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Role of Endothelin-1 in the Migration of Human Olfactory Gonadotropin-Releasing Hormone-Secreting Neuroblasts

Departments of Anatomy Histology and Forensic Medicine (A.P., S.A., M.M., G.B.V.), Clinical Physiopathology and Endocrinology (R.S., C.M.R.), and Andrology Units (M.M., M.L., A.M.), Internal Medicine (R.G.R.), and Preclinical and Clinical Pharmacology (P.F.), University of Florence, I-50134 Florence; Department of Endocrinology (R.M., R.Z.), University of Milan, 20133 Milan; and Experimental and Clinical Medicine (T.B.), University of Catanzaro, 88100 Catanzaro, Italy

Address all correspondence and requests for reprints to: Gabriella B. Vannelli, Department of Anatomy Histology and Forensic Medicine, University of Florence, School of Medicine, V. le Morgagni 85, I-50134 Florence, Italy. E-mail: vannelli{at}unifi.it.

	Abstract

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FNC-B4 neuroblasts that express both neuronal and olfactory markers have been established and cloned. These cells express GnRH and both the endothelin-1 (ET-1) gene and protein and respond in a migratory manner to GnRH in a dose-dependent manner. Previous research has shown that FNC-B4 cells produce and respond to ET-1 by regulating the secretion of GnRH through endothelin type A receptors and by stimulating their proliferation through endothelin type B (ETB) receptors. In this study, we found that FNC-B4 cells are able to migrate in response to ET-1 through the involvement of ETB receptors. Combined immunohistochemical and biochemical analyses showed that ET-1 triggered actin cytoskeletal remodeling and a dose-dependent increase in migration (up to 6-fold). Whereas the ETB receptor antagonist (B-BQ788) blunted the ET-1-induced effects, the ETA receptor antagonist (A-BQ123) did not. Moreover, we observed that FNC-B4 cells were independently and selectively stimulated by ET-1 and GnRH. We suggest that ET-1, through ETB receptor activation, may be required to maintain an adequate proliferative stem cell pool in the developing olfactory epithelium and the subsequent commitment to GnRH neuronal migratory pattern. The coordinate interaction between ET receptors and GnRH receptor participates in the fully expressed GnRH-secreting neuron phenotype.

	Introduction

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HOW CELLS INITIATE, maintain, and stop their migration remains a fascinating problem in many developmental systems. Although the GnRH-secreting neurons are located postnatally within the forebrain, during embryogenesis, they arise from the olfactory placode and then migrate into the hypothalamus (1, 2). Proper spatiotemporal expression of transcription factors plays a crucial role in these processes, both in guiding GnRH-secreting neurons during their migration and in orchestrating their subsequent differentiation (3). Although the endothelin (ET) family was initially characterized as potent vasoactive peptides, a variety of other biological functions were subsequently discovered, such as stimulation of hormone release and regulation of central nervous system activity (4, 5, 6, 7). In particular, ET immunoreactivity (8) and ET receptors (9) are present in median eminence and arcuate nucleus, in which GnRH neurons are mostly concentrated. Interestingly, ET peptides stimulate GnRH release from hypothalamic explants (10), fetal rat hypothalamic neurons (11), and fetal human olfactory cells (12).

