- Email: [email protected]
Cellular estrogen activity: implications for pulsed estrogen therapy
Maturitas 38 Suppl.1 (2001) S7 – S13 www.elsevier.com/locate/maturitas
Cellular estrogen activity: implications for pulsed estrogen therapy Jan Carlstedt-Duke * Department of Medical Nutrition, Karolinska Institutet, Huddinge Hospital, F 60 No6um, SE-141 86 Huddinge, Sweden
Abstract Estrogens exert their principal biological effects through the actions of two different intracellular estrogen receptor (ER) proteins, ERa and ERb. Following the binding of steroid, the protein undergoes a conformational change that results in a transcriptionally active form. The receptor protein is locked into an active state by estradiol, which results in the transition of the receptor through a signal transduction cascade of events, ultimately resulting in the activation of specific genes, thereby inducing the biological events specific for that type of target cell. There is a large variation in the relative expression levels of the two ER isoforms in different target tissues and in different stages of development. In addition, variant forms of the two ER isoforms, the result of splice variation, have been described. ERa and ERb have been shown to differ in specific aspects within the various stages of the signal transduction pathway. Thus, there is a broad spectrum of estrogen response mechanisms as a result of an infinite number of possible combinations of all these factors. In addition, there are gene regulatory mechanisms that are the result of ER–protein interactions instead of ER–DNA interactions. Steroid binding is the key initiating action of the whole pathway, which, in terms of cell biology, is a relatively slow process. The response induced through the action of ER induction can be shown to be dependent on the total dose exposure rather than estradiol concentrations at subsaturating levels. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: ERa; ERb; 17b-estradiol; Aerodiol®
1. Introduction Estrogens, like the other classic steroid hormones, the androgens, glucocorticoids, mineralocorticoids, and progestins, exert their biological actions through a signal transduction system
* Tel.: +46-8-585837, ext. 15; fax: + 46-8-7795171. E-mail address: [email protected] (J. Carlstedt-Duke).
based on an intracellular soluble receptor protein. The steroid receptor proteins were the founding members of a large superfamily of similar signal transduction proteins, the nuclear receptor family [1]. The members of this family are characterized by a unique DNA-binding structural domain and, in many cases, a second ligand-binding domain. In fact, the majority of the members of the nuclear receptor superfamily have not been shown to bind hormone ligand, or similar such molecules, and these members
0378-5122/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 5 1 2 2 ( 0 1 ) 0 0 1 9 9 - 2
S8
J. Carlstedt-Duke / Maturitas 38 Suppl. 1 (2001) S7– S13
are referred to as the orphan receptors. During recent years, the mechanism of action of these signal transduction proteins has been characterized in great detail at the molecular level. The hypothesis of the mechanism of steroid hormone action that was originally postulated concerned the mechanism of estrogen action. Following the synthesis of radiolabeled estradiol with very high specific radioactivity, Jensen and Jacobsen [2] showed that there was a specific retention of radioactivity in estrogen-dependent tissues such as the uterus. In fact, following the injection of a single pulse of radiolabeled estradiol, the retention of radioactivity in estrogen target tissues persisted for more than 6 h. Thus, as early as the time of this seminal finding on estrogen action, it was shown that there was a long-term action following the short-term pulsatile administration of estradiol. Based on this retention of radiolabeled steroid, and the subsequent demonstration that the radioactivity was accumulated within the cell nucleus in the target tissues, a specific intracellular receptor protein for estrogen was hypothesized and subsequently proven. Although specific details of individual systems involving nuclear receptors are now available, the general model for steroid hormone action originally postulated by Jensen and Jacobsen still holds true over 30 years later. The steroid receptor proteins make up one specific subfamily within the superfamily of nuclear receptor proteins. Apart from being the founding members of this superfamily, the steroid receptors are characterized by their ability to recognize and bind the cognate hormone, which in turn induces the receptor to its active state, thereby initiating the whole signal transduction cascade. The interaction with steroid is very much an active process, resulting in a conformational change of the receptor [3,4], thereby enabling it to bind to specific DNA sequences, hormone response elements, and subsequently to interact with the transcriptional apparatus via protein–protein interaction. Thus, the key to the whole signal transduction process is the binding of hormone, but many subsequent steps are involved before the biological effect of the hormone is elicited.
2. The estrogen receptors The original studies indicated that there was one steroid receptor (and one single gene) for each of the major steroid receptors, common to all target tissues. The nature of the biological response induced in a specific target cell through the induction of a steroid receptor seemed to be determined by the network of genes available for interaction with the activated receptor protein [5]. The original estrogen receptor (ER) cDNA was cloned by two groups [6,7]. However, the distribution of this ER could not explain all of the biological responses to estrogens, for instance in the ovary. A significant explanation for the apparent dissociation between the occurrence of ER and response to estrogens in some tissues was obtained when a second ER was cloned [8]. This receptor, the estrogen receptor b (ERb), is completely distinct from the original ER, ERa. The genes for the two ERs are located on two different chromosomes, human chromosomes 6 (ERa) and 14 (ERb). ERb has been cloned from rat, mouse [9], human [10], and fish [11,12]. The basic mechanism of action is identical for the two ER isomers, and many genes can be induced by the action of either isomer (Fig. 1). Specific details of receptor function vary between the two isomers in defined systems. Steroid receptors generally act as homodimers in their functional activated (ligand-bound) form. However, in addition to forming homodimers of each individual ER isomer, these proteins have also been shown to form isomeric heteromers (ERa/b heteromers) [13]. Thus, the varied expression of the two isomers and the potential interaction between the isomers results in a broad spectrum of estrogen response capability. In addition, both ER isomers have been shown to be expressed in variant forms, as a result of splice variation [10,13–18]. There is, therefore, an almost unlimited variation in ER status in target cells. The details of the biological significance of this ER variability with regard to response in specific tissues remains to be determined in many cases.
