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Clomiphene citrate down-regulates estrogen receptor-α through the ubiquitin-proteasome pathway in a human endometrial cancer cell line
Accepted Manuscript Clomiphene citrate down-regulates estrogen receptor-α through the ubiquitinproteasome pathway in a human endometrial cancer cell line Mitsuyoshi Amita, Toshifumi Takahashi, Hideki Igarashi, Satoru Nagase PII:
S0303-7207(16)30077-6
DOI:
10.1016/j.mce.2016.03.029
Reference:
MCE 9464
To appear in:
Molecular and Cellular Endocrinology
Received Date: 9 November 2015 Revised Date:
21 March 2016
Accepted Date: 22 March 2016
Please cite this article as: Amita, M., Takahashi, T., Igarashi, H., Nagase, S., Clomiphene citrate downregulates estrogen receptor-α through the ubiquitin-proteasome pathway in a human endometrial cancer cell line, Molecular and Cellular Endocrinology (2016), doi: 10.1016/j.mce.2016.03.029. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Clomiphene citrate down-regulates estrogen receptor-α through the ubiquitin-proteasome
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pathway in a human endometrial cancer cell line
Mitsuyoshi Amita, Toshifumi Takahashi, Hideki Igarashi, and Satoru Nagase
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Department of Obstetrics and Gynecology, Yamagata University Faculty of Medicine,
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Yamagata 990-9585, Japan.
Correspondence: Toshifumi Takahashi, M.D., Department of Obstetrics and Gynecology, Yamagata University Faculty of Medicine, Yamagata 990-9585, Japan. Phone: +81-23-628-5393,
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Fax: +81-23-628-5396, E-mail: [email protected]
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ABSTRACT We examined how clomiphene citrate (CC) reduces estrogen receptor-α (ERα) in a human endometrial cancer cell line. Ishikawa human endometrial cancer cells were treated with ERα
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ligands such as 17β-estradiol (E2), CC, and the pure antiestrogen, ICI 182,780 (ICI). Thereafter, the expression levels of ERα protein and mRNA were analyzed by western blot and real-time quantitative PCR, respectively, and those of ubiquitinated ERα were analyzed by immunoprecipitation of ERα followed by immunoblotting with an anti-ubiquitin antibody. The
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expression levels of ERα protein after treatment with E2, CC, and ICI were significantly decreased compared to pre-treatment levels without a corresponding decrease in ERα mRNA.
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These ligands significantly increased the levels of ubiquitinated ERα compared to vehicle treatment. Co-treatment with the proteasome inhibitor, MG132, abrogated the decrease in ERα levels caused by treatment with the ligands only. We demonstrated, for the first time, a CC-induced decrease in ERα mediated by the ubiquitin-proteasome pathway in human
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endometrial cancer cells.
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Keywords: clomiphene citrate, estrogen receptor-α, endometrium, ubiquitination, proteasome
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1. Introduction For more than four decades, clomiphene citrate (CC) has been widely used as the first-line treatment for anovulatory infertile women. Although CC restores ovulation in about 80% of
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anovulatory women, the subsequent pregnancy rate is only about 20–40% (Kousta, White and Franks, 1997). The anti-estrogenic effects of CC on the cervix and uterine endometrium have been proposed to cause this low pregnancy rate (Eden, Place, Carter et al., 1989, Gonen and Casper, 1990, Nakamura, Ono, Yoshida et al., 1997, Randall and Templeton, 1991).
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Appropriate endometrial thickness is essential for embryo implantation (Friedler, Schenker, Herman et al., 1996, Senturk and Erel, 2008), with a thickness of at least 6 mm required to
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achieve a clinical pregnancy by artificial insemination (Gonen, Calderon, Dirnfeld et al., 1991). However, CC treatment significantly reduces the endometrial thickness in patients compared to non-CC treatment cycles (Nakamura et al., 1997, Bromer, Aldad and Taylor, 2009, Peeraer, Debrock, De Loecker et al., 2015).
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The anti-estrogenic effects of CC on the endometrium have been explained by two mechanisms: competition with estrogen and reduction of the number of estrogen receptors (ERs) (Kokko, Janne, Kauppila et al., 1981). We recently showed that CC inhibited estrogen-induced estrogen receptor-α (ERα) transactivation by inhibiting the recruitment of
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steroid receptor coactivator-1 in both human endometrial cancer cells and endometrial epithelial cells (Amita, Takahashi, Tsutsumi et al., 2010). However, the mechanism by which CC reduces
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the number of ERs in the endometrium has not been determined. Cellular levels of ERs are maintained by receptor degradation through the ubiquitin-26S
proteasome pathway (Nawaz, Lonard, Dennis et al., 1999, El Khissiin and Leclercq, 1999, Lonard, Nawaz, Smith et al., 2000, Wijayaratne and McDonnell, 2001, Nonclercq, Journe, Body et al., 2004, Laios, Journe, Nonclercq et al., 2005). In the presence of estrogen, ER-heat shock protein (HSP) complexes dissociate to form estrogen-ER complexes and recruit coactivators (Parker, 1995, Wurtz, Bourguet, Renaud et al., 1996, Pratt and Toft, 1997, Robyr, Wolffe and Wahli, 2000). After DNA binding, the estrogen-ER complexes are ubiquitinated and targeted
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for degradation by the 26S proteasome (El Khissiin and Leclercq, 1999, Lonard et al., 2000), which is known as transcription-coupled ER degradation (Berry, Fan and Nephew, 2008). The anti-estrogenic agent ICI 182,780 (ICI) also binds to ERs and stimulates ER release from HSP
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complexes; however, ICI does not recruit coactivators to ERs and blocks receptor transactivation. After ICI binding, ERs are degraded through the ubiquitin-proteasome pathway independent of transactivation (Wijayaratne and McDonnell, 2001, Reid, Hubner, Metivier et al., 2003, Stenoien, Patel, Mancini et al., 2001). Therefore, ICI is a member of an emerging
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category of ER ligands called selective estrogen receptor down-regulators (SERDs) (McDonnell, 2005).
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Because CC reduces the levels of cellular ERs in the endometrium (Kokko et al., 1981, Xia, Zhou and Huang, 1996, Aksel, Saracoglu, Yeoman et al., 1986, Fritz, Holmes and Keenan, 1991), CC might similarly induce ER degradation via the ubiquitin-proteasome pathway. However, to our knowledge, no studies have investigated whether CC induces ER degradation
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in estrogen-targeting cells. In this study, we demonstrate that CC induces ubiquitination and
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subsequent degradation of ERα in a human endometrial cancer cell line.
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2. Materials and Methods 2.1 Reagents CC, 17β-estradiol (E2), and ICI were obtained from Sigma Chemical Co. (St. Louis,
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MO, USA). These reagents were dissolved in ethanol and diluted to their working concentrations with medium. In all experiments, the final concentration of ethanol was 0.1% or less, which did not affect the results (data not shown). The normal rabbit IgG
(sc-2027), anti-ERα (sc-542 and sc-8002), and anti-α-tubulin (sc-5286) antibodies were
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obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-ubiquitin antibody (#3936) for western blotting analysis was obtained from Cell Signaling
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Technology (Beverly, MA, USA). Protein G Plus-Agarose immunoprecipitation reagent (sc-2002) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
2.2 Plasmids
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Green fluorescent protein (GFP)-ERα expression vectors were kind gifts from Dr. Michael A. Mancini (Department of Molecular and Cellular Biology, Baylor College of
2.3 Cell culture
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Medicine, Houston, TX, USA) (Stenoien, Mancini, Patel et al., 2000).
Ishikawa human endometrial cancer cells were kind gift from Dr. Nishida
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(Kasumigaura Medical Center, Tuchiura, Japan). Ishikawa cells were plated at a density of 3 × 104 cells per well in 6-well plates (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ, USA). The cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Sigma Chemical Co., St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA), 100 units/mL penicillin G sodium, and 100 µg/mL streptomycin sulfate in the presence of 5% CO2 at 37 °C.
