DOI 10.1515/hmbci-2013-0034 Horm Mol Biol Clin Invest 2013; 16(1): 7–12
Mini Review Hubertus Jarry*
Estrogen receptor beta and its selective ligands: an option for treatment of menopausal vasomotor symptoms? Abstract: In the human female, menopause is the permanent end of fertility, defined as occurring 12 months after the last menstrual period. During peri- and postmenopausal stages, the vast majority of women experience moderate-to-severe vasomotor symptoms, such as hot flashes and night sweats, which interfere with sleep and may be severe enough to affect quality of life. The only treatment approved by national health authorities is hormone therapy with estrogen alone or in combination with a progestagen. However, this therapeutic regimen is associated with severe side effects, such as stimulation of growth of breast cancer or cardiovascular events. Thus, there is a demand for efficient and safe alternative treatments for menopausal complaints. After the discovery of estrogen receptor beta in many organs, and confirmation of its presence in the brain, many researchers raised the question of whether ERβ-specific ligands may be novel therapeutic agents for treatment of menopausal complaints with the desirable effects of estrogen but without increased risk of tumor incidence. This minireview will briefly summarize the relevance of estrogen receptor beta and its specific ligands for the treatment of menopausal symptoms with a focus on vasomotor menopausal symptoms. At present, estrogen receptor beta-selective ligands do not seem to be active in models of prevention or reversal of osteoporosis. However, data from animal experiments suggest that estrogen receptor beta-selective ligands might be safe therapeutics for the treatment of vasomotor menopausal symptoms. Keywords: β receptor specific ligands; estrogen receptor beta; menopause; vasomotor symptoms. *Corresponding author: Hubertus Jarry, University Medical Center Göttingen Robert-Koch-Strasse 40 37075 Göttingen, Germany, Phone: +49 551 396522, E-mail: [email protected]
Introduction A principle feature of steroid hormone action is binding to and activation of the respective specific receptor in order to regulate gene transcription. The steroid hormone estradiol (E2) is, among other actions, crucial for cellular development and differentiation, and in adult organisms for the maintenance of homoestasis and reproduction. Because of these essential functions not only the ligand E2 but also its interaction partner, the estrogen receptor (ER), is also thought to play a central role in the regulation of a plethora of life processes. In turn, one would expect that the loss of the ER, i.e., missing expression, would be incompatible with life. To support this assumption, Lubahn et al. [1] created an ER-knockout mouse (ERKO) that was suspected to develop a severe phenotype which might even be lethal in utero. Surprisingly, mice of both genders survive to adulthood without apparent macroscopic phenotypes. However, homozygous ERKO females are infertile because their ovaries harbor only cystic follicles but no corpora lutea. With regard to reproduction, ERKO males are subfertile with atrophy of the seminiferous epithelium and seminiferous tubule dysmorphogenesis. In both genders, the most prominent defect observed in non-reproductive tissues was a marked decrease in skeletal bone density [2]. One explanation for this unexpected outcome of the deletion of the gene of the ER was that a second, on a distinct gene encoded ER, must exist. Indeed, Kuiper et al. [3] described the expression of a novel subtype of the ER in rat prostate and ovary. As a consequence of this pioneering discovery the already known ER was renamed ERα while the newly described ER was termed ERβ. The discovery of ERβ in a number of tissues in female but also in male individuals has broadened our knowledge of estrogen signaling, physiology and pathophysiology. The virtually ubiquitous tissue distribution of ERα and ERβ is the molecular basis of why E2 is involved in a plethora of
8 Jarry: Estrogen receptor beta and menopausal symptoms mechanisms in physiology and diseases in both, men and women. The irreversible cessation of ovarian estradiol production is a hallmark in the life of a women. Menopause is the permanent end of menstruation and fertility, defined as occurring 12 months after last menstrual period. In Western societies, average age of menopause is 51 years. During peri- and postmenopausal stages, the vast majority of women (estimates range from 68% to 90%) experience moderate-to-severe vasomotor symptoms (VMS), such as hot flashes and night sweats, that interfere with sleep and which may be severe enough to affect quality of life [4]. The only treatment approved by national food and drug safety authorities is hormone therapy (HT). Estrogen alone or in combination with a progestagen has been the standard therapy for such vasomotor symptoms; however, though observed only in a small number of women, this therapeutic regimen is associated with an increased risk of deadly disease, such as breast cancer or cardiovascular events. Because both physicians and women are concerned with the tolerability and safety profile of estrogen and estrogen plus progestin treatments, alternative menopause therapies are needed. An ideal menopause treatment would relieve menopausal vasomotor, maintain bone mass, has beneficial effects on the cardiovascular system and on lipid metabolism, without stimulation of breast or endometrium cancer. This minireview will briefly summarize the relevance of ERβ and its specific ligands of this ER subtype for the treatment of menopausal symptoms with focus on VMS.