J. Carlstedt-Duke / Maturitas 38 Suppl. 1 (2001) S7– S13
3. Expression of estrogen receptor isomers In rodents, the tissues with the highest expression levels of ERb are the prostate, ovary, mammary glands, and lungs [19,20], all tissues where the mechanism of action of estrogens has been difficult to understand because of the low expression levels of ERa. Not surprisingly, therefore, these are the tissues that exhibit very pronounced dysfunction in ERb knockout mice [21]. ERb is also present in bone, uterus, the central nervous system, and the cardiovascular system, tissues that also express ERa. ERb is widely expressed in developing and adult male urogenital tract [22– 25]. In the testis, both ERa and ERb are expressed, but the expression profiles of these two isomers are very different. Exposure to exogenous estrogen has pronounced effects in developing and adult male urogenital tract [26,27]. One important characteristic of ERb seems to be its capacity to modulate the biological activity of ERa. This quenching effect of ERb on ERa function seems to be exerted particularly efficiently by some of the many isoforms of ERb that exist [13,15].
S9
Not only is there a differential expression of the two ERs in different tissues and expression of various isoforms as the result of splice variation, but there is also a marked temporal variation during different stages of development. This has been shown most clearly in the rat mammary gland [28]. ERa expression was relatively high in prepubertal mammary epithelium, decreased markedly during pregnancy, and was markedly induced during lactation. ERb expression was more constant at a continuous high level. Cells coexpressing ERa and ERb were rare during pregnancy, a proliferative phase, but they represented up to 60% of the epithelial cells during lactation, a postproliferative phase.
4. Ligand interaction with the estrogen receptor The molecular details of the interaction of the two ER isomers with both agonists and antagonists have recently been clarified following the structural analysis of the ligand-binding domain of ERa [29–31] and ERb [32] by X-ray crystallography. The ligand is buried within the protein and
Fig. 1. Mechanism of estrogen action. The two ER isomers, ERa and ERb, are activated following the binding of estradiol (E2). Following steroid binding, the ER forms active homodimers that bind to DNA and activate the transcriptional apparatus. The activated ER can also form ERa/b heterodimers that are in many cases inactive. ER can also interact with other transcription factors (TFX) which can result in gene repression or activation, depending on the system in question.
S10
J. Carlstedt-Duke / Maturitas 38 Suppl. 1 (2001) S7– S13
the ligand–protein interaction actively forms the conformation of the resulting complex. The two receptors bind estradiol with about the same affinity, and the ligand binding affinity of ERa and ERb is overall quite similar for the physiological ligands [20]. However, there is a clear difference in binding affinity between the two receptors for 17a-estradiol, androgen metabolites, phytoestrogens, and a number of pharmaceutical estrogen agonists/antagonists. The latter in particular is of great interest, especially in light of the varying distribution of the ER isomers. This property enables the development of selective estrogen receptor modulators (SERMs), thereby focusing on specific estrogen functions in selected target tissues.
with NFkB [34]. Both ERa and ERb can interact with the fos/jun transcription factor complex on an AP1 site to stimulate gene expression [35,36]. However, estradiol induces the opposite effect with the two receptors in this system, acting as an agonist on ERa. In contrast, in the presence of ERb, the antiestrogens tamoxifen and raloxifene behaved as fully competent agonists in the AP1 pathway while estradiol acted as an antagonist. Thus, the complexity of estrogen action is much more varied than for other steroid hormones and cannot be viewed simply in the context of a specific gene. Gene response to estrogens is dependent on ER status, the balance between the two receptors, the nature of the ligand, and the presence of other transcriptional factors.
5. Gene activation and repression
6. Modulation of steroid hormone action
The classic mechanism of action of steroid hormone receptors follows a sequential pathway including ligand-binding, steroid-dependent activation, and conformational change of the receptor protein, binding to specific DNA sequences (hormone response elements) located in proximity to the promoter region of genes regulated by the hormone in question, and interaction with the transcriptional apparatus by protein– protein interaction. A large group of proteins called coactivators and corepressors are particularly important for this last stage [33]. In fact, the coactivators/ corepressors interact with a number of different transcriptional factors, including the nuclear receptors, thereby integrating a number of signal transduction pathways at the transcriptional level. This mechanism characterizes the major pathway for estrogen action, resulting in the induction of expression of specific genes in different target cells. In addition to the DNA-dependent activation of genes, the two estrogen receptors can also regulate transcriptional activity by protein– protein interaction between ER and other transcriptional factors. This is of particular importance with regard to gene repression, for instance the estradiol-dependent inhibition of interleukin-6 expression by the interaction of ERa
In addition to the conventional activation of the ERs by natural or synthetic hormones, alternative pathways involving the indirect activation of ER through the action of membrane receptors for growth factors and cytokines, located on the surface of the cell, have been described [37–41]. In addition, rapid nongenomic effects of ligands to nuclear receptors have been described. In endothelial cells, ER-mediated estradiol-dependent membrane effects lead to sequential activation of ras, raf, mitogen-activated protein kinase kinase (MEK), and subsequently activation of mitogenactivated protein kinase (MAPK) [42].
7. Pulsatile estrogen administration Up to now, two major routes of administration have been used for hormone replacement therapy: the oral route and the cutaneous route. The main limitation of the oral route is the existence of a marked intestinal and hepatic first pass, which makes necessary the administration of high doses in order to achieve systemic active estrogen concentrations. Furthermore, interindividual variability is quite high and interactions with food and smoking have been reported. Among the nonoral routes, the cutaneous route with, in particular,
J. Carlstedt-Duke / Maturitas 38 Suppl. 1 (2001) S7– S13
S11
Fig. 2. Mean pharmacokinetic profile after administration of a single dose of 300 mg Aerodiol® administered nasally.
transdermal patches, is the most widely used. Hepatic first pass is avoided, but local tolerability, especially in a hot and wet environment, as well as adhesion problems may occur. Furthermore, 2– 3 mg of estradiol must be applied to the skin in order to obtain the passage of 50 mg, and absorption may vary according to the site of application and from patient to patient (poor and high absorbers). The nasal route is an effective and well-established route of drug delivery. The nasal cavity is covered with ciliated pseudostratified columnar epithelium with approximately 160 cm2 of nasal mucosa available for absorption. Beneath the mucosa lies a complex vascular bed, which rapidly conveys any substances absorbed across the epithelium into the systemic bloodstream. This large surface area for absorption combined with a high vascularization confers particularly good absorption capacities on the nasal mucosa. The cilia beat with a powerful forward stroke and this rhythmic activity is responsible for the mucociliary clearance. Any drug deposited locally is, therefore, moved posteriorly, this anteroposterior transit time allowing local absorption, and the nonabsorbed fraction is swallowed.
