. Uteri, vaginae and ovaries of 90-day-old C57BL/6J mice were collected at the estrous stage determined by vaginal smear.

Treatments and histology Female C57BL/6J mice were given daily subcutaneous (s.c.) injections of 10 ␮g ICI 182 780 (Tocris Bioscience, Ellisville, MO, U.S.A.) dissolved in sesame oil or sesame oil alone for 5 or 10 days, starting on the day of birth (⫽day 0). Two-day-old WT and ␤ERKO mice were given a single s.c. injection of 1 mg/10 g body weight (BW) 5-bromo-2⬘deoxyuridine (BrdU, Sigma, St. Louis, MO, U.S.A.) in saline 1 hour before sacrifice. C57BL/6J mice treated with ICI for 5 or 10 days from day 0, intact C57BL/6J mice, WT mice and ␤ERKO mice were ovariectomized at days 90 or 365. Seven days after the surgery, ovariectomized mice were given a single s.c. injection of 0.1 ␮g/25 g BW E2 (Sigma) or sesame oil. Sixteen h after a single injection of E2, uteri and vaginae were collected for real-time RT-PCR. For immunohistochemistry of BrdU, mice were given a single intraperitoneal injection of 1 mg/10 g BW BrdU in saline at 14 hours after a single injection of E2, and uteri and vaginae were collected 2 hours later. Ovaries of 90- and 365-day-old WT and ␤ERKO mice and 90-day-old neonatally ICI-treated C57BL/6J mice were fixed in Bouin’s solution overnight, embedded in paraffin, and serially sectioned at 8 ␮m and stained with hematoxylin and eosin. The uterine epithelial height was measured randomly in 6 areas under a light microscope with ⫻40 objective lens.

Detection of proliferating cells or apoptotic cells Detection of proliferating cells in 2- (n ⫽ 8), 90- (WT: n ⫽ 8, ␤ERKO: n ⫽ 6) and 365-day-old (oil: n ⫽ 5, E2: n ⫽ 8) WT and ␤ERKO mice and in 90-day-old neonatally ICI-treated C57BL/6J mice (n ⫽ 6, E2-treated control mice: n ⫽ 10) by immunohistochemistry for BrdU or apoptotic cells in 2- and 90day-old WT and ␤ERKO mice by TUNEL staining (n ⫽ 5) was performed with minor modifications as described previously

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doi: 10.1210/en.2015-1012

(35). For detection of apoptotic cells, incubation of proteinase K (Wako Pure Chemical Industries, Osaka, Japan) was performed for 10 minutes for 2-day-old mice or for 15 minutes for 90-dayold mice at room temperature. 0.3 U of TdT (Takara Bio, Otsu, Japan) and 4 ␮M biotinylated dCTP (Life Technologies, Carlsbad, CA, U.S.A.) were used at 37°C for 90 minutes. The BrdUpositive cells or apoptotic cells in 200 cells were counted in randomly selected 3 different areas of the uterus and vagina of 2-day-old mice or 6 areas of the uterus and vagina of 90- and 365-day-old mice under a light microscope with ⫻40 objective lens. BrdU-positive cells in the vaginal epithelium of 90- and 365-day-old mice were counted only in the basal layer.

Immunohistochemistry Uteri and vaginae of 90- and 365-day-old WT and ␤ERKO mice and 90-day-old neonatally ICI-treated C57BL/6J mice (n ⫽ 5) were fixed in 10% formalin neutral buffered solution (Wako Pure Chemical Industries) and tissues were then embedded in paraffin and sectioned at 5 ␮m. Sections were deparaffinized and immersed in 3% H2O2 for 10 minutes. Microwave treatment in 10 mM citric acid buffer for 15 minutes at 95°C was performed for antigen retrieval. After washing in PBS, and incubation with 5% goat serum (Vector Laboratories, Burlingame, CA, U.S.A.) for 30 minutes, sections were incubated with anti-p27 rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.) at 1:300 dilution or negative control rabbit Immunoglobulin Fraction (DakoCytomation, Glostrup, Denmark) in PBS containing 5% goat serum at 4°C overnight. After incubation with secondary antibody, p27 was detected by Elite standard ABC kit (Vector Laboratories) and diaminobenzidine (DAB, Sigma). Methyl green was used for counterstaining. The p27-negative cell was defined as a cell having only methyl greenstained or partially DAB-stained nuclei. The p27-negative cells in 200 cells were counted in randomly selected 3 different areas of the uterine epithelium of 90- and 365-day-old mice under a light microscope with ⫻40 objective lens.