Recent gene-inactivation studies of the components of the ET pathway have revealed some other unexpected roles of these peptides, especially during mammalian development. Mice deficient in endothelin-1 (ET-1), endothelin type A (ETA) receptor, and endothelin-converting enzyme-1 have defects in the development of subsets of cephalic and cardiac neural crest derivatives, including branchial arch-derived craniofacial tissues, and great vessel and cardiac outflow structures (13, 14, 15, 16). Mice deficient in endothelin-3 (ET-3) or endothelin type B (ETB) receptor show spotted coat color and aganglionic megacolon due to defects in the development of neural crest-derived melanocytes and enteric neurons, respectively (17, 18). In humans, multigenic Hirschsprung’s disease, or congenital aganglionosis of the distal gut, is caused by a failure of the neural crest cells to form ganglia in the distal part of the gut. This disease is characterized by pigmentation defects, deafness, and megacolon (19, 20, 21). New insights into the pathogenesis and the molecular basis of this abnormality in humans have been gained from the identification of certain critical genes, such as ET-3 and the ETB receptor in mice. These genes play a role in the development of the peripheral nervous system and in that of the connective tissue in the face, neck, and heart (22, 23). Recent research has demonstrated that genetic interactions between mutations in receptor for glial-derived neurotrophic factors (RET, receptor protein tyrosine kinase) and the ETB receptor is one of the underlying mechanisms for this complex disorder (24). Furthermore, the interaction between the RET and ETB receptor loci in humans and mice regulates enteric nervous system development in the distal colon. It also regulates the coordinated and balanced interaction between RET and ETB receptor signaling pathways and controls the development of the mammalian enteric nervous system throughout the intestine (25). Recently, it has been demonstrated that temporally distinct requirements for ETB receptor occur in the proliferation and migration of gut neural crest stem cells (26). In a previous research, we showed that GnRH-secreting neurons, FNC-B4 cells, produce and respond to ET-1 and that this peptide regulates GnRH-secretion or cell proliferation, depending on which subtype of the ET receptor is activated (12). Moreover, GnRH acts in an autocrine/paracrine pattern to promote migration of FNC-B4 neuroblasts (27). The aim of this study was to elucidate the role played by ET-1 in the migratory pattern of GnRH-secreting neurons. FNC-B4 cells dose dependently migrated in response to ET-1 through the involvement of ETB receptor. We also report that GnRH and ET-1 act in a biologically independent and selective manner, and their recruitment is able to elicit a functional crosstalk during migration.

	Materials and Methods

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Cell cultures
The primary human GnRH-secreting neuronal cell line, FNC-B4 cells, has been established, cloned, and propagated in vitro from the fetal olfactory system and cryogenically preserved. FNC-B4 cells have been phenotypically, biochemically, and functionally characterized (12, 27, 28, 29, 30, 31). These cells grow as a monolayer, are nontumorigenic, and have a normal human karyotype. The immortalized GnRH-expressing neuronal cell line, GN11 cells, was used in some experiments (generously provided by S. Radovick, University of Chicago, Chicago, IL). GN11 neuronal cells have been isolated from olfactory bulb tumors of migration-arrested GnRH neurons (32). The human neuroblastoma SH-SY5Y cell line, a clonal derivative from primary tumor of neural crest cells, was used in some experiments [these cells are commercially available from American Type Culture Collection (Manassas, VA) and LGC Promochem (Teddington, UK)]. This cell line has been previously established and extensively characterized both phenotypically and biochemically (33, 34, 35). The primary human GnRH-secreting neuronal cell line FNC-B4, the immortalized GnRH-expressing neuronal cell line GN11, and the SH-SY5Y cell line show different biochemical and functional neuronal features that mimic some of the biochemical and functional heterogeneity that is characteristic of the developing nervous system. These cell types were cultured at 37 C in 5% CO₂ atmosphere in Coon’s modified F-12 medium (Irvine Scientific, Santa Ana, CA), supplemented with 4.5 g/l glucose, 10% fetal bovine serum (Eurobio, Les Ulis, France), and antibiotic/antimycotic solution (penicillin, 100 IU/ml; streptomycin, 100 µg/ml).

