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Molecular Endocrinology, doi:10.1210/me.2006-0270
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Molecular Endocrinology 21 (5): 1163-1174
Copyright © 2007 by The Endocrine Society

Leukemia Inhibitory Factor Induces the Chemomigration of Immortalized Gonadotropin-Releasing Hormone Neurons through the Independent Activation of the Janus Kinase/Signal Transducer and Activator of Transcription 3, Mitogen-Activated Protein Kinase/Extracellularly Regulated Kinase 1/2, and Phosphatidylinositol 3-Kinase/Akt Signaling Pathways

Paolo Magni1, Elena Dozio1, Massimiliano Ruscica, Hajime Watanobe, Anna Cariboni, Roberta Zaninetti, Marcella Motta and Roberto Maggi

Department of Endocrinology (P.M., E.D., M.R., A.C., R.Z., M.M., R.M.), Centre of Excellence on Neurodegenerative Diseases, University of Milan, 20133 Milan, Italy; and Division of Internal Medicine (H.W.), Center for Clinical Research, International University of Health and Welfare, Otawara, Tochigi 324-8501, Japan

Address all correspondence and requests for reprints to: Paolo Magni, Department of Endocrinology, Center of Excellence on Neurodegenerative Diseases, University of Milan, via G. Balzaretti, 9, 20133 Milano, Italy. E-mail: paolo.magni{at}unimi.it.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Leukemia inhibitory factor (LIF) is a pleiotropic cytokine of the IL-6 superfamily. LIF acts through a cell-surface receptor complex formed by two subunits, the specific LIF receptor ß (LIFRß) and the glycoprotein 130. Little is known about LIF involvement in modulating the neuroendocrine circuitry governing the reproductive function and, specifically, the development of GnRH-secreting neurons. In the present study, we evaluated the effect of LIF on the in vitro migration of GN11 cells, a model of immature and migratory GnRH neurons, and the signaling pathways involved in this process. GN11 cells expressed both LIFRß and glycoprotein 130 subunits. Exposure of GN11 cells to 100 ng/ml LIF resulted in activation of the Janus kinases (Jaks)/signal transducer and activator of transcription 3, MAPK/ERK1/2, and phosphatidylinositol 3-kinase/protein kinase B/Akt pathways. The selective inhibition of Jaks, MAPK kinase, and phosphatidylinositol 3-kinase indicated that these signaling pathways were activated independently by LIF and that Jak2 is not the main kinase involved in LIF signaling. Exposure of GN11 cells to LIF for 3 h induced a concentration-dependent chemotactic response, with a plateau at 100 ng/ml LIF. LIF was also found to induce chemokinesis of GN11 cells. Furthermore, LIF-promoted GN11 migration was the result of the partial and independent contribution of all the three signaling pathways activated by LIF. The present data, together with the observation that LIF and LIFRß are expressed prenatally in the mouse nasal compartment, would suggest that LIF might participate in the migration of GnRH neurons.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
LEUKEMIA INHIBITORY FACTOR (LIF) is a pleiotropic cytokine of the IL-6 superfamily, which also includes IL-6, IL-11, oncostatin-M, ciliary neurotrophic factor (CNTF), and cardiotrophin-1. LIF acts through the LIF cell-surface receptor (LIF-R) complex, consisting of two different subunits, the specific low-affinity LIF receptor ß (LIFRß) and the glycoprotein 130 (gp130) (1). After binding to LIFRß, LIF induces a conformational change followed by the heterodimerization of LIFRß and gp130 and the activation of the Janus protein-kinases (Jaks), which phosphorylate tyrosine residues in both receptor subunits. This generates docking sites for the signal transducer and activator of transcription proteins (STATs), primarily for STAT3, but also for STAT1 (2, 3, 4) and for other linker proteins, which propagate the signal to the other pathways, such as the MAPK/ERK1/2 and the phosphatidylinositol 3-kinase (PI3-K)-protein kinase B/Akt pathway (5).

LIF acts on several tissues and cell types to exert a variety of effects, including proliferation of primordial germ cells, spermatocyte differentiation (6), reduction of testosterone synthesis by Leydig cells (7), maintenance of pluripotent embryonic stem cells, endometrial decidualization and blastocyst implantation, hypothalamus-pituitary-adrenal axis activation, normal development of hippocampal and olfactory receptor neurons, pituitary development, osteoblast and osteoclast function, and adipocyte lipid and energy homeostasis (8). In adult animals, LIFRß mRNA was found to be expressed in the mitral cell layer of the olfactory bulb and in different hypothalamic nuclei (9), suggesting a possible involvement of LIF in the control of some neuroendocrine functions, such as reproduction. LIF has indeed been shown to affect the regulation of LH and prolactin secretion by the pituitary (10). Because LIF appears to be structurally and functionally very similar to CNTF and leptin, it is possible that, as shown for these two proteins (11, 12, 13), it could affect the reproductive function and also modulate the secretion of GnRH by hypothalamic neurons; this was indicated by preliminary data suggesting the ability of LIF to stimulate GnRH release by GT1 neurons, which express LIF-R (Dozio, E., M. Ruscica, M. Motta, and P. Magni, in preparation).