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diluted 1:500 or rabbit anti-p27 antibody was diluted 1:500. In order to confirm equal protein loading, the blots were stripped and reprobed with the anti-Histone H3 antibody (Cell Signaling Technology, Beverly, MA, U.S.A.), antiactin antibody (Sigma) or anti-␤-actin antibody (Sigma) as internal controls. One tissue of 90-day-old mice, 3 tissues of 15-day-old mice or 10 tissues of 2-day-old mice were pooled and used for each western blot. Three independent experiments for detection of ER␣, ER␤ and p21 or 2 independent experiments for detection of p27 were carried out.

RNA isolation, RT-PCR and real-time RT-PCR The epithelia and stroma from the uteri and vaginae of 2- and 15-day-old C57BL/6J mice were separated, RNA was isolated from the separated epithelia and stroma, uteri, vaginae and ovaries of 2-, 10-, 15- and 90-day-old C57BL/6J mice and 2- and 90-day-old WT and ␤ERKO mice, and RT-PCR and real-time RT-PCR were performed as described previously (36). An aliquot of cDNA was amplified with specific primers derived from mouse mRNA sequences (Supplemental Table). Peptidylprolyl isomerase A (Ppia) was chosen as an internal standard. One tissue of 90-day-old mice, 3 tissues of 10 and 15-day-old mice or 4 –12 tissues of 2-day-old mice were pooled for each point. Two independent experiments for RT-PCR or 3 independent experiments for real-time RT-PCR were carried out.

Radioimmunoassay (RIA) 2- and 90-day-old C57BL/6J mice were anesthetized with diethyl ether, and blood was collected via the ascending jugular vein into tubes. The serum was isolated by centrifugation at 3000 rpm for 15 minutes at 4°C and stored at – 80°C before use. The serum E2 concentrations were estimated by a commercial RIA kit (DPC 125I RIA kit, 17␤-estradiol double antibody, Siemens Healthcare Diagnostics Inc., Los Angeles, CA, U.S.A). The serum from one 90-day-old mouse or the serum from 10-day-old mice was pooled up to 200 ␮L. Six points for 90-day-old mice or 18 points for 2-day-old mice were examined in each group by RIA.

Western blot For detection of ER␣, 50-day-old C57BL/6J mice were ovariectomized and given a single injection of oil or E2 at 10 days after the surgery. Uteri and vaginae at 2- and 15-day-old mice, and 60-day-old mice at 12 hours after a single injection of oil or E2 were homogenized in 20 mM Tris-HCl buffer (pH 7.5) containing 1% Triton X-100, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA and 250 mM sucrose (buffer A). For detection of ER␤, uteri, vaginae or ovaries in the 2-, 15- and 90-day-old C57BL/6J mice were homogenized in buffer A. The homogenates were centrifuged at 3300 rpm for 10 minutes at 4°C to precipitate nuclei. The precipitations were dissolved in buffer A without sucrose (buffer B) containing 250 mM NaCl and 1% protease inhibitor cocktail (Sigma). For detection of p21 and p27, uteri or vaginae of 2- and 90-day-old WT and ␤ERKO mice were homogenized in the buffer B. Western blot was performed as described previously (36). Briefly, 15 ␮g of proteins or 40 ␮g of proteins were run in 8% SDS-PAGE gels for ER␣ and ER␤ or 14% gels for p21 and p27, respectively. Mouse anti-ER␣ antibody (Leica Biosystems, Newcastle, UK) was diluted 1:1000, rabbit anti-ER-␤ antibody (Zymed Laboratories, San Francisco, CA, U.S.A.) was diluted 1:250, rabbit anti-p21 antibody (Santa Cruz Biotechnology) was

Statistical analysis Data were expressed as the mean ⫾ standard error. Differences were estimated using ANOVA followed by appropriate post hoc tests. Two-tailed Student’s t test or Welch’s t test was used for comparison of two means. Differences were considered significant at P ⬍ .05.

