Activation of Translation in Pituitary
Gonadotrope Cells by
Gonadotropin-Releasing Hormone
Ronald Sosnowski*, Pamela L. Mellon, and Mark A. Lawson
Departments of Reproductive Medicine and Neuroscience and
The Center for Molecular Genetics
University of California, San Diego
La Jolla, California 92093-0674
The neuropeptide GnRH is a central regulator of
mammalian reproductive function produced by a
dispersed population of hypothalamic neurosecretory neurons. The principal action of GnRH is to
regulate release of the gonadotropins, LH and FSH,
by the gonadotrope cells of the anterior pituitary.
Using a cultured cell model of mouse pituitary gonadotrope cells, ␣T3–1 cells, we present evidence
that GnRH stimulation of ␣T3–1 cells results in an
increase in cap-dependent mRNA translation.
GnRH receptor activation results in increased protein synthesis through a regulator of mRNA translation initiation, eukaryotic translation initiation
factor 4E-binding protein, known as 4EBP or PHAS
(protein, heat, and acid stable). Although the GnRH
receptor is a member of the rhodopsin-like family
of G protein-linked receptors, we show that activation of translation proceeds through a signaling
pathway previously described for receptor tyrosine
kinases. Stimulation of translation by GnRH is protein kinase C and Ras dependent and sensitive to
rapamycin. Furthermore, GnRH may also regulate
the cell cycle in ␣T3–1 cells. The activation of a
signaling pathway that regulates both protein synthesis and cell cycle suggests that GnRH may have
a significant role in the maintenance of the pituitary
gonadotrope population in addition to directing the
release of gonadotropins. (Molecular Endocrinology 14: 1811–1819, 2000)
INTRODUCTION
The anterior pituitary consists of several subpopulations of cells identified by their production of specific
hormones and responses to specific releasing factors.
The gonadotrope cells, which produce the heterodimeric glycoprotein gonadotropins LH and FSH,
are responsive to the decapeptide releasing factor
GnRH. Stimulation of gonadotrope cells by pulsatile
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Molecular Endocrinology 14(11): 1811–1819
Copyright © 2000 by The Endocrine Society
Printed in U.S.A.
GnRH from the hypothalamus leads to the increased
production and release of LH and FSH. Both the pulse
frequency and amplitude differentially regulate the
synthesis of gonadotropin mRNA, and the release of
gonadotropins by gonadotrope cells (1–4).
The GnRH receptor expressed in pituitary gonadotropes has been cloned from a number of species (5).
The receptor is a unique member of the rhodopsin
family of G protein-linked seven-transmembrane domain receptors. The receptor lacks the intracellular
carboxyl-terminal tail and contains numerous sequence differences in otherwise highly conserved regions. Ligand binding to the GnRH receptor causes
activation of the G proteins, Gq and G11, although
some evidence exists for the additional activation of
Gi/G0 (6–8). Protein kinase C (PKC) activity is increased by GnRH receptor activation. Additionally,
GnRH receptor activation leads to stimulation of the
mitogen-activated protein kinase (MAPK) pathway (9).
This signaling cascade results in increased transcription of the glycoprotein hormone ␣-subunit and LH
␤-subunit genes (10). Previous studies have demonstrated that GnRH signaling involves activation of the
GTPase Ras (9), but subsequent studies have shown
that this may not be necessary for transcriptional activation via the MAPK signaling cascade (11).
The ␣T3–1 cultured pituitary gonadotrope cell line
expresses the GnRH receptor and is responsive to
GnRH stimulation. This cell line has been a valuable
tool in dissecting the transcriptional regulatory regions
of the ␣-subunit glycoprotein hormone gene (␣-GSU)
that are necessary for pituitary-specific transcription
(12). It has been shown that the mouse and human
genes are transcriptionally responsive to GnRH stimulation in ␣T3–1 cells (13). However, some evidence
suggests that the increase in ␣-GSU expression may
involve a translational component in addition to the
well described transcriptional component. First, although the MAPK cascade mediates increased transcription of the mouse gene in response to GnRH
stimulation (9), the human gene, which is not transcriptionally stimulated by MAPK (14), is also responsive to
GnRH in ␣T3–1 cells. This suggests that other factors
may participate in the GnRH response. Second, in
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transient transfection studies of ␣T3–1 cells with an
␣-subunit promoter-driven reporter gene, GnRH-stimulated reporter enzyme activity peaks within 3 h of
GnRH stimulation, whereas increase of endogenous
␣-subunit mRNA does not reach maximal levels until
12 h (10). This observation suggests that the transcriptional response is delayed with respect to the increase
of reporter gene enzyme activity and may be a result of
increased mRNA stability, increased translation, or
both.