The hypothalamic decapeptide GnRH is the key hormone in the control of reproductive function. The initiation of the reproductive cascade in all mammalian species requires the completion, during embryonal life, of a developmental program consisting in the migration of GnRH neurons from the olfactory placode, along olfactory nerves and through the nasal compartment, into the forebrain and the hypothalamus, where they promote reproductive competence (14, 15). The mechanisms underlying the migration of GnRH neurons to their final destination, in the septo-preoptic region of the hypothalamus, are still not fully understood, although several factors, including anosmin-1 (16), nasal explant GnRH factor (17), adhesion-related kinase (18), and some others (reviewed in Ref. 19) have been reported to play a role in this process. LIF was found to exert chemotactic properties in the nervous system (20) and to function as both a diffusible and extracellular matrix-incorporated molecule (21), therefore making it another good candidate to be studied in this context.

In vivo or ex vivo studies of GnRH neurons are hindered by the very small number of these cells and by their scattered distribution in the septo-hypothalamic region (22). Moreover, experimental attempts to maintain a pure population of GnRH neurons in culture have not been successful (23). An important advancement in this area was represented by the generation of two different cell lines, the GT1 cells (24) and the GN cells (including the GN11 subclone) (25), obtained by genetically targeted tumorigenesis of GnRH neurons in mice. Both cell lines have been shown to express neuronal markers, retain many characteristics of GnRH-secreting neurons (26, 27, 28), and represent good models to study the biology of GnRH neurons in vitro. Whereas GT1 cells represent differentiated postmigratory GnRH neurons, GN11 cells have been shown in our laboratory to retain the characteristics of immature migratory neurons (16, 29, 30, 31). Therefore, GN11 neurons were used as a model system to verify the hypothesis that LIF might modulate the migration of immature GnRH neurons and to evaluate the involvement of the related intracellular signals proximal to LIF-R in this process.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Expression of LIFRß and gp130 in GN11 Cells
The expression of the subunits forming the LIF-R (LIFRß and gp130) in GN11 cells was evaluated at the gene and protein levels. RT-PCR analysis showed that both LIFRß and gp130 genes are expressed in GN11 cells, as indicated by the presence of PCR products of 452 and 687 bp, respectively (Fig. 1Go). The presence of the corresponding proteins was assessed by Western blot and immunofluorescence (IFL) analyses (Fig. 2Go). Immunoreactive bands for LIFRß and gp130 proteins detected by Western blot at 170–190 kDa and 130 kDa, respectively, are evident in extracts derived from human neuroblastoma cells SH-SY5Y used as positive control. The anti-gp130 antibody also recognized a 75-kDa immunoreactive band in these cells. In GN11 cells, LIFRß immunoreactivity showed as a doublet of 170–190 kDa; an additional immunoreactive band, corresponding to a 50-kDa protein, was also detected (Fig. 2AGo). The gp130 immunoreactivity corresponded, as expected, to a band of 130 kDa and to a minor band of smaller size (Fig. 2BGo).


Figure 1
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  		Fig. 1. RT-PCR Analysis of LIFRß and gp130 Gene Expression, the Two Subunits Forming the LIF-R, in Mouse Hypothalamus (Positive Control) and in GN11 Neurons

The transcript for the housekeeping gene GDI-1 is also shown. Mw, Molecular weight markers; control RNA is a control reagent provided with the RT-PCR kit; m hypo, mouse hypothalamus; –RT indicates PCRs done omitting the reverse transcriptase enzyme. Shown is a representative example of three separate experiments.

 

Figure 2
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  		Fig. 2. LIFRß and gp130 Protein Expression

A and B, Western blot immunodetection of LIFRß and gp130 in GN11 cells and human neuroblastoma cells SH-SY5Y (positive control) (50 µg protein/lane of cell extracts was analyzed). C and D, Immunofluorescent detection of LIFRß and gp130 in GN11 neurons cultured in standard conditions. E, Negative control with the primary antibody omitted. F, Negative control with normal rabbit IgG instead of the primary antibody. Scale bar, 10 µm for all images. Shown is a representative example of three or four independent experiments. Ab, Antibody.

 
By IFL, we found a marked specific fluorescent staining distributed along the plasma membranes of GN11 cells, with a clear-cut clustering for both LIFRß and gp130 subunits (Fig. 2Go, C and D). The cell nuclei are not apparently evident, because intact cell preparations have been used. The results of IFL conducted either omitting the primary antibody (Fig. 2EGo) or exposing the cell preparation to normal rabbit IgG (Fig. 2FGo), followed by incubation with the secondary antibody, did not show any significant signal.

Prenatal Expression of LIF and LIFRß in the Mouse Nasal Compartment
To evaluate the prenatal expression of LIF in nasal regions where GnRH neurons originate and start their migration, mouse nose tissue was removed at embryonic d 12.5 (E12.5) (32) and RT-PCR experiments were then performed. The first round of RT-PCR showed that LIF is expressed both in nose tissue and in mouse brain, used as positive control (data not shown). To confirm the specificity of the transcript, nested PCR was then performed. Using 1 µl of the first-round amplicons and a set of nested LIF primers, a strong band of correct size was detected in all samples (Fig. 3AGo). To study a possible relationship between migrating GnRH neurons and LIF, we also performed IFL studies using contiguous heads sections from E12.5 mice. Cryostat serial sections (15 µm) were prepared and incubated with polyclonal anti-GnRH and -LIFRß antibodies. As illustrated in Fig. 3BGo, LIFRß is widely expressed in the nasal mesenchime; in a contiguous section, migrating GnRH cells were seen in the same region that abundantly expresses LIFRß (Fig. 3CGo), indicating the existence of the anatomical prerequisite for the prenatal interaction of such cells with LIF.


