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Original Studies |
Departments of Anatomy Histology and Forensic Medicine (G.F., G.B.V.) and Clinical Physiopathology, Endocrinology (R.M., C.P., C.C., M.S.), and Andrology Units (M.M., M.L.), University of Florence, 50134 Firenze; and Department of Experimental and Clinical Medicine (T.B.), University of Catanzaro, 88100 Catanzaro, Italy
Address correspondence and requests for reprints to: Prof. G. B. Vannelli, M.D., Department Anatomy Histology and Forensic Medicine, Viale Morgagni, 85, 50134 Firenze, Italy. E-mail: vannelli{at}unifi.it
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Introduction |
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We have recently established, cloned, and propagated in vitro primary cell cultures from human fetal olfactory epithelium (4, 5). These neuroblasts exhibit in vitro both olfactory and neuroendocrine properties. In fact, one of these clones (FNC-B4) besides the expression of neuronal markers and olfactory-associated genes is also able to produce and release GnRH, under both odorant and sex steroids control (6). Because peptides of the endothelin (ET) family are well known regulators of GnRH neuron activity, the aim of the present study was to investigate whether olfactory-derived GnRH-secreting neurons produce and respond to ETs. Therefore, we first studied the expression of ET-1 gene and protein in the human fetal olfactory mucosa and then in the GnRH-secreting cell line FNC-B4. The biological effect of ET-1 in this neuronal cell line was also evaluated, and the subtype of receptors involved was characterized. We found that human olfactory neuroblasts, as well as normal fetal mucosa, express ET-1 and that this peptide regulates either GnRH secretion or cell proliferation, depending on which subtype of ET receptors is activated.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human fetal olfactory epithelium specimens were obtained from seven 8- to 12-week-old fetuses after spontaneous or therapeutic abortion and fixed in 4% paraformaldehyde. Legal abortions were performed in authorized hospitals, and certificates of consent were obtained. The study protocols were approved by the university ethical committee.
The isolation, cloning, and characterization of the FNC-B4 cells from primary olfactory human neuroblasts were described previously (4). These cells grow as a monolayer, are nontumorogenic, and have a normal human karyotype. FNC-B4 cells were cultured in Coon’s modified F12 medium supplemented with 10% FCS at 37 C in 5% CO2 atmosphere.
Chemicals
GnRH (2200 Ci/mmol) and
[
-35S-thio]UTP (1300 mCi/mmol) were obtained
from NEN Life Science Products (Milan, Italy).
[125I]ET-1 (2000 Ci/mmol),
[125I]ET-3 (2000 Ci/mmol), and
[32P]CTP (3000 Ci/mmol) were purchased from
Amersham Pharmacia Biotech (Amity PG, Milan, Italy). A
GnRH RIA kit was obtained from Buhlmann Laboratories AG (Allschwil,
Switzerland). Unlabeled ET-1, ET-3, and the ETA-selective antagonist
BQ123 were obtained from NovaBiochem (Laufelfingen, Switzerland). The
ETB-selective agonist IRL 1620 and antagonist BQ788 were purchased from
Alexis (Laufelfingen, Switzerland). The polyclonal antibodies to ET-1
(RAS 6901) and ET-3 (RAS 6911) were purchased from Peninsula Laboratories, Inc. (San Carlos, CA). The polyclonal antibody to
GnRH and synthetic GnRH were obtained from INCSTAR Corp.
(Stillwater, MN). The rabbit polyclonal antibody to ECE-1 was kindly
provided by M. Yanagisawa (Howard Hughes Medical Institute, University
of Texas Southwestern Medical Center, Dallas, TX).
3,4,3',4',tetra-aminodiphenilhydrochloride (Diaminobenzidine) was
obtained from BDH Chemical Ltd. (Poole, UK). Universal immunoperoxidase
staining kits were obtained from Vector Laboratories, Inc.
(Burlingame, CA).