Using the ␣T3–1 cell line as a model system, we
investigated the ability of GnRH receptor activation to
modify translation. In this study, we demonstrate that
GnRH- stimulated synthesis of the ␣-subunit is Ras
dependent. Further, we show GnRH stimulation results in an increase in both phosphorylation of the
translation-regulatory factor 4EBP by the kinase mammalian target of rapamycin (mTOR) and an increase in
cap-dependent translation. These data suggest that
GnRH stimulation of ␣T3–1 cells is partly exerted
through a general increase in cap-dependent translation. We also show that this regulation occurs through
a PKC-dependent mechanism. We conclude that
GnRH stimulates translation through activation of
PKC, Ras, and the mTOR kinase, leading to the direct
phosphorylation of the translational regulatory factor
4EBP.
RESULTS
GnRH Stimulates Glycoprotein
␣-Subunit Synthesis
Previous reports have indicated that ␣-GSU gene expression can be increased by GnRH stimulation
through activation of the MAPK pathway. Although
Ras may participate in the transduction of signals from
the GnRH receptor to MAPK, it is not obligatory and
may not be necessary for stimulation of transcriptional
activity. To demonstrate that GnRH stimulation of
␣T3–1 cells does indeed lead to a Ras-dependent
increase in ␣-GSU protein synthesis, serum-starved
␣T3–1 cells were microinjected with nonimmune IgG
(Fig. 1, A and B) or with anti-Ras IgG (Fig. 1, C and D)
and subsequently stimulated with GnRH analog
(GnRHa). After 24 h of incubation, cells were fixed and
processed for immunohistochemical identification of
␣-GSU. Samples were then examined by fluorescence
microscopy for the presence of the injection marker
cascade blue (Fig. 1, A and C) or for the presence of
␣-subunit (Fig. 1, B and D). Injection with anti-Ras IgG
blocked the GnRH-dependent increase in subunit synthesis seen in control injected cells (Fig. 1, A and B). A
summary of five independent experiments is presented graphically in Fig. 1E. This observation suggests that synthesis of ␣-GSU protein may be partly
dependent on Ras activity. To test this, we examined
the ability of Ras alone to increase ␣-GSU protein in
the absence of GnRH stimulation. Microinjection of
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Fig. 1. GnRH Stimulation of ␣-GSU Protein Synthesis Is Ras
Dependent
Serum-starved ␣T3–1 cells were microinjected with nonimmune IgG (A and B) or with nonimmune IgG plus anti-Ras
IgG (C and D). Cells were simulated with GnRH analog (3 nM)
for 24 h before immunohistochemical detection of ␣-GSU
protein. Injected cells were visualized in panels A and C by
the inclusion of cascade blue in the injection buffer. The
proportion of injected cells staining for ␣-GSU was determined in five separate assays and results are reported in
panel E. The asterisk indicates a significant difference from
control (P ⱕ 0.05) as determined by the method of Fisher.
control nonspecific IgG had no effect on ␣-GSU synthesis in serum-starved ␣T3–1 cells (Fig. 2, A and B),
whereas injection of purified Ras protein caused an
increase in ␣-subunit protein synthesis in the absence
of GnRH stimulation (Fig. 2, C and D). A summary of
five independent determinations is presented graphically in Fig. 2E. The observation that Ras activation
alone can recapitulate the GnRH stimulation of ␣-GSU
protein synthesis strongly suggests the involvement of
Ras in GnRH receptor signal transduction. The further
observation that GnRH action can be blocked by
blocking Ras activation provides strong evidence that
under this paradigm Ras is a component of the GnRH
signaling cascade leading to increased expression in
␣-GSU protein in ␣T3–1 cells.
