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 Children’s Hospital Harvard Medical School Boston, Massachusetts 02115


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {alpha}-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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1AGo, 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. 1BGo). 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.

 
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. 2AGo). 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. 2BGo). 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 Dunnett’s t test.

 
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. 3Go), 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, Student’s unpaired t test.

 
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. 4AGo, 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. 4BGo). 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 [{alpha}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 30–40 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 Dunnett’s t test.

 
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. 5AGo). 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. 5BGo). 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 Dunnett’s t test.

 
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. 6AGo). 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. 6BGo). 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.

 
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. 7AGo). 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. 7BGo). This mutation has been shown to abolish the binding of AP-1 transcription factors to this element (26). As shown in Fig. 7CGo, 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. 7CGo). 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, Student’s unpaired t test.

 
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. 8Go, 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. 8Go, 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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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-{alpha} (TGF{alpha}) (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{alpha} (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-I’s 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-1–1/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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 {gamma}-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{Delta}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{alpha} 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 (30,000–50,000 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 Sanger’s 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 [{alpha}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 (30–40 mg) with 4–6 x 104 cpm of [{alpha}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, Children’s 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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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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