Chemicals
[¹²⁵I]GnRH (2200 Ci/mmol) was obtained from PerkinElmer Life Science Products (Milan, Italy). [¹²⁵I]ET-1 (2000 Ci/mmol) was purchased from Amersham Biosciences (Amity PG, Milan, Italy). A GnRH RIA kit was obtained from Buhlmann Laboratories (Allschwil, Switzerland). Unlabeled ET-1 and the ETA-selective antagonist A-BQ123 were obtained from NovaBiochem (Laufelfingen, Switzerland). The ETB-selective agonist IRL-1620 and antagonist B-BQ788 were purchased from Alexis (Laufelfingen, Switzerland). The polyclonal antibodies to ET-1 (RAS 6901) were purchased from Peninsula Laboratories (San Carlos, CA). Synthetic GnRH was obtained from Incstar (Stillwater, MN). GnRH agonist buserelin (D-tert-butyl-Ser6-des-Gly10-Pro9-ethylamide-GnRH) was purchased from Sigma (St. Louis, MO). GnRH antagonist cetrorelix [Ac-D-Nal (2)1, D-Phe (4Cl)2, D-Pal (3)3, D-Cit6, D-Ala10] was purchased from ASTA Medica (Frankfurt, Germany). The rabbit polyclonal antibodies to the ETA and ETB receptors were provided by Assay Design (Ann Arbor, MI) and were used at 1:100 concentration; the antigoat polyclonal neuron-specific enolase (NSE) antibody (1:100 dilution) sc-7455 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). 3,4,3',4', Tetra-aminodiphenilhydrochloride (diaminobenzidine) was obtained from BDH Chemicals (Poole, UK). Streptavidin-biotin peroxidase complex kits were obtained from Dako (Carpinteria, CA). Other reagents were obtained at the highest grade available from commercial sources or from Sigma.

Isolation of RNA and cDNA synthesis
Total RNA was extracted from cells using RNAase mini kit from Qiagen (Valencia, CA) according to the instructions of the manufacturer. RNA concentration and quality were measured by spectrophotometric analysis at 260 and 280 nm. RNA integrity was assessed by electrophoresis in agarose gel. Total RNA (400 ng) was reverse transcribed to cDNA in a final volume of 80 µl using TaqMan Reverse Transcription kit (Applied Biosystems, Foster City, CA) at the following conditions: 10 min at 25 C, 30 min at 48 C, and 5 min at 95 C.

Real-time quantitative RT-PCR
The mRNA quantitative analysis was performed according to the fluorescent TaqMan methodology, as published previously (36). PCR primers and probe for mRNA quantitation of ET-1 receptors (ETA and ETB) were purchased as an assay-on-demand (AOD) gene expression product from Applied Biosystems. The glyceraldehyde-3-phosphate dehydrogenase gene was chosen as the reference gene, and the corresponding AOD product was provided by Applied Biosystems. The PCR mixture (25 µl final volume) consisted of 1x final concentration of AOD mix, 1x final concentration of Universal PCR Master mix (Applied Biosystems), and 25 ng cDNA. Amplification and detection were performed with an ABI Prism 7700 Sequence Detection System (version 1.7) (Applied Biosystems) with the following thermal cycler conditions: 2 min at 50 C, 10 min at 95 C, and 40 cycles at 95 C for 30 sec and 60 C for 1 min. Each measurement was performed in duplicate. mRNA quantitation was based on the comparative C_T method, according to the instructions of the manufacturer, in which C_T represents the cycle number at which the fluorescent signal, associated with an exponential increase in PCR products, crossed a given threshold. The maximum change in C_T values of the sample (C_{T sample}) was determined by subtracting the average of duplicate C_T values of the reference gene from the average of the duplicate C_T values of the target gene. Human umbilical vascular endothelial cells (HUVEC) (37), classically considered positive targets for ETB ligands, together with the SH-SY5Y cell line, were used as controls.

