Estrogen Receptors

Physiol Rev 87: 905–931, 2007;
doi:10.1152/physrev.00026.2006.
Estrogen Receptors: How Do They Signal

and What Are Their Targets
NINA HELDRING, ASHLEY PIKE, SANDRA ANDERSSON, JASON MATTHEWS, GUOJUN CHENG,
JOHAN HARTMAN, MICHEL TUJAGUE, ANDERS STRÖM, ECKARDT TREUTER, MARGARET WARNER,
AND JAN-ÅKE GUSTAFSSON
Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden; Structural Genomics
Consortium, Botnar Research Centre, University of Oxford, Headington, Oxford, United Kingdom; and
Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada
I. Introduction 906
II. Estrogen Receptors 907
A. Two ER subtypes and several isoforms 907
B. Diverse structures of synthetic, dietary, and environmental estrogens 908
C. Distinct molecular pathways involving ERs 908
III. Estrogen Signaling 910
A. Estrogen regulation of growth factor pathways and branching morphogenesis in the mammary
gland, prostate, and lung 910
B. Imprinting by estrogen 913
C. Estrogen as a trophic factor for neurons 914
D. ERs and cell adhesion 914
IV. Anti-Estrogen Signaling 915
A. Anti-estrogens: benefits and limitations 915
B. Complete blocking of estrogen signaling 916
C. Deleterious effects of anti-estrogens 916
D. Structural basis for ER activity 917
E. Role of helix 12 in the regulation of AF-2 activity 917
F. Classical AF-2 antagonism 919
G. Nonclassical AF-2 antagonism 919
H. Coregulator recognition of antagonist-bound ER 920
I. ER coregulators 920
V. Concluding Remarks 922
Heldring N, Pike A, Andersson S, Matthews J, Cheng G, Hartman J, Tujague M, Ström A, Treuter E, Warner
M, Gustafsson J-Å. Estrogen Receptors: How Do They Signal and What Are Their Targets. Physiol Rev 87: 905–931,
2007; doi:10.1152/physrev.00026.2006.—During the past decade there has been a substantial advance in our under-
standing of estrogen signaling both from a clinical as well as a preclinical perspective. Estrogen signaling is a balance
between two opposing forces in the form of two distinct receptors (ER␣ and ER␤) and their splice variants. The
prospect that these two pathways can be selectively stimulated or inhibited with subtype-selective drugs constitutes
new and promising therapeutic opportunities in clinical areas as diverse as hormone replacement, autoimmune
diseases, prostate and breast cancer, and depression. Molecular biological, biochemical, and structural studies
have generated information which is invaluable for the development of more selective and effective ER ligands. We
have also become aware that ERs do not function by themselves but require a number of coregulatory proteins
whose cell-specific expression explains some of the distinct cellular actions of estrogen. Estrogen is an important
morphogen, and many of its proliferative effects on the epithelial compartment of glands are mediated by growth
factors secreted from the stromal compartment. Thus understanding the cross-talk between growth factor and
estrogen signaling is essential for understanding both normal and malignant growth. In this review we focus on
several of the interesting recent discoveries concerning estrogen receptors, on estrogen as a morphogen, and on the
molecular mechanisms of anti-estrogen signaling.
www.prv.org 0031-9333/07 $18.00 Copyright © 2007 the American Physiological Society 905
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906 HELDRING ET AL.
I. INTRODUCTION raised the question of whether survival of the ER␣ knock-

out mouse was due to ER␤ substituting for the functions
Estrogens play key roles in development and main- of ER␣. ER␤ knockout mice were made, and this was
tenance of normal sexual and reproductive function. In followed by double ER␣␤ knockouts (120). These differ-
addition, in both men and women they exert a vast range ent mouse models revealed that life is possible without
of biological effects in the cardiovascular, musculoskele- either or both ERs but that reproductive functions are
tal, immune, and central nervous systems (73). The most severely impaired (29). In addition, ER␣ and ER␤ were
potent estrogen produced in the body is 17␤-estradiol found to have distinct, nonredundant roles in the immune,
(E2) (Fig. 1). Two metabolites of E2, estrone and estriol, skeletal, cardiovascular, and central nervous systems (29,
although they are high-affinity ligands (121) are much 73, 78). Some of the most remarkable phenotypes of the
weaker agonists on estrogen receptors (ERs). These me- ER␣ and ER␤ knockout mice are the effects on morpho-
tabolites were formally thought to be inactive, but recent genesis. Estrogen is well known to be a morphogen, and
evidence suggests that they may well have tissue-specific its role in morphogenesis is evident from the structure of
roles (71). The ground-breaking findings in the field were the uterus (29), ovary (29), mammary gland (54), prostate
made in the late 1950s when Elwood Jensen (97) discov- (313), lung (181), and brain (301) of ER␣ and ER␤ knock-
ered and started the characterization of an estrogen bind- out mice. At the promoters of some genes, particularly
ing protein, today recognized as ER␣. In 1993, nearly those involved in proliferation, ER␣ and ER␤ can have
three decades later, the first ER␣ knockout mouse was opposite actions (145), a finding which suggests that the
created (150), and the surprising discovery was made that overall proliferative response to E2 is the result of a
life is possible without this receptor which, at the time, balance between ER␣ and ER␤ signaling. Anti-estrogens,
was thought to be the sole mediator of estrogen signaling. designed to block ER␣, are widely and effectively used
Soon after the characterization of the ER␣ knockout clinically in the treatment of breast cancer (28, 43). How-
mouse, ER␤ was discovered (122), and this discovery ever, although synthetic anti-estrogens are mainline ther-
FIG. 1. Molecular structures of the endogenous estro-

gen 17␤-estradiol and of tamoxifen, 4-hydroxy-tamoxifen,
raloxifene, ICI 182,780 (fulvestrant), toremifene, R,R-cis-
diethyl-THC, and genistein.
Physiol Rev • VOL 87 • JULY 2007 • www.prv.org

ESTROGEN RECEPTOR SIGNALING 907
apy for treating ER␣-positive breast cancers, these drugs

have unwanted side effects in nontarget tissues, and after
prolonged treatment, cancers become resistant to anti-
estrogen therapy (197, 256). The idea that ER␤ agonists
may be used to promote growth arrest offers new possi-
bilities for pharmacological intervention in the treatment
of cancers (179, 221). The mechanisms governing the
action and development of resistance to anti-estrogens
are poorly understood. The emerging picture is one in
which ER is capable of adopting a multitude of response
states depending on the nature of the bound ligand
(Table 3 and references therein) and where each ligand
induces a unique receptor conformation capable of re-
cruiting a distinct pattern of coactivators and corepres-
sors to the receptor-transcription complex.
In this review we focus on new insights into estrogen
receptor modulation by synthetic anti-estrogens and co-
regulatory proteins and discuss the molecular concept of
anti-estrogen signaling. We also discuss the roles of ER␣
and ER␤ in mediating growth and morphogenesis.
II. ESTROGEN RECEPTORS
A. Two ER Subtypes and Several Isoforms
Cellular signaling of estrogens is mediated through

two ERs, ER␣ (NR3A1) and ER␤ (NR3A2), both belonging
to the nuclear receptor (NR) family of transcription fac-
FIG. 2. Schematic representation of described estrogen receptor
tors. Like many other members of the NR family, ERs (ER) isoforms. The domains of the receptors include DBD, LBD, and two
contain evolutionarily conserved structurally and func- transcriptional activation functions, AF-1 and AF-2 as indicated for
tionally distinct domains (Fig. 2). The central and most hER␣. Full-length human ER␣ is 595 amino acids long (69). Both short
ER␣ isoforms, hER␣-46 and hER␣-36, lack the NH2-terminal region
conserved domain, the DNA-binding domain (DBD), is harboring AF-1 (52, 305). Percent sequence similarity between hER␣
involved in DNA recognition and binding, whereas ligand wild-type and hER␤ is indicated in hER␤. The 530-amino acid-long hER␤
binding occurs in the COOH-terminal multifunctional li- isoform is currently regarded as the full-length wild-type hER␤ (140).
Note that deletion of the fifth exon giving the hER␤⌬ exon 5 isoform
gand-binding domain (LBD). The NH2-terminal domain is results in a frameshift and a termination of the protein before the LBD
not conserved and represents the most variable domain (93). The striped fill patterns of the 3⬘ end of hER␤2 (also named
both in sequence and length. Transcriptional activation is hER␤cx), hER␤4, and hER␤5 represent the differing COOH-terminal
regions of these isoforms (180, 204, 234). The full-length rat and mouse
facilitated by two distinct activation functions (AF), the ER␤ isoforms are 549 amino acids (46, 296) and have 99% sequence
constitutively active AF-1 located at the NH2 terminus of similarity. The mER␤2 and rER␤2 isoforms contain an 18-amino acid
the receptor and the ligand-dependent AF-2 that resides in insertion in the LBD (25, 149), causing a significant decrease in ligand
binding affinity (148). Deletion of exon 3 results in rat isoforms unable
the COOH-terminal LBD (reviewed in Ref. 194). Both AF to bind DNA (227, 235), and the observed deletion of exon 5 and/or 6 in
domains recruit a range of coregulatory protein com- mice results in isoforms lacking various parts of the LBD (149).
plexes to the DNA-bound receptor. The two ERs share a
high degree of sequence homology except in their NH2-
terminal domains, and they have similar affinities for E2 pathways are active in the cell at the time of hormone
and bind the same DNA response elements. exposure (108, 110, 194).
Ligand-dependent estrogen signaling begins with the The identification of the second estrogen receptor
binding of estrogen to ER. Thereafter, the cell-specific ER␤ and several receptor isoforms has affirmed the com-
transcriptional response to estrogen depends on multiple plex nature of estrogen signaling and helped to explain
factors, the most immediate being the composition of estrogen action in tissues that do not express ER␣. ER␣
coregulatory proteins in a given cell and the characteris- and ER␤ are products of separate genes located on dif-
tics of the promoters of estrogen responsive genes. Since ferent chromosomes (46, 172). Several splice variants
hormones are modulators of transcription, the pattern of have been described for both receptor subtypes (Fig. 2),
modulated genes also depends on what other signaling but whether all the variants are expressed as functional