2.4 Western blot analysis
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After the treatments, the cells were washed twice with PBS and lysed in ice-cold 1× lysis buffer (Cell Signaling Technology, Beverly, MA, USA) containing 20 mM Tris-HCl (pH 7.5), 150 mM sodium chloride, 1% Triton X-100, 1 mM disodium
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ethylenediaminetetraacetic acid (EDTA), 1 mM ethyleneglycoltetraacetic acid (EGTA), 2.5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and 1 µg/mL leupeptin, supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF). The lysates were centrifuged at 14,000 rpm for 10 min at 4 °C, and the protein concentrations of the
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supernatants were determined using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of proteins were separated by
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SDS-polyacrylamide gel electrophoresis (PAGE) (Atto, Tokyo, Japan) and transferred to polyvinylidene difluoride membranes (GE healthcare, Tokyo, Japan). The membranes were blocked in 5% nonfat milk or 5% bovine serum albumin (Sigma Chemical Co., St. Louis, MO, USA) in 1× Tris-buffered saline (Wako Chemical, Osaka,
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Japan). Western blot analyses were performed with various specific primary antibodies. Immunoreactive bands in the immunoblots were visualized with horseradish peroxidase-coupled goat anti-rabbit (sc-2004, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-mouse (sc-2005, Santa Cruz Biotechnology, Santa Cruz, CA, USA)
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immunoglobulin using the enhanced chemiluminescence western blotting system.
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2.5 Real-time quantitative PCR
Total RNA was isolated from Ishikawa cells treated with vehicle (ethanol), E2 (10–8 M), CC (10–6 M), or ICI (10–6 M) for 24 h using ISOGEN (Nippon Gene Co., Ltd., Tokyo, Japan) according to the manufacturer’s instructions. Complementary DNA was generated using the SuperScript II First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Quantitative PCR was performed using the ABI 7300 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) under the following conditions: 10 min at 95 °C
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followed by 40 cycles of 30 s at 95 °C, 1 min at 60 °C, and 1 min at 72 °C. TaqMan Probes for ESR1 (HS00174860_m1) and GAPDH (HS99999905_m1) were purchased from Applied Biosystems (Foster City, CA, USA). The levels of mRNA expression
System SDS software (Applied Biosystems, Foster City, CA, USA).
2.6 Immunoprecipitation
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were quantitated using the standard curve method of relative quantification using 7300
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Cellular lysates were pre-cleaned by incubation with normal rabbit IgG (sc-2027) and Protein G Plus-Agarose immunoprecipitation reagent (sc-2002) for 2 h at 4 °C. The
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lysates were centrifuged at 5,000 rpm for 3 min at 4 °C. After pre-cleaning, the lysates were incubated with the primary antibody against ERα (sc-542) (1:100) overnight at 4 °C. Protein G Plus-Agarose immunoprecipitation reagent (40 µL) was added to the lysate followed by incubation for 4 h at 4 °C. The lysates were centrifuged at 10,000
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rpm for 10 s, and pellets were washed four times with 1× lysis buffer plus 1 mM PMSF and resuspended in 60 µL total volume. After adding SDS loading buffer, the samples were heated for 5 min at 100 °C. The beads were pelleted by centrifugation at 10,000 rpm for 10 s at 4 °C. The supernatants were then analyzed by SDS-PAGE and
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subsequent immunoblotting as described above.
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2.7 DNA transfection
DNA transfection was performed as previously described (Amita et al., 2010). GFP-ERα-expressing vectors were used for live microscopy. The day before the DNA transfection, Ishikawa cells were plated at a density of 1.5 × 105 cells in a 35-mm glass-based dish (Iwaki Science Products, Tokyo, Japan). GFP-ERα DNA (1 µg) and Plus Reagent (6 µL) (Invitrogen, Carlsbad, CA, USA) were diluted in Opti-MEM 1 Reduced Serum Medium (Invitrogen, Carlsbad, CA, USA) without serum and mixed gently for 15 min at room temperature. Lipofectamine (4 µL) (Invitrogen, Carlsbad,
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CA, USA) was diluted in Opti-MEM 1 Reduced Serum Medium without serum and mixed gently for 15 min at room temperature. Both transfection media were mixed gently and kept for 15 min at room temperature before adding 0.8 mL serum-free
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medium. After mixing, 1 mL mixed transfection medium was placed in each well, and the cells were incubated in the presence of 5% CO2 at 37 °C. After 3 h, the medium was
replaced with DMEM supplemented with 10% fetal bovine serum, 100 units/mL
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penicillin G sodium, and 100 µg/mL streptomycin sulfate.
2.8 Live microscopy
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Live microscopy was performed on cells transfected with 1 µg GFP-ERα in a 35-mm glass-based dish. Live cell image acquisition was performed at 37 °C with a confocal microscope (LSM 510 META microscope; Carl Zeiss Co., Ltd., Jena, Germany) after 24 h treatment with ethanol (vehicle), E2 (10–8 M), CC (10–6 M), or ICI (10–6 M). Cell
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images were obtained through a ×63 objective lens by excitation with the 488-nm line from a krypton-argon laser, and the emission was viewed through a 506- to 538-nm band pass filter. The captured images were processed using the ImageJ system software
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(version 1.4) (NIH, Bethesda, MD, USA; http://rsb.info.nih.gov/ij/).
2.9 Statistical analysis
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All data are presented as mean ± SEM. Multiple group comparisons were made by one-way ANOVA followed by Scheffé’s F-test using StatView 5.0 software (Abacus Concepts, Berkeley, CA, USA). Significant differences were defined as those with P < 0.05.
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3. Results 3.1 CC reduces ERα protein expression without decreasing its mRNA level We first examined the effects of E2, CC, and ICI on the ERα protein expression in Ishikawa
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cells (Fig 1). Preliminary data suggested that the expressions of ERα protein were reduced from 1 h to 24 h of treatment with the ligands. Then, cells treated with E2, CC, and ICI for 1, 3, 6, and 24 h were collected, and the protein levels of ERα were analyzed by western blotting. The expression levels of ERα after treatment with E2, CC, and ICI were significantly (P < 0.05)
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decreased within 3 h compared to those before treatment (time = 0). While the levels of ERα in cells treated with E2 and ICI were significantly (P < 0.05) decreased 1 h after treatment, those
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in cells treated with CC were decreased 3 h after treatment. On the other hand, the expression levels of ERα mRNA determined by real-time quantitative PCR analysis 24 h after treatment with E2, CC, and ICI were not significantly (P = 0.82) different compared to vehicle alone.
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3.2 CC degrades ERα via the ubiquitin-proteasome pathway
We next examined whether CC could decrease ERα via the ubiquitin-proteasome pathway. Because the ligand-induced polyubiquitination of ERα followed by proteasome-mediated degradation is very fast (Ben-Nissan and Sharon, 2014), we used MG132, a proteasome
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inhibitor, to prevent ERα degradation by the ligands. We examined the effects of E2, CC, and ICI on the expression of ubiquitinated ERα in the presence of MG132 by immunoprecipitation
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of ERα followed by immunoblotting with an anti-ubiquitin antibody (Fig 2). E2, CC, and ICI significantly increased the levels of ubiquitinated ERα compared to vehicle alone. The amounts of ubiquitinated ERα treated with CC or ICI were significantly larger than those treated with E2. These results suggest that CC induced polyubiquitination of ERα. Therefore, we examined whether CC decreases ERα through proteasomal degradation. The levels of ERα in cells treated with E2, CC, and ICI were significantly decreased compared to those treated with vehicle and MG132 alone. Co-treatment with MG132 and E2, CC, or ICI recovered the levels of ERα
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relative to treatment with the ligands only (Fig 3). Together, these results suggest that CC decreases ERα via the ubiquitin-proteasome pathway.