Measurement of intracellular calcium concentration
Digital video imaging of the intracellular-free calcium concentration ([Ca²⁺]_i) in individual human FNC-B4 cells was performed as described previously (27). Human neurons were grown to subconfluence in complete culture medium on round glass coverslips (25-mm diameter, 0.2-mm thick) for 72 h and then incubated for 48 h in serum-free medium (SFM) at 37 C. Cells were then loaded with 10 µmol/l fura 2-AM and 15% Pluronic F-127 for 30 min at 22 C. [Ca²⁺]_i was measured in fura 2-loaded cells in HEPES-NaHCO₃ buffer containing the following (in mmol/l): 140 NaCl, 3 KCl, 0.5 NaH₂PO₄, 12 NaHCO₃, 1.2 MgCl₂, 1.0 CaCl₂, 10 HEPES, and 10 glucose (pH 7.4). Ratio images (340/380 nm) were collected every 3 sec, and calibration curves were obtained for each cell preparation. ET-1, ETA, and ETB receptor antagonists (A-BQ123 and B-BQ788), GnRH and/or its analog (buserelin), and GnRH antagonist (cetrorelix) (from 0.1 µM to 1 nM) were directly added to the perfusion chamber immediately after recording the [Ca²⁺]_i basal value. In parallel experiments, cells were preincubated with 0.1 µM ETB receptor antagonist for 10 min before addition of ET-1. These experiments were done in triplicate.

Immunocytochemical procedures and confocal laser microscopy
FNC-B4 cells were cultured on glass coverslips in SFM for 18 h and then left untreated or incubated with ET-1. Cells were then fixed with 3.7% paraformaldehyde (pH 7.4) for 10 min and then permeabilized for 10 min with PBS (Ca²⁺/Mg²⁺-free), containing 0.1% Triton X-100. Immunostaining was performed as described previously (12, 23), using polyclonal antibodies to ETA and ETB receptor (1:50), followed by streptavidin-biotin peroxidase complex (LSAB kit; Dako). The specificity of the anti-ETA and ETB receptor antibodies was controlled by preabsorption of the primary antibodies with a membrane preparation from human fetal penile smooth muscle cells (hfPSMC), particularly enriched in the respective receptors, as detected by binding experiments (38), according to a previously described method (39). Briefly, ETA and ETB receptor antibodies (working dilution) were incubated with 1 mg/ml hfPSMC membranes in 50 mM Tris HCl buffer, or with buffer only, overnight at 4 C. After an additional 60-min incubation with 4% polyethylene glycol, the unbound antibodies were separated by rapid centrifugation and used for immunohistochemistry. Filamentous actin (F-actin) was stained with rhodamine phalloidin (1:50; Molecular Probes, Eugene, OR) in the permeabilization solution for 45 min at room temperature. Cells were viewed with a laser scanner confocal microscope (MRC 600; Bio-Rad, Hercules, CA), equipped with a Nikon (Tokyo, Japan) diaphot inverted microscope or with a Nikon Microphot-FX microscope. The percentages shown in Figure 3 concerning various morphologies of actin-based cell deformations (i.e. percentage of cells showing at least two of the following morphological features: actin patches, filopodia, lamellipodia, and membrane ruffles) were obtained by counting the number of activated cells over the total number of stained cells from three different experiments (at least three slides for each experiment and five fields from each slide).

View larger version (92K): [in this window] [in a new window]   FIG. 3. Effects of ETB receptor activation in FNC-B4 neurons on F-actin. FNC-B4 cells were cultured on glass coverslips in SFM for 24 h and then left untreated (CTL) or incubated for 18 h with ET-1 (10–7 M). F-actin, after fixation, was visualized with rhodamine phalloidin (1:50) in the permeabilization solution for 45 min at room temperature. Cells were viewed with a laser scanner confocal microscope. A typical patter of expression of F-actin in unstimulated FNC-B4 cells is shown (CTL). ET-1 (100 nM) exerted striking activation of cell morphology, together with actin stress fiber network, toward a spindle-like shape, with actin-based cell deformation and the presence of actin patches (thin arrow), together with the appearance of lamellipodia (arrowhead) and filopodia (thick arrow) on the edge of cell membranes (ET-1). Simultaneous incubation with ET-1 (100 nM) and BQ123 (100 nM) induced similar actin-based cytoskeletal modifications than ET-1 alone (ET-1+BQ123) (see arrows); vice versa, coincubation of ET-1 (100 nM) and BQ788 (100 nM) did not induce morphological changes different from controls (ET-1+BQ788). Scale bar, 0.15 µm.

