The Estrogen Receptor Enhances AP-1 Activity by Two Distinct Mechanisms with Different Requirements for Receptor Transactivation Functions
Paul Webb,
Phuong Nguyen,
Cathleen Valentine,
Gabriela N. Lopez,
Grace R. Kwok,
Eileen McInerney,
Benita S. Katzenellenbogen,
Eva Enmark,
Jan-Åke Gustafsson,
Stefan Nilsson and
Peter J. Kushner
Metabolic Research Unit (P.W., P.N., C.V., G.N.L., G.R.K.,
P.J.K.) University of California School of Medicine San
Francisco, California 94143
Department of Molecular and
Integrative Physiology (E.M., B.S.K.) University of Illinois
Urbana, Illinois 61801
KaroBio AB (S.N.)
Novum Huddinge, Sweden S-14157
Department of
Medical Nutrition and Biosciences (E.E., J.-A.G.) Karolinska
Institute Huddinge, Sweden S-14186
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ABSTRACT
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Estrogen receptors (ERs
and ß) enhance
transcription in response to estrogens by binding to estrogen response
elements (EREs) within target genes and utilizing transactivation
functions (AF-1 and AF-2) to recruit p160 coactivator proteins. The ERs
also enhance transcription in response to estrogens and antiestrogens
by modulating the activity of the AP-1 protein complex. Here, we
examine the role of AF-1 and AF-2 in ER action at AP-1 sites. Estrogen
responses at AP-1 sites require the integrity of the ER
AF-1 and
AF-2 activation surfaces and the complementary surfaces on the p160
coactivator GRIP1 (glucocorticoid receptor interacting protein 1), the
NID/AF-1 region, and NR boxes. Thus, estrogen-liganded ER
utilizes
the same protein-protein contacts to transactivate at EREs and AP-1
sites. In contrast, antiestrogen responses are strongly inhibited by
ER
AF-1 and weakly inhibited by AF-2. Indeed, ER
truncations that
lack AF-1 enhance AP-1 activity in the presence of antiestrogens, but
not estrogens. This phenotype resembles ERß, which naturally lacks
constitutive AF-1 activity. We conclude that the ERs enhance AP-1
responsive transcription by distinct mechanisms with different
requirements for ER transactivation functions. We suggest that
estrogen-liganded ER enhances AP-1 activity via interactions with p160s
and speculate that antiestrogen-liganded ER enhances AP-1 activity via
interactions with corepressors.
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INTRODUCTION
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Estrogen signal transduction is mediated by two related proteins,
the estrogen receptors
(ER
) and ß (ERß) (1, 2, 3). Both
receptors are conditional transcription factors that belong to the
nuclear receptor superfamily (4, 5, 6) and are comprised of separable
N-terminal (AB), DNA-binding (DBD), and ligand-binding (LBD) domains.
The ERs activate gene expression in two ways. In the best understood
mode of action, or classical pathway, the ERs bind to specific estrogen
response elements (EREs) within target genes and recruit a p160/p300
coactivator complex to the promoter (7, 8). This coactivator complex
enhances gene expression by remodeling chromatin and, perhaps, by
contacting the basal transcription machinery. In a second mode of
action, the ERs utilize unspecified protein-protein interactions to
enhance the activity of heterologous transcription factors. For
example, both ERs enhance the transcription of genes that contain AP-1
sites, the cognate binding site for the Jun/Fos complex (9, 10, 11, 12, 13, 14). The
ERs also enhance the transcription of genes that contain binding sites
for other transcription factors (see Refs. 15, 16, 17, 18, 19, 20 and references
therein).
While the precise mechanism of ER action at AP-1 sites is unknown, our
previous studies have indicated that it shows some striking differences
from ER action at EREs (12, 13). First, antiestrogens, such as the
breast cancer drug tamoxifen, act as potent agonists of ER action at
AP-1 sites, even though they usually block ER action at classical EREs.
This is particularly evident in the case of ERß, which potently
enhances AP-1-dependent transcription in the presence of antiestrogens,
but not estrogen. Second, estrogen action at AP-1 sites does not
require the ER
-DBD and hence does not require specific ERE
recognition (9, 12). ER action at AP-1 does require both the site and
AP-1 proteins (12). Third, the overall strength of ER action at AP-1
sites often bears little relationship to the strength of ER action at
an ERE. For example, ERß is a more potent activator of AP-1-dependent
transcription than ER
(13), but is a weaker activator of classical
estrogen response than ER
(21, 22, 23).
Perhaps more surprisingly, our initial studies of the mechanism of ER
action at AP-1 sites also suggested that estrogen and antiestrogen
effects are products of completely distinct pathways. We (12) and
others (10, 14) found that estradiol effects predominate in several
breast cancer cell lines, but we also found that tamoxifen effects
predominate in other cell lines, including those of uterine and liver
origin. Furthermore, estradiol and tamoxifen action at AP-1 sites
showed distinct structure-function requirements. Estradiol activation
requires the ER
-LBD, whereas tamoxifen activation requires the ER
AB-DBD region. Finally, we found that the estradiol-liganded ER
targeted the VP16 transactivation function to AP-1 sites in mammalian
two-hybrid assays, but the tamoxifen-liganded ER
did not. Later, we
also showed that ERß does not enhance AP-1 activity in the presence
of estrogens, but does enhance AP-1 activity in the presence of
antiestrogens (13). Together, these results suggest that the ERs
participate in distinct sets of protein-protein interactions that lead
to stimulation of AP-1 activity, one promoted by estrogens, the other
promoted by antiestrogens.
It is well established that ER action at classical EREs is mediated by
transactivation functions (4, 5, 6, 24, 25). ER
contains two separate
transactivation functions, AF-1 and AF-2, which synergize strongly to
give the overall level of estrogen response (26, 27). While ERß does
contain a growth factor-inducible AF-1 (28), its activity stems largely
from AF-2 (22, 29). AF-2 of both receptors consists of a small
hydrophobic patch on the surface of the estrogen-liganded LBD (30, 31, 32),
which binds strongly to specific LXXLL motifs (nuclear receptor boxes)
that are found throughout the central region of the p160s (33, 34, 35, 36, 37).
ER
AF-1 consists of a long region of the AB domain that binds the C
terminus of glucocorticoid receptor-interacting protein 1
(GRIP1) (NID/AF-1) and other p160s (29). ERß AF-1 also binds weakly
to SRC-1, but only when AF-1 is phosphorylated (38). Antiestrogens,
including tamoxifen, raloxifene, and ICI 182,780 (ICI), block formation
of AF-2, thereby reducing coactivator complex recruitment (31, 32, 39),
and restrict ER
AF-1 activity by promoting association with
corepressors (40, 41, 42, 43, 44, 45). Tamoxifen, however, does allow some ER
AF-1
activity and, consequently, elicits weak agonist effects at classical
EREs (24).
Because ER transactivation functions play a central role in classical
estrogen response, we ask here whether they might also play a role in
ER action at AP-1 sites. We find that the estrogen/AP-1 pathway
involves ER transactivation functions, but the antiestrogen/AP-1
pathway does not. Instead, the antiestrogen/AP-1 pathway is strongly
inhibited by ER
AF-1 and weakly inhibited by AF-2 of both receptors.
These results reinforce the notion that estrogen and antiestrogen
effects at AP-1 sites are mediated by distinct mechanisms and may
suggest identities for putative estrogen and antiestrogen pathway
targets.
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RESULTS
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The Estrogen/AP-1 Pathway Requires Only the LBD and AF-2
To understand how the ERs enhance AP-1 activity, we examined the
role of ER transactivation functions in estrogen and antiestrogen
action at AP-1 sites. To perform these experiments, we asked whether
transiently transfected wild-type ERs, or ER derivatives bearing
specific mutations in AF-1 or AF-2, would elicit estrogen or
antiestrogen responses at an AP-1-responsive reporter in HeLa
cells.
