Homologous Regulation of the Gonadotropin-Releasing Hormone Receptor Gene Is Partially Mediated by Protein Kinase C Activation of an Activator Protein-1 Element
Brett R. White,
Dawn L. Duval,
Jennifer M. Mulvaney,
Mark S. Roberson and
Colin M. Clay
Animal Reproduction and Biotechnology Laboratory (B.R.W.,
D.L.D., C.M.C.) Department of Physiology College of Veterinary
Medicine and Biomedical Sciences Foothills Campus, Colorado State
University Fort Collins, Colorado 80523
Department of
Biomedical Sciences (J.M.M., M.S.R.) Cornell University Ithaca,
New York 14853
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ABSTRACT
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Homologous regulation of GnRH receptor (GnRHR)
gene expression is an established mechanism for controlling the
sensitivity of gonadotropes to GnRH. We have found that expression of
the GnRHR gene in the gonadotrope-derived
T31 cell line is
mediated by a tripartite enhancer that includes a consensus activator
protein-1 (AP-1) element, a binding site for SF-1 (steroidogenic
factor-1), and an element we have termed GRAS (GnRHR-activating
sequence). Further, in transgenic mice, approximately 1900 bp of the
murine GnRHR gene promoter are sufficient for tissue-specific
expression and GnRH responsiveness. The present studies were designed
to further delineate the molecular mechanisms underlying GnRH
regulation of GnRHR gene expression. Vectors containing 600 bp of the
murine GnRHR gene promoter linked to luciferase (LUC) were transiently
transfected into
T31 cells and exposed to treatments for 4 or
6 h. A GnRH-induced, dose-dependent increase in LUC expression of
the -600 promoter was observed with maximal induction of LUC noted at
100 nM GnRH. We next tested the ability of GnRH
to stimulate expression of vectors containing mutations in each of the
components of the tripartite enhancer. GnRH responsiveness was lost in
vectors containing mutations in AP-1. Gel mobility shift data revealed
binding of fos/jun family members to the AP-1 element of the murine
GnRHR promoter. Treatment with GnRH or phorbol-12-myristate-13-acetate
(PMA) (100 nM), but not forskolin (10
µM), increased LUC expression, which was
blocked by the protein kinase C (PKC) inhibitor, GF109203X (100
nM), and PKC down-regulation (10
nM PMA for 20 h). In addition, a specific
MEK1/MEK2 inhibitor, PD98059 (60 µM), reduced
the GnRH and PMA responses whereas the L-type voltage-gated calcium
channel agonist, ±BayK 8644 (5 µM), and
antagonist, nimodipine (250 nM), had no effect
on GnRH responsiveness. Furthermore, treatment of
T31 cells with
100 nM GnRH stimulated phosphorylation of both
p42 and p44 forms of extracellular signal-regulated kinase (ERK), which
was completely blocked with 60 µM PD98059. We
suggest that GnRH regulation of the GnRHR gene is partially mediated by
an ERK-dependent activation of a canonical AP-1 site located in the
proximal promoter of the GnRHR gene.
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INTRODUCTION
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Upon binding to specific, high-affinity receptors on gonadotrope
cells of the anterior pituitary gland, GnRH stimulates expression of
genes encoding the common
-subunit and specific LHß- and
FSHß-subunits that combine to produce LH or FSH (1, 2, 3, 4). GnRH also
stimulates the secretion of these pituitary gonadotropins that are
essential for normal gonadal function in both males and females (4, 5).
Therefore, the interaction of GnRH with its cognate pituitary receptor
serves as a central point for regulation of reproductive function.
Given the role of GnRH in stimulating gonadotropin synthesis and
secretion, it is not surprising that changes in the secretory rate of
LH are highly dependent on the level of hypothalamic GnRH secretion (6, 7). Additionally, changes in numbers of GnRH receptors (GnRHRs) are
correlated with changes in LH secretion (8). Thus, regulation of LH
secretion appears to be dependent not only on GnRH availability, but
also on the number of GnRHRs and, consequently, the sensitivity of the
pituitary gland to a given dose of GnRH (9). In this regard, a number
of endocrine factors, including 17ß-estradiol, progesterone,
testosterone, inhibin, activin, and GnRH itself, have been implicated
as mediating dynamic changes in the numbers of GnRHRs in the pituitary
gland (10, 11, 12, 13, 14, 15, 16, 17). Of these, perhaps the most dramatic effects are those
mediated by GnRH and 17ß-estradiol. Stimulatory effects of these two
hormones on GnRHR numbers have been demonstrated in several different
species (14, 18, 19, 20). This regulation presumably represents a
physiologically relevant avenue for increasing the sensitivity of the
pituitary gland to GnRH during the periovulatory period (21). More
recently, with the availability of cDNAs for the GnRHR,
researchers have found that changes in GnRHR numbers associated with
GnRH and/or 17ß-estradiol treatment largely correlate with
concomitant fluxes in steady state levels of mRNA (22, 23, 24, 25, 26, 27, 28). Thus,
regulation of GnRHRs by these two hormones may involve a
transcriptional component.
