Estrogen Receptor Alpha (Erα)–Mediated Coregulator Binding And
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Cell Biol Toxicol https://doi.org/10.1007/s10565-020-09516-6 ORIGINAL ARTICLE Estrogen receptor alpha (ERα)–mediated coregulator binding and gene expression discriminates the toxic ERα agonist diethylstilbestrol (DES) from the endogenous ERα agonist 17β-estradiol (E2) Aziza Hussein Bakheit Adam & Laura H. J. de Haan & Ignacio Miro Estruch & Guido J. E. J. Hooiveld & Jochem Louisse & Ivonne M. C. M. Rietjens Received: 1 August 2019 /Accepted: 28 January 2020 # The Author(s) 2020 Abstract Diethylstilbestrol (DES) is a synthetic estro- transcriptomic signatures obtained in the T47D cells gen and proven human teratogen and carcinogen report- were observed. It is concluded that differences observed ed to act via the estrogen receptor α (ERα). Since the in binding of the ERα with several co-repressor motifs, endogenous ERα ligand 17β-estradiol (E2) does not in downregulation of genes involved in histone show these adverse effects to a similar extent, we hy- deacetylation and DNA methylation and in upregulation pothesized that DES’ interaction with the ERα differs of CYP26A1 and CYP26B1 contribute to the differen- from that of E2. The current study aimed to investigate tial effects reported for DES and E2. possible differences between DES and E2 using in vitro assays that detect ERα-mediated effects, including Keywords Estrogen receptor alpha . Diethylstilbestrol . ERα-mediated reporter gene expression, ERα- 17β-estradiol . Coregulator binding . Transcriptomics mediated breast cancer cell (T47D) proliferation and ERα-coregulator interactions and gene expression in T47D cells. Results obtained indicate that DES and E2 Introduction activate ERα-mediated reporter gene transcription and T47D cell proliferation in a similar way. However, Diethylstilbestrol (DES) is a synthetic estrogen that has significant differences between DES- and E2-induced been used from the 1940s to the 1970s to prevent α binding of the ER to 15 coregulator motifs and in premature delivery and fetal death by stimulating the synthesis of estrogen and progesterone in the placenta (IARC 2012). In addition, DES was used in hormonal Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10565-020-09516-6) contains therapy applied for the treatment of prostate and breast supplementary material, which is available to authorized users. cancer (Giusti et al. 1995;IARC2012; Reed and Fenton 2013). From 1971 onwards, the use of DES was * : : : A. H. B. Adam: ( ) L. H. J. de Haan I. M. Estruch prohibited since it was shown to induce rare reproduc- J. Louisse I. M. C. M. Rietjens tive tract cancers in women exposed in utero, while no Division of Toxicology, Wageningen University and Research, PO Box 8000, 6700 EA Wageningen, The Netherlands protective effect against miscarriage and premature de- e-mail: [email protected] livery was actually observed (Titus-Ernstoff et al. 2001). Although DES has been discontinued since 1971, ad- G. J. E. J. Hooiveld verse health effects have later been discovered in wom- Division of Human Nutrition and Health, Wageningen University and Research, PO Box 17, 6700 AA Wageningen, en who had taken DES, as well as in their offspring The Netherlands including even subsequent generations. Adverse effects Cell Biol Toxicol included breast cancer, clear cell adenocarcinoma of the the endogenous ERα agonist E2 does not induce the vagina and cervix, abnormalities in the female genital adverse effects that have been reported for DES to a tract and abnormalities of the male reproductive tract similar extent. This points at the possible existence of (Colton and Greenberg 1982; Palmer et al. 2006). essential differences between ERα activation by DES DES is an analogue of the endogenous female sex on the one hand and E2 on the other hand. Such differ- hormone 17β-estradiol (E2) and binds to both the ences upon ERα binding may be due to possible differ- estrogen receptor α (ERα) and estrogen receptor β ential recruitment of coregulators, including both (ERβ) (Bolger et al. 1998; Nikov et al. 2001). It has coactivators that interact with receptors and enhance been reported that the molecular dimensions of DES their activation, as well as co-repressors that interact are almost identical to those of E2, particularly with with receptors and decrease their activation (Klinge regard to the distance between the terminal hydroxyl 2000; McKenna et al. 1999). groups (Gonzalez et al. 2019)(Fig.1). Molecular So far, it has been reported that, in the presence of docking of E2 and DES into the ligand binding do- DES, the ERα interacts with coregulators NCOA1 main of ERα from mouse and rat revealed similar (Nuclear receptor coactivator 1), NRIP1 (Nuclear binding orientations and confirmed a role for the receptor-interacting protein1) and PNRC2 (Proline- hydroxyl moieties in this interaction (Gonzalez rich nuclear receptor coactivator 1), as indicated by et al. 2019). The ERα agonist action has generally binding to