Body of review Known ERβ selective agonists After the discovery of ERβ in many organs, and confirmation of its presence in the brain, many researchers raised the question of whether ERβ-specific ligands may be novel therapeutic agents for treatment of menopausal complaints with the desirable effects of estrogen but without risk of tumor incidence. To date, only a limited number of compounds with a reasonable selectivity for ERβ have been reported. Basically, two groups of substances have been suggested as putative ERβ-agonists, namely synthetic compounds and phytoestrogens. A thorough review of the known classes of ERβ-selective ligands has been published by Minutolo et al. [5]. Likewise the excellent review by Leitman et al. [6] discusses the state of art
regarding gene regulation and the therapeutic options of compounds of selectively targeting either subtype of ER. Among the synthetic compounds so far developed, diarylpropionitrile (DPN) emerged as one of the most potent and selective ERβ agonists as determined in vitro with ligand binding- and transcriptional assays. Receptor binding efficacy was 70-fold higher for ERβ versus ERα and a 170-fold higher potency was measured in transcriptional assays [7]. Comparable selectivity was reported for the benzoxazole ERB-04. It exerts a 200-fold higher binding affinity for ERβ versus ERα and regulates only ERβ genes [8]. Another example of a synthetic putative ERβ-ligand is the tetrahydrofluorenone ERb-19. The selectivity of ERb-19 for ERβ and not ERα was reflected by the lack of an effect on uterus weight and morphology and, on a molecular level, the observation that it did not stimulate the expression of uterocalin, which is dependent on the activation of ERα [9]. Since the WHI-study the public interest in natural hormones as safe treatment of menopausal complaints increased significantly [10]. Among natural products, there are several examples of ERβ-selective agonists, such as coumestrol, genistein and equol. However, it should be emphasized that the ERβ-selectivity was determined with ligand binding or reporter gene assays, i.e. with biochemical and cell culture methods, respectively. Animal experiments with ovariectomized rats, the standard animal model for the endocrine situation of a menopausal women, did not reveal convincing evidence that phytoestrogens such as soy/red clover isoflavones have beneficial effects on climacteric complaints and osteoporosis. This is in line with the results of clinical studies, as herbal products have not been demonstrated to be as effective as HT or better than placebo in most well-designed trials [4, 10]. A promising exeption might be MF-101. This preparation is derived from 22 herbs that are traditionally used in Chinese medicine for the treatment of menopausal symptoms. MF-101 did not promote the growth of breast cancer cells or stimulate uterine growth in preclinical studies. Though MF101 binds with equal affinity to ERα and ERβ it preferentially regulates genes by ERβ. In a phase II trial MF101 was demonstrated to be safe and more effective in reducing the frequency and severity of hot flashes in postmenopausal women compared with placebo [11]. Thus, MF-101 appears to be a promising therapeutic to treat menopause-associated symptoms. However, at present there is a need for more placebo-controlled studies to convincingly prove beneficial effects of this ERβ-selective herbal product on climacteric complaints with safety in mammary gland and uterus.
Jarry: Estrogen receptor beta and menopausal symptoms 9
Physiology of menopausal vasomotor symptoms The terms ‘hot flashes, hot flushes, climacteric night sweats and VMS’ all describe a progressive spreading of a feeling of heat through the upper body. Individual hot flashes often last between 1 and 5 min, although they may last up to 15 min. Perspiration and palpitations may accompany hot flashes and contribute to the discomfort, inconvenience or anxiety associated with VMS. Fatigue may develop as a result of frequent night awakenings. Hot flashes occur primarily and most intensively in peri- and postmenopausal women. They also may occur when estrogen drops suddenly and rapidly, such as after removal of the ovaries of premenopausal women, with chemically induced menopause, and also in breast cancer patients treated with selective estrogen receptor modulators (SERMs) such as tamoxifen. Men also can experience hot flashes, particularly when testosterone levels fall rapidly, such as in men with prostate cancer treated medically or surgically. Hot flashes are associated with peripheral dilation with increased skin temperature and blood flow, typically within the first few seconds of the flash. Several studies showed that measurement of sternal skin conductance and skin temperature of fingers are the best objective marker of menopausal hot flashes [12]. Estrogen has been studied and used to treat hot flashes for 60 years, but the mechanism by which it works is still in question. Clearly, estrogen plays some role as a mediator of hot flashes. Hot flashes occur as estrogen levels decline and they are alleviated for the most part by treatment with estrogen. There have been examinations of the levels of circulating hormones, such as luteinizing hormone (LH), β-endorphin and adrenocorticotropic hormone, however except for LH, no convincing evidence was reported for a correlation of the occurrence of hot flushes with pulsatile hormone release [13]. The secretion of LH from the pituitary is driven by release of the decapapetide gonadotropin-releasinghormone (GnRH) from hypothalamic GnRH neurons. In both, genders secretion of GnRH occurs in a tightly controlled pulsatile manner. In adult females amplitude and frequency of GnRH release is regulated by E2 and progesterone. Upon gonadectomy GnRH neurons are relieved from the strong negative feedback of E2, resulting in high frequency GnRH-pulses with large amplitudes. This pronounced pulsatile GnRH release pattern is also observed in peri- and menopausal women. Each pulse of LH released from the pituitary is triggered by a pulse of GnRH from the hypothalamus. Thus, the pulse pattern of LH determined in the periphery reflects the pulsatile