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3.3 Effects of ligands on cellular localization of GFP-ERα We examined the effects of ligands on ERα localization using GFP-ERα in Ishikawa cells (Fig 4). The localization of GFP-ERα was categorized into two patterns: nuclear localization was defined by GFP-ERα expression in the nucleus only (Fig 4A, left panel), and nuclear and
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cytoplasmic localization was defined by GFP-ERα localization in both the nucleus and cytoplasm (Fig 4A, middle and right panels). In the absence of ligands (control or vehicle),
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about 80% of GFP-ERα was localized in the nucleus. The pattern of ERα localization was not changed in the cells treated with E2 and CC. However, the ratio of cells expressing GFP-ERα in both the nucleus and cytoplasm was significantly increased by ICI treatment compared to E2 or
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CC treatments.
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4. Discussion In the present study, we demonstrated that CC reduced ERα protein levels without a corresponding decrease of the mRNA through the ubiquitin-proteasome pathway in Ishikawa
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endometrial cancer cells. Several previous reports indicated that CC affects the expression of ERs in the human endometrium (Kokko et al., 1981, Xia et al., 1996, Aksel et al., 1986, Fritz et al., 1991). Cyclic CC treatment lowers both ER and progesterone receptor (PR) concentrations in the
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endometrium of postmenopausal women who receive estrogen replacement therapy (Kokko et al., 1981) and in the luteal-phase endometrium in anovulatory or oligoovulatory women
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compared to normally ovulatory women without CC treatment (Aksel et al., 1986). Conversely, CC treatment does not affect the amount of endometrial ER and PR during the luteal phase in normally ovulatory women (Hecht, Khan-Dawood and Dawood, 1989). Moreover, there is no difference between the cytosolic and nuclear ER concentrations of the luteal-phase
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endometrium during CC treatment cycles and those during spontaneous cycles in normally ovulatory women (Fritz et al., 1991). However, reports regarding changes in endometrial ERs after CC treatment present conflicting results, possibly due to the methodologies used. Because the endometrium consists of glandular epithelial cells and stromal cells and the spatio-temporal
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expression of ER changes according to endometrial dates (Bouchard, Marraoui, Massai et al., 1991), endometrial samples were not separated by cell type in these reports (Kokko et al., 1981,
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Aksel et al., 1986, Fritz et al., 1991, Hecht et al., 1989). In fact, Xia et al. reported that the immunohistological expressions of both ERs and PRs in the late proliferative and secretory phase of CC treatment cycles were significantly lower than those of spontaneous cycles in normally ovulatory infertile women and that ERs are significantly decreased in glandular cells compared to stromal cells (Xia et al., 1996). Despite studies on the in vivo expression of endometrial ERs in CC-treated women, to our knowledge there are no studies investigating the effects of CC on the in vitro expression of ERs in endometrial cells, including both epithelial and stromal cells. Most studies on ligand-induced
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ER expression are performed using breast cancer cell lines such as MCF-7 cells (Wijayaratne and McDonnell, 2001, Reid et al., 2003, Marsaud, Gougelet, Maillard et al., 2003, Yeh, Shioda, Coser et al., 2013). It is well known that ERα protein turnover results from polyubiquitination of
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receptor lysines, K302 and K303, and subsequent proteasomal degradation by the 26S proteasome (Berry et al., 2008, Marsaud et al., 2003). Both K302 and K303 are located within the hinge-region of ERα, which is important for coactivator binding and subsequent transcriptional activity (Kadiyala and Smith, 2014). In the absence of ligands, both K302 and
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K303 preserve ERα stability and protect basal ERα degradation by limiting ubiquitination (Berry et al., 2008). E2 and ICI, an agonist and a pure antagonist of ERα, respectively, in both
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the endometrium and breast cancer cells, induce ERα ubiquitination and subsequent degradation by the 26S proteasome through different mechanisms (Wijayaratne and McDonnell, 2001, Berry et al., 2008). Whereas E2 induces monoubiquitination of K302 by BRCA1/BARD1, a ubiquitin ligase (Eakin, Maccoss, Finney et al., 2007), ICI induces polyubiquitination of both K302 and
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K303 (Berry et al., 2008), and proteasomal degradation leads to the subsequent decrease in ERα. Therefore, the hinge-region lysine residues of ERα, K302 and K303, may be important for E2 and ICI-induced degradation of ERα in breast cancer cells. However, there have been no reports on how ligands such as E2 and ICI decrease ERα protein levels in endometrial cells. In
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the present study, we showed that E2 and ICI decrease the ERα protein levels without decreasing mRNA levels through the ubiquitin-proteasome pathway in Ishikawa endometrial
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cancer cells. The similar ERα degradation pathways after treatment with E2 and ICI may suggest further similarities between the mechanisms in breast cancer and endometrial cancer cells.
To our knowledge, no previous study has reported how CC decreases ERα levels. In the
present study, CC, E2, and ICI, decreased ERα protein levels without decreasing mRNA levels through the ubiquitin-proteasome pathway in Ishikawa endometrial cancer cells. CC is a racemic mixture of two stereoisomers: zuclomiphene and enclomiphene (Crewe, Ghobadi, Gregory et al., 2007). Whereas zuclomiphene has a weak estrogen agonistic effect and potent
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antagonistic effect, enclomiphene shows only an antagonistic effect (Glasier, 1990). Until recently, CC was classified as a selective estrogen receptor modulator (SERM) because several animal studies showed that CC has estrogenic effects in the skeletal and cardiovascular systems
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(Chakraborty, Brown, Ruff et al., 1991, Jimenez, Magee, Bryant et al., 1997, Turner, Evans, Sluka et al., 1998). However, we previously reported that CC has an anti-estrogenic activity in both human endometrial cancer cells and endometrial epithelial cells (Amita et al., 2010); therefore, CC may be classified as a pure antagonist of ERα, like ICI, rather than a SERM.
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Thus, we surmised that the CC-induced decrease of ERα protein might arise from a mechanism similar to that with ICI. However, the precise mechanisms of CC-induced degradation of ERα
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remain unknown.
What is the difference between CC and ICI? We previously showed a dynamic change in the intra-nuclear localization and mobility of ERα in the presence of ligands such as E2, CC, and ICI by live cell microscopy using GFP-ERα in Ishikawa endometrial cancer cells (Amita et
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al., 2010). Although the changes induced in the intra-nuclear GFP-ERα distribution by CC or ICI are similar, the mobile fraction of GFP-ERα with CC treatment is greater than that with ICI based on fluorescence recovery after photobleaching (FRAP) measurements. Ligands decrease GFP-ERα mobility: the mobile fraction decreases in the order ICI, CC, and E2 (ICI > CC > E2)
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(Amita et al., 2010). As treatment with MG132, a proteasome inhibitor, results in the virtual loss of GFP-ERα mobility in FRAP assays, the ubiquitin-proteasome pathway involves
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ligand-induced changes of ERα mobility (Reid et al., 2003, Stenoien et al., 2001). On the other hand, we showed different GFP-ERα cellular localization in Ishikawa endometrial cancer cells treated with CC and ICI. Two studies reported that, whereas ICI treatment results in both nuclear and cytoplasmic distribution of GFP-ERα, E2 and 4-hydroxytamoxifene, a partial antagonist for ERα, have no effect on the nucleocytoplasmic compartmentalization of GFP-ERα in ERα-positive breast cancer cells, such as MCF-7 and T47D cells (Htun, Holth, Walker et al., 1999, Long and Nephew, 2006). These reports are consistent with our results. A similar effect of ICI on nucleocytoplasmic compartmentalization is reported in COS-1 cells transfected with
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untagged ER (Dauvois, White and Parker, 1993). Moreover, RU 58668, a pure ERα antagonist, changes the ERα localization from the nucleus to cytoplasm in COS-7 cells transfected with untagged ER (Devin-Leclerc, Meng, Delahaye et al., 1998). Together, these results suggest that
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ligand-induced changes in ERα localization may be similar in ER-positive and ER-negative cells transfected with GFP-tagged or untagged ERα. Although both CC and ICI have similar anti-estrogenic effects in Ishikawa endometrial cancer cells and endometrial epithelial cells, the
clarify the mechanisms behind this observed effect.
