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Molecular Oncology Group (G.B.T., A.T., V.G.) Royal Victoria
Hospital Montréal, Québec, Canada H3A 1A1
Departments of Biochemistry, Medicine, and Oncology (V.G.)
McGill University Montréal, Québec, Canada
Mammalian Genetics Laboratory (N.G.C., D.J.G., N.A.J.)
ABL-Basic Research Program National Cancer Institute-Frederick
Cancer Research and Development Center Frederick, Maryland
21702
Laboratory of Molecular Endocrinology (F.L.) Centre
Hospitalier de l’Université Laval Research Center Ste-Foy,
Québec, Canada G1V 4G2
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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. Mouse ERß binds to an inverted repeat spaced by three
nucleotides in a gel mobility shift assay and transactivates promoters
containing synthetic or natural estrogen response elements in an
estradiol (E2)-dependent manner. Scatchard
analysis indicates that mERß has slightly lower affinity for
E2 [dissociation constant
(Kd) = 0.5 nM] when
compared with mER (Kd = 0.2
nM). Antiestrogens, including
4-hydroxytamoxifen (OHT), ICI 182,780, and a novel compound, EM-800,
inhibit E2-dependent transactivation
efficiently. However, while OHT displays partial agonistic activity
with ER on a basal promoter linked to estrogen response elements in
Cos-1 cells, this effect is not observed with mERß. Cotransfection of
mERß and H-RasV12 causes enhanced activation
in the presence of E2. Mutagenesis of a serine
residue (position 60), located within a mitogen-activated protein
kinase consensus phosphorylation site abolishes the stimulatory effect
of Ras, suggesting that the activity of mERß is also regulated by the
mitogen-activated protein kinase pathway. Surprisingly, the coactivator
SRC-1 up-regulates mERß transactivation both in the absence and
presence of E2, and in vitro
interaction between SRC-1 and the ERß ligand-binding domain is
enhanced by E2. Moreover, the
ligand-independent stimulatory effect of SRC-1 on ERß transcriptional
activity is abolished by ICI 182,780, but not by OHT. Our results
demonstrate that while ERß shares many of the functional
characteristics of ER, the molecular mechanisms regulating the
transcriptional activity of mERß may be distinct from those of ER. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Additional signals that modulate nuclear receptor function have been described and commonly involve phosphorylation. Steroid receptors, including the progesterone and glucocorticoid receptors, have an increased state of phosphorylation upon ligand binding (7). Similar results have been described for ER where treatment of cells with peptide growth factors or agents that alter cellular cAMP levels result in up-regulation of the receptor coupled with an increase in its phosphorylation state (8, 9). These reports, as well as subsequent studies (10), demonstrated that antiestrogens also caused an increase in receptor phosphorylation albeit to a lesser extent than estradiol (E2), even though they inhibited transactivation. Deletion mapping and mutagenesis of human ER revealed that phosphorylation at Ser118 was required for full activity of AF-1 (10). Furthermore, this residue was shown to be a direct substrate for mitogen-activated protein (MAP) kinase, providing an in vivo link between estrogen action and the Ras-MAP kinase signaling cascade (11, 12). These results have begun to shed light on the molecular events responsible for regulation and proliferation of different cell types by ER in response to estrogens and growth factors.
Transactivation by nuclear receptors has recently been shown to be modulated by a growing family of coregulators (13). These include corepressors such as N-CoR and silencing mediator for retinoid and thyroid hormone receptors (SMRT) (14, 15, 16), which participate in the ligand-independent silencing functions of TR and retinoic acid receptor, and several coactivators: TRIP1/SUG-1 (17, 18), ERAP-140 (19), RIP-140 and RIP-160 (20, 21), TIF1 (22), and the related TIF2 (23), GRIP1 (24), SRC-1, and ERAP-160 (19, 25, 26). The LBD of ER was demonstrated to specifically interact with many of these coactivators, including steroid receptor coactivator-1 (SRC-1), ERAP-160, and RIP-140, and the strength of interaction was increased by E2 but not by the antiestrogens OHT and ICI 164,384 (20, 25, 27). The significance of these coactivators in nuclear receptor function is underscored by recent results demonstrating that SRC-related proteins interact with the nuclear integrators, CBP and p300, to augment nuclear receptor transactivation in mammalian cells (26, 28, 29). The activation domain of CBP can interact with TFIIB (30), thus providing evidence for a link between nuclear receptors and the basal transcription machinery. This growing network of interacting factors has increased our understanding of how steroids such as E2 are able to alter the expression of specific genes at the molecular level.
Although human and mouse ER cDNAs were cloned several years ago (31, 32), RT-PCR of rat prostate mRNA has revealed the presence of a second
ER, referred to as ERß (33). This novel receptor was found to bind
E2 with relatively high affinity and to be capable of
activating transcription of a reporter gene in the presence of this
ligand. In situ hybridization of rat tissues indicated that
ERß is expressed in female animals in primary, secondary, and mature
follicles as well as granulosa cells in the ovary and in the prostate
of male rat. As an initial step toward elucidating the physiological
role of this second ER and understanding its functional relationship
with ER, we report the cloning of the murine homolog of ERß. We
show that mERß is able to bind to an ERE in electrophoretic mobility
shift assays as well as to transactivate, in an
E2-dependent manner, promoters containing either synthetic
or natural EREs in Cos-1 and HeLa cells. In addition,
E2-induced mERß activity is inhibited by several
antiestrogens that have been previously shown to be selective for
ER. We also demonstrate that SRC-1 interacts in vitro
with the mERß LBD in a ligand-dependent manner. However, SRC-1
modulates mERß transcriptional activity in intact cells both in the
presence and absence of ligand. Finally, we show that the
E2-induced transcriptional activity of mERß is enhanced
by cotransfection with Ras. The functional differences observed between
the two ER isoforms are discussed.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). Alignment of mERß with mER indicates that
the receptors share 97% and 60% amino acid similarity in the DBD and
the LBD, respectively (Fig. 2). The AF-2 core sequences
in each receptor are virtually identical; however, no significant
regions of homology could be detected between the two amino-terminal
domains, a region of the protein that contains the AF-1 in ER. Two
potential MAP kinase phosphorylation sites (Ser60 and
Ser94) are present in the amino-terminal region of the
mERß.
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), up to 184 mice were typed for some pairs of markers.
Each locus was analyzed in pairwise combinations for recombination
frequencies using the additional data. The ratios of the total number
of mice exhibiting recombinant chromosomes to the total number of mice
analyzed for each pair of loci and the most likely gene order are:
centromere - Sos2 - 7/184 - Spnb1 - 0/181 -
Estrb 7/173 - Fos. The recombination frequencies
[expressed as genetic distances in centiMorgans (cM) ± the
SE] are - Sos2 - 3.8 ± 1.4 -
[Spnb1, Estrb] - 4.1 ± 1.5 -
Fos. No recombinants were detected between Spnb1
and Estrb in 181 animals typed in common, suggesting that
the two loci are within 1.7 cM of each other (upper 95% confidence
limit).