908 HELDRING ET AL.
proteins with biological functions is not clear. Most ER␣ disruptors. Altered reproductive capacity in wildlife and
variants differ in their 5⬘-untranslated region (UTR), not cancers in the breast and endometrium are thought to be
in the coding sequence. In addition, shorter ER␣ isoforms possible consequences of exposure to endocrine disrup-
lacking exon 1 and consequently the NH2-terminal AF-1 tors (133, 319). On the other hand, epidemiological stud-
(here termed hER␣-46 and hER␣-36) have been isolated ies suggest that diets rich in phytoestrogens, particularly
and identified in different cell lines (52, 305). These re- soy and unrefined grain products, may be associated with
ceptor isoforms have not yet been identified or character- decreased risk of some hormone-induced cancers (re-
ized in tissues, and their involvement in regulating estro- viewed in Ref. 318). Numerous investigations have docu-
gen effects in vivo remains to be determined. They are, mented antiproliferative effects of genistein, the most
however, interesting research tools since they have the abundant phytoestrogen in soy. Genistein (Fig. 1) has a
ability to heterodimerize with the full-length ER␣ and ninefold higher affinity for ER␤ than for ER␣ (5, 121).
thereby repress AF-1-mediated activity (52, 305). Possibly, Because of its ER␤ selectivity, genistein would be ex-
they may also localize to the plasma membrane and may pected to have none of the unwanted side effects of
help to elucidate the mechanisms through which rapid, estrogen replacement on the endometrium. Whether or
“nongenomic” estrogen signaling occurs (144, 305). Un- not genistein by itself is adequate in replacing the effects
like ER␣, several splice variants of ER␤ are expressed in of estrogen on bone, brain, and cardiovascular and im-
tissues. The 530-amino acid (aa)-long human ER␤ isoform mune systems is at present under intense investigation
is currently regarded as the wild-type ER␤ (rat and (reviewed in Refs. 212, 315, 324).
mouse, 549 aa) (140). Several alternative ER␤ isoforms A variety of synthetic estrogen antagonists have been
have been described (Fig. 2), and many of these are developed, and some of them are used clinically to re-
expressed as proteins in tissues (59, 252). Characteriza- verse the growth-promoting effects of estrogens in breast
tion of the functional isoform pattern in human samples is cancers (Fig. 1) (reviewed in Refs. 247, 262). Additional
not complete, but several experiments indicate that ER␤ compounds have been developed and characterized to
isoforms can differentially modulate estrogen signaling obtain more favorable tissue and receptor subtype selec-
and, as a consequence, impact target gene regulation (137, tive effects (reviewed in Refs. 109, 299). The term SERM
167, 242). For example, the human ER␤2 isoform (also (selective estrogen receptor modulator) describes syn-
named ER␤ cx) with 26 unique aa residues replacing the thetic ER ligands that display tissue-selective pharmacol-
COOH-terminal part of the LBD is unable to bind ligand or ogy. As anti-estrogens (or antagonists) they oppose the
coactivators and has no transcriptional activity in re- action of estrogens in certain tissues, while mimicking the
porter assays. ER␤2 dimerizes with preferentially ER␣, action of endogenous estrogens (agonists) in others (108,
thereby silencing signaling via this ER isoform (204). Both 110). The mechanism of anti-estrogen signaling is dis-
species-specific and species-common ER␤ isoforms have cussed in detail below. Hereafter in the review, the term
been identified (reviewed in Ref. 139) (Fig. 2). anti-estrogen will be used rather than SERM, since the
latter term encompasses a wider concept.
B. Diverse Structures of Synthetic, Dietary, and

Environmental Estrogens C. Distinct Molecular Pathways Involving ERs
In contrast to the situation for some NRs, such as the Emerging evidence suggests that there are several
thyroid hormone receptor (TR) or the retinoic acid recep- distinct pathways by which estrogens and ERs may reg-
tor (RAR), where the shape of the ligand-binding cavity is ulate biological processes (74) (Fig. 3). Ligand-bound ERs
well adapted to fit the cognate hormone, ERs⬘ ligand can bind directly to estrogen response elements (ERE), in
cavity appears generous in size for E2 (22). This allows the promoters of target genes or can interact with other
ER to bind a wide range of compounds with strikingly transcription factor complexes like Fos/Jun (AP-1-
diverse structures. In addition to estrogens, ERs also responsive elements) (127) or SP-1 (GC-rich SP-1 motifs)
show affinity for environmental contaminants such as (255) and influence transcription of genes whose promot-
polycyclic aromatic hydrocarbons, phthalates, and pesti- ers do not harbor EREs. Ligand-dependent activation trig-
cides, a class of estrogens termed xenoestrogens (20). gers recruitment of a variety of coregulators to the recep-
Certain plant constituents termed phytoestrogens (101, tor in a complex that alters chromatin structure and fa-
209) also have estrogenic actions that are biologically cilitates recruitment of the RNA polymerase II (Pol II)
relevant in humans as well as in farm animals. transcriptional machinery. The details of ER-transcription
Environmental contaminants with estrogenic activity factor interactions (i.e., ligand specificity, ER subtype
are thought to interfere with endocrine signaling in estro- specificity, interaction domains and motifs) remains to be
gen-responsive tissues and are referred to as endocrine characterized. Interestingly, in certain cell-type and pro-

FIG. 3. Model representing the mechanistically distinct molecular pathways used in the regulatory actions of ERs. The classical (direct) pathway
includes ligand activation and a direct DNA binding to estrogen response elements (ERE) before modulation of gene regulation. The tethered
pathway includes protein-protein interaction with other transcription factors after ligand activation, and thereby gene regulation is affected by
indirect DNA binding. A third mechanism, also called nongenomic with rapid effects, is not as well understood as the genomic mechanism but has
been observed in many tissues. The ligand activates a receptor, possibly associated with the membrane; either it is a classical ER (33, 138, 243), an
ER isoform (141, 305), or a distinct receptor (38) or, alternatively, a signal activates a classical ER located in the cytoplasm. After this rather unclear
event, signaling cascades are initiated via second messengers (SM) that affect ion channels or increase nitric oxide levels in the cytoplasm, and this
ultimately leads to a rapid physiological response without involving gene regulation. The ligand-independent pathway includes activation through
other signaling pathways, like growth factor signaling. In this case, activated kinases phosphorylate ERs and thereby activate them to dimerize, bind
DNA, and regulate genes.
moter contexts, 4-hydroxytamoxifen and raloxifene, sponsive to estrogens and acted as a constitutive tran-
which function as antagonists at EREs, can behave as scriptional activator (111, 291).
agonists through these indirect pathways. ER␤, but not When the two ER subtypes are coexpressed in cells,
ER␣, in the presence of E2 can oppose the actions of ER␤ can antagonize ER␣-dependent transcription (167,
4-hydroxytamoxifen and raloxifene on an AP-1 reporter 168). The molecular mechanisms of ER␤-mediated inhibi-
gene (127, 216, 309, 312). Similar observations have been tion of ER␣ signaling are currently under investigation.
made for the SP-1 pathway (255, 331). For example, it was shown that for ER␣-mediated regu-
In addition to the well-studied transcriptional effects lation of AP-1-dependent transcription, ER␤ expression
of E2, there are rapid effects, i.e., occurring within sec- alters the recruitment patterns of c-Fos to AP-1-regulated
onds or minutes after addition of E2 (283, 284, 307; re- promoters (168). Moreover, expression of ER␤ and the
viewed in Refs. 274, 320). These rapid effects include ER␤ variant ER␤2 increases the proteolytic degradation
activation of kinases and phosphatases and increases in of ER␣ (168). Collectively, these data suggest that the
ion fluxes across membranes. Although these rapid ef- ER␤-mediated inhibition of ER␣ activity involves a com-
fects have been extensively studied, there is still no con- bination of altered recruitment of key transcription fac-
sensus as to whether or not the classical ERs are involved tors and increased ER␣ degradation.
(33, 138, 243) or whether there is a distinct membrane- In addition to ligand-induced transcriptional activi-
associated receptor (38). Tools such as pathway-selective ties of ER, ligand-independent pathways to activate ERs
ligands (77) or cell lines designed to selectively express have been described. Growth factor signaling leads to
ER␣ in the nucleus, cytoplasm, or membrane may prove activation of kinases that may phosphorylate and thereby
useful in studying these extranuclear ER actions (253). activate ERs or associated coregulators in the absence of
Interestingly, evolutionary evidence suggests that early ligand (106). The growth factor activated pathway is
on, estrogen influenced reproduction through ER-inde- thought to significantly contribute to hormone-indepen-
pendent pathways (111) and that the receptor was unre- dent growth in some tumors (30, 269).