GnRH Stimulates Cap-Dependent Translation
Observations by others that ␣-GSU promoter-driven
reporter gene activity peaks well before the endogenous levels of ␣-GSU mRNA, and that the stabilization
effect of GnRH treatment on ␣-GSU mRNA is itself
Regulation of Translation by GnRH
Fig. 2. Ras Alone Can Stimulate ␣-GSU Protein Synthesis
Serum-starved ␣T3–1 cells were microinjected with nonimmune IgG (A and B) or with nonimmune IgG plus purified
wild-type hRas (C and D). Cells were incubated for 24 h
before immunohistochemical detection of ␣-GSU protein. Injected cells were visualized in panels A and C by the inclusion
of cascade blue in the injection buffer. The proportion of
injected cells staining for ␣-GSU, visualized in panels B and
D, was determined in five separate assays and reported in
panel E.
delayed by several hours (10), suggests that mRNA
synthesis and degradation rates, although decreased
by GnRH stimulation, may not fully account for the
GnRH-stimulated increase in ␣-GSU protein synthesis. The contribution of increased translation rates to
GnRH-stimulated ␣-GSU synthesis has not been addressed. To test the hypothesis that GnRH stimulation
of gonadotrope cells includes a translational component, we constructed a bicistronic reporter gene that
would distinguish between changes in transcriptional
and translational activity (Fig. 3A). Similar reporter
plasmids have been used to demonstrate the regulation of cap-dependent translation by insulin (15),
which acts through a receptor tyrosine kinase. The
cytomegalovirus (CMV) immediate early enhancer and
promoter direct synthesis of the bicistronic transcript.
The resultant mRNA contains a firefly luciferase coding
sequence followed by an internal ribosomal entry site
derived from the 5⬘-untranslated region of encephalomyocarditis virus (EMCV) (16) and a second reading
frame encoding the Escherichia coli ␤-galactosidase
gene. Transcription of the reporter plasmid in transfected cells produces a mRNA template for cap-dependent translation of the luciferase reporter, and for
cap-independent translation of the ␤-galactosidase
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Fig. 3. Activation of Cap-Dependent Translation in ␣T3–1
Cells by GnRH
A, Structure of the bicistronic reporter gene pGL3EMCV5⬘-␤. Transfection of the reporter gene results in the
synthesis of the bicistronic mRNA under the control of the
CMV immediate early enhancer/promoter. The resulting
mRNA encodes both firefly luciferase and ␤-galactosidase
reporter genes and are translated by cap-dependent and
independent mechanisms, respectively. Cultured ␣T3–1 or
NIH/3T3 cells transfected with the bicistronic reporter plasmid pGL3-EMCV5⬘-␤ and stimulated with GnRHa or insulin
(B) or with 5 nM GnRHa or 50 ␮g/ml EGF (C) for 8 h. The
histogram represents the ratio of luciferase to ␤-galactosidase activity normalized to the untreated control of at least
three experiments. Error bars represent the SEM of at least
three determinations. Asterisks designate significant difference of means (P ⱕ 0.05) between the treated and the control
as determined by ANOVA and Fisher’s protected least significant difference (PLSD).
gene directed by the EMCV 5⬘-untranslated region.
Measurement of the ratio of reporter gene activity
provides a direct measurement of cap-dependent vs.
cap-independent translation, independent of transcriptional effects. Previous studies have shown that
ratios are consistent independent of reporter gene
order, or overall reporter gene composition, indicating
that reporter enzyme activity is not altered (15). The
bicistronic reporter gene was transfected into the pituitary gonadotrope-derived ␣T3–1 cells, which were
then serum starved for 12 h. Subsequently, cells were
stimulated with GnRHa or insulin for 8 h and assayed
for reporter gene activity. Comparison of the luciferase
to ␤-galactosidase ratio showed that both GnRH and
insulin increased the ratio of luciferase to ␤-galactosi-
MOL ENDO · 2000
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dase activity (Fig. 3B). In contrast, GnRH had no significant effect on NIH/3T3 cells, which do not express
the GnRH receptor, indicating that the effect of GnRH
on translation was specific to the ␣T3–1 cells. These
data demonstrate that both GnRHa and insulin increase cap-dependent translation in ␣T3–1 cells. It
has been demonstrated that epidermal growth factor
(EGF) stimulation, a factor that also activates translation in a manner similar to insulin, increases GnRH
signaling intensity or facilitates GnRH signal transduction (17). To test whether EGF affects GnRH signal
transduction resulting in translational stimulation,
␣T3–1 cells transfected as above were stimulated with
GnRH analog, EGF, or both. The results shown in Fig.
3C indicate that cap-dependent translation is stimulated by EGF but that GnRH and EGF together do not
exhibit synergistic action. No significant increase in
cap-dependent translation was observed with dual
stimulation of GnRHa and insulin (data not shown).
These data strongly suggest that GnRH stimulation of
␣T3–1 cells results in an increase in cap-dependent
translation, and that GnRH, insulin, and EGF stimulation may function through common signaling
intermediates.