SDS-PAGE, Western blot analysis, and immunoblotting
FNC-B4 cells, grown to confluence, were scraped into PBS (Ca²⁺/Mg²⁺-free), centrifuged, and resuspended in lysis buffer [20 mM Tris-HCl, 150 mM NaCl, 0.25% Nonidet P-40, 1 mM Na₃VO₄, 1 mM phenylmethylsulfonylfluoride, 1 mM EGTA (pH 7.4)]. Protein concentration was measured using a Coomassie Bio-Rad protein assay kit. Aliquots containing 30 µg of proteins were diluted in 2x reducing Laemmli’s sample buffer [62.5 mM Tris (pH 6.8), 10% glycerol, 20% SDS, 2.5% pyronin, and 100 mM dithiothreitol] and loaded onto 10% SDS-PAGE. After SDS-PAGE, proteins were transferred to nitrocellulose membranes (Immobilon-P transfer membranes, polyvinylidene difluoride; Millipore). Membranes were blocked overnight at 4 C in 5% BSA-TTBS buffer (0.1% Tween 20, 20 mM Tris-HCl, and 150 mM NaCl), washed in TTBS, and incubated for 2 h with anti-actin primary antibody (1:1000 dilution), followed by peroxidase-conjugated secondary IgG (1:3000). Finally, the reacted proteins were revealed by enhanced chemiluminescence system (ECL; Roche Diagnostics, Basel, Switzerland). For reprobing with different antibodies, the nitrocellulose membranes were washed for 30 min at 50 C in stripping buffer [10 mM Tris (pH 6.8), 2% SDS, 100 mM b-mercaptoethanol] and reprobed with antigoat NSE antibody (1:100 dilution; sc-7455; Santa Cruz Biotechnology).

Chemotactic assay
Cell migration was performed as described previously (27). Briefly, modified Boyden chambers (Nuclepore, Pleasanton, CA), each equipped with a 13-mm diameter and a 5- or 8-µm porosity polyvinylpyrrolidone-free polycarbonate filter were used. The filters were coated with 20 µg/ml human type I collagen or 10 µg/ml fibronectin (Collaborative Biomedical Products, Bedford, MA) for 30 min at 37 C. Confluent FNC-B4 cells, GN11 cells, and SH-SY5Y cells were incubated in SFM for 24 h (control conditions). After mild trypsinization with 0.05% trypsin-EDTA, in 210-µl aliquots, each of which corresponding to 4 x 10⁴ cells/Boyden chamber, the cells were added to the top wells and incubated at 37 C in 5% CO₂ for 6 h. Increasing concentrations of ET-1, or IRL-1620, in the presence or absence of ETA and/or ETB receptor antagonists (A-BQ123 or B-BQ788; 10^–7 M concentration), and GnRH or buserelin, in the presence or absence of cetrorelix (10^–7 M concentration), were added to the bottom chambers. Both ET-1 and GnRH were incubated in the presence of cetrorelix or A-BQ123/B-BQ788, respectively. After incubation, the migrated cells were fixed in 96% methanol and stained with Diff Quick solution (Biomap, Milan, Italy) or Harris’ hematoxylin solution. Chemotaxis was quantitated by randomly counting six chosen fields per filter, and results were expressed as the number of cells per high-power field (HPF).

Statistical analysis
Data are expressed as the mean ± SD. An one-way ANOVA followed by post hoc test (Bonferroni’s correction for multiple comparisons) was performed. The level of P < 0.05 was accepted as statistically significant. Comparisons of percentages were analyzed statistically after conversion through arc-sine transformation from the binomial to the normal distribution. The computer program ALLFIT was used for the analysis of the sigmoidal dose-response curves (40).