To examine the molecular mechanisms underlying transcriptional
regulation of the GnRHR gene, we have cloned the gene encoding the
murine GnRHR (29) and have begun analyzing the regulatory regions
within the promoter of this gene. We have found that expression of the
murine GnRHR gene in the gonadotrope-derived
T31 cell line
(30) is mediated by a tripartite enhancer located within 600 bp of
proximal 5'-flanking region. The components of this enhancer include a
binding site for the nuclear orphan receptor SF-1 (steroidogenic
factor-1) (31), a consensus activator protein-1 (AP-1) element, and a
noncanonical element we have termed the GnRHR-activating sequence or
GRAS (32, 33). In addition, we have constructed transgenic mice
harboring a transgene consisting of approximately 1900 bp of
5'-flanking sequence from the murine GnRHR gene linked to the cDNA
encoding luciferase (LUC) (34). LUC expression in these animals was
confined to the pituitary gland, brain, and testes, all established
sites of expression of the endogenous GnRHR gene. We also found that
pituitary expression of LUC in these transgenic mice was diminished by
immunoneutralization with GnRH antisera and subsequently restored by
administration of a non-cross-reactive GnRH agonist (34). Thus, we
concluded that 1900 bp of proximal promoter from the murine GnRHR gene
contains not only the elements that confer tissue-specific
expression but also one or more regulatory elements that act to confer
GnRH responsiveness in vivo.
Several GnRH responsive elements have been identified in other genes
that are targets for GnRH activation, including the upstream GnRH
response element (GnRH-RE) and pituitary glycoprotein hormone basal
element (PGBE) in the murine
-subunit gene (35), the GnRH-RE of the
human
-subunit gene (36), and two regions referred to as regions A
and B in the rat LHß-subunit gene promoter (37). However, we were not
able to identify homologies to any of these candidate elements in the
GnRHR gene, suggesting that the GnRH-responsive element(s) in
the GnRHR gene may be distinct from those previously defined in either
the glycoprotein hormone
- or LHß-subunit genes. We were intrigued
with the possibility that one or more of the elements comprising the
basal, tripartite enhancer of the GnRHR gene (33) may also be involved
with mediating GnRH responsiveness. For example, AP-1 is a well
established mediator of several signal transduction pathways, including
protein kinase C (PKC) (38, 39, 40). Furthermore, GnRH activates
transcription of fos and jun in pituitary and
T31 cells (41, 42),
and temporal patterns of expression of GnRHR and jun in the pituitary
gland are similar during the ovine estrous cycle (41). Similarly,
several lines of evidence implicate a role for SF-1 in at least
partially mediating GnRH responsiveness. GnRH regulation of SF-1 mRNA
has been demonstrated in the rat pituitary gland (43), and surgical
disconnection of the hypothalamus and pituitary leads to a loss of SF-1
mRNA in the ovine pituitary gland (44). Also, disruption of the
SF-1-binding site in the bovine LHß-subunit gene promoter leads to a
loss of GnRH responsiveness of this promoter in transgenic mice (45).
Finally, since the identity of the protein(s) binding to GRAS is not
yet known, we cannot exclude a potential role for this element in
mediating GnRH responsiveness.
In a similar vein, little is known as to the pathways involved in GnRH
regulation of GnRHR gene expression. In
T31 cells, it appears that
GnRH treatment activates PKC with little effect on intracellular
concentrations of cAMP (46). Also, treatment of rat primary pituitary
cultures with GnRH has revealed a crucial role for mitogen-activated
protein kinase (MAPK) in regulation of GnRHR mRNA levels (47),
an effect likely mediated by PKC (48). Recently, however, Lin and Conn
(49) have suggested that GnRH activation of the GnRHR promoter occurs
via cAMP and protein kinase A (PKA) in a GH3 cell line engineered to
express GnRHRs (GGH3). Thus, the goals of the present studies were
to investigate the relative roles of PKC, PKA, or calcium in mediating
GnRH activation of the GnRHR gene promoter. Additionally, we sought to
identify the GnRH-responsive element(s) located in the proximal
promoter of the murine GnRHR gene. Herein, we report that GnRH
responsiveness of the GnRHR gene promoter in
T31 cells is
dependent on PKC activation of MAPK and is ultimately mediated at a
canonical AP-1 site that binds members of the jun and fos family of
transcription factors.
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RESULTS
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The GnRHR Promoter Is Responsive to Increasing Doses of GnRH in
T31 Cells
To establish the utility of the gonadotrope-derived
T31
cell line as a model for GnRH regulation, we examined the response of
600 bp of proximal promoter from the murine GnRHR gene to increasing
doses of GnRH. A GnRH dose-dependent increase in expression of -600
LUC was observed (Fig. 1
). The
fold-induction of cells treated with 100, 1,000, and 10,000
nM GnRH was higher (P < 0.05) than
in untreated cells, whereas treatment with 0.1, 1, and 10
nM GnRH was not different (P > 0.05) from
controls. Activation at these concentrations is in range with
established affinities of GnRH for its receptor (46). The somewhat
higher concentration of GnRH necessary to activate the GnRHR promoter
may reflect the lower number of receptors on
T31 cells as compared
with bona fide gonadotropes (46). The specificity of the GnRH response
was tested by addition of increasing doses of the competitive GnRH
antagonist Antide (0.001, 0.1, 10, and 1,000 nM) in
the presence of 100 nM GnRH. The ability of 100
nM GnRH to stimulate the -600 promoter was blocked by
inclusion of 0.1 nM Antide (Fig. 1
).

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Figure 1. Expression of Murine GnRHR -600 LUC with
Increasing Doses of GnRH or Antide in T31 Cells
A vector containing approximately 600 bp of 5'-flanking region
from the murine GnRHR gene fused to LUC was cotransfected with
RSV-ßgal using a calcium phosphate-DNA coprecipitation protocol as
described in Materials and Methods. Cells were
transfected and treated with either 0, 0.1, 1, 10, 100, 1,000, or
10,000 nM GnRH. For antagonist treatments, cells were
transfected as above, treated for 30 min with 0, 0.001, 0.1, 10, or
1,000 nM Antide, and treated with 100 nM GnRH.