Aerodiol® is a new 17b-estradiol formulation designed to be administered nasally. It is an aqueous solution of 17b-estradiol combined with a cyclodextrin. After nasal administration, estradiol is rapidly absorbed with a tmax between 10 to 30 min and Cmax around 1000 pg/ml for the 300 mg dose level. Estradiol is rapidly distributed to tissue and returns to levels of untreated postmenopausal women within 12 h after dosing, resulting in a pulsed estrogen therapy (Fig. 2). The maximum plasma concentration of estrone is reached about 15 min later than estradiol and is about three times lower than that of estradiol. The mean estrone-to-estradiol ratio of AUCt ranges from 0.80 to 1.1 and is independent of the dose administered. When exposure obtained with a single dose is compared with the exposure obtained with the same total dose given as two divided doses administered 12 h apart, estradiol exposure (area under the estradiol plasma concentrations versus time curve) is proportional to the dose administered and independent of the administration regimen. Pharmacokinetic profiles obtained with the reference products are flat compared with the Aerodiol® kinetic profile, leading to a constant exposure at low estradiol levels. A 200–300 mg
S12
J. Carlstedt-Duke / Maturitas 38 Suppl. 1 (2001) S7– S13
Aerodiol® dose leads to estradiol exposure similar to that obtained with the reference products: the Estraderm TTS 50 patch and the 2 mg micronized estradiol tablet. The average estrone-to-estradiol ratio of AUCt during patch application (0.8) is similar to that observed after Aerodiol® administration, while after oral dosing this ratio reaches 4.0, illustrating the high intestinal and hepatic first pass of estradiol with oral dosing.
8. Response to pulsed estrogen administration The key to estrogen action in all the mechanisms described above is the binding of the ligand to ER. In the absence of ligand, the receptor is inactive, unless induced by an alternative mechanism (growth factor/cytokine). Following the binding of ligand, the receptor undergoes a conformational change that enables the activated ER to bind to DNA and the proteins involved in the transcriptional apparatus. The specific conformational changes induced within the ligand-binding domain by an agonist results in the formation of a site on the surface of the protein that interacts with coactivators such as SRC-1 or TIFF-2. The bound ligand is buried within the protein and, once activated, the sequential steps of the signal transduction pathway take place. Due to the complexity of the various mechanisms involved, biological response to estrogens occurs with a time delay of hours usually. As early as the time of the seminal finding on estrogen action by Jensen and Jacobsen [2], it was shown that there was a longterm action following the short-term pulsed administration of estradiol. Recently, Vignon et al [43] showed that ER integrates the exposure of a target cell to the total dose of estradiol exposure rather than responding to concentration. Thus, there was no difference in the proliferation of normal and tumor breast cells after pulsed and continuous estradiol treatments when there was the same total 24 h exposure. Since the fate of the ER molecule and its activity is decided following the binding of estradiol, this effect would be expected at subsaturating concentrations of ligand.
References [1] Evans RM. The steroid and thyroid hormone superfamily. Science 1988;240:889 – 95. [2] Jensen EV, Jacobsen HI. Basic guides to the mechanism of estrogen action. Recent Prog Horm Res 1962;18:387 – 414. [3] Darimont BD, Wagner RL, Apriletti JW, Stallcup MR, Kushner PJ, Baxter JD, et al. Structure and specificity of nuclear receptor – coactivator interactions. Genes Dev 1998;12:3343 – 56. [4] Feng W, Ribeiro RC, Wagner RL, Nguyen H, Apriletti JW, Fletterick RJ, et al. Hormone-dependent coactivator binding to a hydrophobic cleft on nuclear receptors. Science 1998;280:1747 – 9. [5] Yamamoto KR. Steroid receptor regulated transcription of specific genes and gene networks. Ann Rev Genet 1985;19:209 – 52. [6] Green S, Walter P, Kumar V, Krust A, Bornert J-M, Argos P, et al. Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A. Nature 1986;320:134 – 9. [7] Greene GL, Gilna P, Waterfield M, Baker A, Hort Y, Shine J. Sequence and expression of human estrogen receptor complementary DNA. Science 1986;231:1150 – 4. [8] Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson J-A, . Cloning of a novel receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 1996;93:5925 – 30. [9] Tremblay GB, Tremblay A, Copeland NG, Gilbert DJ, Jenkins NA, Labrie F, et al. Cloning, chromosomal localization, and functional analysis of the murine estrogen receptor beta. Mol Endocrinol 1997;11:353 – 65. [10] Moore JT, Mckee DD, Slentz-Kesler K, Moore LB, Jones SA, Horne EL, et al. Cloning and characterization of human estrogen receptor beta isoforms. Biochem Biophys Res Commun 1998;247:75 – 8. [11] Tan NS, Lam TJ, Ding JL. The first contiguous estrogen receptor gene from a fish, Oreochromis aureus: evidence for multiple transcripts. Mol Cell Endocrinol 1996;120:177 – 92. [12] Todo T, Adachi S, Yamauchi K. Molecular cloning and characterization of Japanese eel estrogen receptor cDNA. Mol Cell Endocrinol 1996;119:37 – 45. [13] Ogawa S, Inoue S, Watanabe T, Hiroi H, Orimo A, Hosoi T, et al. The complete primary structure of human estrogen receptor b (hERb) and its heterodimerization with ERa in vivo and in vitro. Biochem Biophys Res Comm 1998;243:122 – 6. [14] Chu S, Fuller PJ. Identification of a splice variant of the rat estrogen receptor beta gene. Mol Cell Endocrinol 1997;132:195 – 9. [15] Ogawa S, Inoue S, Watanabe T, Orimo A, Hosoi T, Ouchi Y, et al. Molecular cloning and characterization of human estrogen receptor b: a potential inhibitor of estrogen action in human. Nucleic Acids Res 1998;26:3505 – 12.