Figure 3
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  		Fig. 3. Expression of LIF, LIFRß, and GnRH in the Nasal Compartment of Embryonic Mouse

A, Expression of LIF in the nasal compartment of embryonic mouse at E12.5. Total RNA isolated from dissected nasal region was subjected to nested PCR. A transcript of the expected size (180 bp) for LIF was detected in the nasal region (OP) and in the whole brain embryo (MB), taken as positive control. B and C, Expression of LIFRß during GnRH neuronal migration in the nasal region. Two panels composition of contiguous sections taken from the nose of an E12.5 mouse and stained for LIFRß (B) and GnRH (C) are shown. GnRH immunoreactive cells migrate through the nasal compartment in a region highly positive for LIFRß. MW, Molecular weight. Scale bar, 50 µm.

 
Effect of LIF on Intracellular Signaling Pathways of GN11 Cells
The study of the activation of the intracellular pathways coupled to LIF-R in GN11 cells was focused on the Jaks/STAT3, MAPK/ERK1/2, and PI3-K/Akt systems. GN11 cells were maintained overnight in serum-free medium and then treated with 100 ng/ml LIF for 5–60 min. GN11 cell extracts were then analyzed by Western blot. In basal conditions, no pSTAT3 and pAkt signals were observed in GN11 cells, whereas pERK1/2 was detectable at very low levels. LIF treatment did not modify the STAT3, ERK1/2, and Akt signals, but it resulted in increased levels of the phosphorylated proteins, with maximal levels at 15–30 min for pSTAT3, at 5–15 min for pERK1/2, and at 15–30 min for pAkt. A decrease to low levels was observed for all these phosphorylated proteins thereafter (Fig. 4Go). To get insight into a possible cross talk among these signaling pathways, additional experiments were then carried out using specific inhibitors of the intracellular pathways considered (see Materials and Methods). GN11 cells were maintained overnight in serum-free medium, pretreated with 10 µM of each inhibitor for 30 min, and then treated with 100 ng/ml LIF for 15 min. Western blot analysis showed that pretreatment with Jak inhibitor I blocked LIF-induced phosphorylation of STAT3 (Fig. 5AGo), but did not affect ERK1/2 (Fig. 5BGo) and Akt phosphorylation (Fig. 5CGo), whereas AG490, a selective Jak2 inhibitor, was not able to impair STAT3 phosphorylation (Fig. 5AGo). The MEK (MAPK kinase) inhibitor U0126 blocked LIF-induced phosphorylation of ERK1/2 (Fig. 5BGo), but not of STAT3 (Fig. 5AGo) and Akt (Fig. 5CGo). Moreover, the low, but detectable, pERK1/2 levels found in basal conditions were also completely abrogated by pretreatment with U0126 (Fig. 5BGo). Pretreatment with the PI3-K inhibitor LY-294 prevented the phosphorylation of Akt induced by exposure to LIF (Fig. 5CGo), but did not affect that of STAT3 (Fig. 5AGo) and ERK1/2 (Fig. 5BGo).


Figure 4
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  		Fig. 4. Effect of LIF on STAT3, ERK1/2, and Akt Phosphorylation in GN11 Cells

Cells were exposed to 100 ng/ml LIF for different times. Cell extracts of GN11 cells were prepared and 50 µg protein/lane was analyzed by Western blot with anti-pSTAT3 and anti-STAT3 (A), anti-pERK1/2 and anti-ERK1/2 (B), and anti-pAkt and anti-Akt antibodies (C). The results are representative of three independent experiments.

 

Figure 5
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  		Fig. 5. Effect of Signaling Pathway Inhibitors on LIF-Induced Phosphorylation

STAT3, ERK1/2, and Akt phosphorylations were analyzed by Western blot after 15 min treatment of GN11 cells with 100 ng/ml LIF alone or with 10 µM Jak inhibitor I (JAK I, a pan-specific Jaks inhibitor), 10 µM AG490 (a selective Jak2 inhibitor), 10 µM U0126 (a selective inhibitor of MEK), and 10 µM LY-294 (a selective inhibitor of PI3-K). Cell extracts of GN11 cells were prepared and 50 µg protein/lane was immunoblotted with anti-pSTAT3 and anti-STAT3 (A), anti-pERK1/2 and anti-ERK1/2 (B), and anti-pAkt and anti-Akt antibodies (C). One representative of three independent experiments is shown.

 
Effect of LIF on Chemomigration of GN11 and GT1–7 Cells
The effect of LIF on the chemomigration of GN11 cells was tested by microchemotaxis assay using the Boyden’s chamber. The related cytokine CNTF was used as control of cytokine specificity, because the trimeric CNTF receptor complex includes also the two subunits of LIF-R (33). GN11 neurons were exposed to 0.1–200 ng/ml LIF for 3 h. In the absence of a chemotactic factor, GN11 cells showed a very low spontaneous motility. Because a major migratory response was elicited by exposure of the cells to 1% fetal bovine serum (FBS) (data not shown) (29), this treatment was taken as internal reference. The exposure of GN11 cells to LIF stimulated chemotaxis in a concentration-dependent manner (Fig. 6AGo). In particular, a significant chemotactic response was already evident after 3 h at 10 ng/ml LIF, with a maximal effect at 100 ng/ml LIF (Fig. 6AGo).