Results Ontogenic mRNA and protein expression and localization of ER␣ and ER␤ in female mice reproductive tracts In order to understand the ontogenic changes of ER␣ and ER␤ mRNA expression in the uteri and vaginae of C57BL/6J mice, real-time RT-PCR was performed. ER␣ mRNA expression in the uteri was detected at day 2, and it significantly increased between days 10 and 15, and showed continuous high expression through day 90 (Figure 1A). In vaginae, the mRNA expression of ER␣ signif-

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Neonatal ER␤ persistently inhibits cell proliferation

icantly increased between days 2 and 10, then significantly decreased at day 90. ER␤ mRNA expression in the uteri was detected through days 2 to 90, but was significantly decreased at day 90. In vaginae, ER␤ mRNA expression was high at day 2, and it was then decreased from days 10 to 90. From days 2 to 15, ER␤ mRNA expression in the uteri was significantly lower than that in vaginae, however, it was significantly higher than that in vaginae at day 90. ER␤ mRNA was localized in the uterine and vaginal epithelia by RT-PCR at days 2 and 15, but not in the uterine and vaginal stromal cells (Figure 1B). Both ER␤ transcript variant 1 and 2 mRNAs in uteri and vaginae were detected by RT-PCR from days 2 to 15, but were not

Endocrinology

found at day 90 (data not shown). ER␣ protein (65 kDa) expression in the uteri and vaginae was detected and similar at days 2 and 90 (Figure 1C). ER␤ protein (55 kDa) was detected in the ovaries at day 90, being a positive control. In uteri and vaginae, ER␤ protein expression was high at day 2, and became undetectable at days 15 and 90. Thus, ER␤ mRNA and protein were highly expressed in the uterine and vaginal epithelia at day 2, and decreased with age.

Cell proliferation, apoptosis and gene expression involved in the cell cycle in the uteri and vaginae of ␤ERKO mice at day 2 Proliferative and apoptotic cells in uteri and vaginae of ␤ERKO mice at day 2 were detected by immunohistochemistry for BrdU and TUNEL staining. In ␤ERKO mice at day 2, the BrdU labeling index in the uterine and vaginal epithelia was higher than that in WT mice, but it was not different in the uterine and vaginal stromal cells (Figure 2A-E). The percentage of apoptotic cells was not different in the all tissues of ␤ERKO mice at day 2 (Figure 2F-J). The expression of cyclin A, cyclin D1 and p21 examined by real-time RT-PCR was not different in the uterus and vagina of ␤ERKO mice at day 2 compared with that of WT mice (Figure 2K). The expression of ER␣ and p27 in the vagina of ␤ERKO mice was significantly lower than that in WT mice. The protein levels of p21 and p27 are largely controlled by posttranscriptional mechanisms (37– 41), therefore, the expression of p21 and p27 was examined by Western blot. p21 and p27 protein was not different in uteri and vaginae of ␤ERKO mice at day 2 Figure 1. The expression of estrogen receptor ␣ (ER␣) and ER␤ mRNA in the uteri and vaginae of 2-, 10-, 15- and 90-day-old mice analyzed by real-time RT-PCR (A). Peptidylprolyl isomerase A compared with that of WT mice (Fig(Ppia) was chosen as an internal standard. Data were expressed relative to mRNA expression in ure 2L). The localization of p21 and the uterus of 2-day-old mice (⫽1.0). The expression of ER␤ mRNA in the uterine epithelium (UE), p27 protein in uteri and vaginae of uterine stroma (US), vaginal epithelium (VE) and vaginal stroma (VS) of 2- and 15-day-old mice analyzed by RT-PCR (B). The expression of ER␣ and ER␤ protein in the uteri and vaginae of 2-, ␤ERKO mice at day 2 was analyzed 15- and 90-day-old mice analyzed by Western blot (C). Histone H3 or Actin was chosen as an by immunohistochemistry, but those internal standard. The ovaries at day 90 were used for a positive control of ER␤ expression. One proteins were not detected (data not tissue of 90-day-old mice, 3 tissues of 10 and 15-day-old mice or 12 tissues of 2-day-old mice were pooled for each point. Two independent experiments for RT-PCR, and 3 independent shown). Thus, cell proliferation in experiments for real-time RT-PCR and Western blot were carried out. Different letters (a, b, c, d) the uterine and vaginal epithelia of in the uterus or vagina from days 2 to 90 indicated age-dependent significant changes of the neonatal ␤ERKO mice was stimuexpression (P ⬍ .05). *: P ⬍ .05 compared with the expression of ER␤ in the uteri of agelated but the genes and proteins inmatched mice.