910 HELDRING ET AL.
III. ESTROGEN SIGNALING a 3-wk-old wt mouse, ducts avoid the ER␣⫺/⫺ implant.
This strongly indicates that the ER␣⫺/⫺ fat pad secretes
growth inhibitory substances. At 3 wk of age, the stroma
A. Estrogen Regulation of Growth Factor
of the mammary gland expresses both ER␣ and ER␤
Pathways and Branching Morphogenesis in the
(Fig. 4). We speculate that ER␤ stimulates secretion of a
Mammary Gland, Prostate, and Lung
growth repressor. One possible factor is TGF-␤, which is
an estrogen-regulated gene (94).
Estrogens and androgens are necessary for initiating
The use of knockout mice (119, 133, 152, 161, 225,
programs of morphogenesis at specific times during de-
250, 266) and tissue recombination methods (286) have
velopment, but it is growth factors and their receptors
revealed that ADAM-17 is responsible for processing of
that are needed for communication between epithelium
TGF and HB-EGF and that mammary ductal growth is
and mesenchyme. Without this communication, morpho-
dependent on epithelial amphiregulin and stromal EGFR.
genesis cannot occur. The major players in the cross-talk
ADAM-17 is essential for branching, but other metallopro-
between epithelium and mesenchyme are the epidermal
teinases (MMP) are involved in remodeling of the mam-
growth factor pathway, which includes epidermal growth
mary gland (272, 317). Degradation of ECM proteins by
factor receptor (stromal), epidermal growth factors (epi-
MMPs clears a path for migration of epithelial cells during
thelial), and the proteases which activate them (epithelial
ductal growth. In addition, cleavage of some proteins
and stromal); FGF receptors (epithelial) and their ligands
(elastin, laminin-5, or collagen types IV or VII) releases
(stromal); the transforming growth factor beta family of
growth factors that facilitate growth and migration of
peptides (BMPs) (stromal) and their receptors; and the
epithelial cells (104, 119). Inhibitors of metalloproteinase
Notch signaling pathway (epithelial receptors).
activity, TIMPs, modulate MMP functions. EGF and MMP
Epidermal growth factor receptor (EGFR-1; also
signaling are not independent processes but are essential
called HER-1 and ErbB-1) is essential for epithelial devel-
parts of a well-regulated system. Exposure of breast epi-
opment, tissue repair, tumor growth, tissue-modeling phe-
thelial cells in culture to the EGFR ligand amphiregulin
nomena, angiogenesis, and fibrogenesis. EGFR is acti-
induces expression of MMP2 and MMP9 (173).
vated by six peptide growth factors: EGF, transforming
Tissue recombinant experiments with fetal tissues
growth factor (TGF)-␣, betacellulin, heparin-binding EGF
have shown that stromal, not epithelial, ER␣ is needed
(HB-EGF), amphiregulin, and epiregulin. These are all
during mammary gland morphogenesis (32) and that
expressed as transmembrane precursors that need to be
EGFR phosphorylation and ductal development only oc-
released from the cell surface by proteolysis (79). The
cur when ADAM-17 and amphiregulin are expressed on
proteases which activate EGF receptor ligands are mem-
mammary epithelial cells, whereas EGFR is required in
bers of a disintegrin and metalloproteinase family
the stroma (286). These tissue recombinant experiments
(ADAM) of cell surface proteases. Thus tissue selectivity
indicate that ER␣ is not required for expression of EGFR
in regulation of EGF signaling depends on the type of EGF
ligands or for expression of ADAM-17 in epithelial cells
receptor, the type of protease, and the type of ligand
during morphogenesis. One working scenario for the role
expressed in the tissue.
of ER␣ in mammary gland morphogenesis is that stromal
ER␣ induces ADAM-17, which stimulates release of am-
1. Mammary gland morphogenesis
phiregulin from epithelial cells in the terminal end buds.
Ductal elongation in the mammary gland is abso- Amphiregulin activates EGFR in nearby stromal cells. In
lutely dependent on ER␣ and does not occur in ER␣⫺/⫺ this way epithelium and stroma communicate with each
mice. When pituitary hormones are normalized and pro- other in coordinating ductal growth. This mechanism is
lactin, progesterone, and estrogen are administered to valid during the limited time when ER␣ is expressed in
ER␣⫺/⫺ mice, a truncated mammary structure results. the stroma. Our own data show that this is during weeks
There is abundant alveolar growth but no ductal elonga- 2– 4 of postnatal life (Fig. 5). ADAM-17⫺/⫺ (55, 286),
tion. Kenney et al. (113) transplanted ER␣⫺/⫺ virgin amphiregulin⫺/⫺ (151, 286), and ER␣ ⫺/⫺ mice (113) all
mammary glands into the cleared mammary fat pad of show impaired ductal elongation. EGF receptor⫺/⫺ mice
virgin wild-type (wt) or ER␣⫺/⫺ mice and found that wt do not survive the perinatal period (175).
mammary tissue failed to grow in ER␣⫺/⫺ mammary fat Mammary lobular/alveolar growth does not require
pads. On the other hand, in ER␣⫺/⫺ mammary glands, ER␣ or EGF (314). The requirement for estrogen in alve-
there was lobulo-alveolar growth in wt mouse fat pads. olar growth in ER␣⫺/⫺ mice suggests that ER␤ may be
Treatment of virgin ER␣⫺/⫺ mammary gland with TGF-␣ involved in alveologenesis and epithelial cell differentia-
or heregulin ␤1 stimulated growth and secretory activity tion. Such a role for ER␤ is consistent with the finding of
(113). It is not only in tissue recombinants that wt epithe- incomplete differentiation of the mammary epithelium
lium fails to penetrate ER␣⫺/⫺ fat pad. When 3-wk-old in ER␤⫺/⫺ mice (54). There is a normal ductal tree in
ER␣⫺/⫺ fat pad is implanted into the mammary fat pad of ER␤⫺/⫺ mice, but as these mice age, the epithelium in

FIG. 4. In the adult mouse mammary gland, ⬎60% of the ductal epithelial cells express ER␤ and ⬍20% express ER␣. ER␣ is not found in the
stroma of the adult gland but ER␤ is. In the 2-wk-old mouse, both ER␣ and ER␤ are strongly expressed in both the epithelium and stroma.
the mammary gland continues to express the proliferation manent alterations occur in the male and female genital
marker Ki67 and to proliferate. The gland becomes filled tracts when mice are exposed to DES at certain critical
with large cysts. Mammary epithelial proliferation in times during development. In male mice, prenatal expo-
ER␤⫺/⫺ mice between 1.5 and 2 yr of age appears to be sure to DES is associated with poor semen quality, pros-
independent of estrogen, since at this age the ovaries are tatic disease, cryptorchidism, testicular neoplasia, femini-
depleted of follicles (Fig. 5). Our working hypothesis is zation of the seminal vesicles (induction of the estrogen
that loss of ER␤ leads to loss of TGF-␤ and loss of a major responsiveness and of the estrogen-regulated gene lacto-
growth repressor in the mammary gland. transferrin), and stromal inflammation (9, 190, 240). Im-
printing by DES in the developing prostate is absolutely
2. Prostate morphogenesis dependent on the presence of ER␣, and ER␣⫺/⫺ mice are
Some of the most compelling evidence for a role of resistant to imprinting by DES (238).
estrogen during embryogenesis is the outcome of in utero It is, therefore, not surprising that the structure of the
exposure to the synthetic estrogen diethylstilbestrol prostates of ER␣⫺/⫺ mice are abnormal (206). Surpris-
(DES). Both males and females, who were exposed in ingly, unlike the case in the mammary gland, the ER␣⫺/⫺
utero, develop in adulthood a wide range of abnormalities mouse ventral prostate is characterized by extremely long
in their urogenital tracts. In the 1980s, the McLachlan distended ducts with little branching and very sparse
laboratory pioneered the use of DES in mice for investi- periductal stroma. This phenotype is compatible with the
gating the mechanism of the human DES syndrome. Per- suggestion that ER␣ is a positive regulator of secondary

