Regulation of Gonadotropin-Releasing Hormone (GnRH) Gene Expression by Insulin-Like Growth Factor I in a Cultured GnRH-Expressing Neuronal Cell Line
Shanjun Zhen,
Marjorie Zakaria,
Andrew Wolfe and
Sally Radovick
Division of Endocrinology Childrens Hospital Harvard
Medical School Boston, Massachusetts 02115
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ABSTRACT
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A GnRH-expressing neuronal cell line (NLT) was
used to determine whether insulin-like growth factor I (IGF-I)
regulates GnRH gene expression. A receptor-binding assay demonstrated
the expression of IGF-I receptors on NLT cells. Activation of IGF-I
receptors induced the Ras/Raf-1/mitogen-activated protein kinase
pathway and increased c-fos expression. NLT cells treated
with IGF-I underwent cell proliferation and exhibited a
growth-independent increase in mouse GnRH mRNA expression. In cells
transfected with DNA constructs containing the human GnRH promoter,
which includes a consensus AP-1 binding site fused to the luciferase
reporter gene, a significant increase in reporter activities was
induced by IGF-I, whereas mutation of this AP-1 site significantly
reduced IGF-I-induced promoter activation. These results demonstrate
that IGF-I serves as an important signal in the regulation of both
human and rodent GnRH gene expression.
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INTRODUCTION
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Growth factors exert a profound effect on the proliferation,
differentiation, and functional states of various types of neurons.
However, the mechanisms of their action on GnRH neurons have been
difficult to study due to the scattered distribution and paucity in
GnRH cells in the animal brain (1, 2, 3). Recently, Mellon (4) and our
laboratory (5, 6) have generated immortalized GnRH-expressing neuronal
cell lines by targeted tumorigenesis. The NLT cells established in our
laboratory were generated by targeting the expression of the SV 40-Tag
to GnRH neurons in transgenic mice with the human (h) GnRH gene
promoter (5). These cells express GnRH mRNA as demonstrated by RT-PCR
and ribonuclease (RNase) protection assays, secrete GnRH decapeptide as
demonstrated by RIAs (7), and express markers specific for
differentiated neurons such as microtubule-associated protein 2 and Tau
protein (8, 9, 10). Therefore, these GnRH-expressing neuronal cells
provide a suitable model for studying the effects of extracellular
factors on the regulation of GnRH neuronal activities and the signaling
pathways involved.
Accumulating evidence suggests that IGF-I can regulate the reproductive
axis at several levels. Both IGF-I and its cognate receptors are widely
expressed in the central nervous system, including the hypothalamus
(11, 12, 13). Infusion of IGF-I has been shown to stimulate GnRH release
from the median eminence and accelerate the onset of puberty in female
rats (14, 15). A recent study also indicates that IGF-I exerts a
proliferating effect on the GT1-GnRH-expressing neuronal cells,
although the signaling pathways and the effect of IGF-I on GnRH gene
expression have not been examined (16). In addition to locally produced
IGF-I, circulating levels of IGF-I produced mainly by the liver in
response to GH are also increased during the onset of puberty (17). It
appears that, in addition to its action at the median eminence, which
is outside the blood-brain barrier (14), circulating IGF-I may increase
the responsiveness of gonadotropes to GnRH stimulation because IGF-I
enhances in vitro LH release in response to GnRH stimulation
(18, 19). Therefore, circulating as well as locally produced IGF-I may
serve as an important signal in the regulation of the reproductive
axis.
A common mechanism by which growth factors regulate neuronal function
is through transmembrane receptors with inducible protein-tyrosine
kinase activity. The IGF-I receptor consists of two extracellular
-subunits containing the ligand-binding domains, and two
transmembrane ß-subunits with the ligand-sensitive tyrosine kinase
activity (20). Binding of IGF-I to its cognate receptors stimulates
ß-subunit tyrosine kinase activity, leading to receptor
autophosphorylation and tyrosine phosphorylation of several cellular
substrates, including IRS-1 (21). As a docking protein, IRS-1 can
associate with certain proteins containing Src homology-2 (SH2)
domains, such as growth factor receptor bound-2 (Grb-2), leading to the
activation of Ras (a small GTP-binding protein) (22, 23, 24) and
mitogen-activated protein (MAP) kinase cascades that eventually lead to
phosphorylation of specific transcription factors (25).