Figure 6
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  		Fig. 6. Chemotactic and Chemokinetic Response of GN11 Cells to LIF and CNTF

The assay was performed using a Boyden’s chamber assay. For chemotaxis assay, GN11 cells were exposed to 0.1–200 ng/ml LIF and 100 ng/ml of CNTF for 3 h (A). Chemokinesis was analyzed by exposure of GN11 cells to 100 ng/ml LIF or CNTF for 3 h (B). Data (mean ± SD; n = 8) are expressed as % of the positive control (1% FBS), taken as 100%. *, P < 0.05 vs. untreated cells (ANOVA); CT, chemotaxis; CK, chemokinesis.

 
To better investigate the nature of the effect of LIF on the motility of GN11 cells, they were also exposed to the cytokine added in both compartments of the Boyden’s chamber. The results obtained show that 100 ng/ml LIF induces a significant chemokinesis of GN11 cells with an efficiency similar to that observed in inducing chemotaxis (Fig. 6BGo). No effects on chemotaxis and chemokinesis of GN11 cells were observed using CNTF, indicating the specificity of LIF in the promotion of these processes (Fig. 6Go, A and B). Furthermore, the addition of increasing concentrations of a blocking antibody against gp130 resulted in a dose-related reduction of LIF-induced chemotaxis and chemokinesis (Table 1Go), suggesting the specific involvement of the gp130 component of LIF-R in mediating this process.


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  		Table 1. LIF-Induced Migration of GN11 Cells: Effect of Different Concentration of the Blocking Antibody against gp130 (Anti-g130)

 
In contrast to GN11 cells, GT1–7 cells, which exhibit both morphological and behavioral features of hypothalamic postmigratory GnRH neurons that lost their ability to migrate (29, 30, 31), did not show any chemotactic/chemokinetic response after LIF stimulation (data not shown).

Actin Cytoskeletal Remodeling in LIF-Induced GN11 Migrating Cells
The appearance of filopodia and lamellipodia is a morphological hallmark of cellular motility that can be visualized by F-actin staining. Therefore, we analyzed the morphology of GN11 cells exposed to LIF using a modified protocol of the Boyden’s chamber assay with phalloidin-TRITC F-actin staining (see Materials and Methods). This allows a more physiological approach to analyze the response of the cells to a gradient of concentration of the chemoattractants. Examination of unstimulated GN11 cells 1 h after seeding in Boyden’s chamber showed round cells with little evident actin-positive protrusions (Fig. 7AGo). Exposure to 1% FBS or LIF resulted in reorganization of actin cytoskeleton to a motile phenotype with membrane ruffles and/or lamellipodia in more than 95% of GN11 cells (Fig. 7Go, B and C).


Figure 7
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  		Fig. 7. LIF Stimulates Formation of Filopodia in GN11 Cells

A, GN11 cells were exposed to DMEM, 1% FBS, and LIF (100 ng/ml) for 1 h in the Boyden’s chamber. After fixation, F-actin was visualized with TRITC-phalloidin. Arrows indicate membrane ruffles and lamellipodia, clearly visible in cells exposed to FBS (B) and LIF (C). No actin-positive protrusions were visible in GN11 cells exposed to control medium (DMEM, panel A). Scale bar, 10 µm.

 
Signaling Pathways Involved in LIF-Induced Chemomigration in GN11 Cells
To study which signaling pathways activated by LIF (see above) were involved in GN11 cell migration, another series of chemomigration assays has been carried out, utilizing the specific inhibitors (see Materials and Methods). GN11 cells were pretreated with the inhibitors for 30 min and then exposed to 100 ng/ml LIF for 3 h. Pretreatment of the cells with each inhibitor did not affect the basal motility of GN11 cells. As also reported above, LIF induced a significant chemotactic activity of GN11 cells. Pretreatment with the pan-specific Jak inhibitor I reduced LIF-stimulated chemotaxis, whereas the selective Jak2 inhibitor, AG490, had no effect (Fig. 8AGo). In the presence of the inhibitors of MEK (U0126) and PI3-K (LY-294), LIF-induced chemotaxis of GN11 cells was reduced to a similar extent as with Jak inhibitor I. Because we observed only a partial reduction of the LIF-induced chemotaxis of GN11 cells with the different inhibitors, we tested the effect of the Jak inhibitor I, U0126, and LY-294 used both pairwise and in combination. The inhibitors used pairwise were associated with a reduction of LIF-induced directional motility greater than that obtained with each single substance. Such reduction was even greater when Jak inhibitor I was present in the pair of compounds. Moreover, the addition of a combination of all inhibitors resulted in the complete blockade of LIF-induced chemotaxis (Fig. 8AGo).


Figure 8
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  		Fig. 8. Effect of Signaling Pathway Inhibitors on LIF-Induced GN11 Migration

The assay was performed using a Boyden’s chamber. GN11 cells were pretreated for 30 min with Jak inhibitor I (JAK I), or AG490, or U0126, or LY-294 (all at 10 µM) or the mix of JAK I, U0126, and LY-294 and then exposed to 100 ng/ml LIF for 3 h. Data [mean ± SD; n = 8 are expressed as % of the positive control (1% FBS)], taken as 100%. *, P < 0.05 vs. untreated cells; °, P < 0.05 vs. cells treated with LIF alone (ANOVA). A, Chemotaxis (CT); B, chemokinesis (CK).