We previously showed that estrogen action at AP-1 sites could be
obtained in the absence of the ER
N-terminal domain and DBD,
suggesting that the LBD is necessary for the estrogen/AP-1 pathway
(12). We therefore asked whether the LBD was also sufficient to obtain
an estrogen response at AP-1 sites. Figure 1
shows that the ER
-LBD gave weak
constitutive activation of the AP-1 responsive reporter gene relative
to empty expression vector. Addition of estradiol elicited a further
10-fold increase in the activity of the AP-1-responsive promoter.
Tamoxifen (shown) and raloxifene and ICI (not shown) failed to enhance
AP-1 activity in the presence of the isolated LBD. Thus, the ER
-LBD
is sufficient to obtain a potent estrogen response at AP-1 sites.
We then asked whether the LBD-mediated estrogen response required AF-2.
ER
-LBDs bearing either a specific mutation in AF-2 (LBDmAF-2;
M543A,L544A) or a complete truncation of helix 12 (LBD
AF-2,
truncated at position 537) gave weak constitutive activation at the
AP-1 site, but failed to yield further estradiol activation. We
conclude that AF-2 plays a key role in the estrogen/AP-1 pathway. We
also note that there are strong parallels between the behavior of the
ER
-LBD at AP-1 sites and the behavior of the glucocorticoid receptor
LBD at the herpes simplex virus thymidine kinase promoter (46),
raising the possibility that AF-2-dependent modulation of heterologous
transactivation factor activity could be a common feature of nuclear
receptor action.
Estrogen Responses at AP-1 Sites Require AF-1, but Tamoxifen
Responses Are Independent of AF-1
We next turned our attention to AF-1, which synergises with AF-2
in estrogen response at classical EREs and also mediates weak
antiestrogen agonist effects at classical EREs. In Hela cells, full
ER
AF-1 activity requires a long region of the AB domain (amino
acids 41129, Fig. 2A
) (29, 47, 48, 49, 50). The
two flanking regions (iAF-1A, amino acids 141, iAF-1B, amino acids
129178) weakly inhibit AF-1 activity at EREs (29).

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Figure 2. AF-1 Suppresses Tamoxifen Effects at AP-1 Sites
A, The human ER N-terminal AB domain (amino acids 1178). Amino
acids 141 weakly inhibit AF-1 activity and are designated iAF-1A. The
extent of sequences required for full AF-1 activity in HeLa cells
(amino acids 41129) is marked in gray. A second weak
inhibitory function (amino acids 129178) is designated iAF-1B. B,
Behavior of deletions that eliminate different portions of the ER
N-terminal (AB) domain at classical EREs. The experiment was carried
out using the EREII-LUC reporter gene shown at top.
Included in each transfection were an empty SV40 expression vector, SG5
(none), or SV40 expression vectors for wild-type ER , or ER
truncations lacking the first 101 (N101), 109 (N109), 117 (N117)
amino acids of the AB domain, the entire AB domain ( AB), or
containing an internal deletion of iAF-1B sequences ( 129178). A
schematic of these deletions is shown at the
lefthand side. The effect of ER ligands upon the
activity of the transiently transfected EREII-LUC reporter gene is
shown in the accompanying graph. Individual
points show average luciferase activities, calculated from
triplicate wells in a representative transfection and expressed
relative to the activity of the promoter in the absence of ligand and
ER expression vector that was assigned a value of 1. C, The behavior
of the same ER deletions at an AP-1-responsive reporter gene,
coll73-LUC. The individual points show luciferase activities,
calculated similarly to those of panel B.
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In accordance with our previously published results, ER
N-terminal
deletions lacking AF-1 activity (N101, N109, N117,
AB) elicited
reduced estrogen response and no tamoxifen response at a classical
ERE-responsive reporter (EREII-LUC; Fig. 2B
). Moreover, an ER
internal deletion that retains AF-1, but lacks iAF-1B (
129178),
elicited normal activity in the presence of both ligands and also
showed a modest enhancement of ligand-independent activity.
At an AP-1-responsive reporter (coll73-LUC), the ER
truncations
showed a similar activity profile in the presence of estradiol (Fig. 2C
). Estradiol action was abolished by certain N-terminal deletions
that abolish AF-1 activity (N117,
AB) but was unaffected by deletion
of iAF-1B (ER
129178). In the presence of tamoxifen, however, the
same deletions showed a sharply contrasting phenotype. Two truncations
that abolish AF-1 activity (N101, N109) showed accentuated tamoxifen
response. This result indicates that tamoxifen responses at AP-1 sites
are independent of AF-1 activity per se and may actually be
suppressed by AF-1. Longer N-terminal deletions (N117 or
AB) and the
iAF-1B deletion (
129178) each failed to elicit tamoxifen responses
from the AP-1-responsive reporter. Thus, in the context of full-length
ER
, estrogen responses at AP-1 sites require AF-1, but tamoxifen
responses are independent of AF-1 and require iAF-1B.
Serine 118 Is Dispensable For Tamoxifen Responses at AP-1 Sites
At classical EREs, full ER
AF-1 activity requires a
serine residue (S118) that forms part of a mitogen-activated
protein kinase consensus recognition sequence (PXXSP) and is a
target for phosphorylation (51, 52, 53, 54, 55, 56). At AP-1 sites, ER
-dependent
tamoxifen effects require an unspecified function whose N-terminal
boundary lies between amino acids 109 and 117 of the AB domain (Fig. 2
). We therefore asked whether S118 played any role in ER
action at
AP-1 sites. We changed S118 to either alanine (A), arginine (R), which
has a bulky charged side chain and should disrupt S118-dependent
protein-protein contacts, or glutamic acid (E), which mimics an active
phosphorylated serine residue (55), and asked whether any of these
mutants affected tamoxifen response at EREs or AP-1 sites.
Figure 3A
reveals that ER
-S118E showed
enhanced tamoxifen response at the classical ERE relative to wild-type
ER
and to the S118A and S118R mutants. This result is consistent
with the notion that S118E mimics the phosphorylated, active state of
AF-1 and that S118A and S118R block phosphorylation and do not allow
full AF-1 activity. By contrast, each ER
S118 mutant elicited
tamoxifen responses at AP-1 sites that were comparable to those
obtained with wild type ER
(Fig. 3B
). Similar results (not shown)
were also obtained with an S118A mutation in the context of the
isolated ER
AB-DBD region, which enhances gene expression
constitutively in a manner that resembles tamoxifen-liganded ER
(12, 57). Thus, S118 is dispensable for tamoxifen effects at AP-1 sites. The
fact that ER-S118E showed enhanced tamoxifen activation at the ERE, but
did not affect tamoxifen activation at the AP-1 site, also reinforces
the notion that conventional AF-1 activity is not required for
tamoxifen activation at AP-1 sites.
Removal of ER
AF-1 Creates an ERß-Like Phenotype
Removal of ER
AF-1 leads to enhanced tamoxifen responses at
AP-1 sites in HeLa cells (Fig. 2
). This phenotype is similar to ERß
(13), which naturally lacks constitutive AF-1 activity (29). We
therefore asked whether removal of ER
AF-1 created other ERß-like
phenotypes.