Measurement of intracellular calcium concentration
For intracellular calcium measurements, cells were grown on plastic coverslips (Aclar; Allied Engineering Plastic, Pottsville, PA). During the 24 h before the experiments, cells were maintained in serum-free medium. [Ca2+]i was determined using the calcium-sensitive dye Fura-2/AM as described previously (7). Briefly, cells were loaded with 4 µM Fura-2/AM for 45 min at 37 C, washed and incubated in Fura-2-free medium for another 20 min, and finally resuspended in Krebs-Henseleit HEPES-KHH buffer [1.25 mM CaCl2, 5.36 mM KCl, 0.81 mM MgS04, 130.62 mM NaCl, 5.55 mM glucose, 8.60 mM HEPES sodium salt, 11.7 mM HEPES free acid, and 0.1% BSA (pH 7.4)]. Fluorescence was measured by a spectrofluorimeter (LS50B; Perkin-Elmer Corp., Milan, Italy) using a single-wavelength excitation:emission/340:510 nm. Calibration was performed using ionomycin (8 µM) to obtain Fmax, followed by EGTA (10 mM, pH 10) to obtain Fmin. Fluorescence measurements were converted to [Ca2+]i, assuming a dissociation constant of Fura-2 for calcium of 224 nM.
In situ hybridization
Prepro-ET-1 and ECE-1 messenger RNAs (mRNAs) were detected by in situ hybridization, as described previously (8, 9). FNC-B4 cells and frozen sections (7 µm) from fetal olfactory mucosa were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) and dehydrated in increasing ethanol concentrations. 35S-labeled human prepro-ET-1 and ECE-1 RNA sense and antisense probes were synthesized from specific DNAs using appropriate RNA polymerases (8, 9). Hybridization, removal of nonspecifically bound probes, and autoradiography were performed as described elsewhere (8, 9). Slides were analyzed using a Nikon MICROPHOT FX microscope (Nikon, Tokyo, Japan). Negative controls consisted of: 1) hybridization of sections to relative sense RNA probes; 2) pretreatment of sections with RNase A (20 µg/mL); and 3) addition of 100-fold excess of the relative unlabeled antisense RNA probe to the hybridization mixture containing the antisense 35S-labeled probe.
Immunohistochemistry
Immunohistochemical studies were performed, as described previously (8, 9), on deparaffinized and rehydrated sections or cultured cells fixed in 3.7% paraformaldehyde for 15 min. Primary antibodies, appropriately diluted in PBS were added to the slides and incubated overnight at 4 C. For primary antibodies the following working dilutions were used: 1:1500 for the polyclonal antiserum to ET-1 and 1:2500 for that one to ECE-1. Sections were then incubated with biotinylated secondary antibodies and finally with streptavidin-biotin peroxidase complex (LSAB kit; DAKO Corp. Carpinteria, CA). The development reaction of the product was performed using diaminobenzidine tetrahydrochloride liquid as chromogen. Controls were performed by processing slides lacking the primary antibodies or staining with the corresponding nonimmune serum or preincubating the primary antibodies with the corresponding antigens (ET-1, 100 nM; ECE-1 synthetic 16-amino acid peptide, 1 mg/mL).
The slides were evaluated and photographed using a Nikon MICROPHOT-FX microscope (Nikon).
GnRH and ETs RIA
Immunoreactive GnRH was extracted from conditioned media of FNC-B4 cells with chilled absolute ethanol (-20 C), evaporated to dryness, and subjected to RIA using a commercial kit (Buhlmann Laboratories AG, Allschwil, Switzerland), as described previously (6). Immunoreactive ETs were extracted from conditioned media of FNC-B4 cells using Sep-Pak C18 cartridges (Waters; Millipore Corp., Milford, MA), as described previously (10). The specific RIAs for ET-1 and ET-3 were performed in 0.1 M PBS (pH 7.4) (0.1% triton-X, 0.1% BSA, and 0.01% NaN3) by a two-step incubation procedure. Samples and standards (0.1 mL) were incubated at 4 C overnight with their respective antisera (ET-1: RAS6901, 1:20000; ET-3: RAS6911, 1:40000, 0.1 mL) and further incubated with their respective tracers (0.1 mL, 10 pM) at 4 C overnight. Bound/free separation was performed by a second antibody/PEG procedure. We used two distinct RIAs: an ET-1 RIA (antibody RAS 6901 and [125I]ET-1) and an ET-3 RIA (antibody RAS 6911 and [125I]ET-3). As described previously (10), the first RIA recognizes with equal affinity ET-1 and Big-ET-1, but not ET-3, whereas the second one recognizes ET-3 and ET-1, but shows lower affinity for Big-ET-1.
