Estrogen receptor β: the guardian of the endometrium.

Human Reproduction Update Advance Access published October 10, 2014 Human Reproduction Update, Vol.0, No.0 pp. 1– 20, 2014 doi:10.1093/humupd/dmu053

Estrogen receptor b: the guardian of the endometrium D.K. Hapangama 1,*, A.M. Kamal 1,2, and J.N. Bulmer 3

*Correspondence address. E-mail: [email protected]

Submitted on June 30, 2014; resubmitted on August 20, 2014; accepted on August 27, 2014

table of contents

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Introduction Human endometrial anatomy Human endometrium as an estrogen-receptive organ Methods Estrogen receptors ERb structure ERb receptor activation ERb expression in normal endometrium Evidence from transgenic mice: ERbKO mice Primate studies ERb in the endometrial vasculature ERb in endometrial immune cells ERb in the human uterus Role of ERb in endometrial regeneration ERb expression in the healthy human post-menopausal endometrium ERb in endometrial pathology Endometriosis and adenomyosis Polycystic ovary syndrome Endometrial polyps Endometrial hyperplasia Endometrial cancer Pharmacological regulation of endometrial ERb expression Evidence for regulation of endometrial ERb expression by exogenous hormone treatment Evidence for SERMs (ERb selective pharmacological agents) in endometrium Summary/conclusion

background: The endometrium is the primary target organ for the ‘female’ sex steroid hormone estrogen, which exerts effects in the endometrium via two main classical estrogen receptor (ER) isoforms, ERa and ERb. The main function of the endometrium, embryo implantation, appears unperturbed in ERb knockout mice, which has led researchers to disregard other potentially important functional roles that ERb may have in endometrium. This review focuses on ERb in the human endometrium and its protective role from the undesired effects of ERa.

methods: We conducted a systematic search using PubMed and Ovid for publications between January 1996 and February 2014. All studies that examined ERb expression or function in non-pregnant endometrium or cells derived from the endometrium were considered, including human and animal studies.

& The Author 2014. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: [email protected]

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1 Department of Women’s and Children’s Health, Institute of Translational Medicine, University of Liverpool, Liverpool Women’s NHS Foundation Trust, Liverpool L8 7SS, UK 2The National Center for Early Detection of Cancer, Oncology Teaching Hospital, Baghdad Medical City, Baghdad, Iraq 3 Reproductive and Vascular Biology Group, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne NE2 4HH, UK

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results: Studies of the basic function of ERb isoforms in restraining ERa-mediated cell-specific trophic/mitotic responses to estrogen in other tissues has allowed appreciation of the important potential role of ERb in the regulation of cell fate in the human endometrium. Our current understanding of ERb expression and function in endometrium is, however, incomplete. ERb is dynamically expressed in healthy premenstrual endometrium, persists in post-menopausal atrophic endometrium and may play an important role in endometrial disease. All endometrial cell types express ERb and aberrations in ERb expression have been reported in almost all benign and malignant endometrial proliferative disease. conclusions: The collective evidence suggests that ERb has an important role in normal endometrial function and also in most, if not all, benign and malignant endometrial diseases. However, the conduct of studies of endometrial ERb expression needs to be standardized: agreement is needed regarding the most appropriate control tissue for endometrial cancer studies as well as development of standardized methods for the quantification of ERb immunohistochemical data, similar to those scoring systems employed for other hormonally regulated tissues such as breast cancer, since these data may have direct clinical implications in guiding therapy. Key words: endometrium / estrogen receptor b / hormone receptors / endometrial proliferation / endometrial cancer

The endometrium is the primary target organ for the ‘female’ sex steroid hormone estrogen (Katzenellenbogen, 1984; Greaves et al., 2013). The effects of estrogens are exerted in the endometrium via two main classical estrogen receptor (ER) isoforms, ERa and ERb, and perhaps via the recently described G-protein-coupled estrogen receptor (GPER; formerly GPR30) (Critchley and Saunders, 2009; Holm and Nilsson, 2013). The main function of the endometrium, embryo implantation, appears to be unperturbed in ERb knockout (KO) mice, which has led researchers to disregard other potentially important functional roles that ERb may have in endometrium (Burns and Korach, 2012). Studies from other organs have confirmed the opposing actions of ERb on ERa function (Gustafsson, 2003; Bottner et al., 2014). Evidence for the involvement of ERb has been reported in almost all gynaecological pathologies including menorrhagia, endometriosis, infertility and endometrial cancer (EC), as well as in normal and abnormal pregnancy-related conditions (Fernandez et al., 2012; Ha¨ring et al., 2012a; Hu et al., 2012). The classical trophic effects of ERa are reviewed elsewhere (Arnal et al., 2013) and this review focuses on the less well-described receptor, ERb, and its role in protecting the human endometrium from the undesired effects of ERa.

remains after cessation of ovarian cyclicity as an atrophic, inactive postmenopausal endometrium (Chhieng and Hui, 2011). The germinal layer of the endometrium where the stem cells reside is, therefore, postulated to be the stratum basalis (Padykula et al., 1989; Gargett and Masuda, 2010; Valentijn et al., 2013). The other components of the endometrium, that is the blood vessels and immune cells, exist in both layers (Bulmer et al., 1991a; Spencer et al., 2011). Access to the full thickness of the endometrium containing both stratum basalis and a stratum functionalis usually requires hysterectomy and, therefore, particularly when studying endometrium either from healthy women or those with benign endometrial disease, researchers have mainly considered the stratum functionalis which is easily obtained with an outpatient endometrial biopsy (Hapangama et al., 2008a). However, the hormonal responsiveness, for example, has been postulated to differ between the stratum functionalis and basalis (Prianishnikov, 1978; Padykula et al., 1989). Many studies of endometrial hormone receptors have not only overlooked the structural and functional differences between the stratum basalis and functionalis, but also have discounted the exceptionally dynamic nature of the stratum functionalis during the normal menstrual cycle (Argenta et al., 2014). Consequently, compared with other estrogen-sensitive organs such as the breast, the exact detailed mechanism of estrogen action in the endometrium remains unclear, with seemingly contradictory results.

Human endometrial anatomy The endometrium is the mucosal lining of the uterus and is derived from the inner layer of the embryonic paramesonephric ductal mesenchyme (McCluggage, 2011). Endometrial development and function in menstruating upper order primates (including humans) is complex compared with most other mammals (Slayden and Brenner, 2004; Jabbour et al., 2006). The human endometrium is stratified into two functional layers: the transient superficial stratum functionalis and the permanent deeper stratum basalis adjacent to the myometrium (Ferenczy and Bergeron, 1991). The superficial stratum functionalis is lined by luminal epithelium, contains superficial glandular epithelium and stroma and is completely shed and regenerated during the monthly menstrual cycle and after childbirth (Ferenczy and Guralnicik, 1983; Gargett et al., 2008). It can be divided into the deeper zona spongiosa with a loosely organized stromal zone and a superficial zona compacta with a more compact stroma (Ferenczy, 1980; Wynn, 1989). The stratum basalis contains the terminal part of the endometrial glands and densely organized stroma and is not shed during menses or at parturition; it

Human endometrium as an estrogen-receptive organ Estrogens, progesterone and androgens are the main three classical ovarian steroid hormones that exert their effects on the endometrial cells mainly via their cognate receptors (Hapangama, 2003; Slayden and Brenner, 2004). 17b-estradiol (E2) and estrones are the two main estrogens available for the non-pregnant endometrium, and these exert their cellular functions through nuclear receptors, ERa and ERb, which are hormone-inducible transcription factors (Vani et al., 2008; Blair, 2010; Crandall and Barrett-Connor, 2013). Surprisingly, compared with the large number of studies on breast tissue, for example, the number of studies investigating ERb in endometrium (despite it being a primary target organ for E2) has been modest. There are many reviews of ERb which discuss most other organs and completely disregard endometrium as an organ in which ERb has a functional role (Bottner et al., 2014). Furthermore, there are no recent


Introduction

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Estrogen receptor b in the endometrium

Methods PubMed (Medline) and Ovid searches using the key words ERb, endometrium, endometrial cancer, endometriosis, polycystic ovarian syndrome, infertility, menorrhagia and uterus were carried out systematically for publications from January 1996 until February 2014. All studies examining ERb expression or function in non-pregnant endometrium or primary cells or tissue explants in culture derived from the endometrium, including human and animal studies and endometrial cell lines, were considered.