We previously showed that full-length ER
only enhances AP-1
activity in the presence of tamoxifen and estradiol in HeLa cells and
that ERß strongly enhances AP-1 activity in the presence of a range
of antiestrogens, but not estrogens, in the same cell type (13). Figure 4
shows that, as expected, wild-type
ER
enhanced AP-1 activity in the presence of tamoxifen, estradiol,
and the synthetic agonist diethylstilbestrol (DES), but not raloxifene
or ICI, in HeLa cells. Likewise, ERß enhanced AP-1 activity in the
presence of ICI, raloxifene, and tamoxifen, but not estradiol or DES.
In parallel, short ER
N-terminal truncations (N21, N41) showed
ligand responses that were similar to wild-type ER
. However, longer
truncations that partially (N87), or completely (N109,
AB),
eliminate AF-1 activity showed enhanced antiestrogen effects,
especially with raloxifene and ICI. Thus, ER
AF-1 suppresses
antiestrogen action at AP-1 sites in HeLa cells, and its elimination
allows ER
to behave like ERß in its ligand preferences for action
at AP-1.
We also previously showed that ER
only elicits weak estrogen
responses at AP-1 sites in breast cells (12), but ERß elicits strong
antiestrogen effects (13). Accordingly, wild-type ER
only enhanced
AP-1 activity weakly in the presence of estrogens in MCF-7 cells, and
ERß enhanced AP-1 activity in the presence of antiestrogens, but not
estrogens (Fig. 5A
). In parallel, ER
N-terminal deletions that eliminate AF-1 (N87, N109,
AB) elicited
extremely potent antiestrogen effects. A similar profile was also
obtained in MDA-MB-453 breast cells (data not shown). Thus, AF-1
restricts ER
action in breast cells and, here again, removal of AF-1
allows an ERß-like spectrum of ligand preferences for action at AP-1
sites.
Closer examination revealed some interesting aspects to the
behavior of the ER
N-terminal truncations, which were especially
prominent in MCF-7 cells (Fig. 5B
). Estradiol (and DES, not shown)
effects at AP-1 sites were enhanced by deletion of iAF-1A at the
extreme ER
N terminus and were reduced by deletion of AF-1. These
sequence requirements are very similar to those seen for estrogen
action at EREs (29, 47, 48, 49, 50). Conversely, antiestrogen effects were all
enhanced by elimination of AF-1. However, different truncations showed
distinct ligand preferences. A truncation that partially eliminates
AF-1 activity (N87) showed strongly enhanced tamoxifen effects and
weaker raloxifene and ICI effects. Longer deletions (N109, N117,
AB)
showed stronger ICI and raloxifene effects. Furthermore, tamoxifen
effects were heavily dependent upon the iAF-1B region, but raloxifene
(and ICI, not shown) responses were less dependent upon this region,
and, in fact, were largely mediated by the DBD-LBD region. Thus,
different antiestrogens utilize different regions of ER
to enhance
AP-1 activity. Despite these differences, the stark separation of
estrogen and antiestrogen effects at AP-1 sites underscores our notion
that different ER functions contribute to estrogen and antiestrogen
effects at AP-1 sites.
Do Longer ERß Isoforms Possess a Constitutive AF-1 Function That
Suppresses Antiestrogen Action at AP-1 Sites?
Removal of ER
AF-1 creates a phenotype that resembles ERß.
This result predicts that addition of a constitutive AF-1 activity back
to ERß might suppress its ability to exert antiestrogen effects at
AP-1 sites. We previously showed that a short form of ERß [ERß485,
(1, 58)] lacks a constitutive AF-1 activity (29) and strongly enhances
AP-1 activity in the presence of antiestrogens, but not estrogens (13).
Because a longer form of ERß (ERß530) has now been identified (59, 60), we asked whether it might contain such an AF-1 activity.
Figure 6A
shows that estrogen-liganded
ERß530 gave comparable levels of transcriptional activity to ERß485
at classical EREs. Similar results were also obtained with both forms
of estradiol-liganded ERß in the presence of overexpressed
coactivators, GRIP1 and p300 (+CoAcs). Overexpression of GRIP1 and p300
also enhanced the activity of both forms of ERß in the absence of
ligand, a phenomenon that stems from p160 interaction with the weak
inducible ERß AF-1 function (38), but did not enhance ERß activity
in the presence of tamoxifen (shown) or raloxifene and ICI (not shown).
In parallel, ER
did elicit extremely potent AF-1-dependent tamoxifen
responses in the presence of GRIP1 and p300 (lower panel,
note expanded scale). Thus, ERß530, like ERß485, lacks a
constitutive AF-1 activity.
We also previously showed that the ER
AB domain contains a
constitutive binding site for GRIP1, but the AB domain of ERß485 does
not (29). We therefore generated a glutathione-S-transferase
(GST) fusion protein containing the entire AB domain of the long form
of ERß (amino acids 1144) and asked whether it would bind GRIP1.
Figure 6B
shows that the ER
AB domain bound GRIP1 strongly and the
ERß AB domain did not. Thus, ERß530 lacks a constitutive binding
site for GRIP1.
Finally, we asked whether the longer form of ERß would enhance
AP-1-responsive transcription. Figure 6C
shows that ERß530 actually
enhanced AP-1 activity more potently than ERß485 in the presence of
antiestrogens. Neither form of receptor enhanced AP-1 activity in the
presence of estradiol or DES. Thus, human ERß530 does not possess a
constitutive AF-1 activity that suppresses antiestrogen action at AP-1
sites. It remains to be seen whether even longer forms, such as the 549
amino acid rodent ERß (38), might contain such an activity.
AF-2 Is Dispensable For Antiestrogen Action at AP-1 Sites
Next, we examined the role of AF-2 in antiestrogen action at AP-1
sites. AF-2 consists of a hydrophobic cleft, made up of a cluster of
residues from LBD helices 3, 5, and 12, that forms upon the surface of
the holo-ER (30, 31, 32). To probe the requirement for AF-2 in ER action at
AP-1 sites, we used a mutation (K>A) in a helix 3 lysine residue (61),
which forms key hydrogen bonds with the GRIP1 nuclear receptor box
(32).
We first examined how the AF-2 mutant would affect ER
action at AP-1
sites in HeLa cells. Figure 7A
shows that
the ER
K362A mutant gave reduced estradiol response and normal
tamoxifen response at AP-1 sites. The equivalent ERß AF-2 mutant
(K269A) gave ICI, raloxifene, and tamoxifen responses that were
slightly larger than those obtained with the wild-type ERß (Fig. 7B
).
Likewise, the ER
K362A mutation gave modestly enhanced raloxifene
and ICI responses in the context of the isolated DBD-LBD region at AP-1
sites (not shown). Control transfections revealed that each AF-2 mutant
receptor was inactive at an ERE-responsive reporter (not shown). Thus,
AF-2 is either dispensable or weakly inhibitory for antiestrogen action
at AP-1 sites.

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Figure 7. Mutation of AF-2 Does Not Affect Antiestrogen
Action at AP-1 Sites
A, Effect of tamoxifen or estradiol upon AP-1-responsive transcription
in the presence of either full-length ER , or the AF-2 mutant
(K362A). The data are shown as fold increase of luciferase activity in
the presence of ligand over no hormone. B, Effects of different ligands
upon AP-1-responsive transcription in HeLa cells in the presence of
full-length ERß or the ERß AF-2 mutant (K269A).