Although the intracellular signaling pathways activated by IGF-I have
been studied extensively, the target genes within the central nervous
system (CNS) that are regulated by IGF-I remain to be defined. Our
laboratory has been interested in the study of hGnRH gene transcription
and its regulation. A consensus AP-1 binding site is present in the
promoter of hGnRH gene and is involved in mediating the transcriptional
activation of hGnRH gene by the phorbol ester,
12-0-tetradecanoyl phorbol-13-acetate (TPA) (26). However,
little is known about the possible extracellular ligands that can also
act through this AP-1 binding site to regulate hGnRH transcription and
the signaling pathways involved. In the present study, we provide
evidence that IGF-I receptors are expressed on the NLT cells.
Furthermore, we have examined the effect of IGF-I on the expression of
the mouse (m) GnRH mRNA and hGnRH gene and the signaling pathway
involved. The results from this study provide insights into the
mechanisms involved in the regulation of GnRH neuronal activities by
growth factors.
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RESULTS
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Expression of IGF-I Receptor on NLT Cells
To determine whether NLT cells express type 1 IGF receptors,
receptor- binding assays were performed using [125I]IGF-I
and NLT cells maintained in a serum-free DMEM medium for 24 h to
avoid interference from growth factors in the medium. As shown in Fig. 1A
, competitive inhibition of [125]IGF-I
binding to NLT cells was produced by adding different concentrations of
cold IGF-I, and 50% inhibition (IC50) was achieved by less
than 1 nM of cold IGF-I. Addition of different
concentrations of epidermal growth factor (EGF) or leptin did not
affect IGF-I binding, indicating the specificity of the binding (data
not shown). Scatchard analysis of the binding data revealed the
presence of a single class of high-affinity IGF-I binding sites on the
surface membrane of NLT cells, with a dissociation constant
(Kd) of 5.0 x 10-10 M and
receptor binding sites of 3.2 x 105/cell (Fig. 1B
).
These results indicate that type 1 IGF receptors are expressed on the
surface membrane of NLT cells.

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Figure 1. Expression of IGF-I Receptors in the NLT
GnRH-Expressing Neuronal Cells
A, Competitive inhibition of [125I]IGF-I binding to NLT
cells by unlabeled IGF-I. NLT cells at 90% confluence were maintained
in serum-free DMEM medium for 24 h, washed with PBS containing 1%
BSA, and incubated for 2 h at room temperature with
[125I]IGF-I in the presence or absence of the indicated
concentrations of unlabeled IGF-I. Unbound IGF-I was washed away by
cold PBS, and the percentage of radioactivity bound to cells was
plotted against the concentration of unlabeled IGF-I. B, Scatchard
analysis of IGF-I binding to NLT cells revealed the presence of a
single class of high-affinity IGF-I receptors with Kd value
of 5.0 x 10-10 M. The number of IGF-I
receptors was estimated to be 3.2 x 105/cell.
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IGF-1 Activates p42/p44 MAP Kinases and Induces c-fos
Expression
To characterize the intracellular signaling pathways activated by
IGF-I, induction of MAP kinase activity by IGF-I in NLT cells was
assayed. MAP kinases are a family of serine/threonine kinases that are
activated by threonine and tyrosine phosphorylation by MAP kinase
kinase upon activation of various growth factor receptors (22, 23, 25).
Prominent among them are the p42/p44 MAP kinases, which are involved in
the regulation of transcription factor activity by phosphorylation
(27). To determine whether IGF-I induces p42/p44 MAP kinases in NLT
cells, changes in p42/p44 MAP kinase activities were analyzed by their
ability to phosphorylate a peptide substrate containing the PLS/TP as a
site for phosphorylation (28). IGF-I treatment of NLT cells for 5 min
produced a dose-dependent increase in the p42/p44 MAP kinase activities
(Fig. 2A
). Time course studies indicated that the MAP
kinase activities were significantly increased within 5 min, declined
after 30 min, and remained low (but were still significantly higher
than the control level) during prolonged IGF-I treatment (Fig. 2B
).
These results clearly demonstrate that IGF-I induces p42/p44 MAP
kinases in NLT cells.

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Figure 2. IGF-I Induces p42/p44 MAP Kinases
A, Dose-dependent response of p42/p44 MAP kinases to IGF-I treatment.
NLT cells were cultured in DMEM containing 0.5% FCS overnight. The
cells were treated with the indicated concentrations of IGF-I for 5
min, washed with cold PBS, and lysed in a buffer containing protease
and phosphatase inhibitors. Cellular debris was precipitated at
24,000 x g for 20 min, and the supernatant was
retained to obtain cytoplasmic MAP kinase. The p42/p44 MAP kinase
enzyme assay system (Biotrak, Amersham Life Science, Arlington Heights,
IL) was used for analyzing the MAP kinase activity in the sample.
