Estrogen receptor α AF-2 mutation results in
antagonist reversal and reveals tissue selective
function of estrogen receptor modulators
Yukitomo Araoa, Katherine J. Hamiltona, Manas K. Rayb, Gregory Scottb, Yuji Mishinac, and Kenneth S. Koracha,1
aReceptor Biology Section, Laboratory of Reproductive and Developmental Toxicology andbKnock Out Core, National Institute of Environmental Health
Sciences/National Institutes of Health, Research Triangle Park, NC 27709; andcSchool of Dentistry, University of Michigan, Ann Arbor, MI 48109
Edited by David J. Mangelsdorf, University of Texas Southwestern Medical Center, Dallas, TX, and approved July 22, 2011 (received for review June 10, 2011)
The estrogen receptor (ER) is a ligand-dependent transcription
factor containing two transcriptional activation domains. AF-1 is in
the N terminus of the receptor protein and AF-2 activity is
dependent on helix 12 of the C-terminal ligand-binding domain.
Two point mutations of leucines 543 and 544 to alanines (L543A,
L544A) in helix 12 minimized estrogen-dependent transcriptional
activation and reversed the activity of the estrogen antagonists
ICI182780 (ICI) and tamoxifen (TAM) into agonists in a similar
manner that TAM activated WT ERα through AF-1 activation. To
evaluate the physiological role of AF-1 and AF-2 for the tissue-
selective function of TAM, we generated an AF-2–mutated ERα
knock-in (AF2ERKI) mouse model. AF2ERKI homozygote female
mice have hypoplastic uterine tissue and rudimentary mammary
glands similar to ERα-KO mice. Female mice were infertile as a re-
sult of anovulation from hemorrhagic cystic ovaries and elevated
serum LH and E2 levels, although the mutant ERα protein is
expressed in the AF2ERKI model. The AF2ERKI phenotype suggests
that AF-1 is not activated independently, even with high serum E2
levels. ICI and TAM induced uterotropic and ER-mediated gene
responses in ovariectomized AF2ERKI female mice in the same
manner as in TAM- and E2-treated WT mice. In contrast, ICI and
TAM did not act as agonists to regulate negative feedback of
serum LH or stimulate pituitary prolactin gene expression in a dif-
ferent manner than TAM- or E2-treated WT mice. The functionality
of the mutant ERα in the pituitary appears to be different from
that in the uterus, indicating that ERα uses AF-1 differently in the
uterus and the pituitary for TAM action.
tors (ERs) α and β, which are ligand-dependent transcription
factors. Transcriptional activation is mediated by AF-1 in the N-
terminal domain and AF-2 in the C-terminal ligand-binding
domain (LBD). ER ligands bind to the LBD and induce a con-
formational change of this LBD domain to modulate transcrip-
tional activation. A portion of the AF-2 domain resides in helix
12 and plays a crucial role in determining interactions with
coactivators and corepressors for transcriptional regulation in-
fluencing respective agonist or antagonist effects of the ligand
(1, 2). Helix 12 has conserved hydrophobic amino acids between
species. The mouse ERα residues L543 and L544 are correlated
to the L539 and L540 residues of human ERα helix 12. The
mutation of these residues in mouse and human ERα has been
reported to have similar properties (3–9). Despite these amino
acid mutations, binding to estrogen-responsive DNA sequences
and estradiol (E2) is unaffected (3, 6); however, transcription
activity is markedly lower in the presence of E2 compared with
WT ERα because of the failure to recruit the p160 transcrip-
tional coactivators (4). The mutation of these residues has been
shown to convert the antiestrogens, including ICI164384, RU54876,
and tamoxifen (TAM), into agonists (5, 9). The AF-1 region is
required for a transcriptionally active configuration of this mu-
tant with antagonists (5, 9). TAM is a well known selective ER
modulator (SERM) that is a partial antagonist/weak agonist for
ERα WT (10, 11). Several reports proposed that the N-terminal
AF-1 of ERα WT is required for TAM-mediated partial activity
strogen regulates gene transcription via the estrogen recep-
and that may be related to the cell type specific functionality
of TAM (12). However, it is still not entirely clear how TAM
manifests agonist activities through ERα WT in different tissues.
We focused on the L543A and L544A mutations in the ERα
AF-2 domain (AF2ER) to evaluate the ERα AF-1 and AF-2
functions in vivo and the SERM functionality in the tissues.
The ERα-KO (αERKO) mouse is an established model for
evaluating ERα function in vivo. The αERKO does not express
functional ERα protein as a result of a genetic modification of
Esr1 (13, 14). The αERKO mouse model has revealed various
physiological functions involving ERα (15). However, this model
cannot discern the selective functionality of the ERα AF-1 or AF-
2 in ERα-mediated physiological responses in vivo because no
receptor protein is expressed. The aim of this study was to eval-
uate the physiological function of the ERα AF-1 and AF-2 in vivo.
