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Université de Rennes I, Interactions Cellulaires et Moléculaires, Centre National de la Recherche Scientifique, UMR 6026, Campus de Beaulieu, 35042 Rennes, France
Address all correspondence and requests for reprints to: Dr. M.-L. Thieulant, Equipe d’Information et Programmation Cellulaire, UMR 6026, Batiment 13, Campus de Beaulieu, 35042 Rennes Cedex, France. E-mail: marie-lise.thieulant{at}univ-rennes1.fr
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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regulation has not been fully elucidated. Using transient transfection
assays in T3–1 cells, a cell line of gonadotrope origin, we show
that GnRH stimulates estrogen response element-containing promoters in
an estrogen-independent manner. This effect was strictly ER and GnRH
receptor dependent, as no activation of the reporter gene was observed
in presence of the anti-estrogen ICI 182,780 or a GnRH antagonist.
These data suggest that the GnRH-triggered signaling pathway results in
17ß-estradiol-independent trans-activation of the
ER in T3–1 cells. Furthermore, an additive activation was
achieved when cells were treated with both GnRH and 17ß-estradiol. In
primary pituitary cells, GnRH alone (100 nM) did not cause
a significant stimulation of reporter gene activity, presumingly due to
the low amount of gonadotropes. Interestingly, the combination of
17ß-estradiol and GnRH resulted in a significant increase in ER
trans-activation compared with that in cells treated
with 17ß-estradiol alone. This enhancement was prevented by ICI
182,780, showing an ER requirement. Moreover, we show that the
effects of GnRH on ER transcriptional activity in gonadotrope cell
lines are mediated by the PKC/MAPK pathway. In conclusion, our data
demonstrate that GnRH is an important signal in the regulation of ER
trans-activation in gonadotrope cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and ERß) encoded by two separate genes
have been described (2, 3). These two receptors belong to
the nuclear receptor superfamily of ligand-inducible transcription
factors whose members, the steroid receptors, TRs, and RARs, regulate
gene expression by interacting either in a DNA-protein manner with
cognate DNA sequences called responsive elements (for reviews, see
Refs. 4 and 5) or in a protein-protein manner
with other transcriptional factors (6, 7, 8).
GnRH, produced by neurons in the hypothalamus, plays a predominant role
in gonadotropin regulation. Pulsatile release of GnRH is necessary for
expression of the gonadotropin genes and for hormone secretion (for
review, see Ref. 1). In gonadotrope cells, binding of GnRH
to its seven-transmembrane G protein-coupled receptor (GnRH-R)
initiates a concert of intracellular signaling pathways, including
stimulation of calcium influx and activation of PLC, which leads to
increased protein kinase activities (for review, see Refs.
9, 10, 11). Although it is clear that GnRH is essential for
gonadotropin gene expression, the transcription factors mediating the
effects of GnRH-induced signals on transcription of gonadotropin genes
remain unknown. The PKC/MAPK pathway activated by GnRH preferentially
stimulates -subunit gene transcription, probably by phosphorylation
of the Ets family transcription factors (12). Recent
studies have identified several key factors involved in LHß
gonadotropin gene transcriptional regulation: the nuclear receptor
SF-1, the bicoid-related homeoprotein Ptxl (Pitxl), and the
immediate-early egr-1 gene (13).
In addition to the classical ligand-dependent activation, there is
evidence for cross-coupling between intracellular steroid hormone
receptors and membrane-bound signaling pathways (for review, see Refs.
14 and 15). Stimulation of numerous growth
factor receptors and/or protein kinases leads to ligand-independent or
synergistic increase in ER trans-activation, presumingly
by ER phosphorylation (for review, see Refs. 14, 15, 16).
The possible importance of phosphorylation for ER function was
initially indicated by the finding that the neurotransmitter, dopamine,
can activate ER in the absence of ligand (17). Other
groups have since shown that heregulin (18), insulin
(19), as well as cAMP (14, 20), PKC
(21), and phorbol esters (22) can also
activate ER. Moreover, Turgeon and Waring (23) have
shown a GnRH-dependent activation of rat PR in primary rat pituitary
cells.