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cause of different ERα cellular localization remains unknown. Further examination is needed to
Recently, drugs targeting the ubiquitin-proteasome pathway have been developed for
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cancer therapy (Pellom and Shanker, 2012). The proteasome inhibitor bortezomib has been used to treat multiple myeloma (Merin and Kelly, 2014). However, as proteasome inhibitors can show reproductive toxicity, this type of drug cannot be used in patients treated for infertility. Therefore, another approach is needed to improve the endometrial environment during
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implantation in women taking CC.
In summary, we demonstrated, for the first time to our knowledge, a CC-induced decrease in ERα protein levels through the ubiquitin-proteasome pathway in Ishikawa endometrial cancer cells. In our future work, we will examine the precise mechanisms of ubiquitin-mediated
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degradation of ERα stimulated by CC. This study is clinically relevant to infertile female patients treated with CC and may provide a foundation for improving pregnancy rates of these
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patients in the future.
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Conflict of interest: The authors report no conflict of interest.
Acknowledgments: This study was supported by Grants-in-Aid for General Science Research
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to Toshifumi Takahashi (No. 25462550) and Hideki Igarashi (No. 26462474). The funding source played no role in study design or the collection, analysis, and interpretation of data; in
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the writing of the report; or in the decision to submit the article for publication.
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Lonard, D.M., Nawaz, Z., Smith, C.L. and O'Malley, B.W., 2000. The 26S proteasome is required for estrogen receptor-alpha and coactivator turnover and for efficient estrogen receptor-alpha transactivation, Mol Cell. 5, 939-48. Wijayaratne, A.L. and McDonnell, D.P., 2001. The human estrogen receptor-alpha is a ubiquitinated protein whose stability is affected differentially by agonists, antagonists, and selective estrogen receptor modulators, J Biol Chem. 276, 35684-92. Nonclercq, D., Journe, F., Body, J.J., Leclercq, G. and Laurent, G., 2004. Ligand-independent and agonist-mediated degradation of estrogen receptor-alpha in breast carcinoma cells: evidence for distinct degradative pathways, Mol Cell Endocrinol. 227, 53-65. Laios, I., Journe, F., Nonclercq, D., Vidal, D.S., Toillon, R.A., Laurent, G. and Leclercq, G., 2005. Role of the proteasome in the regulation of estrogen receptor alpha turnover and function in MCF-7 breast carcinoma cells, J Steroid Biochem Mol Biol. 94, 347-59. Parker, M.G., 1995. Structure and function of estrogen receptors, Vitam Horm. 51, 267-87. Wurtz, J.M., Bourguet, W., Renaud, J.P., Vivat, V., Chambon, P., Moras, D. and Gronemeyer, H., 1996. A canonical structure for the ligand-binding domain of nuclear receptors, Nat Struct Biol. 3, 87-94. Pratt, W.B. and Toft, D.O., 1997. Steroid receptor interactions with heat shock protein and immunophilin chaperones, Endocr Rev. 18, 306-60. Robyr, D., Wolffe, A.P. and Wahli, W., 2000. Nuclear hormone receptor coregulators in action: diversity for shared tasks, Mol Endocrinol. 14, 329-47. Berry, N.B., Fan, M. and Nephew, K.P., 2008. Estrogen receptor-alpha hinge-region lysines 302 and 303 regulate receptor degradation by the proteasome, Mol Endocrinol. 22, 1535-51. Reid, G., Hubner, M.R., Metivier, R., Brand, H., Denger, S., Manu, D., Beaudouin, J., Ellenberg, J. and Gannon, F., 2003. Cyclic, proteasome-mediated turnover of unliganded and liganded ERalpha on responsive promoters is an integral feature of estrogen signaling, Mol Cell. 11, 695-707. Stenoien, D.L., Patel, K., Mancini, M.G., Dutertre, M., Smith, C.L., O'Malley, B.W. and Mancini, M.A., 2001. FRAP reveals that mobility of oestrogen receptor-alpha is ligand- and proteasome-dependent, Nat Cell Biol. 3, 15-23. McDonnell, D.P., 2005. The molecular pharmacology of estrogen receptor modulators: implications for the treatment of breast cancer, Clin Cancer Res. 11, 871s-7s. Xia, Y., Zhou, F. and Huang, H., 1996. [Effects of clomiphene citrate treatment on endometrial estrogen and progesterone receptors expressions], Zhonghua Fu Chan Ke Za Zhi. 31, 606-9. Aksel, S., Saracoglu, O.F., Yeoman, R.R. and Wiebe, R.H., 1986. Effects of clomiphene citrate on cytosolic estradiol and progesterone receptor concentrations in secretory endometrium, Am J Obstet Gynecol. 155, 1219-23. Fritz, M.A., Holmes, R.T. and Keenan, E.J., 1991. Effect of clomiphene citrate treatment on endometrial estrogen and progesterone receptor induction in women, Am J Obstet Gynecol. 165, 177-85.
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Figure legends Figure 1. Effects of ligand treatment on ERα protein expression After serum-free starvation for 24 h, Ishikawa cells were treated with vehicle (ethanol), E2 (10–8
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M), CC (10–6 M), or ICI (10–6 M). After 1, 3, 6, and 24 h of treatment, the cell lysates were subjected to SDS-PAGE (5–20% separating gel), and western blotting was performed using an anti-ERα antibody (sc-542). Relative densitometric units of the ERα bands normalized to the levels of α-tubulin are shown in the top panels, with the density of the bands from the lysates
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before treatment (time = 0) set arbitrarily to 1.0. The values shown represent the mean ± SEM of triplicate experiments. The figure is the representative of results from at least three
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independent experiments. Asterisks indicate significant differences between the groups (P < 0.05). N.S., not significant; E2, 17β-estradiol; CC, clomiphene citrate; ICI, ICI 182,780.
Figure 2. Effects of ligand treatment on ERα ubiquitination
After serum-free starvation for 24 h, Ishikawa cells were treated with vehicle (ethanol), E2 (10–8
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M), CC (10–6 M), or ICI (10–6 M). The lysates from cells after 24 h treatment were subjected to immunoprecipitation using an anti-ERα antibody (sc-542). MG132, a proteasome inhibitor, was added to prevent ubiquitinated ERα degradation. The immunoprecipitates were subjected to
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SDS-PAGE (5–20% separating gel), and western blotting was performed using an anti-ubiquitin (middle panel) (#3936) or anti-ERα (lower panel) antibody (sc-8002). Relative densitometric
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units of the ubiquitin bands with the density of the vehicle bands set arbitrarily to 1.0 are shown in the top panel. The values shown represent the mean ± SEM of triplicate experiments. The figure is the representative of results from at least three independent experiments. Asterisks indicate significant differences between the groups (P < 0.05). E2, 17β-estradiol; CC, clomiphene citrate; ICI, ICI 182,780.