We developed a knock-in mouse model with the AF2ER muta-
tions (AF2ERKI). In the AF2ERKI mice, we can definitively
determine that the mutation of helix 12 inactivates the ERα-me-
diated response to endogenous estrogens, making the AF2ERKI
mice comparable to the αERKO mice. Our present studies con-
firmed that estrogen-induced AF-2 activation is critical for regu-
lating female reproductive tissue hormone responses and AF-1 is
not activated independently without AF-2. In addition, this report
shows that the antiestrogens ICI182780 (ICI) or TAM can be
shown to act as agonists in vivo involving AF2ER mutations. Our
in vitro studies indicated the AF-1 activity of ERα is involved
in ICI-mediated AF2ER activation and that activation is similar
to the condition of TAM-mediated ERα WT transactivation.
Therefore, the in vivo observation of ICI-mediated regulation
suggests the ERα AF-1 mediates physiological functions in certain
tissues and may represent tissue specific SERM functions.
Properties of the L543A, L544A Mutant ERα. We demonstrated the
differential functionality of the AF2ER (L543A, L544A mutated
ERα) on the ERα-mediated transcription regulation with in vitro
lines (HepG2 and HeLa cells; Fig. 1 and Fig. S1). First, we
assessed estrogen response element (ERE)-mediated transcrip-
tion activity. As expected, E2 produced strong activation of the
inactive with WT ERα but activated the ERE-mediated tran-
scription of AF2ER in both cell lines. ICI-mediated AF2ER ac-
tivation was more potent than E2 activity. This activation was not
observed in the N-terminal truncated AF2ER (121-AF2ER)-
transfected HepG2 cells, but very weak activity was observed in
Author contributions: Y.A. and K.S.K. designed research; Y.A., K.J.H., M.K.R., and G.S.
performed research; Y.M. contributed new reagents/analytic tools; Y.A., K.J.H., and K.S.K.
analyzed data; and Y.A. and K.S.K. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
1To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
| September 6, 2011
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HeLa cells (Fig. 1B), indicating that the AF2ER mutant has
minimized the AF-2 function and AF-1 is necessary for ICI-me-
diated AF2ER activation. Furthermore, to confirm the level of
ERα AF-1 activity in these cells, C-terminal truncated ERα
(ERa339), which activates ERE-mediated transcription irre-
spective of the presence or absence of ligand, was cotransfected
with the ERE reporter. Coincidentally, the level of ERa339-
mediated activation in these cells is similar to the level of ICI-
mediated AF2ER activity (Fig. 1B), which suggests that the
ICI-dependent AF2ER activity is derived from AF-1. Second, to
assess AF2ER-mediated DNA-tethered transactivation, we tested
AF2ER activity with an AP-1 reporter. ICI activated the AP-1–
mediated transcription through WT ERα as previously reported
(16). However, AF2ER did not activate the AP-1–mediated
transcription with any ligands [E2, ICI, and 4-hydroxy-TAM
(OHT); Fig. 1C]. Furthermore, we assessed the transrepres-
sion activity of AF2ER in NF-κB–dependent gene activation.
AF2ER did not repress the NF-κB–mediated transcription (Fig.
1D). These results suggest that AF2ER is likely to predominantly
regulate the ERE-mediated transcription. OHT, a known ERα
activated AF2ER, but to a lesser extent than ICI (Fig. 1B).
if coregulators for WT ERα were involved in the ICI agonist
activity of the AF2ER, expression vectors for coregulators were
cotransfected, and p300, CREB binding protein (CBP), and
SRC1a were found to increase the ICI-mediated AF2ER activity
in HeLa and HepG2 cells (Fig. 1E and Fig. S1A, Top). We also
determined the effect of coregulators on the OHT agonistic ac-
tivity of WT ERα. Surprisingly, the profile of coregulators’ effect
on OHT-bound WT ERα shows an almost identical pattern to
ICI-liganded AF2ER (Fig. S1A, Middle, and Fig. S1B, Top). In
this condition, however, the profile of coregulators effect on E2-
bound WT ERα is different from OHT-bound WT ERα (Fig.
S1A, Bottom, and Fig. S1B, Bottom). The p300/CBP coactivators
also increased ERa339-mediated ERE activation regardless of
the presence or absence of ICI, but did not enhance 121-AF2ER–
AF2ER activation but did not alter ERa339 transactivation (Fig.
S1 C and D). Furthermore, we found an additive effect of p300/
CBP together with SRC1a on the ICI-dependent AF2ER acti-
vation that was observed in ERa339- but not 121-AF2ER–medi-
ated activation. A similar activation profile was observed for
OHT-mediated WT ERα activation (Fig. S1 E and F). These
results suggest that ICI liganded AF2ER is likely to mimic the
OHT-mediated WT ERα activation.