Rapamycin Inhibits GnRH-Stimulated Translation
The majority of eukaryotic mRNAs bear a 5⬘-cap structure consisting of m7GpppN that is recognized by a
complex of proteins known as the cap-binding complex. A component of this complex, eIF-4E, recognizes
this cap structure and is essential for the interaction of
mRNA with the cap-binding complex and the subsequent initiation of translation (18). The activity of
eIF-4E is repressed by a family of binding proteins,
known as 4EBP or PHAS (protein, heat, and acid stable) (15, 19) that interfere with eIF-4E activation of the
cap-binding complex and thereby inhibit translation
initiation. The repressor activity of 4EBP is regulated
by phosphorylation in response to receptor tyrosine
kinase activity. It has been shown that 4EBP is regulated through the pathway involving a rapamycin-sensitive kinase (20–22) and that 4EBP is directly phosphorylated by mTOR (23). Although stimulation of the
MAPK pathway results in mTOR activation, it is not
clear at which point the MAPK and mTOR cascades
diverge. Activation of mTOR is not dependent on
MAPK activity, as the inhibitor of MAPK activation,
PD098059, does not inhibit activation of mTOR or
phosphorylation of 4EBP (22, 24). To determine
whether GnRH signaling involves mTOR, activation of
cap-dependent translation by GnRHa was analyzed
for sensitivity to rapamycin. Pretreatment of ␣T3–1
cells with rapamycin before stimulation with GnRHa
resulted in attenuation of the activation to 51% that of
untreated cells (Fig. 4A). Further evidence that GnRH
regulates translation via phosphorylation of 4EBP is
obtained by Western blot analysis of 4EBP in protein
extracts isolated from ␣T3–1 cells that have been stimulated with GnRHa alone or in the presence of rapa-
Vol. 14 No. 11
Fig. 4. Stimulation of Translation by GnRH Is Attenuated by
Rapamycin
A, ␣T3–1 cells were transfected with the bicistronic reporter plasmid and incubated in serum-free medium for
12–16 h. Transfected cells were treated with GnRHa for 8 h
after pretreatment with rapamycin for 30 min. The histogram
shows relative levels of stimulation in comparison to untreated controls. The asterisks indicate a significant difference of means (P ⱕ 0.05) by ANOVA and Fisher’s PLSD. B,
Cultured ␣T3–1 cells were incubated for 12 h in the absence
of serum. Cells were vehicle treated (V treated with GnRHa
for 15 min (G), treated with 20 nM rapamycin for 30 min (R), or
both (R⫹G). After harvesting cells, whole-cell protein extracts
were prepared and a Western blot was performed using
4EBP-1 antiserum. The ␣ and ␤ inhibitory, as well as the ␥
noninhibitory, phosphorylated forms of 4EBP-1 are indicated.
mycin (Fig. 4B). In untreated cells, 41% of total 4EBP
detected was present in the nonbinding ␥-form, with
the remainder present in the ␤-form. This is consistent
with observations by others of the phosphorylated
state of wild-type 4EBP in other cell systems (25).
Stimulation of ␣T3–1 cells with GnRHa caused 90% of
the total detected 4EBP to be found in the non-eIF4Ebinding ␥-form after 15 min of stimulation. Rapamycin
treatment causes the conversion of 4EBP to the ␣- and
␤-inhibitory forms, and this conversion is not overcome by GnRH treatment. These data confirm that
GnRH stimulation of cap-dependent translation involves regulation of 4EBP and subsequent derepression of translation through the eIF-4E initiation
pathway.
GnRH Stimulation of Translation Is
Ras Dependent
Insulin, EGF, and other hormones acting through receptor tyrosine kinases activate the MAPK cascade
Regulation of Translation by GnRH
1815
through recruitment and activation of the ubiquitous
GTPase Ras, either by the adapter protein complex of
Grb2/Sos1 or by the activation of phospholipase C-␥.
The MAPK cascade is directly activated by Ras, but
Ras also activates other GTPases and protein kinases.
Previous studies have demonstrated that GnRH ligand
binding leads to activation of Ras, but that this is not
necessary for activation of MAPK (11).
