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and ß1
Departments of Psychiatry and Behavioral Sciences
1 and
Pharmacology
2 , University of Washington, Seattle, Washington
98195
Address all correspondence and requests for reprints to: Robert A. Shapiro, University of Washington, Psychiatry and Behavioral Sciences, Box 356560, Seattle, Washington 98195-6560. E-mail: shapiror{at}u.washington.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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or
ß in response to various treatments. ER and ERß displayed
differential regulation of the AVP promoter. SK-N-SH cells transfected
with ER exhibited increased luciferase activity in response to
estrogen, and the selective estrogen receptor modulators (SERMs),
Tamoxifen, and ICI 182,780. Cells transfected with ERß exhibited a
high constitutive activity, which is unchanged by exposure to SERMs but
can be inhibited by estrogen. Deletion of 1.5 kb from the 5' end or
mutation of a single estrogen response element (ERE)-like sequence
resulted in loss of estrogen-dependent induction by ER and increased
the ability of estrogen to inhibit the high constitutive activity of
ERß. The distal ERE-containing 1.5-kb fragment, when coupled to
luciferase, is able to support both ER and ERß mediated activation
of transcription by estrogen. These results suggest that a single ERE
in the distal 1.5-kb portion of the 5.5-kb fragment contains the
primary positive estrogen responsive sequences for ER and ERß. The
data also suggest that sequences proximal to this element serve to
inhibit transcription mediated by ERß. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In addition, VP is regulated by the steroids estrogen and testosterone.
Numerous studies have demonstrated that gonadectomy dramatically
reduces expression of AVP immunoreactivity and messenger RNA (mRNA)
levels in both the BNST and MA (1, 11, 12, 13, 14, 15), which is
restored in females by estrogen replacement and in males by
testosterone. In contrast, AVP mRNA expression in the SON and the PVN
remains unchanged (unpublished observation). The effects of estrogen
are now known to be mediated by at least two different receptors termed
ER and ERß. These two appear to be colocalized with VP-producing
cells in a region specific fashion. Estrogen receptor- (ER) mRNA
is colocalized in vasopressenergic cells in the BNST, but no
colocalization was observed in the SON or PVN (16).
Interestingly, recent studies using sequences specific for the newly
identified estrogen receptor ß (ERß) subtype (17, 18, 19)
have colocalized ERß mRNA with vasopressinergic cells in the SON
(16, 20, 21, 22), suggesting that changes in VP transcription
may be differentially regulated by the two ER subtypes. In addition,
both ER and ERß have been colocalized in cells of the BNST, MA,
and the periventricular preoptic nucleus (PE) (23).
The ERs have been shown to elicit gene transcription by interacting
with at least two distinct promoter elements, the classical estrogen
response element (ERE), which binds ligand-bound receptor dimers, and
AP1 sites, which involves interaction with fos/jun complexes but does
not require the DNA binding domain of the receptor (24).
Both ER and ERß activate classical EREs in response to
E2 and the antiestrogens or selective estrogen
receptor modulators (SERMs) Tamoxifen and ICI 182,780 can block this
response (24). In contrast to their actions at EREs,
distinct pharmacological profiles have been shown for the actions of
ER and ERß at AP1 sites (25). The SERMs, raloxifene,
Tamoxifen, and ICI 182,780, are transcriptional activators of both
ER and ERß at these sites. In contrast, while
E2 can activate ER transcription at AP1 sites,
it acts as an antagonist of SERM induced transcription by ERß. This
model does not exclude the possibility of other regulatory elements
being directly activated by SERMs (26). It is important to
note that these studies used reporter constructs containing single
response elements in nonneuronal cells (25). It has yet to
be determined whether complex promoters containing multiple response
elements will be regulated in a similar manner. In addition,
ER/ERß heterodimers have been shown to form in vitro
(27, 28); however, their pharmacological profile has yet
to be determined.