912 HELDRING ET AL.
FIG. 5. In wild-type mice at 2 yr of age, there are still follicles and corpora lutea in the ovaries. In the ER␤⫺/⫺ mouse ovary, there are no follicles
and no evidence of corpora lutea. The ducts of mammary glands of 2-yr-old wild-type mice are atrophic (small arrows) with no evidence of the
proliferation marker Ki67. In ER␤⫺/⫺ mice, ducts are large and cystic (long arrows) and many cells express Ki67.
branching and of stromal growth. How might ER␣ be a transient expression of ER␣ observed in prostate epithe-
positive regulator of secondary branching? The ER␣⫺/⫺ lium in the first 2 wk of life (206) leaves open the possi-
prostate is not unlike that of the prostate of mice treated bility that ER␣ may, at this time, regulate follistatin ex-
with EGF, i.e., increased prostatic size, with reduced pression. ER␣ would then induce a repressor of a repres-
ductal branching and swollen ducts. sor and would thus be a positive regulator of notch
Tissue recombinant experiments have shown that signaling and branching. In ER␣⫺/⫺ mice, secretion of
like the mammary gland, prostatic growth requires ER␣ follistatin from the epithelium would be repressed and the
signaling in the mesenchyme, not in the epithelium, and BMP would be free to inhibit branching. BMP7 is ex-
this is compatible with the idea that a mesenchymal pressed in the periurethral urogenital mesenchyme prior
growth factor is ER␣ regulated. Testosterone produced to formation of the prostate buds. In BMP7 null mice,
by the fetal testes drives prostate morphogenesis, but Notch1 signaling is derepressed in the urogenital epithe-
ductal elongation and branching involve a close commu- lium (70, 302, 303). Both ER␣ and EB␤ oppose BMP
nication between the urogenital mesenchyme and epithe- signaling (327).
lium, and this communication involves positive regulation The original description of the ventral prostate of
by ER␣, FGF10, Shh, and repressive activity of BMPs (15, adult, neonatally estrogenized rats [elevated expression
37). Although FGF10 is a prostatic mesenchymal growth of the androgen receptor, a continuous layer of basal
factor, available data show that it is not regulated by cells, an increase in interacinar stromal tissue, disorga-
androgens (290). nized acini with epithelial hyperplasia, luminal sloughing,
BMP7 secreted from the stroma is a repressor of lack of epithelial differentiation, and a remarkable re-
Notch signaling and thus of branching morphogenesis duction in expression of ER␤ (236, 237, 239, 321)] are
(132). Follistatin, an estrogen-regulated gene produced in characteristics of the phenotype of the ER␤⫺/⫺ mouse
the epithelium, is an inhibitor of BMP signaling (105). The ventral prostate (92, 313), and we speculate that these

characteristics of the adult prostates of neonatally es- 3. Lung morphogenesis

trogenized mice are a consequence of the reduction in
The lungs of adult ER␤⫺/⫺ mice are characterized
ER␤ expression in the prostates of these mice (236, 237,
by decreased elastic tissue recoil (164, 165) and an in-
239, 321). creased expression of MMP1 and TIMP2, and cleaved
Recently, the Prins laboratory demonstrated that one (active) MMP2 (181). Alterations in the proteolytic/anti-
of the genes that is downregulated by neonatal estrogen proteolytic balance of the lung have been associated with
exposure is FGF10. This then leads to downregulation of several respiratory diseases characterized by changes in
sonic hedgehog-patched Gli (Shh-ptc-gli) and BMP7. Thus the lung extracellular matrix (ECM). So it is not surprising
ER␣ directly or indirectly is a repressor of BMP7, and the that in the lungs of ER␤⫺/⫺ mice there is accumulation of
branching defects in the dorsolateral prostate of neona- collagen and that suboptimal lung function leads to over-
tally estrogenized mice can be explained by the loss of all hypoxia in these mice (181). ER␤ is abundantly ex-
Shh-ptc-gli and BMP7 signaling (90). pressed in epithelial cells in the adult lung, and the ele-
Interestingly, according to Prins, FGF10, Shh-ptc-gli vated levels of expression of MMP1 and TIMP2 in
and BMP7 are not reduced in the ventral lobes of the ER␤⫺/⫺ mice indicates that ER␤ might normally sup-
prostate following neonatal estrogenization. This differ- press this complex and restrict activation of MMP2.
ence in lobe-specific expression of these factors explains Both EGF and FGF signaling are essential for devel-
why there are more serious branching deficits in the opment of lungs. EGFR is essential for epithelial-mesen-
dorsal than the ventral lobe. It leaves unanswered the chymal interactions, and the lungs of EGFR⫺/⫺ mice are
questions of why the structure and size of the ventral lobe so impaired that mice do not survive the perinatal period
are so profoundly affected by neonatal estrogenization (175). Embryonic lung synthesizes several EGFR ligands
and why FGF10 in the ventral prostate lobe is insensitive and responds to EGF, TGF-␣, and amphiregulin by pre-
to neonatal estrogen. FGF signaling is androgen depen- cocious branching (259, 306). FGF10 is expressed in lung
dent: the urogenital sinus of FGF10⫺/⫺ mice in culture mesenchyme during branching morphogenesis (E10.5–
can be stimulated to produce prostate buds by FGF10 16.5) and is an essential nonredundant growth factor
(107). Transgenic mice expressing a dominant negative
plus testosterone but not by FGF10 alone. The require-
FGF receptor specifically in lung tissue do not develop
ment for both testosterone and FGF10 for the growth of
lungs (226). The severe phenotypes of both the
prostatic buds suggests that testosterone, not estrogen, is
FGF10⫺/⫺ and the EGFR⫺/⫺ mice are not seen in the
essential for expression of FGF receptors in the epithe-
ER␤⫺/⫺ mouse. Lungs of ER␤⫺/⫺ mice are morpholog-
lium. The observed high expression of androgen recep- ically indistinguishable from those of their wt littermates
tors in the ventral prostate of neonatally estrogenized until mice are 5 mo of age. Expression of ER␣ and ER␤ in
mice might help explain the maintenance of FGF10 sig- the embryonic lung has not been reported, and we do not
naling in this tissue. know whether ER␤ plays a role in lung development or
If loss of ER␤ results in profound morphological whether it only functions in remodeling of the adult lung.
changes in the ventral prostate, what are the ER␤ regu- Ryu et al. (249) have shown that fetal human lung is
lated genes that lead to these abnormalities? To answer characterized by high levels of MMP-2 and little TIMP-3
this question we have to examine the epithelium of the expression while the adult human lung exhibits a more
adult ventral prostate for growth factors that are estrogen antiproteolytic profile with decreased MMP-2 and in-
repressed or growth repressors whose expression is increased TIMP-3 expression. The MMP profile in the
creased by estrogen. TGF-␤ is an estrogen-stimulated ER␤⫺/⫺ mouse lung resembles that of the fetal rather
growth repressor (94) that inhibits both testosterone- and than the adult human lung and may reflect a block in
EGF-stimulated prostatic stromal and epithelial cell maturation as is seen in epithelium of other organs in
growth. Epithelial and stromal cells from 20-day-old rat ER␤⫺/⫺ mice.
ventral prostate express the mRNA for TGF-␤1, -2, and -3.
TGF-␤2 mRNA expression is elevated at birth, declines B. Imprinting by Estrogen
during the period of prostate growth, and is elevated
again at 100 days of age (94). Its expression is also in- Several distinct critical periods or “windows in time”
creased in the prostate upon castration of adult rats. This when estrogen influences tissue morphogenesis have
pattern of expression is compatible with a role for TGF-␤ been observed for many organs. In 1975, the Naftolin
in limiting prostate growth (1). Since ER␤ is the only ER laboratory made the astounding observation that in ro-
expressed in the adult ventral prostate epithelium, estro- dents, estrogen masculinizes hypothalamic neurons on
gen regulation of TGF-␤ is most likely mediated by ER␤. embryonic day 18 (185). The estrogen for this masculin-
This would mean that ER␤ has a role in limiting alveolar ization is synthesized in the embryonic brain from testos-
growth in the prostate. terone produced in the testes, and this transient exposure