the coregulator motifs NCOA1_677_700, been associated with stimulation of cell proliferation, NRIP1_173_195 and PNRC2_118_139, respectively, while ERβ activation has been linked with suppres- using the MARCoNI (Microarray Assay for Real- sion of cell proliferation and stimulation of apoptosis time Coregulator-Nuclear receptor Interaction) tech- (Sotoca et al. 2008; Thomas and Gustafsson 2011). nology (Wang et al. 2013). However, no extensive The mode of action by which DES causes its adverse comparison has been carried out between the ERα- effects has not been unraveled yet. It has been reported coregulator interactions in the presence of DES com- that the ERα is required in the mediation of the prolif- pared with E2. This raises the question to what extent erative response to DES in uterus and prostate epithelial DES-mediated coregulator recruitment to the ERα cells in vivo (Chen et al. 2012; Klotz et al. 2000). might be different from that of E2 and whether that Several studies have indicated that a functional ERα is could play a role in the differential biological effects needed for DES-mediated adverse effects, including of these two ERα agonists. The present study inves- phenotypic changes in the reproductive tract and pro- tigates the DES- and E2-dependent modulation of the gressive proliferative lesions and abnormal epithelial interaction of ERα with coregulators using the MAR- cell differentiation in the prostate (Chen et al. 2012). CoNI technology and peptide microarrays containing This is apparent from studies in which these DES- 154 unique nuclear receptor coregulator motifs of 64 induced adverse effects were observed in wild type different coregulators. To provide further information mice, while the effects were absent in ERα knockout on the possible differences between DES- and E2- mice (Couse et al. 2001; Couse and Korach 2004;Prins induced ERα-mediated effects, the present study also et al. 2001). These studies suggest that DES elicits its assesses the relative potency of the two compounds adverse effects on the reproductive tract through an as ERα agonists in a human osteosarcoma U2OS ERα-mediated mechanism. It is of interest to note that ERα reporter gene assay and in a proliferation assay Fig. 1 Chemical structures for E2 and DES Cell Biol Toxicol of human ERα positive T47D breast cancer cells and Reporter gene assay quantifies DES- and E2-induced modulation of gene expression in T47D cells using next generation se- U2OS-ERα cells were seeded in 96-well white plates quencing (RNA-seq) and transcriptome analysis. (PerkinElmer, Groningen, The Netherlands) at a density of 105 cells/ml in phenol red free medium (DMEM/F12) supplemented with DCC-FCS (dextran-coated charcoal- treated fetal calf serum obtained from Gibco (Bleiswijk, Materials and methods The Netherlands) adding 100 μl/well and the cells were incubated at 37 °C and 5% CO2 in a humidified atmo- Cell lines and culture conditions sphere. Twenty-four hours after seeding, medium was changed to phenol red free medium. Forty-eight hours The U2OS (human osteosarcoma) cell line, stably after seeding, cells were exposed to the test compounds expressing ERα in addition to a 3× estrogen- in triplicate, in phenol red free medium (DMEM/F12) responsive element and TATA box binding protein supplemented with DCC-FCS. Exposure medium was combined with a luciferase gene (3x ERE-TATA- prepared to reach the final concentration range of 0.1– luciferase gene) was obtained from Biodetection 100 pM for both DES (Sigma-Aldrich, Zwijndrecht, Systems (BDS) (Amsterdam, The Netherlands). The Netherlands) and E2 (Sigma-Aldrich) using 200- U2OS-ERα cellsweregrowninDMEM:F12,a1:1 time concentrated stock solution in DMSO (Acros, mixture of Dulbecco’s modified Eagle medium Geel, Belgium) diluted in the culture medium. The (DMEM) and Ham’s nutrient mixture F12 (Gibco, maximum concentration of DMSO in exposure medium Bleiswijk, The Netherlands) supplemented with 10% was 0.5%. After removing the medium from the wells, fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, 100 μL of exposure medium containing the test com- Missouri, United States), 0.5% non-essential amino pound were added to the wells and the cells were incu- acids (NEAA) (Gibco, Bleiswijk, The Netherlands), bated for another 24 h at 37 °C and 5% CO2 in a 200 μ/ml geneticin G418 (Gibco, Bleiswijk, humidified atmosphere. After 24 h of exposure, cells The Netherlands) and 50 μg/ml hygromycin (PAA were washed with 0.5× PBS and lysed with 30 μlof Laboratories GmbH, Pasching, Austria). T47D cells, hypotonic low-salt buffer containing 10 mM Tris, 2 mM obtained from the American Type Culture Collection dithiothreitol (DTT, Sigma-Aldrich) and 2 mM 1,2- (Manasssaa, VA, USA), were grown in 1:1 diaminocyclohexane tetraacetic acid monohydrate DMEM:F12/Glutamax culture medium supplement- (CDTA, Sigma-Aldrich) pH 7.8. Plates were kept on ed with 10% FCS. All cells were incubated at 37 °C ice for at least 30 min and subsequently stored at − 80 °C and 5% CO2 in a humidified atmosphere.