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volved in cell cycle were not different compared with that of WT mice. Cell proliferation and apoptosis in the uteri and vaginae of ␤ERKO mice at days 90 and 365 The estrous cycle determined by vaginal smear was not different between WT and ␤ERKO mice at day 90 (data not shown). Corpora lutea in the ovary were found in WT and ␤ERKO mice at days 90 and 365. At days 90 and 365, no histological differences in uteri and vaginae were observed between WT and ␤ERKO mice. In ␤ERKO mice treated with oil, the BrdU labeling index in the uterine luminal epithelium at day 90 was higher

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than that in WT mice (Figure 3A-C, symbol a), whereas it was not different both in the uterine glandular epithelium at day 90 and in uterine luminal and glandular epithelium at day 365. Although a single injection of E2 significantly increased the BrdU labeling index in the uterine luminal and glandular epithelium of WT and ␤ERKO mice at days 90 and 365, ␤ERKO mice showed a much higher index (Figure 3A, D, E, F, I and J, symbol b). In the vaginal epithelium (Figure 3A and F), and uterine and vaginal stroma of oil- or E2-treated ␤ERKO mice at days 90 and 365, the BrdU labeling index was not different compared with that of WT mice (data not shown). The percentage of apoptotic cells in the all tissues was not different between WT and ␤ERKO mice at day 90 (Figure 3K-O). The height of uterine luminal epithelial cells in E2-treated ␤ERKO mice at day 90 was similar to that in WT mice, but it in E2treated ␤ERKO mice at day 365 was significantly taller than that in the WT mice (21.0 ⫾ 0.45 ␮m in WT mice vs. 23.3 ⫾ 0.92 ␮m in ␤ERKO mice). Thus, the uterine epithelial cell proliferation was stimulated in adult ␤ERKO mice, and was enhanced after the E2 treatment.

Figure 2. The BrdU labeling index (A-D and I; n ⫽ 8) and apoptotic cells (E-H and J; n ⫽ 5) in the uterine epithelium (UE), uterine stroma (US), vaginal epithelium (VE) and vaginal stroma (VS) of 2-day-old WT and ␤ERKO mice were analyzed by immunohistochemistry or TUNEL. Magnification: ⫻400. Scale bar: 50 ␮m. The expression of ER␣, cyclin A, cyclin D1, p21 and p27 mRNAs in the uteri (Ut) and vaginae (Vg) of 2-day-old WT and ␤ERKO mice was analyzed by realtime RT-PCR (K). Peptidylprolyl isomerase A was chosen as an internal standard. Data were expressed relative to mRNA expression in the uteri of 2-day-old WT mice (⫽1.0). a: P ⬍ .05 compared with WT mice. The expression of p21 and p27 proteins in the uteri and vaginae of 2day-old WT and ␤ERKO (KO) mice was analyzed by Western blot (L). ␤-Actin was chosen as an internal standard. Twelve tissues of 2-day-old WT or ␤ERKO mice were pooled for each point. Two independent experiments for detection of p27 by Western blot, and 3 independent experiments for real-time RT-PCR and detection of p21 by Western blot were carried out.

mRNA and protein expression involved in cell cycle or growth factors in the uteri and vaginae of ␤ERKO mice at days 90 and 365 A single injection of E2 significantly decreased ER␣ and p27 expression in the uteri and vaginae of WT and ␤ERKO mice at day 90 (Figure 4A). In the vagina of WT and ␤ERKO mice, a single injection of E2 significantly decreased cyclin D1 expression. ER␣, cyclin A, cyclin D1, p21 and p27 expression in the uteri and vaginae of oil- or E2-treated ␤ERKO mice at day 90 was similar to that of WT mice. The protein expression of p21 and p27 in all tissues was not different at day 90 by Western blot (Figure 4B). A single injection of E2 did not decrease p27 protein, although Tong and Pollard (25) show that E2 inhibits p27 protein in the uterine epithelial cell lysate by Western blot and in the uterine epithelia

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Neonatal ER␤ persistently inhibits cell proliferation

Figure 3. The BrdU labeling index in the uterine luminal epithelium (ULE), uterine glandular epithelium (UGE) and vaginal epithelium (VE) of 90-day-old (A-D and M) or 365-day-old (E-H and N) ovariectomized WT and ␤ERKO mice after oil or E2 treatment (90-day-old WT: n ⫽ 8, 90-dayold ␤ERKO: n ⫽ 6, 365-day-old oil-treated mice: n ⫽ 5, 365-day-old E2-treated mice: n ⫽ 8). The apoptotic cells in the uterine luminal epithelium (ULE), uterine glandular epithelium (UGE) and vaginal epithelium (VE) of 90-day-old ovariectomized WT and ␤ERKO mice after oil or E2 treatment (I-L and O) (n ⫽ 5). a: P ⬍ .05 compared with age-matched oil-treated WT mice. b: P ⬍ .05 compared with age-matched E2-treated WT mice. N.D.: not detected. Magnification: ⫻400. Scale bar: 50 ␮m.