	Results

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ETB receptor expression in human FNC-B4 cells
Figure 1A (top) shows ETA and ETB mRNA expression (quantitative RT-PCR) in neuronal compared with endothelial cells, taken as positive controls. Both GnRH-secreting neurons and neuroblastoma cells express, respectively, a 4- and 15-fold higher concentration of ETB transcripts than HUVEC, classically considered positive targets for ETB ligands (37). Western blot analysis with polyclonal anti-ETA and anti-ETB antibodies (Fig. 1A, bottom) shows specific bands for both receptors, at the expected molecular weight, in both FNC-B4 or SH-SY5Y cells. The intracellular immunodistribution of ETA and ETB in FNC-B4 cells is shown in Fig. 1B. Note the positivity for both receptors in the perinuclear cytoplasmic compartment, with a fading localization within the tiny cytoplasmic protrusions (Fig. 1B).

View larger version (50K): [in this window] [in a new window]   FIG. 1. A, Quantitative gene expression of ET-1 receptors in FNC-B4 cells and SH-SY5Y. The top shows the quantitative detection of mRNA (RT-PCR) for ETA and ETB receptors in FNC-B4 and SH-SY5Y neural cells. Expression of the same genes in HUVEC was also shown for comparison. ETB transcripts were definitively higher in GnRH-secreting neurons and neuroblastoma cells than in endothelial cells, used as positive control. Data (mean ± SD; n = 3) are expressed in arbitrary units, calculated according to the comparative CT method and normalized to the glyceraldehyde-3-phosphate dehydrogenase, chosen as reference gene. The bottom shows the expression of ETA and ETB proteins in the same neuronal cells, as determined by Western blot analysis. Specific bands, of the expected sizes, were detected using rabbit polyclonal antibodies against ETA (left) and ETB (right) receptors. B, Immunohistochemistry for ET-1 receptors in FNC-B4 neurons. The intracellular localization of ETA (panel a) and ETB (panel b) receptors in FNC-B4 was revealed by the same antibodies as in A (bottom). Note the positivity for ETA and ETB receptors in the perinuclear cytoplasmic compartment, with a fading localization within the tiny cytoplasmic protrusions (see arrows). Positive staining for both ETA (panel c) and ETB (panel d) receptors disappeared after preabsorbing the two antibodies with a membrane preparation from hfPSMC, enriched in the respective receptors. Scale bar, 0.3 µm.

View larger version (24K): [in this window] [in a new window]   FIG. 2. Measurement of intracellular calcium concentration. Digital video imaging of [Ca2+]i in individual human FNC-B4 cells. ET-1, BQ123 and BQ788, GnRH or buserelin, and cetrorelix were added directly to the perfusion chamber immediately after recording the [Ca2+]i basal value. In parallel experiments, cells were preincubated with 0.1 µM ETB receptor antagonist for 10 min before the addition of ET-1 (black trace). BQ123 was able to almost blunt (70 ± 2%) [Ca2+]i transients (white circles), whereas BQ788 abolished only 30 ± 3% of cases of ET-1-induced [Ca2+]i increases (black circles). Contemporary exposure to both antagonists blunted the effects of ET-1 (gray triangles).

View larger version (25K): [in this window] [in a new window]   FIG. 4. Effects of ETB receptor activation on actin expression in FNC-B4 neurons. FNC-B4 cells were cultured in SFM for 24 h, incubated for 18 h with ET-1 (10–7 M), and then subjected to Western blot analysis of actin (A). A single protein band, migrating at the expected molecular weight, is indicated by the arrowhead. ET-1 stimulated actin expression (*, P < 0.05 vs. control). This enhanced expression was reverted by BQ788 but not by BQ123 (*, P < 0.05 vs. control). Quantification of actin increase is reported in B (percentage of increase in actin expression over the control value, taken as 100%). Experiments were done in triplicate. *, P = 0.05. Nitrocellulose membranes were stripped and reprobed with anti-NSE to assess equal protein loading (C). No modification was observed in NSE expression among lanes (data not shown). CTL, Control.

