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was not induced by E or various selective estrogen responsive modulators (SERMs), ligands that bind ER but exhibit selective tissue dependent activity [17].
Reviews in Endocrine & Metabolic Disorders 2002;3:193±200 # 2002 Kluwer Academic Publishers. Manufactured in The Netherlands.

Estrogen Receptors: Structure, Mechanisms and Function Sylvia Curtis Hewitt and Kenneth S Korach Receptor Biology, LRDT, National Institute of Environmental Health Sciences, NIH, PO Box 12233, Research Triangle Park, NC 27709

Key Words. estrogen receptor, reproductive tract, transgenic mice, estrogen action

Introduction The biological effects of estrogen (E) in mammalian target tissues are important for numerous physiological processes. E is known to induce responses in the reproductive tract, mammary tissue and pituitary but also affects nonreproductive processes such as bone formation and cardiovascular health. Since the initial identi®cation and isolation of the estrogen receptor (ER) molecule decades ago, the mechanism of estrogen (E) action in cells has been intensely studied. At the core, our understanding of the basic mechanism has remained the same, however, the details continue to be de®ned and many new aspects remain to be discovered. The dogma of high af®nity E binding and modulation of transcription via high af®nity estrogen responsive elements (EREs) in target genes still remains the basis of E action in target tissues. However, this model has become much more complex with the understanding that many other factors cooperate and interact to modulate transcription. In addition there are ``twists'' in the ``plot'', in alternatives to E activation of ER as well as ER's ability to modulate transcription of genes that lack EREs. Advances in technology and methods available for molecular, structural, biochemical, genetic and physiological analysis of ER function have allowed for the de®nition of many of the details of ER function in in vitro model systems and the expansion of these models into genetically modi®ed mouse models. This chapter will review the current understanding of the mechanism of ER function and then describe insights gained from use of transgenic mouse models.

Estrogen Receptors: Structure and Mechanism of Action ERs are members of a family of nuclear transcription factors including receptors for sex steroids, thyroid hormone, retinoids as well as many ``orphan'' receptors,

for which no ligands have been identi®ed [1]. A second ER gene was cloned from prostate tissue in 1996 [2], and thus, there are two ER molecules: the original ER ``a'', and the recently discovered ER ``b''. By comparing the ER sequences it is apparent that both share a general domain structure common to ligand modulated nuclear transcription factors [3]. The current understanding of ER mechanisms of action can be summarized with reference to the overall structure of the ER molecules (Fig. 1). The functions of some regions of the ER molecules have been de®ned using deletion and mutation as well as structural analysis. The best characterized functions are a zinc-®nger containing domain (C domain, Fig. 1) that binds with high af®nity and speci®city to EREs in target genes, and a ligand-binding domain (LBD, domain E), which binds E as well as other estrogenic ligands. The ERE is a 13 base pair inverted repeat sequence (GGTCAnnnTGACC), and in vitro DNA binding studies have indicated that the ER binds as a dimer [4], with one ER molecule contacting each 5 base pair inverted repeat [5]. Although the DNA binding is a dimerization stimulus, sequences in the LBD are also involved in dimerization [4], and crystallized truncated ER containing only ligand binding domain is clearly shown to be a dimer in the presence of ligand [6]. In addition, regions in the amino terminus (AF-1) and within the LBD (AF-2) are involved in ligand-independent (AF-1) and ligand-dependent (AF-2) transcriptional activation, as deletion of or introduction of mutations in these regions result in a diminished ability to induce E responsive genes [7,8]. The mechanism by which transcription is mediated by ER is thought to be through interaction of AFs with ``transcriptional machinery'', which is a general term referring to the complex of molecules that assembles and ultimately results in synthesis of mRNA [9]. Much is now known about RNA polymerase II and the enzymes and factors that allow transcription. Transcriptional co-regulators are molecules that mediate the interaction between ERs and the transcriptional machinery. Many co-regulators Address correspondence to: Kenneth S. Korach. E-mail: [email protected]