 
Because we observed LIF ability to stimulate also GN11 cell chemokinesis, we used a similar approach to dissect out the individual contribution of each signaling pathway in mediating this process. Pretreatment with each inhibitor (Jak inhibitor I, U0126, LY-294) reduced LIF-induced chemokinesis, with Jak inhibitor I being the most potent (Fig. 8BGo). The inhibitors used pairwise were associated with a generally greater reduction of LIF-induced chemokinesis than with each inhibitor alone. Moreover, the addition of a combination of all inhibitors resulted in a maximal, although not complete, reduction of LIF-induced chemokinesis (Fig. 8BGo). The inhibitors, used either alone or in combination, did not affect cell viability, as monitored by trypan blue staining (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In the present study, we report the novel evidence of a direct effect of LIF in promoting the chemomigration of immature GN11 neurons via a receptor-mediated mechanism. The LIFRß/gp130 receptor complex was found to be expressed in GN11 cells at both mRNA and protein levels and to be functionally coupled to the Jaks/STAT3, MAPK/ERK1/2, and PI3-K/Akt signaling pathways. Moreover, exposure to LIF induced both chemotaxis and chemokinesis through the independent activation of each of these three signaling pathways. The present results thus strongly support a role of LIF in the migration of GnRH neurons, which is a fundamental process for the development of normal reproductive functions and is under the control of genetic and local signals present along the migratory pathway and represented by adhesion factors and diffusible and extracellular matrix-linked factors (34). Interestingly, we found that LIF was able to stimulate GN11 cell chemotaxis in a dose-dependent manner, with most of the effect in the nanomolar range and a plateau at 100 ng/ml. It is possible that the lack of additional effect at higher doses of LIF might result from the saturation and the down-regulation of the receptor and/or of the signaling pathways. LIF has also been shown to stimulate chemokinesis (intrinsic cell motility) of GN11 neurons, suggesting that it may also play a general role in stimulating the motility of GnRH neurons other than directional migration. The stimulation of migratory activity seems specific for LIF, because the related cytokine CNTF failed to promote GN11 cell migration. In rodents, LIF production is restricted to certain developmental stages and regions, including the olfactory epithelium, allowing us to hypothesize that it might act at this site as a chemokinetic factor to increase the probability to motion (34), to start the migratory activity of GnRH neuronal precursors, and to act as chemoattractant for neurons approaching to the nasal-forebrain junction (19); moreover, LIF binding sites were reported to be mainly limited to specific regions, including the rat olfactory bulb from E18 to birth (35). We report here the novel observation that, at an earlier stage of embryonal development, E12.5, both LIF and LIFRß are expressed in the mouse olfactory compartment and that migrating GnRH cells are present in the same region that abundantly expresses LIFRß, suggesting the potential for a significant action of the cytokine at this level and a possible responsiveness of GnRH cells to LIF. The collective evidence of the presence of LIF binding activity in the olfactory compartment, the observation that in LIF knockout mice an excess of olfactory cell numbers is found (36), and the migration-inducing properties of LIF for macrophages and other cell types (37) may thus represent a strong rationale to explore the possible role of this cytokine in modulating the migration of immature GnRH neurons.

In GN11 cells, LIFRß immunoreactivity was detected as a doublet of about 190 and 170 kDa, presumably representing the fully processed receptor protein, which appears to be mainly localized at the plasma membrane, and an immature form of intracellular LIFRß, respectively (38). Additional LIFRß bands, corresponding to smaller proteins, were also detected and, because an antibody against the C-terminal part of LIFRß was used, they presumably represent LIFRß fragments with a truncated extracellular domain. The expression of the gp130 protein, detected, as expected, as a main band of 130 kDa, was also found in GN11 cells. Additional smaller proteins were detected and may represent immature, less glycosylated variants of gp130 (39). The IFL study confirmed the presence of LIFRß and gp130 proteins in GN11 cells and revealed a cluster pattern of the IFL signal with a punctate distribution, which is suggestive of membrane-bound proteins of receptor nature. LIFRß and gp130 expression may be a feature of both mature and immature GnRH neurons, because it has also been reported in GT1–7 mature immortalized GnRH neurons (13).

We found that the activation of the Jaks/STAT3, MAPK/ERK1/2, and PI3-K/Akt signaling pathways independently mediated the LIF-induced chemomigratory response of GN11 cells. LIF-Rs are known to be coupled to the Jaks/STAT3, MAPK/ERK1/2, and PI3-K/Akt signaling pathways (5, 40, 41, 42), which mediate different effects such as cell proliferation, differentiation, or survival (43). Our data agree with previous studies suggesting the independent activation of the three signaling pathways by LIF in other cell lines (41). The results of cell motility studies suggest that the activation of each of these pathways gives a contribution to LIF-induced chemomigration. Because in both processes, chemotaxis and chemokinesis, the inhibitors, used alone and pairwise, partially reduced LIF-induced motility, with a greater reduction when Jak inhibitor I was present in the pair of compounds, it appears that these pathways are activated separately and operate in parallel and that the JAK-STAT pathway gives a greater contribution to the induction of the motility phenotype. Nevertheless, whether these pathways converge to the same or to different downstream targets to induce cell migration still needs to be clarified. In general, because chemotaxis is considered a more specialized phenomenon, which obviously includes some components of chemokinesis, the observation that the simultaneous inhibition of all of the three signaling pathways completely prevented only chemotaxis, but not chemokinesis, may suggest that these pathways are involved in modulating this specialized phenomenon and that there are other pathways that mediate the random motility (chemokinesis), but not the specialized response to a gradient of a chemoactractant (chemotaxis).

