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Endocrinology Vol. 143, No. 9 3306-3315
Copyright © 2002 by The Endocrine Society

ARTICLE

Hepatocyte Growth Factor/Scatter Factor Facilitates Migration of GN-11 Immortalized LHRH Neurons

P. Giacobini, C. Giampietro, M. Fioretto, R. Maggi, A. Cariboni, I. Perroteau and A. Fasolo

Department of Human and Animal Biology (P.G., C.G., M.F., I.P., A.F.), University of Turin, 10123 Torino, Italy; and Department of Endocrinology (R.M., A.C.), University of Milan, 20133 Milan, Italy

Address all correspondence and requests for reprints to: Prof. Aldo Fasolo, Department of Human and Animal Biology, University of Turin, Via Accademia Albertina 13, 10123 Torino, Italy. E-mail: aldo.fasolo{at}unito.it.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The molecular cues regulating the migratory process of LHRH neurons from the olfactory placode into the brain are not well known, but gradients of chemotropic and chemorepellent factors secreted by the targets are likely to play a key role in guidance mechanisms.

Hepatocyte growth factor/scatter factor (HGF/SF) is a pleiotropic cytokine inducing cell migration. It is involved in a variety of developmental processes through interaction with its receptor c-Met. Here we show that c-Met-antibody labels LHRH migrating neurons in the olfactory mesenchyme of E12 mouse and analyze the potential chemotropic effect of HGF/SF on two immortalized LHRH cell lines, GT1-7 and GN11, isolated from tumors developed in the hypothalamus and in the olfactory bulb, respectively.

By RT-PCR analysis, Western blotting, and immunocytochemistry, we provide evidence for a high level of c-Met expression in GN11, but not in GT1-7, cells. In addition, HGF/SF treatment promotes specific migratory activity of GN11 cells, as demonstrated by collagen gel assay, time-lapse video microscopy, and Boyden’s chamber experiments. Such promotion is inhibited by the neutralizing antibody.

The data reported here represent the first direct evidence of a chemotactic effect of HGF/SF on immortalized LHRH neurons.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
REPRODUCTION IN MAMMALS is under the control of several inputs; one, in particular, is the secretion of anterior pituitary gonadotropins that is regulated by the hypothalamic hormone GnRH, also known as LHRH. LHRH-secreting neurons, like olfactory neurons, originate in the olfactory placode (embryo stage E11.5 in mouse), which will later develop into the olfactory epithelium (1). During development, olfactory neurons send their axons to the olfactory bulb (OB), whereas LHRH neurons migrate into the forebrain along the terminal and vomeronasal nerves to reach their final septohypothalamic localization.

In the last decade, many studies have been performed on the molecular factors seemingly involved in this migratory process. Neuronal targeting is a stepwise process characterized by a series of pathway choices based on adhesive or repulsive cell-to-cell, as well as on cell-to-substrate, interactions. Several adhesion molecules, such as the neural cell-adhesion molecule (NCAM) and its polysialylated form (PSA-NCAM), peripherin, somatostatin, TAG-1, and the recently identified factor NELF (nasal explant LHRH factor; 2) have been shown to be expressed along the olfactory fibers that define the migratory pathway of LHRH neuroendocrine cells (3, 4, 5, 6, 7, 8). However, the molecular cues regulating this migratory process remain incompletely understood, even though gradients of chemotropic and chemorepellent factors secreted by the targets are likely to play a key role in the guidance mechanisms. Previous reports demonstrated the presence of netrin-1 in the hypothalamus and median eminence and suggested that this molecule and its receptor deleted in colorectal cancer (DCC) could play a role in LHRH-neuronal migration (9). Indeed, a recent study has shown that loss of DCC function results in the migration of LHRH neurons to inappropriate destinations (10).

A series of tissue-derived factors and classical growth factors [i.e. reelin, astrotactin, EGF, and others (11, 12, 13, 14, 15, 16)], have been found to exert chemotropic effects on neuronal and nonneuronal cells. Among them, hepatocyte growth factor/scatter factor (HGF/SF) plays a major role. HGF/SF acts via its receptor c-Met, resulting in pronounced effects on certain cell types (17). HGF/SF was initially discovered as a fibroblast-derived effector of epithelial movement (SF) (18, 19), as growth factor for liver (20), and subsequently also as a chemoattractant (21). It was also found to be involved in organ regeneration, angiogenesis, and tumor invasion (22, 23, 24, 25). Of interest for our purposes, HGF/SF recently has been proposed to have an important role in the development and function of the nervous system (26, 27, 28).

Finally, HGF/SF and c-Met are present in olfactory regions such as the OB, the olfactory epithelium, and the olfactory nerve layer (29, 30, 31).