At 4 h post transfection, cells were harvested and cellular
lysates were assayed for LUC and ß-galactosidase activity. LUC
activity was adjusted for ß-galactosidase activity, and values are
expressed as fold increase over the untreated -600 LUC controls.
Values represent the mean ± SEM. An
asterisk indicates values that are greater
(P < 0.05) than that of the untreated -600 LUC
control.
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AP-1 Is Critical for GnRH Responsiveness of the GnRHR Promoter
The retention of GnRH responsiveness to within 600 bp of
5'-flanking sequence is consistent with the possibility that one or
more of the three elements comprising the cell-specific basal enhancer
(33) may also serve to confer GnRH responsiveness. Thus, we next tested
the ability of GnRH to stimulate expression of GnRHR promoters
containing mutations in each of the three components of the tripartite,
basal enhancer alone or in combination in the context of -600 LUC to
determine whether any of these previously identified elements may
mediate GnRH responsiveness. The fold-induction of the wild-type
vector, µSF-1, µGRAS, and the µGRAS/µSF-1 double mutant by GnRH
was greater (P < 0.05) than that of promoterless
control (Fig. 2
) and not different
(P < 0.05) from the -600 wild-type construct.
However, GnRH responsiveness of vectors containing the mutation in
AP-1, either alone or in combination with GRAS or SF-1, was not
different from promoterless control (P > 0.05; Fig. 2
)
and below (P < 0.05) that for the wild-type -600
vector. Finally, basal expression of the GnRHR promoter constructs was
not different (P > 0.05) from promoterless control.
These results indicated that GnRH responsiveness of the GnRHR gene
promoter was acting through the consensus AP-1 element located between
-336 and -330 relative to the start site of translation in the murine
GnRHR gene promoter.

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Figure 2. Effect of Mutations in the Tripartite Enhancer on
GnRH Responsiveness of the Murine GnRHR Gene
Vectors containing mutations in a single element, two elements, or all
three elements of the tripartite, basal enhancer of the murine GnRHR
gene were cotransfected with RSV-ßgal using a calcium phosphate-DNA
coprecipitation protocol as described in Materials and
Methods. Cells were transfected and treated with 100
nM GnRH. At 4 h post transfection, cells were
harvested, and cellular lysates were assayed for LUC and
ß-galactosidase activity. LUC activity was adjusted for
ß-galactosidase activity, and values are expressed as fold increase
over untreated for each mutation. Values represent the mean ±
SEM. An asterisk indicates values that are
greater (P < 0.05) than that of the promoterless
LUC vector.
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Members of the Fos/Jun Family of Transcription Factors Bind to the
Murine GnRHR AP-1 Element
We determined whether a radiolabeled probe containing the
consensus AP-1 element from the murine GnRHR gene promoter could bind
to protein(s) in whole-cell extracts from
T31 cells. Competition
with 10-, 100-, and 500-fold molar excess of homologous unlabeled DNA
identified a sequence-specific complex, whereas the addition of
heterologous competitor (10-, 100-, and 500-fold molar excess) did not
compete for binding (Fig. 3
). To
determine whether the protein complex binding to the consensus AP-1
element was comprised of members of the fos/jun family of transcription
factors, we tested broad-spectrum antibodies that recognize all members
of either the jun or fos families of transcription factors for their
ability to alter binding to this complex. The jun antibody is directed
against the conserved DNA-binding domain of jun family members and thus
blocks accessibility of the DNA to the jun DNA-binding domain resulting
in attenuated binding. In contrast, binding of the fos antibody, which
is directed against a conserved, non-DNA-binding region in each of the
fos family members, results in a higher mol wt complex (i.e.
supershift). The addition of antibodies for either jun (1, 2, or 4
µg) or fos (1 or 2 µg) family members resulted in attenuated
binding or a supershifted complex, respectively (Fig. 3
). Neither the
addition of 1, 2, or 4 µg of an antibody directed against
cAMP-regulatory element binding protein-1 (CREB-1) nor similar
concentrations of rabbit IgG affected binding of cellular proteins to
the radioactive murine AP-1 probe.

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Figure 3. Members of the fos and jun Families of
Transcription Factors bind to the Consensus AP-1 Element in the Murine
GnRHR Gene Promoter
Whole-cell extracts from T31 cells were incubated with a
radiolabeled probe consisting of the consensus AP-1 element from the
murine GnRHR gene promoter. Specificity of DNA-protein interactions was
assessed by competition with 10-, 100-, and 500-fold molar excess of
homologous and heterologous unlabeled DNA. In addition, whole-cell
extracts were incubated with a goat polyclonal antibody directed
against the DNA-binding domain of mouse c-jun p-39 (1, 2, or 4 µg), a
rabbit polyclonal antibody directed against a conserved domain of human
c-fos p62 (1 or 2 µg), a mouse monoclonal antibody directed against
the DNA-binding and dimerization domain of human CREB-1 (1, 2, or 4
µg), or an equal mass of rabbit IgG before the addition of
radiolabeled probe. Binding reactions were subjected to electrophoresis
through polyacrylamide gels as described in Materials and
Methods.
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Activation of the GnRHR Promoter by GnRH Is Mediated by Protein
Kinase C
We next determined whether pharmacological activation of
either PKC [100 nM phorbol-12-myristate-13-acetate (PMA)]
or PKA (10 µM forskolin) could mimic the GnRH response of
the -600 GnRHR promoter. Treatment with GnRH (100 nM),
PMA, or GnRH and PMA in combination increased (P <
0.05) expression of -600 LUC (4.1-, 8.8-, and 18.4-fold, respectively)
as compared with untreated controls (Fig. 4
). Interestingly, while forskolin alone
had no effect on expression of -600 LUC, the inclusion of forskolin
essentially abrogated induction of LUC expression by GnRH, PMA, or the
combined GnRH/PMA treatments (Fig. 4
). None of the treatments resulted
in any change (P > 0.05) in LUC expression from the
promoterless control vector (data not shown).

