Activation of Translation in Pituitary Gonadotrope Cells by Gonadotropin-Releasing Hormone
Ronald Sosnowski1,
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
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ABSTRACT
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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,
T31 cells, we present evidence that
GnRH stimulation of
T31 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
T31 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.
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INTRODUCTION
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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 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, 2, 3, 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, 7, 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 ß-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
T31 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
T31 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
T31 cells. This suggests that other factors
may participate in the GnRH response. Second, in transient transfection
studies of
T31 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
T31 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
T31 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.
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RESULTS
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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
T31 cells does indeed lead to a
Ras-dependent increase in
-GSU protein synthesis, serum-starved
T31 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 control
nonspecific IgG had no effect on
-GSU synthesis in serum-starved
T31 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
T31 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 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
ß-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 ß-galactosidase 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
T31 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
ß-galactosidase ratio showed that both GnRH and insulin increased the
ratio of luciferase to ß-galactosidase 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
T31 cells. These data demonstrate
that both GnRHa and insulin increase cap-dependent translation in
T31 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,
T31 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
T31 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, 21, 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
T31 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
T31 cells that have been stimulated with GnRHa alone
or in the presence of rapamycin (Fig. 4B
). In untreated cells, 41% of
total 4EBP detected was present in the nonbinding
-form, with the
remainder present in the ß-form. This is consistent with observations
by others of the phosphorylated state of wild-type 4EBP in other cell
systems (25). Stimulation of
T31 cells with GnRHa caused 90% of
the total detected 4EBP to be found in the non-eIF4E-binding
-form after 15 min of stimulation. Rapamycin treatment causes the
conversion of 4EBP to the
- and ß-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 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,
T31 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 Gß
-dependent PI 3-kinase activity (27). The
second involves a mechanism dependent on PKC activity and intracellular
calcium (8). To differentiate these mechanisms,
T31 cells
cotransfected with the bicistronic reporter gene and the
dominant-negative mutant rasA15 expression vector were stimulated with
GnRHa or the PKC activator phorbol myristyl-acetate (PMA). Treatment
with PMA stimulated cap-dependent translation in
T31 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
T31 cells by GnRH was also attenuated by the PKC inhibitor
bis-indolylmaleimide, 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 indicates that
the activities of tyrosine kinases or PI3 kinases may not be involved
in GnRH-mediated activation of cap-dependent translation.
 |
DISCUSSION
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The regulation of protein synthesis by endocrine factors such as
insulin, acting through receptor tyrosine kinases, is well described.
However, until recently (28), 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
T31 cells treated with
GnRH analog.
The GnRH-stimulated increase in translational capacity of
T31
cells occurs concurrently with previously reported changes in
-GSU
mRNA stability. It has been shown that
-GSU mRNA half-life is
increased in
T31 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
T31 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 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
T31 cells (34). The
T31 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 ß-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 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.
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MATERIALS AND METHODS
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Plasmids
The bicistronic reporter plasmid pGL3-EMC5'-ß 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 ß-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 ß-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
T31 (12) and NIH/3T3
cells were maintained in DMEM (Life Technologies, Inc.,
Gaithersburg, MD) supplemented with 10% FBS, 4.5 mg/ml glucose, 100
µg/ml of penicillin, and 0.1 mg/ml streptomycin. Cells were grown in
a humidified atmosphere of 5% CO2.
Cells were transfected with 3 µg of reporter plasmid DNA in 35-mm
dishes or plates by the calcium phosphate method (46) for 46 h,
washed twice with PBS, and incubated in fresh serum-free medium for
1216 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 µg/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 µg 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 µl 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 ß-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,
T31 cells were washed
twice with PBS and placed in serum-free medium for 1216 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
ß-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
µg 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 peroxidase-conjugated avidin-biotin
complex (Vector Laboratories, Inc., Burlingame, CA). Blots
were visualized by exposure to Bio-Max 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
T31 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 µg/ml
normal guinea pig IgG, or 5 µg/ml rabbit anti-pan-Ras IgG
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and 5
µg normal guinea pig IgG (26), or 3 µg/ml purified bacterially
expressed wild-type H-Ras protein and 7 µg/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 1530 min in PBS containing 3.7% formaldehyde,
processed for immunohistochemistry using rabbit antirat
-glycoprotein hormone subunit IgG, and visualized by incubation with
fluorescein-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
|
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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.
 |
FOOTNOTES
|
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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 92093-0674. E-mail: mlawson{at}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.
1 Current Address: Nanogen Incorporated, 10398 Pacific Center Court,
San Diego, California 92121. 
Received for publication June 9, 1999.
Revision received June 8, 2000.
Accepted for publication July 27, 2000.
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