Endocrinology

by immunohistochemistry. Therefore, the expression of p21 and p27 in the uteri and vaginae of ␤ERKO mice was analyzed by immunohistochemistry, but p21 was not detected (data not shown). The percentage of p27-negative cells in the uterine luminal and glandular epithelia of ␤ERKO mice treated with oil at days 90 and 365 was higher than that of WT mice (Figure 5A-C and J-L, symbol a, arrows). A single injection of E2 decreased p27 protein in the uterine and vaginal epithelia of WT and ␤ERKO mice. In the uterine luminal and glandular epithelia of ␤ERKO mice treated with E2, many p27-negative cells were observed and the staining intensity of p27 was reduced at days 90 and 365 compared with that of WT mice (Figure 5A, F, G, J, O and P, symbol b, arrows). In the vaginae of ␤ERKO mice at days 90 and 365, p27 protein was not different compared with that of WT mice. Therefore, p27 protein in the uterine epithelium of adult ␤ERKO mice was decreased, especially after treatment of E2. In order to identify the target genes of ER␤ in the uterine epithelia of neonatal and adult mice, the mRNA expression of growth factors and their receptors involved in cell proliferation of adult uterine epithelium (42– 46) was examined (Figure S1). The expression of growth factors and their receptors in the uteri and vaginae of ␤ERKO mice at days 2 and 90 was similar to that of WT mice, except for a decrease of one of the vascular endothelial growth factor (VEGF) receptor, Flt1, in the uteri of ␤ERKO mice. However, the expression of insulin-like growth factor I receptor (Igf1r) in the uteri of ␤ERKO mice was higher than that of WT mice both in days 2 and 90 (but not significant).

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Effects of neonatal ICI treatment in the uteri and vaginae of C57BL/6J mice at day 90 In order to investigate the roles of high levels of ER␤ expression during the neonatal period in epithelial cell proliferation in the uteri and vaginae of adult mice, the effects of neonatal ICI treatment on cell proliferation in the uteri and vaginae at day 90 were examined. The estrous cycle as determined by vaginal smear was similar and corpora lutea in the ovaries were found in the all mice examined (data not shown). In mice neonatally treated with ICI for 5 days, the BrdU labeling index in the uterine luminal epithelium was higher than that in control mice treated with a single injection of oil at day 90 (Figure 6A, symbol a). A single injection of E2 significantly increased the BrdU labeling index in the uterine luminal and glandular, and vaginal epithelia of all mice examined, but mice neonatally treated with ICI for 5 days showed a much higher index in the uterine luminal epithelia (symbol b). In contrast, mice neonatally treated with

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ICI for 10 days showed a lower index in the uterine glandular epithelia (symbol b). The BrdU labeling index in the vaginal epithelial and uterine and vaginal stromal cells was not different in mice neonatally treated with ICI after a single injection of oil or E2 compared with that in control mice (data not shown). In the uterine luminal epithelia of mice neonatally treated with ICI after a single injection of oil, p27 protein was not different compared with that of control mice (Figure 6B-G and Figure S2). A single injection of E2 decreased p27 protein in the uterine and vaginal epithelia of all mice examined. After a single injection of E2, p27 protein in the uterine luminal epithelia of mice neonatally treated with ICI was totally reduced compared with that of control mice. In the vagina of mice neonatally treated with ICI, p27 protein was low regardless of treatment with oil or E2. Thus, cell proliferation in the uterine luminal epithelia was stimulated in mice neonatally treated with ICI, accompanied by a decrease of p27 protein, as well as in ␤ERKO mice.

Figure 4. The expression of ER␣, cyclin A, cyclin D1, p21 and p27 mRNAs in the uterus (Ut) and vagina (Vg) of 90-day-old ovariectomized WT and ␤ERKO mice after oil or E2 treatment was analyzed by real-time RT-PCR. Peptidylprolyl isomerase A was chosen as an internal standard. Data were expressed relative to mRNA expression in the uteri of oil-treated WT mice (⫽1.0). The expression of p21 and p27 protein in the uteri and vaginae of 90-day-old ovariectomized WT and ␤ERKO mice after oil or E2 treatment was analyzed by Western blot (B). ␤-Actin was chosen as an internal standard. One tissue of 90-day-old WT or ␤ERKO mice was used for each point. Two independent experiments for detection of p27 by Western blot, and 3 independent experiments for real-time RT-PCR and detection of p21 by Western blot were carried out. a: P ⬍ .05 compared with oil-treated WT mice. *: P ⬍ .05 compared with genotype-matched oil-treated mice.