On the basis of these considerations, we have investigated c-Met expression in migrating LHRH neurons in vivo and the potential chemotropic effect of HGF/SF on immortalized LHRH neurons in vitro.

Useful tools for the in vitro study of the biology of LHRH neurons are represented by nasal explant cultures (4) and by two immortalized neuronal cell lines, GT1 and GN. GT1 (with the GT1-1, GT1-3, and GT1-7 subclones) and GN (with GN10, GN11, and NLT subclones) cell lines were isolated from tumors induced by genetically targeting the expression of the simian virus-40 large T antigen in mouse LHRH neurons (32, 33). GT1 cell line was derived from a hypothalamic tumor, whereas GN cells were obtained from a tumor developed in the OB. This different derivation is indicative of different maturational stages of the two cell lines, as demonstrated by the fact that GT1 cells retain many characteristics of the mature hypothalamic LHRH neurons (34, 35, 36). On the contrary, it has been found recently that GN11 cells retain the phenotypic characteristics of immature LHRH neurons and show high migratory activity in vitro, responding to fetal bovine serum (FBS) as a chemotactic stimulus (36, 37).

In the present paper, we show that HGF/SF specifically promotes the migratory activity of GN11, but not GT1-7, cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and tissue
The experiments were carried out on CD-1 mice purchased from Charles River Laboratories, Inc. (Calco, Italy). Timed pregnant mice (plug day, E0) were deeply anesthetized and killed at embryonic d 12 (E12). The embryos were harvested and washed in ice-cold Dulbecco’s PBS (Sigma, St. Louis, MO). The heads were fixed in fresh 4% paraformaldehyde (PAF, 4% in 0.1 M phosphate buffer, pH 7.4) overnight at 4 C and cryoprotected the next day. Tissues were then embedded in Killik frozen section medium (Bio Optica, Milan, Italy) and frozen in liquid nitrogen-cooled isopentane. Sagittal sections (8 µm) were cut, mounted on 3-aminopropyl-trietoxysilane-treated slides (Fluka Chemical Co., Milan, Italy) and stored at -20 C until processing for immunohistochemistry.

All animal protocols were approved by the Animal Care and Use Committee of the University of Turin.

Cell cultures
GT1-7 and GN11 cells were grown in monolayer at 37 C in a 5% CO2, in DMEM (Life Technologies, Inc., Grand Island, NY) containing 1 mM sodium pyruvate, 2 mM glutamine (Life Technologies, Inc.), 100 µg/ml streptomycin, 100 U/ml penicillin, 4500 mg glucose (ICN Biomedicals, Inc., Irvine, CA) and supplemented with 10% FBS (Life Technologies, Inc.). The medium was replaced at 2-d intervals. Subconfluent cells were routinely harvested by trypsinization and seeded in 57-cm2 dishes (1 x 106 cells). GT1-7 and GN11 cells within six passages were used throughout the experiments.

Immunocytochemistry
Consecutive E12 mouse sagittal sections (8 µm) were stained for LHRH and c-Met immunoreactivity, respectively. LHRH neurons were labeled using the rabbit polyclonal LR1 anti-LHRH antibody (a generous gift from Dr. Benoit, Montréal, Canada) at a 1:10,000 dilution, whereas c-Met immunoreactivity was detected using the rabbit polyclonal c-Met antiserum (1:500; Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Sections were incubated overnight at 4 C with the mentioned antibodies diluted in 1% normal goat serum and 0.3% Triton X-100 in PBS. Sections were then washed in PBS and incubated with antirabbit biotinylated secondary antiserum and the ABC kit (Vector Laboratories, Inc., Burlingame, CA) with 3,3'-diaminobenzidine (0.015%) as a cryogen. Sections were then washed in PBS, mounted, and cover-slipped.

For immunocytochemical analysis of GN11 and GT1-7, cells were grown on 24-multiwell dishes in 10% FBS DMEM. After 24 h, cells were washed twice with PBS, fixed for 30 min in 4% paraformaldehyde (PAF 4%) at room temperature, and washed twice in PBS. Nonspecific binding was blocked with normal goat serum (10% in PBS, 30 min), and the cells were then incubated overnight with the primary antibody (anti-Met rabbit polyclonal, 1:500; Santa Cruz Biotechnology, Inc.). Antigen was visualized by incubation with antirabbit biotinylated secondary antiserum, the Vector Laboratories, Inc. ABC kit, and 3,3'-diaminobenzidine (0.015%) as a chromogen. Negative controls were run by omitting the primary antibody.