activity of GnRH neurons in the brain. Since the pioneering studies of Tataryn et al., numerous studies have proven that pulsatile GnRH release and the occurrence of hot flashes are causally related [14]. In close vicinity to GnRH neurons in the hypothalamus, yet unidentified neurons are located that regulate body temperature (hypothalamic thermoregulatory center). The current view of the correlation of hot flashes and GnRH-, respectively LH pulses, is that both types of neurons are regulated by neurons that may be estrogen receptive. Upon estrogen deficiency these neurons become overactive, thus causing synchronous hyperactivity of GnRH- and temperature regulating neurons. These neurons may be noradrenalin-, dopamine-, GABA- or serotininergic, which would explain why agonists of these neurotransmitters can be used for treatment of VMS. The mechanisms of how E2 regulates the pulsatile release of GnRH has been investigated intensively, however, the understanding of the molecular and cellular pathways underlying the induction of the phasic and synchronous activity of GnRH neurons is still fragmentary. In all species investigated to date, adult GnRH neurones express ERβ and not ERα. However, in ERβ-KO- and wildtype mice GnRH-mRNA expression was similar, suggesting that GnRH transcript levels were not regulated by ERβ. Thus, the relevance of ERβ for regulation of GnRH expression/secretion remains to be elucidated. What are the effects of ERβ-selective compounds on the activity of GnRH- and temperature regulating neurons? Monitoring tail skin temperatures (TST) of ovariectomized (ovx) rats has generally been used as an animal model for the evaluation of mechanisms of triggering menopausal hot flashes as well for investigation of substances to alleviate VMS. The role of ERα and ERβ in attenuating the increase in TST was assessed in ERα- and ERβ-KO mice, respectively [15]. In both subtype ERKO- and wild-type mice, estrogen depletion by ovx caused an elevation in basal TST. Administration of the ‘non-selective’ agonist E2 suppressed the increase in TST in a dose-dependent manner in wild-type as well as in ERα- and ERβ-deficient mice, thereby suggesting that either ERα or ERβ alone is sufficient for reducing VMS. The ERα-selective agonist propyl-pyrazole-triol (PPT) and the ERβ-selective agonists ERβ-19 alleviated the rise in TST in ovx rats, supporting the findings from the ERknockout mice [16]. Thus, at present, activation of either ERα or ERβ alone is sufficient to normalize the activity of the hypothalamic thermoregulatory center to suppress VMS. This opens the promising perspective that ERβ-agonist might be a therapeutic option for treatment of menopausal VMS.
10 Jarry: Estrogen receptor beta and menopausal symptoms
ERβ and the cardiovascular system Estrogen has pleiotropic effects on the cardiovascular system. Among these actions, E2 modulates vascular function, cardiac myocyte and stem cell survival and the development of cardiac hypertrophy [17]. Expression of both subtypes of ER has been described on mRNA and protein level in isolated rodent and human cardiomyocytes. Cessation of ovarian estrogen production during menopausal transition dramatically increases the risk of cardiovascular events, and in postmenopausal women the risk for heart disease rapidly approaches the risk level observed in men. Wild-type ovx mice and mice lacking ERα showed less cardiac hypertrophy when treated with E2 than with vehicle; while mice lacking ERβ treated with estrogen did not show a reduction in cardiac hypertrophy. These data suggest a role for ERβ in reducing hypertrophy in females [18]. For elucidation of the role of Erβ under physiological conditions, future experiments with ER null mice of both sexes are necessary.
Skeletal system It is well known that estrogen deficiency in postmenopausal women may result in osteoporosis because of a marked increase in bone resorption by osteoclasts that is not compensated for by osteoblast-mediated bone formation. By various analytical methods the presence of both subtypes of ER has been demonstrated in human osteoblasts, osteoclasts and osteocytes [19]. To decipher the relative role of either ER α vs. β, the bone phenotype of the respective knockout mice was studied. Surprisingly, the results of studies of the bone phenotype of both lines of ERKO-mice are less conclusive because, unlike other estrogen target tissues, female bones were not significantly affected by depletion of either or both ER. Thus, on the basis of the published data the relative role of ERα vs. β in the bone can be described as: ERα mediates the growth-promoting effects of estrogens in the maturational bone and transmits the osteoprotective effect of the steroid hormone in adulthood. No conclusive role of ERβ can be deducted from experiments with ERβ-KO mice, at the best it can be summarized that these mice demonstrate few abnormalities in bone phenotype and that ovariectomy has few effects on various bone characteristics [20].
Adipose tissue After transition to menopause an increase in lipid storage and a decline in lipid utilization are observed. Consistently,
as reported for postmenopausal women, the decrease in circulating E2 levels is associated with an increase of visceral fat, lowered lipid utilization and an increase of complications associated with the metabolic syndrome [21]. The observation that ERα and ERβ are expressed in adipose tissue and the fact that E2 treatment counteracts obesity led to the assumption that ERs are mediating this effect. Studies with ER null mice revealed that ERα-KO mice are obese and display impaired insulin responsiveness and glucose tolerance [22]. ERβ-KO animals display a less striking phenotype because only ERβ-KO mice fed with a high-fat diet are characterized by an increase of body weight [23]. However, the involvement of ERβ in the regulation of body weight could be confirmed, as body weights of wild-type mice fed with a high-fat diet and treated with ERβ-selective ligands were lower compared to weights of control animals [24]. Thus, the metabolic findings in knockout mice and use of ER subtype-selective ligands, did not result yet in an unequivocal conclusion about the functions of both subtypes of ER in adipose tissue.