Figure 3. MG132 reverses the ligand-induced degradation of ERα
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After serum-free starvation for 24 h, Ishikawa cells were treated with vehicle (ethanol), MG132 (10–5 M), E2 (10–8 M), CC (10–6 M), ICI (10–6 M), E2 (10–8 M) + MG132 (10–5 M), CC (10–6 M) + MG132 (10–5 M), or ICI (10–6 M) + MG132 (10–5 M) for 24 h. After the various treatments,
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the cell lysates were subjected to SDS-PAGE (5-–20% separating gel), and western blotting was performed using an anti-ERα antibody (sc-542). Relative densitometric units of the ERα bands normalized to the levels of α-tubulin are shown in the top panels, with the density of the bands from vehicle treatment set arbitrarily to 1.0. The values shown represent the mean ± SEM of
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triplicate experiments. The figure is the representative of results from at least three independent experiments. Asterisks indicate significant differences between the groups (P < 0.05). E2,
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17β-estradiol; CC, clomiphene citrate; ICI, ICI 182,780.
Figure 4. Effects of ligands on cellular localization of GFP-ERα A.
Ishikawa
cells
were
transiently transfected with
a green fluorescent
protein
(GFP)-ERα-expressing vector, followed by 24 h of serum starvation and treatment with vehicle
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(ethanol), E2 (10–8 M), CC (10–6 M), or ICI (10–6 M) for 24 h. The cells transfected with GFP-ERα before the treatment were used as a control. The localization of GFP-ERα was categorized into two patterns: nucleus only (left panel) or nucleus and cytoplasm (middle and
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right panels). Bar, 10 µm. B. The ratios of cells with GFP-ERα expression in the nucleus (black bar) and nucleus + cytoplasm (white bar) after various treatments are shown. Each bar
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represents 300 cells expressing GFP-ERα. Numbers inside bars indicate the numbers of cells with GFP-ERα expressions in the nucleus (black bar) or in the nucleus + cytoplasm (white bar)/total number of GFPα-expressing cells examined. E2, 17β-estradiol; CC, clomiphene citrate; ICI, ICI 182,780.
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Highlights Clomiphene citrate decreases estrogen receptor-α in an endometrial cancer cell line. Clomiphene citrate does not decrease estrogen receptor-α mRNA levels.
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Clomiphene citrate increases in the ubiquitination of estrogen receptor-α.
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Proteasome is involved in the degradation of estrogen receptor-α by clomiphene citrate.
S0303-7207(16)30077-6
DOI:
10.1016/j.mce.2016.03.029
Reference:
MCE 9464
To appear in:
Molecular and Cellular Endocrinology
Received Date: 9 November 2015 Revised Date:
21 March 2016
Accepted Date: 22 March 2016
Please cite this article as: Amita, M., Takahashi, T., Igarashi, H., Nagase, S., Clomiphene citrate downregulates estrogen receptor-α through the ubiquitin-proteasome pathway in a human endometrial cancer cell line, Molecular and Cellular Endocrinology (2016), doi: 10.1016/j.mce.2016.03.029. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Clomiphene citrate down-regulates estrogen receptor-α through the ubiquitin-proteasome
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pathway in a human endometrial cancer cell line
Mitsuyoshi Amita, Toshifumi Takahashi, Hideki Igarashi, and Satoru Nagase
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Department of Obstetrics and Gynecology, Yamagata University Faculty of Medicine,
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Yamagata 990-9585, Japan.
Correspondence: Toshifumi Takahashi, M.D., Department of Obstetrics and Gynecology, Yamagata University Faculty of Medicine, Yamagata 990-9585, Japan. Phone: +81-23-628-5393,
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Fax: +81-23-628-5396, E-mail: [email protected]
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ABSTRACT We examined how clomiphene citrate (CC) reduces estrogen receptor-α (ERα) in a human endometrial cancer cell line. Ishikawa human endometrial cancer cells were treated with ERα
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ligands such as 17β-estradiol (E2), CC, and the pure antiestrogen, ICI 182,780 (ICI). Thereafter, the expression levels of ERα protein and mRNA were analyzed by western blot and real-time quantitative PCR, respectively, and those of ubiquitinated ERα were analyzed by immunoprecipitation of ERα followed by immunoblotting with an anti-ubiquitin antibody. The
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expression levels of ERα protein after treatment with E2, CC, and ICI were significantly decreased compared to pre-treatment levels without a corresponding decrease in ERα mRNA.
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These ligands significantly increased the levels of ubiquitinated ERα compared to vehicle treatment. Co-treatment with the proteasome inhibitor, MG132, abrogated the decrease in ERα levels caused by treatment with the ligands only. We demonstrated, for the first time, a CC-induced decrease in ERα mediated by the ubiquitin-proteasome pathway in human
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endometrial cancer cells.
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Keywords: clomiphene citrate, estrogen receptor-α, endometrium, ubiquitination, proteasome
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1. Introduction For more than four decades, clomiphene citrate (CC) has been widely used as the first-line treatment for anovulatory infertile women. Although CC restores ovulation in about 80% of
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anovulatory women, the subsequent pregnancy rate is only about 20–40% (Kousta, White and Franks, 1997). The anti-estrogenic effects of CC on the cervix and uterine endometrium have been proposed to cause this low pregnancy rate (Eden, Place, Carter et al., 1989, Gonen and Casper, 1990, Nakamura, Ono, Yoshida et al., 1997, Randall and Templeton, 1991).
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Appropriate endometrial thickness is essential for embryo implantation (Friedler, Schenker, Herman et al., 1996, Senturk and Erel, 2008), with a thickness of at least 6 mm required to
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achieve a clinical pregnancy by artificial insemination (Gonen, Calderon, Dirnfeld et al., 1991). However, CC treatment significantly reduces the endometrial thickness in patients compared to non-CC treatment cycles (Nakamura et al., 1997, Bromer, Aldad and Taylor, 2009, Peeraer, Debrock, De Loecker et al., 2015).
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The anti-estrogenic effects of CC on the endometrium have been explained by two mechanisms: competition with estrogen and reduction of the number of estrogen receptors (ERs) (Kokko, Janne, Kauppila et al., 1981). We recently showed that CC inhibited estrogen-induced estrogen receptor-α (ERα) transactivation by inhibiting the recruitment of
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steroid receptor coactivator-1 in both human endometrial cancer cells and endometrial epithelial cells (Amita, Takahashi, Tsutsumi et al., 2010). However, the mechanism by which CC reduces
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the number of ERs in the endometrium has not been determined. Cellular levels of ERs are maintained by receptor degradation through the ubiquitin-26S
proteasome pathway (Nawaz, Lonard, Dennis et al., 1999, El Khissiin and Leclercq, 1999, Lonard, Nawaz, Smith et al., 2000, Wijayaratne and McDonnell, 2001, Nonclercq, Journe, Body et al., 2004, Laios, Journe, Nonclercq et al., 2005). In the presence of estrogen, ER-heat shock protein (HSP) complexes dissociate to form estrogen-ER complexes and recruit coactivators (Parker, 1995, Wurtz, Bourguet, Renaud et al., 1996, Pratt and Toft, 1997, Robyr, Wolffe and Wahli, 2000). After DNA binding, the estrogen-ER complexes are ubiquitinated and targeted
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for degradation by the 26S proteasome (El Khissiin and Leclercq, 1999, Lonard et al., 2000), which is known as transcription-coupled ER degradation (Berry, Fan and Nephew, 2008). The anti-estrogenic agent ICI 182,780 (ICI) also binds to ERs and stimulates ER release from HSP
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complexes; however, ICI does not recruit coactivators to ERs and blocks receptor transactivation. After ICI binding, ERs are degraded through the ubiquitin-proteasome pathway independent of transactivation (Wijayaratne and McDonnell, 2001, Reid, Hubner, Metivier et al., 2003, Stenoien, Patel, Mancini et al., 2001). Therefore, ICI is a member of an emerging
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category of ER ligands called selective estrogen receptor down-regulators (SERDs) (McDonnell, 2005).