Labeled sections and cells were observed on an IX50 inverted microscope (Olympus Corp., Hamburg, Germany) equipped with a CCD camera CoolSNAP-Pro (Media Cybernetics, Houston, TX) and images edited with Image Pro-Plus software (Media Cybernetics). Only gray-scale conversion and adjustment to brightness and contrast were used in the preparation of the figures.

RT-PCR and Western blot analysis
Total RNA was isolated by extraction with TRIzol (Life Technologies, Inc.). Single-strand cDNA was synthesized by M-MLV reverse transcriptase from 1 µg total RNA primed with 50 pmol random hexamers (Amersham Biosciences, Buckinghamshire, UK) in a 20-µl reaction. Each reaction consisted of Life Technologies, Inc.’s first-strand cDNA synthesis buffer [50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2], 4 mM deoxynucleotide triphosphate (Amersham Biosciences), 1.8 U/µl RNAsin (Amersham Biosciences), and 10 U/µl M-MLV reverse transcriptase (Life Technologies, Inc.). Samples were incubated at 37 C for 1 h. Negative controls were those to which reverse transcriptase was not added. PCR was carried out using 4 µl cDNA and the appropriate oligonucleotides (0.6–6 µM) in 30-µl PCRs using standard reaction buffer [10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.001% gelatin (Sigma)], 0.8 mM deoxynucleotide triphosphate (Pharmacia), and 0.025 U/µl REDTaq DNA polymerase (Sigma).

The following primers were used: 5'-TGGCATTGTGGAAGGGCTCATGAC-3' and 5'-ATGCCAGTGAGCTTCCCGTTCAGC-3' for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) amplification corresponding to nucleotides 544–732 (accession no. M32599); 5'-GGGACTGCAGCAGCAAAGC-3' and 5'-GTCTGAGCATCTAGAGTTTCC-3' for c-Met amplification corresponding to nucleotides 296–815 (38). The amplification of GAPDH served as a control, with respect to the quality and quantity of RNA that had been retrotranscribed into cDNA. The number of cycles and the annealing temperature used for each primer pair were: 25 cycles and 62 C for c-Met, 25 cycles and 60 C for GAPDH. Amplification products were separated by 1.5% agarose gel electrophoresis and DNA bands visualized by ethidium bromide staining.

For immunoprecipitation and Western-blotting analysis, GT1-7 and GN-11 cells were solubilized in lysis buffer [Tris-HCl (pH 7.4), 100 mM NaCl, 5 mM MgCl2, 0.5% NaDOC, 1% Nonidet P-40, 1 mM phenylmethylsulfonylfluoride, 2 mM orthovanadate] on ice. Lysates were clarified by centrifugation at 16,000 x g for 15 min, and protein content was determined using a bicinchoninic acid kit for protein determination (Sigma). For c-Met immunoprecipitation, 1 mg protein extract was incubated with polyclonal c-Met antiserum (Santa Cruz Biotechnology, Inc.; 1:100) for 1 h at 4 C. Immune complexes were collected with protein-A-Sepharose (Sigma) and washed three times with extraction buffer and once with TNE buffer (50 mM Tris, 140 mM NaCl, 5 mM EDTA). Precipitated proteins were released by boiling in Laemmli buffer [2% sodium dodecyl sulfate, 50 mM Tris-HCl (pH 7.4), 20% ß-mercaptoethanol, 20% glycerol] and subjected to 8% SDS-PAGE. Proteins were blotted onto polyvinylidene difluoride transfer membrane (Millipore, Bedford, MA). After blocking with 5% powder milk in TBST buffer (20 mM Tris; 150 mM NaCl; 0.1% Tween 20, pH 7.4), filters were probed with 1:500 polyclonal c-Met antiserum or 1:500 monoclonal antiphosphotyrosine antibody (Santa Cruz Biotechnology, Inc.) and visualized with the appropriate peroxidase-coupled secondary antibodies using an ECL detection system (Amersham Biosciences).

Cell aggregates (collagen gel assay)
Collagen gel has been found to allow optimal physiological culture conditions for many tissue fragments and normal isolated cells and to study cell migration (39). Cell aggregates were prepared by the so-called hanging drop technique (40). Subconfluent cells were collected by trypsinization, resuspended in complete culture medium, and seeded in 20-µl drops (200,000 cells for both cell lines) on the lid of a culture dish; the lid was then placed on a 35-mm dish filled with 2 ml culture medium and incubated at 37 C for 48 h.