The studies reported here examine the estrogenic regulation of the
complex AVP promoter, which contains several potential ERE and AP1-like
sites, in SK-N-SH cells. SK-N-SH cells are a neuroblastoma cell line
derived from a metastatic bone marrow tumor (29). They
have been shown to express several neuropeptide receptors including µ
and 
-opioid receptors (30), muscarinic acetylcholine
receptors (31), as well as neurotransmitters and
neurotransmitter transporters such as norepinephrine, dopamine, and
their respective transporters (32). In addition, SK-N-SH
cells exhibit neuropeptidergic properties in that they have been
reported to express LH-releasing hormone (LHRH) (33).
To identify the promoter elements important in the estrogenic induction
of the vasopressin gene we have compared the ability of ER and ERß
to activate a series of AVP promoter luciferase reporter constructs in
response to estrogen and SERMs. Differential regulation of the AVP
promoter by ER and ERß was observed.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Plasmids
The complementary DNAs (cDNAs) encoding both human HEO\ER
(34), and rat ERß (17) were subcloned into
the expression vector, pcDNA3 (Invitrogen). A 5.5-kb
fragment containing the rat vasopressin promoter (35) was
excised from the RVP-Sal plasmid (generously provided by Dr. David
Murphy, University of Bristol, UK) with Not1 and
Sal1 and cloned into the Not1 and Xho1
sites of the luciferase reporter plasmid, pGL2-B (Promega Corp.). The resulting plasmid, (p5.5luc) was used to generate
all other constructs.
The G400V mutation present in the original ER clone (HEO) has been
reported to effect the basal activity of ER (36). To
address the possibility that the differences in basal activity we have
observed between ER (HEO) and ERß are due to the G400V mutation,
control experiments were performed comparing the activity of HEO (which
contains a valine at amino acid 400) and HEGO (a glycine at amino acid
400) and ERß on p5.5luc. While HEGO does display a slightly higher
basal activity than HEO, in no case did it approach the high, uninduced
basal activity of ERß. Furthermore, both HEO and HEGO exhibit the
same level estrogen-dependant activation (fold increase) of p5.5luc
(data not shown).
Transient transfections
SK-N-SH cells (
5 x 105 cells)
were transfected in six-well plates with 2 µg/well of each luciferase
reporter and either human ER/hEO plasmid (generously provided by Dr.
Pierre Chambon, Strasbourg, France) or rat ERß (generously provided
by Dr. George Kuiper, Sweden) for 7 h using standard calcium
phosphate procedures described elsewhere (Promega Corp.).
One microgram per well of pCH110, a ß-galactosidase reporter
(Amersham Pharmacia Biotech, Uppsala, Sweden) was
used for normalizing transfection efficiency.
Treatments
After transfection, the media were replaced with phenol red-free
media supplemented 10% charcoal-stripped calf serum and allowed to
recover overnight. Cells were treated in triplicate with various doses
of water soluble 17ß-estradiol (Sigma), 1
µM Tamoxifen (Research Biochemicals International, Natick, MA), or 1 µM ICI 182,780
(Zeneca Pharmaceuticals LTD, London, UK) for 8 h and
harvested in 0.25 ml of reporter lysis buffer (Promega Corp.). For luciferase assays, 100 µl of cell extract was
mixed with luciferase assay reagent (Promega Corp.)
according to the manufacturer’s protocol and read on an E.G.&G LB 953
AutoLumat luminometer for 30 sec/sample as previously described
(37). For ß-galactosidase assays, 50 µl of cell
extract was mixed with 50 µl of Assay 2x buffer (Promega Corp.), incubated at 37 C for 2 h and absorbance at 420
nM determined on a Packard Spectracount 96-well plate
reader as previously described (37). Data are presented as
a ratio of luciferase to ß-galactosidase activity.
Statistics
Statistical analysis was performed using the ANOVA
Scheffé’s F test for posthoc significance.