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, mouse
ovary expressed at least four predominant transcripts of approximately
3.5, 4.6, 7.2, and 9.5 kb and a weaker message of about 4.9 kb. In
contrast, only one transcript could be detected in ovary mRNA when
probed with the mER cDNA. Analysis of total RNA from several other
mouse tissues including liver, heart, kidney, skeletal muscle, thymus,
spleen, and brain were all negative for presence of the ERß
transcripts indicating that expression of ERß was below the level of
detection by Northern blotting in these tissues (data not shown).
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and ß indicated that the
receptors most likely bind to the same type of response element, namely
an inverted repeat spaced by three nucleotides (IR-3). We tested this
by conducting electrophoretic mobility shift assays (EMSA) with both
mouse receptors. Figure 5 demonstrates that both ER
isoforms produced in vitro by rabbit reticulocyte lysates
were able to bind specifically to a vitellogenin A2 (vitA2)-ERE (35)
probe. It was noted that the mERß did not bind as strongly to the
element as mER (compare lanes 2–5 with lanes 7–10) even though
both receptors were present at roughly the same level in the crude
lysates as shown by [35S]methionine-labeled proteins
(data not shown). In addition, both receptors were able to bind to the
pS2-ERE (36) but less efficiently than to the vitA2-ERE (data not
shown), which can most likely be accounted for by the fact that the
pS2-ERE contains an imperfect consensus sequence in the second
half-site.
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, lanes 3 and 8) nor
was there any effect on binding upon addition of specific antagonists
such as OHT (lanes 4 and 9) and ICI 182,780 (lanes 5 and 10), although
the migration rate of the protein/DNA complexes were differentially
altered by each ligand. However, as previously observed with ER
(37), a severe decrease in binding by ERß was observed when the
preincubation step was conducted at room temperature, 37 C, or for
shorter periods of time. The formation of the ERß/DNA complexes could
be partially restored in the presence of E2 at these
temperatures (data not shown).
Transcriptional Activity of mERß on Synthetic and Natural
Promoters
To test whether the vitA2-ERE could mediate mERß transcriptional
activity, we linked one copy of the ERE to either a basal
promoter containing a TATA box (BLuc) or to the more complex viral
thymidine kinase promoter (TKLuc) driving the expression of the
luciferase reporter gene for transactivation studies. In Cos-1 cells,
mERß induced a 5- to 10-fold response on all the ERE-containing
reporters tested when 10 nM E2 was added to the
medium (Fig. 6A). mER produced a slightly larger fold
induction of the ERE-containing reporters when studied under the same
transfection conditions (Fig. 6B). In addition,
E2-dependent transcriptional activity of mERß was also
observed in HeLa cells (Fig. 6C), suggesting a cell type- independent
effect. It is noteworthy that, as already observed in Cos-1 cells, the
levels of E2-induced activation in HeLa cells were somewhat
higher when mER was used as compared with mERß (Fig. 6D), although
it is not known whether similar levels of ER proteins were present in
these transient transfections.
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(36). As
shown in Fig. 6E, mERß was able to transactivate the pS2 promoter in
HeLa cells with a 6-fold induction when E2 was added. In
addition, OHT and ICI 182,780 efficiently blocked the
E2-induced effect of mERß on pS2. This interaction is
mediated through the ERE since its deletion abolished the response to
E2 (Fig. 6E).
Ligand Binding
To ascertain whether there was a dose dependency of E2
for mERß and mER, we tested the activity of both receptors in the
presence of increasing E2 concentrations using the
vitA2-ERE-TKLuc reporter in Cos-1 cells. Comparison of the
dose-response curves of Fig. 7A shows a shift of
approximately 4-fold of E2 concentration required to
achieve half of the maximal level of induction between the two
receptors. These results suggested that mERß may have lower affinity
for E2 than measured for mER. To verify if the
difference in E2 responsiveness was due to a difference in
ligand binding, we performed a binding analysis on both mERß and
mER. [3H[E2 was used to conduct binding
studies with mERß, and results were plotted by the method of
Scatchard. As shown in Fig. 7B, this analysis yielded an average
dissociation constant (Kd) of 0.5 nM for
E2 when performed on receptor prepared from rabbit
reticulocyte lysates. This value is comparable to that obtained for the
rat ERß, which was reported to be 0.6 nM (33). We
obtained an average Kd of 0.2 nM for mER
(Fig. 7C), which is well within the range of previously published
determinations for the cloned human receptor (38). Therefore, this
slightly reduced affinity of mERß for E2 may provide an
explanation for the shift in E2 responsiveness indicated by
the dose-response curves (Fig. 7A).
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by blocking its transcriptional activity. More
precisely, OHT is a mixed agonist-antagonist and blocks the activity of
AF-2 but not of AF-1, whereas ICI 164,384 and 182,780 are pure
antagonists that block both AF-1 and AF-2 activities (Refs. 6 and 39
and see below). We tested the effects of OHT and ICI 182,780 along with
other antagonist compounds on mERß transactivation, and the results
are shown in Fig. 8. All antagonists tested, including
OHT, ICI 182,780, hydroxy-toremifen, raloxifene, and EM-652 (the active
derivative of the novel nonsteroidal antiestrogen EM-800), effectively
inhibited E2 activity (Fig. 8A). In contrast to its
stimulatory effect on mER, OHT did not display an agonistic activity
on mERß when tested with a vitA2-ERE-BLuc reporter in either Cos-1
(Fig. 8B) and HeLa cells (data not shown).
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). Furthermore, when added at 10
nM and above, ICI 182,780 and OHT lowered mERß activity
even below its basal level (compared with untreated in Fig. 8C). We
observed a similar dose response for OHT and ICI 182,780 using either
receptor (Fig. 8, D and E). However, for OHT, there was an apparent
shift in the dose response toward the lower concentrations for mERß
as compared with mER (Fig. 8D).
Ras Enhances E2-Induced Transcriptional
Activity of mERß
Phosphorylation of serine residues, in particular
Ser118, has been shown to be necessary for maximal activity
of AF-1 in the hER and to mediate the effect of the
Ras-Raf-1-mitogen-activated protein kinase (MAPK) kinase and MAPK
pathway on the transcriptional activity of the ER (10, 11, 12, 40, 41). In
an attempt to investigate the effect of the activation of this pathway
on mERß activity, we used H-RasV12, a dominant active
form of H-Ras, in transactivation studies. As shown in Fig. 9A, H-RasV12 acted to further increase by a
factor of 3 the E2-induced activation of mERß using the
vitA2-ERE-TKLuc reporter in Cos-1 cells. ICI 182,780, but not OHT,
completely abolished the E2-dependent induction of mERß
(Fig. 9A) and mER (Fig. 9B) by Ras, suggesting that the effect of
Ras on mERß activity is mediated by a putative AF-1 present in the
amino-terminal domain. These results also show that ICI 182,780
suppresses transactivation mediated by both AF-1 and AF-2.
