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Keywords:

  • aromatase;
  • knock-out;
  • nuclear receptor;
  • oestradiol;
  • oestrogen receptor

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. The oestrogen receptor
  5. Oestrogen receptor β
  6. ArKO, ERKO and BERKO – oestrogen and oestrogen receptor knock-outs
  7. Receptor interplay
  8. Conclusions
  9. Acknowledgements
  10. References

Enmark E, Gustafsson J-A (Karolinska Institutet, Huddinge, Sweden). Oestrogen receptors – an overview (Review). J Intern Med 1999; 246: 133–138.

The oestrogen receptor (ER) is a ligand-activated transcription factor that mediates the effects of the steroid hormone 17β-oestradiol in both males and females. Since the isolation and cloning of ER, the prevailing opinion has been that only one such receptor exists. The finding of a second subtype of ER (ERβ) has caused considerable excitement and has forced endocrinologists to re-evaluate many aspects of the actions of oestrogens. In this article, we will try to summarize the current knowledge about the two oestrogen receptor subtypes, with the emphasis on oestrogen receptor β (ERβ), and to comment on the observations in mice lacking either receptor or the hormone itself.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. The oestrogen receptor
  5. Oestrogen receptor β
  6. ArKO, ERKO and BERKO – oestrogen and oestrogen receptor knock-outs
  7. Receptor interplay
  8. Conclusions
  9. Acknowledgements
  10. References

Oestrogens are steroid hormones traditionally connected with female reproduction. These hormones are mainly synthesized in the ovary and testis, but also in peripheral tissues through aromatization of androgens [1]. During the recent decade, important roles for oestrogens have also been reported in the male reproductive system [2] and in a number of non-reproductive tissues exemplified by, but not limited to, bone tissue, the cardiovascular system and the central nervous system (CNS).

Oestrogens are also extremely important clinically and are commonly used for prevention and treatment of postmenopausal symptoms and as contraceptives. Oestrogen antagonists are used in the treatment of hormone-dependent breast cancer and have occasionally been tried also in the treatment of prostate cancer.

One aspect of oestrogen action that has hitherto often been overlooked is that these hormones are of importance in several tissues not traditionally considered as ‘oestrogen targets’. Amongst these, the gastrointestinal tract and the immune system are of particular interest. For several years it has been claimed that oestrogens may protect against colon cancer, and similar claims have also been made for diets containing soy, a product rich in phytoestrogens.

As for the immune system, it has long since been known that oestrogens have important effects. Most autoimmune diseases are more common in women than in men and quite frequently begin under conditions when oestrogen levels change dramatically, e.g. during puberty, menopause or pregnancy. In animal models, physiological doses of oestradiol have been shown to improve diseases involving T-cell-mediated immunity, e.g. arthritis, but to aggravate diseases such as SLE or immune complex-mediated glomerulonephritis, which are connected to the B-cell-mediated immune response. The recent discoveries in our laboratory have revealed a mechanism through which these oestrogenic effects may perhaps be mediated.

The oestrogen receptor

  1. Top of page
  2. Abstract
  3. Introduction
  4. The oestrogen receptor
  5. Oestrogen receptor β
  6. ArKO, ERKO and BERKO – oestrogen and oestrogen receptor knock-outs
  7. Receptor interplay
  8. Conclusions
  9. Acknowledgements
  10. References

Most, if not all, of the known effects of oestrogens are mediated by ligand-activated transcription factors, called oestrogen receptors (ERs). They have a structure characteristic of members of the nuclear receptor superfamily, one of the largest protein families known to date with more than 70 currently recognized members, the number still increasing every year [3]. Other members of this protein family are the receptors for testosterone, progesterone, corticoids, thyroid hormone, vitamins A and D3, and a large group of proteins referred to as ‘orphan’ receptors, for which specific ligands are still unknown.

For many other members of the nuclear receptor superfamily, multiple receptor subtypes have been identified, usually two or three, as for instance in the case of the thyroid hormone and retinoic acid receptors as well as for many of the orphan receptors [4].

Analysis of the oestrogen receptor and other steroid receptors shows that they can be subdivided into several functional domains [5]. There is a highly conserved DNA-binding domain (C) containing two zinc fingers, which are involved in specific DNA binding and receptor dimerization, and a less well conserved ligand-binding domain (E) harbouring regions important for ligand binding, receptor dimerization, nuclear localization and interactions with transcriptional coactivators and corepressors.