Collagen gel was obtained as previously described (37), and 200 µl were pipetted onto the bottom of a 24-well culture dish and left to set at room temperature. Cells aggregates were transferred over the cushion and then overlaid with an additional 200 µl collagen. As the overlaid collagen was set, it was covered with 400 µl DMEM (0% FBS), or supplemented with recombinant human HGF/SF (50 ng/ml, Sigma), or with HGF/SF 50 ng/ml preincubated overnight with the blocking antibody to HGF/SF (10–20 µg/ml) (antihuman HGF/SF monoclonal antibody, Sigma) and transferred to the cell culture incubator. After 24 h, the aggregates were fixed in PAF 4%, and phase-contrast microscope pictures were taken. For the quantitative analysis, the aggregate surface was subdivided radially into 8 compartments and, within each compartment, the distance separating the cell body of the most distant 10 cells, from the border of the aggregate, were measured (80 measurements for each experiment). The mean distance of migration for each experimental condition was then calculated and the data obtained from 4 independent wells for each group (serum free, HGF/SF, anti-HGF/SF) were compared by ANOVA and Student Newman-Keuls test, P < 0.05.

Time-lapse video microscopy
Living cells were observed under an inverted fluorescent microscope (Eclipse TE 200; Nikon, Tokyo, Japan) equipped with an incubation chamber for constant temperature and CO2 regulation. Time-lapse pictures were acquired with a CCD gray-scale camera (PCO Computer Optics GmbH, Kelheim, Germany), controlled by the Image Pro-Plus software.

GN11 cells were grown in serum-free conditions for 24 h before the recordings.

Time-lapse images of GN11 cell lines, exposed to serum-free medium (SFM) with or without HGF/SF (50 ng/ml), were taken at 1-h intervals for 8 h.

To ensure an accurate assessment of cell motility, only picture frames containing isolated cells were examined.

Chemomigration assay
The assay was performed using a 48-well Boyden’s microchemotaxis chamber according to manufacture instructions (Neuroprobe, Cabin John, MD). Briefly, the cells grown in complete medium until subconfluence were harvested, and the suspension (105 cells/50 µl serum-free DMEM for both cell lines) was placed in the open-bottom wells of the upper compartment. Each pair of wells were separated by a poly-vinylpyrrolidone-free polycarbonate porous membrane (8-µm pores) precoated with gelatin (0.2 mg/ml in PBS). For chemotaxis (the directed migration of cells toward regions of higher concentration of chemotactic factors) experiments, the chemoattractants (DMEM FBS 1%, DMEM FBS 0%, HGF/SF at 6.2, 12.5, 25, 50, 100 ng/ml) were placed into the wells of the lower compartment of the chamber. Chemokinesis (stimulation of increased random cell motility) was distinguished from chemotaxis by placing the same concentration of chemoattractant in both the upper and lower wells of the Boyden’s chamber, thereby eliminating the chemical gradient.

The Boyden’s chamber was then kept for 3 h in the cell culture incubator. The cells were allowed to migrate through the membrane for 3 h. After incubation, the cells were fixed and stainted (Diff-Quick stain kit, Dade Behring AG, Dudingen, Switzerland) and mounted onto glass slides. For quantitative analysis, the cells were observed using an oil immersion 100x objective on a light microscope. Six random objective fields of stained cells were counted for each well, and the mean number of migrating cells per square millimeter was calculated. The number of migrated cells, obtained from 10 independent wells for each group, was compared by ANOVA and Dunnett’s tests.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
c-Met expression in E12 primary olfactory system and in GT1-7 and GN11 cell lines
Immunohistochemistry indicated c-Met protein expression in the olfactory neuroepithelium as well as in cells migrating through the nasal mesenchyme of E12 embryos (Fig. 1Go). To establish whether c-Met-positive cells were LHRH-migrating neurons, single immunohistochemical stainings were performed on consecutive sagittal sections (Fig. 1Go).



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  		Figure 1. LHRH and c-Met immunohistochemistry performed on consecutive sagittal sections of a 12-d mouse embryo (E12). LHRH-immunoreactive cells emerged from the developing olfactory epithelium and migrate through the olfactory mesenchyme (arrows) toward the forebrain. Met-immunoreactivity in the adjacent section resembles the staining pattern of LHRH. Note that the Met-antibody (a-Met) likely labels the same groups of migrating elements (arrows) stained with the LHRH antiserum (a-LHRH). OE, Olfactory epithelium; L, lumen. Scale bar, 250 µm.

 
Even though it was not possible to perform simultaneous double-labeling experiments, c-Met expression seems localized to the LHRH neuronal migratory pathway, and the neuroendocrine cells migrating through the nasal regions seem to express the c-Met-receptor.

To ascertain whether GT1-7 and GN11 cell lines express the c-Met receptor, RT-PCR analysis was undertaken. Because previous studies revealed that c-Met is expressed in the murine adult OB (30), OB extracts were used as positive control (for the negative control, sterilized water was used to replace the DNA sample).