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). To determine whether one or both serine residues were
involved in the Ras-mediated induction, we mutated Ser60 to
Ala (S60A) and Ser94 to Ala (S94A) in mERß and used both
mutants in transactivation studies. Figure 9D shows that H-ras had no
inducing effect on the S60A mutant in the presence of E2
while the S94A mutant retained its responsiveness to H-ras as compared
with the wild type mERß (Fig. 9A). This suggests that
Ser60 in mERß is a potential target for phosphorylation
by the Ras-Raf-1-MAPK kinase-MAPK pathway.
SRC-1 Interacts with and Augments Transcriptional Activity of
mERß
The contribution of the AF-2 domain to mERß activity was
investigated by examining the effect of the coactivator SRC-1, which
has been shown to interact with a number of nuclear receptors including
hER (25, 43). Glutathione-S-transferase (GST) fusion proteins were
generated with both the mERß and mER LBDs (Fig. 10A) and tested in a GST-pull down experiment (Fig. 10B). GST-mERßEF and GST-mERDEF were expressed in
Escherichia coli, purified with GST-Sepharose, and incubated
with [35S]methionine-labeled SRC-1. As shown in Fig. 10B, the LBD of mERß interacted weakly with SRC-1 in the absence of
E2 (lane 3), whereas addition of E2 caused an
increase in interaction between the two proteins (lane 4). As expected,
estrogen antagonists that affect the AF-2 of ER, namely OHT (lane 5)
and ICI 182,780 (lane 6), do not promote a ligand-dependent SRC-1
interaction. The E2-dependent interaction with SRC-1 was
also efficiently blocked in the presence of 10-fold higher
concentration of the various antagonists (data not shown).
Protein-protein interactions between SRC-1 and the mER LBD
paralleled those observed with mERß (see Fig. 10B, lanes 8–12).
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). A similar
response was also seen with mER in the presence of E2
(Fig. 10C). However, SRC-1 alone efficiently increased the basal level
of transcriptional activity by mERß, suggesting a ligand-independent
effect of SRC-1 on mERß (Fig. 10C). The basal transcriptional level
of mER was not affected by the addition of SRC-1. We also studied
the effect of SRC-1 on the pS2 promoter in HeLa cells and found a
similar ligand-independent activation of mERß by SRC-1 as well as a
potentiation of the E2-induced activity (Fig. 10D). These
results suggest that SRC-1 does not act on mERß in a promoter- and
cell-specific manner. Furthermore, ICI 182,780, but not OHT, was able
to abrogate the ligand-independent effect of SRC-1 on mERß
transcriptional activity (Fig. 10, C and D). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, which, for many
years, has remained the only nuclear receptor known to have
E2 as a ligand. Recently, it has been shown that a second
receptor, ERß, is also able to respond to this hormone. ERß was
cloned by degenerative PCR from rat prostate (33) and more recently
from human testes (44), but its physiological role remains to be
elucidated. As a first step toward investigating the role of ERß in
development and homeostasis, we report the cloning of the murine
homolog. The availability of the murine cDNA provides us with the means
to characterize this receptor utilizing both biochemical and genetic
methodologies and will allow us to study its relationship, if any, with
ER in normal reproductive physiology. We first used a fragment of the LBD of the mouse ERß cDNA to establish the localization of the gene to chromosome 12. We have compared our interspecific map of chromosome 12 with a composite mouse linkage map location of many uncloned mouse mutations (provided from Mouse Genome Database, computerized database maintained at The Jackson Laboratory, Bar Harbor, ME). Estrb mapped in a region of the composite map that lacks mouse mutations with a phenotype that might be expected for an alteration in this locus (data not shown). The central region of mouse chromosome 12 shares a region of homology with human chromosome 14q, suggesting that Estrb will reside on 14q as well.
The mERß cDNA was used in a Northern analysis on murine ovary mRNA
that revealed that the mERß gene generates several transcripts as
compared with a single message for mER in this tissue. Partial cDNAs
obtained from an ovary library all contained a 3'-untranslated region
(UTR) of 657 bp followed by a polyA+ tail, suggesting that
the different transcripts seen in the Northern analysis may differ at
their 5'-ends or in the coding region. Although we were unable to
detect any messages in the total RNA from other mouse tissues, the
hERß homolog appears to be expressed in mRNA isolated from testes,
spleen, and thymus in addition to ovary (44).
The two mouse ER isoforms are closely related to each other in their
LBD and DBD, indicating that these receptors may regulate common gene
networks and respond to similar ligands. Using EMSA, a ligand-binding
assay, and transient transfection experiments, we showed that mERß
has slightly lower affinities than mER for ERE and E2
binding in vitro, but that mERß can transactivate reporter
genes driven by synthetic and natural E2-responsive
promoters in vivo as efficiently as mER in both Cos-1 and
HeLa cells. However, the two ERs display no sequence homology in their
amino-terminal domains, suggesting that each receptor may possess
distinct transactivation functions. We made use of the differential
mode of action of the estrogen antagonists OHT and ICI 182,780 and
activation of receptor activity by the Ras-Raf-1-MAPK pathway to
demonstrate functional similarities and differences between mERß and
mER. We first showed that, when assayed in Cos-1 cells with an
E2-responsive reporter gene driven by a basal promoter, OHT
does not display an agonistic activity with ERß. It has recently been
shown that a specific region of the mER AF-1 is required for OHT
agonism (45): the absence of a corresponding domain in mERß may
explain the present observation. We demonstrated that mERß, as
previously shown for ER (11, 12), can be activated by the
Ras-Raf-1-MAPKK-MAPK pathway. We have identified a serine residue in
mERß that could be the target of phosphorylation upon cotransfection
with Ras. This was demonstrated by the fact that a mutation at
Ser60 in the amino terminus of mERß eliminates the effect
seen with Ras. Although another serine residue at position 94 also
matches the MAP kinase consensus, mutation at this position has no
effect on Ras-mediated activation. Moreover, alignment of the mouse,
rat, and human amino acid sequences in this region indicates that
Ser60 is conserved in all species whereas Ser94
is replaced by a glycine in the human ERß. Finally, we showed that
the induction of the E2-dependent activation of mERß by
Ras can be abolished by ICI 182,780, but not OHT, an observation that
provides further evidence of the involvement of an AF-1-like domain in
regulating mERß functions.
Antiestrogens play an important role in the treatment of breast cancer.
We therefore monitored the efficacy of a series of antiestrogens
previously shown to be selective for ER on mERß and found that all
compounds tested, including OHT, ICI 182,780, hydroxy-toremifen,
raloxifen, and the novel nonsteroidal antiestrogen EM-800, inhibited
E2-dependent activation by mERß. We showed that OHT is
also AF-2 selective on mERß, and that ICI 182,780 inhibits both
activation functions, displays no estrogenic activity, and thus can be
considered as a pure antagonist of mERß activity. However, in
dose-response studies, OHT proved to be a more potent inhibitor of
mERß than of mER. In addition, we show that the mode of action of
ICI 182,780, as previously observed for ICI 164,384, involves the
inhibition of both AF-1 and AF-2 activity of ER.