Whereas ER appears to play a critical role in the control of pituitary
gland and gonadotropin regulation, the mechanisms responsible for
pituitary ER regulation (for the gene as well as the protein) have not
been fully elucidated. In the adult anterior pituitary gland,
estrogen-binding sites are localized within gonadotrope, lactotrope,
somatotrope, and thyrotrope cells, with the highest amounts in
gonadotrope cells. More recently, several groups (24, 25, 26, 27, 28)
have demonstrated that ER is the predominant species in the adult
rat pituitary. Concerning the presence of ERß mRNA and protein in
pituitary cells, conflicting data have been published
(24, 25, 26, 27, 28). Nevertheless, it seems that the amount of ERß
protein in the pituitary would be extremely low (28). It
is well established that the level of ER expression is closely
correlated to the magnitude of the ER-mediated response. Among the
factors that may alter the level of ER expression, estrogens
themselves appear to be the primary negative or positive regulators in
many E2-responsive tissues (reviewed in Ref. 29). On the
other hand, there is evidence that the pituitary ER expression level
is dependent on trophic factors from the hypothalamus, because median
eminence lesions result in a reduction of the receptor in this gland
(30). Moreover, Singh and Muldoon (31) showed
that exposure to GnRH significantly increased nuclear E2 binding, both
in vivo and in vitro, and suggested a direct
effect of GnRH on pituitary ER. Cocultures of pituitary cells with
hypothalamic explants for 16 h allowed us to confirm the release
of factors able to modulate ER or LHß gene expression in pituitary
cells (unpublished data). Moreover, pulsatile GnRH treatment of
pituitary cell aggregates (10 nM/1 min·h)
increased ER mRNA levels after 4 h of pulses (32).
We thus hypothesized that the previously observed actions of GnRH on
ER expression may result from the effects of functional cross-talk
between GnRH and ER in the gonadotrope cells.
The purpose of the present study was to explore the possibility of
involvement of multiple signal transduction pathways in regulating
ER transcriptional activity. As our in vitro and in
vivo data suggested an interrelation between GnRH and pituitary
ER (32) (our unpublished data), we studied ER
transcriptional activity in response to GnRH to further identify the
mechanisms by which the peptide could exert its effect on ER
regulation. In this study we provide evidence that ER can be
transcriptionally activated in gonadotrope cells in an
estrogen-independent manner. In view of the importance of PKC and MAPK
in GnRH signaling, we were interested to determine whether the
hormone-independent activation occurs through these PKC- and MAP-kinase
dependent pathways.
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Materials and Methods |
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Cell cultures and transfection
Pituitary cell monolayers. Anterior pituitary cells were
obtained by trypsin dispersion from glands as previously described
(32). For transfection studies, dispersed cells were
plated in six-well dishes at 1 x 106
cells/well in a humidified atmosphere of 5% CO2
in MEM containing d-valine (Sigma, St. Louis,
MO), 0.1 mg/ml kanamycin sulfate, 2.5 µg/ml fungizone
(Sigma), and 10% FCS (24). After 3 d,
cells were washed, incubated for 2 h in MEM-d-valine
supplemented with 2% steroid free Ultroser (Biosepra SA, Cergy-St
Christophe, France), and transfected as indicated.
T3–1 cells. The gonadotrope-derived clonal cell line
expressing the -subunit and containing GnRH receptors was provided
by Dr. P. Mellon (33). Cells were grown in DMEM in high
glucose with L-glutamine and sodium pyruvate (Life Technologies, Inc., Gaithersburg, MD), 10% FBS, 0.1 mg/ml
kanamycin sulfate, and 2.5 µg/ml fungizone. Three days before
transfection, cells (1–2 x 105 cells) were
plated on six-well plates. Two hours before transfection, cells were
washed, and medium was replaced by serum-free medium supplemented with
2% USF.
For all studies cells were transfected with estrogen response element (ERE)-luciferase reporter genes (1 µg/well) using Fugene-6 (Roche Molecular Biochemicals, Indianapolis, IN) according to manufacturer’s instructions, washed, and treated with E2 (10 nM) or vehicle alone for 48 h. GnRH (Sigma) or des-Gly10-[D-Ala6]LHRH ethylamide (Sigma) were applied for the last 8 h before harvesting the cells. All transfection experiments were performed in triplicate. After treatments, cells were washed and lysed by thawing in 150 µl lysis buffer (Promega Corp., Charbonnières, France). Luciferase activity was determined with reagents from Promega Corp. according to the manufacturer using a luminometer (ML 3000, Dynatech Corp., Chantilly, VA). Protein concentrations in cell lysates were determined using the colorimetric protein assay system (Bio-Rad Laboratories, Inc., Richmond, CA). Results were expressed as luciferase activity normalized per 100 µg proteins.