RT-PCR reactions, carried out using specific c-Met oligoprimers, yielded the predicted 519-bp amplicons in OB samples; a strong expression level was found in GN11 cell line, whereas c-Met was seemingly absent in GT1-7 cells (Fig. 2AGo). RT-PCRs, performed using GAPDH primers, yielded a 188-bp product and confirmed that equal amounts of RNA, extracted from adult mouse OB, GT1-7, and GN11 cells, were included in each reaction (Fig. 2AGo).



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  		Figure 2. A, c-Met expression in OB, GT1-7, and GN11 cell line extracts. Detected by RT-PCR, mRNA yields the predicted 519-bp amplicons. C-met expression is detected in OB and GN11 cells; mRNA is not detectable in GT1-7 cells. The predicted size of the GAPDH PCR product is 188 bp. B, C-met was immunoprecipitated from GN11 and GT1-7 cells and probed with anti-c-Met. GN11 cells express both the precursor protein and the ß-chain, p145met, whereas GT1-7 cells do express the 145-kDa band only at a barely detectable level. C, C-met was immunoprecipitated (IP) from GN11 and GT1-7 cells and was detected (wb) with antiphosphotyrosine antibody either after no incubation or after additional incubation for 15 min in 50 ng/ml HGF. The upper band indicates the c-Met precursor protein, p170 met. The lower band indicates the ß-chain, p145met. Incubation of GN11 cells with HGF enhances the phosphorylative state of the receptor.

 
Immunoprecipitation and Western blot experiments were performed to identify the expression of the c-Met protein in both cell lines. c-Met was immunoprecipitated, using a rabbit polyclonal antibody, from cells either treated or not with HGF/SF (50 ng/ml for 15 min). The same antibody was also used to detect the protein (Fig. 2BGo). GN11 cells express HGF/SF receptor. As shown by other groups, both the precursor protein p170met and the p145met were detected (41).

A band of 145-kDa molecular mass was barely detectable in GT1-7 cells (Fig. 2BGo).

Following the immunoprecipitation protocol, an antiphosphotyrosine antibody was also used to detect the phosphorylation state of the receptor in both cell lines. c-Met was found to be constitutively tyrosine-phosphorylated in GN11 cells, even though at barely detectable levels (Fig. 2CGo). Subsequent incubation with HGF enhanced the phosphorylation in GN11 cells, whereas antiphosphotyrosine did not detect any specific band in GT1-7 cells (Fig. 2CGo).

As a further test of the plasma-membrane localization of c-Met in GN11 and GT1-7 cells, immunocytochemical experiments were performed by incubating nonpermeabilized cells with the c-Met antibody. A plasma-membrane-associated staining pattern was clearly evident in GN11 cells (Fig. 3AGo). According to RT-PCR and Western blot analyses, GT1-7 cells did show only a weak c-Met immunoreactivity, near to background levels (Fig. 3BGo).



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  		Figure 3. Immunolocalization of c-Met protein in nonpermeabilized GN11 (A) and GT1-7 (B) cells. GN11 cells exhibit a strong immunoreactivity for c-Met, whereas GT1-7 cells show only a weak labeling, near to background levels (see particular). Scale bar, 100 µm.

 
Collagen gel assay
Previous studies have shown that GN11, but not GT1-7, cells show significant migratory activity and actively respond to the chemotactic stimulus represented by FBS (37).

Considering that the expression of c-Met in GN11 cells might be linked to their ability to respond to HGF/SF, a series of migratory assays were performed. The collagen gel assay was chosen as the first good experimental tool, because it is a generally accepted procedure to study cell migration in a three-dimensional matrix. In these experiments, both the morphology of migrating cells and the distance which these cells cover under 24 h of HGF/SF stimulation were evaluated. Cellular aggregates of GT1-7 and GN11 cells were prepared by the hanging-drop technique (40) and incubated for 24 h in collagen gel prepared in SFM (DMEM 0% FBS) with or without 50 ng/ml HGF/SF. In addition, GN11 cell aggregates were also exposed to HGF/SF preincubated with a specific anti-HGF/SF (20 µg/ml).

