Clomiphene citrate down-regulates estrogen receptor-α through the ubiquitin-proteasome pathway in a human endometrial cancer cell line

https://doi.org/10.1016/j.mce.2016.03.029Get rights and content

Highlights

  • Clomiphene citrate decreases estrogen receptor-α in an endometrial cancer cell line.

  • Clomiphene citrate does not decrease estrogen receptor-α mRNA levels.

  • Clomiphene citrate increases in the ubiquitination of estrogen receptor-α.

  • Proteasome is involved in the degradation of estrogen receptor-α by clomiphene citrate.

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 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 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. 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 endometrial cancer cells.

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.

References (48)

  • D.M. Lonard et al.

    The 26S proteasome is required for estrogen receptor-alpha and coactivator turnover and for efficient estrogen receptor-alpha transactivation

    Mol. Cell.

    (2000)
  • X. Long et al.

    Fulvestrant (ICI 182,780)-dependent interacting proteins mediate immobilization and degradation of estrogen receptor-alpha

    J. Biol. Chem.

    (2006)
  • Y. Nakamura et al.

    Effects of clomiphene citrate on the endometrial thickness and echogenic pattern of the endometrium

    Fertil. Steril.

    (1997)
  • D. Nonclercq et al.

    Ligand-independent and agonist-mediated degradation of estrogen receptor-alpha in breast carcinoma cells: evidence for distinct degradative pathways

    Mol. Cell Endocrinol.

    (2004)
  • M.G. Parker

    Structure and function of estrogen receptors

    Vitam. Horm.

    (1995)
  • J.M. Randall et al.

    Cervical mucus score and in vitro sperm mucus interaction in spontaneous and clomiphene citrate cycles

    Fertil. Steril.

    (1991)
  • G. Reid et al.

    Cyclic, proteasome-mediated turnover of unliganded and liganded ERalpha on responsive promoters is an integral feature of estrogen signaling

    Mol. Cell.

    (2003)
  • A.L. Wijayaratne et al.

    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.

    (2001)
  • M. Amita et al.

    Molecular mechanism of the inhibition of estradiol-induced endometrial epithelial cell proliferation by clomiphene citrate

    Endocrinology

    (2010)
  • G. Ben-Nissan et al.

    Regulating the 20S proteasome ubiquitin-independent degradation pathway

    Biomolecules

    (2014)
  • N.B. Berry et al.

    Estrogen receptor-alpha hinge-region lysines 302 and 303 regulate receptor degradation by the proteasome

    Mol. Endocrinol.

    (2008)
  • S. Dauvois et al.

    The antiestrogen ICI 182780 disrupts estrogen receptor nucleocytoplasmic shuttling

    J. Cell Sci.

    (1993)
  • J. Devin-Leclerc et al.

    Interaction and dissociation by ligands of estrogen receptor and Hsp90: the antiestrogen RU 58668 induces a protein synthesis-dependent clustering of the receptor in the cytoplasm

    Mol. Endocrinol.

    (1998)
  • C.M. Eakin et al.

    Estrogen receptor alpha is a putative substrate for the BRCA1 ubiquitin ligase

    Proc. Natl. Acad. Sci. U. S. A

    (2007)
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