A growing network of coactivators that interact with ER has now been
cloned. These include RIP140 (20), TIF2 (23), and SRC-1 (25). However,
only SRC-1 has been shown to up-regulate ER-stimulated transcription
(25, 43). mERß transcriptional activity could also be stimulated by
SRC-1 in a ligand-dependent manner in cotransfection assays as well as
in vitro where we observed a very strong ligand-dependent
interaction with a GST-mERß LBD fusion protein. Surprisingly, we also
observed ligand-independent SRC-1 enhancement of mERß transcriptional
activity. These results suggest that SRC-1 may interact with a region,
other than the AF-2, of the mERß protein. McInerney et al.
(43) have shown that constructs expressing the ABCD (no LBD) and EF
(LBD only) domains of hER separately are able to transactivate in
the presence of E2, and that SRC-1 can enhance the level of
activation under these conditions. These results led them to suggest
that SRC-1 may act as an adapter to promote AF-1 and AF-2 receptor
activities and are in agreement with our suggestion that SRC-1 may
interact with several regions of nuclear receptor proteins.
The identification of a second ER in mammals is very exciting, and
these and previous studies have only begun to define the putative role
that it plays in the mediation of estrogen action. Clearly, further
studies into the mechanism of action of ERß are required if we are to
understand how it functions in vivo. The results we present
in this paper indicate that ERß and may have both redundant and
distinct functions, as exemplified by the manner with which they
respond to OHT, Ras, and SRC-1, and suggest that ERß plays an unique
role in the physiological actions of natural estrogens.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Chromosomal Localization of the ERß Gene
Interspecific backcross progeny were generated by mating
(C57BL/6J x M. spretus) F1 females and C57BL/6J males
as described (34). A total of 205 N2 mice were used to map
the Estrb locus (see text for details). DNA isolation,
restriction enzyme digestion, agarose gel electrophoresis, Southern
blot transfer, and hybridization were performed essentially as
described (46). All blots were prepared with Hybond N+
nylon membrane (Amersham). The probe, a 550-bp
HindIII/EcoRI fragment of the mouse cDNA, was
labeled with [32P]--dCTP using a random-primed
labeling kit (Stratagene); washing was done to a final stringency of
0.8x SSCP, 0.1% SDS, at 65 C. Fragments of 13.5 and 5.5 kb were
detected in EcoRI-digested C57BL/6J DNA, and fragments of
8.4 and 5.1 kb were detected in EcoRI-digested M.
spretus DNA. The presence or absence of the 8.4- and 5.1-kb
EcoRI M. spretus-specific fragments, which
cosegregated, was followed in the backcross mice.
A description of the probes and RFLPs for the loci linked to Estrb including Sos2, Spnb1, and Fos has been reported previously (47). Recombination distances were calculated as described (48) using the computer program SPRETUS MADNESS. Gene order was determined by minimizing the number of recombination events required to explain the allele distribution patterns.
Northern Analysis
Five micrograms of mouse ovary poly-A+ RNA were
electrophoresed on a 1% formaldehyde-agarose gel and blotted onto
Hybond-N+. The entire mouse ER cDNA (49) and the 550-bp
PCR product encoding mERß were radiolabeled with
[32P]--dCTP (Amersham, Arlington Heights, IL) by
random priming (Pharmacia). Prehybridization was carried out for 4
h in 50% formamide, 5xSSPE, 5x Denhardt’s solution, 1% glycine,
and 100 µg/ml denatured salmon sperm DNA at 42 C. Hybridization was
conducted overnight at the same temperature in 50% formamide, 5x
SSPE, 1x Denhardt’s solution, 10% dextran sulfate, 0.3% SDS, 100
µg/ml denatured salmon sperm DNA, and 2 x 106
cpm/ml denatured probe. The membrane was washed to high stringency
(0.1x SSC, 0.1% SDS at 65 C for 30 min) and exposed to X-OMAT AR film
(Kodak) overnight at -85 C. The positions of the 18S and 28S rRNAs
were determined by the electrophoresis of total mouse ovary RNA on the
same gel.
Plasmids
The expression vector pCMX-mER was constructed by ligating a
1.9-kb NaeI-EcoRI fragment of mER (49) into
the appropriate sites of the eukaryotic expression vector, pCMX (50).
pCMX-mERß was constructed in a similar fashion by subcloning the
2.1-kb SalI-EcoRI fragment described above into
pCMX. Fusion proteins were generated between the mERß and mER LBDs
and GST by subcloning fragments of the mouse cDNAs into the pGEX-2T
expression vector (Pharmacia). Briefly, a unique PvuII site
located at position 678 and an EcoRI site in the vector were
used to excise the LBD (domains E and F) from the full-length mERß
cDNA. The fragment was then subcloned into the SmaI site of
pGEX to produce pGST-mERßEF. For mER, the hinge region and the LBD
(domains D, E, and F) were amplified from the full length mER cDNA
using PCR and subcloned directionally into the BamHI and the
blunted EcoRI sites of the pGEX vector (pGST-mERDEF) (see
Fig. 9A). Both constructs were verified to be in-frame with GST by
sequencing. vitA2-ERE-BLuc and vitA2-ERE-TKLuc were constructed by
ligating the vitA2-ERE oligonucleotide (see below for sequence) into
SalI-BamHI-digested TKLuc vector. pS2-ERELuc
contains the 
1050-bp pS2 promoter (36) preceding the luciferase
reporter of pGL3 (Promega, Madison, WI). The pS2
ERELuc, in which the
ERE was replaced by sequences encoding
EcoRI-EcoRV sites, was generated by PCR
mutagenesis using the ExSite kit from Stratagene as described by the
manufacturer. The serine to alanine mutants at positions 60 and 94 of
mERß were also generated by PCR mutagenesis. The oligonucleotides
used were: S60A,
5'-CTCTATGCAGAACCTCAAAAGGCTCCTTGGTGTGAAGC-3'; S94A,
5'-GGTTGTGCCAGCCCTGTTACTGCTCCAAGCGCCAAGAGG-3'. The
H-RasV12 expression plasmid was a generous gift from Dr.
Morag Park, McGill University.
Chemicals
E2 was obtained from Sigma Chemical Co. (St. Louis,
MO). EM-800, EM-652, ICI-182780, and OH-toremifene were synthesized in
the medicinal chemistry division of the Laboratory of Molecular
Endocrinology, CHUL Research Center, Québec, Canada. OHT was
kindly provided by Dr. D. Salin-Drouin, Besins-Iscovesco, Paris,
France.
EMSA
mERß and mER proteins were synthesized by in
vitro transcription-translation using rabbit reticulocyte lysates
(Promega) and pCMX-mERß and pCMX-mER, respectively, as templates.