High-performance liquid chromatography (HPLC)
Reverse-phase HPLC was performed on a BIO-SIL column (250 x 4 mm; Bio-Rad Laboratories, Inc. Richmond, CA), as described previously (10). After extraction, samples or standards were injected and eluted with a linear gradient of acetonitrile in 0.1% trifluoroacetic acid with a flow rate of 1 mL/min. The acetonitrile gradient was from 30–90% for 45 min for ET-1 and from 10–70% for 40 min for GnRH. Fractions (1 mL) were collected, evaporated, and subjected to specific RIAs. The reproducibility of the elution pattern of each HPLC run was verified by adding about 3000 cpm labeled ligands.
Binding studies
Binding studies were performed as described previously (7). Confluent FNC-B4 cells were washed once with 20 mM HEPES, 10 mM MgSO4, 0.5% BSA (pH 7.4), and incubated in 200 µL of the same binding medium at 22 C for 60 min with fixed concentrations (15–50 pM) of [125I]ET-1 or [125I]ET-3 in the presence or absence of increasing concentrations of unlabeled ET-1 or ET-3 (10-11–10-7 M). After incubation, cells were extensively washed with ice-cold PBS, 0.1% BSA, solubilized in 0.5 N NaOH, and the cell-bound radioactivity was determined. Measurements were obtained in triplicate. Cell counts routinely varied less than 10% between wells.
DNA synthesis
DNA synthesis was measured as the amount of [methyl-3H]thymidine ([3H]TdR) incorporated into trichloroacetic acid-precipitable material. Cells were plated in 24-well dishes at a density of 2 x 104 cells/well in complete culture medium containing 10% FCS. Confluent cells (approximately 1 x 105 cells/well) were made quiescent by incubation in serum-free medium for 48 h. The cells were then incubated with vehicle or increasing concentrations of IRL1620 for 20 h. Experiments were also performed incubating FNC-B4 cells with fixed (100 nM) concentration of ET analogs (ET-1, ET-3, IRL1620, ET-1+BQ-123). Thereafter, FNC-B4 cells were pulsed for an additional 4 h with 1.0 µCi/mL of [3H]TdR (6.7 Ci/mmol) (New England Nuclear, Boston, MA). At the end of the pulsing period, [3H]TdR incorporation into cellular DNA was determined with a ß-counter.
In-gel kinase assay
Mitogen-activated protein kinase (MAPK) activity was evaluated
as the ability of kinases present in total cell lysates to
phosphorylate myelin basic protein (MBP; 0.5 mg/ml) copolymerized in
SDS-polyacrylamide gels, as described previously (11). Cells grown on
6-well plates, washed, and resuspended in serum-free medium were
stimulated with ET-1 or ET-3 (0.1 µM) and BQ123 or BQ788
(1 µM) for 5 min. At the end of incubation, cells were
scraped in cold PBS-1 mM
Na3VO4, centrifuged, and
the obtained pellets resuspended in lysis buffer [20 mM
Tris (pH 7.4), 150 mM NaCl, 0.25% Nonidet P40, 1
mM Na3VO4, and
1 mM PMSF]. Aliquots containing 10 µg of proteins
evaluated by Bio-Rad Laboratories, Inc. (Hercules, CA)
protein assay reagent were boiled in 2x Laemmli’s sample buffer,
separated on a 10% SDS-polyacrylamide gel containing 0.5 mg/mL MBP.