914 HELDRING ET AL.
to estrogen initiates a critical period of sexual differenti- affect neurite elongation and differentiation directly
ation of the brain (reviewed in Ref. 184). We now know through its regulation of cytoskeletal genes that are re-
that there are even earlier time points in development quired for neurite growth (49, 154, 260).
when estrogen can influence brain function. In 1992 Beyer Both ER␣ and ER␤ are expressed in the adult rat
et al. (17) showed that sexual differentiation in prolactin brain with ER␤ mRNA-containing cells more widely dis-
expression of certain diencephalic neurons occurs before persed throughout the brain than those expressing ER␣
the testosterone surge, and in 2001 studies on the brains mRNA (12, 36, 129, 213, 270). Comparison of ER mRNA
of ER␤⫺/⫺ mice revealed that ER␤ influences neuronal expression patterns in rat, mouse (125, 270), monkey (72),
survival as early as embryonic day 15 (301). For the and human (214, 215) indicates that, although the general
uterus, vagina, prostate, and lung, one critical period distribution is similar, data cannot be extrapolated from
when estrogen influences morphogenesis is between em- one species to another. The paraventricular nucleus of
bryonic days 9 and 16. There is a second critical period rats has been reported to express only ER␤, while in the
between postnatal days 1 and 6 when the prostate and the mouse it contains cells that also express ER␣. The human
skeleton are imprinted (176). In the developing mammary paraventricular nucleus has been reported to express
gland on the other hand, estrogen influences morphogen- mRNA for both ER␣ and ER␤, with ER␣ predominating.
esis at puberty. In addition to the well-characterized role of estrogen
on feedback regulation of hypothalamic and pituitary hor-
mone secretion (102) and facilitation of female reproduc-
C. Estrogen as a Trophic Factor for Neurons tive behavior (157), estrogen also has effects on the mod-
ulation of motor behavior (8) and on mood and mental
Some 30 years ago, it was found that estrogen en- state (50, 76). More recently, estrogen has been recog-
hances the growth and arborization of axons and den- nized as a neuroprotective agent. The use of estrogen to
drites in hypothalamic neurons grown in organotypic cul- protect against Alzheimer’s disease, Parkinson’s disease,
tures (294). Neonatal estrogen treatment of rats has been schizophrenia, and ischemic stroke (16, 153, 258, 275, 276)
shown to increase synapse formation in the arcuate nu- is being widely debated at present. Multiple epidemiolog-
cleus during postnatal development (3) and to restore ical reports have suggested that estrogen replacement
deafferentation-induced loss of axodendritic synapses in may delay the onset of and slow the cognitive decline
adults (166). Synapse remodeling in adults does not occur associated with Alzheimer’s disease (reviewed in Ref.
only in response to axonal injury but is a continuously 264). In some studies estrogen showed no beneficial ef-
ongoing process involved in memory and learning. Syn- fects on cognition (182, 271). Suggestions for possible
apses in the arcuate nucleus and in the CA1 region of the mechanisms through which estrogen might exert positive
hippocampus exhibit a phasic remodeling, coordinated effects in the brain include prevention of amyloid
with fluctuation in hormone levels during the estrous ␤-plaques (10, 67, 325) and regulation of apoptosis. Sev-
cycle (205). Growth-promoting effects of estrogen have eral studies have shown that estrogen can modulate ex-
also been described in the midbrain (244), cortex (62), pression of the antiapoptotic proteins Bcl-2 and/or Bcl-xL
hippocampus (68), spinal cord (298), and pituitary (26). (the long isoform of Bcl-x) in hippocampal, cortical, and
It is generally accepted that in the early postnatal dorsal root ganglion neurons (41, 193, 222). In addition,
period as well as in adults, changes in the morphology of estrogen can downregulate expression of the proapop-
synapses and increased number of dendritic spines can totic Bad or Nip-2, which is a negative regulator of Bcl-2
account for many of the estrogen-induced changes in (21, 66).
brain morphology, sexual behavior, and memory and Perhaps the optimal strategy for estrogen replace-
learning. ment still has to be worked out. It seems that there is a
Effects of estrogens on neuronal growth are medi- critical period around the menopause, and if estrogen
ated via regulation of endogenous neurotrophic sub- replacement therapy is used early after menopause it
stances. Estrogen regulates nerve growth factor (NGF) delays the onset of and slows the cognitive decline. How-
and its receptors in cholinergic neurons (293), TGF-␤ in ever, estrogen replacement therapy at a later stage does
the hypothalamus (155), brain-derived neurotrophic fac- not seem to improve the cognitive function. It also still
tor in the cortex (281), NGF receptors in sensory neurons remains to be determined whether selective ER␣ and ER␤
(280), and the insulin-like growth factor and its receptor agonists might be more effective than the conjugated
in the hypothalamus (42, 233). estrogens currently used.
As in peripheral tissues, the effects of estrogen on
growth factor signaling are reciprocated, and growth fac- D. ERs and Cell Adhesion
tors can influence estrogen signaling. Thus NGF has been
shown to regulate ER action in the forebrain, possibly via Adhesion between neighboring cells and cell adhe-
phosphorylation events (177). However, estrogen can also sion to the ECM influence the structure of normal epithe-

lium as well as cell migration and invasion during tumori- The potential of ER␤ as a predictive factor in breast
genesis and metastasis. Estrogen signaling plays a signif- cancer has been under intense scrutiny. Several publica-
icant role in the regulation of these processes, particularly tions have evaluated ER␤ mRNA in breast cancer, but the
in the mammary gland. In the MCF7 breast cancer cell usefulness of ER␤ as a diagnostic marker is still not clear
line, estrogen stimulates morphological changes associ- (39, 219, 251, 285). While ER␤ is the predominant receptor
ated with the rearrangement of intermediate and actin in the normal breast, its levels are reduced in breast
filaments and modifications in the formation of cell-cell tumors, and some laboratories have reported on a corre-
and cell-ECM adhesion plaques (35, 254). During tumori- lation between the presence of ER␤ and lower tumor
genesis there are alterations in expression of adhesion stage and grade (96, 183, 207, 208). Others have found no
molecules and proteins involved in cell migration (re- correlation of ER␤ protein with clinical parameters (60).
viewed in Ref. 31). Expression of the cadherin and catenin The unwanted agonistic effects of anti-estrogens in
families, CD44, integrins and metalloproteases are all the breast and uterus have traditionally been linked to a
changed in cancer. E-cadherin is highly expressed in ER␣- strong AF-1 activity of ER␣, since an effective and specific
positive, low-grade tumors (220, 282), while P-cadherin is structural inhibition of AF-2 activity is seen with these
present in ER␣-negative, high-grade tumors (128, 218). ligands (Fig. 6). Although the tissue-selective agonist ac-
From studies on the ER␤⫺/⫺ mouse mammary gland tivity of anti-estrogens may be a function of the cell
(54), it appears that ER␤ positively regulates the terminal type-specific set of coregulators, surprisingly few of the
differentiation of the mammary gland epithelium. The loss well-known NR and ER coactivators (reviewed in Refs. 6,
of ER␤ results in a reduction of the ECM and lamina 163, 279) have been reported to be directly involved in
basalis and, frequently, in an increase of the interepithe- anti-estrogen signaling (Tables 1, and 2). The contribution
lial cell space, characterized by decreased levels of the of ER␤ to the tissue-selective agonistic effects of tamox-
adhesion molecules, E-cadherin, connexin 32, occludin, ifen is considered minimal, since the activity of the AF-1
and integrin ␣2. domain in this receptor subtype is almost negligible in
During tumor progression, epithelial-to-mesenchy- reporter-mediated gene expression (75).
mal transition is associated with the loss of adherens Anti-estrogens were traditionally thought to occupy
junctions, profound morphological changes, and en- the ligand-binding pocket blocking access to E2 and freez-
hanced migratory and invasive capabilities (reviewed in ing ER in “inactive” conformations that are unfavorable to
Refs. 31, 103). Loss of E-cadherin is a key event in this coactivator interactions. Current evidence suggests that
progression. The E-box binding transcriptional repressors cell type-specific profiles of coregulators determine tran-
Snail, Slug, SIP1, and Twist are key repressors of E- scriptional activities of antagonist-bound ERs (108, 110,
cadherin transcription. Snail in turn is repressed by an E2 263). Furthermore, different antagonists induce ligand-
regulated gene, the metastatic tumor antigen 3 (MTA3), a specific conformational changes in ERs, allowing expo-
component of the Mi-2/NuRD nucleosome-remodeling sure of unique surfaces for coregulator interactions (82,
and histone deacetylation complex (58). In mammary ep- 199, 217). These findings, together with recent gene ex-
ithelial cells, ER␣ is necessary for maintenance of mam- pression profiling data (56, 57, 84), extend the traditional
mary epithelial architecture by constraining Snail repres- view of ER antagonist function to include “active anti-
sion of E-cadherin. ER␣ with its corepressors NCoR and estrogen signaling.”
SAFB1 can bind to and repress transcription of the E-
cadherin promoter (201). Thus E-cadherin expression is
regulated by the cellular ER content, coactivators/core- A. Anti-estrogens: Benefits and Limitations
pressors, and active signaling from other pathways.
When clinically employed as antiproliferative agents
in the treatment of breast cancer, the agonist action of
IV. ANTI-ESTROGEN SIGNALING antiestrogens is desirable in certain tissues like bone and
cardiovascular system, while highly inappropriate in tis-
Many of the growth-stimulatory effects of estrogens sues like uterus or breast. Optimization of therapeutic
in breast cancer have been linked to ER␣ (14, 248). Today, strategies for targeting hormone-dependent tumors is fo-
ER␣ expression is routinely checked in pathological di- cused on agents with slightly modified pharmacological
agnosis, and if ER␣ is expressed in the tumor, the patient profiles. Raloxifene and toremifene are therapeutically
receives the anti-estrogen tamoxifen. Seventy percent of established anti-estrogens, available for prevention of os-
women with ER␣-positive breast cancer benefit from ta- teoporosis (18) and treatment of advanced hormone-sen-
moxifen (43a, 100). The usefulness and importance of sitive breast cancer (80). Since the mechanism of action
tamoxifen in breast cancer therapy is well established, of tamoxifen, raloxifene and toremifene are closely re-
and the disease-free survival rate has increased drasti- lated, similarities in their therapeutic profiles are ex-
cally due to the widespread use of tamoxifen (24). pected. Although raloxifene and toremifene do exhibit the