Expert opinion It was originally hoped that ERβ might be an effective target for reversing the physiological changes that occur in the menopause, without increasing the risk factors of estrogens, namely stimulation of breast and endometrium cancers. At present, ERβ-selective ligands do not seem to be active in models of prevention or reversal of osteoporosis. However, at least data from animal experiments suggest that ERβ-selective ligands might be safe therapeutics for treatment of VMS.
Outlook The future demographical changes in Western societies will have a marked impact on the costs for the treatment of age-associated diseases, in particular osteoporosis, cardiovascular insults, metabolic disorders and neurodegenerative processes. Based on the currently available data, ERβ-specific ligands have the potential for efficient treatment of these diseases in both genders without the known severe side effects, i.e., stimulation of growth of steroid dependent cancer. Thus, treatment of VMS remains a significant indication for the use of ERβ-ligands but the safe treatment of the above mentioned age-related diseases will gain importance.
Jarry: Estrogen receptor beta and menopausal symptoms 11
Highlights –– Cessation of ovarian function at menopause results in permanently low estrogen levels. –– Currently two types of estrogen receptors (ER) are known, ERα and ERβ. –– ERβ is expressed in many organs, among others, in various brain areas. –– Hormone therapy (HT) with estrogen or a combination of estrogen plus progestins is an efficient treatment of menopausal complaints, such as vasomotor symptoms (VMS).
–– Because of side effects of HT, ERβ-specific ligands are considered a new alternative treatment of the symptoms of estrogen deficiency. –– Based on animal experiments it is suggested that the currently available ERβ-selective ligands might be safe therapeutics for treatment of VMS. Acknowledgments: The author declares that there is not a conflict of financial or other interest. Received July 4, 2013; accepted July 18, 2013; previously published online August 20, 2013
References 1. Lubahn DB, Moyer JS, Golding TS, Couse JF, Korach KS, Smithies O. Alteration of reproductive function but not prenatal sexual development after insertional disruption of the mouse estrogen receptor gene. Proc Natl Acad Sci USA 1993;90:11162–6. 2. Korach KS, Couse JF, Curtis SW, Washburn TF, Lindzey J, Kimbro KS, Eddy EM, Migliaccio S, Snedeker SM, Lubahn DB, Schomberg DW, Smith EP. Estrogen receptor gene disruption: molecular characterization an eyperimental and clinical phenotypes. Recent Prog Horm Res 1996;51:159–86. 3. Kuiper GG, Enmark E, Pelto-Huikko M, Nisson S, Gustafsson JA. Cloning of a novel receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 1996;93:5925–30. 4. Pinkerton JA, Stovall DW, Kightlinger RS. Advances in the treatment of menopausal symptoms. Women’s Health 2009;5:361–84. 5. Minutolo F, Macchia M, Katzenellenbogen BS, Katzenellenbogen JA. Estrogen receptor β ligands: recent advances and biomedical applications. Med Res Rev 2011;31:364–442. 6. Leitman DC, Paruthiyil S, Vivar OI, Saunier EF, Herber CB, Cohen I, Tagliaferri M, Speed TP. Regulation of specific target genes and biological responses by estrogen receptor subtype agonists. Curr Opin Pharmacol 2010;10:629–36. 7. Meyers MJ, Sun J, Carlson KE, Marriner GA, K atzenellenbogen BS, Katzenellenbogen JA. Estrogen receptor β potency-selective ligands: Structure-activity relationship studies of diarylpropionitriles and their acetylene and polar analogues. J Med Chem 2001;44: 4230–51. 8. Harris HA, Albert LM, Leathurby YL, Malamas MS, Mewshaw RE, Miller CP, Kharode YP, Marzolf J, Komm BS, Winneker RC, Frail DE, Henderson RA, Zhu Y, Keith JC, Jr. Evaluation of an estrogen receptor-ß agonist in animal models of human disease. Endocrinology 2003;144:4241–9. 9. Jackson RL,Greiwe JS, Schwen RJ. Emerging evidence of the health benefits of S-equol, an estrogen receptor β agonist. Nutrition Reviews 2011;69:432–48.