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Because CC reduces the levels of cellular ERs in the endometrium (Kokko et al., 1981, Xia, Zhou and Huang, 1996, Aksel, Saracoglu, Yeoman et al., 1986, Fritz, Holmes and Keenan, 1991), CC might similarly induce ER degradation via the ubiquitin-proteasome pathway. However, to our knowledge, no studies have investigated whether CC induces ER degradation
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in estrogen-targeting cells. In this study, we demonstrate that CC induces ubiquitination and
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subsequent degradation of ERα in a human endometrial cancer cell line.
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2. Materials and Methods 2.1 Reagents CC, 17β-estradiol (E2), and ICI were obtained from Sigma Chemical Co. (St. Louis,
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MO, USA). These reagents were dissolved in ethanol and diluted to their working concentrations with medium. In all experiments, the final concentration of ethanol was 0.1% or less, which did not affect the results (data not shown). The normal rabbit IgG
(sc-2027), anti-ERα (sc-542 and sc-8002), and anti-α-tubulin (sc-5286) antibodies were
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obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-ubiquitin antibody (#3936) for western blotting analysis was obtained from Cell Signaling
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Technology (Beverly, MA, USA). Protein G Plus-Agarose immunoprecipitation reagent (sc-2002) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
2.2 Plasmids
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Green fluorescent protein (GFP)-ERα expression vectors were kind gifts from Dr. Michael A. Mancini (Department of Molecular and Cellular Biology, Baylor College of
2.3 Cell culture
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Medicine, Houston, TX, USA) (Stenoien, Mancini, Patel et al., 2000).
Ishikawa human endometrial cancer cells were kind gift from Dr. Nishida
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(Kasumigaura Medical Center, Tuchiura, Japan). Ishikawa cells were plated at a density of 3 × 104 cells per well in 6-well plates (Falcon, Becton Dickinson Labware, Franklin Lakes, NJ, USA). The cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Sigma Chemical Co., St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA), 100 units/mL penicillin G sodium, and 100 µg/mL streptomycin sulfate in the presence of 5% CO2 at 37 °C.
2.4 Western blot analysis
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After the treatments, the cells were washed twice with PBS and lysed in ice-cold 1× lysis buffer (Cell Signaling Technology, Beverly, MA, USA) containing 20 mM Tris-HCl (pH 7.5), 150 mM sodium chloride, 1% Triton X-100, 1 mM disodium
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ethylenediaminetetraacetic acid (EDTA), 1 mM ethyleneglycoltetraacetic acid (EGTA), 2.5 mM sodium pyrophosphate, 1 mM sodium orthovanadate, and 1 µg/mL leupeptin, supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF). The lysates were centrifuged at 14,000 rpm for 10 min at 4 °C, and the protein concentrations of the
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supernatants were determined using the Bio-Rad protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of proteins were separated by
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SDS-polyacrylamide gel electrophoresis (PAGE) (Atto, Tokyo, Japan) and transferred to polyvinylidene difluoride membranes (GE healthcare, Tokyo, Japan). The membranes were blocked in 5% nonfat milk or 5% bovine serum albumin (Sigma Chemical Co., St. Louis, MO, USA) in 1× Tris-buffered saline (Wako Chemical, Osaka,
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Japan). Western blot analyses were performed with various specific primary antibodies. Immunoreactive bands in the immunoblots were visualized with horseradish peroxidase-coupled goat anti-rabbit (sc-2004, Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-mouse (sc-2005, Santa Cruz Biotechnology, Santa Cruz, CA, USA)
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immunoglobulin using the enhanced chemiluminescence western blotting system.
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2.5 Real-time quantitative PCR
Total RNA was isolated from Ishikawa cells treated with vehicle (ethanol), E2 (10–8 M), CC (10–6 M), or ICI (10–6 M) for 24 h using ISOGEN (Nippon Gene Co., Ltd., Tokyo, Japan) according to the manufacturer’s instructions. Complementary DNA was generated using the SuperScript II First-Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Quantitative PCR was performed using the ABI 7300 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA) under the following conditions: 10 min at 95 °C
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followed by 40 cycles of 30 s at 95 °C, 1 min at 60 °C, and 1 min at 72 °C. TaqMan Probes for ESR1 (HS00174860_m1) and GAPDH (HS99999905_m1) were purchased from Applied Biosystems (Foster City, CA, USA). The levels of mRNA expression
System SDS software (Applied Biosystems, Foster City, CA, USA).
2.6 Immunoprecipitation
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were quantitated using the standard curve method of relative quantification using 7300
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Cellular lysates were pre-cleaned by incubation with normal rabbit IgG (sc-2027) and Protein G Plus-Agarose immunoprecipitation reagent (sc-2002) for 2 h at 4 °C. The
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lysates were centrifuged at 5,000 rpm for 3 min at 4 °C. After pre-cleaning, the lysates were incubated with the primary antibody against ERα (sc-542) (1:100) overnight at 4 °C. Protein G Plus-Agarose immunoprecipitation reagent (40 µL) was added to the lysate followed by incubation for 4 h at 4 °C. The lysates were centrifuged at 10,000
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rpm for 10 s, and pellets were washed four times with 1× lysis buffer plus 1 mM PMSF and resuspended in 60 µL total volume. After adding SDS loading buffer, the samples were heated for 5 min at 100 °C. The beads were pelleted by centrifugation at 10,000 rpm for 10 s at 4 °C. The supernatants were then analyzed by SDS-PAGE and
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subsequent immunoblotting as described above.
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2.7 DNA transfection
DNA transfection was performed as previously described (Amita et al., 2010). GFP-ERα-expressing vectors were used for live microscopy. The day before the DNA transfection, Ishikawa cells were plated at a density of 1.5 × 105 cells in a 35-mm glass-based dish (Iwaki Science Products, Tokyo, Japan). GFP-ERα DNA (1 µg) and Plus Reagent (6 µL) (Invitrogen, Carlsbad, CA, USA) were diluted in Opti-MEM 1 Reduced Serum Medium (Invitrogen, Carlsbad, CA, USA) without serum and mixed gently for 15 min at room temperature. Lipofectamine (4 µL) (Invitrogen, Carlsbad,
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CA, USA) was diluted in Opti-MEM 1 Reduced Serum Medium without serum and mixed gently for 15 min at room temperature. Both transfection media were mixed gently and kept for 15 min at room temperature before adding 0.8 mL serum-free
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medium. After mixing, 1 mL mixed transfection medium was placed in each well, and the cells were incubated in the presence of 5% CO2 at 37 °C. After 3 h, the medium was
replaced with DMEM supplemented with 10% fetal bovine serum, 100 units/mL
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penicillin G sodium, and 100 µg/mL streptomycin sulfate.
2.8 Live microscopy
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Live microscopy was performed on cells transfected with 1 µg GFP-ERα in a 35-mm glass-based dish. Live cell image acquisition was performed at 37 °C with a confocal microscope (LSM 510 META microscope; Carl Zeiss Co., Ltd., Jena, Germany) after 24 h treatment with ethanol (vehicle), E2 (10–8 M), CC (10–6 M), or ICI (10–6 M). Cell
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images were obtained through a ×63 objective lens by excitation with the 488-nm line from a krypton-argon laser, and the emission was viewed through a 506- to 538-nm band pass filter. The captured images were processed using the ImageJ system software
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(version 1.4) (NIH, Bethesda, MD, USA; http://rsb.info.nih.gov/ij/).
2.9 Statistical analysis
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All data are presented as mean ± SEM. Multiple group comparisons were made by one-way ANOVA followed by Scheffé’s F-test using StatView 5.0 software (Abacus Concepts, Berkeley, CA, USA). Significant differences were defined as those with P < 0.05.