Serum E2 levels and mRNA expression of the essential enzyme for E2 synthesis in the ovaries, uteri and vaginae from days 2 to 90 The serum E2 levels were determined in C57BL/6J mice at days 2, 10 and 90 by RIA. In mice in estrus at day 90, serum E2 levels were 2.7 ⫾ 0.35 pg/mL, whereas serum E2 was not detected at days 2 and 10. In the uteri and vaginae, the expression of cytochrome P450 side-chain cleavage enzyme (Cyp11a1) and aromatase (i.d. essential enzymes for E2 synthesis) was significantly lower than that in the ovaries at day 2 (Figure S3, symbol a). In the ovaries, the expression of Cyp11a1 and aromatase at day 2 was dramatically lower than that at day 90 (symbol b). Thus, serum E2 levels and the expression of Cyp11a1 and aromatase in the ovaries, uteri and vaginae were very low in neonatal mice.

Discussion It has been reported that ER␤ inhibits epithelial cell proliferation in the

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Neonatal ER␤ persistently inhibits cell proliferation

Endocrinology

adult mouse uterus, regardless of E2 treatment (7, 10, 31). In the present study, we demonstrated that ER␤ is impor-

tant for the inhibition of cell proliferation not only in the adult but also in the neonatal uterine epithelium. In the uteri and vaginae of ␤ERKO mice at day 2, epithelial cell proliferation was stimulated. In addition, the expression of ER␤ mRNA and protein in the uteri and vaginae of neonatal mice was high and localized in the epithelium, suggesting that ER␤ in the uterine and vaginal epithelia of neonatal mice inhibits cell proliferation. Cyclin A in the uteri and vaginae of neonatal WT mice was significantly higher (3.8-fold and 2.1-fold) and p27 in the uteri of neonatal WT mice was significantly lower (0.37-fold) than that in E2-treated adult mice by the ddCt method. These differences in the gene expression levels between neonatal and adult uteri and vaginae may result in high cell proliferation in the neonatal epithelial cells. However, gene and protein expression levels involved in the cell cycle at day 2 were similar in the uteri and vaginae of WT and ␤ERKO mice. Therefore, the inhibiting mechanism of cell proliferation by ER␤ is not due to p27 expression levels in the neonatal uteri and vaginae. Epithelial cell proliferation was stimulated both in the uterus and vagina of neonatal ␤ERKO mice, whereas at days 90 and 365, it was further stimulated only in the uterus of ␤ERKO mice. Therefore, ER␤ in the uteri and vaginae may have a different mechanism in regulating cell proliferation. In the uteri of ␤ERKO mice treated with oil or E2 at days 24 and 64, epithelial cell proliferation is much stimulated compared with that of WT mice (7, 10). Although p27 mRNA expression was not different as compared with that of WT mice, p27-expressing cells decreased in the uterine epithelium of ␤ERKO mice Figure 5. The percentage of p27-negative cells in the uterine luminal epithelium (ULE), uterine at days 90 and 365. It is known that glandular epithelium (UGE) (A,B, E, F, I, J, M, N, S and T) and vaginal epithelium (C, D, G, H, K, L, O and P) of 90-day-old (A-H and S) or 365-day-old (I-P and T) ovariectomized WT and ␤ERKO the level of p27 is largely controlled mice after oil or E2 treatment was detected by immunohistochemistry (n ⫽ 5). A negative control by translational mechanisms (38, in the uteri (Q) and vaginae (R). Arrows indicated p27-negative cells. a: P ⬍ .05 compared with 39) and degradation via ubiquitinage-matched oil-treated WT mice. b: P ⬍ .05 compared with age-matched E2-treated WT mice. Scale bar: 50 ␮m. proteasome pathway (37). In pros-

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doi: 10.1210/en.2015-1012

tate cancer cell line, ER␤1 induces down-regulation of p45Skp2 and CCNE2/CCNA2 expression (ie, mediators of p27 degradation) and results in the concomitant increase of p27 protein. ER␤2 induces up-regulation of p45Skp2, p27 and protected form of T198- phosphorylated p27 from p45Skp2-mediated degradation (47). Therefore, ER␤-induced inhibition of cell proliferation in the uterine epithelium is partially mediated by the accumulation of p27 protein in adult mice. ER␣ mRNA expression was not different in the uterus of ␤ERKO mice at days 2 and 90 compared with that of WT mice, indicating that this inhibition in the uterine epithelium is not due to changes in the ER␣ expression levels. On the contrary, ER␣ expression in the vagina of ␤ERKO mice at day 2 was lower than that of WT mice. ER␤ transcript variant 1, acts as a dominant negative form of ER␣ (48), is expressed both in the