Typically 5 µl of programmed lysate were used in each binding
reaction. DNA-binding reactions were carried out in binding buffer (5
mM Tris, pH 8.0, 40 mM KCl, 6% glycerol, 1
mM dithiothreitol, 0.05% Nonidet P-40), 2 µg of
poly(deoxyinosinic-deoxycytidylic)acid, 0.1 µg of denatured salmon
sperm DNA, and 10 µg of BSA with 0.1 ng probe that was labeled by
end-filling with Klenow in the presence of
[32P]--dCTP. Preincubations containing ligand and/or
cold competitor as indicated were conducted on ice for 30 min, after
which the probe was added and allowed to bind for 30 min at room
temperature. The entire reaction (20 µl) was loaded onto a 4%
polyacrylamide gel and electrophoresed at 150 V at room temperature.
Gels were dried and exposed overnight at -85 C. The following
oligonucleotides and their compliments were used as probes and
competitors: vitA2-ERE,
5'-TCGACAAAGTCAGGTCACAG-TGACCTGATCAAG-3' (51);
pS2-ERE,
5'TCGACCCTGCAAGGTCACGGTGGCCA-CCCCGTG-3' (36);
IR3,
5'-TCGACGTGTAGGTCA-CAGTGACCTCTTCA-3'.
Scatchard Analysis
Ligand binding studies were conducted essentially as previously
described (52) with the following modifications. mERß and mER were
produced using rabbit reticulocyte lysates, diluted 12-fold in TEG
buffer (10 mM Tris, pH 7.5, 1.5 mM EDTA, 10%
glycerol) and kept on ice until use. One hundred microliters of this
dilution were used in each binding reaction at 4 C overnight containing
[2,4,6,7-3H]17ß-estradiol concentrations ranging from
0.01–3 nM. Nonspecific binding was assessed by including
200-fold excess E2 in a parallel set of samples. Unbound
steroids were removed with dextran-coated charcoal and counts per min
were determined by liquid scintillation counting.
Cell Culture, DNA Transfection, and Luciferase Assay
For transfection, Cos-1 and HeLa cells were seeded in six-well
plates in phenol red-free DMEM (GIBCO BRL, Gaithersburg, MD)
supplemented with 10% charcoal dextran-treated FBS, and 100 µg/ml
penicillin and 100 µg/ml streptomycin. At 50–60% confluency, cells
were transfected with 1–2 µg of reporter plasmid, 0.5–1 µg
receptor expression vector, 1 µg CMX-ßgal or RSV-ßgal, and 6–7.5
µg pBluescriptKSII, using the calcium phosphate/DNA precipitation
method (53). After 8–16 h, cells were washed and typically 10
nM E2 or 100 nM antiestrogens,
unless otherwise stated, were added to the growth medium for 16 h.
For luciferase assay, cells were lysed in potassium phosphate buffer
containing 1% Triton X-100, and light emission was detected using a
luminometer after addition of luciferin. Values are expressed as
arbitrary light units normalized to the ß-galactosidase activity of
each sample.
In Vitro Protein-Protein Interactions
Fusion proteins were expressed in E. coli DH5 as
follows. An overnight culture was diluted 10-fold in 500 ml prewarmed
LB containing 100 µg/ml ampicillin and incubated for 1 h at 37
C. Isopropyl-ß-D-thiogalactopyranoside was added to a
final concentration of 1 mM to induce expression, and the
culture was allowed to grow for a further 3 h. Cells were cooled
on ice for 10 min and centrifuged at 2500 x g for 20
min. The pellet was resuspended in 12 ml ice-cold PBS and sonicated.
After the addition of one tenth volume of 10% triton, the extract was
centrifuged at 12,000 x g for 20 min at 4 C. Clarified
extracts were aliquoted in 1-ml samples and frozen at -85 C.
The 3.3-kb SRC-1 cDNA encoding the predicted 1061 amino acid open reading frame as originally cloned (25) was digested with XbaI and SalI, blunt ended with Klenow, ligated into pCMX, and labeled with [35S]methionine (Amersham) in vitro using rabbit reticulocyte lysates as described above. Approximately 300 µg of total protein from extracts containing the GST fusion proteins were loaded onto glutathione-Sepharose 4B (Pharmacia) for 30 min at 4 C with gentle agitation. After a short centrifugation, the beads were resuspended in 150 µl IPAB buffer (20) containing 10 nM ligand or antagonists and 4 µl of labeled SRC-1 crude lysate and allowed to bind for 90 min at 4 C. Beads were washed twice in the presence of IPAB and twice with IPAB without BSA, dried briefly, and resuspended in 30 µl loading buffer. Bound proteins were analyzed by SDS-PAGE. The gel was treated with Amplify (Amersham), dried, and exposed at -85 C.
|
ACKNOWLEDGMENTS |
|---|
and human SRC-1
cDNAs and M. Park for the gift of the H-RasV12 expression
vector. We are grateful to L. McBroom and R. Sladek for their
suggestions and comments on the manuscript. |
FOOTNOTES |
|---|
Financial support was provided by the Medical Research Council of Canada, the National Cancer Institute of Canada, and the Cancer Research Society, Inc., to V. Giguère. G. B. Tremblay is a Postdoctoral fellow and V. Giguère a Scientist of the Medical Research Council of Canada. This work was also supported by EndoRecherche Inc.