Generation of AF2ERKI Mice. To assess the effect of loss of AF-2
function in vivo, we generated AF2ERKI mice through homolo-
gous recombination in mouse ES cells. The construct targeted the
ninth exon of mouse Esr1, which contains helix 12, possessing the
AF-2 region of the LBD as well as the stop codon. Leucines 543
and 544 were mutated to alanines, and a 6xHis-tag was added to
the C-terminal end of the mouse ERα protein (Fig. 2A, SI Mate-
rials and Methods, and Fig. S2). The expression of AF2ER mutant
protein in the AF2ERKI mice was evaluated by Western blot
analysis of uterine tissues by using the anti-mouse ERα antibody
and anti–His-tag antibody. The uterine tissue was collected from
AF2ERKI homozygote (AF2ERKI/KI), heterozygote (AF2ERKI/
+), and WT female mice. As shown in Fig. 2B, we detected a 66-
kDa signal using the anti ERα antibody. After stripping, the
membrane was probed with the anti–His-tag antibody. A 66-kDa
signal was detected in the AF2ERKI/KIbut not in WT, and a faint
signal was detected in the AF2ERKI/+. Furthermore, we per-
formed immunohistochemistry using the AF2ERKI/KIand WT
female uteri. As shown in Fig. 2C, anti-ERα antibody–derived
signal was detected in all the tissue compartments of the uterus,
includingtheendometrial epithelial cellsandstromalcellsof both
genotypes. Taken together, these results indicate that the mutant
AF2ER protein is being expressed in the mice.
Phenotype of AF2ERKI Female Is Similar to That of αERKO Female.
Continuous breeding studies indicated that AF2ERKI/KIfemale
mice were infertile. We evaluated the estrous cycle of WT,
AF2ERKI/+, and AF2ERKI/KIfemale mice by daily vaginal lavage
for nine consecutive days. WT and AF2ERKI/+female mice
showed a normal estrous cycle. On the contrary, the AF2ERKI/KI
female mice showed no estrous cycle (Fig. S3). Another endpoint
linked to reproductive cyclicity and responsiveness is serum lu-
teinizing hormone (LH) levels. The serum LH level of AF2ERKI/KI
(5.39 ± 2.33 ng/mL) was elevated fourfold vs. WT (1.29 ± 2.12 ng/
mL), and the serum E2 level of AF2ERKI/KI(96.8 ± 10.1 pg/mL)
L544A mutated ERα). (A) Schematic illustration of
the ERα mutants. (B) HepG2 or HeLa cells were
transfected with the reporter gene (3xERE-TATA-
luc) and expression vectors for WT or mutated
receptors were maintained with or without ligands.
(C) HepG2 or HeLa cells were transfected with the
reporter gene (7xAP1-TATA-luc) and expression
vectors for WT or AF2ER. The cells were maintained
with or without ligands. The luciferase activities for
the each treatment were represented as fold
change for the empty expression vector, pcDNA3
(no ERα). (D) Transrepression function of WT ERα
and AF2ER. HeLa cells were transfected with the NF-
κB reporter gene (3xMHC-luc), and expression vec-
tors for p65/RelA and WT ERα or AF2ER were
maintained with or without ligands. The luciferase
activities were expressed relative to p65 activity in
the absence of ligand (100%). (E) The effect of
cofactors on ICI- or OHT-dependent AF2ER activa-
tion. HeLa cells were transfected with the 3xERE
reporter gene and expression vectors for cofactors
and AF2ER. The cells were maintained with or
without ligands (100 nM E2, ICI, or OHT were used
for treatments). Luciferase activity is represented as
the mean ± SD of three independent experiments.
Transcription function of AF2ER (L543A,
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was twofold higher than WT (44.2 ± 9.43 pg/mL). These hormone
levels were similar to those from a pool of αERKO serum from
several female mice (LH, 5.78 ng/mL; E2, 87.6 pg/mL). Such el-
evated LH levels suggested that the negative feedback of the
hypothalamic–pituitary–gonadal axis in AF2ERKI/KIis disrupted,
as has been reported in the αERKO animals (17), consistent with
the infertility. Female reproductive tract organs from WT and
AF2ERKI/KImice were analyzed for morphological and histo-
logical parameters. AF2ERKI/KIuteri were significantly smaller
and more hypoplastic than those of their WT littermates (Fig.
2D). AF2ERKI/KIuterine tissues possess luminal epithelium, but
fewer glandular structures were evident compared with WT uteri
(Fig. 2E, Top). We evaluated the expression level of uterine
Foxa2, a gene that is implicated in uterine gland development
(18). The level of uterine Foxa2 mRNA was significantly lower in
AF2ERKI/KIthan WT (Fig. 2F), which is consistent with less
glandular morphology. The ovaries from the AF2ERKI/KIfemale
mice show cystic and hemorrhagic follicles, reminiscent of the
αERKO ovarian phenotype. A few primary follicles can be seen,
but no corpora lutea were observed in AF2ERKI/KIovaries (Fig.
2E, Middle). Mammary gland whole-mounts were analyzed for
evidence of ductal proliferation and differentiation. In 8-wk-old
WT mice, ductal trees extended past the lymph node and had
enlarged terminal end buds. AF2ERKI/KImammary glands never
developed beyond a rudimentary epithelial ductal tree (Fig. 2E,
Bottom), similar to αERKO females. Taken together, these re-
sults clearly suggest that estrogen-dependent AF-2–mediated
female reproductive functions.