916 HELDRING ET AL.
FIG. 6. Major conformations induced by ER agonists and antagonists. Schematic representation of ERs bound to E2 (A), E2/LxxLL NR-box (B),
raloxifen (C), genistein (D), and ICI (E). The location of AF-2 between H3-H5 and H12 is indicated in A. The position of H12 is indicated by a green
cylinder. The LxxLL peptide is shown as a purple cylinder with leucines in stick form in B.
same beneficial effects as tamoxifen on bone and on bances and may render neurons more sensitive to stress
serum lipoprotein profile, unlike tamoxifen, raloxifene and excitatory neurotoxins.
and toremifene do not induce uterine growth. Unfortu- Aromatase inhibitors are effective in advanced breast
nately, raloxifene causes an increase in hot flushes and cancer, but clinical trials have indicated that resistance
also increases the incidence of thromboembolic diseases. develops with this strategy (reviewed in Ref. 40). Despite
Toremifene has not been in clinical use long enough to intense research, optimal agents targeting ER signaling
yield sufficient follow-up data. However, the few safety have not yet been identified. Further investigation is likely
data that are accumulated suggest a favorable safety pro- to increase our understanding of the complexities of ER
file for toremifene (80). signaling and increase the chances of rational design of
more optimal ER ligands for targeting estrogen-depen-
dent tumors.
B. Complete Blocking of Estrogen Signaling
There are two therapeutic strategies for completely C. Deleterious Effects of Anti-estrogens
blocking estrogen signaling; one is the use of a pure ER
antagonist, having no agonistic properties, and the other The acquired tamoxifen resistance developed in tu-
one is blocking of estrogen synthesis with the use of mors after some time of treatment is a major problem and
aromatase inhibitors. Fulvestrant (ICI 182,780) binds, in- cause of serious concern to clinicians. Recent data indi-
hibits, and promotes degradation of ERs (47, 162). Pre- cate that growth factor signaling is likely to be involved in
clinical studies have indicated that fulvestrant in combi- the development of tamoxifen resistance. There is recip-
nation with growth factor receptor inhibition is an effec- rocal cross-talk between estrogen and growth factor sig-
tive treatment option for postmenopausal women with naling: estrogens regulate growth factor signaling and
advanced breast cancer. Because estrogen is essential for growth factors can activate ER (191, 257). The HER2
maintenance of brain function, complete blocking of es- downstream signaling molecules ERK1 and ERK2 can
trogen receptors may be associated with cognitive distur- phosphorylate ER, leading to enhanced sensitivity of the
TABLE 1. Coactivators reported to be involved in tamoxifen signaling
Gene Product Mechanism of Activation Involved ER Domain Reference Nos.
AIB1 Recruit HATs, NR boxes ER␣-AF-1 and LBD 2, 210, 311

SRC-1 Recruit HATs, NR boxes ER␣-AF-1 and LBD 263, 278
L7SPA Not known ER␣ hinge 95
p68 Enhance tamoxifen activity when S118 phosphorylated, can recruit HATs ER␣-AF-1 45
ER, estrogen receptor; LBD, ligand-binding domain; AF, activation function.

TABLE2. Estrogen receptor corepressors involved in LBDs of both ER subtypes bound to a range of different
antagonist activity ligands and coactivator fragments (see Table 3). These
studies have provided valuable information regarding the
Mechanism of Involved ER
Gene Product Interaction Domain Reference Nos. structural basis of receptor agonism/antagonism and the
determinants that influence ER␣’s and ER␤’s ligand-bind-
NCoR Class I ER␣-LBD 95, 98, 112, 134, 263 ing preferences (229, 232). The crystal structures dem-
SMRT Class I ER␣-LBD 51, 95, 112, 263, 278
ER␤-LBD
onstrate that ligand binding to a buried cavity in the
RTA Class III ER␣-AF1 198 interior of the ER-LBD stabilizes distinct receptor con-
SAF B1/SAF B2 Class III ER␣-DBD 200, 202, 203, 295 formations that are subsequently interpreted by the
HDAC 4 Class III ER␣-AF1 136
SHP Class III or IV Not known 116
transcriptional apparatus.
NEDD 8 Class III or IV Not known 47 One of the main weaknesses of crystal structure
SAP 30 Class IV Indirect 81, 130, 186 analysis is that this technique typically provides a static
picture/snapshot of the molecules being analyzed. How-
Class I corepressors, CoRNR box recruitment motif; class III, lack
an identified interaction motif; class IV, indirect recruitment. ever, the sheer variety of complexes that have been de-
termined for ER-LBD has allowed us to build a compre-
hensive dynamic picture of the characteristic structural
receptor to its cognate hormone and potentially to ligand- alterations that accompany binding of particular classes
independent receptor activation (106). Interestingly, of receptor ligands.
ERK1/2 expression and activity are increased in several
estrogen nonresponsive human breast cancer cell lines
E. Role of Helix 12 in the Regulation
(30, 269). Coregulators are also subject to posttransla-
of AF-2 Activity
tional modifications by the signaling kinases and thereby
these enzymes may indirectly affect ER activity (53, 85,
134, 322). Data from xenograft models and cell lines show ER’s ligand-dependent transcriptional activation
that overexpression of the growth factor receptor HER2 function (AF-2) is localized in a conformationally dy-
may constitute a primary mechanism of tumor resistance namic region of the LBD. The key element of the AF-2
to tamoxifen (13, 117, 170, 228). Recent clinical evidence conformational switch is a short helical region (helix 12;
shows that patients with tumors expressing high levels of H12) located at the carboxy terminus of the LBD. Differ-
the transcriptional coactivator AIB1 in combination with ent classes of receptor ligands influence H12’s orientation
HER2 have a poor response to tamoxifen. Those patients with respect to the rest of the LBD (Fig. 6). Agonist
with tumors expressing high HER2 or AIB1 separately had ligands stabilize a receptor conformation that is optimal
a good disease-free survival (210). These data suggest that for efficient interaction with coactivators and thereby
increased growth factor signaling via HER2 activates ki- facilitates transcriptional activation. In such a “transcrip-
nases that lead to phosphorylation of ER and AIB1, cir- tionally active” conformation, the helical elements that
cumstances under which the agonistic activity of tamox- comprise the AF-2 region (helices H3–5 and H12) assem-
ifen might be enhanced (reviewed in Refs. 211, 256). ble to form a shallow hydrophobic binding site for the
leucine-rich LxxLL motifs of NR coactivators (Fig. 6, A
and B). In this conformation, H12 is positioned across the
D. Structural Basis for ER Activity entrance to the ligand-binding pocket and constitutes a
key part of the coactivator docking surface (22, 195, 267).
Structural analysis of ER domains over the past de- In contrast, receptor antagonists interfere with position-
cade has greatly enriched our understanding of receptor ing of H12 through a variety of mechanisms resulting in
function (232). While elucidation of the intact receptor’s ER conformations in which the coactivator recruitment
three-dimensional architecture continues to pose formi- site is incomplete (Fig. 6, C–E).
dable challenges to structure determination techniques In cases where H12 is not stabilized in the “agonist
due to interdomain flexibility and the poorly structured conformation,” the helix is reoriented to bind along and
nature of the NH2-terminal region, analysis of the individ- occlude the AF-2 groove (Fig. 6, C and D), thereby phys-
ual DNA- and ligand-binding domains has provided a ically blocking the recruitment site (22, 267). The com-
wealth of information about sequence-specific DNA target monly held view is that H12 is able to bind along the
recognition (27, 114) and the ligand-induced conformational coactivator groove by mimicking the consensus LxxLL
changes that underpin receptor activation (188, 232). motif with its own intrinsic related sequence (267). Our
The vast majority of structural studies of ER have recent analysis, however, suggests an alternate and more
focused on its COOH-terminal LBD due to the desire to compelling explanation: ER’s H12 region contains an
develop subtype-specific ligands (115, 159). Consequently, extended corepressor (CoRNR) box sequence that oc-
numerous crystal structures have been determined for the cludes the AF-2 site and prevents unwanted interaction