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Fig. 1. Comparison of domain structures of ERa and ERb. The estrogen receptors are members of the nuclear receptor superfamily, and share a domain structure, which is depicted schematically. The ERs have 6 domains, A±F, and the number of amino acids in these domains as well as the functions associated with these domains are indicated for each form of ER. AF-1 and AF-2 refer to regions that mediate the transcriptional activation functions of the ERs. The degree of homology between ERa and ERb in the C and E domains are indicated below these domains.

have been isolated that interact with ER in a ligand dependent manner [9±11]. The most well-characterized co-regulators interact with the AF-2 of the ER, although recently molecules that interact with the N-terminus have been described. Notably, the co-activator, p68, which interacts with the AF-1 region of ERa, has RNA helicase activity and associates with SRA, a RNA molecule with co-activator activity [12±14]. Tsai's review in this volume describes in greater detail the roles and mechanisms of co-activators in steroid hormone action. The general mechanism of ER activation is depicted in Fig. 2. Upon binding to ligand, the conformation of the LBD is altered, allowing interaction with co-activators if the ligand is an agonist, but preventing this interaction in the case of an antagonist [6,9]. Interestingly, crystal structures of the liganded-ERa LBD indicate that the binding of antagonist results in ER helix 12 shifting to a conformation that interferes with binding of co-activators by allowing an LXXLL-like sequence in helix 12 to bind the co-activator binding pocket of the LBD [15]. Conversely, the binding of agonist repositions helix 12, exposing the co-activator binding site of the LBD. ER dimers bind ERE sequences in target genes, and if agonist and co-activator are associated, induce transcription by interacting with and activating necessary components of the transcriptional apparatus.

Alternative Mechanisms of ER Activity In addition to the classical mechanism of ER function, several alternative mechanisms have been elucidated

Fig. 2. Mechanisms of ER mediated transcription. ER-mediated transcription is initiated following E binding or ligand-independent activation resulting from growth factor receptor pathway signaling and cross-talk with ER. Once activated, ER dimers recruit coactivators and can mediate transcription of genes via direct binding to EREs in target genes (``classical'' mechanism). Alternatively, ER can recruit coactivators to an AP-1 complex or remove repressors from the AP-1 complex (AP-1 mediated mechanism). Finally, ER can interact with promoters containing Sp1 binding sequences and ERE-half sites (Sp1…N†x ERE(1/2) mediated).

(Fig. 2). First, ER can regulate expression of genes that lack EREs. One such mechanism that has been studied in some detail is the ER regulation of genes containing AP1 elements that bind the fos/jun dimer, which recruits ERa and co-activators [16]. In addition, ER may also sequester repressors away from the fos/jun transcription apparatus, resulting in activation through AP-1 [16]. This alternative activity of ER adds complexity to the actions and roles of estrogen. The insulin-like growth factor (IGF-1) gene is induced by E via an AP-1 element. Studies using aERKO mice indicated the IGF-1 pathway was not induced by E or various selective estrogen responsive modulators (SERMs), ligands that bind ER but exhibit selective tissue dependent activity [17]. This observation indicates that this ER/AP-1 pathway functions in vivo and also illustrates the requirement for ERa for this ERE-independent regulation. Estrogen regulation is also seen in genes such as cFOS and TGFa, which lack a full ERE sequence. This regulation is mediated by an interaction between ERa and SP1 proteins, which bind ERE-half sites and GC rich sequences, respectively, in the regulatory regions of these genes [18]. Another alternative mechanism of ER action involves ligand-independent ER activation, in which growth factor receptor signaling results in ER target gene induction. Both in vivo and in vitro studies have indicated that ER mediated responses can be induced or increased by EGF and IGF-1 [19±24]. Thus, cross-talk and signal ampli®cation between membrane-bound growth factor