Estrogen receptors The first known ER subtype, ERa, was identified in the rat uterus in 1966 (Toft and Gorski, 1966), followed by the cloning of human ERa cDNA (gene ESR1) in 1985 (Walter et al., 1985). This was followed by the discovery of a second ER subtype, ERb, in the prostate and ovaries of rats in 1996 (Kuiper et al., 1996). The human ERb gene ESR2 was cloned first in 1996 from the testis (Mosselman et al., 1996) and subsequent work has increased understanding of the physiological and pathological action of E2 in human cells and tissues. E2 may also bind to transmembrane GPER, which mediates rapid signalling events traditionally associated with G-protein-coupled receptors; this receptor is reviewed elsewhere (Prossnitz and Barton, 2014), and it is therefore outside the remit of this review. Interestingly, the pivotal accepted role of ERs in reproduction is a late evolutionary development, as there is evidence suggesting that in early order invertebrates, the reproductive role of E2 is not mediated by ER and may take place through ancient, ER-independent pathways (Thornton et al., 2003; Keay et al., 2006). Cloning, genome mapping and phylogenetic analysis studies have indicated that ER isoforms are likely to have been generated by duplication of the Esr gene early in the vertebrate lineage (Thornton 2001; Wu et al., 2003). It is proposed that the functions of an ancestral gene are partitioned among duplicate genes by complementary loss of tissue-specific expression (Thornton, 2001; Wu et al., 2003). The tissue-specific expression of two ER isoforms

and their splice variants, therefore, provides the potential for very flexible regulation of target tissues by E2. ERb can have opposing actions to ERa on the same gene promoter in response to E2 (Smith et al., 2004; Thomas and Gustafsson, 2011). These inhibitory effects of ERb on ERa activity may be exerted through a combination of altered recruitment of key transcription factors and increased ERa degradation (Matthews et al., 2006). In contrast, ERb expression is induced by E2 acting via ERa and may be suppressed by hypermethylation of the ERb promoter (Rody et al., 2005). The pro-proliferative function of ERa is essential for reproduction, yet is associated with obvious E2-associated health risks in the endometrium (and in other organs) (Koos, 2011; May, 2014). Therefore, the ERa opposing activity of ERb has been of particular interest with the emergence of new receptor isotype-specific pharmacological modulators.

ERb structure ERb is a member of the Class I nuclear hormone receptor superfamily of ligand-inducible transcription factors and shares the common, evolutionarily conserved structural and functionally distinct domains of other superfamily members (Matthews et al., 2006) (Fig. 1A ). This includes a central, highly conserved DNA binding domain (DBD), which binds to the same estrogen responsive element (ERE) as ERa in the target gene promoters; a multifunctional ligand-binding domain (LBD) at the C-terminal; the ligand-dependent activation function 2 (AF2) at the C-terminal; and the constitutively active AF1 at the N-terminal and flexible-hinge D-domain between the LDB and the DBD (Fig. 1A) (Nilsson et al., 2001; Harnish, 2006). Transcriptional activation of ERb is facilitated by two acidic activation domains, AF1 and AF2, which recruit a range of specific co-regulatory protein complexes to the DNAbound receptor (Fig. 1B) (Benecke et al., 2001). Although there is close homology in the DBD (97%) and LBD (60%) between the two ER subtypes (Fig. 1A), significant divergence exists between the N-terminal regions, where only 20% of amino acid identity is shared. ER subtypespecific (Mosselman et al., 1996), promoter-specific and cell-specific E2 actions on target genes are therefore thought to be due to this highly variable N-terminal domain and ligand-independent AF1 (Katzenellenbogen et al., 2001; Hawse et al., 2008; Kumar et al., 2012). Despite their close homology, the ERb (ESR2) gene is located on chromosome 14, whereas ERa protein is coded by a different gene (ESR1) located on chromosome 6 (Menasce et al., 1993; Enmark et al., 1997). The human ERb (ESR2) gene is highly conserved with that of other higher order primates such as chimpanzee, rhesus monkey and orang-utan (Lewandowski et al., 2002). Five alternatively spliced transcript variants of the ERb (ESR2) gene have been described to date as ERb1–5 (Moore et al., 1998) (Fig. 1A, and published primer sequences for splice variants included in Supplementary data. Table SI), although the characterization of the functional isoform pattern in human endometrium is not complete. The 530-amino acid human ERb isoform is currently regarded as the wildtype ERb1 (Leygue et al., 1998). Unlike ERa, in addition to ERb1 at least two other splice variants, ERb2 and ERb5, are transcribed with all three proteins being identical except for the C-terminus; all are expressed as proteins and have been described in the endometrium (Collins et al., 2009). ERb2 does not bind to the ligand or make homodimers and the C-terminus truncations of both ERb2 and ERb5 proteins may affect their ligand binding capacities, although they can form


reviews that specifically discuss the current evidence regarding the potential roles of ERb in the endometrium. Although estrogens are responsible for many physiological functions in both females and males (Couse and Korach, 1999; Harris, 2006), the evidence from ERa, ERb double KO mice confirms that life is possible without E2 action, although ERb is almost universally expressed in all human organs (Lubahn et al., 1993; Dupont et al., 2000; Weihua et al., 2000). Furthermore, E2 is essential for reproductive function in females, yet only ERa seems to be essential in preserving fertility (Lubahn et al., 1993). The main function of ERb is therefore thought to be particularly in preventing undesired ERa-mediated actions of E2 (Hall and McDonnell, 2005; Pettersen, 2011). In this review, we focus on the expression and function of ERb in non-pregnant endometrium. We discuss the available evidence regarding the involvement of ERb in pre- and post-menopausal healthy endometrium, in benign premenopausal endometrial pathologies and in EC, with reference to animal data where appropriate. ERb expression and its possible action on the stratum functionalis and the germinal stratum basalis will also be examined in detail. The effects and expression of ERa on the endometrium are reviewed elsewhere (Brosens et al., 2004; Jabbour et al., 2006; Critchley and Saunders, 2009).

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LBD and activation function 1 and 2 (AF1/AF2) regions. ERb1 is the wild-type ERb with 530 amino acids (aa). (B) Co-activators and suppressors of ERb. A graphical illustration of some known co-activators and repressors or ERb. ER, estrogen receptor; AF1/AF2, activation function 1 and 2; SRA, steroid receptor RNA activator; p68, the DEAD box proteins DDX5; SRC 1, 2 and 3, steroid receptor co-activator 1 – 3; CBP, CREB-binding protein; p300, E1A-associated protein p300; TRAP220, mediator complex subunit 1; DRIP, vitamin D3 receptor interacting protein; TRAP, thyroid hormone receptor; ASC-1 and 2, activating signal cointegrator 1, 2; RTA, repressor of tamoxifen transcriptional activity; NCoR, nuclear receptor corepressor; SMRT, silencing mediator for retinoic acid and thyroid hormone receptors; RIP140, receptor-interacting protein 140; E6-AP, E6-associated protein; RPF1, ribosome production factor 1 homologue; PGC1, peroxisome proliferator-activated receptor g coactivator 1-a; CAPER, coactivator of activating protein-1 and estrogen receptors; CoAA, coactivator activator; CARM1, coactivator-associated arginine methyltransferase 1; PRMT1, protein arginine methyltransferase 1; CoCoA, calcium binding and coiled-coil domain containing protein 1; mSiah2, seven in absentia 2.

heterodimers with either ERb1 or ERa (Ogawa et al., 1998; Fujimura et al., 2001). Very little is known about ERb5, which has only been described in the context of malignant endometrium (Collins et al., 2009). The isoforms may differentially modulate E2 signalling and, as a consequence, impact target gene regulation (Ramsey et al., 2004). Tissue and species-specific expression of the isoforms of the splice variants of ERb mRNA and products has been described and may have functional consequences in ERb-mediated responses (Weiser et al., 2008). Some of the existing contradictory reports of ERb expression may be explained by the fact that studies may either have employed non-specific

primers which did not distinguish the splice variants or examined a single splice variant in isolation, disregarding the potential collective effect of co-existing variants. The lack of commercially available specific antibodies to these alternatively spliced variants of ERb has been the major obstacle for researchers investigating their specific function in vivo. Reliable and specific antibodies for these splice variants are therefore urgently required to unravel the important information on the functional and clinically relevant involvement of ERb and its splice variants in human diseases. However, most of the available functional data only mention either ERb or ERb1, with little reference to the other variants.