918 HELDRING ET AL.
TABLE 3. ER ligand-binding domain (LBD) crystal structures available from RCSB (November 2006)
Ligand Ligand Class Coregulator PDB Code* Comments Reference Nos.
ER␣ E2 Agonist IERE First structure of steroid receptor with 22

agonist
IA52 Intermolecular disulfide crosslink 44, 61
generates nonphysiological
conformation of H12
IQKT Agonist conformation 115
IQKU Agonist conformation 289
IG50 Agonist conformation 108
SRC2-3 IGWR Agonist conformation with CoA peptide
Cyclic LxxLL IPCG Complex with cyclic antagonistic LxxLL
peptide
DES Agonist mSRC2-2 JERD First illustration of CoA recruitment with 267
LxxLL peptide bound along AF2
RAL core Agonist SRC2-2 IGWQ Agonist conformation 308
GEN Agonist SRC2-2 IX7R Agonist conformation. Provides clues to 160
origins of ER␣/␤ selectivity of GEN
THC Agonist mSRC2-2 IL21 Agonist conformation 268
WAY-244 Agonist SRC2-2 1X7E Agonist conformation with ER␤ selective 159
agonist
Benzopyran Agonist 210J Antagonist H12 conformation with ER␤ 196
selective agonist
Oxabicyclophenols Agonist mSRC2-2 1ZKY/2BIV/2FAI Agonist conformation with ER␤ selective 86
agonist based on oxabicylic scaffold
Dihydroequilenin Agonist mSRC2-2 2BIZ Agonist conformation unpub
RAL Antagonist 1ERR Complex with SERM highlighting 22
archetypal “antagonist conformation” of
H12 with AF2 groove occluded
4-OHT Antagonist Affinity selected JERT Antagonist conformation 118
peptide
2BJ4 Complex with ER␣-specific phage-selected 267
peptide antagonist highlighting novel
putative coregulator docking site
Tetrahydro, Antagonist IUOM/IXQC ER␣ selective SERMs (SERAMs): 245, 246
isoquinolines antagonist conformation
GW56J8 Antagonist IR5K Antagonist conformation with orientation
of H12 that differs slightly from the
archetypal RAL/OHT structures
Dihydrobenzoxanthines Antagonist ISJ0/IXP1/IXP6/ Series of ER␣-selective SERMs; antagonist 19, 115
IXP9/IXPC conformation
Chromanes Antagonist IYIM/IYIN Series of ER␣-selective SERMs 288
Aryl-naphthalene Antagonist 2AYR Complex with SERM effective against 91
uterine leiomyoma
ER␤ E2 Agonist NcoA5 peptide 2J7X LxxLL-stabilized agonist conformation This study
17-Epiestriol Agonist NcoA5 peptide 2JZY Ligand orientation dictated by hydroxyl This study
positioning on D ring
ERB041 Agonist SRC1-1 IX7B Series investigating ER␤-selective agonist 159
binding
WAY-J97 Agonist SRC1-1 1U9E Series investigating ER␤-selective agonist 159
binding
WAY-697 Agonist SRC1-1 1X76 Series investigating ER␤-selective agonist 159
binding
WAY-244 Agonist SRC1-1 1X78 Series investigating ER␤-selective agonist 159
binding
CL-272 Agonist SRC1-1 1O3Q Series investigating ER␤-selective agonist 158
binding
WAY-338 Agonist SRC1-1 IU3R Series investigating ER␤-selective agonist 158
binding
WAY-297 Agonist SRC1-1 1U3S Series investigating ER␤-selective agonist 158
binding
2-arylindene-1-one Agonist SRC1-1 1ZAF Series investigating ER␣/␤-selective 171
agonist binding
2-Phenyl-naphthalene Agonist SRC1-1 1YY4/1YYE Series investigating ER␤-selective agonist 174
binding
Tetrahydrofluorenone Agonist 2GIU ER␤-selective agonist; H12 bound along 316
AF2 in suboptimal orientation
Benzopyran Agonist 210G H12 bound along AF2 in suboptimal 196
orientation with ER␤ selective agonist.
Binding mode

TABLE 3. —Continued
Ligand Ligand Class Coregulator PDB Code Comments Reference Nos.
GEN Partial agonist SRC1-1 1QKM H12 bound along AF2 in suboptimal 160, 230
orientation
1X7J Presence of LxxLL peptide forces H12 to
adopt classic agonist orientation
RAL Antagonist 1QKN Antagonist conformation 230
OHT Antagonist 2FSZ Nonnative H12 orientation stabilized by 304
crystal contacts; additional OHT
molecule bound to AF2 cleft
Triazine Antagonist INDE ERb-selective SERM; antagonist 83
conformation
THC Antagonist 1L2J Antagonist conformation related to that 268
seen in 1QKM. First demonstration of
“passive⬙/indirect antagonism
ICI 164.384 Full 1HJI Only pure antagonist structure; ICI side 231
Antagonist chain sterically blocks both agonist and
antagonist docking of H12. AF2
accessible
Coregulator peptide nomenclature is given as p160 subtype followed by NR box number. E2, 17␤-estradiol; DES, diethylstilbestrol; RAL,
raloxifene; OHT, 4-hydroxytamoxifen; THC, 5R-11R-diethyl-5,6,11,12-tetrahydrochrysene-5-ol; GEN, genistein; ER␤041, 2-(3-fluoro-4-hydroxyphenyl)-7-
vinyl-1, 3-benzoxozol-5-ol; ICI 164.384, (7␣,17␤)-n-butyl-3,17-dihydroxy-N-methyl-estra-1,3,5(10)triene-7-undecanamide. * Released PDB entries
(www.resb.org) as of November 2006.
with traditional NR corepressors (Heldring, unpublished that all structural studies have been carried out with the
data). This would explain why removal of H12 greatly isolated LBD, and we have little idea how other regions of
enhances ER’s ability to interact with traditional NR core- the receptor influence H12 dynamics. Instead, the “ago-
pressors such as NCoR and SMRT (89, 310). nist” and “antagonist” states of H12 observed in the LBD
structures most likely represent stable end points in a
continuum of possible conformations that reflect the di-
F. Classical AF-2 Antagonism verse agonist/antagonist character of ER ligands.
The AF-2 antagonists tamoxifen/raloxifene (Fig. 6C)

and pure antagonist ICI 164,384 (Fig. 6E) all harbor a G. Nonclassical AF-2 Antagonism
bulky side chain of varying length that cannot be con-
tained within the ligand-binding pocket. As a conse- AF-2 antagonism can also be achieved without a
quence, H12 is sterically hindered from aligning in the bulky substituent (268). Such indirect antagonism occurs
proper agonist conformation. While tamoxifen and ralox- when a ligand fails to make the appropriate contacts
ifene are observed to induce similar receptor conforma- within the ligand-binding cavity so that the antagonist
tions, the rER␤-ICI structure (231) is slightly different rather than the agonist orientation of H12 is preferred.
since ICI’s long alkylamido side chain itself binds along The crystal structure of hER␤ bound to genistein reveals
the AF-2 recruitment site and completely abolishes the that even though genistein binds within the ligand pocket
association between H12 and the rest of the LBD in a similar manner to estrogen, H12 adopts an antagonist
(Fig. 6E). Such differences in H12 positioning and con- position rather than the typical agonist position (230)
comitant AF-2 groove accessibility may underlie the pure (Fig. 6D). The origin of this destabilizing effect on H12 is
antagonist effects seen with ICI (189, 231). Subtle differ- unclear even though it is known that the agonist position-
ences in the positioning and stabilization of H12 along ing of H12 is more unstable in ER␤ than in ER␣ possibly
AF-2 may underlie the difference in tissue-specific actions due to altered residues at positions involved in anchoring
of certain anti-estrogens. For example, a recent structure the NH2-terminal end of H12. The degree of destabili-
of hER␣ in complex with GW5638, an anti-estrogen that is zation appears to be marginal and the agonist orienta-
mechanistically distinct from raloxifene and 4-hydroxyta- tion can be promoted by crystallizing ER␤-GEN in the
moxifen, exhibits a novel orientation for H12 along the presence of an LxxLL peptide (160). In contrast, THC,
AF-2 binding groove (323). which acts as a full agonist on ER␣ while being a potent
It is worth noting that the structural snapshots pro- antagonist on ER␤, strongly favors the antagonist H12
vided by the various ligand complexes create a rather conformation when bound to ER␤. THC appears to
false impression that the orientation of H12 is limited to a antagonize ER␤ by affecting key residues in the ligand-
few positions. This situation is exaggerated by the fact binding pocket that leads to the stabilization of tran-