Phosphorylated peptide substrate was separated from unincorporated
label on a binding paper, and the extent of phosphorylation was
detected by scintillation counting. B, Time course of IGF-I-induced
p42/p44 MAP kinase activities. NLT cells were treated with 10 ng/ml
IGF-I for the indicated duration, and p42/p44 MAP activities were
measured. *, P < 0.05 vs. control,
analyzed using ANOVA followed by Dunnetts t test.
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Activation of Ras/Raf-1/MAP kinase signal pathway by growth factors has
been shown to induce the expression of specific transcription factors
including c-fos in various cell lines (16, 26, 29). Recent
studies from this laboratory demonstrated that treatment with TPA, a
phorbol ester, also increases c-fos expression and
stimulates hGnRH gene transcription in the Gn11 cells (26). To
determine whether c-fos gene expression is activated in
response to IGF-I stimulation in NLT cells, a reporter plasmid
containing -720 bp of the mouse c-fos promoter linked to
the bacterial chloramphenicol acetyltransferase (CAT) reporter gene was
transiently transfected into the NLT cells. After 12 h of culture
in a DMEM medium containing 0.5% serum, these transfected cells were
treated with 10 ng/ml of IGF-I for 6 h. A significant increase in
CAT reporter activity was induced by IGF-I treatment of the NLT cells
transfected with the reporter plasmid (Fig. 3
), whereas
the reporter activity generated by the Rous sarcoma virus (RSV)
promoter was not affected by IGF-I. This result indicates that
activation of IGF-I receptor stimulates c-fos expression in
NLT cells.

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Figure 3. IGF-I Stimulates c-fos Gene
Transcription
A CAT reporter plasmid containing -720 bp of the mouse
c-fos promoter or a RVS-CAT plasmid was transiently
transfected to NLT cells. After 48 h of transfection, the cells
were maintained in DMEM containing 0.5% serum for 12 h before
IGF-I treatment (10 ng/ml for 6 h). *, P <
0.05 vs. control, Students unpaired t
test.
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IGF-1 Stimulates Mouse GnRH mRNA Expression
To determine whether IGF-I regulates the expression of endogenous
mGnRH mRNA in NLT cells, RNase protection assays were performed using a
32P-labeled antisense mGnRH mRNA probe generated by
in vitro transcription of a cDNA template containing exons
1, 2, 3, and 4 of the mGnRH gene. As a control, antisense actin probe
was also prepared at the same time. Total cellular RNA was obtained
from NLT cells cultured in a DMEM medium containing 0.5% serum for
12 h and treated with or without IGF-I at a concentration of 10
ng/ml for 4 or 12 h. As shown in Fig. 4A
, treatment
of NLT cells with IGF-I for 4 and 12 h enhanced mGnRH mRNA
expression. Quantification of the mGnRH mRNA levels with a
PhosphorImager demonstrated that a 1.5-fold and 2.5-fold increase in
mGnRH mRNA expression was induced by IGF-I at 4 and 12 h,
respectively. In contrast, actin mRNA levels were comparable among
cells treated with or without IGF-I (Fig. 4B
). This result indicates
that IGF-I regulates GnRH gene expression independent of its growth-
promoting effect on NLT cells.

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Figure 4. IGF-I Stimulates mGnRH mRNA Expression in NLT as
Demonstrated by RNase Protection Assay
A, Template cDNA containing exons 1, 2, 3, and 4 of the mGnRH gene
inserted in the pGEM-T plasmid between the NcoI and
PstI sites was digested with NcoI, and
in vitro transcription was performed by using
[ 32P]UTP and SP6 polymerase to generate antisense
mGnRH probes. As a control, mouse actin antisense probe was also
synthesized. The riboprobes were hybridized with 3040 mg/tube of
total cellular RNA obtained from NLT cells treated with IGF-I at a
concentration of 10 ng/ml for 4 or 12 h. The hybridized RNA
fragments were precipitated and electrophoresed in a 6% denaturing
polyacrylamide gel. The positions of the GnRH and actin mRNA signals
are indicated on the right. B, Phosphorimager
quantification of relative GnRH mRNA density (GnRH mRNA
vs. actin mRNA signals) in response to IGF-I treatment
(10 ng/ml) for the indicated durations. Values are expressed as
mean ± SEM. *, P < 0.05
vs. control, analyzed using ANOVA followed by Dunnetts
t test.
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IGF-1 Promotes NLT Cell Proliferation
To determine whether IGF-I has any effect on DNA synthesis
and NLT cell proliferation, [3H]thymidine incorporation
into DNA in NLT cells was investigated. After culture for 3 days in a
DMEM medium containing 0.5% serum in the presence or absence of
different concentrations of IGF-I, the NLT cells were pulse labeled
with [3H]thymidine for 4 h, and the rate of
thymidine incorporation was determined. IGF-I at a concentration of 50
ng/ml or above significantly increased thymidine incorporation compared
with those treated with 0.5% serum alone (Fig. 5A
).