Data analyses
Results are expressed as the mean ± SEM of
3–10 experiments, each performed in triplicate. Statistical analysis
were carried out by one- or two-way ANOVA. Significant differences
between treatment groups were determined by Fisher’s test. Statistical
significance was inferred at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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by GnRH in the
gonadotrope-derived T3–1 cells mRNA levels (32) could also
regulate ER-dependent transcription from an ERE reporter gene. To
examine the transcriptional activity of the ER in response to GnRH,
T3–1, a gonadotrope-derived cell line (33) was chosen.
Cells were transfected with an ERE-luciferase reporter construct
(ERE-SV-LUC) and treated with ethanol vehicle (control) or E2 (10
nM) for 48 h. GnRH (100 nM) was added for
the last 8 h before harvesting. Luciferase activity was normalized
for protein content. E2 treatment significantly stimulated ERE-SV-LUC
activity by 2.3-fold vs. untreated control (Fig. 1
; P < 0.01
vs. control; n = 10). Neither GnRH nor E2 enhanced
transcription from the control reporter plasmid lacking the ERE
sequence (SV-LUC; data not shown). GnRH alone induced a stimulation of
reporter gene activity (4.5-fold vs. untreated control;
n = 10), which was significantly more potent than that seen with
E2 alone (
2-fold; P < 0.05 vs. E2;
n = 10). Treatment of the cells with E2 for 48 h coupled with
GnRH administration during the last 8 h resulted in an 8-fold
increase vs. the untreated control value (Fig. 1; n =
10; P < 0.001 vs. GnRH alone). In addition,
ANOVA showed an additive effect between E2 and GnRH. Similar results
were obtained in T3–1 cells transfected with a ERE-tk-LUC reporter
vector (data not shown), demonstrating that the enhancement of
transcription by GnRH was not promoter dependent.
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T3–1 cells transiently transfected with ERE-SV-LUC showed a
dose-dependent increase in luciferase activity in response to both GnRH
and GnRH/E2 (Fig. 2). GnRH stimulated
luciferase activity at the smaller dose tested (1 nM), with
maximal stimulation occurring at 100 nM. No further
increase was observed with 1 µM GnRH (not shown). A
similar pattern was seen for GnRH/E2 cotreatment (Fig. 2). In both
cases the effects were linear in the tested range (r = 0.955
for GnRH alone or GnRH/E2, respectively; n = 3). Stimulation of
T3–1 cells with GnRH/E2 clearly resulted in an additive effect on
the stimulation of LUC activity, as the calculated sum of the
individual inductions was similar to the measured inductions of E2/GnRH
cotreatment at three doses (sum calculated of individual inductions by
E2 and GnRH vs. measured induction by E2/GnRH cotreatment,
2.9 vs. 4.1, 4.7 vs. 6.9, and 7.8 vs.
10.6, respectively for 1, 10, and 100 nM GnRH;
n = 3). A dose-response study with the GnRH agonist,
des-Gly10-[D-Ala6]LHRH
ethylamide, showed a similar pattern (not shown). As E2 was used at a
saturating concentration, those results suggest that E2 and GnRH
mechanisms of ER activation are different.
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-mediated transcription by antiestrogen or GnRH
antagonist in T3–1 cells in GnRH-mediated activation, the
pure antiestrogen, ICI 182,780, was employed. In Fig. 3A, T3–1 cells were transfected with
ERE reporter and subsequently treated with ethanol vehicle, E2 (10
nM), or ICI 182,780 (100 nM) for 48 h.
Cells were stimulated with GnRH (100 nM) for the final
8 h. The effects of GnRH or GnRH/E2 on ERE-SV-LUC expression were
completely blocked by ICI 182,780, showing the requirement for ER in
this mechanism. To test the involvement of GnRH-R in ER-mediated
transcription in response to GnRH, a GnRH antagonist
[Ac-D-(2)NAL(1),pF-D-Phe2,D-Trp3,D-Arg6]LRF
(luteinizing releasing hormone) (Dr. Rivier) was added to T3–1
cells 60 min before GnRH. The ability of GnRH to
trans-activate ER was abolished at the control level by
the presence of GnRH antagonist (Fig. 3B), indicating that activation
by GnRH is mediated by its own receptor. In addition, the stimulatory
effect of GnRH/E2 was reduced by 70% at approximately the level of
estrogen-treated cells. Thus, the data show that transcriptional
effects were dependent on both ER and GnRH-R.