Gel was washed first with 20% 2-propanol in buffer A [50
mM Hepes (pH 7.4) and 5 mM
2-ßmercaptoethanol] for 1 h, then denatured with 6
M guanidine-HCl in buffer A for 1 h, and finally
renatured in buffer A-0.04% Tween 20 for 16 h at 4 C. After a 1-h
preincubation in buffer B [25 mM Hepes (pH 7.4), 10
mM MgCl2, 100 mM
Na3VO4, 5 mM
2-ßmercaptoethanol, and 0.5 mM EGTA], the kinase assay
was carried out by incubating the gel at 25 C for 2 h in 5 mL
buffer B containing 40 µM ATP and 50 µCi of
[
-32P]ATP. The gel was washed with 5%
trichloroacetic acid-1% sodium pyrophosphate, dried, and subjected to
autoradiography.
Statistical analysis
Data are expressed as the mean ± SE. The significance of the difference was estimated by Student’s t test, with a level of P < 0.05 accepted as statistically significant. The computer program ALLFIT (12) was used for the analysis of sigmoidal dose response curves. The binding data were evaluated quantitatively with nonlinear least square curve fitting using the computer program LIGAND (13).
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Results |
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shows the expression of ET-1
gene (A) and protein (E) in the olfactory mucosa from an 11-week-old
human fetus. With both in situ hybridization and
immunohistochemistry we found an intense positivity for ET-1 in the
epithelial cells lining the nasal cavity and in the endothelial cells
of the surrounding blood vessels. Fig. 1B shows results obtained with
the antisense [35S]-labeled RNA probe for
ECE-1, the converting enzyme involved in the synthesis of the
biological active form of ET-1. The immunolocalization of ECE-1 protein
is reported in Fig. 1F. Note that silver grains and immunostaining for
ECE-1 are present in the same epithelial and endothelial cells
expressing ET-1. The same figure (C and D) shows results with sense
[35S]-labeled RNA probes for human prepro-ET-1
and ECE-1, as control. Only a weak background signal was observed.
Control sections for ET-1 and ECE-1 proteins were virtually unstained
(data not shown). These results essentially indicate that olfactory
cells of the nasal cavity express all the genes necessary for ET-1
synthesis and do contain the immunoreactive protein.
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, A and B, respectively). A control
section using the sense probe for ET-1 is also shown in Fig. 2C.
Similar results were obtained with the sense probe for ECE-1 (data not
shown). An intense immunoreactivity for ET-1 and ECE-1 was observed in
the cytoplasm of FNC-B4 cells (Fig. 2, D and E, respectively),
indicating that the mature protein is actively synthesized. A control
section for ET-1-like immunoreactivity is shown in Fig. 2F. To verify
whether FNC-B4 cells were able to secrete authentic ET-1 in the spent
medium, we subjected a 24-h conditioned medium from FNC-B4 cells to
combined HPLC and RIAs. A typical elution profile of FNC-B4
cell-conditioned media is reported in Fig. 3. With the ET-1 RIA we found two
distinct peaks of immunoreactivity: the first peak eluted with Big ET-1
standard and the second one with ET-1 standard. With the ET-3 RIA we
found just a single peak, corresponding to ET-1.