J. Carlstedt-Duke / Maturitas 38 Suppl. 1 (2001) S7– S13 [16] Fuqua SAW, Chamness GC, Mcguire WL. Estrogen receptor mutations in breast cancer. J Cell Biochem 1993;51:135 – 9. [17] Friend KE, Resnick EM, Ang LW, Shupnik MA. Specific modulation of estrogen receptor mRNA isoforms in rat pituitary throughout the estrous cycle and in response to steroid hormones. Mol Cell Endocrinol 1997;131:147 – 55. [18] Zhang QX, Hilsenbeck SG, Fuqua SA, Borg A, . Multiple splicing variants of the estrogen receptor are present in individual human breast tumors. J Steroid Biochem Mol Biol 1996;59:251 – 60. [19] Enmark E, Pelto-Huikko M, Grandien K, Fried G, Lagerkrantz S, Lagerkrantz J, et al. Human estrogen receptor b- gene structure, chromosomal localisation and expression pattern. J Clin Endocrinol Metab 1997;82:4258 – 65. [20] Kuiper GG, Carlsson B, Grandien K, Enmark E, Ha¨ ggblad J, Nilsson S. Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta. Endocrinology 1997;138:863 – 70. [21] Krege JH, Hodgin JB, Couse JF, Enmark E, Warner M, Mahler JF, et al. Generation and reproductive phenotypes of mice lacking estrogen receptor b. Proc Natl Acad Sci USA 1998;95:15667 – 82. [22] Brandenberger AW, Tee MK, Lee JY, Chao V, Jaffe RB. Tissue distribution of estrogen receptors alpha (ER-alpha) and beta (ER-beta) mRNA in the midgestational human fetus. J Clin Endocrinol Metab 1997;82:3509 –12. [23] Hess RA, Gist DH, Bunick D, Lubahn DB, Farrell A, Bahr J, et al. Estrogen receptor (alpha and beta) expression in the excurrent ducts of the adult male rat reproductive tract. J Androl 1997;18:602 –11. [24] Shughrue PJ, Lane MV, Scrimo PJ, Merchenthaler I. Comparative distribution of estrogen receptor-alpha (ERalpha) and beta (ER-beta) mRNA in the rat pituitary, gonad, and reproductive tract. Steroids 1998;63:498 –504. [25] Van Pelt AM, de Rooij DG, van der Burg B, van der Saag PT, Gustafsson J-A, , Kuiper GG. Ontogeny of estrogen receptor-beta expression in rat testis. Endocrinology 1999;140:478 – 83. [26] Singh J, Handelsman DJ. Morphometric studies of neonatal estrogen imprinting in the mature mouse prostate. J Endocrinol 1999;162:39 –48. [27] Eddy EM, Washburn TF, Bunch DO, Goulding EH, Gladen BC, Lubahn DB, et al. Targeted disruption of the estrogen receptor gene in male mice causes alteration of spermatogenesis and infertility. Endocrinology 1996;137:4796 – 805. [28] Saji S, Jensen EV, Nilsson S, Rylander T, Warner M, Gustafsson J-A, . Estrogen receptors alpha and beta in the rodent mammary gland. Proc Natl Acad Sci USA 2000;97:337– 42. [29] Brzozowski AM, Pike AC, Dauter Z, Hubbard RE, Bonn T, Engstro¨ m O, et al. Molecular basis of agonism and
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42] [43]
S13
antagonism in the oestrogen receptor. Nature 1997;389:753 – 8. Tanenbaum DM, Wang Y, Williams SP, Sigler PB. Crystallographic comparison of the estrogen and progesterone receptor’s ligand binding domains. Proc Natl Acad Sci USA 1998;95:5998 – 6003. Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, Agard DA, et al. The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 1998;95:927 – 37. Pike AC, Brzozowski AM, Hubbard RE, Bonn T, Thorsell AG, Engstro¨ m O, et al. Structure of the ligandbinding domain of oestrogen receptor beta in the presence of a partial agonist and a full antagonist. EMBO J 1999;18:4608 – 18. Glass CK, Rosenfeld MG. The coregulator exchange in transcriptional functions of nuclear receptors. Genes Dev 2000;14:121 – 41. Galien R, Garcia T. Estrogen receptor impairs interleukin-6 expression by preventing protein binding on the NF-kB site. Nucleic Acids Res 1997;25:2424 – 9. Paech K, Webb P, Kuiper GG, Nilsson S, Gustafsson J-A, , Kushner PJ, et al. Differential ligand activation of estrogen receptors ERalpha and ERbeta at AP1 sites. Science 1997;277:1508 – 10. Webb P, Nguyen P, Valentine C, Lopez GN, Kwok GR, McInerney E, et al. The estrogen receptor enhances AP-1 activity by two distinct mechanisms with different requirements for receptor transactivation functions. Mol Endocrinol 1999;13:1672 – 85. Power RF, Mani SK, Codina J, Conneely OM, O’Malley BW. Dopaminergic and ligand-independent activation of steroid hormone receptors. Science 1991;254:1636 – 9. Cavailles V, Garcia M, Rochefort H. Regulation of the cathepsin-D and pS2 gene expression by growth factors in MCF7 human breast cancer cells. Mol Endocrinol 1989;3:552 – 8. Bunone G, Briand PA, Miksicek RJ, Picard D. Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation. EMBO J 1996;15:2174 – 83. Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchiyama S, Sasaki H, et al. Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science 1995;270:1491 – 4. Cenni B, Picard D. Ligand-independent activation of steroid receptors: new roles for old players. Trends Endocrinol Metab 1999;10:41 – 6. Collins P, Webb C. Estrogen hits the surface. Nat Med 1999;5:1130 – 1. Vignon F, Gompel A, Siromachkova M, Pre´ bois C, Varin C, Roste`ne W. Effects of pulsed or continuous estradiol administration on proliferation of normal and tumoral human breast cells. Menopause 1999;6:362.
