Endocrine disruptor bisphenol A strongly binds to human estrogen-related receptor γ (ERRγ) with high constitutive activity
Introduction
Bisphenol A (BPA1), 2,2-bis(4-hydroxyphenyl)propane, has a symmetrical chemical structure of HO-C6H4-C(CH3)2-C6H4-OH. BPA, with a worldwide production of approximately 3.2 million t per year, is used mainly in the production of polycarbonate plastics and epoxy resins. It has been long acknowledged to be an estrogenic chemical able to interact with human estrogen receptors (ER) (Dodds and Lawson, 1938, Krishnan et al., 1993, Olea et al., 1996), and recently to act as an antagonist for a human androgen receptor (AR) (Sohoni and Sumpter, 1998, Xu et al., 2005). Both ER and AR belong to the group III steroid hormone receptors, a subfamily of 48 human nuclear receptors (NRs) (Nuclear Receptors Nomenclature Committee, 1999, Robinson-Rechavi et al., 2001).
Various so-called “low dose effects” of BPA have recently been reported in vivo for reproductive organ tissues and systems in mice and rats. For instance, very low dose levels of BPA were shown to have an increase in size and weight of the fetal mouse prostate (Nagel et al., 1997, Gupta, 2000), and a decrease in daily sperm production and fertility in male mice (Gupta, 2000, vom Saal et al., 1998). All of these low dose effects of BPA have been explained as the output effects of steroid hormone receptors (Welshons et al., 2003). It should be noted, however, that BPA's binding to ER and AR and hormonal activity is extremely weak, 1000–10,000 times lower than for natural hormones, making the intrinsic significance of low dose effects intangible and obscure (National Toxicology Program, 2001, Safe et al., 2002, Gray et al., 2004; vom Saal and Hughes, 2005). This discrepancy on low dose effects prompted us to enquire whether BPA may interact with NRs other than ER and AR.
The estrogen-related receptors (ERRs) are a subfamily of orphan NRs closely related to ERs, ERα and ERβ (Giguère, 2002, Horard and Vanacker, 2003). There are three ERR family members, ERRα, ERRβ and ERRγ, with ERRγ the most recently identified third member (Eudy et al., 1998, Hong et al., 1999). ERRs and ERs show a considerable level of amino acid sequence similarity and identity in both their DNA-binding (DBD) and ligand-binding (LBD) domains. Although 17β-estradiol (E2), a natural ligand of ERs, does not bind to any of the ERR family, ERRs can bind to functional estrogen response elements (EREs) in ER target genes, suggesting a possible overlap between ERR and ER action (Huppunen and Aarnisalo, 2004). ERRs also bind to ERR-response element (ERRE), but as monomers.
ERRs are all orphan receptors, while efforts to discover synthetic compounds that might modify the activities of the ERRs have identified only a few chemicals that suppress ERRs’ spontaneous transcriptional activities. For instance, diethylstilbestrol (DES) was found to repress the molecular activities of ERRs (Tremblay et al., 2001, Coward et al., 2001), although DES is considerably weaker in inhibiting ERR activities compared with its action as an ER-activator. 4-Hydoroxytamoxifen (4-OHT) has also been identified as an inverse agonist of ERRγ, deactivating the receptor by decreasing the very high level of spontaneous constitutive activity (Coward et al., 2001). From evidence in a receptor binding assay in which [3H]4-OHT was used as a tritium-labeled receptor tracer (Coward et al., 2001), 4-OHT binds strongly to ERRγ. Collectively, these results reveal that E2, DES and 4-OHT all bind to ERs very strongly, but that their binding abilities to ERRγ vary.
When we compared the chemical structures of these ligands, it became clear that they share only the phenol group usually acknowledged as a key structural element for receptor recognition (Fig. 1). Since BPA in a compact minimum-energy conformation also shares this phenol group, we assumed that BPA is a potent binder to ERRγ. In the present study, we first established the competitive receptor-binding assay using [3H]4-OHT as a tracer. The reported binding assay (Coward et al., 2001) utilized glutathione-coated beads to cargo GST-ERRγ-LBD, namely ERRγ-LBD fused to glutathione S-transferase (GST), for B/F separation of the tracer [3H]4-OHT. Instead of expensive glutathione-coated beads, we used 1% dextran-coated charcoal (DCC) to absorb and remove receptor-free [3H]4-OHT. This worked successfully, and we eventually could demonstrate that BPA binds strongly to ERRγ. This initial result has led us further to detailed examination of BPA in the reporter gene assay for ERRγ, and here we report that BPA retains extremely high ERRγ's constitutive activity.
Section snippets
Test compounds
Bisphenol A (BPA), CAS no. 80-05-7, purity 99%, Tokyo Kasei Kogyo Co. Ltd., Tokyo, Japan. 4-Hydroxytamoxifen (4-OHT), CAS no. 68047-06-3, purity 98%, Sigma–Aldrich Inc., St. Louis, MO. Diethylstilbestrol (DES), CAS no. 56-53-1, purity 97%, Wako Pure Chemical Industries Ltd., Osaka, Japan. Nonylphenol, CAS no. 84852-15-3, Technical grade, Sigma–Aldrich. Estrone (E1), CAS no. 53-16-7, 98%, Wako. 17β-estradiol (E2), CAS no. 50-28-2, 98.9%, Research Biochemicals International, Natick, MA. Estriol
Saturation binding [3H]4-OHT to ERRγ
For the present study, we first attempted to establish a routine radio-labeled receptor binding assay. As mentioned earlier, instead of expensive glutathione-coated beads carrying GST-ERRγ-LBD (Coward et al., 2001), we utilized 1% dextran-coated charcoal (DCC) to absorb and remove receptor-free [3H]4-OHT. We used this B/F separation method successfully for [3H]E2 and the estrogen receptor (Nakai et al., 1999). In the present study, B/F separation of the tracer [3H]4-OHT also worked successfully
Discussion
ERRγ, unlike ERα, shows extremely high constitutive activity in the reporter gene assay (Fig. 3b). At present, neither the target gene of ERRγ's constitutive transcriptional activity, nor its natural ligand are known. If ERRγ were to possess an agonistic natural ligand, 4-OHT could act as an antagonist of that natural ligand, and BPA as an antagonist. If the endogenous ligand were to function as a natural inverse agonist, 4-OHT would be interpretable as an analogue of such an agonist. In that
Acknowledgements
We thank Prof. Ian A. Meinertzhagen, Dalhousie University, Canada, for reading the manuscript. This study was supported by Health and Labour Sciences Research Grants for Research on Risk of Chemical Substances from the Ministry of Health, Labor and Welfare of Japan to YS. This work was also supported in part by grants-in-aid from the Ministry of Education, Science, Sports and Culture in Japan to YS.
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