10. Seidlova-Wuttke D, Jarry H, Wuttke W. Plant derived alternatives for Hormone Replacement Therapy (HRT). Horm Mol Biol Clin Invest 2013;16:35–45. 11. Stovall DW, Pinkerton JV. MF-101, an estrogen receptor β agonist for the treatment of vasomotor symptoms in periand postmenopausal women. Curr Opin Investig Drugs 2009;10:365–71. 12. Kronenberg F. Menopausal hot flashes: A review of physiology and biosociocultural perspectives on methods of assessment. J Nutrition 2010;140:1380–5. 13. Casper RF, Yen SS, Wilkes MM. Menopausal flushes: a neuroendocrine link with pulsatile luteninizing hormone secretion. Science 1979;205:823–5. 14. Tataryn IV, Meldrum DR, Lu KH, Frumar AM, Judd HL. LH, FSH and skin temperature during the menopausal hot flash. J Clin Endocrinol Metab 1979;49:152–4. 15. Opas EE, Gentile MA, Kimmel DB, Rodan GA, Schmidt A. Estrogenic control of thermoregulation in ER alpha KO and ER beta KO mice. Maturitas 2006;53:210–16. 16. Opas EE, Scafonas A, Nantermet PV, Wilkening RR, Birzin ET, Wilkinson H, Colwell LF, Schaeffer JM, Towler DA, Rodan GA, Schmidt A. Control of rat skin temperature regulation by estrogen receptor beta selective ligand. Maturitas 2009;64:46– 51. 17. Murphy E. Estrogen signaling and cardiovascular disease. Circ Res. 2011;109:687–96. 18. Skavdahl M, Steenbergen C, Clark J, Myers P, Demianenko T, Mao L, Rockman HA, Korach KS, Murphy E. Estrogen receptorbeta mediates male-female differences in the development of pressure overload hypertrophy. Am J Physiol Heart Circ Physiol 2005;288:H459–76. 19. Bland R. Steroid hormone receptor expression and action in bone. Clin Sci 2000;98:217–40. 20. Seidlova-Wuttke D, Nguyen BT, Wuttke W. Long-term effects of ovariectomy on osteoporosis and obesity in estrogen-receptorβ-deleted mice. Comp Med 2012;62:8–13. 21. Ferrara CM, Lynch NA, Nicklas BJ, Ryan AS, Berman DM. Differences in adipose tissue metabolism between postmen-
12 Jarry: Estrogen receptor beta and menopausal symptoms opausal and perimenopausal women. J Clin Endocrinol Metab 2002;87:4166–70. 22. Swanson HI, Wada T, Xie W, Renga B, Zampella A, Distrutti E, Fiorucci S, Kong B, Thomas AM, Guo GL, Narayanan R, Yepuru M, Dalton JT, Chiang JY. Role of nuclear receptors in lipid dysfunction and obesity-related diseases. Drug Metab Dispos 2013;41:1–11. 23. Foryst-Ludwig A, Clemenz M, Hohmann S, Hartge M, Sprang C, Frost N, Krikov M, Bhanot S, Barros R,
Morani A, Gustafsson JA, Unger T, Kintscher U. Metabolic actions of estrogen receptor beta (ERbeta) are mediated by a negative cross-talk with PPARgamma. PLoS Genet 208;4:e1000108. 24. Yepuru M, Eswaraka J, Kearbey JD, Barrett CM, Raghow S, Veverka KA, Miller DD, Dalton JT, Narayanan R. Estrogen receptor-beta-selective ligands alleviate high-fat dietand ovariectomy-induced obesity in mice. J Biol Chem 2010;285:31282–303.
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Mini Review Hubertus Jarry*
Estrogen receptor beta and its selective ligands: an option for treatment of menopausal vasomotor symptoms? Abstract: In the human female, menopause is the permanent end of fertility, defined as occurring 12 months after the last menstrual period. During peri- and postmenopausal stages, the vast majority of women experience moderate-to-severe vasomotor symptoms, such as hot flashes and night sweats, which interfere with sleep and may be severe enough to affect quality of life. The only treatment approved by national health authorities is hormone therapy with estrogen alone or in combination with a progestagen. However, this therapeutic regimen is associated with severe side effects, such as stimulation of growth of breast cancer or cardiovascular events. Thus, there is a demand for efficient and safe alternative treatments for menopausal complaints. After the discovery of estrogen receptor beta in many organs, and confirmation of its presence in the brain, many researchers raised the question of whether ERβ-specific ligands may be novel therapeutic agents for treatment of menopausal complaints with the desirable effects of estrogen but without increased risk of tumor incidence. This minireview will briefly summarize the relevance of estrogen receptor beta and its specific ligands for the treatment of menopausal symptoms with a focus on vasomotor menopausal symptoms. At present, estrogen receptor beta-selective ligands do not seem to be active in models of prevention or reversal of osteoporosis. However, data from animal experiments suggest that estrogen receptor beta-selective ligands might be safe therapeutics for the treatment of vasomotor menopausal symptoms. Keywords: β receptor specific ligands; estrogen receptor beta; menopause; vasomotor symptoms. *Corresponding author: Hubertus Jarry, University Medical Center Göttingen Robert-Koch-Strasse 40 37075 Göttingen, Germany, Phone: +49 551 396522, E-mail: [email protected]
Introduction A principle feature of steroid hormone action is binding to and activation of the respective specific receptor in order to regulate gene transcription. The steroid hormone estradiol (E2) is, among other actions, crucial for cellular development and differentiation, and in adult organisms for the maintenance of homoestasis and reproduction. Because of these essential functions not only the ligand E2 but also its interaction partner, the estrogen receptor (ER), is also thought to play a central role in the regulation of a plethora of life processes. In turn, one would expect that the loss of the ER, i.e., missing expression, would be incompatible with life. To support this assumption, Lubahn et al. [1] created an ER-knockout mouse (ERKO) that was suspected to develop a severe phenotype which might even be lethal in utero. Surprisingly, mice of both genders survive to adulthood without apparent macroscopic phenotypes. However, homozygous ERKO females are infertile because their ovaries harbor only cystic follicles but no corpora lutea. With regard to reproduction, ERKO males are subfertile with atrophy of the seminiferous epithelium and seminiferous tubule dysmorphogenesis. In both genders, the most prominent defect observed in non-reproductive tissues was a marked decrease in skeletal bone density [2]. One explanation for this unexpected outcome of the deletion of the gene of the ER was that a second, on a distinct gene encoded ER, must exist. Indeed, Kuiper et al. [3] described the expression of a novel subtype of the ER in rat prostate and ovary. As a consequence of this pioneering discovery the already known ER was renamed ERα while the newly described ER was termed ERβ. The discovery of ERβ in a number of tissues in female but also in male individuals has broadened our knowledge of estrogen signaling, physiology and pathophysiology. The virtually ubiquitous tissue distribution of ERα and ERβ is the molecular basis of why E2 is involved in a plethora of