Cellular estrogen activity: implications for pulsed estrogen therapy Jan Carlstedt-Duke * Department of Medical Nutrition, Karolinska Institutet, Huddinge Hospital, F 60 No6um, SE-141 86 Huddinge, Sweden
Abstract Estrogens exert their principal biological effects through the actions of two different intracellular estrogen receptor (ER) proteins, ERa and ERb. Following the binding of steroid, the protein undergoes a conformational change that results in a transcriptionally active form. The receptor protein is locked into an active state by estradiol, which results in the transition of the receptor through a signal transduction cascade of events, ultimately resulting in the activation of specific genes, thereby inducing the biological events specific for that type of target cell. There is a large variation in the relative expression levels of the two ER isoforms in different target tissues and in different stages of development. In addition, variant forms of the two ER isoforms, the result of splice variation, have been described. ERa and ERb have been shown to differ in specific aspects within the various stages of the signal transduction pathway. Thus, there is a broad spectrum of estrogen response mechanisms as a result of an infinite number of possible combinations of all these factors. In addition, there are gene regulatory mechanisms that are the result of ER–protein interactions instead of ER–DNA interactions. Steroid binding is the key initiating action of the whole pathway, which, in terms of cell biology, is a relatively slow process. The response induced through the action of ER induction can be shown to be dependent on the total dose exposure rather than estradiol concentrations at subsaturating levels. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: ERa; ERb; 17b-estradiol; Aerodiol®
1. Introduction Estrogens, like the other classic steroid hormones, the androgens, glucocorticoids, mineralocorticoids, and progestins, exert their biological actions through a signal transduction system
* Tel.: +46-8-585837, ext. 15; fax: + 46-8-7795171. E-mail address: [email protected] (J. Carlstedt-Duke).
based on an intracellular soluble receptor protein. The steroid receptor proteins were the founding members of a large superfamily of similar signal transduction proteins, the nuclear receptor family [1]. The members of this family are characterized by a unique DNA-binding structural domain and, in many cases, a second ligand-binding domain. In fact, the majority of the members of the nuclear receptor superfamily have not been shown to bind hormone ligand, or similar such molecules, and these members
0378-5122/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 5 1 2 2 ( 0 1 ) 0 0 1 9 9 - 2
S8
J. Carlstedt-Duke / Maturitas 38 Suppl. 1 (2001) S7– S13
are referred to as the orphan receptors. During recent years, the mechanism of action of these signal transduction proteins has been characterized in great detail at the molecular level. The hypothesis of the mechanism of steroid hormone action that was originally postulated concerned the mechanism of estrogen action. Following the synthesis of radiolabeled estradiol with very high specific radioactivity, Jensen and Jacobsen [2] showed that there was a specific retention of radioactivity in estrogen-dependent tissues such as the uterus. In fact, following the injection of a single pulse of radiolabeled estradiol, the retention of radioactivity in estrogen target tissues persisted for more than 6 h. Thus, as early as the time of this seminal finding on estrogen action, it was shown that there was a long-term action following the short-term pulsatile administration of estradiol. Based on this retention of radiolabeled steroid, and the subsequent demonstration that the radioactivity was accumulated within the cell nucleus in the target tissues, a specific intracellular receptor protein for estrogen was hypothesized and subsequently proven. Although specific details of individual systems involving nuclear receptors are now available, the general model for steroid hormone action originally postulated by Jensen and Jacobsen still holds true over 30 years later. The steroid receptor proteins make up one specific subfamily within the superfamily of nuclear receptor proteins. Apart from being the founding members of this superfamily, the steroid receptors are characterized by their ability to recognize and bind the cognate hormone, which in turn induces the receptor to its active state, thereby initiating the whole signal transduction cascade. The interaction with steroid is very much an active process, resulting in a conformational change of the receptor [3,4], thereby enabling it to bind to specific DNA sequences, hormone response elements, and subsequently to interact with the transcriptional apparatus via protein–protein interaction. Thus, the key to the whole signal transduction process is the binding of hormone, but many subsequent steps are involved before the biological effect of the hormone is elicited.
2. The estrogen receptors The original studies indicated that there was one steroid receptor (and one single gene) for each of the major steroid receptors, common to all target tissues. The nature of the biological response induced in a specific target cell through the induction of a steroid receptor seemed to be determined by the network of genes available for interaction with the activated receptor protein [5]. The original estrogen receptor (ER) cDNA was cloned by two groups [6,7]. However, the distribution of this ER could not explain all of the biological responses to estrogens, for instance in the ovary. A significant explanation for the apparent dissociation between the occurrence of ER and response to estrogens in some tissues was obtained when a second ER was cloned [8]. This receptor, the estrogen receptor b (ERb), is completely distinct from the original ER, ERa. The genes for the two ERs are located on two different chromosomes, human chromosomes 6 (ERa) and 14 (ERb). ERb has been cloned from rat, mouse [9], human [10], and fish [11,12]. The basic mechanism of action is identical for the two ER isomers, and many genes can be induced by the action of either isomer (Fig. 1). Specific details of receptor function vary between the two isomers in defined systems. Steroid receptors generally act as homodimers in their functional activated (ligand-bound) form. However, in addition to forming homodimers of each individual ER isomer, these proteins have also been shown to form isomeric heteromers (ERa/b heteromers) [13]. Thus, the varied expression of the two isomers and the potential interaction between the isomers results in a broad spectrum of estrogen response capability. In addition, both ER isomers have been shown to be expressed in variant forms, as a result of splice variation [10,13–18]. There is, therefore, an almost unlimited variation in ER status in target cells. The details of the biological significance of this ER variability with regard to response in specific tissues remains to be determined in many cases.