Antiestrogens ICI and TAM Induce Uterotropic Responses in AF2ERKI
Homozygote. Based on our in vitro studies, to assess the func-
tionality of AF2ER in vivo, ovariectomized (OVX) AF2ERKI/KI
female mice were injected in a 3-d bioassay with vehicle, ICI,
TAM, or E2, and uteri were collected 24 h after the last treat-
ment. E2 and TAM significantly induced the uterine growth of
WT mice, and ICI treatment was ineffective. In contrast, ICI and
TAM treatment increased the uterine wet weight of AF2ERKI/KI
mice (Fig. 3 A and B and Fig. S4). We detected the incorporation
of 5-ethynyl-29-deoxyuridine (EdU) into newly synthesized DNA
in the ICI- and TAM-treated AF2ERKI/KIuterine endometrial
epithelial cells (Fig. 3C), suggesting that ICI or TAM induces the
proliferation of AF2ERKI/KIendometrial epithelial cells. Fur-
thermore, we evaluated the regulation of some uterine estrogen-
responsive genes—lactotransferrin (Ltf) (19), insulin-like growth
factor 1 (Igf1) (20), and cytochrome c oxidase subunit VIIa
polypeptide 2-like (Cox7a2l) (21)—by quantitative PCR. As
shown in Fig. 3D, the expression of these genes was regulated by
E2 and TAM in the WT uterus and not by ICI treatment. In
contrast, the expression of those genes was regulated by ICI and
TAM in the AF2ERKI/KIuterus but not by E2. These results
indicate that the functionality of the AF2ER mutant in the
in vivo AF2ERKI mouse model is similar to the in vitro findings.
We also assessed the AF2ER function in the receptive uterus
model. In the receptive uterus, estrogen induces stromal cell but
not epithelial cell proliferation (22). We detected that ICI in-
creased proliferation of stromal cells but not epithelial cells in
the AF2ERKI/KIuterus, as did E2 in WT (Fig. S5).
One of the major antagonistic effects of ICI has been shown to
result from loss of ERα protein in vivo and in vitro, which
involves a proteasome-mediated proteolysis of ERα (23, 24). As
ICI was an agonist in AF2ERKI/KI, we examined the effect of ICI
on the level of AF2ER protein by Western blot. The level of
AF2ER protein was not reduced, whereas the level of WT ERα
was markedly reduced, after ICI treatment (Fig. 3E). It appears
that, in contrast to WT, the pure antiestrogen does not accel-
erate the turnover and loss of the AF2ER protein.
ICI and TAM Do Not Regulate Pituitary Gene Expression in AF2ERKI
Homozygote. It is known that ovariectomy releases negative
feedback, resulting in increased serum LH level. Additionally, E2
replacement down-regulates serum LH level. As expected, serum
LH levels were high in the vehicle-treated OVX WT female mice
and were decreased by E2 treatment to 25% of the vehicle level.
mutation. Diagrams show the WT ERα locus, targeting construct, targeted mutant allele in the ES cells/chimera mice, and F1 mutant allele after ACN cassette
self-excision. The targeting construct contained ERα exon 9 (light gray boxes show coding sequence and dark gray box shows 39 UTR), the L543A, L544A
mutations (“mutation”), an extra XbaI site (Xb), and a 6xHis-tag epitope (6xHis). The ACN cassette was flanked at the 59 and 39 ends by loxP sites (closed
arrowheads). Open box suggests the position of 59 external probe for Southern blot (59Ex), and pairs of open arrowheads suggest PCR primer sets for 39
external PCR (39Ex PCR), His-tag PCR, and PCR genotyping. D, DrdI; Xm, XmnI; B, BamHI; Xh, XhoI; H, HindIII. (B) Representative results of Western blot probed
for the ERα, His-tagged ERα (His-Tag), and β-tubulin in the 8-wk-old individual mouse uterus are shown. β-Tubulin was used as a loading control.+/+, WT; KI/+,
heterozygote; KI/KI, homozygote. (C) Uterine ERα immunohistochemistry of 8-wk-old representative mice. (D) Morphology of AF2ERKI female reproductive
organs in the 8-wk-old representative mice. (E) Histology of 8-wk-old representative AF2ERKI female mice. Uterine (Top) and ovarian (Middle) tissue H&E
staining from WT (Left) and AF2ERKI homozygote (Right) mice. (Scale bar: 100 μm.) Mammary gland (Bottom) whole-mount Carmine alum staining from
8-wk-old representative mice. (Scale bar: 1 cm.) (F) The mRNA level of Foxa2 was quantified by real-time PCR. The mRNA levels were represented as fold
change for the WT. Values are presented as mean ± SD.
Targeting strategy and confirmation of L543A, L544A ERα knock-in mutation. (A) Schematic illustration of the targeting strategy used to introduce
| www.pnas.org/cgi/doi/10.1073/pnas.1109180108Arao et al.
This level was similar to that seen in intact (i.e., non-OVX) WT
also down-regulated serum LH level, similar to E2 treatment. In
contrast, the serum LH level of AF2ERKI/KIwas not regulated by
ICI, TAM, or E2 treatment (Fig. 4A). To correlate serum LH
levels to pituitary gene regulation, we analyzed the expression of
the LH β-subunit gene (Lhb). The level of Lhb mRNA in the
OVX WT pituitary was reduced by E2 or TAM treatment.
However, the expression of Lhb was not changed by ICI, TAM, or
E2 in the AF2ERKI/KIpituitary (Fig. 4B). The basal level of Lhb
expression in the AF2ERKI/KIpituitary was 1.5 times higher than
also analyzed the regulation of the prolactin gene (Prl), a well
known estrogen-responsive gene in the pituitary (26) (Fig. 4C).