Jak1, Jak2, and tyrosine kinase 2 are the main kinases of the Jak family activated by LIF (40). Our observation that the selective inhibition of Jak2 by AG490 did not block STAT3 phosphorylation and did not affect LIF-induced migration suggests that Jak2 is not the main kinase involved in LIF signaling in GN11 cells. The present data agree with previous studies, which reported that Jak1 appears to be essential for STAT3 phosphorylation (44) and that targeted disruption of the Jak1 gene abrogates gp130-mediated signaling, whereas the targeted disruption of the Jak2 gene does not abolish the responsiveness to LIF (45, 46). Our data on PI3-K agree with a previous observation showing that PI3-K activation promotes cell migration in inflammatory cells, such as eosinophils, macrophages, neutrophils, and T lymphocytes (47). Akt is one of the downstream effectors described for PI3-K, and recent evidence has implicated Akt in the efficient chemotactic response to chemoattractants (48). In our study, Akt appeared to be activated by PI3-K, as demonstrated by the blockade of LIF-induced phosphorylation of Akt in the presence of the PI3-K inhibitor LY-294. Because LY-294 treatment also reduced LIF-stimulated migration of GN11 cells, future studies will be necessary to clarify whether Akt is one of the downstream effectors of PI3-K involved in cytoskeletal reorganization. Several studies indicated that LIF-induced ERK1/2 activation can be inhibited by PI3-K inhibitors, suggesting the involvement of PI3-K in regulating ERK activation, whereas STAT3 phosphorylation was mostly unaffected (49). In our study, we observed that not only STAT3, but also ERK1/2, is activated independently of PI3-K. The precise role of ERK1/2 activation in facilitating cell movement remains unclear. This pathway was described not to be essential for Gas adhesion-related kinase-induced GnRH neuronal migration (50) but is involved in stromal-derived factor-1, macrophage inflammatory protein 3{alpha}, and eotoxin-induced actin polymerization, in migration of T cells or eosinophils (51, 52, 53) and in IL-6-mediated migration of T cells (54). The different sensitivity of cell migration to MEK inhibition suggests that there are multiple pathways leading to migration and, although different receptors can share the same signaling pathways, some of these are preferentially activated in response to one selected agent.

In conclusion, the present results, showing that LIF is able to promote the chemomigration of immature GnRH neurons via receptor-mediated mechanisms, support the concept that this cytokine may play an important role in the development of the hypothalamic compartment and the related reproductive competence. LIF thus appears to participate to the complex control of the reproductive function at different times and sites, with central (development and function of the hypothalamus-pituitary system) and peripheral (blastocyst implantation) actions. This study provides a novel evidence for future studies aimed at clarifying relevant issues on the role of LIF in the GnRH migratory process, such as the regulation of LIF/LIF-R expression in the nervous system, the specific timing of LIF action during the development of the GnRH-secreting system, and the potential effect of this cytokine in the adult olfactory epithelium, which has been considered a potential reservoir of GnRH neurons (55).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Chemicals
Recombinant LIF, CNTF, Jak2 inhibitor tyrphostin AG490, PI3-K inhibitor LY-294, Phalloidin-tetramethylrhodamine B isothiocyanate (TRITC) and 4',6-diamidino-2-phenylindole dihydrochloride were obtained from Sigma-Aldrich (Milan, Italy); the MEK inhibitor U0126 was from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA), and the Jak inhibitor I was from Calbiochem (San Diego, CA).

Cell Cultures
GN11 (a kind gift of Dr. S. Radovick, Children’s Hospital, Division of Endocrinology, Boston, MA), GT1–7 (a kind gift of Dr. R. I. Weiner, San Francisco, CA), and SH-SY5Y human neuroblastoma cells [kindly provided by Dr. J. Biedler (Memorial Sloan-Kettering Cancer Center, New York, NY), and selected as a positive control for the expression of the two LIF-R subunits (56)], were routinely grown in monolayer at 37 C in a humidified CO2 incubator. GN11 and GT1–7 cells were cultured in DMEM (Biochrom, Berlin, Germany) supplemented with 1 mM sodium pyruvate, 100 µg/ml streptomycin, 100 U/ml penicillin, 2 mM L-glutamine, and 10% FBS (Life Technologies, Inc., Gaithersburg, MD). SH-SY5Y cells were grown in MEM (Biochrom) supplemented with 1 mM sodium pyruvate, 100 µg/ml streptomycin, 100 U/ml penicillin, 2 mM glutamine, and 10% FBS. The medium was replaced at 3-d intervals for all cell lines. Subconfluent cells were routinely harvested with 0.05% trypsin/0.02% EDTA (Biochrom) and seeded in 100-mm Petri dishes at 0.1 x 106 cells per dish for GN11, 3.5 x 106 cells per dish for GT1–7, and 1 x 106 cells per dish for SH-SY5Y. For the study of migration, GN11 and GT1–7 cells were seeded in a Boyden’s chamber at 0.15 x 106 cells per well.

Immunohistochemistry
All animal procedures were performed in accordance with institutional guidelines. Paraformaldehyde (4%)-fixed heads from embryonic mice (E12.5) were cut at 15-µm sections with a cryostat. Contiguous sections were blocked with 10% normal goat serum in PBS/0.3% Triton X-100 for 1 h and then incubated with primary antibodies (GnRH, 1:400; Immuno-Star, Hudson, WI; LIFRß, 1:100; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) at room temperature overnight, followed by Alexa488 antirabbit IgG (Molecular Probes, Eugene, OR). Nuclei were counterstained with 4',6-diamidino-2-phenylindole dihydrochloride. Preparations were then examined with a fluorescent microscope (Carl Zeiss, Thornwood, NY), and images were taken.