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3. Results 3.1 CC reduces ERα protein expression without decreasing its mRNA level We first examined the effects of E2, CC, and ICI on the ERα protein expression in Ishikawa
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cells (Fig 1). Preliminary data suggested that the expressions of ERα protein were reduced from 1 h to 24 h of treatment with the ligands. Then, cells treated with E2, CC, and ICI for 1, 3, 6, and 24 h were collected, and the protein levels of ERα were analyzed by western blotting. The expression levels of ERα after treatment with E2, CC, and ICI were significantly (P < 0.05)
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decreased within 3 h compared to those before treatment (time = 0). While the levels of ERα in cells treated with E2 and ICI were significantly (P < 0.05) decreased 1 h after treatment, those
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in cells treated with CC were decreased 3 h after treatment. On the other hand, the expression levels of ERα mRNA determined by real-time quantitative PCR analysis 24 h after treatment with E2, CC, and ICI were not significantly (P = 0.82) different compared to vehicle alone.
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3.2 CC degrades ERα via the ubiquitin-proteasome pathway
We next examined whether CC could decrease ERα via the ubiquitin-proteasome pathway. Because the ligand-induced polyubiquitination of ERα followed by proteasome-mediated degradation is very fast (Ben-Nissan and Sharon, 2014), we used MG132, a proteasome
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inhibitor, to prevent ERα degradation by the ligands. We examined the effects of E2, CC, and ICI on the expression of ubiquitinated ERα in the presence of MG132 by immunoprecipitation
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of ERα followed by immunoblotting with an anti-ubiquitin antibody (Fig 2). E2, CC, and ICI significantly increased the levels of ubiquitinated ERα compared to vehicle alone. The amounts of ubiquitinated ERα treated with CC or ICI were significantly larger than those treated with E2. These results suggest that CC induced polyubiquitination of ERα. Therefore, we examined whether CC decreases ERα through proteasomal degradation. The levels of ERα in cells treated with E2, CC, and ICI were significantly decreased compared to those treated with vehicle and MG132 alone. Co-treatment with MG132 and E2, CC, or ICI recovered the levels of ERα
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relative to treatment with the ligands only (Fig 3). Together, these results suggest that CC decreases ERα via the ubiquitin-proteasome pathway.
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3.3 Effects of ligands on cellular localization of GFP-ERα We examined the effects of ligands on ERα localization using GFP-ERα in Ishikawa cells (Fig 4). The localization of GFP-ERα was categorized into two patterns: nuclear localization was defined by GFP-ERα expression in the nucleus only (Fig 4A, left panel), and nuclear and
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cytoplasmic localization was defined by GFP-ERα localization in both the nucleus and cytoplasm (Fig 4A, middle and right panels). In the absence of ligands (control or vehicle),
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about 80% of GFP-ERα was localized in the nucleus. The pattern of ERα localization was not changed in the cells treated with E2 and CC. However, the ratio of cells expressing GFP-ERα in both the nucleus and cytoplasm was significantly increased by ICI treatment compared to E2 or
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4. Discussion In the present study, we demonstrated that CC reduced ERα protein levels without a corresponding decrease of the mRNA through the ubiquitin-proteasome pathway in Ishikawa
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endometrial cancer cells. Several previous reports indicated that CC affects the expression of ERs in the human endometrium (Kokko et al., 1981, Xia et al., 1996, Aksel et al., 1986, Fritz et al., 1991). Cyclic CC treatment lowers both ER and progesterone receptor (PR) concentrations in the
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endometrium of postmenopausal women who receive estrogen replacement therapy (Kokko et al., 1981) and in the luteal-phase endometrium in anovulatory or oligoovulatory women
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compared to normally ovulatory women without CC treatment (Aksel et al., 1986). Conversely, CC treatment does not affect the amount of endometrial ER and PR during the luteal phase in normally ovulatory women (Hecht, Khan-Dawood and Dawood, 1989). Moreover, there is no difference between the cytosolic and nuclear ER concentrations of the luteal-phase
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endometrium during CC treatment cycles and those during spontaneous cycles in normally ovulatory women (Fritz et al., 1991). However, reports regarding changes in endometrial ERs after CC treatment present conflicting results, possibly due to the methodologies used. Because the endometrium consists of glandular epithelial cells and stromal cells and the spatio-temporal
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expression of ER changes according to endometrial dates (Bouchard, Marraoui, Massai et al., 1991), endometrial samples were not separated by cell type in these reports (Kokko et al., 1981,
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Aksel et al., 1986, Fritz et al., 1991, Hecht et al., 1989). In fact, Xia et al. reported that the immunohistological expressions of both ERs and PRs in the late proliferative and secretory phase of CC treatment cycles were significantly lower than those of spontaneous cycles in normally ovulatory infertile women and that ERs are significantly decreased in glandular cells compared to stromal cells (Xia et al., 1996). Despite studies on the in vivo expression of endometrial ERs in CC-treated women, to our knowledge there are no studies investigating the effects of CC on the in vitro expression of ERs in endometrial cells, including both epithelial and stromal cells. Most studies on ligand-induced
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ER expression are performed using breast cancer cell lines such as MCF-7 cells (Wijayaratne and McDonnell, 2001, Reid et al., 2003, Marsaud, Gougelet, Maillard et al., 2003, Yeh, Shioda, Coser et al., 2013). It is well known that ERα protein turnover results from polyubiquitination of
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receptor lysines, K302 and K303, and subsequent proteasomal degradation by the 26S proteasome (Berry et al., 2008, Marsaud et al., 2003). Both K302 and K303 are located within the hinge-region of ERα, which is important for coactivator binding and subsequent transcriptional activity (Kadiyala and Smith, 2014). In the absence of ligands, both K302 and
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K303 preserve ERα stability and protect basal ERα degradation by limiting ubiquitination (Berry et al., 2008). E2 and ICI, an agonist and a pure antagonist of ERα, respectively, in both
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the endometrium and breast cancer cells, induce ERα ubiquitination and subsequent degradation by the 26S proteasome through different mechanisms (Wijayaratne and McDonnell, 2001, Berry et al., 2008). Whereas E2 induces monoubiquitination of K302 by BRCA1/BARD1, a ubiquitin ligase (Eakin, Maccoss, Finney et al., 2007), ICI induces polyubiquitination of both K302 and
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K303 (Berry et al., 2008), and proteasomal degradation leads to the subsequent decrease in ERα. Therefore, the hinge-region lysine residues of ERα, K302 and K303, may be important for E2 and ICI-induced degradation of ERα in breast cancer cells. However, there have been no reports on how ligands such as E2 and ICI decrease ERα protein levels in endometrial cells. In
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the present study, we showed that E2 and ICI decrease the ERα protein levels without decreasing mRNA levels through the ubiquitin-proteasome pathway in Ishikawa endometrial
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cancer cells. The similar ERα degradation pathways after treatment with E2 and ICI may suggest further similarities between the mechanisms in breast cancer and endometrial cancer cells.
To our knowledge, no previous study has reported how CC decreases ERα levels. In the
present study, CC, E2, and ICI, decreased ERα protein levels without decreasing mRNA levels through the ubiquitin-proteasome pathway in Ishikawa endometrial cancer cells. CC is a racemic mixture of two stereoisomers: zuclomiphene and enclomiphene (Crewe, Ghobadi, Gregory et al., 2007). Whereas zuclomiphene has a weak estrogen agonistic effect and potent
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antagonistic effect, enclomiphene shows only an antagonistic effect (Glasier, 1990). Until recently, CC was classified as a selective estrogen receptor modulator (SERM) because several animal studies showed that CC has estrogenic effects in the skeletal and cardiovascular systems
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(Chakraborty, Brown, Ruff et al., 1991, Jimenez, Magee, Bryant et al., 1997, Turner, Evans, Sluka et al., 1998). However, we previously reported that CC has an anti-estrogenic activity in both human endometrial cancer cells and endometrial epithelial cells (Amita et al., 2010); therefore, CC may be classified as a pure antagonist of ERα, like ICI, rather than a SERM.
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Thus, we surmised that the CC-induced decrease of ERα protein might arise from a mechanism similar to that with ICI. However, the precise mechanisms of CC-induced degradation of ERα
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remain unknown.