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Figure 4. Effects of GnRH and Activators of the Protein
Kinase A or C Second Messenger Pathways on Expression of Murine GnRHR
-600 LUC in T31 Cells
A vector containing approximately 600 bp of proximal 5'-flanking region
from the murine GnRHR gene fused to LUC was cotransfected with
RSV-ßgal using a calcium phosphate-DNA coprecipitation protocol as
described in Materials and Methods. Cells were
transfected and treated with 100 nM GnRH, 100
nM PMA, 10 µM forskolin (FSK), or each
combination of these treatments. At 4 h post transfection, cells
were harvested and cellular lysates were assayed for LUC and
ß-galactosidase activity. LUC activity was adjusted for
ß-galactosidase activity, and values are expressed as fold increase
over untreated -600 LUC. Values represent the mean ±
SEM. An asterisk represents values that are
greater (P < 0.05) than that of the untreated
-600 LUC control.
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Activation of the -600 murine GnRHR promoter by PMA and not forskolin
suggests a PKC-mediated pathway for induction of GnRHR gene expression.
To further examine this possibility, we added 100 nM of
GF109203X (Bisindolylmaleimide I), a PKC inhibitor, in the presence of
100 nM GnRH or PMA. Consistent with PKC-mediated
activation, the inclusion of 100 nM GF109203X completely
blocked both GnRH- and PMA-mediated induction of -600 LUC (Fig. 5
). The specificity of this inhibition
was tested by examining the response of 1500 bp of proximal promoter
from the human glycoprotein hormone
-subunit gene, a well
established PKA-responsive promoter (50), to forskolin in the presence
of GF109203X. The fold-induction of human
-1500 LUC by forskolin
was not affected by GF109203X, indicating that the inhibition of GnRH
and PMA induction of the murine GnRHR promoter by GF109203X was
specific to PKC (Fig. 5
).

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Figure 5. Effect of GF109203X on GnRH and PMA Responsiveness
of the Murine GnRHR Promoter
Vectors containing approximately 600 bp of 5'-flanking region from the
murine GnRHR gene or 1500 bp of proximal 5'-flanking region from the
human glycoprotein hormone -subunit gene fused to LUC were
cotransfected with RSV-ßgal using a calcium phosphate-DNA
coprecipitation protocol as described in Materials and
Methods. Cells were transfected, treated with or without 100
nM GF109203X (GFX) for 15 min, and treated with either 100
nM GnRH, 100 nM PMA, both GnRH and PMA, or 10
µM forskolin (FSK). At 4 h post transfection, cells
were harvested and cellular lysates were assayed for LUC and
ß-galactosidase activity. LUC activity was adjusted for
ß-galactosidase activity, and values are expressed as fold increase
over untreated -600 LUC. Values represent the mean ±
SEM. An asterisk represents values that are
greater (P < 0.05) than that of the untreated
-600 LUC control.
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As further confirmation of PKC dependence of the GnRHR gene response to
GnRH,
T31 cells were treated with 10 nM PMA for
20 h to down-regulate PKC (51). After 20 h of 10
nM PMA, the GnRH, PMA, and combined responses were
completely blocked (Fig. 6
).

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Figure 6. Effect of PKC Down-Regulation on GnRH and PMA
Responsiveness of the Murine GnRHR Promoter
Vectors containing approximately 600 bp of 5'-flanking region from the
murine GnRHR gene fused to LUC were cotransfected with RSV-ßgal using
a calcium phosphate-DNA coprecipitation protocol as described in
Materials and Methods. Approximately 20 h before
experimental treatments, one-half of the cells were treated with 10
nM PMA to induce PKC down-regulation, and the other half
remained untreated for controls. Cells were transfected and treated
with either 100 nM GnRH, 100 nM PMA, or both
GnRH and PMA. At 6 h post transfection, cells were harvested and
cellular lysates were assayed for LUC and ß-galactosidase activity.
LUC activity was adjusted for ß-galactosidase activity, and values
are expressed as fold increase over untreated -600 LUC. Values
represent the mean ± SEM]. An
asterisk represents fold increases that are greater
(P < 0.05) than that of the untreated -600 LUC
control.
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GnRH Activation of the GnRHR Promoter Is Mediated by a
Mitogen-Activated Protein Kinase Pathway
At least two possible pathways downstream of PKC have been
identified as being involved in GnRH responsiveness. One pathway
results in the activation of jun-N-terminal kinase (JNK), and the other
results in the activation of extracellular signal-regulated kinase
(ERK), also known as MAPK (51, 52). Since no specific inhibitors of JNK
were available, we examined a specific MEK1/MEK2 (MAPK kinase family
members) inhibitor (PD98059) to determine the role of the MAPK pathway
on GnRH induction of GnRHR gene expression. As shown previously, GnRH
and PMA, alone or in combination, stimulated (P <
0.05) LUC expression (Fig. 7
). The
addition of 60 µM PD98059 resulted in a dramatic
reduction (P < 0.05) in the GnRH, PMA, and combined
responses (Fig. 7
). Thus, these results indicate that GnRH
responsiveness may be conferred through PKC activation of a MAPK
pathway. If correct, then GnRH should lead to ERK activation that in
turn can be inhibited by PD98059.