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trans-activation in the primary pituitary
cells shows that E2 treatment resulted in a
weak, but significant, increase in reporter luciferase activity
(1.64 ± 0.10-fold vs. control; n = 4;
P < 0.05). Treatment with 100 nM
GnRH alone caused a slight stimulation of reporter gene activity over
the control level (1.33 ± 0.15-fold vs. control;
n = 4); however, this variation was not statistically significant.
Interestingly, GnRH significantly increased ER
trans-activation when combined with E2. The ER
trans-activation by the combination of GnRH/E2 was enhanced
approximately 3-fold vs. the unstimulated control value
(n = 4; P < 0.0001). The increase was 1.8-fold
over that seen with E2 alone (n = 4; P < 0.005).
Moreover, a significant interaction between E2 and GnRH was observed on
ER trans-activation (by two-ANOVA, P <
0.0001). In addition, the ability of GnRH or GnRH/E2 to
trans-activate the ER was blocked by ICI 182,780 (100
nM; Fig. 4), suggesting that the transcriptional
effects of GnRH were ER dependent. Taken together, these results
demonstrate that ER can be activated in primary pituitary cells by
GnRH in addition to estrogens.
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involves PKC and MAPK
pathways, -ß1, -ßII,
-
, -
, and -
isozymes), was tested. T3 cells were
transfected with ERE-SV-LUC and treated for 48 h with E2 (10
nM) or vehicle. The cells were pretreated for 60 min with
or without GF109203X (10 µM) before treatment with GnRH
(100 nM). As shown in Fig. 5A, GF109203X caused a complete
inhibition of ER trans-activation in response to GnRH,
whereas the ability of GnRH/E2 to enhance the ER
trans-activation was partly reduced, indicating that this
pathway is involved in the GnRH-induced enhancement of ER
transcriptional activity.
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T3–1 gonadotroph cell line via PKC
(10, 12), we investigated whether MAPK could play a role
in the cross-talk between GnRH and ER. We tested the effect of
PD98059, a specific MAPKK inhibitor, on GnRH-mediated ER
trans-activation in the T3–1 gonadotroph cell line.
Cells were transfected with ERE-SV-LUC reporter and then treated with
ethanol or E2 (10 nM). PD98059 (50
µM) was introduced 1 h before adding GnRH
(100 nM). Figure 5B
shows that the MAPKK
inhibitor induced a 60% inhibition of GnRH-mediated activation of the
ERE reporter (n = 2). The GnRH/E2 activation of ERE reporter was
reduced 65% by PD98059. This remaining activity could correspond to
E2-dependent activation. These results suggest that ER activation by
GnRH proceeds via a MAPK dependent-pathway. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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in the
pituitary gland. Several arguments support the hypothesis that GnRH
could affect the transcriptional activation function of the ER in
gonadotrope cells. Singh and Muldoon (31) have shown that
exposure to GnRH significantly increased nuclear E2 binding. Weisenberg
et al. (30) reported that the number of
pituitary ER-binding sites is dependent on trophic factors from the
hypothalamus. In a previous study we have demonstrated that GnRH
pulsatile administration enhances ER mRNA expression in rat
pituitary cells (32). In this report we have further shown
that GnRH activates the estrogen signaling pathway in the pituitary
gland.
Ligand-independent activation of the ER is indeed observed in
gonadotrope-derived cell lines (T3–1 cells) transiently transfected
with estrogen-inducible reporter constructs (ERE-SV-LUC and ERE-tk-LUC)
and treated with GnRH. The magnitude of the ligand-independent
activation of the ER by GnRH that we observed was 2-fold higher than
that seen with E2. The stimulatory effect of GnRH on ER-mediated
reporter gene transcription was enhanced in the presence of E2,
resulting in an additive effect. The ability of GnRH to
trans-activate the ER in presence or absence of E2 was
blocked both by a pure antiestrogen (ICI 182,780) and a GnRH
antagonist. Thus, our results demonstrate that the expression of
estrogen-responsive reporter genes may be stimulated by a peptide
factor, GnRH, in the absence of E2 and in a manner dependent upon the
presence of ER and GnRH-R in gonadotrope-derived cell lines. The
fact that addition of GnRH in the presence of a saturating dose of E2
results in a further and additive increase in reporter gene expression
indicates that there are basic differences between the mechanisms of
ligand-independent and ligand-dependent activation of ER, suggesting
the involvement of two different pathways.