RNA Extraction and RT-PCR
For RNA studies, cells were washed with cold PBS, collected with a rubber policeman, snap-frozen in liquid nitrogen, and stored at –80 C until RNA extraction. Total cellular RNA was extracted with the phenol-chloroform method using the Tri Reagent solution (Sigma-Aldrich). Mouse hypothalamus, collected from adult animals and whole mouse brain, collected from E12.5 mice, were homogenized in Tri Reagent (1 ml for 50–100 mg tissue), and then RNA was extracted as described above. RT-PCR analysis for the detection of the gene expression of the two LIF-R subunits (LIFRß and gp130) and the constitutively expressed gene Rab GDP-dissociation inhibitor (GDI-1) was performed on 1 µg total RNA for each sample, quantified after an initial DNAase digestion step using the deoxyribonuclease I kit (Sigma-Aldrich). For, LIFRß, gp130, and GDI-1 sequences, the following oligonucleotides were used: LIFRß: forward, 5'-TCAgTTTCAgCCAggAgTAA-3'; reverse, 5'gCAATAATCAATCCCACAgA-3' (57); gp130: forward, 5'CAgCgTACACTgATgAAggTgggAAAgA3'; reverse, 5'gCTgACTgCAgTTCTgCTTgA-3' (58); GDI-1: forward, 5'-gAggCCTTgCgTTCTAATCTg-3'; reverse, 5'TgAggATACAgATgATgCgA-3' (59). The reverse transcription (RT) reaction was carried out in 50 µl volume using a commercially available GeneAmp kit and a DNA thermal cycler, both from Applied Biosystems (Milan, Italy), at 25 C/10 min, and then 48 C/30 min, and a final hold at 95 C/5 min. The PCR included an initial cDNA denaturating step (95 C/5 min) followed by 30 cycles (94 C/1 min + 56 C/1 min + 72 C/1 min for LIFRß; 94 C/1 min + 65 C/1 min + 72 C/3 min for gp130; 94 C/1 min + 54 C/1 min + 72 C/1 min for GDI-1) and a final step incubation at 72 C for 7 min. Amplification products were separated by 1.5% agarose gel electrophoresis and detected by ethidium bromide fluorescence on a UV transilluminator. The identity of the PCR products has been confirmed by sequencing (Istituto FIRC di Oncologia Molecolare, Milan, Italy). The correct functioning of the RT-PCR was confirmed by including a control RNA provided with the RT-PCR kit, the reagent pAW 109, which, after reverse transcription and amplification using the specific primers (DM151 and DM152) also supplied with the kit, yields a PCR product of 308 bp.

For LIF expression analysis, nasal regions (mainly represented by olfactory placode and surrounding vomeronasal epithelia) were dissected from E12.5 mouse heads in ice-cold buffer, and total RNA extraction and RT analysis were performed as described above. PCR was carried out using the appropriate oligonucleotides (0.6–6 µM) in 25 µl reaction mix and the following conditions: 35 cycles of denaturing at 94 C for 1 min, annealing at 60 C for 1 min, and extension at 72 C for 2 min. To confirm the specificity of the transcripts, nested PCR with a second pair of specific primers was performed on 1 µl of the first round reaction, under the same conditions. PCR products were analyzed by electrophoresis on a 2% agarose gel, and bands were visualized under UV illumination after ethidium bromide staining. The primers used were as follows. LIF sense: 5-CCTTACTgCTgCTggTTCTg-3'; LIF antisense: 5'-gCTCCACCAACTTggTCTTC-3'; LIF sense nested: 5'-TCCCATCACCCCTgTAAATg-3'; LIF antisense nested: 5'-TTAggCgCACATAgCTTTTC-3'.

Western Blot Analysis
Cells were collected in 100 µl radioimmunoprecipitation assay buffer containing 1% protease inhibitor (Sigma-Aldrich). The cell suspension was transferred into a tube, kept on ice for 20 min, and then centrifuged at 13,000 rpm for 15 min at 4 C. Protein concentration was quantified with the BCA Protein Assay (Pierce Chemical Co., Rockford, IL). Protein samples (resuspended in Laemmli sample buffer) and molecular mass markers (Amersham Biosciences, Milan, Italy) were separated on a sodium dodecylsulfate-polyacrylamide gel. Proteins were transferred from the gel to a nitrocellulose membrane overnight at 4 C. The membrane was washed with Tris-buffered saline Tween 20/0.1% Tween 20 for 30 min and blocked with 5% dry milk in Tris-buffered saline Tween-20/0.1% Tween 20 for 1 h at room temperature. The blot was then incubated overnight at 4 C with a diluted solution of the primary antibody (anti-LIFRß, 1:500; anti-gp130, 1:200; anti-pSTAT3, 1:100; anti-STAT3, 1:100; anti-pERK1/2, 1:100; anti-ERK1/2, 1:800; anti-pAkt, 1:100; anti-Akt, 1:100; all from Santa Cruz Biotechnology). The subsequent incubation with a secondary antibody conjugated with peroxidase was performed at room temperature for 2 h. Immunoreactivity was detected by the SuperSignal West Pico Substrate working solution (Pierce Chemical Co.) and exposure of the membrane to photographic film at room temperature for the required time. For STAT3, ERK1/2, and Akt phosphorylation studies, cells were maintained in DMEM/10% FBS until 70–80% confluence was reached, after which the medium was removed and replaced by fresh serum-free medium overnight before exposure to LIF alone or in combination with selective inhibitors (the pan-specific Jak inhibitor, Jak inhibitor I; the Jak2-selective inhibitor tyrphostin AG490; the MEK inhibitor U0126; the PI3-K inhibitor LY-294; all at the concentration of 10 µM) for different times. Western blot analysis was then conducted as described above.

