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Reviews in Endocrine & Metabolic Disorders 2002;3:193±200
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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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Hewitt and Korach
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
Estrogen Receptors
195
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,
196
Hewitt and Korach
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.
Estrogen Receptors
197
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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Hewitt and Korach
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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