The ligand-independent trans-activation of ER by GnRH was
not detected in primary pituitary cells, probably due to the
heterogeneity of the cell types present in the pituitary and the low
amount of gonadotropes (10%). However, the simultaneous presence of an
optimally inducing concentration of E2 and GnRH results in a level of
reporter gene expression significantly higher than that obtained with
E2. Moreover, the results suggest an interaction between E2 and GnRH in
primary pituitary cells. Previous studies have shown that in rat
pituitary cells E2 can increase GnRH receptor number and amplify
GnRH-stimulated gonadotropin secretion (for review, see Ref.
34). Moreover, E2 has been shown to increase anterior
pituitary levels of PKC (35). Thus, although the
mechanisms of E2 action on gonadotropes are not well defined, it
appears to modulate GnRH action by exerting coordinated effects at and
distal to the GnRH receptor. We show that the effect of GnRH/E2 is
completely blocked by ICI 182,780, indicating that the response is
dependent upon a functional ER. These results imply cross-talk between
the ER and GnRH signal transduction pathways and a role for GnRH in
E2 signaling in the gonadotropes.
GnRH binding triggers activation of the MAPK pathway through a
mechanism involving PKC in primary rat pituitary cells as well as in
the gonadotrope-derived T3–1 cells (9, 11). Given this
evidence, we assessed the involvement of the PKC and MAPK signaling
pathways in the cross-talk between GnRH-R and ER in T3–1 by
treating cells with protein kinase inhibitors. The PKC inhibitor,
GF109203X, completely blocked the reporter trans-activation
resulting from GnRH action, whereas the combined action of GnRH/E2 was
partly inhibited, suggesting the involvement of the PKC pathway.
Although the PKA pathway does not appear to be involved in
GnRH-mediated effect in T3 cells (9, 11), we cannot
totally exclude that with the concentration of GF109203X used in this
study the PKA pathway is not also partly affected. Treatment with
PD098059, which inhibits the MAPK pathway, decreased GnRH- or
GnRH/E2-mediated activation of the ERE reporter by 60% and 65%,
respectively. Thus, our data demonstrate that the effects of GnRH on
ER activation in gonadotrope cells are mediated by the PKC/MAPK
pathway.
To our knowledge, this is the first report on the modulation of ER
transcriptional activation by GnRH in gonadotrope cells, suggesting
that a GnRH-triggered signaling cascade can result in E2-independent
trans-activation of the ER. There are numerous findings
of ligand-independent activation of mammalian ER in transiently
transfected cells as well as in vivo (14, 15, 16).
Several studies have now shown that ER is subject to phosphorylation
and activation induced by various peptide growth factors
[e.g. epidermal growth factor (EGF) (36),
TGF (36) IGF-I (22), and heregulin
(18)], events that can subsequently initiate ERE-mediated
gene expression. Functional analyses of ER have shown that the two
transcriptional activation functions, AF-1 and AF-2, are differentially
activated by those factors; AF-1 is more responsive to EGF and TGF
signaling, and IGF-I preferentially targets AF-2 (36, 37).
Three serine residues Ser104,
Ser106, and Ser118 are the
targets of the cyclin-dependent kinases and MAP kinases (for reviews,
see Refs. 14, 15, 16). More detailed studies have confirmed
that phosphorylation of Ser118 is required for
full activity of AF-1 and is mediated via the Ras/MAPK pathway in
EGF-stimulated cells (36, 38, 39, 40). Recent studies provide
evidence that the cross-talk between EGF and IGF-I with ER in MCF-7
breast cancer cells involves Akt (41). A potential PKA
phosphorylation site does exist within the ER at
Ser236 (20). Phosphorylation of
ER by casein kinase II and pp90rsk1 on
Ser167 in vitro has also been
demonstrated (for review, see Ref. 14). PKC has been
reported to phosphorylate the receptor on residue
Ser122 (21), whereas tyrosine 537 is
phosphorylable by members of the Src family of tyrosine kinases
in vitro (42, 43). Although activation of the
ER has been shown to be associated with phosphorylation of the AF-1
and AF-2 domains, the precise roles of individual phosphorylation sites
are not known.
