Activation, estrogen receptor alpha

Some sections of the KE description were derived and adapted from the External Scientific Report (Viviani et al., 2023).

Biological state

Estrogen Receptor Alpha (ERα) is a receptor covalently bound by estrogens, which following the dimerization can translocate to the nucleus (Björnström and Sjöberg, 2005), where it can bind to estrogen responsive elements and recruit co-activators or co-repressors, which can attract co-regulatory proteins, like histone acetyltransferase, ubiquitin ligases, and protein remodelers (Thomas and Gustafsson, 2011) (Fig.1). A non-genomic signalling of Erα is described (Fig. 7), not requiring the dimerization for the induction of kinases and calcium flux (Levin, 2002; Vasudevan and Pfaff, 2008). The non-genomic action of ERα is able to regulate more genes (Gu et al., 2014). Both signalling pathways are important for the human organism (Pedram et al., 2014; Pedram et al., 2016).

The ER structure is composed by different domains (A-F) which are responsible for the binding to the ligands, the dimerization, the binding to the DNA and for the activation of transcription (Nilsson et al., 2001) (Fig. 8). The A/B domain, or activation function 1 (AF1) is responsible for transactivation and protein-protein interaction, and it acts independently of ligand binding. Then, there is the C-domain, which is responsible for DNA binding and receptor dimerization. The D-domain instead is the phosphorylation site or ER and has nuclear localization sequences. The E-domain, or activation function 2 (AF2) is the ligand binding domain and the site for the binding with co-activators and co-repressors (Ellmann et al., 2009). Lastly, the F-domain that prevents improper ligand activation and dimerization (Yang et al., 2008).

Therefore, ligand binding, dimerization and DNA binding processes are the first steps to inducing the transcription of target genes. But the ER activity largely depends also on the presence and recruitment of different co-activators and co-repressors. Once ER is bound to estrogen responsive elements, it can recruit different proteins that can favour or obstruct the action of the receptor (Thomas and Gustafsson, 2011). The main co-activators are the steroid receptor co-activators (SRC-1 and SRC-3), which are able to recruit co-regulatory proteins (Heldring et al., 2007). The main transcription factors that can be regulated by ER are activating protein 1 (AP1), specificity protein 1 (SP1), cAMP response element-binding protein (CREB), nuclear factor-κB (NF-κB) and p53 (Biswas et al., 2005; Björnström and Sjöberg, 2005; Fox et al., 2009).

Instead in absence of ligands, ER can be activated by the phosphorylation from protein kinases which are stimulated by hyperactive growth factor receptors (Britton et al., 2006). ER is also able to rapidly activate other pathways, namely MAPK, PI3K, EGFR, and SRC (Kousteni et al., 2001; Song et al., 2002; Razandi et al., 2004; Shupnik, 2004).

Biological compartment

ERα is mainly expressed in uterus, prostate (stroma), ovary (theca cells), testes (Leydig cells), epididymis, bone, breast, various regions of the brain, liver, and white adipose tissue (Dahlaman-Wright et al., 2006).

At the subcellular compartments, estrogen receptors (ERs), are localized in cytoplasm where they exist as monomers bound to heat shock proteins (HSPs). Estrogen binding alters receptor conformation and triggers release from the HSPs, thereby allowing receptor dimerization and translocation in the nucleus where these dimers bind to specific DNA sequences and recruit numerous co-factors to regulate gene transcription. Unliganded ER are also characterized as monomers in the nucleus and at the plasma membrane (Gourdy et al., 2018)

General role in biology

The main function of ERα is to mediate the action of estrogens, known to be involved in physiological pathological conditions (endometrial proliferation in menstrual cycle and endometrial carcinoma in postmenopausal women). This receptor is also involved in apoptotic and proliferative functions involving the MAPK/ERK pathway, mainly in breast cancer cells (Zheng et al., 2007; Lin et al., 2010; Zhang et al., 2012; Li et al., 2013). Another player involved in the increased proliferation induced by ERα is c-myc (Dubik and Shiu, 1992). The increased proliferation induced by ER has been linked to tumours (Thomas and Gustafsson, 2011). The interaction between estrogen and ERα and the increased proliferation has been proved in breast and uterine tissues (Ellmann et al., 2009).