J. Carlstedt-Duke / Maturitas 38 Suppl. 1 (2001) S7– S13
3. Expression of estrogen receptor isomers In rodents, the tissues with the highest expression levels of ERb are the prostate, ovary, mammary glands, and lungs [19,20], all tissues where the mechanism of action of estrogens has been difficult to understand because of the low expression levels of ERa. Not surprisingly, therefore, these are the tissues that exhibit very pronounced dysfunction in ERb knockout mice [21]. ERb is also present in bone, uterus, the central nervous system, and the cardiovascular system, tissues that also express ERa. ERb is widely expressed in developing and adult male urogenital tract [22– 25]. In the testis, both ERa and ERb are expressed, but the expression profiles of these two isomers are very different. Exposure to exogenous estrogen has pronounced effects in developing and adult male urogenital tract [26,27]. One important characteristic of ERb seems to be its capacity to modulate the biological activity of ERa. This quenching effect of ERb on ERa function seems to be exerted particularly efficiently by some of the many isoforms of ERb that exist [13,15].
S9
Not only is there a differential expression of the two ERs in different tissues and expression of various isoforms as the result of splice variation, but there is also a marked temporal variation during different stages of development. This has been shown most clearly in the rat mammary gland [28]. ERa expression was relatively high in prepubertal mammary epithelium, decreased markedly during pregnancy, and was markedly induced during lactation. ERb expression was more constant at a continuous high level. Cells coexpressing ERa and ERb were rare during pregnancy, a proliferative phase, but they represented up to 60% of the epithelial cells during lactation, a postproliferative phase.
4. Ligand interaction with the estrogen receptor The molecular details of the interaction of the two ER isomers with both agonists and antagonists have recently been clarified following the structural analysis of the ligand-binding domain of ERa [29–31] and ERb [32] by X-ray crystallography. The ligand is buried within the protein and
Fig. 1. Mechanism of estrogen action. The two ER isomers, ERa and ERb, are activated following the binding of estradiol (E2). Following steroid binding, the ER forms active homodimers that bind to DNA and activate the transcriptional apparatus. The activated ER can also form ERa/b heterodimers that are in many cases inactive. ER can also interact with other transcription factors (TFX) which can result in gene repression or activation, depending on the system in question.
S10
J. Carlstedt-Duke / Maturitas 38 Suppl. 1 (2001) S7– S13
the ligand–protein interaction actively forms the conformation of the resulting complex. The two receptors bind estradiol with about the same affinity, and the ligand binding affinity of ERa and ERb is overall quite similar for the physiological ligands [20]. However, there is a clear difference in binding affinity between the two receptors for 17a-estradiol, androgen metabolites, phytoestrogens, and a number of pharmaceutical estrogen agonists/antagonists. The latter in particular is of great interest, especially in light of the varying distribution of the ER isomers. This property enables the development of selective estrogen receptor modulators (SERMs), thereby focusing on specific estrogen functions in selected target tissues.
with NFkB [34]. Both ERa and ERb can interact with the fos/jun transcription factor complex on an AP1 site to stimulate gene expression [35,36]. However, estradiol induces the opposite effect with the two receptors in this system, acting as an agonist on ERa. In contrast, in the presence of ERb, the antiestrogens tamoxifen and raloxifene behaved as fully competent agonists in the AP1 pathway while estradiol acted as an antagonist. Thus, the complexity of estrogen action is much more varied than for other steroid hormones and cannot be viewed simply in the context of a specific gene. Gene response to estrogens is dependent on ER status, the balance between the two receptors, the nature of the ligand, and the presence of other transcriptional factors.
5. Gene activation and repression
6. Modulation of steroid hormone action
The classic mechanism of action of steroid hormone receptors follows a sequential pathway including ligand-binding, steroid-dependent activation, and conformational change of the receptor protein, binding to specific DNA sequences (hormone response elements) located in proximity to the promoter region of genes regulated by the hormone in question, and interaction with the transcriptional apparatus by protein– protein interaction. A large group of proteins called coactivators and corepressors are particularly important for this last stage [33]. In fact, the coactivators/ corepressors interact with a number of different transcriptional factors, including the nuclear receptors, thereby integrating a number of signal transduction pathways at the transcriptional level. This mechanism characterizes the major pathway for estrogen action, resulting in the induction of expression of specific genes in different target cells. In addition to the DNA-dependent activation of genes, the two estrogen receptors can also regulate transcriptional activity by protein– protein interaction between ER and other transcriptional factors. This is of particular importance with regard to gene repression, for instance the estradiol-dependent inhibition of interleukin-6 expression by the interaction of ERa
In addition to the conventional activation of the ERs by natural or synthetic hormones, alternative pathways involving the indirect activation of ER through the action of membrane receptors for growth factors and cytokines, located on the surface of the cell, have been described [37–41]. In addition, rapid nongenomic effects of ligands to nuclear receptors have been described. In endothelial cells, ER-mediated estradiol-dependent membrane effects lead to sequential activation of ras, raf, mitogen-activated protein kinase kinase (MEK), and subsequently activation of mitogenactivated protein kinase (MAPK) [42].
7. Pulsatile estrogen administration Up to now, two major routes of administration have been used for hormone replacement therapy: the oral route and the cutaneous route. The main limitation of the oral route is the existence of a marked intestinal and hepatic first pass, which makes necessary the administration of high doses in order to achieve systemic active estrogen concentrations. Furthermore, interindividual variability is quite high and interactions with food and smoking have been reported. Among the nonoral routes, the cutaneous route with, in particular,
J. Carlstedt-Duke / Maturitas 38 Suppl. 1 (2001) S7– S13
S11
Fig. 2. Mean pharmacokinetic profile after administration of a single dose of 300 mg Aerodiol® administered nasally.
transdermal patches, is the most widely used. Hepatic first pass is avoided, but local tolerability, especially in a hot and wet environment, as well as adhesion problems may occur. Furthermore, 2– 3 mg of estradiol must be applied to the skin in order to obtain the passage of 50 mg, and absorption may vary according to the site of application and from patient to patient (poor and high absorbers). The nasal route is an effective and well-established route of drug delivery. The nasal cavity is covered with ciliated pseudostratified columnar epithelium with approximately 160 cm2 of nasal mucosa available for absorption. Beneath the mucosa lies a complex vascular bed, which rapidly conveys any substances absorbed across the epithelium into the systemic bloodstream. This large surface area for absorption combined with a high vascularization confers particularly good absorption capacities on the nasal mucosa. The cilia beat with a powerful forward stroke and this rhythmic activity is responsible for the mucociliary clearance. Any drug deposited locally is, therefore, moved posteriorly, this anteroposterior transit time allowing local absorption, and the nonabsorbed fraction is swallowed.