What is the difference between CC and ICI? We previously showed a dynamic change in the intra-nuclear localization and mobility of ERα in the presence of ligands such as E2, CC, and ICI by live cell microscopy using GFP-ERα in Ishikawa endometrial cancer cells (Amita et
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al., 2010). Although the changes induced in the intra-nuclear GFP-ERα distribution by CC or ICI are similar, the mobile fraction of GFP-ERα with CC treatment is greater than that with ICI based on fluorescence recovery after photobleaching (FRAP) measurements. Ligands decrease GFP-ERα mobility: the mobile fraction decreases in the order ICI, CC, and E2 (ICI > CC > E2)
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(Amita et al., 2010). As treatment with MG132, a proteasome inhibitor, results in the virtual loss of GFP-ERα mobility in FRAP assays, the ubiquitin-proteasome pathway involves
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ligand-induced changes of ERα mobility (Reid et al., 2003, Stenoien et al., 2001). On the other hand, we showed different GFP-ERα cellular localization in Ishikawa endometrial cancer cells treated with CC and ICI. Two studies reported that, whereas ICI treatment results in both nuclear and cytoplasmic distribution of GFP-ERα, E2 and 4-hydroxytamoxifene, a partial antagonist for ERα, have no effect on the nucleocytoplasmic compartmentalization of GFP-ERα in ERα-positive breast cancer cells, such as MCF-7 and T47D cells (Htun, Holth, Walker et al., 1999, Long and Nephew, 2006). These reports are consistent with our results. A similar effect of ICI on nucleocytoplasmic compartmentalization is reported in COS-1 cells transfected with
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untagged ER (Dauvois, White and Parker, 1993). Moreover, RU 58668, a pure ERα antagonist, changes the ERα localization from the nucleus to cytoplasm in COS-7 cells transfected with untagged ER (Devin-Leclerc, Meng, Delahaye et al., 1998). Together, these results suggest that
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ligand-induced changes in ERα localization may be similar in ER-positive and ER-negative cells transfected with GFP-tagged or untagged ERα. Although both CC and ICI have similar anti-estrogenic effects in Ishikawa endometrial cancer cells and endometrial epithelial cells, the
clarify the mechanisms behind this observed effect.
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cause of different ERα cellular localization remains unknown. Further examination is needed to
Recently, drugs targeting the ubiquitin-proteasome pathway have been developed for
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cancer therapy (Pellom and Shanker, 2012). The proteasome inhibitor bortezomib has been used to treat multiple myeloma (Merin and Kelly, 2014). However, as proteasome inhibitors can show reproductive toxicity, this type of drug cannot be used in patients treated for infertility. Therefore, another approach is needed to improve the endometrial environment during
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implantation in women taking CC.
In summary, we demonstrated, for the first time to our knowledge, a CC-induced decrease in ERα protein levels through the ubiquitin-proteasome pathway in Ishikawa endometrial cancer cells. In our future work, we will examine the precise mechanisms of ubiquitin-mediated
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degradation of ERα stimulated by CC. This study is clinically relevant to infertile female patients treated with CC and may provide a foundation for improving pregnancy rates of these
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Conflict of interest: The authors report no conflict of interest.
Acknowledgments: This study was supported by Grants-in-Aid for General Science Research
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to Toshifumi Takahashi (No. 25462550) and Hideki Igarashi (No. 26462474). The funding source played no role in study design or the collection, analysis, and interpretation of data; in
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the writing of the report; or in the decision to submit the article for publication.
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Figure legends Figure 1. Effects of ligand treatment on ERα protein expression After serum-free starvation for 24 h, Ishikawa cells were treated with vehicle (ethanol), E2 (10–8
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M), CC (10–6 M), or ICI (10–6 M). After 1, 3, 6, and 24 h of treatment, the cell lysates were subjected to SDS-PAGE (5–20% separating gel), and western blotting was performed using an anti-ERα antibody (sc-542). Relative densitometric units of the ERα bands normalized to the levels of α-tubulin are shown in the top panels, with the density of the bands from the lysates
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before treatment (time = 0) set arbitrarily to 1.0. The values shown represent the mean ± SEM of triplicate experiments. The figure is the representative of results from at least three
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independent experiments. Asterisks indicate significant differences between the groups (P < 0.05). N.S., not significant; E2, 17β-estradiol; CC, clomiphene citrate; ICI, ICI 182,780.
Figure 2. Effects of ligand treatment on ERα ubiquitination
After serum-free starvation for 24 h, Ishikawa cells were treated with vehicle (ethanol), E2 (10–8
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M), CC (10–6 M), or ICI (10–6 M). The lysates from cells after 24 h treatment were subjected to immunoprecipitation using an anti-ERα antibody (sc-542). MG132, a proteasome inhibitor, was added to prevent ubiquitinated ERα degradation. The immunoprecipitates were subjected to
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SDS-PAGE (5–20% separating gel), and western blotting was performed using an anti-ubiquitin (middle panel) (#3936) or anti-ERα (lower panel) antibody (sc-8002). Relative densitometric
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units of the ubiquitin bands with the density of the vehicle bands set arbitrarily to 1.0 are shown in the top panel. The values shown represent the mean ± SEM of triplicate experiments. The figure is the representative of results from at least three independent experiments. Asterisks indicate significant differences between the groups (P < 0.05). E2, 17β-estradiol; CC, clomiphene citrate; ICI, ICI 182,780.
Figure 3. MG132 reverses the ligand-induced degradation of ERα
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After serum-free starvation for 24 h, Ishikawa cells were treated with vehicle (ethanol), MG132 (10–5 M), E2 (10–8 M), CC (10–6 M), ICI (10–6 M), E2 (10–8 M) + MG132 (10–5 M), CC (10–6 M) + MG132 (10–5 M), or ICI (10–6 M) + MG132 (10–5 M) for 24 h. After the various treatments,
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the cell lysates were subjected to SDS-PAGE (5-–20% separating gel), and western blotting was performed using an anti-ERα antibody (sc-542). Relative densitometric units of the ERα bands normalized to the levels of α-tubulin are shown in the top panels, with the density of the bands from vehicle treatment set arbitrarily to 1.0. The values shown represent the mean ± SEM of
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triplicate experiments. The figure is the representative of results from at least three independent experiments. Asterisks indicate significant differences between the groups (P < 0.05). E2,
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17β-estradiol; CC, clomiphene citrate; ICI, ICI 182,780.
Figure 4. Effects of ligands on cellular localization of GFP-ERα A.
Ishikawa
cells
were
transiently transfected with
a green fluorescent
protein
(GFP)-ERα-expressing vector, followed by 24 h of serum starvation and treatment with vehicle
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(ethanol), E2 (10–8 M), CC (10–6 M), or ICI (10–6 M) for 24 h. The cells transfected with GFP-ERα before the treatment were used as a control. The localization of GFP-ERα was categorized into two patterns: nucleus only (left panel) or nucleus and cytoplasm (middle and
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right panels). Bar, 10 µm. B. The ratios of cells with GFP-ERα expression in the nucleus (black bar) and nucleus + cytoplasm (white bar) after various treatments are shown. Each bar
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represents 300 cells expressing GFP-ERα. Numbers inside bars indicate the numbers of cells with GFP-ERα expressions in the nucleus (black bar) or in the nucleus + cytoplasm (white bar)/total number of GFPα-expressing cells examined. E2, 17β-estradiol; CC, clomiphene citrate; ICI, ICI 182,780.
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Highlights Clomiphene citrate decreases estrogen receptor-α in an endometrial cancer cell line. Clomiphene citrate does not decrease estrogen receptor-α mRNA levels.
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Clomiphene citrate increases in the ubiquitination of estrogen receptor-α.
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Proteasome is involved in the degradation of estrogen receptor-α by clomiphene citrate.