Figure 1 (A) Schematic illustration of the comparative structures of ERb splice variants and ERa isoform. All receptor isoforms contain the distinct DBD,

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This review of the functional role of ERb therefore mainly focuses on ERb1, unless otherwise stated.

ERb receptor activation

ERb expression in normal endometrium Evidence from transgenic mice: ERbKO mice E2 action is essential for normal development of female sexual characteristics; ERa is the main receptor responsible for these actions, as evidenced by the phenotype of the ERaKO mouse, which demonstrated a nonfertile and infantile phenotype (Lubahn et al., 1993). In contrast the

Figure 2 Schematic diagram of ER activation. ER binds to ligand, dimerizes, translocates to the nucleus and binds to the ERE located in the respective gene promoters to initiate recruitment of co-activators (COA), co-repressors (COR) and chromatin-remodelling factors to either activate or repress transcription of target genes (Classical pathway). Ligand-bound ER can also interact directly with other transcription factor (TF) complexes and influence the transcription of genes that do not possess the ERE in their promoter (non-classical pathway).


ERb binds E2 with similar affinity to that of ERa and upon binding to the ligand, the activated receptor may exert effects involving the classical hormone signalling pathway (Pastore et al., 2012). This involves ERb dimerizing, translocating to the nucleus, binding to the ERE located in the respective gene promoters to initiate recruitment of co-activators, co-repressors and chromatin-remodelling factors to either activate or repress transcription of target genes (McDonnell and Norris, 2002; Saxon and Turner, 2005; Zhang and Trudeau, 2006; Zhao et al., 2008; Fig. 2). DNA binding of ERb, and hence its nuclear localization, is reported by some to be rapidly lost at body temperature when the ligand, E2, is absent (Pace et al., 1997; Tan et al., 1999). Although these findings have been described in the context of the classical hormonal pathway, recent literature examining a new class of ERb-selective compounds has demonstrated activation of multiple endogenous genes through ERb by selectively recruiting ERb and co-activators to target genes without binding to ERb (Vivar et al., 2010). This novel pathway of ligand-independent ERb activation may play an important role and needs to be explored further in the future to understand the collective and the full impact of both classical and non-classical pathways in ERb function. ERb1 can form homodimers or heterodimers with ERa and ERb splice variants (e.g. ERb2), and therefore, the expression and

availability of each ER subtype in a cell will influence the cell-specific response to E2 (Kuiper and Gustafsson, 1997; Tremblay et al., 1999). At a subcellular level, ERb is localized in the nucleus, cytoplasm and mitochondria and this is regulated by both the availability of the ligand and by the co-expression of ERa/ERb2 (Chen et al., 2007; Milanesi et al., 2009). Furthermore, ligand-bound ERb can interact directly with other transcription factor complexes and influence the transcription of genes that do not possess the ERE in their promoter (Kushner et al., 2000). In the presence of E2, ERb is able to oppose the effects of tissue-specific ER modulators such as tamoxifen via these indirect pathways (Paech et al., 1997). Therefore, the availability and type of the estrogenic agonist, the cellular expression of steroid receptors (including ERs and their respective isoforms, as well as other hormone receptors) and the expression of co-regulators, all influence cellular expression of ERb and ERb-mediated gene expression in response to estrogen in any given tissue type.

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Primate studies Primates menstruate and have an endometrial cycle and structure similar to that of humans. In primates such as macaques, ERb expression does

not change across the menstrual cycle in either the stratum functionalis or the stratum basalis. Only ERb has been reported to be expressed in the endometrial endothelial cells throughout the menstrual cycle and ERb has been proposed to regulate the angiogenic and vascular changes that occur in embryo implantation, early placentation and the maintenance of pregnancy (Slayden and Brenner, 2004). In the marmoset monkey, ERb was highly expressed in endometrial epithelial cells throughout the menstrual cycle and in pregnancy. Increased stromal ERb expression was observed in the late proliferative phase with the staining index decreasing by half as the secretory phase progressed and remaining low in pregnancy (Silvestri and Fraser, 2007). Treatment with GnRH agonists or ovariectomy caused significant reductions in PR and ERb expression, but not in ERa when compared with the late proliferative phase of the normal menstrual cycle. In rhesus macaques, ERb expression was increased with E2 treatment in simulated cycles and the levels decreased in the epithelial cells in the stratum functionalis with the subsequent combined treatment with E2 and progesterone (Critchley et al., 2001). These authors also reported static expression of both ER isoforms in the stratum basalis across the menstrual cycle (Critchley et al., 2001). When compared with other animals, primate endometrium is the closest to that of humans with obvious similarities. However, there are subtle yet striking differences in endocrinology; for example, E2 levels rise in the mid-secretory phase of the cycle in humans, but this is not observed in non-human primates (Narkar et al., 2006). Therefore, caution should be exercised when extrapolating primate data to human endometrial physiology, including ERb expression and function.

ERb in the endometrial vasculature Benign angiogenesis is a unique property of endometrium which is essential for normal regeneration of the stratum functionalis after menstrual shedding and is also a fundamental feature of the E2-dominant proliferative endometrium (Nayak and Brenner, 2002). Most described trophic effects of E2 on this benign endometrial angiogenic process are exerted via ERa either directly or indirectly, acting on endometrial epithelial and stromal cells to secrete angiogenic growth factors (Rees and Bicknell, 1998). The evidence that E2 has a direct action on endometrial vessels is suggested by reports describing the presence of ER isoforms in the endometrial vasculature. Nevertheless, there is significant controversy regarding cyclical variation in expression of ER subtypes, including ERb, in the endometrial vascular cells. Although all studies report expression of ERb by endometrial vessels (Critchley et al., 2001; Lecce et al., 2001; Greaves et al., 2013), some have suggested that endometrial endothelial ERb expression may be dynamically regulated during the menstrual cycle (Lecce et al., 2001), while others reported non-cyclical constitutive expression (Critchley et al., 2001). Lecce et al. (2001) reported expression of both ERa and ERb in endometrial endothelial and vascular smooth muscle cells at different phases of the menstrual cycle, although in the menstrual phase, when ovarian hormone levels are at a nadir, both ER isoforms were reported to be absent from the vascular compartment (Lecce et al., 2001). The ERb expression by the vascular smooth muscle cells was highest in the late secretory phase, whereas those who reported detection of ERa in the endothelium noted the highest endothelial ERa expression to be in E2-dominant midcycle endometrium (Lecce et al., 2001). There are also conflicting reports of PR expression by vascular endothelium with some reporting


ERbKO mouse was fertile; this observation may have led to a relative lack of interest from reproductive biologists to investigate ERb expression and function in the endometrium (Weihua et al., 2000). Although fertile, ERbKO mice did display some reproductive deficiencies with a subfertile phenotype and an exaggeration of the endometrial epithelial proliferative response to E2, suggesting a suppressive role of ERb on the actions of ERa (Wada-Hiraike et al., 2006). However, there is evidence that ERb could partially compensate for the loss of ERa in the genital tract since the uterine phenotype of ERab double KO mice has been described to be similar to that of an aggravated ERaKO uterine phenotype, whereas the ERbKO genital tract appeared to be normal (Dupont et al., 2000). The decrease in ovarian production of E2 was thought to be responsible for the reduced fertility and smaller litter size seen in ERbKO animals and therefore, these animals initially were not thought to have a defect in implantation or placentation (Weihua et al., 2000). Normal endometrial epithelial cell maturation was associated with the attainment of apical–basal polarity, which relied on formation of intercellular adherent junctions that provides a structural foundation for normal epithelial architecture (Valentijn et al., 2013). E-cadherin, localized to the lateral membrane of differentiated epithelia, is essential for the maintenance of functional junctions and was deficient in ERbKO mouse endometrium (Wada-Hiraike et al., 2006), resulting in deformed glandular genesis and loss of glandular differentiation. Recent work has highlighted the importance of endometrial glands in implantation (Filant and Spencer, 2013), suggesting that, despite earlier beliefs, ERb may indeed have an important role to play in the reproductive function of the endometrium. Treatment with E2 increased both stromal and epithelial ERa and ERb expression in wild-type, ovariectomized mice, whilst combined E2 and progesterone treatment decreased expression of ERb in endometrial epithelium (Wada-Hiraike et al., 2006). Conversely, ERbKO mice showed up-regulation of progesterone receptor (PR) in response to E2 compared with the wild-type mice, suggesting that ERb represses epithelial PR expression (Wada-Hiraike et al., 2006). There are no reports of detailed examination of the proliferative effect of endometrial epithelial cells in ERa, ERb double KO mice in response to E2 and the published studies only describe the uterine phenotype in comparison with wild-type mice (Dupont et al., 2000). ER subtype-specific ligand studies have also indicated that ERb can modulate ERa activity in a response specific manner (Frasor et al., 2003). When ERa was selectively deleted in the mouse uterine epithelium, although the E2-induced initial epithelial mitogenic response remained intact, prolonged E2 treatment induced an increase in epithelial apoptosis, indicating that the protective effect of E2 against uterine epithelial apoptosis is mediated via ERb (Winuthayanon et al., 2010). These observations further suggest that the direct action of E2 on endometrial epithelial cells via ERb is to induce apoptosis. Ovariectomized ERbKO mice showed an aberrant hyper-proliferative response in the absence of E2 (Weihua et al., 2000). These mice would have low E2 and absent progesterone, but relatively high androgen levels of adrenal origin. In breast epithelium, androgen up-regulated ERb via androgen receptor (AR) and inhibited proliferation (Rizza et al., 2014); if a similar mechanism exists in the endometrium, the action of androgens in the absence of ERb may be to stimulate proliferation.