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Table 1. The Phenotypes reported in mice with disrupted ERa, ERb, or both ERa and ERb Tissue

a

b

ab

Mammary Fertility

Immature-ductal rudiment Both sexes are infertile

Immature-ductal rudiment Both sexes are infertile

Pituitary Ovary

Elevated LH production, low prolactin E and T elevated Follicles don't mature, hemorrhagic cystic follicles begin developing at puberty as a result of chronic elevated LH. LH and FSH receptors expressed Reduced ovulations in superovulation trial, ``trapped follicle'' phenotype after superovulation Immature. Insensitive to E-no mitosis or induction of E responsive genes PR present, P responsive genes induced, decidualization is E independent No implantation Progressive ¯uid retention and dilation of seminiferous tubules, eventual loss of sperm Disrupted mating behavior E protection retained in vascular injury study

Normal structure and lactation Fertile males Subfertile females: Infrequent pregnancies, small litter sizes Normal Appears Normal, inef®cient ovulation in superovulation trial, ``trapped follicle'' phenotype at superovutation

Uterus: E resposiveness Uterus: Progesterone responsiveness Uterus: Implantation Testes

Mating behaviors Cardiovascular responses Bone

Both sexes shorter. Female:smaller diameter male: lower density

receptor pathways and nuclear receptors has been proposed (reviewed in Yee and Lee [25] and Cenni and Picard [26]). In support of this hypothesis, the estrogen receptor a knockout (aERKO) mouse, which lacks ERa, has been used to show that this receptor is essential for EGF-induced DNA synthesis in uterine epithelium [27]. Further complicating these alternative modes of ER activity, EGF and IGF-1 can stimulate a reporter gene driven by GC rich promoter element from the cathepsin D gene [18].

Animal Models for the Study of ER The structure and mechanisms of ER-mediated regulation of various target genes have been extensively characterized using isolated components or in vitro cell models. The ability to produce transgenic mice with disruption of the ER genes has allowed study of the physiological processes that require ERs in live animals and provided insight into differing physiological roles of ERa and ERb. Table 1 summarizes many of the phenotypes reported in mice with disrupted ERa (aERKO), ERb (bERKO) or both ERa and ERb (abERKO) (reviewed in Couse and Korach [28]). The most signi®cant effects, as might be expected, are seen in reproductive systems of these animals. Both male and female aERKO and abERKO mice are infertile, whereas

Elevated LH production Progressive degeneration of germ cells, transdifferentiation of granualosa cells into Sertoli cells

Normal responses to E

Insensitive to E-like aERKO

nd

nd

Normal; pups carried to term Normal

nd Progressive ¯uid retention and dilation of seminiferous tubules, eventual loss of sperm Disrupted mating behavior E protection lost in vascular injury study Shorter

Normal mating behavior E protection retained in vascular injury study Increased density in females, no effect in males

the bERKO males are fertile and the bERKO females are subfertile [29,30].

Role of ER in Female Fertility The most signi®cant phenotype in the aERKO and bERKO females is complete infertility in the aERKO and decreased fertility of the bERKO as a result of failure of several components necessary for successful reproduction. Normally, gonadotropin production by the pituitary is regulated by a negative feedback loop in which rising E levels down-regulate transcription of the LHb gene, and thus, withdrawal of E by ovariectomy causes an increase in serum LH. The aERKO female also has elevated LH transcript in the pituitary as well as elevated serum LH [31]. In contrast, LH levels are normal in the bERKO (unpublished), indicating that the negative feedback is mediated by ERa rather than ERb. Immunohistochemical analysis of the expression of ERa and ERb protein in a normal ovary indicate that, although both ERs are present, their distribution differs, with ERb predominantly in the granulosa cells of the follicles and ERa localized in the thecal and interstitial regions of the ovary (Fig. 3). Therefore, one might expect the a or b ERKO mouse ovaries to exhibit different phenotypes. The aERKO ovaries develop hemorrhagic cysts [29], and lack mature follicles and corpora lutea,

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ance (Fig. 4), yet a similar result was obtained in a superovulation trial in the bERKO (6 oocytes/trial bERKO vs. 33.7 oocytes/trial WT), indicating a role for ERb in ovulation as well. Analysis of the structures in the ovary following the superovulation trial indicated that although fewer ovulations occurred in the aERKO and bERKO animals, several pre-ovulatory follicles were present (Fig. 5). This is similar to the ``trapped follicle'' phenotype that has been reported in several other mouse models including RIP140 (an estrogen receptor interacting co-represser) knockout [35], progesterone receptor knockout [36,37] and the Cox2 (cycloxygenase 2) knockout [38]. Further studies, however, have not yet revealed why these follicles fail to rupture in the aERKO and bERKO. It is clear that one cause of the infertility in the aERKO female is dysregulation of LH. The decreased response of the follicles in both the aERKO and bERKO animals even when gonadotropins are provided exogenously may underlie their ovulatory defects.