After a 24-h incubation, no spontaneous motility of GN11 cells was observed in the absence of HGF/SF (Fig. 4AGo). However, GN11 cells, stimulated with HGF/SF, consistently migrated out of the aggregate and became arranged in chains in the collagen matrix (Fig. 4BGo). No migration of GN11 cells was observed in the presence of HGF/SF immunoneutralized with the specific antibody (Fig. 4CGo). Figure 3DGo shows, at higher magnification, that GN11 migrating cells displayed a bipolar morphology and typically migrated radially. These results thus demonstrate that GN11 cells specifically responded to HGF/SF stimulation with increased migratory activity. GT1-7 cells did not show any migration into the collagen matrix under the same experimental conditions (Fig. 4Go, E and F). Figure 4FGo also shows, at higher magnification, the absence of GT1-7 cells outside the aggregate after 24 h of incubation with 50 ng/ml HGF/SF in the culture medium.



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  		Figure 4. GN11 cell aggregates, cultured for 24 h in collagen gel matrix, respectively, in SFM (DMEM 0% FBS, A), in the presence of 50 ng/ml HGF/SF containing medium (B and D) and in the presence of specific anti-HGF/SF antibody (+Ab) (C). Cell migration is prevented in A and C experimental conditions. D, Brightfield picture of GN11 cell aggregate (higher magnification of B), showing the chain of migrating cells that emerge from the aggregate under HGF/SF stimulation (cresyl violet staining). E, GT1-7 cell aggregate, cultured for 24 h in the presence of 50 ng/ml HGF/SF, showing the absence of migration (F, higher magnification of E). Scale bars: A, B, C, and E, 125 µm; D, 12.5 µm; F, 25 µm.

 
A quantitative analysis of the migratory activity of GN11 cells, under the three previously described experimental conditions, was also performed by measuring the mean distances between the front of migration of the cells and the border of the aggregate (Fig. 5Go). When the cells were cultured in the presence of HGF/SF, GN11 cells migrated off the edge of the aggregates 5-fold more, with respect to the control conditions.



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  		Figure 5. Chemotactic response of GN11 cells cultured for 24 h, in serum-deprived medium, before the chemomigration assay. This was performed using the collagen gel assay. Cell aggregates were cultured for 24 h in serum-deprived medium, or supplemented with 50 ng/ml HGF/SF, or with HGF/SF preincubated with the neutralizing anti-HGF/SF antibody. The mean distance covered by the cells in the 3 experimental conditions was measured. In each experiment, 80 measurements were performed, and the results presented are expressed as mean ± SD (n = 4). All values shown were statistically significant, as determined by the Student’s Newman-Keuls test. *, Significant P < 0.05 vs. controls (SFM; anti-HGF).

 
All of these results strongly suggest that the observed chemotropic response was specifically attributable to the presence of HGF/SF.

Time-lapse video microscopy
Time-lapse video microscopy was used to evaluate the motility response induced by HGF/SF after 24 h of serum deprivation, a procedure that slows down the spontaneous motility of GN11 cells (37). Cells were monitored for 8 h in an incubation chamber at constant temperature and CO2 regulation. Time-lapse microscopy images of these cells were taken at 1-h intervals. Figure 6Go illustrates the behavior of six GN11 cells in control conditions, in the absence of HGF/SF, during an 8-h recording. Time-lapse microscope images show the lack of spontaneous motility of these cells that kept the same positions during all the time of the monitoring.



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  		Figure 6. Time-lapse video microscopy of GN11 cells, monitored for 8 h in SFM. The cells were cultured in SFM for 24 h before the recording. Time-lapse microscopy images were taken at 1-h intervals and show the lack of cell motility of four representative cells. Scale bar, 50 µm.

 
Figure 7Go shows the behavior of three representative cells exposed to 50 ng/ml HGF/SF. Cells A and B displayed an elongated morphology and moved in opposite directions, sliding over each other. The cell indicated as C in Fig. 7Go began to extend a leading process after 1 h of HGF-exposition. Over the next hours, this process continued to extend and broaden, directing toward the other cells. Contraction of the trailing process can be appreciated in the final two frames. These results indicate that HGF was sufficient to induce a motility response in the absence of FBS.



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  		Figure 7. Time-lapse video microscopy of GN11 cells under HGF/SF stimulation (50 ng/ml). The cells were starved in SFM for 24 h before the recording. The picture frames, taken at 1-h intervals, show the behavior of three cells exposed to HGF/SF for 8 h. Cells A, B, and C, after 1 h of exposure, begin to assume a fusiform shape and, over the next hours, display high cell motility. Scale bar, 50 µm.