To examine the potential role of Ras in GnRH regulation of translation, ␣T3–1 cells were cotransfected
with the bicistronic reporter plasmid and an expression vector encoding the dominant-negative mutant
Ras A15 (26). If Ras is a component of the signaling
pathway leading to translational stimulation by GnRH,
the presence of dominant negative Ras would be expected to impair the ability of GnRH stimulation to
cause an increase in cap-dependent translation. Indeed, as shown by the results presented in Fig. 5A, the
presence of dominant-negative Ras (A15) limited
GnRH stimulation of translation to approximately 50%
of that observed in cells cotransfected with a null
expression vector (⫺). The ability of dominant-negative Ras to attenuate GnRH stimulation of cap-dependent translation indicates that Ras may participate in
the GnRH signaling cascade leading to the regulation
of 4EBP.
PKC Activity Is Necessary for GnRH Action
The stimulation of the MAPK cascade by GnRH signaling is dependent on protein tyrosine kinase activity
(11). However, at least two mechanisms are possible
by which tyrosine kinase activity can be induced by a
G protein-coupled receptor. The first is through activation of Ras via G␤␥-dependent PI 3-kinase activity
(27). The second involves a mechanism dependent on
PKC activity and intracellular calcium (8). To differentiate these mechanisms, ␣T3–1 cells cotransfected
with the bicistronic reporter gene and the dominantnegative mutant rasA15 expression vector were stimulated with GnRHa or the PKC activator phorbol myristyl-acetate (PMA). Treatment with PMA stimulated
cap-dependent translation in ␣T3–1 cells. Additionally,
the action of PMA was inhibited by the presence of
dominant-negative RasA15, similar to the inhibition of
GnRH action (Fig. 5A). As a further demonstration of
the involvement of PKC in the activation of translation
by GnRH, stimulation of translation in ␣T3–1 cells by
GnRH was also attenuated by the PKC inhibitor bisindolylmaleimide, providing further evidence that PKC
activity is essential for GnRH-mediated translational
regulation (Fig. 5B). These observations suggest that
Ras activation proceeds through a PKC-dependent
mechanism and that translational regulation is dependent on Ras activation. Interestingly, we were unable
to detect significant inhibition of translational stimulation by the tyrosine kinase inhibitor genistein, or wortmannin, the PI3 kinase inhibitor (data not shown). This
is consistent with observations reported by others for
GnRH activation of the MAPK pathway (11) and indi-
Fig. 5. Involvement of PKC and Ras in Stimulation of Translation by GnRH
A, ␣T3–1 cells were cotransfected with the bicistronic reporter gene and a null expression vector (⫺) or cotransfected
with a vector expressing the dominant negative Ras mutant
A15 (A15). Cells were treated with PMA or GnRHa for 8 h and
harvested. The ratio of luciferase-␤-galactosidase activity of
the Ras A15 cotransfected cells is plotted relative to the
activity of null vector control. Error bars represent the SEM of
at least three experiments. Asterisks indicate a significant
difference (P ⱕ 0.05) between treated null and treated Ras
A15 cotransfected cells as determined by ANOVA and Fisher’s PLSD. B, Cultured ␣T3–1 cells transfected with the bicistronic reporter and treated with GnRHa with or without 30
min pretreatment with the PKC inhibitor bis-indolylmaleimide
(Bis-IM). The histogram indicates the relative induction of
luciferase to ␤-galactosidase activity of the treated cells normalized to the untreated control. Error bars indicate the SEM
of at least three experiments. The asterisk indicates a significant difference from the control mean (P ⱕ 0.05) as determined by ANOVA and the Fisher’s PLSD.
cates that the activities of tyrosine kinases or PI3
kinases may not be involved in GnRH-mediated activation of cap-dependent translation.
DISCUSSION
The regulation of protein synthesis by endocrine factors such as insulin, acting through receptor tyrosine
kinases, is well described. However, until recently (28),
MOL ENDO · 2000
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similar actions have not been described for hormones
that utilize G protein-coupled receptors. Here we
present evidence that the G protein-linked GnRH receptor regulates translation by a mechanism involving
PKC, Ras, the rapamycin-sensitive mTOR kinase, and
the translational inhibitor 4EBP. We demonstrate this
using a novel reporter gene that distinguishes between
regulated, cap-dependent translation and unregulated
cap-independent translation. We show that the stimulation of translation is similar to that observed by
insulin and EGF stimulation. We also show that the
effect on translation can be attenuated by the PKC
inhibitor bis-indolylmaleimide and can be mimicked by
direct stimulation of PKC activity with phorbol ester.
Additionally, the involvement of Ras in this aspect of
GnRH signaling is demonstrated by the ability of the
dominant-negative mutant ras A15 to attenuate the
GnRH response, as well as the ability of anti-Ras IgG
to attenuate the response in microinjected ␣T3–1 cells
treated with GnRH analog.