The N-terminal A/B domain is highly variable in sequence and length and usually contains a transactivation function, which activates target genes by directly interacting with components of the core transcriptional machinery or with coactivators that mediate signalling to downstream proteins. The hinge (D) domain contributes flexibility to the DNA- versus the ligand-binding domain and has also, in some cases, been shown to influence the DNA-binding properties of individual receptors; it may also serve as an anchor for certain corepressor proteins. Finally, the C-terminal F domain has been shown to contribute to the transactivation capacity of the receptor, but its other functions, if any, are to a large extent unknown.

The ‘classical’ oestrogen receptor (ERα) was discovered by Elwood Jensen in 1958 and cloned in 1986 from uterus [6], and for many years it remained a dogma that only one such receptor existed.

It was previously assumed that ERα was indispensable for maintenance of life, since no cases had been reported of humans or animals with an inactivated or deleted receptor created by naturally occurring mutations.

However, in 1994, a patient study was published describing a man lacking functional oestrogen receptor(α) [7]. This person had severe osteoporosis and reduced fertility. This finding effectively invalidated the notion that deletion of this oestrogen receptor would be lethal.

Furthermore, in 1993, development of an oestrogen receptor (ERα) knock-out (ERKO) mouse strain was reported [8]. In this mouse strain, however, specific oestrogen binding could still be observed in some tissues. In retrospect, this suggested the existence of a second receptor for oestrogen.

In 1996, our laboratory reported on the isolation of the first cDNA clone for a second oestrogen receptor, which we called, and which is now recognized as, oestrogen receptor β (ERβ) [9]. This discovery has raised a number of questions regarding the respective physiological roles of ERα and ERβ, and some of these issues will be addressed below.

Oestrogen receptor β

  1. Top of page
  2. Abstract
  3. Introduction
  4. The oestrogen receptor
  5. Oestrogen receptor β
  6. ArKO, ERKO and BERKO – oestrogen and oestrogen receptor knock-outs
  7. Receptor interplay
  8. Conclusions
  9. Acknowledgements
  10. References

ERβ is homologous to the previously identified mammalian oestrogen (ERα), particularly in the DNA-binding domain (95% amino acid identity) and in the ligand-binding domain (55% amino acid identity) ( Fig. 1).

image

Figure 1. Percentage amino acid identity between human ERβ and other oestrogen receptors: rat ERβ (rERβ), mouse ERβ (mERβ), human ERα (hERα) and rainbow trout ER (rtER). Numbers above each box refer to amino acid numbers, whereas numbers inside each box refer to percentage amino acid identity. Alignment was performed using the Clustal alignment tool and MegAlign/DNAStar software.

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The human ERβ gene has been mapped to band q22–24 of chromosome 14 [10]. Since the human ERα gene has been mapped to the long arm of chromosome 6, this definitely excludes the possibility of differential splicing to explain the formation of the ERβ isoform. 14q22–24 is close to a gene associated with early onset of Alzheimer’s disease [11]. The risk of Alzheimer’s disease is reduced in women by oestrogen replacement therapy and, furthermore, this treatment appears to improve the condition in some cases. Future genetic analysis will clarify if there is a genetic link between Alzheimer’s disease and the ERβ gene. Since ERβ is the major oestrogen receptor in the brain, this remains at least an exciting possibility.

ERβ has also been cloned from human [1012], mouse [13], marmoset monkey (Callitrix jacchus) (Gaughan, J. unpublished, Genbank no. Y09372), Japanese quail [14] and Japanese eel [15]. Human ERβ shows approximately 89% identity to rat ERβ, 88% identity to mouse ERβ, and 47% identity to human ERα, in its translated portion, a degree of similarity well in accordance with that observed for species homologues of other nuclear receptors. It can, however, be noted that the degree of homology between ERα and ERβ is low for two receptor subtypes, particularly in the ligand-binding domain. In fact, ERα and ERβ are not more similar than some receptor pairs with different ligands, e.g. the glucocorticoid and progesterone receptors. This indicates that it should be possible to design synthetic oestrogens specific for one of the two receptor subtypes, a fact with obvious clinical implications. The three-dimensional crystal structure of the ERα ligand-binding domain [16] has been determined and is currently in progress for ERβ[17]. These structures will of course be useful tools in designing subtype-specific agonists and antagonists.

Regarding natural oestrogens as ligands, ERβ binds oestradiol with a Kd similar to that of ERα, 0.6 n m, and the binding is also similar for many other oestrogens, although some differences have been noted, most importantly for some phytoestrogens [18] (see below).