The basal level of Prl expression in the AF2ERKI/KIpituitary was
20% lower than in WT and was similar to that in the αERKO
pituitary (17, 25). The expression of the Prl gene was increased by
E2 but not TAM in the WT pituitary, as opposed to Lhb gene
regulation. In the AF2ERKI/KIpituitary, the Prl gene expression
was not regulated by ICI, TAM, or E2. Because Pit1 is a key
regulator for Prl expression, we measured the Pit1 mRNA level
and found no differences between genotypes (Fig. 4D). Lack of
pituitary responsiveness of the AF2ERKI to ICI or TAM raised
the question whether AF2ER protein was expressed in the
AF2ERKI/KIpituitary. AF2ER protein expression was confirmed
by Western blot in the AF2ERKI/KImouse pituitary. Although the
level of AF2ER protein in the AF2ERKI/KIpituitary appeared
lower than in WT, the ratio of uterine and pituitary receptor
protein levels is similar between WT and AF2ERKI/KImice.
Furthermore, the molecular sizes of the uterine and pituitary
AF-1 in the pituitary compared with the uterus.
In the present study, we report the initial generation and char-
acterization of AF2ERKI mice, which have L543A and L544A
mutations in the AF-2 region of helix 12 of the LBD. First, we
demonstrated the properties of this mutation by using HepG2
and HeLa cells (Fig. 1 and Fig. S1). We suggested here that the
ICI activity of the AF2ER mutant is mediated through the N-
terminal AF-1 predominantly, as the AF-1–truncated AF2ER
mutant (i.e., 121-AF2ER) reduces the ICI-mediated activation,
and the level of ICI-mediated AF2ER activity is coincident with
the level of AF-2–truncated mutant (i.e., ERa339)–mediated
activation in each cell type. However, we were not able to ex-
clude the possibility of AF-2 contribution from ICI-mediated
AF2ER activation, because 121-AF2ER in the HeLa cells was
weakly activated by ICI. Furthermore, we found that the p300/
CBP and SRC1a cooperatively activate the ICI-dependent
AF2ER activation in the HeLa cells, and it was observed in AF-
1- but not AF-2–mediated activation. Interestingly, p300/CBP
E2 2 mg/kg treatments for three consecutive days. Values are presented as mean ± SEM. (B) Uterine histology after vehicle, ICI, TAM, or E2 (10 μg/kg)
treatments for three consecutive days in representative mice. (C) ICI and TAM induce the proliferation of endometrial epithelial cells in AF2ERKI uterus.
Uterine EdU incorporation was analyzed. Hoechst was used as a counterstain to visualize tissue. (D) The mRNA levels of Ltf, Igf1, and Cox7a2l genes were
quantified by real-time PCR. The mRNA levels were represented as fold change vs. vehicle treatment of WT. Values are shown as mean ± SD; *P < 0.05 vs.
vehicle in each genotype. (E) Representative results of Western blots probed for ERα and β-tubulin from the vehicle- and ICI-treated individual mouse uteri are
shown. The ERα level was normalized to the level of β-tubulin in each sample and presented as fold change vs. vehicle in each group.
Assessment of AF2ER function in the AF2ERKI mouse uterus. (A) Uterine wet weight after vehicle (Veh), ICI (2 mg/kg), TAM (2 mg/kg), E2 10 μg/kg, or
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increased AF-1 activity but not AF-2 activity. On the contrary,
SRC1a increased ICI-dependent AF-2 activity modestly, but not
that of AF-1. These results may suggest that the ICI-liganded
AF2ER induces modest activation of AF-2 and may involve
enhancing the AF-1 activity in such cells or tissues. It may ex-
plain the differential tissue response of AF2ER with ICI. We
also tested the OHT-mediated WT ERα activation to compare
ICI-mediated AF2ER activation because the OHT is known as
a WT ERα AF-1–activating SERM (27). In our experimental
condition, the activation functions of coregulators for E2 or
OHT bound WT ERα were different. This result may suggest
that OHT-bound WT ERα makes a different coactivator in-
terface for E2-bound WT ERα (28). Interestingly, the profile of
coactivators’ effect on ICI-mediated AF2ER activation is almost
identical to that of OHT-mediated WT ERα activation. These
results suggest that the ICI-liganded AF2ER is mimicking the
OHT-bound WT receptor. To analyze the involvement of co-
regulators in the ICI-dependent AF2ER activation of various
tissues, it will be helpful to understand AF-1–dependent tissue-
selective functionality of SERMs.