Received for publication December 6, 1996. Revision received December 23, 1996. Accepted for publication December 30, 1996.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
expressed in the mouse embryo. Mol Cell Biol 10:2335–2340This article has been cited by other articles:
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|
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|
|
|
|
|
|
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|
|
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|
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|
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|
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|
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|
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|
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|
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|
|
|
|
 |
|
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|
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|
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|
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|
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|
|
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|
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|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
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|
|
|
|
 |
|
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|
|
|
|
 |
|
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|
|
|
|
 |
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
 |
|
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|
|
|
|
|
|
U. Ohnemus, M. Uenalan, F. Conrad, B. Handjiski, L. Mecklenburg, M. Nakamura, J. Inzunza, J.-A. Gustafsson, and R. Paus Hair Cycle Control by Estrogens: Catagen Induction via Estrogen Receptor (ER)-{alpha} Is Checked by ER{beta} Signaling Endocrinology, March 1, 2005; 146(3): 1214 - 1225. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
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|
|
|
|
|
|
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|
|
|
|
|
|
K. Stokes, B. Alston-Mills, and C. Teng Estrogen response element and the promoter context of the human and mouse lactoferrin genes influence estrogen receptor {alpha}-mediated transactivation activity in mammary gland cells J. Mol. Endocrinol., October 1, 2004; 33(2): 315 - 334. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A.G.B. Hurst, D.W. Goad, M. Mohan, and J.R. Malayer Independent Downstream Gene Expression Profiles in the Presence of Estrogen Receptor {alpha} or {beta} Biol Reprod, October 1, 2004; 71(4): 1252 - 1261. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A Bardin, N Boulle, G Lazennec, F Vignon, and P Pujol Loss of ER{beta} expression as a common step in estrogen-dependent tumor progression Endocr. Relat. Cancer, September 1, 2004; 11(3): 537 - 551. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. E. Aiyar, J.-l. Sun, A. L. Blair, C. A. Moskaluk, Y.-z. Lu, Q.-n. Ye, Y. Yamaguchi, A. Mukherjee, D.-m. Ren, H. Handa, et al. Attenuation of estrogen receptor {alpha}-mediated transcription through estrogen-stimulated recruitment of a negative elongation factor Genes & Dev., September 1, 2004; 18(17): 2134 - 2146. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. H. Smith, S. R. Coats, H. Qin, M. S. Petrie, J. W. Covington, M. Su, M. Eren, and D. E. Vaughan Differential and Opposing Regulation of PAI-1 Promoter Activity by Estrogen Receptor {alpha} and Estrogen Receptor {beta} in Endothelial Cells Circ. Res., August 6, 2004; 95(3): 269 - 275. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. B. Hawkins and P. Thomas The Unusual Binding Properties of the Third Distinct Teleost Estrogen Receptor Subtype ER{beta}a Are Accompanied by Highly Conserved Amino Acid Changes in the Ligand Binding Domain Endocrinology, June 1, 2004; 145(6): 2968 - 2977. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Laganiere, G. B. Tremblay, C. R. Dufour, S. Giroux, F. Rousseau, and V. Giguere A Polymorphic Autoregulatory Hormone Response Element in the Human Estrogen-related Receptor {alpha} (ERR{alpha}) Promoter Dictates Peroxisome Proliferator-activated Receptor {gamma} Coactivator-1{alpha} Control of ERR{alpha} Expression J. Biol. Chem., April 30, 2004; 279(18): 18504 - 18510. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Labrie, P. Champagne, C. Labrie, J. Roy, J. Laverdiere, L. Provencher, M. Potvin, Y. Drolet, M. Pollak, L. Panasci, et al. Activity and Safety of the Antiestrogen EM-800, the Orally Active Precursor of Acolbifene, in Tamoxifen-Resistant Breast Cancer J. Clin. Oncol., March 1, 2004; 22(5): 864 - 871. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. D. Toran-Allerand Minireview: A Plethora of Estrogen Receptors in the Brain: Where Will It End? Endocrinology, March 1, 2004; 145(3): 1069 - 1074. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Labrie, M. El-Alfy, L. Berger, C. Labrie, C. Martel, A. Belanger, B. Candas, and G. Pelletier The Combination of a Novel Selective Estrogen Receptor Modulator with an Estrogen Protects the Mammary Gland and Uterus in a Rodent Model: The Future of Postmenopausal Women's Health? Endocrinology, November 1, 2003; 144(11): 4700 - 4706. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Geraldes, M. G. Sirois, and J.-F. Tanguay Specific Contribution of Estrogen Receptors on Mitogen-Activated Protein Kinase Pathways and Vascular Cell Activation Circ. Res., September 5, 2003; 93(5): 399 - 405. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Otsuki, H. Gao, K. Dahlman-Wright, C. Ohlsson, N. Eguchi, Y. Urade, and J.-A. Gustafsson Specific Regulation of Lipocalin-Type Prostaglandin D Synthase in Mouse Heart by Estrogen Receptor {beta} Mol. Endocrinol., September 1, 2003; 17(9): 1844 - 1855. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. S. A. Samy, R. Zheng, T. Matsutani, L. W. Rue III, K. I. Bland, and I. H. Chaudry Mechanism for normal splenic T lymphocyte functions in proestrus females after trauma: enhanced local synthesis of 17{beta}-estradiol Am J Physiol Cell Physiol, July 1, 2003; 285(1): C139 - C149. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Greco, L. S. Lubbers, and J. D. Blaustein Estrogen Receptor {beta} Messenger Ribonucleic Acid Expression in the Forebrain of Proestrous, Pregnant, and Lactating Female Rats Endocrinology, May 1, 2003; 144(5): 1869 - 1875. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Bai and V. Giguere Isoform-Selective Interactions between Estrogen Receptors and Steroid Receptor Coactivators Promoted by Estradiol and ErbB-2 Signaling in Living Cells Mol. Endocrinol., April 1, 2003; 17(4): 589 - 599. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. A. Orihuela, A. Parada-Bustamante, P. P. Cortes, C. Gatica, and H. B. Croxatto Estrogen Receptor, Cyclic Adenosine Monophosphate, and Protein Kinase A Are Involved in the Nongenomic Pathway by Which Estradiol Accelerates Oviductal Oocyte Transport in Cyclic Rats Biol Reprod, April 1, 2003; 68(4): 1225 - 1231. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. W. Cousins, M. E. Marin-Castano, D. G. Espinosa-Heidmann, A. Alexandridou, L. Striker, and S. Elliot Female Gender, Estrogen Loss, and Sub-RPE Deposit Formation in Aged Mice Invest. Ophthalmol. Vis. Sci., March 1, 2003; 44(3): 1221 - 1229. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Lu, H. K. Surks, H. Ebling, W. E. Baur, D. Brown, D. C. Pallas, and R. H. Karas Regulation of Estrogen Receptor alpha -mediated Transcription by a Direct Interaction with Protein Phosphatase 2A J. Biol. Chem., February 7, 2003; 278(7): 4639 - 4645. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Maira, C. Martens, E. Batsche, Y. Gauthier, and J. Drouin Dimer-Specific Potentiation of NGFI-B (Nur77) Transcriptional Activity by the Protein Kinase A Pathway and AF-1-Dependent Coactivator Recruitment Mol. Cell. Biol., February 1, 2003; 23(3): 763 - 776. [Abstract] [Full Text]