This apparent increase in NLT cell proliferation in response to IGF-I
was confirmed by examining the number of cells in the culture. A 4-fold
increase in NLT cell number was produced by IGF-I at 50 ng/ml for 3
days, compared with those treated with 0.5% serum alone (Fig. 5B
).
These results clearly indicate that IGF-I is neurotrophic to NLT cells
in that it stimulates DNA synthesis and promotes cell
proliferation.

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Figure 5. IGF-I Stimulates DNA Synthesis and NLT Cell
Proliferation
A, IGF-I increases [3H]thymidine incorporation into DNA
in NLT cells. NLT cells were maintained in DMEM containing 0.5% serum
in the presence or absence of different concentrations of growth
factors for 48 h, and [3H]thymidine was added to a
final concentration of 0.5 mCi/ml for 4 h. Cells were lysed, and
[3H]thymidine incorporated was counted by liquid
scintillation for 1 min. B, IGF-I stimulates NLT cell proliferation.
Cells were cultured in a 0.5% serum DMEM with or without IGF-I (50
ng/ml) for 3 days, and the numbers of surviving cells were compared
(the number of cells at the beginning of the experiment was designated
as control). *, P < 0.05 vs.
control, analyzed using ANOVA followed by Dunnetts t
test.
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IGF-1 Stimulates hGnRH Gene Transcription
To determine whether IGF-I has any effect on hGnRH gene
transcription, NLT cells were transfected with a reporter plasmid
containing -412 bp (containing the AP-1 binding site) of the hGnRH
gene promoter fused to the luciferase reporter gene (LUC), together
with a neomycin-encoding plasmid. After selection with G418, stable NLT
cells expressing LUC activity were employed for subsequent studies. The
expression of the reporter gene was measured by LUC assay. Significant
increases in LUC reporter activities were produced by IGF-I treatment
of the stable cells containing -412 bp of the hGnRH promoter.
Dose-response studies demonstrated that optimal stimulation of the
promoter activity was obtained by 10 ng/ml of IGF-I for 6 h (Fig. 6A
). Further increasing IGF-I concentrations diminished
the response. Time course studies demonstrated that the response was
quick in onset because a significant increase in the reporter
activities was induced after 1 h, and peak activities were
achieved by 6 h of IGF-I treatment (Fig. 6B
). Prolonging IGF-I
treatment for 24 h decreased the reporter activity below the
control level, presumably due to receptor desensitization and
down-regulation as have been demonstrated for neuropeptide receptors
(30). These data indicate that the -412 bp of the hGnRH gene is
involved in mediating IGF-I induction of hGnRH gene transcription.

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Figure 6. IGF-I Stimulates the Luciferase Reporter Activities
of Construct Containing -412 bp of the hGnRH Gene Sequence
A, A dose-response study of IGF-I action on NLT cells stably
transfected with -412-LUC construct. NLT cells were maintained in
serum-free medium for 12 h before either IGF-I or vehicle
treatment for 6 h. B, A time course study of IGF-I action on NLT
cells stably transfected with -412-LUC construct. IGF-I at a final
concentration of 10 ng/ml was added to the medium for the indicated
duration. Data are expressed as mean ± SEM of
triplicate measurements.
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AP-1 Binding Site Mediates IGF-I-Induced hGnRH Gene
Transcription
A consensus AP-1 binding site (TGACTCA) is present
between -402 to -396 bp of the hGnRH gene 5'-flanking promoter region
(Fig. 7A
). Previous study from this laboratory
demonstrated that this AP-1 binding site mediates the stimulatory
effect of TPA on hGnRH gene transcription (26). To determine whether
this AP-1 binding site participates in mediating IGF-I-induced
transcriptional activation of hGnRH gene, site-directed mutagenesis of
the AP-1 binding sites was done by substituting nucleotide A with T at
position -398 (Fig. 7B
). This mutation has been shown to abolish the
binding of AP-1 transcription factors to this element (26). As shown in
Fig. 7C
, mutation of this AP-1 binding site significantly decreased the
IGF-I-stimulated increase in the LUC activity in cells transfected with
the mutant construct, indicating the importance of the AP-1 binding
site in mediating the effect of IGF-I on hGnRH gene transcription (Fig. 7C
). IGF-I treatment of NLT cells transfected with the RSV promoter-LUC
plasmid had no effect on the expression of reporter activity,
indicating that changes in reporter activity were not due to IGF-I
induced cell growth.

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Figure 7. Involvement of the AP-1 Binding Site in Mediating
the Effect of IGF-I on hGnRH Gene Transcription
A, Schematic representation of the hGnRH-LUC fusion gene used to
generate 5'-deletion plasmids at the indicated nucleotide positions.