Note: considering the AOPs under development the stressors interacting with the estrogen metabolism should be tested negative in all the in vitro assays reported below.  Additional proof of concept supporting the chain of the events herein described is given by a negative result in the in vitro assays but positive outcome in the Uterotrophic Bioassays. Uterotrophic Bioassay is indeed indicative of a single endocrine mechanism i.e., estrogenicity that could be related to mechanism other than direct binding to ER alpha or ER beta receptor.

In the regulatory area methods are available to measure ER receptor activity.  OECD in the “Revised Guidance document 150” give insightful information, including limits on their use, on validated and/or widely accepted assays with estrogenic active substance specific endpoints.

OECD in vitro assays

• OECD TG 493 (July 2015): Performance-Based Test Guideline for Human Recombinant Estrogen Receptor (HRER) In Vitro Assays to Detect Chemicals with ER Binding Affinity
• OECD TG 455 (June 2021): Performance-Based Test Guideline (PGBT) For Stably Transfected Transactivation In Vitro Assays to Detect Estrogen Receptor Agonists and Antagonists. It comprises several mechanistically and functionally similar test methods for the identification of estrogen receptor (i.e., ERα, and/or ERβ). The fully validated reference test methods that provide the basis for this PBTG are: 1) The Stably Transfected TA (STTA) assay using the (h) ERα-HeLa-9903 cell line; and 2) The VM7Luc ER TA assay (3) using the VM7Luc4E2 cell line1 which predominately expresses hERα with some contribution from hERβ.
• OECD TG 457 (October 2012): BG1luc estrogen receptor transactivation test method for identifying estrogen receptor agonists and antagonists.

OECD in vitro screens assays (non-mammalian)

• OECD TG 250 (June 2021): EASZY assay - Detection of Endocrine Active Substances, acting through estrogen receptors, using transgenic tg (CYP19A1b:GFP) Zebrafish embryo
• OECD TG 230 (September 2009): 21-Day Fish Assay a Short-Term Screening for Oestrogenic and Androgenic Activity, and Aromatase Inhibition

 OECD in vivo mammalian screens and test

• OECD TG 440 (October 2007): Uterotrophic bioassay in rodents

Others non-OECD tests

• US EPA (2009) Estrogen Receptor Binding Assay Using Rat Uterine Cytosol: This assay identifies chemicals that have the potential to interact with the estrogen receptor (ER) in vitro.  Principle of this particular assay is based on the competitive protein-binding methods. A radiolabelled ligand and an unlabelled ligand are presented together to a specific receptor. The radioactivity measurement provides the quantitative estimation of the bound and unbound fraction of the ligand with the receptor. All cytosolic estrogen receptor subtypes that are expressed in the specific tissue, including ERα and ERβ are used for the determination of estrogen receptor binding. This assay is simple and rapid to perform when optimal conditions for binding are determined. Assay determines if a ligand/chemical can interact and displace the endogenous hormone 17β-oestradiol (Freyberger et al., 2010, from KE ID 1046, AOP Wiki)
• Yeast estrogen screen (YES) (ISO 19040-1 and 19040-2)
• T47D-Kbluc assay (Wilson et al., 2004);
• ToxCast Estrogen Receptor Agonist Pathway Model: The ToxCast estrogen receptor (ER) pathway model is a mathematical model that combines the results from 18 high-throughput screening (HTS) assays from the ToxCast and Tox21 research programs. The HTS assays measure ER binding, dimerization, chromatin binding, transcriptional activation and ER-dependent cell proliferation. The model uses activity patterns across the in vitro assays to predict whether a chemical is an ER agonist or antagonist or is otherwise influencing the assays through a manner dependent on the physics and chemistry of the technology platform (“assay interference”). The output of the model provides an area under the curve (AUC) value for the potential of a chemical to cause ER agonism, normalized with respect to the positive control chemical, oestradiol.
• QSAR models for ER interaction are available at the website of Danish (https://qsar.food.dtu.dk/) and US (https://www.epa.gov/tsca-screening-tools/epi-suitetm-estimation-program-interface) EPA and OECD (https://www.oecd.org/chemicalsafety/risk-assessment/oecdquantitativestructure-activityrelationshipsprojectqsars.htm).