The ERβ protein is capable of stimulating transcription of an oestrogen receptor target gene in a manner similar to ERα, but in many cell types the degree of activation is lower than that of ERα.

Sequence comparison of all known oestrogen receptors shows that these receptors form three groups, where the receptors cloned from fish constitute a separate subgroup ( Fig. 2). The exception in the fish subgroup is the ER cloned from japanese eel, which actually represents an ERβ homologue [15]. Mice with double knock-outs of both the ERα and ERβ genes (see below) will be helpful to clarify if the ‘fish’ subtype of oestrogen receptor actually represents an ‘ERγ’ also present in mammalian species.

image

Figure 2. Summary of all oestrogen receptors cloned from various species. The phylogeny was created using the Clustal alignment tool and MegAlign/DNAStar software, and includes the DNA-, hinge- and ligand-binding domains of the respective receptors . For receptors marked with an asterisk, the available sequence information is incomplete. In addition to the receptors included in the alignment, short sequences of oestrogen receptors have also been reported in the Genbank database from Callitrix jacchus (ERβ subtype), cat fish (‘fish’ subtype) cat, red deer, Arabian camel, anole and Oryctolagus cuniculus (all ERα subtype).

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In the rat, ERα shows highest expression in uterus, testis, pituitary, ovary, kidney, epididymis and adrenal, whereas ERβ is most expressed in brain, prostate, ovary, lung, bladder and epididymis [9].

Analysis of the tissue distribution of ERβ in the human indicates some important differences as compared with rodents [10]. The most striking difference is seen in the prostate, where the expression of ERβ is very high in the rat but is lower in the human, as judged from Northern blotting and in situ hybridization. However, the expression of ERβ in the human testis seems to be higher than in rat testis, and particularly high in the developing spermatocytes. It has to be kept in mind, however, that these data refer to mRNA and not protein levels and thus need to be substantiated by immunohistochemistry or similar techniques.

The expression of ERβ may have some relevance in the intensive debate concerning alleged effects of different xenobiotics on the reproductive ability of animals, particularly in fish and humans [19], where compounds called ‘environmental oestrogens’, including, for example, PCBs, have been in particular focus. We have shown that both ERα and ERβ may bind at least some of these compounds [18]. The affinity is very low, about one-thousandth of that of oestradiol. Still, ERβ binds at least two such compounds, methoxychlor and bisphenol A, with considerably higher affinity than ERα, and this may possibly be of biological significance.

In the ovaries, the stroma of the cortex expresses ERβ in the human but not in the rat. The expression of ERβ is very high in human but not in rat thymus, a finding (cf. above) of possible relevance to the issue of oestrogens and the immune system.

Finally, the high levels of ERβ seen in the gastrointestinal tract of the human are not seen in the rat. ERβ has a relatively high affinity for some substances with oestrogenic activity derived from soy and other plants, in particular genistein and coumestrol, considerably higher affinity than that exhibited by ERα[18]. For several years it has been claimed that oestrogens, including these phytoestrogens, may protect against colon cancer [20] and it is possible that ERβ may mediate some of these effects.

ArKO, ERKO and BERKO – oestrogen and oestrogen receptor knock-outs

  1. Top of page
  2. Abstract
  3. Introduction
  4. The oestrogen receptor
  5. Oestrogen receptor β
  6. ArKO, ERKO and BERKO – oestrogen and oestrogen receptor knock-outs
  7. Receptor interplay
  8. Conclusions
  9. Acknowledgements
  10. References

Recently, three very interesting mouse strains have been created, in which either of the two known oestrogen receptors or the hormone oestradiol itself has been eliminated. The two oestrogen receptors were eliminated through targeted disruption of the respective ER gene, and oestradiol was removed by inactivation of the enzyme aromatase (cyp19), which converts testosterone to oestradiol. These mice are very interesting and informative concerning the functions of oestrogens and its two receptors.

The aromatase knock-out mice, termed ArKO [21], are characterized by infertile females, but not males. This is caused by a defect in ovulation and is furthermore accompanied by underdeveloped external genitalia, uteri and mammary glands. Examination of the hormonal levels shows, as expected, that oestradiol levels are below detection, whereas testosterone levels are markedly elevated. The ArKO is furthermore characterized by dull fur coat and increased size of mammary and gonadal fat pads. Nothing has yet been published regarding the bone phenotype of the ArKO. Aromatase deficiency in humans, however, is characterized by osteopenia with failure of epiphyseal closure.