Aerodiol® is a new 17b-estradiol formulation designed to be administered nasally. It is an aqueous solution of 17b-estradiol combined with a cyclodextrin. After nasal administration, estradiol is rapidly absorbed with a tmax between 10 to 30 min and Cmax around 1000 pg/ml for the 300 mg dose level. Estradiol is rapidly distributed to tissue and returns to levels of untreated postmenopausal women within 12 h after dosing, resulting in a pulsed estrogen therapy (Fig. 2). The maximum plasma concentration of estrone is reached about 15 min later than estradiol and is about three times lower than that of estradiol. The mean estrone-to-estradiol ratio of AUCt ranges from 0.80 to 1.1 and is independent of the dose administered. When exposure obtained with a single dose is compared with the exposure obtained with the same total dose given as two divided doses administered 12 h apart, estradiol exposure (area under the estradiol plasma concentrations versus time curve) is proportional to the dose administered and independent of the administration regimen. Pharmacokinetic profiles obtained with the reference products are flat compared with the Aerodiol® kinetic profile, leading to a constant exposure at low estradiol levels. A 200–300 mg
S12
J. Carlstedt-Duke / Maturitas 38 Suppl. 1 (2001) S7– S13
Aerodiol® dose leads to estradiol exposure similar to that obtained with the reference products: the Estraderm TTS 50 patch and the 2 mg micronized estradiol tablet. The average estrone-to-estradiol ratio of AUCt during patch application (0.8) is similar to that observed after Aerodiol® administration, while after oral dosing this ratio reaches 4.0, illustrating the high intestinal and hepatic first pass of estradiol with oral dosing.
8. Response to pulsed estrogen administration The key to estrogen action in all the mechanisms described above is the binding of the ligand to ER. In the absence of ligand, the receptor is inactive, unless induced by an alternative mechanism (growth factor/cytokine). Following the binding of ligand, the receptor undergoes a conformational change that enables the activated ER to bind to DNA and the proteins involved in the transcriptional apparatus. The specific conformational changes induced within the ligand-binding domain by an agonist results in the formation of a site on the surface of the protein that interacts with coactivators such as SRC-1 or TIFF-2. The bound ligand is buried within the protein and, once activated, the sequential steps of the signal transduction pathway take place. Due to the complexity of the various mechanisms involved, biological response to estrogens occurs with a time delay of hours usually. As early as the time of the seminal finding on estrogen action by Jensen and Jacobsen [2], it was shown that there was a longterm action following the short-term pulsed administration of estradiol. Recently, Vignon et al [43] showed that ER integrates the exposure of a target cell to the total dose of estradiol exposure rather than responding to concentration. Thus, there was no difference in the proliferation of normal and tumor breast cells after pulsed and continuous estradiol treatments when there was the same total 24 h exposure. Since the fate of the ER molecule and its activity is decided following the binding of estradiol, this effect would be expected at subsaturating concentrations of ligand.
References [1] Evans RM. The steroid and thyroid hormone superfamily. Science 1988;240:889 – 95. [2] Jensen EV, Jacobsen HI. Basic guides to the mechanism of estrogen action. Recent Prog Horm Res 1962;18:387 – 414. [3] Darimont BD, Wagner RL, Apriletti JW, Stallcup MR, Kushner PJ, Baxter JD, et al. Structure and specificity of nuclear receptor – coactivator interactions. Genes Dev 1998;12:3343 – 56. [4] Feng W, Ribeiro RC, Wagner RL, Nguyen H, Apriletti JW, Fletterick RJ, et al. Hormone-dependent coactivator binding to a hydrophobic cleft on nuclear receptors. Science 1998;280:1747 – 9. [5] Yamamoto KR. Steroid receptor regulated transcription of specific genes and gene networks. Ann Rev Genet 1985;19:209 – 52. [6] Green S, Walter P, Kumar V, Krust A, Bornert J-M, Argos P, et al. Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A. Nature 1986;320:134 – 9. [7] Greene GL, Gilna P, Waterfield M, Baker A, Hort Y, Shine J. Sequence and expression of human estrogen receptor complementary DNA. Science 1986;231:1150 – 4. [8] Kuiper GG, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson J-A, . Cloning of a novel receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 1996;93:5925 – 30. [9] Tremblay GB, Tremblay A, Copeland NG, Gilbert DJ, Jenkins NA, Labrie F, et al. Cloning, chromosomal localization, and functional analysis of the murine estrogen receptor beta. Mol Endocrinol 1997;11:353 – 65. [10] Moore JT, Mckee DD, Slentz-Kesler K, Moore LB, Jones SA, Horne EL, et al. Cloning and characterization of human estrogen receptor beta isoforms. Biochem Biophys Res Commun 1998;247:75 – 8. [11] Tan NS, Lam TJ, Ding JL. The first contiguous estrogen receptor gene from a fish, Oreochromis aureus: evidence for multiple transcripts. Mol Cell Endocrinol 1996;120:177 – 92. [12] Todo T, Adachi S, Yamauchi K. Molecular cloning and characterization of Japanese eel estrogen receptor cDNA. Mol Cell Endocrinol 1996;119:37 – 45. [13] Ogawa S, Inoue S, Watanabe T, Hiroi H, Orimo A, Hosoi T, et al. The complete primary structure of human estrogen receptor b (hERb) and its heterodimerization with ERa in vivo and in vitro. Biochem Biophys Res Comm 1998;243:122 – 6. [14] Chu S, Fuller PJ. Identification of a splice variant of the rat estrogen receptor beta gene. Mol Cell Endocrinol 1997;132:195 – 9. [15] Ogawa S, Inoue S, Watanabe T, Orimo A, Hosoi T, Ouchi Y, et al. Molecular cloning and characterization of human estrogen receptor b: a potential inhibitor of estrogen action in human. Nucleic Acids Res 1998;26:3505 – 12.