Figure 6. The BrdU labeling index (A) in the uterine luminal epithelium (ULE), uterine glandular epithelium (UGE) and vaginal epithelium (VE) of ovariectomized control mice (C) or 90-day-old ovariectomized neonatally ICI-treated mice for 5 days (neo-ICI5) or 10 days (neo-ICI10) from day 0, after oil or E2 treatment. a: P ⬍ .05 compared with oil-treated control mice. b: P ⬍ .05 compared with E2treated control mice. The immunohistochemistry of p27 in the uteri (B-G) of 90-day-old ovariectomized control (B and E), neo-ICI5 (C and F) or neo-ICI10 (D and G) mice, after oil (B-D) or E2 (E-G) treatment (n ⫽ 5). Scale bar: 50 ␮m.

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9

neonatal uterine and vaginal epithelia, however, ER␣ is expressed in the neonatal vaginal epithelium, but not in the neonatal uterine epithelium (49). Since stimulated ER␣ signaling by E2 induced down-regulation of ER␣ mRNA expression (Figure 4), ER␤ in the vaginal epithelium of neonatal mice may indirectly inhibit cell proliferation through the inactivation of ER␣. In the prostatic epithelium of adult ␤ERKO mice, epithelial cell apoptosis is reduced (8, 9), however, the percentage of apoptotic cells in the uteri and vaginae of ␤ERKO mice at days 2 and 90 was not different compared with that of WT mice. This result suggests that ER␤ is not involved with apoptosis in the uteri and vaginae from neonatal to adult mice. MPP (1,3-Bis(4-hydroxyphenyl)-4-methyl-5-[4-(2piperidinylethoxy)phenol]-1H-pyrazole hydrochloride), the selective ER␣ receptor antagonist, has more than 200fold binding selectivity for ER␣ over ER␤ (50), whereas few selective ER␤ receptor antagonists have been reported (eg, PHTPP (4-[2-Phenyl-5, 7-bis (trifluoromethyl) pyrazolo [1,5-␣] pyrimidine-3- yl] phenol) has only 36-fold binding selectivity for ER␤ over ER␣ and shows a weak antagonize transcriptional activity of ER␣) (51, 52). Since ICI is a pure and robust antagonist for both ER␣ and ER␤ (4, 32, 33), roles of neonatal ER␤ in the uterus and vagina were investigated by comparison of neonatal ICI-treated mice with ␤ERKO mice. In the uterine luminal epithelium at day 90, the inhibition of neonatal ERs activity by ICI treatment for 5 days increased cell proliferation and decreased p27 expression, as well as in ␤ERKO mice. Thus, inhibition of neonatal ER␤ function in the uterine epithelium results in the stimulation of cell proliferation accompanied with a decrease of p27 protein in adult mice. In order to identify the target genes of ER␤ in uterine epithelia of neonatal mice and irreversibly affected the genes to adulthood, the mRNA expression of growth factors and the receptors involved in cell proliferation of adult uterine epithelium (42– 46) was examined, however, almost all the expression in the uteri and vaginae of ␤ERKO mice from days 2 and 90 was not different compared with that of WT mice. Only Igf1r expression in the uteri of ␤ERKO mice was higher than that of WT mice at days 2 and 90 (but not significantly), therefore, the increase of Igf1r expression might be involved in permanent stimulation of uterine epithelial cell proliferation induced by ␤ERKO or neonatal ICI treatment. Neonatal ICI treatment for 10 days also decreased p27 expression in the uterine luminal epithelium, but cell proliferation was not altered. In contrast to ␤ERKO mice, neonatal ICI treatment for 10 days decreased cell proliferation in the uterine glandular epithelium at day 90. Since ICI antagonizes both ER␣ and ER␤ (32, 33), ICI also inhibits the effects of ER␣ on cell proliferation in the uterine