We confirmed that the AF2ERKI mouse expresses the
AF2ER mutant ERα protein in the uterus (Fig. 2) and pituitary
(Fig. 4) by identifying the His-tag incorporated into the knock-in
allele, using an anti–His-tag antibody. Even though the in vitro
analysis indicated weak activation of AF2ER by E2, the pheno-
type described herein of the AF2ERKI female is similar to that
of the αERKO female (15), indicating that the mutation of AF-2
is unable to mediate physiological responses to E2. We also
confirmed that treatment with a pharmacological level of E2 (2
mg/kg) does not induce the uterotropic growth in the AF2ERKI/KI
female (Fig. 3 and Fig. S4). These results clearly suggested that
the AF-1 function is not activated by E2 independently. Our
preliminary results suggest that growth factors (IGF-1 or EGF)
are unable to activate uterine proliferation, specifically DNA
synthesis, as reflected by lack of incorporation of the deoxyur-
idine analogue EdU in the AF2ERKI/KIfemale (Fig. S6A). This
result is different from those in a similar mouse model in which
the ERα AF-2 is mutated: the ENERKI mouse. The ERα in the
ENERKI mouse is mutated in the ligand-binding pocket
(G525L), is unable to bind E2, and exhibits reduced E2-de-
pendent transcription activation while maintaining helix 12
structure (29). In the ENERKI homozygous female, IGF-1 in-
duced the proliferation of uterine endometrial epithelial cells
without E2, as reflected by an increase in the general cell cycle
marker Ki67. However, progression to S-phase as reflected by
DNA synthesis was not evaluated in the ENERKI mouse. These
differences between AF2ERKI and ENERKI may suggest that
helix 12 also contributes toward regulating the growth factor-
mediated ERα activation.
It has been suggested from in vitro cell studies that the dif-
ferential functionality of AF-1 in different cell types and on
different gene promoters is an explanation for the mechanism of
SERM action (12, 30). We demonstrated here that TAM regu-
lated uterine responses (Fig. 3) and serum LH level but not pi-
tuitary Prl gene expression in the OVX WT female (Fig. 4). On
the contrary, ICI or TAM regulated uterine responses in the
OVX AF2ERKI female, like the response seen in TAM-treated
WT female mice (Fig. 3); however, it had no response in the
pituitary (Fig. 4). Differential tissue actions of ERα have been
proposed as being dependent on the ERα isoforms ERα66 and
ERα46 (31). ERα66 is the full-length ERα (harboring AF-1 and
AF-2) and ERα46 is the shorter AF-1–deficient isoform. In this
report, we confirmed that the full-length ERα is expressed in the
uterus and pituitary by using the anti-ERα antibody and anti–
His-tag antibody (Fig. 4). This result clearly suggests that the
differences in the TAM-mediated transcriptional activity of the
ERα in the uterus and pituitary are results of the differential
AF-1 functionality, not differential isoform action.
The TAM responses in WT female pituitary suggest that TAM
affects the pituitary functions differently than E2. A study that
used the ERα−/AA(NERKI) mouse, which lacks ERE-dependent
transactivation but retains ERE-independent activity (32), iden-
tified that the negative feedback action of estrogen on LH is
mediated by ERE-independent mechanisms and that Prl gene
regulation is mediated by ERE-dependent mechanisms (33).
Taken together, these findings suggested that TAM affects ERE-
independent responses (serum LH regulation) but not ERE-
dependent actions (Prl gene regulation) in the pituitary. This
result implies that the ERα AF-1 is not functioning to regulate
Prl gene expression. Neither ICI nor TAM were able to regulate
any responses in AF2ERKI female pituitary (Fig. 4). Our in vitro
results indicated that the AF2ER mutant regulates ERE-mediated
but not non–ERE-mediated transcription (Fig. 1). These findings
suggest that our results are consistent with previous reports and
the AF2ERKI mouse model is able to distinguish the differential
TAM functionality in tissues in vivo.
Recently, Börjesso et al. reported generation of AF-1 truncated
mouse (ERαAF-10) and helix 12 (AF-2) truncated mouse (ERαAF-
2 (34). The report used a loss-of-function approach of truncated
the AF2ERKI mouse pituitary. (A) Serum
LH levels were determined after vehicle
(Veh), ICI (2 mg/kg), TAM (2 mg/kg), or E2
(10 μg/kg) treatments for three consecu-
tive days. Values are presented as mean ±
SEM; *P < 0.05 vs. vehicle in each geno-
type. The pooled serum from the non-
OVX WT and αERKO female mice was
used as reference. (B) The mRNA level of
Lhb was quantified by real-time PCR. (C)
The mRNA level of Prl was quantified by
real-time PCR. The mRNA levels are pre-
sented as fold change for vehicle of WT.
Values are presented as mean ± SD; *P <
0.05 vs. vehicle in each genotype. (D) The
mRNA level of Pit1 was quantified by real-
time PCR. The mRNA levels are presented
as fold change vs. WT. Values are pre-
sented as mean ± SD. (E) Representative
results of Western blots probed for the
ERα, His-tagged ERα, and β-tubulin from
WT and AF2ER homozygote individual
mouse uteri and pituitaries are shown.
Functional difference of AF2ER in
| www.pnas.org/cgi/doi/10.1073/pnas.1109180108Arao et al.
mutants for comparison with WT ERα and observed varied E2
responses in different tissues of the ERαAF-10, but no responses to
E2 in the ERαAF-20tissues, similar to αERKO mice lacking the
receptor protein (34), indicating that ERα AF-1 function is tissue
dependent and AF-2 is required for the estrogenic responses. Our
AF2ERKI phenotypes and differential stimulated tissue activities
with ICI or TAM would be consistent with those observations and
allow assignment of a differential activity of female neuroendocrine
raloxifene, a new-generation SERM, does not activate AF2ER
function in vitro (Fig. S6B) and in vivo uterine response (Fig. S6C).