 
Microchemotaxis assay
Boyden’s chamber assay gives quantitative information regarding the cell population that migrates toward a concentration gradient of a chemotropic factor. Therefore, the cellular response of GN11 cells to HGF was tested through a series of microchemotaxis assays using Boyden’s chambers (see Materials and Methods). GN11 cells did not show a significant migratory activity in the absence of any chemotactic factor (Fig. 8Go), whereas the exposure to 1% FBS induced a significant chemomigration of these cells. The results also show that exposure to a gradient of HGF induced a significant strong chemotactic response of GN11 cells (Fig. 8Go). HGF exerted its effect in a concentration-dependent manner, with a maximal increase in responsiveness at 50 ng/ml (Fig. 8AGo). At this concentration, HGF was able to induce a chemotactic response comparable with that obtained by 1% FBS stimulus (Fig. 8AGo). At higher concentration (100 ng/ml), however, HGF produced no further increase in migration of GN11 cells (Fig. 8AGo).



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  		Figure 8. Chemotactic response of GN11 cells (A) and GT1-7 cells (B), cultured for 24 h in serum-deprived medium before the chemomigration assay. This was performed using Boyden’s chamber, in the absence or in the presence of increasing concentrations of HGF. Results are expressed as mean ± SEM. *, Significant P < 0.01 vs. control (SFM). C, Chemotactic (CT) and chemokinetic (CK) responses of GN11 cells were calculated in the presence of 25 ng/ml HGF. Results are expressed as mean ± SEM. *, Significant P < 0.05 vs. control (SFM).

 
GT1-7 cells did not respond to increasing concentrations of HGF (Fig. 8BGo); in addition, these cells did not present any chemotactic response, even in the presence of a high concentration of FBS (10% FBS; data not shown).

To better investigate whether the effect of HGF on the motility of GN11 cells was specifically directional (chemotaxis) or caused by the induction of random locomotor activity (chemokinesis), the cells were exposed to HGF present in the lower (chemotaxis) or in both (chemokinesis) compartments of the Boyden’s chamber. In these conditions, the induction of the cell motility was significantly less efficient in chemokinesis experiments than that induced by the same concentration of HGF/SF placed exclusively in the lower compartment of the chamber (chemotaxis; Fig. 8CGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Although HGF/SF has been shown to be present in the brain in several areas (29, 30, 31, 42), the function of HGF/SF in the nervous system is largely unknown. It has been found to exhibit neurotrophic actions (21, 43, 44) as well as to function as an axonal chemoattractant (21, 45). Recently, HGF/SF has been also proposed as a mitogen in the trans-telencephalic migration of interneurons from the ganglionic eminence to the cerebral cortex (27) and as a novel mitogen for the olfactory ensheathing cells derived from adult rats (28). The data reported here are therefore the first evidence of a direct chemotactic effect of HGF/SF on LHRH-immortalized neurons.

GN11 and GT1-7 neurons displayed different migratory activity under HGF/SF stimulation. This effect is well correlated with the strong c-Met expression found in the GN11 cell line, demonstrated by RT-PCR, Western blot, and immunocytochemical analyses. Immunocytochemical results of c-Met localization in nonpermeabilized GN11 cells confirmed its membrane localization. On the contrary, the same set of experiments indicated that the HGF/SF receptor was absent or only at a barely detectable level in GT1-7 cells.

C-Met expression conferred to GN11 cells chemotactic responsiveness to HGF/SF. A collagen gel assay was used to assess the motility and the phenotype of these cells exposed to exogenous HGF/SF. After 24 h of HGF/SF exposure, the migration of GN11 cells typically extended from the aggregates 5-fold more than in control conditions. The motility response was blocked by neutralizing antibody as well as in serum-free conditions (DMEM 0% FBS). On the other hand, GT1-7 cells did show a refractory response to the induced motility stimulus of HGF/SF; indeed, they did not leave the aggregates under different concentrations of the growth factor. Time-lapse video microscopy shows that GN11 cells, cultured for 24 h in serum-deprived medium and subsequently recorded for 8 h in the presence of HGF/SF, assumed a typical morphology of migrating neurons with an elongated cell body and the extension of leading processes. In addition, these cells often moved, sliding over each other. This also confirms the importance of cellular contacts during their movements and demonstrates, once more, that preincubation in serum-deprived medium does not influence the chemotactic response of GN11 cell line (37). Time-lapse recording in SFM without addition of HGF/SF demonstrates that GN11 cells failed to show any spontaneous motility and reinforces the hypothesis of a specific role of HGF/SF in the induction of a migratory response of this LHRH immortalized cell line.

In other model systems, cells expressing c-Met, indeed, seem to move toward a concentration gradient of HGF/SF (27, 46). Therefore, we have investigated the quantitative response of both GT1-7 and GN11 cell lines by microchemotaxis assays, performed using the Boyden’s chamber, under HGF/SF stimulation. We have observed that GN11, but not GT1-7 cells, significantly respond to this chemotactic stimulus in a dose-related fashion. Most important is the finding that HGF/SF was significantly efficient in stimulating chemotaxis, but not chemokinesis, as demonstrated by a strong reduction of cell migration after exposure of the cells to a uniform concentration of the chemoattractant. The magnitude of maximal stimulation was comparable with that obtained using 1% FBS that, to date, represented the strongest stimulus for these cells (37).