Role of ER in the Uterus

Fig. 3. Localization of ERa and ERb expression in the ovary. Immunohistochemical staining for ERa or ERb in a normal ovary shows the differential expression pattern of these ERs.

indicating the absence of ovulation (Fig. 4). The ovaries also produce elevated levels of E in the serum [29,32]. This is similar to the ovaries observed in LH overexpressing transgenic mice [33], indicating that perhaps the chronically elevated LH level in the aERKO leads to the ovarian phenotype. In support of this hypothesis, treatment of aERKO females with GnRH antagonist decreases the serum LH level and also prevents the ovaries from developing hemorrhagic cysts [34]. Attempts to superovulate the aERKO using exogenous gonadotropins prior to the development of hemorrhagic cysts resulted in ovulations, but with a signi®cantly lower ef®ciency than in WT mice (14.5 oocytes/trial aERKO vs. 40 oocytes/trial WT) [34], although LH and FSH receptors are expressed in the aERKO ovaries. Oocytes from both WT and aERKO animals were fertilized in vitro with equal ef®ciency, indicating that ERa has a role in ovulation but is not required for proper oocyte development. The bERKO ovary has a normal appear-

The rodent uterus is essential for the implantation and support of developing embryos during pregnancy. In addition, the initiation and maintenance of the pregnancy is dependant on ovarian hormones. The pre-ovulatory peak of E is important in proliferation of the uterine epithelium in preparation for implantation, while rising progesterone (P) levels following ovulation are important for implantation of the embryo and the formation of decidual tissue in the stroma. The uterus of the aERKO is hypoplastic, similar in appearance to be a pre-pubertal uterus (Fig. 4), and does not respond to E in terms of weight increase, epithelial proliferation, or induction of E responsive genes [29,32]. Furthermore, the aERKO does not support the implantation of donor embryos (SCH, unpublished). All of these responses are normal in the bERKO, and the bERKO can carry a pregnancy to term [30]. Thus, the dysfunction of the ovary is not the only component that causes the aERKO infertility, while the inef®cient ovarian function in the bERKO appears to account for the subfertility. Interestingly, deciduomas can be induced experimentally in the aERKO uteri, indicating that this aspect of uterine function does not require ERa [39]. Overall, it is apparent that ERa is the ER subtype required for uterine reproductive function. Although the phenotypes in the aERKO uterus and ovary are severe enough to account for the infertility, it is important to note that lack of ERa also leads to severe disruption in mating behaviors, while the bERKO exhibits normal mating behaviors [40,41]. This indicates that ERa, but not ERb, has a requisite role in regulation of female mating behavior.

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Fig. 4. Ovarian histology, reproductive tracts and mammary gland whole mounts from WT, aERKO, bERKO and abERKO tissues. Arrows indicate rudimentary ductal tree in mammay glands.

Role of ER in Mammary Glands At birth the mouse mammary gland consists of a rudimental ductal tree, which, as the female mouse matures, elongates in response to E and branches in response to progesterone (P) to eventually ®ll the stroma. In the aERKO, the ductal rudiment fails to elongate [42], while in the bERKO the gland develops normally (Fig. 4). Moreover, the bERKO mother successfully nurses her young, indicating normal lactation function as well. These observations indicate ERa is required for normal mammary gland maturation and development. Interestingly, the rudimentary ductal tree in the aERKO can be stimulated with P to develop lobular alveolar structures, indicating that the ducts retain their ability to respond to P.

Role of ER in Male Fertility Fig. 5. aERKO and bERKO ovaries following superovulation. ``Trapped'' follicles are indicated by arrows. Note the corpus lutea (CL) in the bERKO section.