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). One site binds
with virtual equal affinity ET-1 (Kd = 93.1
± 43 pM) and ET-3 (Kd = 308 ±
148 pM) and, therefore, corresponds to the ET unselective
receptor ETB, whereas the other site binds with higher affinity ET-1
(Kd = 285.8 ± 122 pM) than ET-3
(Kd = 150.4 ± 60 nM) and,
therefore, corresponds to the ETA receptor. The density of the ETA
receptor (Bmax = 16,420 ± 4,760 sites/cell)
was 2-fold higher than the density of the ETB receptor
(Bmax = 8,710 ± 2,003 sites/cell). Because
ET-1 mobilizes intracellular calcium concentration
([Ca2+]i) in
GnRH-secreting immortalized hypothalamic neurons (14), we tested the
effect of increasing concentration of ET-1 and specific receptor
antagonists in FNC-B4 cells. Fig. 5 shows
a representative tracing of
[Ca2+]i waveforms evoked
by ET-1 (top). We found that nanomolar concentration of ET-1
induced a dramatic increase in
[Ca2+]i. The stimulatory
effect of ET-1 (1 nM) was completely abolished by
the ETA antagonist BQ123 (100 nM), but was not
affected by the ETB antagonist BQ788 (100 nM;
Fig. 5, bottom). These findings indicate that the ETA
subtype is involved in the ET-1-induced
[Ca2+]i mobilization in
FNC-B4 cells. The effect of increasing concentrations of ET-1 and ET-3
on GnRH secretion is showed in Fig. 6. A
24-h incubation with ET-1 and ET-3 was able to stimulate GnRH secretion
from FNC-B4 cells, although with different EC50s.
Indeed, mathematical analysis using the program ALLFIT indicates that
the EC50 for ET-1 was 5.8
nM (n = 7), whereas the
EC50 for ET-3 was 205 nM
(n = 4). The difference between the EC50s
was statistically significant (P < 0.005). In three
independent experiments, the stimulatory effect of 100
nM ET-1 on GnRH secretion (64.6 ± 11.6%
over the control value), was significantly blunted by the simultaneous
incubation with an equimolar concentration of the selective ETA
antagonist BQ-123 (5.2 ± 6% over the control value,
P < 0.05). These findings indicate that ETA receptors
are not only involved in
[Ca2+]i mobilization, but
also in the ET-1-induced GnRH secretion in FNC-B4 cells. We then
investigated whether ETA and ETB receptors were involved in the control
of FNC-B4 proliferation (Fig. 7, top). We found that incubation with 100
nM ET-1 or 100 nM ET-3
increased DNA synthesis to a similar extent [54.6 ± 5.3% over
the basal level (n = 9) and 77.3 ± 14% (n = 15),
respectively] and that the ET-1 stimulation was not counteracted by
100 nM BQ-123 (41.17 ± 7%, n = 9). In
addition, 100 nM IRL-1620, a selective ETB
agonist, also increased thymidine incorporation (78 ± 14%,
n = 15; Fig. 7). This effect was dose dependent, with an
EC50 = 0.12 nM (Fig. 7, bottom). Taken together, these results strongly indicate
that ETB and not ETA receptors mediate the mitogenic effects of ETs on
FNC-B4 cells. Because in most proliferating cells the mitogenic
signaling is mediated by an activation of MAPKs, also called
extracellular signal-regulated kinases, we studied the effect of ET-1
on p44ERK1 activity. As shown by in-gel kinase
assay (Fig. 8), both ET-1 (100
nM) and ET-3 (100 nM)
induced a rapid stimulation of p44ERK1 activity
(5-min treatment). Again, this effect seems to be mediated by
interaction with ETB but not ETA receptors as ET-1 activation of the
enzyme is blunted to unstimulated control level when ET-1 is
administrated in the presence of BQ788, but not by BQ123. Hence, we
found two distinct functions for ET receptors in FNC-B4 cells: ETA
regulates [Ca2+]i
mobilization and GnRH secretion, whereas ETB regulates
p44ERK1 activity and DNA synthesis.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In conclusion, our study extends to a human model previous observations in animal models on a dual role of ET receptors in neuronal cells: 1) regulating neurosecretion (ETA); and 2) stimulating proliferation (ETB). In addition, the present results provide the first evidence that in humans ET-1 does not deserve just a peripheral role (7, 8, 10, 15, 16) but also a central role in controlling reproductive functions, through the regulation of GnRH-secreting neurons. These findings shed new light on the possibility to use selective ET agonists to target and modulate the activity of GnRH-producing cells.
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Footnotes |
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Received October 13, 1999.
Revised December 29, 1999.
Accepted January 7, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The arrows indicate the elution position of
ET standards.