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The Estrogen/AP-1 Pathway Requires ER
Recognition Sites within
the p160 Coactivator GRIP1
Estradiol effects at AP-1 sites require ER
transactivation
functions. Because the actions of the ER transactivation functions at
classical EREs are mediated by p160s, we next asked whether the actions
of the ER transactivation functions at AP-1 sites were also mediated by
p160s. Figure 8
shows that overexpression
of the p160 GRIP1 enhanced basal AP-1 activity and ER action at the
AP-1 site. By contrast, overexpression of a GRIP1 derivative with
mutations in NR boxes II and III (NR box mutant), which is unable to
bind AF-2, enhanced basal AP-1 activity but showed weakened estradiol
and DES effects. Overexpression of another GRIP1 derivative with a
truncation of NID/AF-1 region (
1121C), which is unable to bind AF-1,
completely failed to potentiate estradiol and DES effects at the AP-1
site. Thus, estrogen action at AP-1 sites requires the integrity of
ER
binding regions within the GRIP1 molecule. It therefore appears
that estrogen action at AP-1 sites, like estrogen action at EREs, is
mediated by contacts with p160s (see Discussion).
The behavior of the GRIP1 truncation (
1121C) requires further
comment. First, it failed to enhance basal AP-1 activity, suggesting
that the GRIP1 C-terminal region contains unspecified functions that
are essential for p160 coactivation at the AP-1 site. Second, it
strongly potentiated antiestrogen effects, especially those of
tamoxifen. We have previously shown that this GRIP1 truncation acts as
a specific dominant negative for ER
AF-1 (29). The ability of
GRIP1
1121C to enhance antiestrogen effects at AP-1 sites is
therefore consistent with our notion that AF-1 suppresses antiestrogen
action at AP-1 sites. Accordingly, GRIP1
1121C failed to enhance
antiestrogen effects in the presence of ER
truncations that lack
AF-1 (N109,
AB, not shown). Thus, our studies with GRIP1
overexpression also provide further support for the notion that the
antiestrogen/AP-1 pathway is independent of ER transactivation
functions and is strongly suppressed by AF-1.
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DISCUSSION
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Activation Function-Mediated and Independent Pathways of ER Action
at AP-1 Sites
Several lines of evidence indicate that the ER can modulate the
activity of heterologous transcription factors via estrogen- and
antiestrogen-specific mechanisms. Differences in the cell specificity,
structure-function requirements, and behavior in mammalian two-hybrid
assays led us to propose that ER
-dependent estradiol and tamoxifen
effects at AP-1 sites are products of distinct mechanisms (12). Later,
we showed that ERß does not enhance AP-1-dependent transcription in
the presence of estrogens, but does enhance AP-1-dependent
transcription in the presence of antiestrogens (13). Other genes have
also been described that respond weakly to estrogens, and strongly to
antiestrogens, in the presence of either form of ER (15, 17, 62, 63).
Here, we have shown that different ER functions contribute to estrogen
and antiestrogen effects at AP-1 sites. Estrogen effects only require
the isolated ER
-LBD and its AF-2 function. Tamoxifen effects require
the proximal part of the ER
B region (iAF-1B), along with an intact
DBD (12), and raloxifene and ICI effects require the ER
DBD-LBD
region, but not AF-2. Thus, our study reinforces the notion that ER
activates AP-1-dependent transcription via estrogen- and
antiestrogen-specific mechanisms.
Even though the estrogen/AP-1 pathway does not require the ER-DBD, it
does share some features with classical estrogen response. Estrogen
response at AP-1 sites requires AF-1 and AF-2 in the context of the
full-length ER and AF-2 in the context of the isolated ER-LBD.
Furthermore, estrogen effects at AP-1 sites require the integrity of
the GRIP1 NR boxes, which bind AF-2, and the NID/AF-1 region, which
binds AF-1. By contrast, the antiestrogen/AP-1 pathway is independent
of ER transactivation functions and is strongly suppressed by ER
AF-1. We also suggest that the lack of a constitutive AF-1 in ERß,
perhaps coupled with its relatively weak AF-2 (64), accounts for its
ability to enhance AP-1 activity in the presence of antiestrogens, but
not estrogens. Based on these results, we propose that the
estrogen/AP-1 pathway should be renamed the activation function
(AF)-mediated/AP-1 pathway and the antiestrogen/AP-1 pathway
should be renamed the AF-independent/AP-1 pathway. This new
nomenclature reflects important mechanistic features of each pathway
and makes no assumptions about ligand preference.
We expect that estrogen enhancement of AP-1 activity will always occur
through the AF-mediated pathway and that antiestrogen enhancement of
AP-1 activity will largely occur through the AF-independent pathway.
However, most of our studies have been conducted in HeLa cells that
exhibit low AF-1 activity, and high AF-2 activity. Because tamoxifen is
an AF-1 releasing agonist, we are presently exploring the idea that
tamoxifen, but not raloxifene and ICI, will be able to work through the
AF-mediated/AP-1 pathway in cell types that exhibit high AF-1
activity.
Mutations That Block One Pathway of ER Action at AP-1 Sites Enhance
ER Action in the Other Pathway
One recurring observation of our studies has been that mutations
that block one pathway of ER action at AP-1 sites enhance the other
(12). Accordingly, we showed here that ER
AF-1 promotes estrogen
effects at AP-1 sites but also inhibits antiestrogen effects. We also
note that the ER
DBD-LBD region elicits potent AF-2-independent
antiestrogen effects at AP-1 sites, but the LBD alone elicits only
strong AF-2-dependent estrogen effects at AP-1 sites. We have also
recently identified an ER
-DBD point mutant that blocks antiestrogen
effects at AP-1 sites and, instead, allows ER
to elicit extremely
potent AF-2-dependent estrogen effects at AP-1 sites (R. M. Uht,
C. A. Anderson, P. Webb, D. B. Starr, and
P. J. Kushner, unpublished data). Together, these
results suggest that the ER-DBD promotes antiestrogen effects at AP-1
sites but also inhibits estrogen effects. Thus, disruption of the ERs
ability to participate in one pathway often frees the receptor to
participate in the other.
A Model to Explain the Two Pathways of ER Action at AP-1 Sites
What are the molecular interactions that underlie ER action at
AP-1 sites? ER/p160 contacts play an important role in the
AF-mediated/AP-1 pathway, just as they do in classical estrogen
response. Nonetheless, we suspect that there may be significant
differences between the role of ER/p160 contacts at AP-1 sites and at
EREs. The isolated ER
-LBD enhances AP-1-responsive transcription in
an AF-2-dependent manner, even though it does not bind jun/fos (12).
Thus, the ER-LBD cannot enhance AP-1 activity by binding jun/fos and
recruiting p160s to the jun/fos complex, as the ERs do at EREs. It is
known that AP-1 proteins activate transcription by recruiting a
p300-p160 complex to the promoter via direct contacts with
p300/CREB-binding protein (34). We therefore propose that the ER
transactivation functions may serve to recognize p160s within the
Jun/Fos-coactivator complex and to bring the ER to the AP-1-responsive
promoter (Fig. 9A
). The ER, positioned on
the complex, would then enhance its activity. We are presently testing
this hypothesis by asking whether ER can enhance the activity of
isolated coactivator proteins.

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|
Figure 9. Models for the Two Pathways of ER Action at AP-1
Sites
A, The AF-mediated/AP-1 pathway. Estrogen-liganded ER uses its own
transactivation functions (AF-1 and AF-2) to recognize the cognate
surfaces (NID/AF-1; NR boxes) upon p160s that are present in the
jun/fos (J/F)-associated complex. From this location, ER enhances
AP-1-dependent transcription. The ER-DBD (striped) is
dispensable for this process. B, The AF-independent/AP-1 pathway.