|
|
|
|
|
|
M. E. Marin-Castano, S. J. Elliot, M. Potier, M. Karl, L. J. Striker, G. E. Striker, K. G. Csaky, and S. W. Cousins Regulation of Estrogen Receptors and MMP-2 Expression by Estrogens in Human Retinal Pigment Epithelium Invest. Ophthalmol. Vis. Sci., January 1, 2003; 44(1): 50 - 59. [Abstract] [Full Text] [PDF]
|
|
|
|
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 A. J. Jakimiuk, S. R. Weitsman, H.-W. Yen, M. Bogusiewicz, and D. A. Magoffin Estrogen Receptor {alpha} and {beta} Expression in Theca and Granulosa Cells from Women with Polycystic Ovary Syndrome J. Clin. Endocrinol. Metab., December 1, 2002; 87(12): 5532 - 5538. [Abstract] [Full Text] [PDF]
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D. A. Schreihofer, D. F. Rowe, E. F. Rissman, E. M. Scordalakes, J.-a. Gustafsson, and M. A. Shupnik Estrogen Receptor-{alpha} (ER{alpha}), But Not ER{beta}, Modulates Estrogen Stimulation of the ER{alpha}-Truncated Variant, TERP-1 Endocrinology, November 1, 2002; 143(11): 4196 - 4202. [Abstract] [Full Text] [PDF]
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N. Vasudevan, S. Ogawa, and D. Pfaff Estrogen and Thyroid Hormone Receptor Interactions: Physiological Flexibility by Molecular Specificity Physiol Rev, October 1, 2002; 82(4): 923 - 944. [Abstract] [Full Text] [PDF]
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H. T. Kim, B. C. Kim, I. Y. Kim, M. Mamura, D. H. Seong, J.-J. Jang, and S.-J. Kim Raloxifene, a Mixed Estrogen Agonist/Antagonist, Induces Apoptosis through Cleavage of BAD in TSU-PR1 Human Cancer Cells J. Biol. Chem., August 30, 2002; 277(36): 32510 - 32515. [Abstract] [Full Text] [PDF]
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P. Yi, S. Bhagat, R. Hilf, R. A. Bambara, and M. Muyan Differences in the Abilities of Estrogen Receptors to Integrate Activation Functions Are Critical for Subtype-Specific Transcriptional Responses Mol. Endocrinol., August 1, 2002; 16(8): 1810 - 1827. [Abstract] [Full Text] [PDF]
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N. Sharifi, A. E. Reuss, and S. Wray Prenatal LHRH Neurons in Nasal Explant Cultures Express Estrogen Receptor {beta} Transcript Endocrinology, July 1, 2002; 143(7): 2503 - 2507. [Abstract] [Full Text] [PDF]
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M. J. Evans, K. Lai, L. J. Shaw, D. C. Harnish, and C. C. Chadwick Estrogen Receptor {alpha} Inhibits IL-1{beta} Induction of Gene Expression in the Mouse Liver Endocrinology, July 1, 2002; 143(7): 2559 - 2570. [Abstract] [Full Text] [PDF]
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F. Gay, P. Barath, C. Desbois-Le Peron, R. Metivier, R. Le Guevel, D. Birse, and G. Salbert Multiple Phosphorylation Events Control Chicken Ovalbumin Upstream Promoter Transcription Factor I Orphan Nuclear Receptor Activity Mol. Endocrinol., June 1, 2002; 16(6): 1332 - 1351. [Abstract] [Full Text] [PDF]
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P. Yang, A. Kriatchko, and S. K. Roy Expression of ER-{alpha} and ER-{beta} in the Hamster Ovary: Differential Regulation by Gonadotropins and Ovarian Steroid Hormones Endocrinology, June 1, 2002; 143(6): 2385 - 2398. [Abstract] [Full Text] [PDF]
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A. Menuet, E. Pellegrini, I. Anglade, O. Blaise, V. Laudet, O. Kah, and F. Pakdel Molecular Characterization of Three Estrogen Receptor Forms in Zebrafish: Binding Characteristics, Transactivation Properties, and Tissue Distributions Biol Reprod, June 1, 2002; 66(6): 1881 - 1892. [Abstract] [Full Text] [PDF]
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J. M. Hall, D. P. McDonnell, and K. S. Korach Allosteric Regulation of Estrogen Receptor Structure, Function, and Coactivator Recruitment by Different Estrogen Response Elements Mol. Endocrinol., March 1, 2002; 16(3): 469 - 486. [Abstract] [Full Text] [PDF]
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S. Raghow, M. Z. Hooshdaran, S. Katiyar, and M. S. Steiner Toremifene Prevents Prostate Cancer in the Transgenic Adenocarcinoma of Mouse Prostate Model Cancer Res., March 1, 2002; 62(5): 1370 - 1376. [Abstract] [Full Text] [PDF]
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J. R. Schultz, M. A. Loven, V. M. S. Melvin, D. P. Edwards, and A. M. Nardulli Differential Modulation of DNA Conformation by Estrogen Receptors alpha and beta J. Biol. Chem., March 1, 2002; 277(10): 8702 - 8707. [Abstract] [Full Text] [PDF]
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A. A. Kowalski, L. G. Graddy, D. S. Vale-Cruz, I. Choi, B. S. Katzenellenbogen, F. A. Simmen, and R. C.M. Simmen Molecular Cloning of Porcine Estrogen Receptor-{beta} Complementary DNAs and Developmental Expression in Periimplantation Embryos Biol Reprod, March 1, 2002; 66(3): 760 - 769. [Abstract] [Full Text] [PDF]
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 C. A. Lamartiniere, J. Wang, M. Smith-Johnson, and I.-E. Eltoum Daidzein: Bioavailability, Potential for Reproductive Toxicity, and Breast Cancer Chemoprevention in Female Rats Toxicol. Sci., February 1, 2002; 65(2): 228 - 238. [Abstract] [Full Text] [PDF]
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P. H. Driggers, J. H. Segars, and D. M. Rubino The Proto-oncoprotein Brx Activates Estrogen Receptor beta by a p38 Mitogen-activated Protein Kinase Pathway J. Biol. Chem., December 7, 2001; 276(50): 46792 - 46797. [Abstract] [Full Text] [PDF]
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 B. S. McEwen Genome and Hormones: Gender Differences in Physiology: Invited Review: Estrogens effects on the brain: multiple sites and molecular mechanisms J Appl Physiol, December 1, 2001; 91(6): 2785 - 2801. [Abstract] [Full Text] [PDF]
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B. Greco, E. A. Allegretto, M. J. Tetel, and J. D. Blaustein Coexpression of ER{beta} with ER{alpha} and Progestin Receptor Proteins in the Female Rat Forebrain: Effects of Estradiol Treatment Endocrinology, December 1, 2001; 142(12): 5172 - 5181. [Abstract] [Full Text] [PDF]
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A. Warnmark, A. Wikstrom, A. P. H. Wright, J.-A. Gustafsson, and T. Hard The N-terminal Regions of Estrogen Receptor alpha and beta Are Unstructured in Vitro and Show Different TBP Binding Properties J. Biol. Chem., November 30, 2001; 276(49): 45939 - 45944. [Abstract] [Full Text] [PDF]