View larger version (44K): [in this window] [in a new window]   FIG. 5. Biological effects of ETB receptor activation in FNC-B4 neurons. A, Cell migration of FNC-B4 cells toward ET-1 was performed according to Materials and Methods. Increasing concentrations of ET-1, with BQ123 or BQ788 (10–7 M), were added to the bottom chambers. Effect of IRL-1620 is also shown. FNC-B4 cells responded to SFM conditions, showing spontaneous random migration [7 ± 1.4 cells per HPF after 6 h of incubation; n = 30]. ET-1 induced a 4- to 6-fold increase in migratory pattern in FNC-B4 cells, in a dose-dependent manner (from 10–9 to 10–7 M; *, P < 0.0005 vs. control conditions, SFM). IRL-1620 (10–7 M) induced a similar increase to an equimolar concentration of ET-1 (*, P < 0.0005 vs. control). BQ788 counteracted ET-1 effects, whereas BQ123 did not reduce ET-1-induced migration (*, P < 0.0005; n = 12). B, A representative pattern of migrating cells on Boyden chamber filters, under different experimental conditions, is shown. Scale bar, 0.03 µm.

View larger version (16K): [in this window] [in a new window]   FIG. 6. Migration of GN11 or SH-SY5Y cells toward ET-1. Effect of ET-1 (10–8 and 10–7 M), with or without BQ123 or BQ788 (10–7 M), on cell migration of GN11 (A) or SH-SY5Y (B), performed according to Materials and Methods. ET-1 at all the concentration tested induced in both GN11 cells and in the human neuroblastoma SH-SY5Y cells a significant (*, P < 0.0005) increase in migratory pattern. ET-1 (10–7 M)-induced cell migration was completely counteracted by an equimolar concentration of BQ788 but not of BQ123 (*, P < 0.0005 vs. control).

View larger version (14K): [in this window] [in a new window]   FIG. 7. Crosstalk of ET-1 and GnRH peptides in FNC-B4 neurons. Effects of GnRH on ET-1 production in FNC-B4 neurons. RIA of ET-1 in conditioned media from FNC-B4 cells. In the presence of increasing concentrations of buserelin (from 10–12 to 10–6 M), a dose-dependent 9-fold increase in ET-1 secretion (EC50 of 0.95 ± 0.17 nM; n = 3) was observed.

View larger version (36K): [in this window] [in a new window]   FIG. 8. Effects of ET-1 and GnRH agonists and antagonists on GnRH release from FNC-B4 cells. ET-1 (10–7 M) and buserelin (10–7 M) induced 3-fold and 4-fold increase, respectively, in GnRH secretion (both *, P < 0.0005 vs. control). ET-1 stimulation on GnRH release was unaffected by BQ788 (10–7 M; *, P < 0.0005 vs. control) and cetrorelix (10–7 M; *, P < 0.0005 vs. control) but almost completely blunted by BQ123 (10–7 M). Buserelin stimulation was unaffected by both ET-1 antagonists (both *, P < 0.0005 vs. control) but completely abrogated by cetrorelix. Columns indicate the percentage of increase in GnRH secretion over the control value (100%), as measured by RIA. Experiments were done in triplicate. CTL, Control.

View larger version (18K): [in this window] [in a new window]   FIG. 9. Effects of ET-1 and GnRH agonists and antagonists on FNC-B4 migration. ET-1 or GnRH (10–7 M), with or without BQ123, BQ788, or cetrorelix (10–7 M), were added to the bottom chambers. ET-1 and GnRH elicited a sustained increase in migration (both *, P < 0.0005 vs. control). ET-1 effect was completely blocked by BQ788 but not by BQ123 and cetrorelix (both *, P < 0.0005 vs. control). GnRH-induced migration was completely counteracted by cetrorelix but not by the two ET-1 antagonists (both *, P < 0.0005 vs. control). Experiments were performed in triplicate. CTL, Control.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Previous researches has demonstrated that FNC-B4 cells express ET-1 and respond to ET-1 through a GnRH release, which, in turn, was able to induce FNC-B4 cells to migrate (12, 27). Here, we demonstrate that GnRH stimulated ET-1 release. Because ET-1 stimulated motility and GnRH release, we investigated whether these GnRH-related effects may be, at least partially, mediated by a GnRH-induced activation of ET-1 signaling. Our findings suggest a crosstalk between these two neuropeptides. In fact, functional data, by using selective antagonists, indicate that FNC-B4 cells were independently and selectively stimulated by the two different peptides. Thus, ET-1, through ETA/ETB and GnRH receptor crosstalk, may participate in differentiation of the neuroendocrine phenotype of FNC-B4 olfactory neuroblasts.