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ERb in endometrial immune cells Immune cells are a major component (.20% of the stromal cells in the late secretory phase) of the endometrium; the cell numbers change in the stratum functionalis according to the ovarian hormone cycle with higher levels seen in the secretory phase, particularly in late secretory phase endometrium (Bulmer et al., 1991b; Berbic and Fraser, 2013). The majority of endometrial leucocytes in the stratum functionalis consist of three cell types: T cells, macrophages and uterine natural killer (uNK) cells, with very few neutrophils except in menstrual endometrium, and rare B cells (Vassiliadou and Bulmer, 1996), although B lymphocytes are seen in the stratum basalis (Bulmer et al., 1988; Marshall and Jones, 1988). The observation that the stromal leucocytes increase in number during the window of implantation is a compelling reason for implicating endometrial leucocytes with a key role in the implantation process, and the immunological maintenance of pregnancy (Blois et al., 2011; Evans et al., 2011). Because of their frequency in the late secretory phase and early pregnancy, as well as the dramatic increase in numbers around the time of expected implantation in a fertilized cycle, many studies have focused on the uNK cells. Recent work has focused on their potential role in spiral artery remodelling in early pregnancy (Robson et al., 2012), although other roles suggested relate to control of trophoblast invasion, immunosuppression and cytokine secretion, amongst others (reviewed in Lash et al., 2010). The available evidence regarding endometrial stromal immune cell numbers and composition in the endometrial stratum basalis and in post-menopausal endometrium is particularly scarce and, for the reasons previously alluded to, evidence on any aberrations of these cell numbers or their function in endometrial pathologies is generally limited to the stratum functionalis and at best is confusing, due to the lack of established and agreed methods of assessment. In humans, uNK cells increase in number dramatically in the midsecretory phase of the menstrual cycle, although the explanation for this increase remains uncertain. This has led to investigation of expression of steroid hormone receptors by uNK cells and in particular the expression of PR since uNK cells are also prominent in progesterone-treated endometrium. Mouse uNK cells are devoid of both ER receptor subtypes

(Borzychowski et al., 2003), but human uNK cells purified from early pregnancy decidua contained mRNA for both ERb1 and ERb2, whilst not expressing ERa or PR transcripts; using immunohistochemistry only ERb1 protein was expressed in uNK cells of non-pregnant endometrium, with no ERb2 protein (Henderson et al., 2003). Expression levels for ERb splice variants in mRNA from purified uNK cells from nonpregnant endometrium are not yet known. This highlights the speciesspecific differences in regulation and function of the various endometrial cell types. Furthermore, ERb expression and specific functions have been described in T-cells (Rider et al., 2006) and macrophages (Kramer and Wray, 2002), yet the ERb expression of endometrial T-cells and macrophages has not been specifically examined. Studies in KO mice and in other murine models have suggested that in T-cells, E2 modulation of the immune response may depend on the origin of the T-cells, and may be tissue-specific (Maret et al., 2003; Wu et al., 2013). Hence, there is a need for further studies to characterize steroid receptor expression by human endometrial immune cells.

ERb in the human uterus Expression of ERb mRNA and proteins has been documented in human endometrium across the menstrual cycle and in post-menopausal endometrium. The method employed in most studies of ERb expression in endometrium has been polymerase chain reaction (PCR), and the level of mRNA detected for ERb at any time in the menstrual cycle has been reported by most researchers to be significantly lower than that for ERa (Matsuzaki et al., 2000; O’Neill et al., 2004). However, many studies either using immunohistochemistry to localize ERb protein at the cellular level with analysis by a variety of semi-quantitative methods or using western blotting reported that the levels of ERb protein in human endometrium are either comparable with or in excess of those of ERa in the endometrium (Villavicencio et al., 2006; Wu et al., 2012). ERb is expressed in all endometrial cell types, including glandular epithelium and stromal cells. ERb is reported by some groups to be the sole ER expressed in many specific cell types within the endometrium, including the endometrial endothelium (Taylor and Al-Azzawi, 2000; Critchley et al., 2001) and uNK cells (Henderson et al., 2003), although as stated before (in the ERb in endometrial immune cells section), others have reported conflicting results (Lecce et al., 2001).

ERb expression in the developing uterus Studies in ERb KO mice suggest that ERb may play an important role in maintaining endometrial quiescence, particularly in the immature uterus (Weihua et al., 2000). Similarly in the pre-pubertal human uterus, ERa and ERb are expressed in both endometrial epithelium and stroma during early development before maturity, whereas menarche (maturation)-associated proliferation coincides with an exclusive increase in ERa expression (Spencer et al., 2011).

ERb expression in healthy adult human endometrium across the menstrual cycle The dynamic expression pattern of ovarian steroid receptor proteins and mRNA in the endometrium according to menstrual cycle phase has been well established. Ligand-activated ERa induces expression of both ER subtypes, PR and AR in the endometrium (Brosens et al., 2004). Progesterone, working through PR, counteracts these effects of E2 on steroid receptor expression, and also prevents proliferation of endometrial epithelial cells, promoting epithelial differentiation and stromal


PR mRNA to be present (Krikun et al., 2005), whilst all existing reports assert that PR protein is not expressed by these cells (Critchley et al., 2001). In summary, the human data suggest that ERb is the main ER subtype in endometrial endothelial cells and that expression may be hormonally regulated, although precise receptor expression remains to be fully clarified (Critchley et al., 2001; Lecce et al., 2001; Kayisli et al., 2004; Krikun et al., 2005). Subsequently, in an elegant set of rodent experiments, Masuda et al. (2007) demonstrated that bone marrow-derived endothelial stem/progenitor cells preferentially express ERa, and that physiological post-natal vascular regeneration is E2 regulated via ERa (Masuda et al., 2007). In the endometrium of rhesus macaques (Macaca mulatta), ERb is the only steroid receptor to be expressed by endothelial cells and perivascular smooth muscle cells, but the perivascular stromal cells expressed all types of steroid receptors (Slayden and Brenner, 2004). The exact ER isoform regulating the vascular remodelling and neovasculogenesis that occurs in regular human endometrial regeneration during the menstrual cycle and after parturition is therefore not yet conclusively established, although there is no doubt that ERb exists in these cells, with evidence that it is likely to play a pivotal role in that process.

8

et al., 2008; Fig. 3). There are no current studies that examine simultaneously the expression (and therefore interplay) of all ovarian steroid receptors (ERa, ERb, PR and AR) in these two functionally very distinct endometrial layers of healthy endometrium (Fig. 3). Therefore, data on spatial and temporal differences in the expression of all steroid hormone receptor types in human endometrium remain inconclusive.