Gel shift assay
Nuclear extracts were prepared from cultured MCF-7 cells as
described (38, 39). Briefly, protein extracts (10 µg)
were incubated with 1 µg poly(dI-dC) in 20 µl buffer [10
mM HEPES (pH 7.9), 1 mM dithiothreitol, 80
mM KCl, 2 mM MgCl2, 10% glycerol, and 3
µg/ml BSA] plus 32P 5'-end-labeled
oligonucleotides (20,000 cpm) for 15 min at room temperature
(40) with or without 100-fold excess of specific or
nonspecific competitor. Complexes were resolved by electrophoresis at 4
C on a 6% nondenaturing acrylamide gel equilibrated in 0.25x TBE (5
mM Tris and 0.5 mM EDTA), dried and exposed to
film for 2–3 days. Specific oligonucleotides used: ERE-1:
5'-AGTGGGCAGCCTCACCCCTA-3'; ERE-2: 5'-GCAGGGCCAGCCTGACCGTGT-3'; c-ERE:
5'-GATCCAGGTCACTGTGACCTG.
Mutagenesis
The distal 1.5-kb fragment (p5'-1.5luc) was excised from p5.5luc
with Not1 and BglII and cloned into the
Not1 and BglII site of pGL2-B. p4.0luc was
generated by linearizing p5.5luc with Not1, partially
digesting with BglII, filling in the ends with Klenow
and religating the plasmid. The 1.1 kb proximal portion of the promoter
was amplified by PCR from p5.5luc, cloned into TAPCR
(Invitrogen), excised with Xho1 and
HindIII and cloned into the Xho1 and
HindIII sites of pGL2-B (p1.1luc).
Oligonucleotide-directed mutagenesis
Two point mutations were introduced into p5.5luc by Sequence
Overlap Extension procedure (41). One mutation
(pcon5.5luc) changes ERE-2 to a consensus ERE, the other (pmut5.5luc)
changes two nucleotides in ERE-2 which disrupt the 5 base palindrome.
The two changes have each been previously shown to functionally disrupt
estrogen regulation of an ERE reporter gene in transient transfection
experiments (42, 43). Overlapping PCR fragments were first
generated in separate reactions and then reannealed and extended in a
second PCR. In the first step, two oligonucleotides (complement and
reverse complement) containing the desired nucleotide changes were
synthesized for each mutation. Two fragments were then synthesized by
PCR using a 5'oligo (5'-CATACatgcatGGACGCAC-3') and the reverse
complement to generate the 5' fragment and with the complement (see
below) and a 3' oligonucleotide (5'AGCAcccgggAGACAGAGTC-3') to generate
the 3' fragment (95 C for 45 sec; 55 C for 60 sec; 72 C for 90 sec; 35
cycles). After gel purification, 10 ng of each fragment were mixed and
used as template in a second PCR with the two outer oligonucleotides.
The resulting 800-bp fragment was digested with Nsi1
and Sma1 and used to replace the corresponding fragment in
5.5luc. All nucleotide changes were confirmed by DNA sequencing.
Oligonucleotides used are shown below. Mutated nucleotides in
the ERE palindrome (underlined) are in bold
type.
pmut5.5luc: 5'-AGCGCCAGCCTGGCCGTGTGTGCCAGAAGATCTTC-3'.
pcon5.5luc: 5'-GCTTCTGCAGGGTCAGCCTGACCGT-3'
DNA sequencing
Sequencing of the 5.5-kb promoter fragment was performed at the
University of Washington Genome Center by fluorescent dye termination
on a PE-ABI 373 using the ABI Prism Dye primer cycle sequencing kit and
custom designed primers according to the standard protocol recommended
by the manufacturer. The complete sequence has been deposited in
GenBank (Accession No. AF112362).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). SK-N-SH cells were then transiently
transfected with plasmids encoding either human ER (HEO) or rat
ERß to examine the role of estrogen in mediating expression of the
vasopressin gene.