920 HELDRING ET AL.
scriptionally nonproductive conformations (268). These interacting with corepressors. Both peptides bind along
findings provide key information for the rational design the AF-2 region in a manner similar to the PPAR-SMRT
of novel ER ligands with enhanced subtype selectivity complex confirming that the general structural principles
and diverse physiological effects. Yet another example of corepressor motif binding to AF-2 are conserved within
of nonclassical AF-2 antagonism may emerge from a the NR superfamily. However, an internal CoRNR-box
recent structure observation that 4-hydroxytamoxifen sequence motif within H12 serves as an effective “core-
occupies a second binding site on the ER␤ AF-2 surface pressor surrogate” and provides a considerable barrier to
(304). If this weak-affinity nonvalidated binding site is binding.
pharmacologically significant, antagonists might directly Alternatively, the AF-2 region may not serve as the
interfere with receptor-coactivator interactions. dominant protein-protein interaction site in the anti-estro-
gen-bound state, and putative ER cofactors may target
other alternative binding surfaces that are revealed in a
H. Coregulator Recognition of ligand-specific manner. Recently, we characterized one
Antagonist-Bound ER such novel recruitment site for another phage-derived,
ER␣-specific interaction motif that lies on the opposite
face of the LBD (118). Whilst the question remains as to
As described above, coactivators (and in some cases whether this motif acts as a fortuitous conformational
corepressors) are able to bind to the LBD conformation probe or is an actual mimic of an ER interaction partner,
stabilized by agonists via LxxLL motifs. However, recep- the docking site on the surface of ER appears to be a bona
tor antagonists induce a conformation in which AF-2 ex- fide control surface involved in regulating receptor
hibits little, if any, affinity for such motifs. It is well activity.
established, at least for certain members of the NR super-
family, that the AF-2 region also serves as the docking site
for NR corepressors (87, 187, 224). In the case of antag- I. ER Coregulators
onist-bound PPAR, the leucine-rich CoRNR box motif of
SMRT binds as an extended helix along the AF-2 binding Studies in the past decade have provided much novel
site (326). In the case of ER, the receptor conformation information on the general and cell-type specific actions
induced by anti-estrogens such as raloxifene and 4-hy- of distinct coregulators involved in chromatin remodel-
droxytamoxifen, in which AF-2 is completely occluded, is ing, histone modifications, transcription initiation and
difficult to reconcile with the known binding mode of the elongation, splicing, and coordinated degradation. Most
CoRNR motif. Recently, we have determined the crystal of these proteins are bona fide coregulators for many
structures of two affinity-selected peptides, specific for members of the NR family and have been studied exten-
the 4-hydroxytamoxifen-bound receptor conformation, in sively using ERs (reviewed in Refs. 146, 223, 279; see also
complex with the antagonist-bound ER␣ LBD (Heldring, http://www.nursa.org). Coregulators are recognized to be
unpublished data). The two structures demonstrate that critical for proper function of ER signaling in develop-
the AF-2 region of ER is, in principle, capable of directly ment, reproduction, and physiology, and alterations in
TABLE 4. Estrogen receptor corepressors which decrease estrogen stimulatory effects
Gene Product Mechanism of Interaction Involved ER Domain Reference Nos.
LCoR Class II ER␣-LBD 48

SHP Class II ER␣-LBD ER␤-LBD 99
RIP 140 Class II ER␣-LBD 23
DAX-1 Class II ER␣-LBD ER␤-LBD 131, 329
MTAIs Possibly class II ER␣-AF1, AF2, and DBD 124
DP97 Class III ER␣-LBD 241
REA Class III ER␣-LBD 34, 273
MTA1 Class III ER␣-LBD 123, 169, 192
BRCA1 Class III ER␣-LBD 156, 330
LIM domain only 4 Class III ER␣-DBD and LBD 277
Smad4 Class III ER␣-AF1 142
MRF-1 Class III ER␣-AF1 and LBD 64
pp32 Class III ER␣-DBD 147
CtBP Class IV Indirect 7, 300
HER2/neu Class IV Indirect 126, 210
SHARP Class IV Indirect 265
Class II, LxxLL recruitment motif; class III, lack an identified interaction motif; class IV, indirect recruitment.

coregulator function and expression are associated with opposite function, found in the same protein complexes
cancer and other diseases. (4, 143, 178, 287, 297).
The best-defined structure-function coregulator in- With regard to corepressors, a number of recent
teraction is with the steroid receptor coactivator SRC studies have demonstrated that both agonist- and antag-
(p160) family of coactivators. The important role of the onist-bound ERs are able to recruit a variety of proteins
p160 family members in ER signaling has been demon- that apparently repress activity (Tables 2 and 4). Simpli-
strated with loss of function studies where ER activation fied, these putative ER corepressors can be grouped
is heavily affected, and severe phenotypes have been into four major classes based on the reported interac-
observed in estrogen target tissues when p160s are absent tion mechanisms (Fig. 7): those that contain a classical
(63, 325). Interestingly, increased expression of AIB1 corepressor (CoRNR-box; class I) interaction motif
(SRC-3) has been observed in breast tumors, suggesting (263), those that contain an LxxLL motif and are re-
a possible involvement in carcinogenesis (2). However, cruited in an estrogen-dependent manner acting as anti-
many other proteins are also involved in ER signaling. coactivators (class II) (23, 48, 116, 329), those with less
Coactivators and corepressors have been shown to ex- defined interaction mechanisms but most likely differ-
ist in multifunctional protein complexes (81, 88, 261, ent from NR/CoRNR box type of interactions and pos-
292, 328). Even though the components of several com- sibly including an interaction-domain outside LBD
plexes are identified, less is known about the processes (class III) (34, 47, 64, 124, 142, 169, 198, 203, 241, 330),
controlling the recruitment by transcription factors. and those that have indirect effects and possibly are
One model suggests that distinct coactivator and core- recruited via complexes (class IV) (130, 210, 265, 300).
pressor complexes are present in a preformed state and However, the possibility also exists that some corepres-
are recruited to the transcription site depending on the sors are recruited through multiple independent mech-
state of the transcription factor/NR/ER (65, 81). However, anisms and therefore would qualify to belong to several
some coactivators and corepressors are, despite their classes. For example, small heterodimer partner (SHP)
FIG. 7. Schematic representation of ER corepres-

sors based on the reported interaction mechanism.
Class I: corepressors interacting through classical
CoRNR-box motifs. Class II: corepressors interacting
through NR boxes, thereby blocking coactivator bind-
ing. Class III: corepressors that most likely display
interactions different from NR and CoRNR-box type.
Class IV: corepressors with indirect effects possibly
recruited via complexes.

922 HELDRING ET AL.
has been shown, in addition to acting as an anti-coac- basis of anti-estrogen signaling which is invaluable for the
tivator (class II) in an estrogen-dependent manner, also development of more selective and effective ER ligands
to repress the agonist activity of tamoxifen (116), and and for future improvements of clinical applications.
this function is most likely achieved through a different
mechanism of recruitment. Several of the corepressors ACKNOWLEDGMENTS
shown to affect estrogen signaling may have the poten- Address for reprint requests and other correspondence:
tial to be involved in antagonist signaling as well, pro- J.-Å. Gustafsson, Dept. of Biosciences and Nutrition, Karolinska
vided that their mechanisms of repression are not due Institutet, Novum, SE-141 57 Huddinge, Sweden (e-mail:jan-
to anti-coactivator function by occupying the hydro- ake.gustafsson@mednut.ki.se).
phobic NR box site.
GRANTS
Some of the research discussed in this review was sup-
V. CONCLUDING REMARKS ported by grants from the Swedish Cancer Research Fund,
Swedish Research Council, KaroBio AB, and the European Com-
The writing of this review coincides with a heated mission funded network of Excellence CASCADE.
debate about the clinical use of estrogens and anti-estro-
DISCLOSURES
gens provoked by the results of two large epidemiological
studies, the Women’s Health Initiative (WHI) investigating J.-Å. Gustafsson is share holder and consultant of KaroBio AB.
the pros and cons of hormone replacement therapy (HRT)
and the Early Breast Cancer Trialist’s Collaborative
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Estrogen Receptors

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Estrogen Receptors

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Physiol Rev 87: 905–931, 2007;

Estrogen Receptors: How Do They Signal

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I. INTRODUCTION raised the question of whether survival of the ER␣ knock-

FIG. 1. Molecular structures of the endogenous estro-

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apy for treating ER␣-positive breast cancers, these drugs

II. ESTROGEN RECEPTORS

A. Two ER Subtypes and Several Isoforms

Cellular signaling of estrogens is mediated through

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B. Diverse Structures of Synthetic, Dietary, and

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characteristics of the adult prostates of neonatally es- 3. Lung morphogenesis

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TABLE 1. Coactivators reported to be involved in tamoxifen signaling

Gene Product Mechanism of Activation Involved ER Domain Reference Nos.

AIB1 Recruit HATs, NR boxes ER␣-AF-1 and LBD 2, 210, 311

ER, estrogen receptor; LBD, ligand-binding domain; AF, activation function.

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Ligand Ligand Class Coregulator PDB Code* Comments Reference Nos.

ER␣ E2 Agonist IERE First structure of steroid receptor with 22

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Ligand Ligand Class Coregulator PDB Code Comments Reference Nos.

The AF-2 antagonists tamoxifen/raloxifene (Fig. 6C)

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TABLE 4. Estrogen receptor corepressors which decrease estrogen stimulatory effects

Gene Product Mechanism of Interaction Involved ER Domain Reference Nos.

LCoR Class II ER␣-LBD 48

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FIG. 7. Schematic representation of ER corepres-

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Physiol Rev • VOL 87 • JULY 2007 • www.prv.org