The relative position and the sequence of the promoter region
containing the AP-1 binding site is depicted. B, Mutation of the AP-1
binding site was done by substituting A with T at nucleotide -400. The
mutation was confirmed by sequencing the construct. C, Comparison of
the reporter activities elicited by -412, -412 mut-, or RSV-LUC in
response to IGF-I stimulation in NLT cells stably transfected with the
constructs. Data are expressed as mean ± SEM of
triplicate measurements. **, P < 0.01
vs. control, Students unpaired t
test.
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AP-1 is a family of nuclear transcription factors composed of either
homodimers of c-Jun or heterodimers of c-Jun with other proteins such
as c-Fos (31). The AP-1 binding site is also known as the TPA response
element because it mediates gene induction by phorbol esters such as
TPA. To test whether NLT cells contain nuclear proteins that bind to
the AP-1 site, gel mobility shift assays were performed using
32P-radiolabeled DNA fragment containing the AP-1 binding
site and nuclear extracts from NLT cells under either basal, TPA, or
IGF-I-induced conditions. As shown in Fig. 8
, a specific
complex was formed in the gel mobility shift assay using the AP-1
binding site with the nuclear extracts of NLT cells stimulated with
either IGF-I or TPA. Addition of 100-fold cold competitor AP-1 sequence
diminished the complex formation, while the mutant AP-1 sequence had no
effect on the protein-DNA interaction. To determine the identity of the
transcription factors in the complexes, preimmune or anti c-Fos rabbit
antiserum was applied in an attempt to supershift the protein-DNA
complexes. As shown in Fig. 8
, antiserum directed against c-Fos
supershifted the complex. These results indicate that c-Fos is one of
the transcription factors binding to the AP-1 site.

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Figure 8. Gel-Mobility Assay
Nuclear extracts for gel-shift assays were obtained from NLT cells
treated with either TPA (120 nM) or 10 ng/ml IGF-I for
6 h, and reacted with a double-strand
[32P]dCTP-labeled probe containing AP-1 binding sequence.
For competition studies, 100x molar excess of unlabeled DNA fragment
containing either the intact AP-1 binding site or its mutant form was
added to the reaction mixture simultaneously with the labeled probe.
For supershift assay, polyclonal rabbit anti-Fos antibody or normal
rabbit serum was added to the reaction mixture.
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DISCUSSION
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Although studies have shown that IGF-I and its cognate receptors
are widely expressed in the CNS and are essential for CNS development,
few target genes in the brain have been defined. In this study, a
pathway by which the IGF-I signal is transduced from the plasma
membrane to the nucleus to induce hGnRH gene transcription has been
characterized. We show that IGF-I induces Ras/Raf-1/MAP kinase pathway
and enhances c-fos expression in the NLT neuronal cell line.
Mutation of the AP-1 binding site in the hGnRH gene promoter decreases
the stimulatory effect of IGF-I, indicating the importance of the
interaction between immediate-early genes and the AP-1 site in
mediating the effect of IGF-I on hGnRH gene transcription. IGF-I also
enhances mGnRH mRNA expression and promotes NLT cell proliferation. Our
studies, therefore, provide the first evidence that IGF-I serves as an
important signal in regulating GnRH gene expression.
The analysis of extracellular signals in the regulation of GnRH neurons
has been facilitated by the development of GnRH-expressing neuronal
cell lines (4, 5) that allow us to monitor the effects of extracellular
factors on the proliferation and differentiation of GnRH neurons under
chemically defined conditions. The NLT cells established in this
laboratory have been shown to display neuronal morphology and express
neuron-specific markers and are able to secrete GnRH decapeptide (7).
Therefore, these cells provide a new in vitro model with
which to explore the extracellular factors and intracellular signaling
pathways involved in the regulation of GnRH neurons. This is
particularly important for the study of GnRH neurons because their
scattered distribution in the animal forebrain and paucity in cell
number make in vivo analysis difficult (1, 2, 3).
To determine the role of growth factors in the regulation of GnRH
neurons, we examined the expression of growth factor receptors on the
NLT GnRH-expressing neuronal cell line. Our findings demonstrate that
receptors for a number of growth factors, including IGF-I and EGF
receptors, are expressed on the NLT cells (32). These findings are
interesting because both IGF-I and transforming growth factor-
(TGF
) (which is the endogenous ligand for EGF receptors in the
hypothalamus) have been shown to regulate GnRH neuronal activities
under in vivo conditions and are believed to play an
important role in the regulation of pubertal development (14, 15, 33, 34).