8 Jarry: Estrogen receptor beta and menopausal symptoms mechanisms in physiology and diseases in both, men and women. The irreversible cessation of ovarian estradiol production is a hallmark in the life of a women. Menopause is the permanent end of menstruation and fertility, defined as occurring 12 months after last menstrual period. In Western societies, average age of menopause is 51 years. During peri- and postmenopausal stages, the vast majority of women (estimates range from 68% to 90%) experience moderate-to-severe vasomotor symptoms (VMS), such as hot flashes and night sweats, that interfere with sleep and which may be severe enough to affect quality of life [4]. The only treatment approved by national food and drug safety authorities is hormone therapy (HT). Estrogen alone or in combination with a progestagen has been the standard therapy for such vasomotor symptoms; however, though observed only in a small number of women, this therapeutic regimen is associated with an increased risk of deadly disease, such as breast cancer or cardiovascular events. Because both physicians and women are concerned with the tolerability and safety profile of estrogen and estrogen plus progestin treatments, alternative menopause therapies are needed. An ideal menopause treatment would relieve menopausal vasomotor, maintain bone mass, has beneficial effects on the cardiovascular system and on lipid metabolism, without stimulation of breast or endometrium cancer. This minireview will briefly summarize the relevance of ERβ and its specific ligands of this ER subtype for the treatment of menopausal symptoms with focus on VMS.
Body of review Known ERβ selective agonists After the discovery of ERβ in many organs, and confirmation of its presence in the brain, many researchers raised the question of whether ERβ-specific ligands may be novel therapeutic agents for treatment of menopausal complaints with the desirable effects of estrogen but without risk of tumor incidence. To date, only a limited number of compounds with a reasonable selectivity for ERβ have been reported. Basically, two groups of substances have been suggested as putative ERβ-agonists, namely synthetic compounds and phytoestrogens. A thorough review of the known classes of ERβ-selective ligands has been published by Minutolo et al. [5]. Likewise the excellent review by Leitman et al. [6] discusses the state of art
regarding gene regulation and the therapeutic options of compounds of selectively targeting either subtype of ER. Among the synthetic compounds so far developed, diarylpropionitrile (DPN) emerged as one of the most potent and selective ERβ agonists as determined in vitro with ligand binding- and transcriptional assays. Receptor binding efficacy was 70-fold higher for ERβ versus ERα and a 170-fold higher potency was measured in transcriptional assays [7]. Comparable selectivity was reported for the benzoxazole ERB-04. It exerts a 200-fold higher binding affinity for ERβ versus ERα and regulates only ERβ genes [8]. Another example of a synthetic putative ERβ-ligand is the tetrahydrofluorenone ERb-19. The selectivity of ERb-19 for ERβ and not ERα was reflected by the lack of an effect on uterus weight and morphology and, on a molecular level, the observation that it did not stimulate the expression of uterocalin, which is dependent on the activation of ERα [9]. Since the WHI-study the public interest in natural hormones as safe treatment of menopausal complaints increased significantly [10]. Among natural products, there are several examples of ERβ-selective agonists, such as coumestrol, genistein and equol. However, it should be emphasized that the ERβ-selectivity was determined with ligand binding or reporter gene assays, i.e. with biochemical and cell culture methods, respectively. Animal experiments with ovariectomized rats, the standard animal model for the endocrine situation of a menopausal women, did not reveal convincing evidence that phytoestrogens such as soy/red clover isoflavones have beneficial effects on climacteric complaints and osteoporosis. This is in line with the results of clinical studies, as herbal products have not been demonstrated to be as effective as HT or better than placebo in most well-designed trials [4, 10]. A promising exeption might be MF-101. This preparation is derived from 22 herbs that are traditionally used in Chinese medicine for the treatment of menopausal symptoms. MF-101 did not promote the growth of breast cancer cells or stimulate uterine growth in preclinical studies. Though MF101 binds with equal affinity to ERα and ERβ it preferentially regulates genes by ERβ. In a phase II trial MF101 was demonstrated to be safe and more effective in reducing the frequency and severity of hot flashes in postmenopausal women compared with placebo [11]. Thus, MF-101 appears to be a promising therapeutic to treat menopause-associated symptoms. However, at present there is a need for more placebo-controlled studies to convincingly prove beneficial effects of this ERβ-selective herbal product on climacteric complaints with safety in mammary gland and uterus.
