Clomiphene citrate down-regulates estrogen receptor-α through the ubiquitin-proteasome pathway in a human endometrial cancer cell line
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 anovulatory women, the subsequent pregnancy rate is only about 20–40% (Kousta et al., 1997). The anti-estrogenic effects of CC on the cervix and uterine endometrium have been proposed to cause this low pregnancy rate (Eden et al., 1989, Gonen and Casper, 1990, Nakamura et al., 1997, Randall and Templeton, 1991). Appropriate endometrial thickness is essential for embryo implantation (Friedler et al., 1996, Senturk and Erel, 2008), with a thickness of at least 6 mm required to achieve a clinical pregnancy by artificial insemination (Gonen et al., 1991). However, CC treatment significantly reduces the endometrial thickness in patients compared to non-CC treatment cycles (Nakamura et al., 1997, Bromer et al., 2009, Peeraer et al., 2015).
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 et al., 1981). We recently showed that CC inhibited estrogen-induced estrogen receptor-α (ERα) transactivation by inhibiting the recruitment of steroid receptor coactivator-1 in both human endometrial cancer cells and endometrial epithelial cells (Amita et al., 2010). However, the mechanism by which CC reduces 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 et al., 1999, El Khissiin and Leclercq, 1999, Lonard et al., 2000, Wijayaratne and McDonnell, 2001, Nonclercq et al., 2004, Laios 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 et al., 1996, Pratt and Toft, 1997, Robyr et al., 2000). After DNA binding, the estrogen-ER complexes are ubiquitinated and targeted for degradation by the 26S proteasome (El Khissiin and Leclercq, 1999, Lonard et al., 2000), which is known as transcription-coupled ER degradation (Berry et al., 2008). The anti-estrogenic agent ICI 182,780 (ICI) also binds to ERs and stimulates ER release from HSP 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 et al., 2003, Stenoien et al., 2001). Therefore, ICI is a member of an emerging category of ER ligands called selective estrogen receptor down-regulators (SERDs) (McDonnell, 2005).
Because CC reduces the levels of cellular ERs in the endometrium (Kokko et al., 1981, Xia et al., 1996, Aksel et al., 1986, Fritz et al., 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 in estrogen-targeting cells. In this study, we demonstrate that CC induces ubiquitination and subsequent degradation of ERα in a human endometrial cancer cell line.
Section snippets
Reagents
CC, 17β-estradiol (E2), and ICI were obtained from Sigma Chemical Co. (St. Louis, 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 obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The
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 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) decreased within 3 h
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 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 endometrium of postmenopausal women
Conflict of interest
The authors report no conflict of interest.
Acknowledgments
This study was supported by Grants-in-Aid for General Science Research 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 the writing of the report; or in the decision to submit the article for publication.
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