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(Fig. 2A) or ERß (Fig. 2B) were transfected
with p5.5luc and treated with 50 nM 17ß-estradiol
(E2). ER transfected cells exhibited a plasmid
dependant increase in E2-induced promoter
activity. ERß transfected cells exhibited a marked receptor-dependant
increase in constitutive activity. No significant activity was observed
in the absence of transfected receptor. At low ERß concentrations,
addition of E2 increased promoter activity,
however, at high receptor concentrations (
2 µg/well), ERß
mediated transcription is inhibited by E2. No
receptor-dependent increases in luciferase expression were observed in
cells transfected with the control luciferase plasmid, pGL2b (Fig. 2C).
In fact, a small decrease was observed.
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or ERß. As observed in
Fig. 2A
, E2 increased ER mediated
transcription while E2 inhibited the high
constitutive activity of ERß (Fig. 3A).
Both ICI and Tamoxifen also demonstrated agonist activity in ER
transfected cells, an effect consistent with action at AP1-like
elements of the AVP promoter but had no effect on the constitutive
activity of ERß (Fig. 3, B and C). No ligand-induced increase in
luciferase activity was observed in cells transfected with the control
plasmid, pcDNA3.1 (data not shown). Thus, in contrast to ER, which
is able to activate the AVP promoter in response to
E2 and SERMs, transfection with ERß results in
a high transcriptional activity with only a minimal response to added
ligands.
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and
ERß-mediated induction by estrogen, deletion mutations were
constructed (p4.0luc and p1.1luc), fused to the luciferase gene (Fig. 1) and tested for estrogen-mediated luciferase activity (Fig. 4). At receptor concentrations (1 µg)
that exhibit E2 stimulation for both ER and
ERß (see Fig. 2B), deletion of the distal 1.5 kb (p4.0luc) resulted
in a complete loss of E2 activation when
cotransfected with ER (Fig. 4a). In fact, a small but significant
inhibition is observed, indicating that the critical sequences for
ER activation by E2 reside within a 1.5 kb
fragment located 4.0 kb upstream of the AVP coding region. Deletion of
an additional 3.0 kb (p1.1luc) resulted in the loss of significant
inhibition by estrogen, suggesting the presence of inhibitory sequences
within the 3.0-kb segment.
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), deletion of the
distal 1.5 kb (p4.0luc) increased the level of constitutive activity,
and resulted in E2-induced inhibition. Further
deletion (p1.1luc) reduced the constitutive activity but retained
significant inhibition. These data suggest that sequences located in
the 1.1-kb portion of the promoter are responsible for the high
constitutive activity of ERß in the absence of
E2 and the inhibition of transcription in
response to E2.
To confirm that sequences within the 5' 1.5 kb fragment were capable of
mediating E2 activation by ER, p5'-1.5luc was
tested with both ER and ERß for estrogen-mediated luciferase
activity (Fig. 5A).
E2 increased luciferase activity in cells
expressing either ER or ERß (1 µg), confirming the importance of
these sequences in mediating E2 activation of the
AVP gene. Cells transfected with ERß and p5'-1.5luc responded more
robustly to E2 when compared with the 5.5-kb
construct, possibly due to a lower constitutive activity (Fig. 5A).
Removal of the proximal 4.0 kb dramatically altered the effects of
SERMs on promoter activation by the two receptor subtypes. In ER
transfected cells, ICI was able to inhibit E2
activation to the same level as ICI alone. Tamoxifen was less potent
than E2, but its effect in combination with
E2 was additive (Fig. 5B). Both ICI and Tamoxifen
antagonized activation of ERß by E2 but had
little agonist activity of their own (Fig. 5C), consistent with a
classical ERE response. These results suggest that the sequences
required for SERM-mediated transcription of VP by ER and the high
constitutive activity of ERß reside primarily in the proximal 4.0-kb
region of the promoter, whereas E2-mediated
transcription requires the 1.5-kb distal region.