The GnRH-stimulated increase in translational capacity of ␣T3–1 cells occurs concurrently with previously reported changes in ␣-GSU mRNA stability. It
has been shown that ␣-GSU mRNA half-life is increased in ␣T3–1 cells with GnRH treatment from 1.2
to 8 h (10). However, in untreated cultured rat pituitary
cells the half-life of mRNA is about 6 h (29). It is
possible that factors in addition to GnRH contribute to
␣-GSU mRNA stability. However, the ability of GnRH
to modify ␣-GSU mRNA stability in vitro suggests that
this mechanism is physiologically relevant in vivo. Although our initial observation of the effect of GnRH
stimulation on protein synthesis in ␣T3–1 cells was
made monitoring ␣-GSU protein synthesis, the mechanism characterized in this report is more general in its
effect. In addition to specific transcriptional activation,
a more general activation of cap-dependent translation would serve to amplify stimulatory response signals rapidly. The translational effects of stimulation are
long lasting and have been measured as much as 20 h
after stimulation (15). Therefore, GnRH can modulate
transcriptional, posttranscriptional, and translational
mechanisms to effect changes in target cell metabolism that enhance hormone biosynthesis.
The data presented provide evidence that GnRH
receptor activation leads to signaling targets normally
associated with growth factor receptor activation and
results in activation of translation in a manner similar to
insulin receptor and EGF receptor activation. GH-releasing hormone was shown to stimulate translation in
GH3 cells through a calcium-dependent pathway resulting in regulation of eIF2, not 4EBP, as described
here for GnRH (30, 31). The involvement of the eIF2
pathway in GnRH stimulation of translation has not
been investigated. A significant difference between
translational regulation by GnRH vs. that by insulin via
4EBP is the potential role of Ras. Previous studies
examining the regulation of 4EBP by the insulin receptor have not implicated Ras in the signaling cascade
regulating translation, although Ras is a downstream
Vol. 14 No. 11
component of insulin receptor signaling (32). Our results show that Ras has a role in the regulation of
translation by GnRH receptor activation. The MAPK
cascade can independently phosphorylate eIF4E, as
can PKC, and this stimulates cap-dependent translation (33). It is not likely that our observations are solely
a result of direct regulation of eIF4E by PKC because
the effect was attenuated by dominant negative Ras
and rapamycin. Additionally, the MEK inhibitor
PD098059, which prevents MAPK activation and subsequent phosphorylation of eIF4E, did not inhibit
GnRHa-induced translational stimulation (M. A. Lawson, unpublished observations). Roles for other signaling pathways regulating translation cannot be ruled
out because the inhibitors tested were not capable of
completely abolishing the stimulatory effect of GnRH,
consistent with the participation of multiple signal cascades in translation regulation.
The demonstration that GnRH influences cell metabolism through stimulation of cap-dependent
translation strongly suggests that the gonadotrope
population can be dynamically regulated by GnRH
stimulation, as can the amount of hormone synthesized and released in response to stimulation. Activation of translation in response to releasing factor stimulation is a rapid and simple mechanism to replenish
protein levels before an increase in mRNA synthesis.
In concert with the reported increased ␣-GSU mRNA
stability in response to GnRH stimulation, significant
increase in protein synthetic capacity can be attained.
The demonstration that GnRH increases protein synthesis in gonadotrope cells through a transduction
pathway normally associated with growth factor activation provides evidence that releasing factors may
have a significant role in the maintenance of pituitary
cell subpopulations. Others have reported that GnRH
does regulate cell cycle in ␣T3–1 cells (34). The ␣T3–1
cell line represents an immature, proliferating gonadotrope cell that expresses the definitive marker GnRH
receptor and steroidogenic factor-1 genes, but not the
LH and FSH ␤-subunit genes expressed in fully mature
gonadotropes (35). The possibility that GnRH could
act through an insulin-like signaling mechanism as
reported here in a cultured cell model system has
important implications for the role of GnRH in the
development of tissues expressing the GnRH receptor. GnRH-expressing neurons can be detected as
early as embryonic day 11.5 and are established in the
hypothalamus before the development of the gonadotrope population (36). It can be postulated that GnRH
expression is a gonadotrope-specific signal that affects the proliferation of GnRH receptor-expressing
cell types. Precedence can be found in the parallel
GH-releasing hormone (GHRH)/somatotrope endocrine axis. Overexpression of human GHRH in transgenic mice leads to pituitary somatotropes hyperplasia (37). Mutation of the GHRH receptor in the little
mouse leads to a paucity of somatotrope in the anterior pituitary (38), and evidence exists that receptor
function is necessary for normal development of the
Regulation of Translation by GnRH
somatotrope cell population (39). These observations
suggest that GHRH receptor signaling is necessary for
proper proliferation of the somatotrope population.