In the olfactory epithelium, proliferation of neural precursor cells and differentiation of their progeny into olfactory receptor neurons begin during embryogenesis and continue throughout life (43, 44). The ET-3/ETB pathway has the potential to differentiate neural crest cells to multipotent precursors (45, 46, 47). Thus, exposure of FNC-B4 neuroblasts to ET-1 may lead to different stages of differentiation of functional activation. The first one is characterized by an immature and undifferentiated stage, with a huge proliferative commitment, and the second one, more differentiated, is characterized by morphological and cytoskeletal activation, the acquisition of clear chemotactic properties, and the recruitment of both subtypes of ET and GnRH receptors. These receptors, together with functional recruitment of both GnRH and ET-1 molecules, are able to crosstalk and interact with each other (the so-called "loop activity between ET and GnRH"). We suggest that the onset of this activity loop plays a role underlying the development of the specialized neuroendocrine cells as they migrate through nasal regions.

Maggi et al. (12) have shown that, during early embryonic life, ET-1 gene and protein, as well as endothelin-converting enzyme-1, are present in cells in the olfactory epithelium. From these data, together with our in vitro data, we speculate that, in the in vivo situation, ET-1 interacts with growth and differentiating factors not only to maintain a reservoir of GnRH-secreting neural cells but also to stimulate cell migration and a fully activated phenotype. Therefore, ET-1 could play a role in both guiding GnRH-secreting human neurons during their migration and orchestrating their subsequent differentiation.

During early embryogenesis, brain areas that undergo rapid neurogenic development and migration of cells through a differentiating matrix require very high energy for nutrients and the disposal of waste products. These requirements are best met by a rich blood supply. These needs constitute a possible trigger for the development of an elaborate "cellulo-vascular bridge" from the placode epithelium across the nasal septum and into the ventromedial forebrain (48). A close association between these angiogenic mechanisms and migration of GnRH neurons has been reported in embryonic mice, in early stage human embryos, and in those fetuses affected by Kallmann’s syndrome (48). Angiogenesis is an important early event not only in normal development but also in tumor progression, beginning in premalignant lesions (49). The endothelin system represents one of the most studied growth factor families that has been involved in modulating angiogenesis (50). Moreover, a close association between the loss of ETB receptor mRNA expression and the onset of a metastatic phenotype of neuroblastoma tumors, strictly associated with the disease progression, has been described previously (51). The blunted migration observed through specific ETB receptor antagonism in a human-derived neuroblastoma cell line suggested the possible role of ETB receptor activation in neoplastic differentiation. We thus hypothesize that ETB receptor signaling may be required in in vivo conditions to maintain a mature neuronal differentiation. Additional studies are needed to clarify whether the loss of ETB receptor expression during development can represent one of the underlying molecular mechanisms for both the hypogonadotropic hypogonadism as well as the onset of olfactory-derived tumors.

	Acknowledgments

 
We thank Prof. Vieri Boddi for his helpful supervision in statistical analysis.

	Footnotes

 
This work was supported by grants from the Ministero dell’Istruzione, dell’Università e della Ricerca, Telethon (number E523), and the University of Florence.

First Published Online June 30, 2005

Abbreviations: AOD, Assay-on-demand; [Ca²⁺]_i, intracellular calcium; ET-1, endothelin-1; ET-3, endothelin-3; ETA, endothelin type A; ETB, endothelin type B; F-actin, filamentous actin; hfPSMC, human penile smooth muscle cells; HPF, high-power field; HUVEC, human umbilical vascular endothelial cells; ns, not significant; NSE, neuron-specific enolase; SFM, serum-free medium.

Received January 14, 2005.

Accepted for publication June 15, 2005.

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