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Figure 7. Effect of PD98059 on GnRH and PMA Responsiveness of
the Murine GnRHR Promoter
Vectors containing approximately 600 bp of 5'-flanking region from the
murine GnRHR gene fused to LUC were cotransfected with RSV-ßgal using
a calcium phosphate-DNA coprecipitation protocol as described in
Materials and Methods. Cells were transfected, treated
with or without 60 µM PD98059 for 15 min, and treated
with either 100 nM GnRH, 100 nM PMA, or both
GnRH and PMA. In addition, cells were also treated with 60
µM PD98059 at 3 h after GnRH treatment. At 6 h
post transfection, cells were harvested and cellular lysates were
assayed for LUC and ß-galactosidase activity. LUC activity was
adjusted for ß-galactosidase activity, and values are expressed as
fold increase over untreated -600 LUC. Values represent the mean
± SEM. An asterisk represents values that
are greater (P < 0.05) than that of the untreated
-600 LUC control. A dagger represents values that are
less (P < 0.05) than those for cells treated with
GnRH, PMA, or both GnRH and PMA in the absence of PD98059.
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To test this possibility,
T31 cells were treated with the GnRH
agonist, buserelin (10 nM), for 0, 15, or 30 min in the
presence or absence of 60 µM PD98059 and examined for ERK
activation by Western blot analysis using an antibody recognizing
phosphorylated p42 and p44 ERK. Phosphorylation of both the p42 and p44
forms of ERK was evident at 15 and 30 min after 10 nM
buserelin treatment (Fig. 8
). At each
time point, phosphorylation of both forms of ERK was blocked by
PD98059. To control for potential loading differences, the
antiphospho-ERK immunoblot was stripped and reprobed using an antisera
recognizing ERK protein independent of phosphorylation state (Fig. 8
).

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Figure 8. The Specific MEK1/MEK2 Inhibitor, PD98059, Blocks
GnRH Activation of ERK1 and ERK2 in T31 Cells
T31 cells were serum starved for approximately 2 h. Cells
received dimethylsulfoxide (Control) or PD98059 (60 µM)
15 min before and for the duration of buserelin administration (0, 15,
or 30 min). Cells were then lysed and debris cleared by centrifugation.
Lysates were resolved by SDS-PAGE and transferred to polyvinylidene
difluoride membrane. Initially, the blot was probed with a
phospho-specific ERK antibody (p-ERK), which recognizes the dual
phosphorylated (thus activated) forms of ERK1 and ERK2. The blot was
then stripped and reprobed with an ERK antibody (ERK) that detects
relative amounts of ERK protein independent of phosphorylation state.
The arrows identify the p44 (upper arrow)
and p42 (lower arrow) forms of ERK.
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GnRH Activation of the GnRHR Promoter Is Not Affected by an
Activator or an Inhibitor of L-Type Voltage-Gated Calcium Channels
In addition to MAPK, L-type voltage-gated calcium channels have
also been implicated in mediating GnRH regulation of the common
-
and specific LHß-subunit genes. To assess the potential role of
L-type voltage-gated calcium channels, we tested the ability of either
an agonist (±BayK 8644) or antagonist (nimodipine) of L-type
voltage-gated calcium channels to alter the responsiveness of the -600
GnRHR promoter to GnRH in
T31 cells. Neither the addition of 5
µM ±BayK 8644 nor 250 nM nimodipine, alone
or in combination with GnRH, affected (P < 0.05) the
GnRH response of -600 LUC expression compared with that of controls
(Fig. 9
, A and B). In contrast,
consistent with others (53, 54), treatment of the human
-1500
promoter with ±BayK 8644 increased LUC activity (Fig. 9A
), whereas
nimodipine decreased GnRH-induced LUC activity (Fig. 9B
).
 |
DISCUSSION
|
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The binding of GnRH to its pituitary receptor not only stimulates
but is obligatory for the synthesis and secretion of LH. Thus, the
GnRHR is the site of the primary stimulatory input to gonadotrope cells
and represents a potential control point in regulation of gonadotropin
secretion and, consequently, gonadal function in mammals. In fact,
changes in the number of pituitary GnRHRs have been implicated as an
important mechanism underlying the regulation of LH secretion (8, 9, 10, 26, 27). Consistent with this, several hormones, most notably
17ß-estradiol and GnRH itself, have been shown to regulate both GnRHR
numbers and mRNA in the pituitary gland (22, 23, 25, 26, 27, 28).
For the past several years, our laboratory has focused on the molecular
mechanisms underlying regulation of GnRHR gene expression. Based on
transient expression assays in the gonadotrope-derived
T31 cell
line, we have suggested that cell-specific expression of the murine
GnRHR gene is mediated by a tripartite enhancer located within 600 bp
of proximal 5'-flanking region. The components of this enhancer include
a consensus AP-1 element, a binding site for SF-1, and a noncanonical
element we have termed GRAS (33). Furthermore, approximately 1900 bp of
proximal promoter from the GnRHR gene are capable of conferring
tissue-specific expression and GnRH responsiveness on a heterologous
reporter gene in transgenic mice (34). In the present study, we have
been able to recapitulate GnRH responsiveness in vitro in
the
T31 cell line, thus allowing a more refined analysis of the
region(s) of the GnRHR gene that confer GnRH regulation.