More strikingly, it has also been observed that CRF
and EGF can serve as mitogenic factors for the corticotrope population (40). By analogy, similar action can
be postulated for the regulation of the gonadotropes.
At least one model system suggests that this is indeed
possible. The hypogonadal hpg (41) mouse bears a
deletion in the GnRH gene that renders it nonfunctional (42). Pituitary function can be recovered by
transplantation of tissue containing GnRH neurons or
by implantation of immortalized GnRH-secreting cells
(43). Although the increase in circulating gonadotropin
content has been documented (44), the effect on cell
number in the pituitary has not been examined. Future
studies will examine the potential role of GnRH in
gonadotrope cell proliferation more closely.
In summary, activation of the GnRH receptor stimulates cap-dependent translation through the phosphorylation of the translational regulatory factor 4EBP.
This action suggests that GnRH signaling regulates
both transcriptional and translational activity in the
target cell population. Use of a regulatory pathway
associated with growth factor signaling also suggests
that GnRH signaling may play a role in the maintenance of cell types expressing the GnRH receptor and
provide a specific mechanism for controlling the activity of GnRH-responsive cell populations.
MATERIALS AND METHODS
Plasmids
The bicistronic reporter plasmid pGL3-EMC5⬘-␤ was constructed in the reporter gene vector pGL3 Basic (Promega
Corp., Madison, WI). The plasmid contains the 664-bp SpeI
fragment of pCDNAI (Invitrogen, San Diego, CA) containing
the cytomegalovirus immediate early enhancer and promoter
inserted into the NheI site of the multiple cloning site. The
692-bp ClaI to NcoI fragment from the plasmid pTM1 (45)
containing the 5⬘-untranslated region and internal ribosomal
entry site of EMCV, the 3.2-kb NcoI to BamHI fragment of
pSDKLacZ containing the coding sequence of ␤-galactosidase, and the SV40 T-antigen splice and polyadenylation
sites were also inserted after the luciferase coding region.
The resultant plasmid contained the luciferase coding region
followed by the EMCV 5⬘-untranslated region containing the
internal ribosome entry site and the ␤-galactosidase coding
sequence. The dominant-negative Ras expression plasmid
used in cotransfection studies contained the KpnI to BamHI
cDNA fragment of pCEP4-RasA15 encoding the dominant
negative Ras mutant A15 (26) inserted into the multiple cloning site of the expression plasmid pcDNAI (Invitrogen).
Cell Culture and Transfection
The pituitary gonadotrope cell line ␣T3–1 (12) and NIH/3T3
cells were maintained in DMEM (Life Technologies, Inc.,
Gaithersburg, MD) supplemented with 10% FBS, 4.5 mg/ml
glucose, 100 ␮g/ml of penicillin, and 0.1 mg/ml streptomycin.
Cells were grown in a humidified atmosphere of 5% CO2.
Cells were transfected with 3 ␮g of reporter plasmid DNA
in 35-mm dishes or plates by the calcium phosphate method
1817
(46) for 4–6 h, washed twice with PBS, and incubated in fresh
serum-free medium for 12–16 h. Transfected cells were then
treated with 5 nM (im-Bzl-D-His6, Pro9-N-ethylamide) GnRH
analog (GnRHa, kindly provided by Jean Rivier), insulin (80
nM), or EGF (50 ␮g/ml) for 8 h. Transfected cells were pretreated with the inhibitors bis-indolylmaleimide or rapamycin
(Calbiochem, La Jolla, CA) at 100 nM or 20 nM, respectively,
for 30 min before stimulation with GnRHa. In cotransfection
experiments, 1 ␮g of reporter plasmid DNA was used with
two molar equivalents of empty vector or Ras A15 expression
plasmid. Constant DNA concentration was maintained by
supplementation with nonspecific plasmid DNA. Cells were
harvested by scraping into 1 ml of 150 mM NaCl, 1 mM EDTA,
and 40 mM Tris-Cl (pH 7.4 at 25 C). Harvested cells were
pelleted in a 5415C centrifuge (Eppendorf, Madison, WI) and
resuspended in 50 ␮l of 100 mM potassium phosphate (pH
8.0) at 25 C, 0.2% Triton X-100. The resultant extracts were
clarified by further centrifugation for 5 min and assayed immediately for luciferase activity (Analytical Luminescence
Laboratory, Ann Arbor, MI) and ␤-galactosidase activity
(Tropix, Inc., Bedford, MA) using a MicroLumat 96P luminometer (EG&G Berthold, Gaithersburg, MD). Results are reported as a mean of at least three experiments. Error is
reported as SEM.