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Neonatal ER␤ persistently inhibits cell proliferation

luminal and glandular epithelia of neonatal mice. ER␣ expression in the uteri was increased from days 2 to 15, suggesting that ICI showed high antagonizing effects on ER␣ and/or less antagonizing effects on ER␤ from days 5 to 10 compared with those from days 0 to 5. Disruption of ER␣ causes uterine hypoplasia and inhibits development of endometrial glands (53), therefore, suppression of ER␣ function by ICI treatment for 10 days might cause a decrease in the uterine cell proliferation. On the contrary, neonatal exposure to estrogen also causes uterine hypoplasia through ER␣ (16, 54), indicating that uterine epithelial cell proliferation is suppressed by excess ER␣ signaling in the neonatal mice. Exposure to neonatal diethylstilbestrol in ␤ERKO or selective ER␣ agonist in C57BL/6 mice induces uterine hypoplasia and inhibition of development of endometrial glands as well as neonatal DES or estrogen treatment in WT mice (55–56). Indeed, the perinatal exposure to estrogen up-regulates ER␣ expression in the uterine epithelia of neonatal mice but down-regulates ER␤ in the prenatal rat uteri (11, 12, 57), therefore, exogenous estrogen in neonatal rodents may result in reduced uterine epithelial cell proliferation through an increase of ER␣ rather than a decrease of ER␤ signaling. In the ovaries of ␤ERKO mice at days 60 and 150, atretic follicles are increased but corpora lutea are decreased (58, 59). In ␤ERKO mice at days 730, tumors of the ovaries and gonadotropes in the pituitaries are observed, and neuropeptide Y expression that stimulates secretion of GnRH is high in the hypothalamus (31). These data indicate that the hypothalamus-pituitary-gonadal axis is affected by loss of ER␤. However, histology of the ovaries and estrous cycles in the ␤ERKO at days 90 and 365, and mice neonatally treated with ICI at day 90 were similar to those in WT mice. In the ovaries of neonatal ␤ERKO mice, follicle formation and development and Cyp11a1 expression is not different compared with that of WT mice (60, 61), suggesting that serum E2 levels in ␤ERKO mice are similar to those in WT mice during the neonatal period. Although Lei et al (62) show that mouse serum E2 levels from day 0 to 7 were ca. 20 – 80 pg/mL, serum E2 levels were not detected in this study and the expression of Cyp11a1 and aromatase was very low in neonatal mice. Therefore, changes in cell proliferation of uterine and vaginal epithelium in ␤ERKO mice may not be indirect effects of hypothalamus-pituitary-gonadal axis in ␤ERKO mice. Furthermore, ligand-independent ER␤ transcriptional activity is stimulated through phosphorylation of AF-1 domain by MAP kinase pathway (63, 64), and some genes are regulated by ligand-independent manner of ER␤ in breast cancer cell line (65). Thus, it may be considered that ligand-independent ER␤ actions can af-

Endocrinology

fect cell proliferation in the neonatal uterine and vaginal epithelium. In conclusion, neonatal ER␤ is important for the persistent inhibition of epithelial cell proliferation in the neonatal and adult mouse uterus. This inhibition is partially mediated through the accumulation of p27 protein in adult mouse uterus. Extreme endometrial hyperplasia with epithelial invasion of the stroma is found in some of 2 years old ␤ERKO mice (31). In the human endometrial cancer cells and tissues, E2 stimulates cell proliferation via the degradation of p27 (66, 67). Therefore, suppression of ER␤ function in the uterine epithelium during the neonatal period may cause destabilization of p27 in adult and be involved with a higher risk for proliferative diseases of human endometrium.

Acknowledgments We thank Dr. Raphael Guzman, Department of Molecular Cell Biology and Cancer Research Laboratory of University of California at Berkeley, for his critical reading of this manuscript. Address all correspondence and requests for reprints to: Dr. Tomomi Sato, Graduate School of Nanobioscience, Yokohama City University, 22–2 Seto, Kanazawa-ku, Yokohama 236 – 0027, Japan. Tel.: ⫹81– 45–787–2394; fax: ⫹81– 45–787– 2413; e-mail: [email protected]. This work was supported by a Grant-in-Aid for Scientific Research (B) (T.I.), a Grant-in-Aid for Encouragement of Young Scientists and a Grant-in-Aid for Scientific Research (C) (T.S.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Grants for Support of the Promotion of Research at Yokohama City University (No. W18005, K2109, G2314, G2401 and IR2502 to T.S.), a Health Sciences Research Grant from the Ministry of Health, Labor and Welfare, Japan (to T.I).. Disclosure Summary: None of the authors have any potential conflicts of interest associated with this research.

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Neonatal Estrogen Receptor β Is Important in the Permanent Inhibition of Epithelial Cell Proliferation in the Mouse Uterus.

Estrogen receptor α (ERα) plays a pivotal role in the mouse uterine and vaginal epithelial cell proliferation stimulated by estrogen, whereas ERβ inhi...
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