We find that IGF-I is highly neurotrophic to NLT cells in that it
stimulates DNA synthesis and strongly promotes NLT cell proliferation.
The proliferating effect of IGF-I on NLT cells is similar to the
effects of IGF-I on the GT1 GnRH-expressing cell line (16), which is
consistent with the observation that the brains of transgenic mice that
overexpress IGF-I were larger than those of controls owing to an
increase in cell size and cell number as well as the increase in myelin
content (35). In contrast, transgenic mice with targeted disruption of
the IGF-I gene display severe growth retardation (36). These mice were
also infertile when they reached adult age, due in part to
abnormalities in the development of the gonads (37). IGF-I is also
required for the normal development of the CNS because mice with null
mutation of IGF-I receptor die at birth, exhibiting a severe growth
deficiency including abnormalities of the CNS (38). Because less than
1500 GnRH neurons are present in adult brain (1, 2, 3), a delicate
regulatory mechanism must be present in the CNS to regulate the
proliferation, survival, and differentiation of GnRH neurons during
different stages of development. The survival and growth-promoting
effects of IGF-I and basic fibroblast growth factor on immortalized
GnRH-expressing neurons (16, 39), the ability of IGF-I (present study),
and TGF
(33, 34) to regulate GnRH gene expression and to increase
GnRH release from the hypothalamus (14, 15, 33, 34) suggest that growth
factors may be important in regulating the GnRH neuronal network.
An important finding of this study is that the IGF-I signal is
transmitted to the nucleus to regulate GnRH gene expression. The
stimulation of hGnRH promoter activities and enhancement of mGnRH mRNA
expression by IGF-I suggests that one of IGF-Is functions in the
brain is to enhance GnRH gene expression. This finding is interesting
because recent studies suggest that growth factors are involved in the
control of pubertal development (33, 34, 40), which is preceded by
enhanced GnRH gene expression (41) and a subsequent increase in GnRH
secretion into the hypothalamic-hypophyseal portal blood (42, 43).
Female sexual precocity can also be induced experimentally in both
primates and rodents by lesions of the hypothalamus (9, 42, 43, 44). It is
suggested that instead of removing an inhibitory restraint on GnRH
neurons, hypothalamic injuries may induce precocious puberty by
increasing neurotrophic/mitogenic activities surrounding the site of
injury through increasing the release of growth factors (33, 34, 40).
Our present results suggest that IGF-I may directly act on GnRH neurons
in the hypothalamus to enhance GnRH gene transcription and, therefore,
play a crucial role in controlling the onset of puberty. The IGF-I
concentrations required for transcriptional activation of hGnRH gene
and enhancement of mGnRH mRNA expression are much lower than that
needed for promoting NLT cell proliferation. This difference may
reflect a divergence in the intracellular signaling pathways, such as
the kinetics and extent of MAP kinase phosphorylation, that may be
relevant to distinct biological effects.
Although IGF-I signaling has been studied extensively using various
peripheral cell lines, to our knowledge, this is the first study
defining its signal transduction in a centrally derived neuronal cell
line. We show that IGF-I rapidly induces p42/p44 MAP kinase activities
and increases c-fos transcription in NLT cells. Although the
precise mechanism by which IGF-I enhances c-fos
transcription is not known, studies in PC12 cells and other cell lines
have demonstrated that activation of MAP kinase cascades down-stream of
Ras by EGF or nerve growth factor can lead to immediate-early gene
expression (27, 45). MAP kinases, when activated, can phosphorylate
TCF/Elk-1, resulting in induction of c-fos transcription. It
has also been demonstrated that an activated MAP kinase other
than p42/p44 can phosphorylate cAMP response element-binding protein at
Ser133, which then binds to cAMP-response element in c-fos
promoter to turn c-fos transcription (24). Another
Ras-dependent signaling pathway leading to the induction of
c-fos expression is through c-jun kinase-mediated
TCF/Elk-1 phosphorylation (46). c-jun Kinase has also been
shown to phosphorylate c-Jun (47, 48). Since the Ras/Raf-1/MAP kinase
pathway is important for neuronal survival, proliferation, and
differentiation, the growth-promoting and enhancement of hGnRH
transcription by IGF-I in NLT cells suggest that this same signaling
pathway may also be important for the normal development and proper
functioning of the GnRH neurons.