Western Blot Analysis
Twenty-four hours after plating, ␣T3–1 cells were washed
twice with PBS and placed in serum-free medium for 12–16
h. Control and rapamycin-pretreated cells were then stimulated with GnRHa at 5 nM for 15 min and immediately harvested in ice-cold buffer as described above. After pelleting,
cells were lysed in a buffer of 50 mM Tris-Cl (pH 7.4 at 25 C),
100 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, 50 mM ␤-glycerolphosphate, 1 mM EGTA, 50 mM NaF, 10 mM Na4P2O7, 0.1
mM Na3VO4, and 50 nM okadaic acid and subjected to three
cycles of freeze-thaw. Extracts were clarified by centrifugation and assayed for protein content by the method of Bradford (47). For each sample, 50 ␮g of protein were boiled in
Laemmli sample buffer and run on a 15% denaturing polyacrylamide gel. Protein was blotted to Immobilon-P membrane (Millipore Corp., Bedford, MA) by semidry transfer.
Detection of 4EBP was performed using rabbit antiserum (15)
diluted 1:4000 and enhanced chemiluminescence (Amersham Pharmacia Biotech, Arlington Heights, IL) with biotinylated secondary antibody and horseradish peroxidaseconjugated avidin-biotin complex (Vector Laboratories, Inc.,
Burlingame, CA). Blots were visualized by exposure to BioMax film and by storage phosphorimaging on a Molecular
Imager GS-525 (Molecular Dynamics, Inc., Sunnyvale, CA).
Stored images were analyzed with Molecular Analyst 1.5
software (Bio-Rad Laboratories, Inc., Hercules, CA).
Microinjection and DNA Synthesis Assays
Trypsinized ␣T3–1 cells were seeded on glass coverslips at
75% confluence and starved for 24 h in serum-free DMEM
(Life Technologies, Inc.). For microinjection, the culture medium was replaced with serum-free DMEM containing vehicle
or GnRH analog (3 nM) and incubated a further 24 h. Cells
were injected with 0.5X Tris-Borate buffer containing either
10 ␮g/ml normal guinea pig IgG, or 5 ␮g/ml rabbit anti-panRas IgG (Santa Cruz Biotechnology, Inc., Santa Cruz, CA)
and 5 ␮g normal guinea pig IgG (26), or 3 ␮g/ml purified
bacterially expressed wild-type H-Ras protein and 7 ␮g/ml
normal guinea pig IgG. Injected cells were marked by the
inclusion of cascade blue in the injection buffer. After incubation for 24 h, Cells were fixed and stained as described
previously (48). Briefly, after incubation, cells were fixed for
15–30 min in PBS containing 3.7% formaldehyde, processed
for immunohistochemistry using rabbit antirat ␣-glycoprotein
hormone subunit IgG, and visualized by incubation with flu-
MOL ENDO · 2000
1818
orescein-conjugated goat-antirabbit IgG secondary antibody. Results are reported as percentage of injected cells
staining for ␣-GSU. Between 35 and 152 injected cells were
counted per experiment. Error is reported as SEM proportion
by the method of Fisher. Significance is reported at
P ⬍ 0.05.
Acknowledgments
We thank Teri Williams and Brian Powl for excellent technical
assistance and Wade Blair and Zvi Naor for helpful discussion. We also thank Bert Semler for the gift of the EMCV
cDNA and Nahum Sonenberg for the gift of the 4EBP-1
antiserum.
Vol. 14 No. 11
10.
11.
12.
13.
Received June 9, 1999. Revision received June 8, 2000.
Accepted July 27, 2000.
Address requests for reprints to: Mark A. Lawson, Ph.D.,
Department of Reproductive Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 920930674. E-mail: mlawson@ucsd.edu.
This work was supported by NIH Grants R03 DK-52284
and R01 HD-37568 to M.A.L. and by U54 HD-12303 and R01
HD-20377 to P.L.M.
* Current Address: Nanogen Incorporated, 10398 Pacific
Center Court, San Diego, California 92121.
14.
15.
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