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). Oligonucleotides containing both sequences were synthesized
and tested in gel mobility shift assays with nuclear extracts of MCF7
carcinoma cells, which contain ER (44, 45). Only one
sequence (ERE-2) demonstrated specific binding (Fig 6). This ERE sequence was inactivated
(pmut5.5luc) by site-directed mutagenesis (Fig. 1) and tested for
luciferase activity in a cotransfection assay with 1 µg of ER
(Figs. 4A and 7A) or ERß (Figs. 4B and 7B).
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and pmut5.5luc showed no
activation by E2 (Figs. 4A and 7A) confirming the
requirement for this distal ERE in mediating ER activation by
E2. Mutation of the same ERE to a consensus ERE
(pcon5.5luc, Fig. 1) resulted in increased promoter activity compared
with p5.5luc (Fig. 4A). The mutation had no effect on the ability of
Tamoxifen or ICI to activate the promoter (Fig. 7A), further suggesting that SERM
activation is not ERE mediated, but rather by sequences within the
proximal 4.0-kb region. Indeed, sequence analysis of this region of the
promoter revealed several potential AP-1 like sequences.
Cells transfected with ERß and pmut5.5luc maintained the high
constitutive activity previously observed with p5.5luc and p4.0luc. In
addition, E2 inhibited luciferase activity to the
same extent as was previously observed using the p4.0luc construct
(Figs. 4B and 7B). In the same context, the consensus ERE exhibited
increased luciferase activity when compared with either p5.5luc or
p5.5luc cotransfected with ER. Thus, the distal ERE provides
positive ERß-mediated regulation by E2. When
the ERE is removed, either by deletion or point mutation,
E2 treatment results in ERß-mediated inhibition
of AVP transcription. Similar to the results observed with ER, the
mutation had no effect on the ability of Tamoxifen or ICI to activate
the promoter in ERß transfected cells (Fig. 7B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ERß might interact with these various
elements to give rise to the complex regulation observed in
vivo. In this report, we provide evidence for the differential
regulation of AVP transcription by ER and ERß in a neuronal cell
line.
ER activates the AVP promoter in response to
E2 by using the distal ERE element in a manner
consistent with the classical model of estrogen receptor activation.
Mutations that remove the ERE lose the ability to respond to
E2 but have no effect on the agonist activity of
Tamoxifen or ICI 182,780, demonstrating that SERM activation of the VP
promoter does not require an ERE element. While the data (Fig. 4A)
suggests the possible presence of inhibitory sequences for ER within
the p4.0luc construct, which can be removed by further deletion
(p1.1luc), the inhibition was not consistently observed in all
experiments (Fig. 7A). This inconsistency may be due to variation in
the expression of unknown nuclear coactivator proteins that maybe
involved. The enhancement of transcription by Tamoxifen and ICI 182,780
indicates that elements located elsewhere in the promoter are also
major contributors to transcriptional activation by ER. Agonist
effects of Tamoxifen and ICI 182,780 mediated by ER have been
reported for AP1 sites (24) and also cAMP and CRE mediated
gene transcriptional effects (37). Iwasaki et
al. (10) have described a proximal CRE-like element
capable of supporting this mode of regulation. It is thought that
cAMP-dependent regulation of the gene is central to the osmotic
regulation of magnocellular AVP gene transcription
(46).
Cells transfected with ERß exhibit a more complex transcriptional
profile. At low levels of transfected ERß (Fig. 2B),
E2 is able to induce expression of luciferase
from the full-length promoter. Transfection with increasing amounts of
ERß results in high constitutive activity that is inhibited by
E2 (Figs. 2B and 3A) and unaffected by the
addition of SERMs. At low receptor levels (1 µg), constructs that
delete or mutate the distal ERE maintain the constitutive activity, but
are inhibited by E2, suggesting that the
constitutive activity may be due to transcription from AP1 like
sequences (25) present within the 4.0-kb construct. When
cotransfected with the 1.5 kb distal promoter, which contains the ERE
element, the constitutive activity is decreased and
E2 increases ERß-mediated transcription. Thus,
in cells expressing low levels ERß, the opposing effects of
E2 stimulation at the ERE and inhibition at
AP1-like sequences results in relatively constant vasopressin
transcription.