Immunofluorescence Analysis
The presence of LIFRß and gp130 immunoreactivity in GN11 cells was also assessed by IFL. Cells were grown at 0.01 x 106 cells on 12-mm polylysine-coated glass slides and fixed in 4% paraformaldehyde for 5 min at 4 C. Preparations of paraformaldehyde-fixed GN11 cells were preincubated for 30 min with 10% normal serum in PBS and then incubated with a diluted solution of the primary antibodies indicated above (anti-LIFRß, 1:100; anti-gp130, 1:100) for 1 h at room temperature. Cells were then washed in PBS and incubated for 1 h with Alexa Fluor 488 F(ab')2 fragment of antirabbit antibodies (1:2000; Molecular Probes). Negative controls were run by either omitting the primary antibody or using normal rabbit IgG instead of the primary antibody (Santa Cruz Biotechnology).

GN11 Cell Migration Study
The assay was performed using a 48-well Boyden’s microchemotaxis chamber, according to manufacturer’s instruction (Neuro Probe, Gaithersburg, MD) and as described previously (29, 31). Briefly, cells, grown in complete medium until subconfluence, were collected by PBS/0.02% EDTA, and the suspension (0.15 x 106 cells/50 µl of DMEM/0.1% BSA) was placed in the open-bottom wells of the upper compartment. Each pair of wells was separated by a polyvinyl-pyrrolidone-free polycarbonate porous membrane (8-µm pores) precoated with gelatin (0.2 mg/ml in PBS). For chemotaxis experiments (the directed migration of cells toward regions of higher concentration of chemotactic factors), 28 µl of the control experimental medium (DMEM) or of chemoattractants (1% FBS or different concentrations of LIF in DMEM) were placed into the wells of the lower compartment of the chamber. Chemokinesis (the stimulation of increased random cell motility) was distinguished from chemotaxis by placing the same concentration of chemoattractant in both the upper and the lower wells of the chamber, thereby eliminating the chemical gradient. To assess whether the signaling pathways activated by LIF were involved in LIF-mediated migration and to evaluate the specific involvement of the gp130 component of LIF-R in mediating this process, cells were collected as described above, resuspended in FBS-free medium, aliquoted in culture tubes, and treated either for 30 min with the inhibitors described above, all at the concentration of 10 µM, or for 60 min with different concentrations (1, 5, 10 ng/ml) of the antimouse gp130 antibody (R&D Systems, Minneapolis, MN) before transfer into the Boyden’s chamber. The chamber was then kept in the cell culture incubator for 3 h. At the end of the incubation period, the cells migrated through the pores and adherent to the underside of the membrane, were fixed, stained using the Diff-Quik kit (Biomap, Milan, Italy), and mounted onto glass slides. For quantitative analysis, cells were observed using a x100 objective on a light microscope. Six random objective fields of stained cells were counted for each well, and the mean number of migrating cells/mm2 was calculated and expressed as percent with respect to positive control (1% FBS was taken as 100%).

For F-actin stainings, GN11 cells were seeded at a density of 1000 cells per well in the open-bottom wells of the upper compartment of the Boyden’s chamber and exposed to DMEM, DMEM plus 1% FBS or 100 ng/ml of LIF, loaded in the lower compartment. After 1 h incubation, cells located on the upper part of the membrane, representative of migrating cells, were fixed in 4% paraformaldehyde-PBS for 30 min at RT and incubated with phalloidin-TRITC (1:500, in PBS) for 30 min at 37 C. Preparations were then examined with a fluorescent microscope (Zeiss), and images were taken.

Analysis of the Data
Statistical analysis was performed using the Prism statistical analysis package (GraphPad Software, San Diego, CA). Data are given as mean ± SD. Differences between treatment groups were evaluated by ANOVA, followed by post hoc Tukey and Dunnett’s test and considered significant at P < 0.05.


   ACKNOWLEDGMENTS
 
We thank Ms. Paola Assi and Giovanna Miccichè for their skillful technical collaboration.


   FOOTNOTES
 
This work was supported by the University of Milan and the Ministero dell’Università e della Ricerca [FIRB 2001, Grant RBNE01JKLF_005 (to P.M.); PRIN 2005, Grant 2005051740_003 (to R.M.)] and grants-in-aid (Nos. 12671072 and 14571071) from the Japan Society for the Promotion of Science (to H.W.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online February 13, 2007

1 P.M. and E.D. contributed equally to this study. Back

Abbreviations: CNTF, Ciliary neurotrophic factor; GDI-1, Rab GDP-dissociation inhibitor; gp130, glycoprotein 130; IFL, immunofluorescence analysis; Jak, Janus kinase; LIF, leukemia inhibitory factor; LIF-R, LIF receptor complex; LIFRß, LIF receptor ß subunit; MEK, MAPK kinase; PI3-K, phosphatidylinositol 3-kinase; RT, reverse transcription; STAT, signal transducer and activator of transcription; TRITC, tetramethylrhodamine B isothiocyanate.

Received for publication June 30, 2006. Accepted for publication February 6, 2007.


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