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E. R. Levin Genome and Hormones: Gender Differences in Physiology: Invited Review: Cell localization, physiology, and nongenomic actions of estrogen receptors J Appl Physiol, October 1, 2001; 91(4): 1860 - 1867. [Abstract] [Full Text] [PDF]
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R. H. Karas, H. Schulten, G. Pare, M. J. Aronovitz, C. Ohlsson, J.-A. Gustafsson, and M. E. Mendelsohn Effects of Estrogen on the Vascular Injury Response in Estrogen Receptor {alpha},{beta} (Double) Knockout Mice Circ. Res., September 14, 2001; 89(6): 534 - 539. [Abstract] [Full Text] [PDF]
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 P. Devanesan, R. J. Santen, W. P. Bocchinfuso, K. S. Korach, E. G. Rogan, and E. Cavalieri Catechol estrogen metabolites and conjugates in mammary tumors and hyperplastic tissue from estrogen receptor-{alpha} knock-out (ERKO)/Wnt-1 mice: implications for initiation of mammary tumors Carcinogenesis, September 1, 2001; 22(9): 1573 - 1576. [Abstract] [Full Text] [PDF]
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D. Lu, Y. Kiriyama, K. Y. Lee, and V. Giguere Transcriptional Regulation of the Estrogen-inducible pS2 Breast Cancer Marker Gene by the ERR Family of Orphan Nuclear Receptors Cancer Res., September 1, 2001; 61(18): 6755 - 6761. [Abstract] [Full Text] [PDF]
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D. A. Schreihofer, E. M. Resnick, V. Y. Lin, and M. A. Shupnik Ligand-Independent Activation of Pituitary ER: Dependence on PKA-Stimulated Pathways Endocrinology, August 1, 2001; 142(8): 3361 - 3368. [Abstract] [Full Text] [PDF]
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C. M. Klinge Estrogen receptor interaction with estrogen response elements Nucleic Acids Res., July 15, 2001; 29(14): 2905 - 2919. [Abstract] [Full Text] [PDF]
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 I. Leav, K.-M. Lau, J. Y. Adams, J. E. McNeal, M.-E. Taplin, J. Wang, H. Singh, and S.-M. Ho Comparative Studies of the Estrogen Receptors {beta} and {{alpha}} and the Androgen Receptor in Normal Human Prostate Glands, Dysplasia, and in Primary and Metastatic Carcinoma Am. J. Pathol., July 1, 2001; 159(1): 79 - 92. [Abstract] [Full Text] [PDF]
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H. Cardenas, K.A. Burke, R.M. Bigsby, W.F. Pope, and K.P. Nephew Estrogen Receptor {beta} in the Sheep Ovary During the Estrous Cycle and Early Pregnancy Biol Reprod, July 1, 2001; 65(1): 128 - 134. [Abstract] [Full Text] [PDF]
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L. O'Donnell, K. M. Robertson, M. E. Jones, and E. R. Simpson Estrogen and Spermatogenesis Endocr. Rev., June 1, 2001; 22(3): 289 - 318. [Abstract] [Full Text] [PDF]
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S. F. Palter, A. B. Tavares, A. Hourvitz, J. D. Veldhuis, and E. Y. Adashi Are Estrogens of Import to Primate/Human Ovarian Folliculogenesis? Endocr. Rev., June 1, 2001; 22(3): 389 - 424. [Abstract] [Full Text] [PDF]
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K. S. Bramlett, Y. Wu, and T. P. Burris Ligands Specify Coactivator Nuclear Receptor (NR) Box Affinity for Estrogen Receptor Subtypes Mol. Endocrinol., June 1, 2001; 15(6): 909 - 922. [Abstract] [Full Text] [PDF]
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C. Guo, L. Savage, K. D. Sarge, and O.-K. Park-Sarge Gonadotropins Decrease Estrogen Receptor-{beta} Messenger Ribonucleic Acid Stability in Rat Granulosa Cells Endocrinology, June 1, 2001; 142(6): 2230 - 2237. [Abstract] [Full Text] [PDF]
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 G. V. Childs, G. Unabia, and S. Komak Differential Expression of Estradiol Receptors Alpha and Beta by Gonadotropes During the Estrous Cycle J. Histochem. Cytochem., May 1, 2001; 49(5): 665 - 666. [Abstract] [Full Text]
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J. Takeyama, T. Suzuki, S. Inoue, C. Kaneko, H. Nagura, N. Harada, and H. Sasano Expression and Cellular Localization of Estrogen Receptors {{alpha}} and {beta} in the Human Fetus J. Clin. Endocrinol. Metab., May 1, 2001; 86(5): 2258 - 2262. [Abstract] [Full Text]
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G. B. Tremblay, T. Kunath, D. Bergeron, L. Lapointe, C. Champigny, J.-A. Bader, J. Rossant, and V. Giguère Diethylstilbestrol regulates trophoblast stem cell differentiation as a ligand of orphan nuclear receptor ERR{beta} Genes & Dev., April 1, 2001; 15(7): 833 - 838. [Abstract] [Full Text]
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H. O. D. Critchley, R. M. Brenner, T. A. Henderson, K. Williams, N. R. Nayak, O. D. Slayden, M. R. Millar, and P. T. K. Saunders Estrogen Receptor {beta}, But Not Estrogen Receptor {{alpha}}, Is Present in the Vascular Endothelium of the Human and Nonhuman Primate Endometrium J. Clin. Endocrinol. Metab., March 1, 2001; 86(3): 1370 - 1378. [Abstract] [Full Text]
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G. LECCE, G. MEDURI, M. ANCELIN, C. BERGERON, and M. PERROT-APPLANAT Presence of Estrogen Receptor {beta} in the Human Endometrium through the Cycle: Expression in Glandular, Stromal, and Vascular Cells J. Clin. Endocrinol. Metab., March 1, 2001; 86(3): 1379 - 1386. [Abstract] [Full Text]
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M. J. Weyant, A. M. Carothers, N. N. Mahmoud, H. L. Bradlow, H. Remotti, R. T. Bilinski, and M. M. Bertagnolli Reciprocal Expression of ER{{alpha}} and ER{beta} Is Associated with Estrogen-mediated Modulation of Intestinal Tumorigenesis Cancer Res., March 1, 2001; 61(6): 2547 - 2551. [Abstract] [Full Text]
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R. Clarke, F. Leonessa, J. N. Welch, and T. C. Skaar Cellular and Molecular Pharmacology of Antiestrogen Action and Resistance Pharmacol. Rev., March 1, 2001; 53(1): 25 - 72. [Abstract] [Full Text] [PDF]
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 R. K. Dubey and E. K. Jackson Estrogen-induced cardiorenal protection: potential cellular, biochemical, and molecular mechanisms Am J Physiol Renal Physiol, March 1, 2001; 280(3): F365 - F388. [Abstract] [Full Text] [PDF]
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S. K. Kang, K.-C. Choi, C.-J. Tai, N. Auersperg, and P. C. K. Leung Estradiol Regulates Gonadotropin-Releasing Hormone (GnRH) and its Receptor Gene Expression and Antagonizes the Growth Inhibitory Effects of GnRH in Human Ovarian Surface Epithelial and Ovarian Cancer Cells Endocrinology, February 1, 2001; 142(2): 580 - 588. [Abstract] [Full Text] [PDF]
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H. A. Harris, R. A. Henderson, R. A. Bhat, and B. S. Komm Regulation of Metallothionein II Messenger Ribonucleic Acid Measures Exogenous Estrogen Receptor-{beta} Activity in SAOS-2 and LNCaPLN3 Cells Endocrinology, February 1, 2001; 142(2): 645 - 652. [Abstract] [Full Text] [PDF]
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