In this study we demonstrate that mutation of the AP-1 binding site
located between -402 and -396 bp decreases IGF-I-induced hGnRH
transcription, indicating that signaling through IGF-I receptors can
activate hGnRH gene transcription through this AP-1 site. This finding
is interesting because although increased expression of
c-fos has been demonstrated in GnRH neurons during the
preovulatory LH surge (49), the precise role of c-Fos within the GnRH
neurons remains unclear. Our studies, therefore, demonstrate the
importance of the interaction between immediate-early genes and the
AP-1 binding site in mediating IGF-I-stimulated hGnRH gene
transcription in the NLT cells. In contrast to hGnRH, rodent GnRH gene
expression is inhibited by TPA treatment of the GT1 GnRH neurons (22, 23, 50, 51, 52). Although lacking a consensus AP-1 binding site, the rodent
GnRH gene contains several regions in the proximal promoter sequence
that confer responsiveness to TPA (50, 53). Therefore, a species
difference exists between the human and rodent GnRH genes in their
response to phorbol ester stimulation.
In summary, the results in the present studies have established that
the Ras/Raf-1/MAP kinases and c-fos are components of the
signal transduction pathways by which IGF-I regulates hGnRH gene
transcription in NLT cells. Future investigations by targeted
disruption of the Ras/Raf-11/MAP kinase signaling pathway in
transgenic mice will provide critical information about this pathway on
the normal development and functional regulation of GnRH neurons under
in vivo conditions.
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MATERIALS AND METHODS
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Cell Culture
NLT cells were maintained in DMEM supplemented with 10% FBS.
For receptor-binding assays, cells were transferred to 60-mm culture
dishes. For transfections, cells were plated into 35-mm six-well
plates.
Receptor-Binding Assay
Cells were allowed to grow to 90% confluence at the time of
binding assay. To avoid interference from growth factors in the FBS,
the medium was replaced with a serum-free DMEM medium overnight before
binding assays. After changing to a binding buffer (DMEM containing 1%
BSA and 20 mM HEPES), [125I]IGF-I at 50,000
cpm/ml medium (specific activity of 221 mCi/mg, New England Nuclear,
Boston, MA), and cold IGF-I (GIBCO BRL, Gaithersburg, MD) at indicated
concentrations were added simultaneously. The cells were incubated at
24 C for 1 h, washed twice with cold PBS/1% BSA, and lysed with
0.1% SDS. The bounded radioactivity was counted in a
-counter.
Nonspecific binding was calculated as the percentage of
[125I]IGF-I binding in the presence of 1 mM
insulin (GIBCO BRL). The dissociation constant (Kd) was
calculated by Scatchard analysis (54) and the cells in the dish were
counted to calculate IGF-I binding sites per cell.
Plasmids
Promoter fragments of the hGnRH gene (55, 56) were inserted as
HindII fragments into the promoterless vector, pSVOAL
5
(57). All GnRH fragments contained 5 bp of 5'-untranslated sequence and
the indicated 5'-endpoint. The RSV promoter-LUC and neo-resistant
plasmids were described previously (26). The fosCAT reporter plasmid
contains -720 bp of the c-fos promoter linked to the CAT
reporter gene (from G. M. Cooper, Dana-Farber Cancer Institute, Boston,
MA).
Stable NLT Cell Lines
Reporter plasmids (3 mg/well) were cotransfected with the
neo-resistant plasmid at a ratio of 10:1 to NLT cells with
lipofectamine (GIBCO BRL). After transfection (48 h), cells were
treated with genticin (400 mg/ml) for 2 days. The cells were plated at
various dilutions. Genticin-resistant clones were selected again with
genticin for 2 days, and positive clones were identified by luciferase
assay as described previously. For studies of IGF-I and TPA induction,
cells were maintained in DMEM containing 0.5% FBS overnight, and
either IGF-I or TPA was added at the indicated concentrations and
duration. Values are expressed as mean ± SEM of
triplicate measurements.
Incorporation of [3H]Thymidine into DNA
in NLT Cells
IGF-I-stimulated thymidine incorporation was assayed as
described previously (58). Briefly, cells were cultured in a DMEM
containing 0.5% FBS with or without different concentrations of IGF-I
or TGF
for 48 h. [3H]thymidine (NEN Life Science
Products, Boston, MA) was added to a final concentration of 0.5 mCi/ml,
and incubation was continued for 4 h. Cells were collected onto
glass microfibers and lysed, and unincorporated nucleotide was removed
by repeated washing with water. Filters were dried and counted in
scintillation fluid for 1 min.