Antiestrogen-liganded ER (with AF-2 inactive, as designated by a
cross) binds a nuclear receptor corepressor (CoR) and
sequesters unspecified repressors (R) from the AP-1 complex. This
interaction is mediated by regions of ER that do not interact with
coactivators including the ER-DBD (striped). AP-1
activity is thereby enhanced by ERs that do not directly participate in
the AP-1 complex.
|
|
Our studies do not directly address the mechanism of the
AF/independent AP-1 pathway. However, any hypothesis to account for
antiestrogen effects at AP-1 sites must account for one puzzling
observation. Tamoxifen-liganded ER
and isolated ER
AB-DBD region
both enhance AP-1 activity yet do not target the strong VP16
transactivation function to AP-1 sites in mammalian two-hybrid assays
(12). Thus, antiestrogen-liganded ER
seems to be able to enhance
AP-1 activity without directly participating in the AP-1 complex. How
is this possible? One attractive idea is that the antiestrogen-liganded
ER enhances AP-1 activity by binding, and sequestering, AP-1-associated
repressors (Fig. 9B
). Two pieces of evidence are consistent with this
notion. First, nuclear receptor corepressors preferentially associate
with receptor/antihormone complexes (43, 65). Second, in some
circumstances, the iAF-1B region represses ER
action at classical
EREs, suggesting that it recruits a repressor (29). The same iAF-1B
region plays an important role in antiestrogen responses at AP-1 sites
(Figs. 2
, 4
, and 5
). Thus, corepressors are possible agents in
antiestrogen action at AP-1 sites. We are presently exploring this
hypothesis by asking whether ER/N-CoR interactions might play a role in
antiestrogen action at AP-1 sites. We stress that N-CoR itself need not
be present in the AP-1 complex. Rather, other functional components of
the repressor complex, such as histone deacetylases, could be
associated with the jun/fos-coactivator complex and be diverted from
that location by formation of the antiestrogen-liganded ER/N-CoR
complex.
We also note that our model suggests an explanation for the apparent
mutual exclusivity of the two pathways. The estrogen- and
antiestrogen-liganded ERs must be present in completely distinct
subnuclear locations, either at or away from the promoter, to enhance
AP-1 activity. Furthermore, nuclear receptors are unable to interact
with coactivators and corepressors simultaneously (7, 65). Disruption
of ER/coactivator or ER/corepressor interactions would free ER to
participate more strongly in the other type of interaction.
Differences between Tamoxifen and Raloxifene Action at AP-1
Sites
There are several differences between the behavior of tamoxifen
and raloxifene at AP-1 sites. Tamoxifen-liganded ER
enhances AP-1
activity in HeLa cells, but raloxifene-liganded ER
does not (13).
More subtly, ER
AF-1 partially suppresses tamoxifen effects at AP-1
sites, but completely suppresses raloxifene action at AP-1 sites, and
different regions of AF-1 suppress tamoxifen and raloxifene effects
(Fig. 5B
). Furthermore, tamoxifen effects at AP-1 sites are heavily
dependent upon iAF-1B, and raloxifene effects are less dependent upon
this region, and, in fact, are largely mediated by the DBD-LBD region.
On the basis of our model, we speculate that the tamoxifen-liganded
ER
relies on iAF-1B to bind corepressors, and work through the
AF-independent pathway at AP-1 sites, and that raloxifene-liganded
ER
relies on the DBD-LBD region to achieve the same effect.
Tamoxifen and raloxifene also show different properties at classical
EREs. Antiestrogens all permit ER
to bind DNA (66, 67), yet
tamoxifen allows high AF-1 activity in some contexts (24), and ICI and
raloxifene do not (67, 68). Thus, antiestrogens exert distinct effects
upon ER
LBD/AB domain cross-talk in diverse gene regulation assays.
Perhaps understanding the way that different ligands influence
ER/corepressor interactions will clarify both the ligand specificity of
ER action at AP-1 sites and release of AF-1 activity at EREs.
Do AF-Mediated and AF-Independent Antiestrogen Effects Occur
in Vivo?
Antiestrogens block estrogen response by blocking AF-2 activity.
They also possess the capacity to enhance gene expression at EREs and
AP-1 sites, but with completely opposite requirements for AF-1. At
EREs, tamoxifen releases AF-1 activity, which can result in quite
substantial cell and promoter-specific tamoxifen effects (57). However,
raloxifene releases considerably less AF-1 activity and ICI none (67).
At AP-1 sites, as we have shown here, tamoxifen, raloxifene, and ICI
enhance AP-1 activity via an activation function-independent
mechanism.
Because each type of antiestrogen effect shows characteristic ligand
preferences, it may be possible to guess which mechanisms are active
in vivo. In breast, antiestrogens block estrogen action and
show few agonist effects (69, 70). Thus, AF-2 may play an important
role in breast physiology. In uterus, tamoxifen acts as an ER agonist,
but raloxifene does not. Thus, ER
AF-1 may play an important role in
uterine physiology. We stress, however, that ER
AF-1 lacks strong
activity at simple EREs in a tamoxifen-responsive uterine cell line
(12), suggesting that any putative uterine AF-1 activity must be of a
type that is only observed at more complex promoters, perhaps
containing spaced ERE half-sites (71), and binding sites for
heterologous transcription factors. Finally, in bone and cardiovascular
system, tamoxifen and raloxifene both act as ER agonists. Even ICI,
which has the additional effect of promoting ER protein degradation
in vivo (72), shows some agonist activity in bone (73).
Thus, these effects are candidates for an AF-independent mechanism.
We recognize that ER action in vivo will likely prove to be
much more complex than ER action at our simple transiently transfected
reporter genes. As a first step to understanding the contributions of
AF-1-mediated and AF-1-independent antiestrogen effects in
vivo, it will be interesting to determine whether both types of
effect are active upon chromosomally integrated target genes or, even
better, upon native ER target genes in their normal chromosomal
context. Should both types of mechanism indeed prove to be active
in vivo, an informed strategy to block or promote specific
antiestrogen effects will require an understanding of the contributions
of each mechanism to regulation of key ER target genes.
 |
MATERIALS AND METHODS
|
---|
Mammalian Reporter Genes and Expression Vectors
Coll73-LUC and ERE-II-LUC have been previously described (12, 13). The ER
expression vectors have been previously described but,
for ease of comparison, we have given them a consistent nomenclature.
The previous names and sources of each construct are as follows:
SG5-ER = HEG0; SG5-ERV400 = HE0, n101 = HE302; n117
= HE303;
129178 = HE316 (49), CMV-ER; n21 = n21;
n41 = E41; n87 = A87; n109 = M109; ER
AB (50). For
some experiments the effects of ER-N109 were compared with SV40-driven
ER expression vectors. For these experiments, the ER N109 cDNA was
moved out of the CMV expression vector as an
EcoRI/BamHI fragment and recloned into pSG5. The
coactivator expression vectors (GRIP1, GRIP1 NR box II and III mutant,
and GRIP1
1121C) have each been previously described (29, 36).
The ER
-LBD expression vector SG5-LBD and its mutant derivatives were
constructed from pKCR2-HE14 (74), which encodes human ER-LBD amino
acids 282595. First, the LBD-coding sequence was moved into pSG5 as
an EcoRI fragment. A V400 mutation, which was present in the
original human cDNA clone (75), was corrected by incorporating
wild-type human sequences from the full-length pSG5-ER
as a
HindIII/BamHI fragment. The resulting ER
-LBD
expression vector was subjected to point mutagenesis with a PCR-based
method designed to incorporate oligonucleotides into the LBD cDNA
(Stratagene). The mutations converted methionine 543 and leucine
544 to alanine residues (LBDmAF-2) or introduced a stop codon after
tyrosine 537 (LBD
AF-2). ERs bearing specific amino acid
substitutions at serine 118 and at lysine 362 (ER
) or lysine 269
(ERß) were derived by similar methods. The nature of each mutant ER
was confirmed by sequence analysis.