Our results so far suggest that AF2ERKI mice can experimentally
demonstrate tissue selective function and response of ERα AF-1 in
estrogen target tissues for evaluating tissue-selective SERM func-
tions and identifying the beneficial actions of SERMs as potential
hormonal therapeutic agents.
Materials and Methods
Cell Culture and Luciferase Assay. Cells were cultured in phenol red-free
medium supplemented with 10% charcoal-stripped FBS for transient trans-
fection. The cells were transfected for 6 h, then changed to fresh media
supplemented with 100 nMof E2, ICI, or OHT. Luciferase assay was performed
18 h after treatments. Details are described in SI Materials and Methods.
Generation of Knock-In Animals. AtargetingvectorcontainingmouseERαexon
9 with an additive XbaI site in exon 9, an18-bp 6xHis-tag epitope sequence,
the L543A and L544A mutations, and the ACN cassette was used for the se-
lection of ES cells. The targeted ES clones were injected into C57BL/6 blasto-
cysts to generate chimeric mice. Male chimeras were bred to C57BL/6 female
mice to establish germline transmission, and the resulting heterozygous mice
were interbred. Further details are described in SI Materials and Methods.
ER Ligand Treatments. OVX WT or AF2ERKI female mice (n = 6 per group)
were injected s.c. daily for 3 d with 10 μg/kg or 2 mg/kg E2 dissolved in
sesame oil, intraperitoneally with 2 mg/kg ICI, or 2 mg/kg TAM dissolved in
DMSO. DMSO (50 μL) was used as vehicle in all experiments. The tissues and
serum were collected 24 h after the last injection. All procedures involving
animals were conducted in accordance with National Institutes of Health
guidelines and were in compliance with a National Institute of Environ-
mental Health Sciences-approved animal protocol.
Quantitative PCR. For quantitative PCR, tissues were homogenized and total
RNA was extracted. The RNA was reverse-transcribed, and then real-time PCR
was performed. The primer sets are described in Table S1. Samples were
analyzed in triplicate, and the 18s rRNA was used as an internal control for
Statistical Analysis. Statistical analysis was performed with two-way ANOVA
with GraphPad Prism software (GraphPad), and a P value lower than 0.05
was considered statistically significant. Other methods are described in SI
Materials and Methods.
ACKNOWLEDGMENTS. We thank Dr. GeoffreyGreene for the gift of the ACN
cassette plasmid, Sandra Burkett (Mouse Cancer Genetics Program/National
Cancer Institute) for conducting the FISH analysis, and David Monroy and
members of the National Institute of Environmental Health Sciences
Comparative Medicine Branch staff for animal care. We also thank Drs.
Karina Rodriguez, Joy Winuthayanon, and April Binder for the hormone
analysis; Sylvia Hewitt for critical reading of the manuscript; and other
members of the Receptor Biology Group for helpful discussions. We also
value the early contributions of Trisha Castranio and Dr. Deborah Swope to
this project. This study was funded by Z01ES70065 from the Division of
Intramural Research of the National Institute of Environmental Health
1. Brzozowski AM, et al. (1997) Molecular basis of agonism and antagonism in the
oestrogen receptor. Nature 389:753e758.
2. Wu YL, et al. (2005) Structural basis for an unexpected mode of SERM-mediated ER
antagonism. Mol Cell 18:413e424.
3. Danielian PS, White R, Lees JA, Parker MG (1992) Identification of a conserved region
required for hormone dependent transcriptional activation by steroid hormone re-
ceptors. EMBO J 11:1025e1033.
4. Valentine JE, Kalkhoven E, White R, Hoare S, Parker MG (2000) Mutations in the es-
trogen receptor ligand binding domain discriminate between hormone-dependent
transactivation and transrepression. J Biol Chem 275:25322e25329.
5. Mahfoudi A, Roulet E, Dauvois S, Parker MG, Wahli W (1995) Specific mutations in the
estrogen receptor change the properties of antiestrogens to full agonists. Proc Natl
Acad Sci USA 92:4206e4210.
6. Ince BA, Zhuang Y, Wrenn CK, Shapiro DJ, Katzenellenbogen BS (1993) Powerful domi-
nant negative mutants of the human estrogen receptor. J Biol Chem 268:14026e14032.
7. Ince BA, Schodin DJ, Shapiro DJ, Katzenellenbogen BS (1995) Repression of endoge-
nous estrogen receptor activity in MCF-7 human breast cancer cells by dominant
negative estrogen receptors. Endocrinology 136:3194e3199.
8. Schodin DJ, Zhuang Y, Shapiro DJ, Katzenellenbogen BS (1995) Analysis of mechanisms
that determine dominant negative estrogen receptor effectiveness. J Biol Chem 270:
9. Montano MM, Ekena K, Krueger KD, Keller AL, Katzenellenbogen BS (1996) Human
estrogen receptor ligand activity inversion mutants: receptors that interpret anti-
estrogens as estrogens and estrogens as antiestrogens and discriminate among dif-
ferent antiestrogens. Mol Endocrinol 10:230e242.