Cells transfected with ERß and the pGL2b plasmid vector occasionally
showed small but highly variable increases in luciferase activity. In
contrast, cells transfected with ERß (1 µg) and p5.5luc
consistently increased luciferase activity. Given the magnitude of
ERß-mediated luciferase expression, we were initially concerned that
these effects might be derived from sequences in the luciferase vector
backbone. Vector sequences have been reported to confound the
interpretation of data from promoter studies due to either position
effects of enhancer-like sequences or the presence of promoter elements
contained within the vector sequences (47). Sequence
analysis of the pGL2b vector failed to reveal either ERE or AP-1 like
sequences. The strongest argument against the involvement of vector
sequences is that both point mutations within the ERE, which do not
change the length of the promoter, and 1.5-kb deletion of the ERE
(which does change the length of the promoter) are able to abolish the
increase in transcription observed in both ER and ERß transfected
cells.
These observations suggest a model for the differential regulation of
AVP transcription by ER and ERß. According to this model, cells
expressing ER could respond to E2 by
increasing VP mRNA transcription, while in cells expressing ERß, the
opposing actions of E2 on ERE and AP1-like
elements would result in relatively stable AVP mRNA levels. In
addition, these results suggest that SERMs may activate vasopressin
expression in cells expressing ER.
The physiological relevance of this model is demonstrated by the
regulation of VP gene expression by hormones in vivo.
Vasopressin is expressed in the PVN, SON, BNST, and the MA. It has been
previously shown that gonadectomy virtually abolishes VP mRNA and
peptide expression in the BNST, and this effect can be reversed by
local implants (3) of testosterone or estrogen. The post
castration decline of VP mRNA in the BNST is not rapid (3–7 days), and
interestingly, the peptide remains detectable for up to 8 weeks
(13). While we have previously provided evidence of a
transcriptional effect measured by intronic probe detection of VP
primary transcript in situ hybridization studies
(48), others have suggested that changes in mRNA
poly(A)-metabolism may also play a role (49). Consistent
with the loss of VP mRNA, is the fact that both ER and ERß are
highly expressed in the BNST and the MA. The mix of ER and ERß
might explain the slow decline of VP mRNA and peptide levels in these
regions. In cells expressing ER, estrogen deficiency would tend to
reduce transcription, but in cells expressing ERß the higher
constitutive activity may tend to oppose this effect. Because cells in
the BNST, MA, and PE have been recently shown to express both receptor
subtypes (23), it will be interesting to determine the
effect of E2 on ER/ERß heterodimers on VP
mRNA expression. Most significantly, relatively little change in VP
mRNA is observed in the SON and PVN, regions that express exclusively
ERß (16, 21, 22). Thus, the opposing effects of
E2 at ERE and AP1 sites could explain the
relative lack of change in AVP mRNA in these neurons.
In summary, Paech et al. (25), have recently
described differential ligand activation of ER and ERß at AP1
sites. In their model, using a consensus AP1 reporter construct derived
from the collagenase promoter, estrogen had no effect on ERß
activation of AP1 sites but was able to antagonize SERM activation. In
the present analysis of the AVP gene, we are able to test this model on
a complex, natural promoter. We report a constitutive activity of ERß
and observe estrogen acting as an inverse agonist in cells expressing
relatively high levels of ERß or in our ERE-deficient constructs. A
recent report by Forman et al. (50) describes a
similar example of inverse agonism by androstane metabolites of the
constitutive androstane receptor-ß (CAR-ß), another member of the
nuclear steroid receptor family. Although no increased constitutive
activity by ERß at AP1 sites was reported by Paech et al.