Role of ERb in endometrial regeneration The hypothesis that endometrium regenerates from the hormonally resistant stratum basalis came initially from Prianishnikov (1978). Other studies suggested that although the initial part of post-menstrual endometrial regeneration is independent of estrogen, subsequent postmenstrual repair growth is estrogen-dependent (Ferenczy, 1976). The stratum basalis is widely accepted as the germinal compartment of the endometrium where stem progenitor cells reside (Padykula et al., 1989; Valentijn et al., 2013) and ERb1 and ERb2 are expressed by all cell types in the stratum basalis (Critchley et al., 2002). Many investigators of endometrial stem progenitor cells have reported a reduction or lack of ERa and PR in the more primitive cells with progenitor activity (Chan and Gargett, 2006; Valentijn et al., 2013). In mouse endometrium, the endometrial epithelial progenitor cell pool expanded dramatically despite the lack of expression of both ERa and PR (Janzen et al., 2013). However, none of the descriptive papers of endometrial stromal/epithelial stem/progenitor populations in either animal or human studies examined or commented on ERb expression (Chan and Gargett, 2006; Cervello et al., 2010; Masuda et al., 2010; Janzen et al., 2013), and postpartum vascular regeneration (thought to involve stem cells), although E2-dependent, has been reported to be mediated via ERa rather than ERb (Masuda et al., 2007). Furthermore, studies on mouse endometrial regeneration provide evidence that the primitive label-retaining cells are estrogen responsive (Gargett et al., 2012). Therefore, further work is needed to examine expression of ERb and other hormone receptors in the primitive epithelial and stromal stem/progenitor population, since the observed (possible direct) stimulatory effect of E2 on the progenitor population may be mediated via ERb.

ERb expression in the healthy human post-menopausal endometrium Normal post-menopausal endometrium is the remaining stratum basalis after the cessation of the ovarian hormonal cycle. Similarities between the premenopausal stratum basalis and post-menopausal endometrium have been described, both in the gene expression profile (Nguyen et al., 2012) and in expression of epithelial markers (Valentijn et al., 2013). However, the hormonal milieu of pre- and post-menopausal endometrium is clearly different (Labrie, 2014). Post-menopausal endometrium is exposed to relatively low E2 levels, absent progesterone and relatively unchanged androgen levels of adrenal origin (Yasui et al., 2012). ERb expression in post-menopausal endometrium was reported to be weaker than ERa expression in both stromal and epithelial compartments (Zang et al., 2008). Another small immunohistochemical study which included only 11 post-menopausal patients reported downregulation of both ERs in post-menopausal endometrium compared with the late proliferative phase (Mylonas et al., 2007). Others have reported moderate to strong ERb immunostaining in all cell types in normal post-menopausal endometrium which did not change in hormone-treated (continuous combined E2 and progestagen hormone


decidualization in preparation for embryo implantation (Critchley and Saunders, 2009). ERb mRNA expression across the menstrual cycle is much lower than that of ERa, despite the high levels of ERb protein expression throughout the cycle (Matsuzaki et al., 1999). ERb mRNA is present in epithelial, stromal and endothelial cells, with the highest levels seen in the epithelial cells (Matsuzaki et al., 1999; Critchley et al., 2002). In the estrogendominant proliferative phase, nuclear ERa protein levels are high in all endometrial cell types with moderate levels of nuclear ERb protein. Expression of both ER subtypes increases in the late proliferative and early secretory phases and subsequently decreases in the mid-late secretory phase, yet ERb is the predominant ER subtype in the late secretory phase endometrial stroma (Critchley et al., 2001; Lecce et al., 2001). Whilst stromal ERb expression is increased or maintained in the late secretory phase, epithelial expression of ERb protein decreases in common with ERa (Critchley et al., 2001, 2002; Lecce et al., 2001). Therefore, in the mid-late secretory phase in particular, where endometrial ERa expression diminishes, ERb remains as the predominant ER isoform in the stratum functionalis (Lecce et al., 2001). Interestingly, ERb2 is also expressed in both stromal and epithelial cell compartments, similar to ERb1, but there is a significant decrease in ERb2 in the glandular epithelium of the stratum functionalis in the mid-secretory phase, whilst ERb1 persists (Critchley et al., 2002). This may suggest that ERb1 is able to largely form homo-dimers and become dominant in response to E2 in the mid-secretory phase endometrial stratum functionalis when circulating E2 levels are still high. Intriguingly, the mid-secretory phase of the cycle in humans is also known to be associated with increased E2 levels and a reduction in endometrial glandular proliferative activity (Critchley et al., 2006; Narkar et al., 2006; Cooke et al., 2013). ERb expression in the epithelial compartment of the stratum functionalis correlates with the time when highest E2 levels are seen in the menstrual cycle (late proliferative, early secretory phases) (Lecce et al., 2001), whereas stromal and vascular ERb levels peak in the late secretory phase, with the plateaued second peak of circulating E2 (Lecce et al., 2001) (Fig. 3). Since both E2 via ERa and progesterone via PR have been shown to increase ERb transcripts, we postulate that ERb is the main safety mechanism whereby the potent mitogenic action of E2 is restricted in the healthy endometrium. It has been postulated by many authors that the stratum basalis of the endometrium may be less responsive to ovarian hormone regulation than the stratum functionalis (Prianishnikov, 1978; Padykula et al., 1984). Expression of ERb in the stratum basalis has been reported to remain static across the menstrual cycle (Critchley et al., 2001): this particular study included a modest 32 full-thickness endometrial samples at different time points in the cycle, utilized an immune scoring method that examined only the intensity of the staining and also did not examine co-expression of other hormone receptors such as PR and AR. ERb2 expression was very low in the stratum basalis compared with ERb1, suggesting that ERb1 is the dominant splice variant in the stratum basalis (Critchley et al., 2002). The reason why the stratum functionalis of the endometrium but not the stratum basalis responds to ovarian hormonal signals, despite both layers expressing all classical ovarian hormone receptors, is not fully understood. ERa and PR are present, but their levels in the stratum basalis and the cyclical changes in their expression levels are controversial with reports ranging from alterations in the secretory phase to no change across the menstrual cycle (Critchley et al., 2001, 2002; Leyendecker

Hapangama et al.

9



Figure 3 Expression of ERs in normal, healthy human endometrium. Immunohistochemical staining of paraffin-embedded full thickness human endometrial tissue sections demonstrating brown (DAB) positive nuclear ERb (mouse monoclonal antibody PPG5/10 MCA1974S, Serotec, Kidlington, UK; pretreatment pressure cook in citrate pH6; incubation 1:50 overnight at 48C) (A– F) and ERa (rabbit polycolonal ab37438, Abcam, Cambridge, UK; pretreatment pressure cook in citrate pH6; incubation 1:50 for 2 h at 208C) (G– L) in the stratum functionalis (A, B, G and H) and stratum basalis (D, E, J and K) of proliferative (A, D, G and J), secretory (B, E, H and K) and post-menopausal (C, F, I and L) endometrium. Magnification ×200 except C, I ×100.

replacement therapy) post-menopausal endometrium (Vani et al., 2008). A further study described expression of ERb1 and ERb2 in up to 29 and 21% of healthy post-menopausal endometrium, respectively, and concluded that there is likely to be an antiproliferative role in both endometrium and breast tissue (Cheng et al., 2007). Combined treatment with E2 and testosterone, however, increased ERb expression in

post-menopausal endometrium (Zang et al., 2008), and the antiproliferative effect of androgen treatment on the endometrium is thought to be via increased ERb expression. However, androgen metabolites generated by the aromatase-independent enzymes activate both ER subtypes and ERb activation by these metabolites may represses ERa in the postmenopausal endometrium (Hanamura et al., 2014).

10

ERb in endometrial pathology Endometriosis and adenomyosis

endometrial stratum functionalis of women with endometriosis may also have a reduction in ERb expression compared with healthy controls (Hapangama et al., 2008b). Further studies have shown higher ERa mRNA levels in endometriotic lesions compared with the eutopic endometrium (Matsuzaki et al., 2000, 2001; Fig. 4). Furthermore, ERb promoter methylation has been proposed as the primary defect resulting in differential ERb expression between ectopic and eutopic endometrium (Xue et al., 2007). It is of interest that the pattern of ERb expression reported in both adenomyosis and ectopic endometriotic lesions was similar to that of the endometrial stratum basalis (Mehasseb et al., 2011), which in turn is proposed to be unresponsive to hormones. There are suggestions from primate (Donnez et al., 2012; Sourial et al., 2014) and human studies (Leyendecker et al., 2002; Valentijn et al., 2013) that endometrial stratum basalis plays an essential role in the pathogenesis of endometriosis. This may suggest that the regulation of cell fate in these pathological lesions is distinct from that of the differentiated stratum functionalis in the eutopic endometrium but rather is similar to the stratum basalis that contains the germinal capacity. Further exploration of their similarities to the germinal stratum basalis may improve our understanding of endometrial cellular growth and regeneration.