Jarry: Estrogen receptor beta and menopausal symptoms 9
Physiology of menopausal vasomotor symptoms The terms ‘hot flashes, hot flushes, climacteric night sweats and VMS’ all describe a progressive spreading of a feeling of heat through the upper body. Individual hot flashes often last between 1 and 5 min, although they may last up to 15 min. Perspiration and palpitations may accompany hot flashes and contribute to the discomfort, inconvenience or anxiety associated with VMS. Fatigue may develop as a result of frequent night awakenings. Hot flashes occur primarily and most intensively in peri- and postmenopausal women. They also may occur when estrogen drops suddenly and rapidly, such as after removal of the ovaries of premenopausal women, with chemically induced menopause, and also in breast cancer patients treated with selective estrogen receptor modulators (SERMs) such as tamoxifen. Men also can experience hot flashes, particularly when testosterone levels fall rapidly, such as in men with prostate cancer treated medically or surgically. Hot flashes are associated with peripheral dilation with increased skin temperature and blood flow, typically within the first few seconds of the flash. Several studies showed that measurement of sternal skin conductance and skin temperature of fingers are the best objective marker of menopausal hot flashes [12]. Estrogen has been studied and used to treat hot flashes for 60 years, but the mechanism by which it works is still in question. Clearly, estrogen plays some role as a mediator of hot flashes. Hot flashes occur as estrogen levels decline and they are alleviated for the most part by treatment with estrogen. There have been examinations of the levels of circulating hormones, such as luteinizing hormone (LH), β-endorphin and adrenocorticotropic hormone, however except for LH, no convincing evidence was reported for a correlation of the occurrence of hot flushes with pulsatile hormone release [13]. The secretion of LH from the pituitary is driven by release of the decapapetide gonadotropin-releasinghormone (GnRH) from hypothalamic GnRH neurons. In both, genders secretion of GnRH occurs in a tightly controlled pulsatile manner. In adult females amplitude and frequency of GnRH release is regulated by E2 and progesterone. Upon gonadectomy GnRH neurons are relieved from the strong negative feedback of E2, resulting in high frequency GnRH-pulses with large amplitudes. This pronounced pulsatile GnRH release pattern is also observed in peri- and menopausal women. Each pulse of LH released from the pituitary is triggered by a pulse of GnRH from the hypothalamus. Thus, the pulse pattern of LH determined in the periphery reflects the pulsatile
activity of GnRH neurons in the brain. Since the pioneering studies of Tataryn et al., numerous studies have proven that pulsatile GnRH release and the occurrence of hot flashes are causally related [14]. In close vicinity to GnRH neurons in the hypothalamus, yet unidentified neurons are located that regulate body temperature (hypothalamic thermoregulatory center). The current view of the correlation of hot flashes and GnRH-, respectively LH pulses, is that both types of neurons are regulated by neurons that may be estrogen receptive. Upon estrogen deficiency these neurons become overactive, thus causing synchronous hyperactivity of GnRH- and temperature regulating neurons. These neurons may be noradrenalin-, dopamine-, GABA- or serotininergic, which would explain why agonists of these neurotransmitters can be used for treatment of VMS. The mechanisms of how E2 regulates the pulsatile release of GnRH has been investigated intensively, however, the understanding of the molecular and cellular pathways underlying the induction of the phasic and synchronous activity of GnRH neurons is still fragmentary. In all species investigated to date, adult GnRH neurones express ERβ and not ERα. However, in ERβ-KO- and wildtype mice GnRH-mRNA expression was similar, suggesting that GnRH transcript levels were not regulated by ERβ. Thus, the relevance of ERβ for regulation of GnRH expression/secretion remains to be elucidated. What are the effects of ERβ-selective compounds on the activity of GnRH- and temperature regulating neurons? Monitoring tail skin temperatures (TST) of ovariectomized (ovx) rats has generally been used as an animal model for the evaluation of mechanisms of triggering menopausal hot flashes as well for investigation of substances to alleviate VMS. The role of ERα and ERβ in attenuating the increase in TST was assessed in ERα- and ERβ-KO mice, respectively [15]. In both subtype ERKO- and wild-type mice, estrogen depletion by ovx caused an elevation in basal TST. Administration of the ‘non-selective’ agonist E2 suppressed the increase in TST in a dose-dependent manner in wild-type as well as in ERα- and ERβ-deficient mice, thereby suggesting that either ERα or ERβ alone is sufficient for reducing VMS. The ERα-selective agonist propyl-pyrazole-triol (PPT) and the ERβ-selective agonists ERβ-19 alleviated the rise in TST in ovx rats, supporting the findings from the ERknockout mice [16]. Thus, at present, activation of either ERα or ERβ alone is sufficient to normalize the activity of the hypothalamic thermoregulatory center to suppress VMS. This opens the promising perspective that ERβ-agonist might be a therapeutic option for treatment of menopausal VMS.