The female ERKO mouse, where oestrogen receptor α has been inactivated, has cystic haemorrhagic follicles and no corpora lutea, and the males have testicular atrophy, decreased spermatogenesis and inactive sperm [8]. As a consequence, both sexes of this mouse strain are infertile. Furthermore, the ERKO mouse shows abnormalities in reproductive behaviour [22] and breast development [23]. The bone tissue is mildly affected [24]. Interestingly, the protective effects of oestrogen in the cardiovascular system are seemingly unaffected, as tested by the carotid arterial injury model [25].

The levels of oestradiol are grossly elevated in this mouse strain and it should be kept in mind that some of the effects seen, e.g. in the ovary, may well be a consequence of this. For this reason, it will be of great interest to study ERKO mice in which the level of oestradiol has been lowered, e.g. by creating tissue-specific knock-out mice.

The oestrogen receptor β knock-out mouse (BERKO) was created less than 1 year ago. Thus, only limited information is available regarding its phenotype. However, the reproductive phenotype is known [26]. Interestingly, it bears a striking resemblance to that of the aromatase knock-out mouse. The male BERKO mice are, as far as is currently known, fully fertile . The female mice, however, have a reduced fertility due to a block in the last step of follicle development before ovulation. Hence, few or no corpora lutea are present. This block may be, at least partly, overcome by treatment with external follicle-stimulating hormone (FSH) and luteinizing hormone (LH). We have previously reported the ERβ levels to be very high in the rodent prostate [9]. Interestingly, our preliminary analysis indicates signs of prostate hyperplasia in BERKO mice, findings which will be of great interest to follow up in older animals. Quite surprisingly, these mice appear to have reduced abdominal fat, pointing to a hitherto overlooked aspect of oestrogen function.

Receptor interplay

  1. Top of page
  2. Abstract
  3. Introduction
  4. The oestrogen receptor
  5. Oestrogen receptor β
  6. ArKO, ERKO and BERKO – oestrogen and oestrogen receptor knock-outs
  7. Receptor interplay
  8. Conclusions
  9. Acknowledgements
  10. References

Several groups have reported that ERα and ERβ may form functional heterodimers [27]. Hence, three different pathways through which oestrogens may activate target genes are possible. In cells expressing only one of the two receptor subtypes, homodimers of ERα or ERβ may interact with their respective response elements in target genes. In cells expressing both receptors, heterodimers containing one molecule each of ERα and ERβ may also be formed. It is still an open question whether specific response elements exist that are selectively recognized by only one or two of the three possible combinations. It already seems clear that the two receptors may act differently on the same response element when using such ‘non-classical’ response elements as, for example, AP-1 sites [28].

Studies of the two mouse strains in which either of the two known oestrogen receptor subtypes has been eliminated, ERKO and BERKO, suggest that the two receptors may compensate for each other at least to some extent, since the phenotype in some cases is less severe than expected. This is probably the case in, for example, the testis of the BERKO, and in bone of both ERKO and BERKO.

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. The oestrogen receptor
  5. Oestrogen receptor β
  6. ArKO, ERKO and BERKO – oestrogen and oestrogen receptor knock-outs
  7. Receptor interplay
  8. Conclusions
  9. Acknowledgements
  10. References

Oestrogens have important functions both in the reproductive system and in other tissues such as bone and the cardiovascular system.

A large number of different pathological conditions are associated with changes in the production of oestrogen and/or the cellular response to these stimuli. Osteoporosis, cancer in the breast, endometrium and prostate, and atherosclerosis are some examples of diseases where oestrogen receptors may be involved. Our recent discovery of ERβ shows that the mechanisms behind the effects of oestrogen are far more complicated than previously assumed and gives unique opportunities to gain a better understanding of these phenomena [29].

Studies of the receptor expression patterns and the effects of eliminating one or both oestrogen receptor subtypes help in understanding the mechanism of action of oestrogen, both in the known target tissues and in other tissues.

Finally, development of ERα- and ERβ-specific ligands may open up new possibilities for treatment, e.g. of postmenopausal symptoms and breast cancer.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. The oestrogen receptor
  5. Oestrogen receptor β
  6. ArKO, ERKO and BERKO – oestrogen and oestrogen receptor knock-outs
  7. Receptor interplay
  8. Conclusions
  9. Acknowledgements
  10. References
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Received 19 January 1999; accepted 23 February 1999.