Polycystic ovary syndrome The clinical phenotype of polycystic ovary syndrome (PCOS) includes reproductive and hormonal aberrations. The endometrial consequences are those of a high and unopposed effect of estrogen, and possibly androgens (Hapangama and Bulmer, 2014). The aetiology of PCOS is not fully understood and using genotyping a +1730 G/A polymorphism in the ERb gene has been proposed to be associated with susceptibility to PCOS (Kim et al., 2010). Homozygous ERbKO mice have defective ovulation reminiscent of PCOS in humans (Imamov et al., 2005). It has been postulated that women with PCOS exhibit a lower pregnancy rate secondary to decreased endometrial expression of both ERa and ERb during the window of implantation, potentially decreasing endometrial receptivity, reducing conception and hence lowering fertility (Wang et al., 2011). However, investigation of the differential ER subtype expression in women with PCOS compared with healthy women has not revealed a definitive pattern (Maliqueo et al., 2003). Anovulatory PCOS is associated with a 3- to 4-fold increased risk of developing EC compared with unaffected women and the effect is postulated to be due to the progesterone unopposed action of E2 (Fearnley et al., 2010; Hapangama and Bulmer 2014), presumed to be via ERa. A gradual increase in ERb levels from anovulatory endometrium to endometrial hyperplasia (EH) was seen in women with PCOS, suggesting either direct involvement of ERb in endometrial proliferation or an indirect effect due to the action of ERa which can cause increased expression of all steroid receptors including ERb (Villavicencio et al., 2006). Further studies are needed to evaluate the involvement of ER subtypes and the endometrial proliferative aberration that occurs in PCOS.

Endometrial polyps Endometrial polyps are commonly occurring outgrowths of the endometrium consisting of a monoclonal overgrowth of endometrial stromal cells with inclusion of a non-neoplastic glandular component (Indraccolo et al., 2013). Endometrial polyps are usually benign but are occasionally associated with focal atypical hyperplasia or even


Endometriosis is a common benign gynaecological condition defined by the presence of endometrium-like tissue outside of the endometrial cavity (Bernardi and Pavone, 2013; Sourial et al., 2014). Adenomyosis is defined as the presence of endometrium within the myometrium (Hapangama and Bulmer, 2014). Aberrant cell proliferation and altered cell fate have been described in eutopic and ectopically grown endometrium and in adenomyosis; estrogen dependence may play a role in the pathophysiology of both conditions (Yang et al., 2007; Hapangama et al., 2009). Although this may immediately suggest a trophic estrogenic effect via ERa, ectopic endometrial lesions and adenomyosis have been reported to express high levels of ERb (.100×) compared with the eutopic endometrium (Bulun et al., 2012). The available evidence regarding levels of ERa and ERb expression in ectopic endometriotic lesions is, however, contradictory (Shao et al., 2014). Factors that are likely to contribute to the variable results include the heterogeneity of the lesions studied, as well as the fact that when surgically excised ectopic endometriotic lesions are examined using techniques such as PCR and western blotting which homogenize the whole tissue the contribution from endometrial-gland and stroma-like tissue to the levels reported can vary widely. Relative over-expression of ERb mRNA (therefore decreased ERa:ERb mRNA ratio) in ovarian endometriomas compared with either peritoneal ectopic lesions (Bukulmez et al., 2007) or eutopic endometrium (Brandenberger et al., 1999; Fujimoto et al., 1999; Bukulmez et al., 2007) has been described suggesting a unique E2-dependent growth of ovarian endometriomas. Deficient methylation of the ERb promoter resulting in pathological overexpression of ERb in endometriotic stromal cells has been suggested by some authors who also report relatively low ERa expression in these cells (Bulun et al., 2012). A high level of ERb has been proposed as the reason for low ERa expression, resulting in low PR levels contributing to progesterone resistance and inflammation. ERb knockdown has been reported to significantly increase ERa mRNA and protein levels in endometriotic stromal cells (Trukhacheva et al., 2009; Bulun et al., 2012). This theory is reviewed extensively (Bulun et al., 2012). Conversely, ERb overexpression in endometrial stromal cells was reported to decrease ERa mRNA and protein levels and ERb knockdown significantly decreased proliferation of endometriotic stromal cells (Trukhacheva et al., 2009). In human endometriotic lesions (Fujimoto et al., 1999; Bukulmez et al., 2007) and in induced lesions in endometriosis models in baboons (Fazleabas et al., 2003), and in rodents (Han et al., 2012), there was decreased ERa, while ERb was maintained. Compared with healthy women, women with endometriosis and adenomyosis showed a decrease in the ERa/ERb ratio in proliferative phase eutopic endometrium, suggesting ERb dominance (Juhasz-Bass et al., 2011; Mehasseb et al., 2011), although there is no conclusive evidence suggesting differential proliferative activity in the proliferative phase endometrium of women with endometriosis. Most cellular aberrations described in the eutopic endometrium have been observed in the stratum functionalis in the secretory phase where a persistence in proliferative activity is detected (Hapangama et al., 2009, 2012) and ERb expression in women with endometriosis in the secretory phase has been reported to be unchanged (Hudelist et al., 2005) or decreased (Hapangama et al., 2008b). In the window of implantation,

Hapangama et al.

11


benign, active, peritoneal human endometriosis lesions (A and B) and in Grade 1 (C and D) and Grade 3 (E and F) endometrioid EC. Antibodies, pretreatments and incubation conditions are as detailed in Fig. 3. Magnification ×200.

adenocarcinoma, and these findings are more common in postmenopausal women (Costa-Paiva et al., 2011). Endometrial polyps lack the cyclical changes seen in the adjacent endometrium and E2 stimulation is postulated as the main driving force for endometrial polyp formation (Van Bogaert, 1988). This is supported by the observation that the use of tamoxifen, which acts as an ER agonist on the endometrium, increases the risk of endometrial polyps (Erdemoglu et al., 2008; Tokyol et al., 2009). ERb mRNA expression in benign endometrial polyps has been reported to be similar to the adjacent normal endometrium (Zitao et al., 2010), whilst protein expression in the stromal compartment was increased (Ye et al., 2006). In tamoxifentreated endometrial polyps, glandular ERb expression appeared to be lower than that of ERa, suggesting a lack of ERb mediated opposition

of ERa activity (Hachisuga et al., 2003). Further sufficiently powered studies are necessary to evaluate the involvement of ERb in the pathogenesis of benign endometrial polyps.

Endometrial hyperplasia EH is histologically defined as the abnormal overgrowth of endometrial glands in relation to the endometrial stroma (Ordi et al., 2013). Several classification schemes have been proposed and reproducibility is variable (Yang et al., 2012; Li and Song, 2013; Ordi et al., 2013). Unopposed estrogen stimulation, usually associated with anovulation or occasional ovulation in peri-menopausal women, is a common cause of EH, which may also be seen in 20% of women with PCOS with oligomenorrhoea


Figure 4 Expression of ERs in pathological human endometrium. Positive, brown nuclear immunostaining for ERb (A, C and E) and ERa (B, D and F) in

12 (Prakansamut et al., 2014). When cytological atypia is present, EH is associated with approximately a 30% risk of developing into or co-existing with EC (Lacey et al., 2007; Park et al., 2011). Transcripts of ERb splice variants have been described in EH (Witek et al., 2001), and ERb protein expression in EH without cytological atypia in women without PCOS has been reported to remain unchanged compared with normal proliferative endometrium, whilst in pre-cancerous atypical hyperplasia, ERb expression decreases (Hu et al., 2005). This suggests that the loss of ERb regulation of ERa-mediated proliferation results in EH.