10 Jarry: Estrogen receptor beta and menopausal symptoms
ERβ and the cardiovascular system Estrogen has pleiotropic effects on the cardiovascular system. Among these actions, E2 modulates vascular function, cardiac myocyte and stem cell survival and the development of cardiac hypertrophy [17]. Expression of both subtypes of ER has been described on mRNA and protein level in isolated rodent and human cardiomyocytes. Cessation of ovarian estrogen production during menopausal transition dramatically increases the risk of cardiovascular events, and in postmenopausal women the risk for heart disease rapidly approaches the risk level observed in men. Wild-type ovx mice and mice lacking ERα showed less cardiac hypertrophy when treated with E2 than with vehicle; while mice lacking ERβ treated with estrogen did not show a reduction in cardiac hypertrophy. These data suggest a role for ERβ in reducing hypertrophy in females [18]. For elucidation of the role of Erβ under physiological conditions, future experiments with ER null mice of both sexes are necessary.
Skeletal system It is well known that estrogen deficiency in postmenopausal women may result in osteoporosis because of a marked increase in bone resorption by osteoclasts that is not compensated for by osteoblast-mediated bone formation. By various analytical methods the presence of both subtypes of ER has been demonstrated in human osteoblasts, osteoclasts and osteocytes [19]. To decipher the relative role of either ER α vs. β, the bone phenotype of the respective knockout mice was studied. Surprisingly, the results of studies of the bone phenotype of both lines of ERKO-mice are less conclusive because, unlike other estrogen target tissues, female bones were not significantly affected by depletion of either or both ER. Thus, on the basis of the published data the relative role of ERα vs. β in the bone can be described as: ERα mediates the growth-promoting effects of estrogens in the maturational bone and transmits the osteoprotective effect of the steroid hormone in adulthood. No conclusive role of ERβ can be deducted from experiments with ERβ-KO mice, at the best it can be summarized that these mice demonstrate few abnormalities in bone phenotype and that ovariectomy has few effects on various bone characteristics [20].
Adipose tissue After transition to menopause an increase in lipid storage and a decline in lipid utilization are observed. Consistently,
as reported for postmenopausal women, the decrease in circulating E2 levels is associated with an increase of visceral fat, lowered lipid utilization and an increase of complications associated with the metabolic syndrome [21]. The observation that ERα and ERβ are expressed in adipose tissue and the fact that E2 treatment counteracts obesity led to the assumption that ERs are mediating this effect. Studies with ER null mice revealed that ERα-KO mice are obese and display impaired insulin responsiveness and glucose tolerance [22]. ERβ-KO animals display a less striking phenotype because only ERβ-KO mice fed with a high-fat diet are characterized by an increase of body weight [23]. However, the involvement of ERβ in the regulation of body weight could be confirmed, as body weights of wild-type mice fed with a high-fat diet and treated with ERβ-selective ligands were lower compared to weights of control animals [24]. Thus, the metabolic findings in knockout mice and use of ER subtype-selective ligands, did not result yet in an unequivocal conclusion about the functions of both subtypes of ER in adipose tissue.
Expert opinion It was originally hoped that ERβ might be an effective target for reversing the physiological changes that occur in the menopause, without increasing the risk factors of estrogens, namely stimulation of breast and endometrium cancers. At present, ERβ-selective ligands do not seem to be active in models of prevention or reversal of osteoporosis. However, at least data from animal experiments suggest that ERβ-selective ligands might be safe therapeutics for treatment of VMS.
Outlook The future demographical changes in Western societies will have a marked impact on the costs for the treatment of age-associated diseases, in particular osteoporosis, cardiovascular insults, metabolic disorders and neurodegenerative processes. Based on the currently available data, ERβ-specific ligands have the potential for efficient treatment of these diseases in both genders without the known severe side effects, i.e., stimulation of growth of steroid dependent cancer. Thus, treatment of VMS remains a significant indication for the use of ERβ-ligands but the safe treatment of the above mentioned age-related diseases will gain importance.
Jarry: Estrogen receptor beta and menopausal symptoms 11
Highlights –– Cessation of ovarian function at menopause results in permanently low estrogen levels. –– Currently two types of estrogen receptors (ER) are known, ERα and ERβ. –– ERβ is expressed in many organs, among others, in various brain areas. –– Hormone therapy (HT) with estrogen or a combination of estrogen plus progestins is an efficient treatment of menopausal complaints, such as vasomotor symptoms (VMS).
–– Because of side effects of HT, ERβ-specific ligands are considered a new alternative treatment of the symptoms of estrogen deficiency. –– Based on animal experiments it is suggested that the currently available ERβ-selective ligands might be safe therapeutics for treatment of VMS. Acknowledgments: The author declares that there is not a conflict of financial or other interest. Received July 4, 2013; accepted July 18, 2013; previously published online August 20, 2013
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