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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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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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T3–1 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 T3–1 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 T3–1 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. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-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 T3–1 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 T3–1 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 T3–1 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 T3–1 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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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T3–1 CellsT3–1
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 T3–1 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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) 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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T3–1 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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). 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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). 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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T3–1 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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). 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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T3–1 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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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 T3–1 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).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 T3–1 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 T3–1 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 T3–1 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 Gq/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 T3–1 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 T3–1 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 T3–1 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 T3–1cells 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 T3–1 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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 T3–1 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 manufacturer’s 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 T3–1 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 1–2 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
T3–1 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
Dunnett’s 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 Tukey’s studentized range
test.
|
ACKNOWLEDGMENTS |
|---|
T3–1 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 |
|---|
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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