GN11 cell line was derived from a tumor developed in the OB (33); therefore, these cells are representative of LHRH neurons arrested during their transit to the brain, and they keep many features of migrating neurons (36, 37). On the contrary, GT1-7 cells, derived from a hypothalamic tumor, exhibit both morphological and behavioral features of the hypothalamic postmigratory neurons that lost their ability to migrate (36, 37, 47). The unresponsiveness of GT1-7 cells to HGF/SF may be then correlated with a more differentiated phenotype, with respect to GN11, including the development of higher adhesive properties that would block their motility.

Based on the previous assumption that GN-derived cells are representative of immature migrating LHRH neurons, we propose HGF/SF as one of the possible chemical factors inducing the migration of LHRH neurons in vivo as well. Although the several studies focused on LHRH neuronal migration have identified the preferential association between these neurons and the olfactory fibers in the nasal mesenchyme, the migratory guidance molecules involved in such a process remain mostly unknown. Schwarting et al. (10) have recently presented evidences that a versatile guidance molecule, such as netrin-1, and its receptor DCC, play a role in this migratory process. Loss of DCC function results, indeed, in the migration of many LHRH neurons to inappropriate destinations.

Unfortunately, mice lacking either HGF/SF or its receptor die during early embryogenesis, with defects in placenta, liver, and muscle (46, 48, 49, 50); and this embryonic lethality makes it difficult to study the role of HGF/SF in the LHRH migratory process. Nevertheless, several lines of evidence support the hypothesis that HGF/SF could be involved in the control of the migration of LHRH neurons.

HGF/SF and c-Met mRNA transcripts were identified in the mouse developing OB, by Northern analysis (29, 30, 31). In addition, HGF/SF transcript has been evidenced in the forebrain as early as E11.5 (27), which is a stage corresponding to the beginning of the LHRH migratory process (1, 51). HGF/SF expression in the target tissue of this neuronal migration suggests that molecular signaling between forebrain and the olfactory placodes might influence olfactory pathway development. In the developing olfactory system, HGF/SF and c-Met distributions are distinct. As cited above, both molecules are localized in the developing and adult OB (29, 30, 31); and, more precisely, HGF/SF is seemingly expressed in the glomerular layer, whereas c-Met mRNA is distributed in the mitral cell layer (30). In addition, in the embryonic olfactory mucosa, c-Met mRNA is localized in the olfactory epithelium as well as in the olfactory nerve layer (30). Coincident with the appearance of c-Met in the embryonic olfactory epithelium, HGF/SF and its activator, tissue plasminogen activator (tPA), can be detected in the lamina propria underlying the epithelium and in the epithelium, respectively (30). Furthermore, tPA have been found to be expressed in migrating cells of the olfactory neuroepithelium during mice embryogenesis (52). Because LHRH neurons represent a subpopulation of these migrating cells (together with olfactory ensheathing and olfactory marker protein-positive cells), as shown by Tarozzo et al. (53), and tPA expression overlapped with the migratory pathway of these migrating cells, we cannot exclude that tPA is expressed by LHRH cells themselves. We demonstrate here, for the first time, the expression of both the c-Met mRNA and protein in the GN11 LHRH-secreting cell line but not in GT1-7 cells. Moreover, we do show that HGF/SF represents a chemotactic factor for GN11 neurons.

Lastly, an intriguing result was our observation, by immunohistochemical experiments, that, during embryonic development, c-Met expression correlates its temporal and spatial distribution with the LHRH migratory pathway.

Based on the preexisting literature and on the present results, it is possible to postulate that HGF/SF may play a main role as a chemoattractant for elongating olfactory axons and for migrating neuroendocrine (LHRH) cells. The present study provides a new basis for additional studies aimed at clarifying the roles of HGF/SF in the LHRH migratory process in vivo.


   Acknowledgments
 


   Footnotes
 
This work was supported by CNR, M.U.R.S.T., Compagnia di San Paolo, Telethon (Grant E.523). Paolo Giacobini is the recipient of a fellowship from "Accademia Nazionale dei Lincei," Rome.

Abbreviations: DCC, Deleted in colorectal cancer; FBS, fetal bovine serum; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HGF, hepatocyte growth factor; OB, olfactory bulb; PAF, paraformaldehyde; SF, scatter factor; SFM, serum-free medium; tPA, tissue plasminogen activator.

Received February 6, 2002.

Accepted for publication May 8, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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