(25), examination of the results presented suggests a
small inhibitory effect of E2. This may reflect
differences in the ability of ERß to activate a single AP1 reporter
construct as opposed to a complex promoter containing multiple AP-like
sequences. Alternatively, the presence of AP1-like variants may be
preferentially activated in the presence of ERß. It has also been
reported (24, 25) that ER is able to activate
transcription at AP1 elements in response to E2.
In the AVP promoter, no E2-mediated transcription
was observed in constructs that removed the distal ERE even though
there are several AP1-like elements present that should be able to
mediate SERM activation of the promoter. Nevertheless, our results are
consistent with the model proposed by Paech et al.
(25) in that estrogen can negatively regulate nonERE
transcription by ERß. Experiments are in progress to identify those
sequences responsible for the high constitutive activity of ERß and
the negative regulation by E2.
|
Acknowledgments |
|---|
cDNA
clones HEO and HEGO, and George Kuiper (Karolinska Institute, Sweden)
for the Rat ERß cDNA, Zeneca Pharmaceuticals for the
gift of ICI 182,780, Ms. Zhou Yang for technical assistance, Pam
McMillan Ph.D. for critical review of the manuscript and Sherry Neher
for help with the preparation of this manuscript. |
Footnotes |
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Received May 23, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ß mRNA in the rat CNS. J
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immunoreactivity in neurons of the rat forebrain.
Endocrinology 139:5267–5270 and ERß at AP1 sites. Science 277:1508–1510 and ß form heterodimers on DNA. J Biol
Chem 272:19858–19862. Mol Endocrinol 11:1486–1496This article has been cited by other articles:
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 C. K. Cheng, B. K. C. Chow, and P. C. K. Leung An Activator Protein 1-Like Motif Mediates 17{beta}-Estradiol Repression of Gonadotropin-Releasing Hormone Receptor Promoter via an Estrogen Receptor {alpha}-Dependent Mechanism in Ovarian and Breast Cancer Cells Mol. Endocrinol., December 1, 2003; 17(12): 2613 - 2629. [Abstract] [Full Text] [PDF]
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J. G. Ramos, J. Varayoud, L. Kass, H. Rodriguez, L. Costabel, M. Munoz-de-Toro, and E. H. Luque Bisphenol A Induces Both Transient and Permanent Histofunctional Alterations of the Hypothalamic-Pituitary-Gonadal Axis in Prenatally Exposed Male Rats Endocrinology, July 1, 2003; 144(7): 3206 - 3215. [Abstract] [Full Text] [PDF]
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S. J. Somponpun and C. D. Sladek Osmotic Regulation of Estrogen Receptor-{beta} in Rat Vasopressin and Oxytocin Neurons J. Neurosci., May 15, 2003; 23(10): 4261 - 4269. [Abstract] [Full Text] [PDF]
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 A. C. Scallet, M. Wofford, J. C. Meredith, W. T. Allaben, and S. A. Ferguson Dietary Exposure to Genistein Increases Vasopressin but Does Not Alter {beta}-Endorphin in the Rat Hypothalamus Toxicol. Sci., April 1, 2003; 72(2): 296 - 300. [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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S. Somponpun and C. D. Sladek Role of Estrogen Receptor-{beta} in Regulation of Vasopressin and Oxytocin Release in Vitro Endocrinology, August 1, 2002; 143(8): 2899 - 2904. [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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C. B. Wade, S. Robinson, R. A. Shapiro, and D. M. Dorsa Estrogen Receptor (ER){{alpha}} and ER{beta} Exhibit Unique Pharmacologic Properties When Coupled to Activation of the Mitogen-Activated Protein Kinase Pathway Endocrinology, June 1, 2001; 142(6): 2336 - 2342. [Abstract] [Full Text] [PDF]
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