The additive activity displayed by GnRH and E2 is not easily
comprehensible. As it is known that the ER can be phosphorylated on
different residues, it could be postulated that the ER hyperactivity
observed in the presence of GnRH and E2 is due to hyperphosphorylation
of the receptor protein. At this stage we do not know whether ER is
the direct target of the phosphorylation events induced by the PKC/MAPK
pathways. We cannot exclude that a coactivator, interacting with ER,
functions in a MAPK-induced phosphorylation way. Such a hypothesis may
help to explain the additive increase in reporter gene activity that is
activated by addition of GnRH in the presence of a saturating
concentration of E2.
The data presented here provide evidence that the transcription factor
ER may mediate some of the effects of the hypothalamic factor, GnRH,
suggesting that the interaction between ER and GnRH signaling could
have important physiological consequences. Feedback regulation by E2 at
the level of both the hypothalamus and the pituitary is of fundamental
importance for controlling reproduction in females. One of E2’s
effects is to sensitize the pituitary to hypothalamic signals such as
GnRH. Signaling cascades initiated by GnRH binding to its receptor may
modulate the transcriptional activity of ER, ligand-occupied or not,
and thereby increase the magnitude of target gene expression.
Alternatively, it is possible that GnRH contributes to maintain
moderate levels of ER activity in gonadotropes when estrogen levels
are low, as observed in males or in females during diestrus. Indeed, it
is conceivable that cross-talk pathways may sensitize the ER to
suboptimal stimulation by low levels of estrogens and thereby promote
biologically meaningful responses under conditions in which ligand
stimulus alone would be unable to generate a significant signal. It is
not known whether the target genes activated by ER in response to
GnRH or E2 are identical or whether there are genes preferentially or
selectively regulated by one or the other pathway. Although -subunit
promoter activity is dramatically affected by estrogen, no high
affinity ERE has been identified in this promoter (1, 44).
On the other hand, an ERE was identified in the rat LHß gene
(1). Identification of such a mechanism may be of critical
importance in the understanding the involvement of ER in
gonadotropin gene transcription. Although it is clear that GnRH is
essential for gonadotropin gene expression, the transcription factors
that ultimately are targets of GnRH action remain unknown. The PKC/MAPK
pathway activated by GnRH preferentially stimulates -subunit gene
transcription by phosphorylation of the Ets family transcription
factors, whereas induction of the LHß gene is dependent on PKC and
calcium influx (45). Recent studies have identified key
factors involved in LHß gonadotropin gene transcription: the nuclear
receptor SF-1, the bicoid-related homeoprotein Ptxl (Pitxl), and the
immediate-early egr-1 gene (13). A marked
increase in gonadotropin mRNA levels has been observed in ER
gene-disrupted female mice (46), consistent with
previously described transcriptional suppression of gonadotropin
subunit gene expression in response to sustained administration of E2
in wild-type mice. These results provide genetic evidence that ER
plays a critical role in gonadotropin gene transcription.
In summary, it appears that the mechanisms regulating pituitary ER
action are certainly complex. The data presented here extend previous
observations regarding the regulation of pituitary ER and show for
the first time that rat pituitary ER activity could be under
hypothalamic control. Our data demonstrate that the rat ER can be
activated in the absence of estrogen through an alternate signaling
pathway involving the GnRH receptor. This activation is dependent upon
the presence of ER and GnRH-R and mainly occurs via PKC and MAPK
pathways. In addition, the mechanism of ER activation by GnRH could
differ from that used by E2. Cross-talk between the GnRH signal
transduction system and ER in gonadotrope cells may represent an
important modulating mechanism for the estrogen responsiveness of
gonadotrope cells.
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Acknowledgments |
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T3–1 cells,
and to Dr. Rivier for [Ac-D-(2)NAL(1),
pF-D-Phe2,D-Trp3,D-Arg6]LRF.
We thank Dr. W. Wahli (IBM, University of Lausanne, Lausanne,
Switzerland) for the plasmid ERE-tk-LUC. |
Footnotes |
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1 Association pour la Recherche sur le Cancer fellow. 
Abbreviations: E2, 17ß-Estradiol; EGF, epidermal growth factor; ERE, estrogen response element; GnRH-R, G protein-coupled receptor; LRF, luteinizing releasing hormone.
Received February 20, 2001.
Accepted for publication April 23, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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protein and
estrogen responsiveness. Endocrinology 141:2174–2184This article has been cited by other articles:
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