Endometrial cancer

disease outcome (Sastre-Serra et al., 2013). Nevertheless, the existing data on the correlation between clinical outcome and the ERa/ERb ratio are conflicting (Jongen et al., 2009; Zannoni et al., 2013). Methodological differences (sample size and scoring methods) are likely to explain the observed discrepancies in the available literature. There are over 160 publications describing ERb involvement in EC, yet the data on ERb expression, correlation with disease stage and grade of histological differentiation remain controversial (Table I). Another reason for this is that not all studies included healthy control tissue comparators. Some studies have only described ERb expression in neoplastic cells and the surrounding niche (Sˇmuc and Rizˇner, 2009). When healthy controls were used, they included either pre- or post-menopausal women, although in some studies, the menopausal status of the healthy control women was not even mentioned (Jarzabek et al., 2013; Knapp et al., 2013). When premenopausal control tissue was used, attention was not given to the known differences between menstrual cycle phase (Jazaeri et al., 2001), or in full thickness endometrial samples from hysterectomies, the differences between stratum basalis and functionalis, with no information provided regarding which layer was included in the analysis (Chakravarty et al., 2007; Knapp et al., 2013). Early studies also suffered from deficiencies in antibody and primer specificities, particularly when splice variants were not appreciated. Breast and prostatic carcinogenesis has been reported to be associated with a decrease or loss of ERb expression, and this is the basis for the hypothesis that ERb plays a tumour suppressor role (Bottner et al., 2014). In breast and ovarian cancer, ERb exerted anti-proliferative effects via prevention of ERa transcriptional complexes from activating c-myc, cyclin-1 and cyclin-A genes and induction of cyclin-dependent kinase inhibitors leading to arrest of the cell cycle in G2 phase (Paruthiyil et al., 2004). Similarly, in the endometrium (as described above), some authors have reported a decrease in ERb expression in EC suggesting an analogous function. A decrease in both ERb mRNA levels and protein (immunohistochemistry) has been reported in endometrioid EC compared with either adjacent normal endometrium or normal proliferative endometrium from healthy premenopausal control women (Paul et al., 2004; Hong-bing et al., 2008; Sˇmuc and Rizˇner, 2009). However, a potential oncogenic role has also been proposed by other reports showing an up-regulation of ERb5 transcript in high-grade endometrioid EC, which was also associated with HER2 and MYBL2 oncogene expression (Skrzypczak et al., 2004; Ha¨ring et al., 2012b). Since ovarian steroid hormone receptor expression is a feature of differentiated endometrial cells, with primitive or undifferentiated cells not expressing ER or PR, the diminishing steroid receptor expression levels in EC, including ERb, may merely be a sign of loss of cellular differentiation and/or cellular transformation (Fig. 4). Alternatively, dysregulation of steroid receptor homeostasis owing to loss of PR and ERa with possible persistant expression and up-regulation of ERb isoforms (ERb5) and splice variants, particularly in high-grade and advanced stage endometrioid EC, may reflect a tumour-promoting role for ERb in high-grade ECs. Data from ERabKO mice may suggest ERb could partially compensate for the loss of ERa (Dupont et al., 2000) and therefore the reported increase in ERb in high-grade ECs may have a tumour-promoting effect similar to ERa. However, conclusive data on the functional role of ERb or the splice variants on regulating EC growth or metastasis does not exist to date. The reason for the apparent contradictory data for ERb to be a tumour suppressor and a promotor may be due to a tissue-specific dual role of ERb; a tumour suppressor role in healthy


EC is the most common gynaecological malignancy and is usually a disease of the post-menopausal endometrium (Murali et al., 2014). In contrast with atrophic and inactive post-menopausal endometrium (McCluggage, 2011), the hallmark of EC is dysregulated and inexhaustible cellular proliferation (Owings and Quick, 2014). At least Type-I endometrioid EC is thought to be E2-dependent (although recent results indicate that Types I and II ECs have similar risk factors), and unopposed E2 action is known to increase the risk of endometrial carcinogenesis (Setiawan et al., 2012). Therefore, conditions such as PCOS and obesity, where there is an excessive extra-ovarian conversion of adrenal androgens to estrogens, as well as HRT use, increase the risk of EC (Thanapprapasr and Thanapprapasr, 2013). Available data on ERb protein and mRNA expression in EC subtypes are largely confined to endometrioid EC (Table I). The general consensus that the carcinogenesis process in Type II ECs is E2-independent is likely to be the reason for the lack of data on ERb expression in Type II ECs in particular, with virtually no studies examining the ERb expression in serous and clear cell carcinoma. Nevertheless, there are emerging data suggesting involvement of ERb in carcinogenesis of other tissues, such as the intestine, that are typically not regarded as E2-responsive (Hogan et al., 2009), and hence future studies examining ERb in type II ECs are warranted. In addition to the wild-type ERb1, the expression of splice variant isoforms ERb2, b3, b4, b5 and exon deletes (bD1, b2D5, bD4, bD6) has been described in endometrioid EC (Saegusa and Okayasu, 2000; Utsunomiya et al., 2000; Sakaguchi et al., 2002; Paul et al., 2004; Skrzypczak et al., 2004; Mylonas et al., 2005; Chakravarty et al., 2007; Collins et al., 2009; Zannoni et al., 2013). Similar expression patterns to ERa have been reported for ERb variants (ERb1, ERb2 and ERb5) in endometrioid EC samples: ERb1 and ERb2 immuno-expression was higher in low-grade ECs, whereas ERb5 expression was constitutively intense regardless of the grade (Collins et al., 2009). In tamoxifen-associated ECs, ERb expression was particularly prominent compared with spontaneous ECs (Negoita and Mihailovici, 2011). In a different study that included a semi-quantitative scoring system similar to the quickscore hormone receptor evaluation in breast cancer, ERb1and ERb2 expression did not correlate with tumour grade, FIGO (International Federation of Gynecology and Obstetrics) stage or myometrial invasion, but the ERa/ERb2 ratio was reported to be an independent prognostic factor of overall survival (Zannoni et al., 2013). The ERa/ERb ratio has been used to evaluate the imbalance in the relative expression of ER isoforms in endometrial (Fujimoto et al., 2000; Jazaeri et al., 2001; Takama et al., 2001; Jongen et al., 2009; Sˇmuc and Rizˇner, 2009) and other hormonal-regulated malignancies, such as breast cancer, and has been suggested as a potential predictor of

Hapangama et al.

Reference

Isoform studied

Patients

...........................................................

Study groups (n samples)

Age (years)

Control

Design

...............................................................................

Methods

IHC scoring method

ERb antibody

Results ERb expression in ECs

.......................................................................................................................................................................................................................................................... % positive

Polyclonala Imunnotech1,a

ERb Protein +, mRNA + ERb mRNA + in the cytoplasm of malignant cells

Utsunomiya et al. (2000)

ERb

45 endometrioid G1–3

30–85

No control

RT–PCR ISH IHC

Jazaeri et al. (2001)

ERb

7 endometrioid G1 7 carcino-sarcoma

55–63

7 pre M 16 PM

RT–PCR Western blot

ERb mRNA ↔ in PM and cancer groups ERb/ERa ratio in cancer

Takama et al. (2001)

ERb1

33 endometrioid 1 clear 1 serous

36–81

1 SP 1 EHNA

Multiplex RT-qPCR western blot

ERb/ERa ratio (in advanced stage, with myometrial invasion)

Fujimoto et al. (2000)

ERb1

20 stage I 20 stage II 20 stage III

31–48

20 normala

RT–PCR IHC

Paul et al. (2004)

ERb1 ERbD6

14 endometrioid G1, G2 7 ECHA 38 EHNA

41–67

11 PP 12 SP

RT-qPCR

ERb1 in EC compared with PP ERbD6 in complex hyperplasia and EC compared with PP

Skrzypczak et al. (2004)

ERb1 –5

19 stage I (A,B) 8 stage IC, IIB

39–69

6 PM 14 PP 1 SP

RT-qPCR

ERb1, b2 and b5 + in EC ERb3 and ERb4 +ERb5 in EC ERb2D5 mRNA in EC

Chakravarty et al. (2007)

ERb1 ERb2/bcx

26 endometrioid cancer 20 EHNA

a

22 PP 15 SP

RT–PCR IHC

IRS

b1 MonoclonalEMR02, Novocastra3 b2/bcx rabbit poly clonal in house

ERb1 + ↔ in benign and EC ERb2/bcx in EC (protein and mRNA) compared with PP

Jongen et al. (2009)

ERb1

315 endometrioid

32–89

No control

TMA IHC

Positive if .10% has intensity of 2+ or more

Mouse monoclonal, ppg5/10, Serotec4

ERb/ERa ratio .1 associated with disease free and overall survival

Hong-Bing et al. (2008)

ERb1

68 endometrioid 93 EHNA 86 ECHA

a

Normal adjacent

IHC

photo analysis

Rabbit polyclonal, Santa Cruza,2

ERb in atypical hyperplasia and EC (ERb inversely correlate with ki67)

Collins et al. (2009)

ERb1 ERb2 ERb5


a

No control

RT-qPCR IHC

Not quantified

Monoclonal, b1 ppg5/10 b2 57/3 b5 5/25, Serotec4

ERb1, ERb2 and ERb5 (mRNA and protein) + in EC ↔ between the grades

Sˇmuc and Rizˇner (2009)

ERb

24 endometrioid G1–3 1 serous

37–83

Normal adjacent

RT-qPCR

H-score

Goat polyclonal L-20, Santa Cruz2


Table I Published studies examining ERb expression in EC.