Based on several lines of evidence, we suggest that PKC-mediated
activation of an AP-1 element in the proximal promoter of the GnRHR
gene is an important component underlying GnRH regulation of GnRHR gene
expression. First, retention of GnRH responsiveness to within 600 bp of
proximal 5'-flanking region is consistent with the location of AP-1
between -336 and -330 relative to the start site of translation in
the GnRHR gene promoter. Second, mutation of AP-1 alone or in
combination with the other two components of the tripartite enhancer of
the GnRHR gene leads to loss of GnRH responsiveness. Third,
pharmacological activation of PKC, but not PKA, mimics GnRH induction
of the -600 GnRHR gene promoter. Fourth, a specific PKC inhibitor
(GF109203X) blocks both GnRH and PMA activation of the GnRHR promoter.
Finally, down-regulation of the PKC second messenger system
dramatically reduces the GnRH and PMA responses of the GnRHR
promoter.
Our result indicating that GnRH-induced GnRHR gene expression is
mediated by PKC is consistent with the proposed mechanism(s) of action
of GnRH. In both gonadotropes and
T31 cells, GnRH-induced signal
transduction partially occurs via coupling of the bound GnRHR with
Gq
/G11
, leading to stimulation of
multiple phospholipase activities, formation of inositol
1,4,5-trisphosphate and diacylglycerol, elevation of intracellular free
calcium concentrations, and activation of PKC (46, 55). Recently,
however, others have reported that GnRH regulation of the GnRHR gene in
a heterologous cell line (GH3) is mediated by cAMP and PKA (49). In the
present study, neither forskolin nor (Bu)2-cAMP (data not
shown) had any detectable stimulatory effect on activity of the GnRHR
promoter. In fact, the most striking effect of these compounds was a
complete inhibition of both GnRH and PMA induction of the GnRHR gene
promoter. Recently, investigators have reported that coupling of a G
protein-coupled receptor to G
q/phospolipase C can be
inhibited by cAMP (56). Thus, a relative lack of phospholipase C
activity may represent a potential mechanism for attenuation of GnRH
responsiveness by forskolin. Alternatively, inhibition of PKC signaling
by forskolin could occur by inhibition of the MAPK pathway (57, 58) or
phosphorylation of CREB and subsequent inhibition of c-jun (59).
Certainly other possibilities exist for cross-talk among these signal
transduction pathways (60); however, regardless of the precise
mechanisms, it is clear that our results regarding the role of PKA in
affecting GnRHR gene expression in
T31 cells is fundamentally
different from those obtained in GH3 cells (49). As the mouse GnRHR
promoter was common to both of these studies, it would seem that the
discrepancy is most likely due to the different cell lines used to
detect GnRH regulation.
It is abundantly clear that GnRH can activate a myriad of intracellular
signaling pathways, including MAPK and changes in intracellular
concentrations of calcium (46, 60). In fact, both of these pathways
have been implicated in GnRH regulation of gene expression.
Transcriptional induction of the murine glycoprotein hormone
-subunit gene by GnRH requires activated MAPK (61). In contrast, the
GnRH response of the human
-subunit gene may be more dependent on
calcium (32, 62, 63, 64). The picture in regard to the LHß-subunit gene
is not entirely clear with conflicting reports as to the relative
dependency of MAPK or voltage-gated calcium channels in mediating
GnRH responsiveness of the rat LHß-subunit gene promoter (53, 54).
Herein, we provide evidence that PKC activation of a MAPK pathway, and
not L-type voltage-gated calcium channels, is largely involved in GnRH
responsiveness of the GnRHR gene promoter. In support of this, we find
that a specific MEK1/MEK2 inhibitor (PD98059) not only reduces the GnRH
and PMA responses of the GnRHR promoter but also blocks GnRH-induced
ERK phosphorylation in
T31 cells. In contrast, treatment with a
calcium channel agonist (±BayK 8644) or antagonist (nimodipine) had
little effect on GnRH responsiveness of the GnRHR gene promoter. Thus,
these data are consistent with a recent report demonstrating
MAPK-dependent induction of GnRHR mRNA levels by GnRH in primary
cultures of rat pituitary cells (47).
In addition to activation of MAPK and calcium channels, GnRH has
recently been shown to activate the JNK pathway in
T31 cells (51).
Due to the absence of specific JNK inhibitors, we were not able to
directly address the potential contribution of this pathway to GnRH
activation of the GnRHR promoter. However, since GnRH induction of the
GnRHR gene is mediated at a canonical AP-1 site that clearly binds one
or more jun family members, it is not at all unlikely that the JNK
pathway may also play a major role in conferring GnRH responsiveness of
the GnRHR gene. If correct, then GnRH induction of GnRHR gene
expression may require functional activation of both ERK- and
JNK-mediated signaling cascades that ultimately converge at AP-1.
In contrast to GnRH regulation of the
- and LHß-subunit gene
promoters in which multiple elements appear to serve as targets for
GnRH signaling, our results indicate that GnRH responsiveness of the
GnRHR gene may be largely conferred through a single element. GnRH
responsiveness of the murine
-subunit gene is conferred by an
element termed the pituitary glycoprotein basal element or PGBE and an
upstream GnRH-RE (35). More specifically, binding sites for a
LIM-homeodomain transcription factor (LH-2) within the PGBE (65) and an
Ets factor-binding motif within the GnRH-RE (61) appear to be the
operative sites for GnRH induction of murine
-subunit gene
expression. The presence of the Ets-binding site is consistent with the
involvement of MAPK in mediating GnRH regulation of the murine
-subunit gene. In regard to GnRH regulation of the LHß-subunit
gene, two separate regions contained within 490 bp of proximal promoter
appear to interact to confer GnRH responsiveness of the rat
LHß-subunit gene in GH3 cells engineered to express GnRHRs; however,
the identity of the functional GnRH response element(s) contained
within these regions is not known (37). Using a transgenic mouse model,
Keri et al. (66) found that 776 bp of 5'-flanking sequence
from the bovine LHß-subunit gene were sufficient for GnRH
responsiveness. Although the operative GnRH response elements in the
bovine LHß-subunit gene have not been conclusively identified,
mutation of the SF-1 binding site in the bovine gene led to a
significant reduction in basal activity as well as GnRH responsiveness
in transgenic mice (45). Thus, regulation of SF-1 binding activity may
represent one avenue for GnRH regulation of LHß-subunit gene
expression. We were not able, however, to detect any significant role
for the SF-1-binding site in the GnRHR gene in mediating GnRH
responsiveness. Rather, our data indicate that virtually all of the
GnRH responsiveness of the GnRHR promoter is mediated at a single AP-1
site. Thus, while common pathways may underlie GnRH regulation of its
primary target genes in gonadotropes, different elements in the
promoters of the
-subunit, LHß-subunit, and GnRHR genes
ultimately mediate the GnRH response.