Endocrine systems with respect to hormone structure, receptors, synthesis pathways, hormonal axes and degradation pathways are well conserved across vertebrate taxa especially in the case of estrogen, androgen and thyroid hormones and steroidogenesis (OECD TG 150)

Taxonomic applicability: mammals

Life stage Applicability: This KE is applicable to adulthood; reproductive and post reproductive (menopausal, aging) phases

Sex Applicability: This KE is applicable to females.

Björnström L and Sjöberg M, 2005. Mechanisms of estrogen receptor signaling: convergence of genomic and nongenomic actions on target genes. Molecular endocrinology, 19 4:833-842

Dahlman-Wright K, Cavailles V, Fuqua SA, Jordan VC, Katzenellenbogen JA, Korach KS, Maggi A, Muramatsu M, Parker MG and Gustafsson JA, 2006. International Union of Pharmacology. LXIV. Estrogen receptors. Pharmacol Rev, 58:773-781. doi: 10.1124/pr.58.4.8

Gu Y, Chen T, López E, Wu W, Wang X, Cao J and Teng L, 2014. The therapeutic target of estrogen receptor-alpha36 in estrogen-dependent tumors. J Transl Med, 12:16. doi: 10.1186/1479-5876-12-16

Levin ER, 2002. Cellular functions of plasma membrane estrogen receptors. Steroids, 67:471-475. doi: 10.1016/s0039-128x(01)00179-9

Lin SL, Yan LY, Zhang XT, Yuan J, Li M, Qiao J, Wang ZY and Sun QY, 2010. ER-alpha36, a variant of ER-alpha, promotes tamoxifen agonist action in endometrial cancer cells via the MAPK/ERK and PI3K/Akt pathways. PLoS One, 5:e9013. doi: 10.1371/journal.pone.0009013

Pedram A, Razandi M, Blumberg B and Levin ER, 2016. Membrane and nuclear estrogen receptor α collaborate to suppress adipogenesis but not triglyceride content. Faseb j, 30:230-240. doi: 10.1096/fj.15-274878

Pedram A, Razandi M, Lewis M, Hammes S and Levin ER, 2014. Membrane-localized estrogen receptor α is required for normal organ development and function. Dev Cell, 29:482-490. doi: 10.1016/j.devcel.2014.04.016

Razandi M, Pedram A, Merchenthaler I, Greene GL and Levin ER, 2004. Plasma membrane estrogen receptors exist and functions as dimers. Mol Endocrinol, 18:2854-2865. doi: 10.1210/me.2004-0115

Thomas C and Gustafsson J-Å, 2011. The different roles of ER subtypes in cancer biology and therapy. Nature Reviews Cancer, 11:597-608. doi: 10.1038/nrc3093

Vasudevan N and Pfaff DW, 2008. Non-genomic actions of estrogens and their interaction with genomic actions in the brain. Front Neuroendocrinol, 29:238-257. doi: 10.1016/j.yfrne.2007.08.003

Viviani B, Bernardini E, Galbiati V, Maddalon A, Melzi A, Midali M, Serafini M, Corsini E, Melcangi RC, Scanziani E, 2023. Development of Adverse Outcome Pathways relevant for the identification of substances having endocrine disruptors properties. EFSA supporting publication 2023:EN-7748 47 pp. doi:10.2903/sp.efsa.2023.EN-7748.