J. Carlstedt-Duke / Maturitas 38 Suppl. 1 (2001) S7– S13 [16] Fuqua SAW, Chamness GC, Mcguire WL. Estrogen receptor mutations in breast cancer. J Cell Biochem 1993;51:135 – 9. [17] Friend KE, Resnick EM, Ang LW, Shupnik MA. Specific modulation of estrogen receptor mRNA isoforms in rat pituitary throughout the estrous cycle and in response to steroid hormones. Mol Cell Endocrinol 1997;131:147 – 55. [18] Zhang QX, Hilsenbeck SG, Fuqua SA, Borg A, . Multiple splicing variants of the estrogen receptor are present in individual human breast tumors. J Steroid Biochem Mol Biol 1996;59:251 – 60. [19] Enmark E, Pelto-Huikko M, Grandien K, Fried G, Lagerkrantz S, Lagerkrantz J, et al. Human estrogen receptor b- gene structure, chromosomal localisation and expression pattern. J Clin Endocrinol Metab 1997;82:4258 – 65. [20] Kuiper GG, Carlsson B, Grandien K, Enmark E, Ha¨ ggblad J, Nilsson S. Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta. Endocrinology 1997;138:863 – 70. [21] Krege JH, Hodgin JB, Couse JF, Enmark E, Warner M, Mahler JF, et al. Generation and reproductive phenotypes of mice lacking estrogen receptor b. Proc Natl Acad Sci USA 1998;95:15667 – 82. [22] Brandenberger AW, Tee MK, Lee JY, Chao V, Jaffe RB. Tissue distribution of estrogen receptors alpha (ER-alpha) and beta (ER-beta) mRNA in the midgestational human fetus. J Clin Endocrinol Metab 1997;82:3509 –12. [23] Hess RA, Gist DH, Bunick D, Lubahn DB, Farrell A, Bahr J, et al. Estrogen receptor (alpha and beta) expression in the excurrent ducts of the adult male rat reproductive tract. J Androl 1997;18:602 –11. [24] Shughrue PJ, Lane MV, Scrimo PJ, Merchenthaler I. Comparative distribution of estrogen receptor-alpha (ERalpha) and beta (ER-beta) mRNA in the rat pituitary, gonad, and reproductive tract. Steroids 1998;63:498 –504. [25] Van Pelt AM, de Rooij DG, van der Burg B, van der Saag PT, Gustafsson J-A, , Kuiper GG. Ontogeny of estrogen receptor-beta expression in rat testis. Endocrinology 1999;140:478 – 83. [26] Singh J, Handelsman DJ. Morphometric studies of neonatal estrogen imprinting in the mature mouse prostate. J Endocrinol 1999;162:39 –48. [27] Eddy EM, Washburn TF, Bunch DO, Goulding EH, Gladen BC, Lubahn DB, et al. Targeted disruption of the estrogen receptor gene in male mice causes alteration of spermatogenesis and infertility. Endocrinology 1996;137:4796 – 805. [28] Saji S, Jensen EV, Nilsson S, Rylander T, Warner M, Gustafsson J-A, . Estrogen receptors alpha and beta in the rodent mammary gland. Proc Natl Acad Sci USA 2000;97:337– 42. [29] Brzozowski AM, Pike AC, Dauter Z, Hubbard RE, Bonn T, Engstro¨ m O, et al. Molecular basis of agonism and
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42] [43]
S13
antagonism in the oestrogen receptor. Nature 1997;389:753 – 8. Tanenbaum DM, Wang Y, Williams SP, Sigler PB. Crystallographic comparison of the estrogen and progesterone receptor’s ligand binding domains. Proc Natl Acad Sci USA 1998;95:5998 – 6003. Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, Agard DA, et al. The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 1998;95:927 – 37. Pike AC, Brzozowski AM, Hubbard RE, Bonn T, Thorsell AG, Engstro¨ m O, et al. Structure of the ligandbinding domain of oestrogen receptor beta in the presence of a partial agonist and a full antagonist. EMBO J 1999;18:4608 – 18. Glass CK, Rosenfeld MG. The coregulator exchange in transcriptional functions of nuclear receptors. Genes Dev 2000;14:121 – 41. Galien R, Garcia T. Estrogen receptor impairs interleukin-6 expression by preventing protein binding on the NF-kB site. Nucleic Acids Res 1997;25:2424 – 9. Paech K, Webb P, Kuiper GG, Nilsson S, Gustafsson J-A, , Kushner PJ, et al. Differential ligand activation of estrogen receptors ERalpha and ERbeta at AP1 sites. Science 1997;277:1508 – 10. Webb P, Nguyen P, Valentine C, Lopez GN, Kwok GR, McInerney E, et al. The estrogen receptor enhances AP-1 activity by two distinct mechanisms with different requirements for receptor transactivation functions. Mol Endocrinol 1999;13:1672 – 85. Power RF, Mani SK, Codina J, Conneely OM, O’Malley BW. Dopaminergic and ligand-independent activation of steroid hormone receptors. Science 1991;254:1636 – 9. Cavailles V, Garcia M, Rochefort H. Regulation of the cathepsin-D and pS2 gene expression by growth factors in MCF7 human breast cancer cells. Mol Endocrinol 1989;3:552 – 8. Bunone G, Briand PA, Miksicek RJ, Picard D. Activation of the unliganded estrogen receptor by EGF involves the MAP kinase pathway and direct phosphorylation. EMBO J 1996;15:2174 – 83. Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchiyama S, Sasaki H, et al. Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science 1995;270:1491 – 4. Cenni B, Picard D. Ligand-independent activation of steroid receptors: new roles for old players. Trends Endocrinol Metab 1999;10:41 – 6. Collins P, Webb C. Estrogen hits the surface. Nat Med 1999;5:1130 – 1. Vignon F, Gompel A, Siromachkova M, Pre´ bois C, Varin C, Roste`ne W. Effects of pulsed or continuous estradiol administration on proliferation of normal and tumoral human breast cells. Menopause 1999;6:362.