ERß530 was generated from a CMV vector containing a full-length cDNA
clone (S. Nilsson, unpublished). An EcoRI fragment
spanning the 5'-end of the longer cDNA was obtained and substituted
into our existing ERß485 expression vector (13). The orientation of
the insert was confirmed by restriction analysis and sequencing. The
GST-ERß AB domain fusion protein was generated by amplification of
the sequences homologous to the AB domain (amino acids 1144) by PCR
and subcloning the resulting fragment into the
BamHI/EcoRI sites of pGEX-5X-3.
Cell Culture and Transfection
HeLa cells were maintained and transfected as previously
described (12), except that 2 µg of luciferase reporter were
employed. Also included in each transfection was 1 µg of pJ3
ß-galactosidase control. All cells were grown and transfected in
phenol red-free medium. Cell lysates were prepared 3640 h after
transfection, and luciferase and ß-galactosidase assays were
performed using the standard methods described in the reference above.
Where indicated, luciferase activities were corrected for variations in
transfection efficiency using ß-galactosidase activity.
Protein Binding Assays
Fusions of GST to various human ER
domains were prepared as
described (76). Bacteria expressing the fusion proteins were
resuspended in buffer IPAB-80 (20 mM HEPES, 80
mM KCl, 6 mM MgCl2, 10% glycerol,
1 mM dithiothreitol, 1 mM ATP, 0.2
mM phenylmethylsulfonyl fluoride, and protease inhibitors;
pH 7.9) and sonicated mildly, and the debris was pelleted at 12,000 rpm
for 1 h in an ss34 rotor. The supernatant was incubated for 2
h with 500 µl of glutathione Sepharose 4B beads that had been
previously washed with 5 vol of PBS 0.2% Triton X-100 and equilibrated
with 5 vol of IPAB 80. GST-fusion proteins beads were then washed with
5 vol of PBS 0.05% Nonidet P-40 and resuspended in 1 ml of IPAB-80 for
storage at 4 C until use. All procedures were performed at 4 C.
Assays of GST-ER fusions were carried out in a volume of 100 µl that
contained 40 µl of bead suspension (volume equivalent to 10 µl of
compact beads) and 1 µl of 35S in vitro
translated GRIP1 in IPAB-80 2.5% nonfat milk and incubated for
1.5 h at 4 C. Beads were washed five to six times with IPAB-80
containing 0.05% NP-40. Input-labeled proteins, proteins bound to GST,
and the ER fusion beads were subjected to SDS-PAGE in 10% acrylamide
and then to autoradiography.
 |
ACKNOWLEDGMENTS
|
---|
We would like to thank Drs. P. Chambon, D. Metzger (Strasbourg,
France), and D. Leitmann (San Francisco, CA) for gifts of ER expression
vectors; Carol Anderson for technical assistance and helpful
discussions; and Dr. R. Uht for critical reading of the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Peter Kushner/Dr. Paul Webb, Metabolic Research Unit, University of California San Francisco School of Medicine, MRU 1119 HSW, San Francisco, California 94122-0540.
This work was supported by the State of California Breast Cancer
Research Program Grant 1KB-0188 to P.W.; by the US Army Breast
Cancer Research Program and by NIH Grants CA-80210 and DK-51083 to
P.J.K.; by NIH Grant CA-18119 to B.S.K.; and by grants from the
Swedish Cancer Fund to J.-A.G.
Peter Kushner is a consultant and director with significant financial
holdings in KaroBio AB, a Swedish pharmaceutical development company
with interests in nuclear receptors.
Received for publication February 25, 1999.
Revision received June 7, 1999.
Accepted for publication June 30, 1999.
 |
REFERENCES
|
---|
-
Kuiper GGJMA, Enmark E, Pelto-Huikko M, Nilsson S,
Gustafsson JA 1996 Cloning of a novel estrogen receptor expressed in
rat prostate and ovary. Proc Natl Acad Sci USA 93:59255930[Abstract/Free Full Text]
-
Katzenellenbogen BS, Korach KS 1997 Editorial: A new actor in
the estrogen receptor dramaenter ER ß. Endocrinology 138:861862[Free Full Text]
-
Giguere V, Tremblay A, Tremblay GB 1998 Estrogen receptor
ß: re-evaluation of estrogen and antiestrogen signaling. Steroids 63:335339[CrossRef][Medline]
-
Tsai MJ, OMalley BW 1994 Molecular mechanisms of action of
steroid/thyroid receptor superfamily members. Annu Rev Biochem 63:451486[CrossRef][Medline]
-
Ribeiro RC, Kushner PJ, Baxter JD 1995 The nuclear hormone
receptor gene superfamily. Annu Rev Med 46:443453[CrossRef][Medline]
-
Katzenellenbogen BS 1996 Estrogen receptors: bioactivities
and interactions with cell signaling pathways. Biol Reprod 54:287293[Abstract]
-
Horwitz KB, Jackson TA, Bain DL, Richer JK, Takimoto GS, Tung
L 1996 Nuclear receptor coactivators and corepressors. Mol Endocrinol 10:11671177[Abstract]
-
Glass CK, Rose DW, Rosenfeld MG 1997 Nuclear receptor
coactivators. Curr Opin Cell Biol 9:222232[CrossRef][Medline]
-
Gaub MP, Bellard M, Scheuer I, Chambon P, Sassone CP 1990 Activation of the ovalbumin gene by the estrogen receptor involves the
fos-jun complex. Cell 63:12671276[Medline]
-
Philips A, Chalbos D, Rochefort H 1993 Estradiol increases and
anti-estrogens antagonize the growth factor-induced activator protein-1
activity in MCF7 breast cancer cells without affecting c-fos and c-jun
synthesis. J Biol Chem 268:1410314108[Abstract/Free Full Text]
-
Umayahara Y, Kawamori R, Watada H, Imano E, Iwama N, Morishima
T, Yamasaki Y, Kajimoto Y, Kamada T 1994 Estrogen regulation of the
insulin-like growth factor I gene transcription involves an AP-1
enhancer. J Biol Chem 269:1643316442[Abstract/Free Full Text]
-
Webb P, Lopez GN, Uht RM, Kushner PJ 1995 Tamoxifen activation
of the estrogen receptor/AP-1 pathway: potential origin for the
cell-specific estrogen-like effects of antiestrogens. Mol Endocrinol 9:443456[Abstract]
-
Paech K, Webb P, Kuiper GGJM, Nilsson S, Gustafsson JA,
Kushner PJ, Scanlan TS 1997 Differential ligand activation of estrogen
receptors ER
and ER ß at AP1 sites. Science 277:15081510[Abstract/Free Full Text]
-
Philips A, Teyssier C, Galtier F, Rivier-Covas C, Rey JM,
Rochefort H, Chalbos D 1998 FRA-1 expression level modulates regulation
of activator protein-1 activity by estradiol in breast cancer cells.
Mol Endocrinol 12:973985[Abstract/Free Full Text]
-
Yang N, Venugopalan M, Hardikar S, Glasebrook A 1996 Identification of an estrogen response element activated by metabolites
of 17-ß-estradiol and raloxifene. Science 273:12221225[Abstract]
-
Elgort MG, Zou A, Marschke KB, Allegretto EA 1996 Estrogen and
estrogen receptor antagonists stimulate transcription from the human
retinoic acid receptor-alpha 1 promoter via a novel sequence. Mol
Endocrinol 10:477487[Abstract]
-
Montano MM, Jaiswal AK, Katzenellenbogen BS 1998 Transcriptional regulation of the human quinone reductase gene by
antiestrogen-liganded estrogen receptor-
and estrogen receptor-ß.