10. McDonnell DP (1999) The Molecular Pharmacology of SERMs. Trends Endocrinol
11. Smith CL, Nawaz Z, O’Malley BW (1997) Coactivator and corepressor regulation of
the agonist/antagonist activity of the mixed antiestrogen, 4-hydroxytamoxifen. Mol
12. Berry M, Metzger D, Chambon P (1990) Role of the two activating domains of the
oestrogen receptor in the cell-type and promoter-context dependent agonistic ac-
tivity of the anti-oestrogen 4-hydroxytamoxifen. EMBO J 9:2811e2818.
13. Lubahn DB, et al. (1993) Alteration of reproductive function but not prenatal sexual
development after insertional disruption of the mouse estrogen receptor gene. Proc
Natl Acad Sci USA 90:11162e11166.
14. Hewitt SC, et al. (2010) Biological and biochemical consequences of global deletion of
exon 3 from the ER alpha gene. FASEB J 24:4660e4667.
15. Couse JF, Korach KS (1999) Estrogen receptor null mice: what have we learned and
where will they lead us? Endocr Rev 20:358e417.
16. Jakacka M, et al. (2001) Estrogen receptor binding to DNA is not required for its
activity through the nonclassical AP1 pathway. J Biol Chem 276:13615e13621.
17. Couse JF, Yates MM, Walker VR, Korach KS (2003) Characterization of the hypotha-
lamic-pituitary-gonadal axis in estrogen receptor (ER) Null mice reveals hyper-
gonadism and endocrine sex reversal in females lacking ERalpha but not ERbeta.
Mol Endocrinol 17:1039e1053.
18. Jeong JW, et al. (2010) Foxa2 is essential for mouse endometrial gland development
and fertility. Biol Reprod 83:396e403.
19. Lee MO, Liu Y, Zhang XK (1995) A retinoic acid response element that overlaps an
estrogen response element mediates multihormonal sensitivity in transcriptional ac-
tivation of the lactoferrin gene. Mol Cell Biol 15:4194e4207.
20. Hewitt SC, Li Y, Li L, Korach KS (2010) Estrogen-mediated regulation of Igf1 tran-
scription and uterine growth involves direct binding of estrogen receptor alpha to
estrogen-responsive elements. J Biol Chem 285:2676e2685.
21. Bourdeau V, et al. (2004) Genome-wide identification of high-affinity estrogen re-
sponse elements in human and mouse. Mol Endocrinol 18:1411e1427.
22. O’Brien JE, et al. (2006) Estrogen-induced proliferation of uterine epithelial cells is
independent of estrogen receptor alpha binding to classical estrogen response ele-
ments. J Biol Chem 281:26683e26692.
23. Gibson MK, et al. (1991) The mechanism of ICI 164,384 antiestrogenicity involves rapid
loss of estrogen receptor in uterine tissue. Endocrinology 129:2000e2010.
24. Dauvois S, Danielian PS, White R, Parker MG (1992) Antiestrogen ICI 164,384 reduces
cellular estrogen receptor content by increasing its turnover. Proc Natl Acad Sci USA
25. Scully KM, et al. (1997) Role of estrogen receptor-alpha in the anterior pituitary
gland. Mol Endocrinol 11:674e681.
26. Maurer RA, Notides AC (1987) Identification of an estrogen-responsive element from
the 59-flanking region of the rat prolactin gene. Mol Cell Biol 7:4247e4254.
27. McInerney EM, Katzenellenbogen BS (1996) Different regions in activation function-1
of the human estrogen receptor required for antiestrogen- and estradiol-dependent
transcription activation. J Biol Chem 271:24172e24178.
28. McDonnell DP, Clemm DL, Hermann T, Goldman ME, Pike JW (1995) Analysis of es-
trogen receptor function in vitro reveals three distinct classes of antiestrogens. Mol
29. Sinkevicius KW, et al. (2008) An estrogen receptor-alpha knock-in mutation provides
evidence of ligand-independent signaling and allows modulation of ligand-induced
pathways in vivo. Endocrinology 149:2970e2979.
30. Danielian PS, White R, Hoare SA, Fawell SE, Parker MG (1993) Identification of resi-
dues in the estrogen receptor that confer differential sensitivity to estrogen and
hydroxytamoxifen. Mol Endocrinol 7:232e240.
31. Penot G, et al. (2005) The human estrogen receptor-alpha isoform hERalpha46 an-
tagonizes the proliferative influence of hERalpha66 in MCF7 breast cancer cells. En-
32. Jakacka M, et al. (2002) An estrogen receptor (ER)alpha deoxyribonucleic acid-bind-
ing domain knock-in mutation provides evidence for nonclassical ER pathway sig-
naling in vivo. Mol Endocrinol 16:2188e2201.
33. Glidewell-Kenney C, et al. (2007) Nonclassical estrogen receptor alpha signaling me-
diates negative feedback in the female mouse reproductive axis. Proc Natl Acad Sci
34. Börjesson AE, et al. (2011) Roles of transactivating functions 1 and 2 of estrogen
receptor-alpha in bone. Proc Natl Acad Sci USA 108:6288e6293.
Arao et al.PNAS
| September 6, 2011
| vol. 108
| no. 36