In Vitro Assays for p42/p44 MAP Kinase Activity
Cells were maintained in DMEM with 0.5% FBS for 12 h,
stimulated with IGF-I at indicated concentrations and duration. Cells
were washed twice with cold PBS and lysed in an ice-cold buffer (10
mM Tris, 150 mM NaCI, 2 mM
dithiothreitol, 1 mM orthovanadate, 1 mM
phenylmethylsulfonylfluoride, 10 mg/ml leupeptin, 10 mg/ml aprotinin,
pH 7.4). Cellular debris was precipitated at 25,000 x
g for 20 min and the supernatant was retained for MAP kinase
assay. The BIOTRAK p42/p44 MAP kinase enzyme assay system (Amersham,
Arlington Heights, IL) was used to measure p42/p44 MAP kinase
activities. The substrate peptide contains a PLS/TP sequence as
phosphorylation site for p42/p44 MAP kinases. Briefly, 15 ml of sample
or lysis buffer and 10 ml of substrate buffer were mixed with 5 ml
Mg2+ [32P]ATP (1 mCi/tube). The reaction was
incubated at 30 C for 30 min. Phosphorylated peptide was separated from
unincorporated label on binding paper. After washing the binding paper
with 1% acetic acid and distilled water, the extent of
32P-phosphorylation of the substrate peptide was detected
by scintillation counting. Values are expressed as mean ±
SEM of triplicate measurements.
Gel-Mobility Shift Assay
Nuclear extracts for gel-shift assays were obtained from NLT
cells treated with either TPA (120 nM) or 10 ng/ml IGF-I
for 6 h, using a procedure as described (59). Double-strand
[32P]dCTP-labeled probe containing AP-1 binding sequence
was obtained using Klenow and primers complementary to the probe
sequence at 5'- and 3'-ends. Binding was performed in 15 ml of binding
buffer (20% glycerol, 20 mM HEPES, pH 7.4, 50
mM KCl), 1 mM dithiothreitol, 1 mg
poly(deoxyinosinic-deoxycytidylic)acid, 0.1 mg salmon sperm DNA. Probe
(30,00050,000 cpm) was added to each reaction with NLT nuclear
extract. For competition studies, 100 x molar excess of unlabeled
DNA fragment containing either the intact AP-1 binding site or its
mutant form was added to the reaction mixture simultaneously with the
labeled probe. For supershift assay, polyclonal rabbit anti-Fos
antibody or normal rabbit serum was added to the reaction mixture.
Reaction was carried out at room temperature for 30 min.
Electrophoresis was performed on a 5% nondenaturing gel containing 5%
glycerol.
RNase Protection Assay
The assay was performed with the RPA II ribonuclease Protection
Assay Kit (Ambion, Austin, TX). Template cDNA containing exons 1, 2, 3,
and 4 of the mGnRH gene was obtained by RT-PCR of total cellular RNA
from NLT cells. First-strand cDNA synthesis was performed by using
oligo-d(T)16 as the primer and Moloney murine leukemia
virus reverse transcriptase (Boehringer Mannheim). PCR was performed by
using primers complementary to sequences in exon 1 and exon 4 of the
mGnRH gene. The 5'-primer sequence is: 5'-GAAGTACTCAACCTACCAA-3', and
the 3' primer is 5'- GCCATAACAGGTCACAAGCCTC-3'. The cDNA fragment was
cloned into pGEM-T plasmids (Promega, Madison, WI) and sequenced by
Sangers method using T7/SP6 primers and Sequenase according to the
manual (United States Biochemical, Cleveland, OH). The template plasmid
was digested with the restriction enzyme NcoI, and in
vitro transcription was performed by using
[
32P]UTP and SP6 polymerase to generate the antisense
mGnRH probe. As a control, mouse actin antisense probe was also
synthesized. Solution hybridization was performed by mixing sample RNA
(3040 mg) with 46 x 104 cpm of
[
32P]UTP-labeled riboprobe in a final volume of 30 ml
of hybridization solution. Hybridization was carried out at 45 C
overnight, and the mixture was digested with RNase at 37 C for 1
h. The hybridized RNA fragments were precipitated, resuspended in 8 ml
of gel-loading buffer and heated at 95 C for 5 min before
electrophoresis in a 6% denaturing polyacrylamide gel. The amount of
radioactivity in the samples was counted using a PhosphorImager
(Molecular Dynamics, Sunnyvale, CA).
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs. Fredric E. Wondisford (Thyroid Unit, Beth Israel
Hospital/Harvard Medical School, Boston, MA) for helpful discussion and
assistance in the gel-shift assay, G. M. Cooper (Dana-Farber Cancer
Institute, Boston, MA) for the fosCAT plasmid, and Laurie Cohen for
critically reviewing the manuscript.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Sally Radovick, M.D., Division of Endocrinology, Childrens Hospital, Harvard Medical School, Enders 409, 300 Longwood Avenue, Boston, Massachusetts 02115.
This work was supported by NIH Grant HD-30040 and by American Cancer
Society Grant DB-73786 (to S.R.). Shanjun Zhen is a recipient of an
Individual National Research Service Award.
Received for publication February 21, 1997.
Accepted for publication April 10, 1997.
 |
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