J Biol Chem 273:2544325449[Abstract/Free Full Text]
-
Liu HL, Golder-Novoselsky E, Seto MH, Webster L, McClary J,
Zajchowski DA 1998 The novel estrogen-responsive B-box protein (EBBP)
gene is tamoxifen-regulated in cells expressing an estrogen receptor
DNA-binding domain mutant. Mol Endocrinol 12:17331748[Abstract/Free Full Text]
-
Xie W, Duan R, Safe S 1999 Estrogen induces adenosine
deaminase gene expression in MCF-7 human breast cancer cells: role of
estrogen receptor-Sp1 interactions. Endocrinology 40:219227
-
Xing W, Archer TK 1998 Upstream stimulatory factors mediate
estrogen receptor activation of the cathepsin D promoter. Mol
Endocrinol 12:13101321[Abstract/Free Full Text]
-
Kuiper GG, Carlsson B, Grandien K, Enmark E, Haggblad J,
Nilsson S, Gustafsson JA 1997 Comparison of the ligand binding
specificity and transcript tissue distribution of estrogen receptors
and ß. Endocrinology 138:863870[Abstract/Free Full Text]
-
McInerney EM, Weis KE, Sun J, Mosselman S, Katzenellenbogen BS 1998 Transcription activation by the human estrogen receptor subtype
ß (ERß) studied with ERß and ER
receptor chimeras.
Endocrinology 139:45134522[Abstract/Free Full Text]
-
Tremblay GB, Tremblay A, Labrie F, VG 1999 Dominant activity
of activation function 1 (AF-1) and differential stoichiometric
requirements for AF-1 and -2 in the estrogen receptor
-ß
heterodimeric complex. Mol Cell Biol 19:19191927[Abstract/Free Full Text]
-
Gronemeyer H, Benhamou B, Berry M, Bocquel MT, Gofflo D,
Garcia T, Lerouge T, Metzger D, Meyer ME, Tora L, Vergezac A, Chambon P 1992 Mechanisms of antihormone action. J Steroid Biochem Mol Biol 41:217221[CrossRef][Medline]
-
Parker MG 1993 Steroid and related receptors. Curr Opin Cell
Biol 5:499504[Medline]
-
Tora L, White J, Brou C, Tasset D, Webster N, Scheer E,
Chambon P 1989 The human estrogen receptor has two independent
nonacidic transcriptional activation functions. Cell 59:477487[Medline]
-
Lees JA, Fawell SE, Parker MG 1989 Identification of
constitutive and steroid-dependent transactivation domains in the mouse
oestrogen receptor. J Steroid Biochem 34:3339[CrossRef][Medline]
-
Tremblay GB, Tremblay A, Copeland NG, Gilbert DJ, Jenkins NA,
Labrie F, Giguere V 1997 Cloning, chromosomal localization, and
functional analysis of the murine estrogen receptor ß. Mol Endocrinol 11:353365[Abstract/Free Full Text]
-
Webb P, Nguyen P, Shinsako J, Anderson C, Feng W, Nguyen MP,
Chen D, Huang S-M, Subramanian S, McInerney E, Katzenellenbogen BS,
Stallcup MR, Kushner PJ 1998 Estrogen receptor activation function 1
works by binding p160 coactivator proteins. Mol Endocrinol 12:16051618[Abstract/Free Full Text]
-
Feng W, Ribeiro RC, Wagner RL, Nguyen H, Apriletti JW,
Fletterick RJ, Baxter JD, Kushner PJ, West BL 1998 Hormone-dependent
coactivator binding to a hydrophobic cleft on nuclear receptors.
Science 280:17471749[Abstract/Free Full Text]
-
Brzozowski AM, Pike AC, Dauter Z, Hubbard RE, Bonn T, Engstrom
O, Ohman L, Greene GL, Gustafsson JA, Carlquist M 1997 Molecular
basis of agonism and antagonism in the oestrogen receptor. Nature 389:753758[CrossRef][Medline]
-
Shiau AK, Barstad D, Loria PM, Cheng L, Kushner PJ, Agard DA,
Greene GL 1998 The structural basis of estrogen receptor/coactivator
recognition and the antagonism of this interaction by tamoxifen. Cell 95:927937[Medline]
-
Voegel JJ, Heine MJS, Tini M, Vivat V, Chambon P, Gronemeyer H 1998 The coactivator TIF2 contains three nuclear receptor-binding
motifs and mediates transactivation through CBP binding dependent and
independent pathways. EMBO J 17:507519[Abstract/Free Full Text]
-
Torchia J, Rose DW, Inostroza J, Kamei Y, Westin S, Glass CK,
Rosenfeld MG 1997 The transcriptional co-activator p/CIP binds CBP and
mediates nuclear-receptor function. Nature 387:677684[CrossRef][Medline]
-
Heery DM, Kalkhoven E, Hoare S, Parker MG 1997 A signature
motif in transcriptional co-activators mediates binding to nuclear
receptors. Nature 387:733736[CrossRef][Medline]
-
Ding XF, Anderson CM, Ma H, Hong H, Uht RM, Kushner PJ,
Stallcup MR 1998 Nuclear receptor-binding sites of coactivators
glucocorticoid receptor interacting protein1 (GRIP1) and steroid
receptor coactivator 1 (SRC-1): multiple motifs with different binding
specificities. Mol Endocrinol 12:302313[Abstract/Free Full Text]
-
Kalkhoven E, Valentine JE, Heery DM, Parker MG 1998 Isoforms
of steroid receptor co-activator 1 differ in their ability to
potentiate transcription by the oestrogen receptor. EMBO J 17:232243[Abstract/Free Full Text]
-
Tremblay A, Tremblay GB, Labrie F, Giguere V 1999 Ligand-independent recruitment of SRC-1 to estrogen receptor ß
through phosphorylation of activation function AF-1. Mol Cell 3:513519[Medline]
-
Darimont BD, Wagner RL, Apriletti JW, Stallcup MR, Kushner PJ,
Baxter JD, Fletterick RJ, Yamamoto KR 1998 Structure and specificity of
nuclear receptor-coactivator interactions. Genes Dev 12:33433356[Abstract/Free Full Text]
-
Smith CL, Nawaz Z, OMalley BW 1997 Coactivator and
corepressor regulation of the agonist/antagonist activity of the mixed
antiestrogen, 4-hydroxytamoxifen. Mol Endocrinol 11:657666[Abstract/Free Full Text]
-
Lavinsky RM, Jepsen K, Heinzel T, Torchia J, Mullen TM, Schiff
R, Del-Rio AL, Ricote M, Ngo S, Gemsch J, Hilsenbeck SG, Osborne CK,
Glass CK, Rosenfeld MG, Rose DW 1998 Diverse signaling pathways
modulate nuclear receptor recruitment of N-CoR and SMRT complexes. Proc
Natl Acad Sci USA 95:29202925[Abstract/Free Full Text]
-
Jackson TA, Richer JK, Bain DL, Takimoto GS, Tung L, Horwitz
KB 1997 The partial agonist activity of antagonist-occupied steroid
receptors is controlled by a novel hinge domain-binding coactivator
L7/SPA and the corepressors N-CoR or SMRT. Mol Endocrinol 11:693705[Abstract/Free Full Text]
-
Wagner BL, Norris JD, Knotts TA, Weigel NL, McDonnell DP 1998 The nuclear corepressors N-CoR and SMRT are key regulators of both
ligand- and 8-bromo-cyclic AMP-dependent transcriptional activity of
the human progesterone receptor. Mol Cell Biol 18:13691378[Abstract/Free Full Text]
-
Zhang X, Jeyakumar M, Petukhov S, Bagchi MK 1998 A nuclear
receptor corepressor modulates transcriptional activity of
antagonist-occupied steroid hormone receptor. Mol Endocrinol 12:513524[Abstract/Free Full Text]
-
Laherty CD, Billin AN, Lavinsky RM, Yochum GS, Bush AC, Sun
JM, Mullen TM, Davie JR, Rose DW, Glass CK, Rosenfeld MG, Ayer DE,
Eisenman RN 1998 SAP30, a component of the mSin3 corepressor complex