In summary, the past several years have witnessed enormous
progress in our understanding of the molecular mechanisms underlying
GnRH regulation of
- and LHß-subunit gene expression (46). In
contrast, GnRH regulation of the GnRHR gene has remained relatively
unexplored. Results from the present studies suggest that GnRH
regulation of GnRHR gene expression is partially mediated by PKC/MAPK
activation of a canonical AP-1 site located in the proximal promoter of
the GnRHR gene. Also, it is clear that both jun and fos family members
are present in
T31cells and are capable of binding to the AP-1
site in the GnRHR promoter. In this regard, it is important to note
that while both jun and fos are established targets for GnRH regulation
both in vivo and in
T31 cells (41, 42), a clear role
for these proteins in mediating GnRH responsiveness has been lacking.
As such, these data provide a functional, candidate target element for
jun and fos in a well established GnRH-responsive gene and contribute
to our expanding knowledge of the repertoire of elements and factors
used by GnRH to communicate with its primary target genes in
gonadotropes.
 |
MATERIALS AND METHODS
|
---|
Materials
Forskolin, PMA, and GnRH antagonist (Antide) were purchased from
Sigma Chemical Co. (St. Louis, MO). The GnRH was obtained from Bachem
(Philadelphia, PA). The GF109203X (Bisindolylmaleimide I), nimodipine,
±BayK 8644, and PD98059 were purchased from Calbiochem (La Jolla, CA).
Antibodies for jun (c-Jun/AP-1 [D]-G, catalog no. sc-44-G), fos
(c-Fos [K-25], catalog no. sc-253), and CREB (CREB-1 [X-12],
catalog no. sc-240) were obtained from Santa Cruz Biotechnology (Santa
Cruz, CA).
Plasmids
The plasmid -600 LUC consisted of 600 bp of 5'-flanking region
from the murine GnRHR gene fused to the cDNA encoding LUC in the
pGL3 basic vector (Promega, Madison, WI) (29). The
construction of vectors containing mutations in the individual elements
of the tripartite basal enhancer (µGRAS, µAP-1, and µSF-1) were
described previously (33). The mutant vectors contained either a
NotI site (µGRAS and µSF-1) or an EcoRI site
(µAP-1) in place of the wild-type sequence. Double and triple mutants
that included all combinations of these mutated elements were also
constructed (33). The human
-1500 LUC vector consisted of
approximately 1500 bp of 5'-flanking region from the human
-subunit
gene promoter linked to LUC (50). The control vector used to test for
transfection efficiency in all experiments contained the Rous sarcoma
virus promoter linked to the cDNA encoding ß-galactosidase
(RSV-ßgal).
Cell Culture and Transient Transfections
Cultures of
T31 cells were maintained at 37 C in a
humidified 5% CO2 in air atmosphere. Cells were cultured
before transfection in high-glucose DMEM containing 2 mM
glutamine, 5% FBS, 5% horse serum, 100 U/ml penicillin, and 100
µg/ml streptomycin sulfate (Mediatech, Herndon, VA). After
transfection, the cells were cultured in the same medium without FBS.
Transient transfections were carried out using a calcium phosphate/DNA
coprecipitation method as previously described (50). Briefly, the day
before transfection, 2 x 106 cells were plated in
100-mm tissue culture dishes. Complete media were removed, and calcium
phosphate/DNA precipitates in a total volume of 1 ml were added to the
plates. At 30 min, posttransfection media were added, and cells were
treated for either 4 or 6 h with either GnRH or the treatment as
indicated. Results of a 4-h treatment are depicted in Figs. 1
, 2
, 4
, and 5
; however, further analysis of the time course of the GnRH
response revealed a greater response at 6 h; therefore, Figs. 6
, 7
, and 9
show results of a 6-h treatment. In Fig. 1
, the GnRH
antagonist Antide (67) was added 30 min before GnRH. In Fig. 5
, the PKC
inhibitor GF109203X (68) was added 15 min before GnRH, PMA, or
forskolin. In Fig. 7
, the MEK1/MEK2 inhibitor PD98059 was added 15 min
before GnRH or PMA and again at 3 h after treatment. In Fig. 9
, the L-type calcium channel antagonist nimodipine was added 30 min
before GnRH. Within each assay, treatments were performed in
triplicate, and different plasmid preparations were used for each
assay. After either 4 or 6 h of treatment, cells were washed twice
with ice-cold PBS, harvested in 1 ml of ice-cold PBS containing 1
mM EDTA, concentrated by centrifugation at 300 x
g for 5 min, and lysed in 200 µl of 25 mM
glycyl-glycine (pH 7.8), 15 mM MgSO4, 1%
Triton-X100, and 1 mM dithiothreitol. Lysates were cleared
by centrifugation at 16,000 x g for 2 min. Lysates (20
and 50 µl for LUC and ß-galactosidase, respectively) were assayed
according to manufacturers instructions for LUC (Promega, Madison,
WI) and ß-galactosidase (Topix, Bedford, MA) activity using a Turner
20D luminometer (Turner Designs, Sunnyvale, CA). LUC values were
divided by ß-galactosidase activity to normalize for transfection
efficiency (33).