ERb (protein and mRNA) were significantly lower than ERa in normal and cancer tissue ERb/ ERa ratio ↔ non-metastatic EC ERb/ERa in metastatic EC

ERb (and ERa) mRNA ERb/ERa ratio (not significant)

Continued

13


14

G1 –3, Grade 1 –3; PP, proliferative phase; SP, secretory phase; PM, post-menopausal; pre M, premenopausal; EHNA, endometrial hyperplasia with no atypia; ECHA, endometrial complex hyperplasia with atypia; IHC, immunohistochemistry; TMA, tissue microarray; ISH, in situ hybridization; IRS, immunoreactive score; +, expressed; +, minimal expression levels; , increased; , decreased; ↔, not changed; 1Marseille, France; 2Biotechnology, Inc., Santa Cruz, CA, USA; 3Newcastle upon Tyne, UK; 4Serotec Ltd, Oxford, UK; 5Glostrup, Denmark. a Important information is not reported.

ERb/ERa in low-grade EC ERb1 and ERb2 /ERa ratio .1 independently associate with higher risk of death (ERa alone does not predict the outcome) IHC No control ERb1 ERb2 Zannoni et al. (2013)


35–88

Western blot ERb Knapp et al. (2013)


44–76

29 normala

Allred

Monoclonal b1 ppg5/10, DAKO5 b2 57/3, Serotec4

ERb and ERa in EC ERb and ERa in G1

ERb1, ERb2 ↔ ERbD1 in G1, 2 EC; ERbD4 in G2, 3 EC (ERb1, ERb2, ERb5 correlate with HER2; 6 exon skip isoforms of ERb correlate with HER2, MYBL, cyclin B1,D1,A1) RT-qPCR 15 PP 6 SP 17 PM 54–82 46 endometrioid G1–3 ERb1 –5 ERbD1 ERbD2 ERbD3 Ha¨ring et al. (2012b)

Results

..........................................................................................................................................................................................................................................................

ERb antibody IHC scoring method

...............................................................................

Design

Methods Control Age (years)

...........................................................

Study groups (n samples)

Patients

Isoform studied Reference

Table I Continued

endometrium which expresses the full complement of other ovarian hormone receptor types and particularly ERa, and a potential tumour promoter role in high-grade EC cells that have lost other receptor subtypes. This is an interesting possibility that needs to be explored in future studies. In summary, the exact role of ERb in endometrial carcinogenesis and progression of EC is not yet fully known. Therefore, comprehensive characterization of ERb expression in EC subtypes and further functional studies are required to reveal the involvement and contribution of ERb in endometrial carcinogenesis. Since at least some ECs are likely to respond to hormonal chemotherapeutic agents, a detailed examination of all steroid receptor types and standardization of the methodology for quantification of hormone receptors in EC in a similar approach to that used for other hormone responsive cancers, such as in breast cancer, is urgently needed.

Pharmacological regulation of endometrial ERb expression Evidence for regulation of endometrial ERb expression by exogenous hormone treatment There is evidence from both in vivo and in vitro studies that ERb plays an important role in determining cellular responses to estrogens and antiestrogens (Hall and McDonnell, 1999). Progesterone induced ERb transcription whilst down-regulating the PR gene in telomerase immortalized endometrial epithelial cells (Hombach-Klonisch et al., 2005). In contrast, treatment of women with an intrauterine system containing the androgenic progestagen, Levenorgestrel, decreased epithelial and stromal ERb (and also ERa and PR) expression after 3 months (Engemise et al., 2011). In non-human primates, the non-specific progestogen, MPA increased endometrial stromal ERb expression in ovariectomized animals (Wang et al., 2002). The available evidence therefore suggests that the endometrial ERb expression in response to exogenous hormones depends on the duration and dose as well as the presence of other steroid hormones and their receptors.

Evidence for SERMs (ERb selective pharmacological agents) in endometrium Studies in rodents have shown that Puerarin, a selective estrogen receptor modulator (SERM) with ERb preferential agonist activity, may affect endometrial expression of implantation-specific proteins and may have a contraceptive effect (Saha et al., 2012). In agreement with these data, a Phase 2 trial investigating the use of a selective ERb agonist for climacteric symptoms in 164 post-menopausal women reported an increased incidence of endometrial thickening (18 versus 6% in the placebo group) but with benign histology (Grady et al., 2009). In contrast, others have reported non-uterotrophic activity by ERb selective agonists in ovariectomized rats (Hertrampf et al., 2008). Therefore, the expected uterine/ endometrial neutral effect of novel ERb selective SERMs has not been conclusively confirmed in long-term studies and should be explored prior to administering these agents for their other potential health benefits. However, studies of these agents in other tissues have highlighted the complexities of interplay between ERb and other hormonal nuclear receptor functions. Differential actions of these agents in the endometrial epithelial and stromal compartments also have to be fully


ERb expression in ECs

Hapangama et al.

15


elucidated in health and in endometrial pathologies prior to considering the therapeutic potential of the many available selective pharmacological modulators of ERb for patient benefit.

Summary/conclusion

Supplementary data Supplementary data are available at http://humupd.oxfordjournals.org/ online.

Acknowledgements The authors are grateful for Dr Nicola Tempest and Mrs Jo Drury for their assistance in manuscript preparation.

D.K.H. conceived the manuscript, D.K.H. and A.M.K. prepared the first draft and J.N.B. revised the manuscript critically for important intellectual content. A.M.K. drafted the figures and tables. All authors revised and read the manuscript and approved the submitted final version.

Funding We acknowledge the support by Wellbeing of Women project grant RG1073 (D.K.H.), RG1342 (J.N.B.) and RG1487 (D.K.H.), Iraqi Government (A.M.K.) and BBSRC BB/E016790/1 (J.N.B.).

Conflict of interest None declared.

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Despite early predictions to the contrary, the collective evidence suggests that ERb has an important role in normal endometrial tissue homeostasis, cell turnover and regeneration and also in most if not all benign and malignant endometrial diseases. Information gained from studies of other tissues on the basic function of ERb isoforms in restraining the ERa-mediated cell-specific trophic/mitotic responses to E2 has allowed appreciation of the important role of ERb in regulation of cell fate in human endometrium. Our current understanding of ERb expression and function in endometrium is, however, far from complete. ERb is dynamically expressed in healthy premenstrual endometrium and persists in post-menopausal atrophic endometrium. The possible dysregulation of ERb expression that has been reported in benign endometrial proliferative diseases, such as endometriosis and PCOS, as well as in EC highlights the importance of exploring the potential use of this ER isoform for targeted therapy with estrogenSERMs. However, there are several voids in our current knowledge; for example, the existence of ERb splice variants and their influence on ERb1 function and E2 responsiveness in health and in endometrial disease. Research clarifying these areas and functional studies may explain conflicting data that exist in the current literature. Future studies should specifically examine the inter-regulatory effect of the ERb splice variants, whereby each of the splice variants regulates the effect of the other variants, as well as the co-regulatory effect of ERb with other steroid hormone receptors and their combined effects on endometrial cellular responses to steroid hormone signals, as these are all pivotal for predicting the effects of pharmacological hormone receptor modulators in endometrial function. Our current understanding of the role of ERb in the endometrium from many animal studies remains to be confirmed by functional studies (both in vitro and in vivo) on human endometrium, or human endometrial epithelial cells in particular. However, there is also an urgent need to standardize the way that immunohistochemical studies of steroid receptor expression in benign and malignant endometrium are conducted and assessed, especially in EC; agreement is needed regarding the most appropriate control tissue (post- or premenopausal endometrium), especially in the light of increased levels of obesity and the associated estrogenic effects on the endometrium, as well as the development of a robust method for quantification/assessment of immunohistochemical data on ERb expression, similar to the scoring systems that are employed in other hormonally regulated tissues, such as breast cancer, since these data may have obvious direct clinical implications in guiding therapy.

Authors’ roles

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The effect of estrogen on the estrogen receptor-immunoreactive cells in the rat medial preoptic nucleus.

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The mitosis-differentiation checkpoint, another guardian of the epidermal genome.