involved in N-CoR-mediated repression by specific transcription
factors. Mol Cell 2:3342[Medline]
-
Boruk M, Savory JG, Hache RJ 1998 AF-2-dependent potentiation
of CCAAT enhancer binding protein ß-mediated transcriptional
activation by glucocorticoid receptor. Mol Endocrinol 12:17491763[Abstract/Free Full Text]
-
Imakado S, Koike S, Kondo S, Sakai M, Muramatsu M 1991 The
N-terminal transactivation domain of rat estrogen receptor is localized
in a hydrophobic domain of eighty amino acids. J Biochem (Tokyo) 109:684689[Abstract]
-
Danielian PS, White R, Lees JA, Parker MG 1992 Identification
of a conserved region required for hormone dependent transcriptional
activation by steroid hormone receptors. EMBO J 11:10251033[Abstract]
-
Metzger D, Ali S, Bornert JM, Chambon P 1995 Characterization
of the amino-terminal transcriptional activation function of the human
estrogen receptor in animal and yeast cells. J Biol Chem 270:95359542[Abstract/Free Full Text]
-
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 271:2417224178[Abstract/Free Full Text]
-
Ali S, Metzger D, Bornert JM, Chambon P 1993 Modulation of
transcriptional activation by ligand-dependent phosphorylation of the
human oestrogen receptor A/B region. EMBO J 12:11531160[Abstract]
-
Le Goff P, Montano MM, Schodin DJ, Katzenellenbogen BS 1994 Phosphorylation of the human estrogen receptor. Identification of
hormone-regulated sites and examination of their influence on
transcriptional activity. J Biol Chem 269:44584466[Abstract/Free Full Text]
-
Lahooti H, White R, Danielian PS, Parker MG 1994 Characterization of ligand-dependent phosphorylation of the estrogen
receptor. Mol Endocrinol 8:182188[Abstract]
-
Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchiyama S, Sasaki H,
Masushige S, Gotoh Y, Nishida E, Kawashima H, Metzger D, Chambon P 1995 Activation of the estrogen receptor through phosphorylation by
mitogen-activated protein kinase. Science 270:14911494[Abstract]
-
Bunone G, Briand PA, Miksicek RJ, Picard D 1996 Activation of
the unliganded estrogen receptor by EGF involves the MAP kinase pathway
and direct phosphorylation. EMBO J 15:21742183[Abstract]
-
Trowbridge JM, Rogatsky I, Garabedian MJ 1997 Regulation of
estrogen receptor transcriptional enhancement by the cyclin A/Cdk2
complex. Proc Natl Acad Sci USA 94:1013210137[Abstract/Free Full Text]
-
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 activity of the anti-oestrogen 4-hydroxytamoxifen.
EMBO J 9:28112818[Abstract]
-
Kuiper GG, Gustafsson JA 1997 The novel estrogen
receptor-ß subtype: potential role in the cell- and
promoter-specific actions of estrogens and anti-estrogens. FEBS
Lett 410:8790[CrossRef][Medline]
-
Moore JT, McKee DD, Slentz-Kesler K, Moore LB, Jones SA, Horne
EL, Su JL, Kliewer SA, Lehmann JM, Willson TM 1998 Cloning and
characterization of human estrogen receptor ß isoforms. Biochem
Biophys Res Commun 247:7578[CrossRef][Medline]
-
Ogawa S, Inoue S, Watanabe T, Hiroi H, Orimo A, Hosoi T, Ouchi
Y, Muramatsu M 1998 The complete primary structure of human estrogen
receptor ß (hER ß) and its heterodimerization with ER
in
vivo and in vitro. Biochem Biophys Res Commun 243:122126[CrossRef][Medline]
-
Henttu PM, Kalkhoven E, Parker MG 1997 AF-2 activity and
recruitment of steroid receptor coactivator 1 to the estrogen receptor
depend on a lysine residue conserved in nuclear receptors. Mol Cell
Biol 17:18321839[Abstract]
-
Montano MM, Katzenellenbogen BS 1997 Identification of a novel
cis-acting element in the promoter of an estrogen-responsive gene
that modulates sensitivity to hormone and antihormone. Proc Natl Acad
Sci USA 94:25812585[Abstract/Free Full Text]
-
Zou A, Marschke KB, Arnold KE, Berger EM, Fitzgerald P, Mais
DE, Allegreto EA 1999 Estrogen receptor ß activates the human
retinoic acid receptor
-1 promoter in response to tamoxifen and
other estrogen receptor antagonists, but not in response to estrogen.
Mol Endocrinol 13:418430[Abstract/Free Full Text]
-
Hanstein B, Liu H, Yancisin MC, Brown M 1999 Functional
analysis of a novel estrogen receptor-ß isoform. Mol Endocrinol 13:129137[Abstract/Free Full Text]
-
Torchia J, Glass C, Rosenfeld MG 1998 Co-activators and
co-repressors in the integration of transcriptional responses. Curr
Opin Cell Biol 10:373383[CrossRef][Medline]
-
Reese JC, Katzenellenbogen BS 1992 Examination of the
DNA-binding ability of estrogen receptor in whole cells: implications
for hormone-independent transactivation and the actions of
antiestrogens. Mol Cell Biol 12:45314538[Abstract]
-
McDonnell DP, Clemm DL, Hermann T, Goldman ME, Pike JW 1995 Analysis of estrogen receptor function in vitro reveals
three distinct classes of antiestrogens. Mol Endocrinol 9:659669[Abstract]
-
Willson TM, Norris JD, Wagner BL, Asplin I, Baer P,
Brown HR, Jones SA, Henke B, Sauls H, Wolfe S, Morris DC, McDonnell DP 1997 Dissection of the molecular mechanism of action of GW5638, a
novel estrogen receptor ligand, provides insights into the role of
estrogen receptor in bone. Endocrinology 138:39013911[Abstract/Free Full Text]
-
Gradishar WJ, Jordan VC 1997 Clinical potential of new
antiestrogens. J Clin Oncol 15:840852[Abstract]
-
Jordan VC 1998 Antiestrogenic action of raloxifene and
tamoxifen: today and tomorrow. J Natl Cancer Inst 90:967971[Free Full Text]
-
Norris JD, Fan DJ, Wagner BL, McDonnell DP 1996 Identification
of the sequences within the human complement 3 promoter required for
estrogen responsiveness pro- vides insight into the mechanism of
tamoxifen mixed agonist activity. Mol Endocrinol 10:16051616[Abstract]
-
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 89:40374041[Abstract]
-
Sibonga JD, Dobnig H, Harden RM, Turner RT 1998 Effect of the
high-affinity estrogen receptor ligand ICI 182,780 on the rat tibia.
Endocrinology 139:37363742[Abstract/Free Full Text]
-
Kumar V, Green S, Stack G, Berry M, Jin JR, Chambon P 1987 Functional domains of the human estrogen receptor. Cell 51:941951[Medline]
-
Tora L, Mullick A, Metzger D, Ponglikitmongkol M, Park I,
Chambon P 1989 The cloned human oestrogen receptor contains a mutation
which alters its hormone binding properties. EMBO J 8:19811986[Abstract]
-
Sadovsky Y, Webb P, Lopez G, Baxter JD, Fitzpatrick PM, Gizang
GE, Cavailles V, Parker MG, Kushner PJ 1995 Transcriptional activators
differ in their responses to overexpression of TATA-box-binding
protein. Mol Cell Biol 15:15541563[Abstract]