The infertility of the aERKO males indicated for the ®rst time that E has an essential role in male reproduction. Evaluation of the testis histopathology indicated dilation of the seminiferous tubules and lack of sperm in mature

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animals [43]. When germ cells from aERKO males were transplanted into testis of germ-cell depleted WT recipients, normal offspring were produced by the recipients [44,45]. This experimental observation combined with the ability of ERa-disrupted sperm from ERa heterozygous (ERa + ) males to produce offspring indicate that ERa is not needed for sperm function, but is required in the male reproductive tract to allow maturation of sperm. Although the lack of sperm alone results in male infertility, it should be noted that the aERKO and abERKO males also exhibit de®cient mating behaviors [46,47], while the bERKO mating behaviors are normal [41], indicating the importance of ERa in normal male mating behaviors.

Phenotypes in the abERKO The phenotypes exhibited by the abERKO are similar to those of the aERKO (Table 1) [48], emphasizing the importance of ERa in both male and female reproduction. One exception is the phenotype observed in the ovary (Figs. 4 and 6) [48]. The abERKO ovary exhibits normal

follicles, containing an oocyte, a normal complement of granulosa cells, and surrounding theca. However, there is also a marked presence of abnormal follicles that, when viewed in a 2-dimensional section, appear similar to seminiferous tubules. These follicles most often lack an oocyte and granulosa cells, but rather possess Sertoli-like cells, located along the basement membrane and exhibiting the characteristic cytosolic extensions and tripartite nucleoli. Further analysis indicates the presence of what may be intermediate structures, i.e., follicles containing a degenerating oocyte, surrounded by both granulosa cells and Sertoli-like cells. Because this phenotype emerges progressively with age, it is currently thought that these tubular structures represent ``ghosts'' of follicles in which the germ cell has died and a population of remaining granulosa cells is ``transdifferentiated'' into Sertoli-like cells.

Phenotypes in Non-Reproductive Tissues Epidemiological data showing increased risks for osteoporosis and cardiovascular disease in postmenopausal

Fig. 6. Details of follicles from abERKO ovary. All three representative follicles are from the same ovary. The top panel shows a normal developing follicle, with higher power detail to right showing granulosa cells. The bottom panel shows a seminiferous tubule-like structure with higher power detail to right. Note the Sertoli-like cells along the basement membrane. The middle panel shows an intermediate follicle with characteristics of both.

Estrogen Receptors

women indicates a possible role for E in bone and cardiovascular tissue. Therefore the phenotypes of the ERKO mice in these tissues have been studied. Analysis of bone tissues has shown aERKO femurs are shorter [49] as are male aERKO and abERKO but not bERKO femurs [50]. The shorter femurs are associated with lower serum IGF1 levels. aERKO femurs are also of smaller diameter in the female and lower density in the male [28]. An increase in bone mineral content and density was seen in bERKO females, with no change in bERKO males [51,52], indicating that ERb may actually exert a negative effect on bone density in the female mouse. Cardiovascular tissues from ERKO mice were analyzed by studying the protective effectiveness of estradiol in an aortic injury model. The protective effect of E was retained in the bERKO tissues (53). Initial studies indicated the protective effect was also present aERKO tissue (54). However, studies utilizing an ERa de®cient mouse derived in another laboratory (ERaKOSt) indicated a lack of estrogen protection. In addition, the abERKO tissues lacked estrogen protection as well (55). The initial ®nding of retained estrogen protection in the original aERKO model is most likely due to the expression of an ERa splice variant resulting in residual truncated ERa (32) combined with the superphysiological dose of estradiol required in the study (55). Overall, the studies indicate that ERa mediates the estrogen protective response observed in this model.

Summary This brief overview has described the well-established mechanisms and physiological functions of ER as well as the frontiers of study that continue to illustrate new details of the mechanisms of action and roles of ER as an important transcriptional modulator. Future investigation promises continuing advances that are eagerly awaited and will have many important applications in diagnostic and therapeutic aspects of reproduction and cancers of reproductive tissues.

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