Gel-Shift Assays
Whole-cell extracts from
T31 cells were prepared by the
method of Manley et al. (69). Gel-shift assays were
conducted as previously described (29). Briefly, whole-cell extracts
(5.1 µg of protein) were incubated for 10 min at 4 C in 20 µl of
Dignam buffer D [20 mM HEPES (pH 7.9), 20% glycerol
(vol/vol), 0.1 M KCl, 0.2 mM EDTA, 0.5
mM dithiothreitol] containing 2 µg of poly(dI-dC)
(Pharmacia Biotech, Piscataway, NJ) and, where indicated, either a goat
polyclonal antibody directed against the DNA-binding domain of mouse
c-jun p-39 (1, 2, or 4 µg), a rabbit polyclonal antibody directed
against a conserved domain of human c-fos p62 (1 or 2 µg), a mouse
monoclonal antibody directed against the DNA-binding and dimerization
domain of human CREB-1 (1, 2 or 4 µg), or an equal mass of rabbit
IgG. After incubation, the radiolabeled probe (100,000 cpm) was added,
and, where indicated, unlabeled competitor. Reactions were incubated at
room temperature for 30 min, and free probe was separated from bound
probe by electrophoresis for 12 h at 35 mA in 6% polyacrylamide gels
that were prerun at 100 V for 30 min in 25 mM Tris, 190
mM glycine, and 1 mM EDTA, pH 8. Gels were
transferred to blotting paper, dried, and exposed to Hyperfilm MP
(Amersham, Arlington Heights, IL) for approximately 16 h at -70 C
with Dupont Cronex intensifying screens (Dupont, Boston, MA).
Radiolabeled probes were prepared by labeling the antisense strand with
[
-32P]ATP (4500 Ci/mmol; ICN, Irvine, CA) and T4
poly-nucleotide kinase followed by annealing to the complementary
strand. Double-stranded DNA probes were purified by centrifugation on a
G-25 Microspin column (Pharmacia Biotech, Piscataway, NJ).
ERK Activation Assays
T31 cells were grown to approximately 70% confluence and
serum starved for 2 h before drug treatment and lysis. The
specific MEK1/MEK2 inhibitor PD98059 (60 µM) or control
vehicle (dimethyl sulfoxide) was applied to the cells in DMEM 15 min
before and during treatment with the GnRH agonist buserelin
([D-SER(tBU)6,Pro9-ethylamide]GnRH (10
nM). After treatment, cells were washed with ice-cold
buffer containing 0.15 M NaCl and 10 mM HEPES
(pH 7.5) and lysed in RIPA buffer containing 20 mM Tris (pH
8.0), 137 mM NaCl, 10% glycerol, 1% NP-40, 0.1% SDS,
0.5% deoxycholate, 2 mM EDTA, 5 mM sodium
vanadate, 5 mM benzamidine, and 1 mM
phenylmethylsulfonyl fluoride on ice. The cell lysates were
collected and debris cleared by centrifugation. Proteins were resolved
using denaturing PAGE followed by transfer to polyvinylidene
difluoride membrane by electroblotting. Samples were analyzed for ERK
phosphorylation by Western blotting using an antibody to the dual
phosphorylated forms of ERK1 and ERK2 (Promega, Madison, WI). The blot
was then stripped and reprobed with an antibody that detects relative
amounts of ERK protein independent of phosphorylation state (Santa Cruz
Biotechnology).
Statistical Analysis
Data were analyzed using SAS (70). Means for GnRH-treated cells
were expressed as fold increases over nontreated cells. Means for LUC
activity in Figs. 1
, 7
, and 9
were logarithmically transformed due to
nonnormality and then analyzed. In Figs. 1
, 4
, 5
, and 6
, means for LUC
activity were analyzed by ANOVA and compared with control values with
Dunnetts two-tailed t-test. Least-squares means for LUC
activity in Fig. 2
were analyzed with the General Linear Models
procedure and compared using least significant differences. Since the
response of the pGL3 basic vector varied across assays, the
mean GnRH response for the pGL3 basic vector within each
assay was included as a covariable in the model used for calculation of
least-squares means for all vectors in Fig. 2
. In Figs. 7
and 9
, means
for LUC activity were compared using Tukeys studentized range
test.
 |
ACKNOWLEDGMENTS
|
---|
The
T31 cells were a generous gift from Dr. Pam Mellon
(Salk Institute, La Jolla, CA). The authors would like to thank Ann
Burns, Buffy Ellsworth, Anthony Guillen, Meredith Holtzen, Dr. Scott
Nelson, and Mark Riccardi for their time and efforts toward completion
of this study.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Colin M. Clay, Animal
Reproduction and Biotechnology Laboratory, Department of Physiology,
College of Veterinary Medicine and Biomedical Sciences, Foothills
Campus, Colorado State University, Fort Collins, Colorado 80523.
This work was supported by NIH Grants R29HD-32416 to C.M.C. and
R29HD-34722 to M.S.R. B.R.W. was supported by NIH Training Grant
HD-07031, and D.L.D. was supported by NIH Postdoctoral Fellowship
National Research Service Award 1F32HD-08169.
Received for publication June 11, 1998.
Revision received December 21, 1998.
Accepted for publication December 28, 1998.
 |
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