From the Department of Pharmacology and the Bruno
Ceccarelli Center, University of Milan, the Consiglio Nazionale delle
Ricerche Center of Molecular and Cellular Pharmacology, and DIBIT,
Department of Neuroscience, San Raffaele Institute, 20132 Milan, Italy,
the ** Department of Genetics and Microbiology, University of Pavia,
27100 Pavia, Italy, the
National Cancer
Institute, 20129 Milan, Italy, the
Department of Biology,
University of Bologna, 40126 Bologna, Italy, and the
¶¶ Department of Pharmacology, School of Pharmacy,
University of Calabria, 87036 Arcavacata di Rende, Italy
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ABSTRACT |
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SK-N-BE neuroblastoma cell clones transfected
with p75NTR and lacking Trk neurotrophin receptors,
previously reported to undergo extensive spontaneous apoptosis and
to be protected by nerve growth factor (NGF) (Bunone, G., Mariotti, A.,
Compagni, A., Morandi, E., and Della Valle, G. (1997)
Oncogene 14, 1463-1470), are shown to exhibit (i)
increased levels of the pro-apoptotic lipid metabolite ceramide and
(ii) high activity of caspases, the proteases of the cell death
cascade. In the p75NTR-expressing cells, these parameters
were partially normalized by prolonged NGF treatment, which, in
addition, decreased apoptosis, similar to caspase blockers. Conversely,
exogenous ceramide increased caspase activity and apoptosis in both
wild-type and p75NTR-expressing cells. A new
p75NTR-expressing clone characterized by low spontaneous
apoptosis exhibited high endogenous ceramide and low caspase levels. A
marked difference between the apoptotic and resistant clones concerned
the very low and high activities of nitric-oxide (NO) synthase,
respectively. Protection from apoptosis by NO was confirmed by
results with the NO donor S-nitrosoacetylpenicillamine and
the NO-trapping agent hemoglobin. We conclude that the
p75NTR receptor, while free of NGF, triggers a cascade
leading to apoptosis; the cascade includes generation of ceramide
and increased caspase activity; and the protective role of NO occurs at
step(s) in between the latter events.
Apoptosis (programmed cell death) is now recognized to play
fundamental roles in both the physiology and pathology of the brain. On
the one hand, apoptosis is instrumental in the selection of neurons,
working coordinately with neuroprotection (1, 2); on the other hand, it
plays central roles in the development of specific diseases (3).
Understanding of factors and mechanisms governing the apoptotic program
of nerve cells represents therefore a key issue in modern biomedical sciences.
Among the neuronal pro-apoptotic factors, considerable attention is
presently focused on NGF1 and
its receptors. In classical studies, this neurotrophin has been shown
to afford protection to various families of neurons, working through
its high affinity receptor, TrkA. As far as the low affinity NGF
receptor, p75NTR, initial studies suggested its role to be
cooperative to TrkA (see, for example, Refs. 4 and 5). Recently,
however, homologies have been noticed between the transduction domain
of p75NTR and those of well known death receptors,
i.e. the tumor necrosis factor- Compared with the other death receptors, p75NTR appears
unique inasmuch as only in some cases, apoptosis was shown to develop following NGF binding, whereas in other cases, it occurred in the
absence of the specific ligand (spontaneous apoptosis). The signaling events triggered in the first condition are beginning to be
elucidated (11-14), whereas those of the second remain undefined. Whatever their nature, these events appear to operate within the cell
under the control of various modulators, among which is the short-lived
messenger NO (15). Based on results in a number of cell types, from
macrophages to neurons, NO was initially suggested to work as a
stimulator of apoptosis, whereas recently, it has been shown to play a
protective role (16-19).
In this work, the signaling events responsible for
p75NTR-induced apoptosis and their regulation by NO were
investigated in a panel of transfected SK-N-BE human neuroblastoma
clones, some of which had already been shown to undergo spontaneous
apoptosis (20). The results demonstrate (i) that both ceramide and
caspases play crucial roles in the death signaling cascade induced by
p75NTR and (ii) that NO protects against the
apoptotic program, working downstream of ceramide generation and
upstream of the operative caspase cascade.
Materials--
The rabbit polyclonal antibody specific for human
p75NTR (9651) was kindly provided by Dr. M. Chao. The
rabbit polyclonal antibody specific for human caspase-1 (pan-ICE) and
the mouse monoclonal antibody specific for human caspase-3 (clone
10C1.C9) were purchased from Oncogene Research Products (Cambridge,
United Kingdom). The mouse monoclonal antibody specific for human Bcl-2
(clone 100) was from Ancell Corp. (Bayport, MN). The rabbit polyclonal
antibody recognizing TrkA, TrkB, and TrkC (C14) was from Santa Cruz
Biotechnology (Santa Cruz, CA). Culture sera and media were from Life
Technology Inc. (Basel, Switzerland), Hygromycin, human recombinant
NGF, and fluorescein isothiocyanate-annexin V were from Roche Molecular Biochemicals (Mannheim, Germany). C2-ceramide from BIOMOL
(Bremen, Germany). The caspase fluorogenic substrate Ac-DEVD-AMC and
inhibitors Ac-YVAD-CMK, DEVD-CHO, and Z-DEVD-FMK, SNAP, and
L-NAME were from Calbiochem (San Diego, CA). Silica Gel 60 was from Merck (Milan, Italy). Bicinchoninic acid was from Pierce.
L-[3H]Arginine and the enhanced
chemiluminescence kit were from Amersham International
(Buckinghamshire, United Kingdom). Hemoglobin, 8-Br-cGMP, propidium
iodide, Nonidet P-40, and the remaining chemicals were from Sigma (Milan).
Clone Generation and Cell Culture--
The various
p75NTR-expressing clones were developed as described (20).
In brief, after transfection with a pC4SLR vector containing the coding
sequence of the human p75NTR gene and the hygromycin
resistance gene, selection was carried out in a medium supplemented
with 300 µg/ml hygromycin, and colonies were isolated 4 weeks after
transfection. The level of p75NTR expression in the clones
and its localization in the plasma membrane were established by Western
blotting as well as by immunofluorescence. Compared with the classical
model of neurotrophin action, i.e. PC12 cells, they were of
the order of 280% in the BEp75B, BEp75H, and BEp75AR clones (see also
Bunone et al. (20)), whereas the BEp75Hy
Growth of wild-type SK-N-BE and clone cells was carried out as
described (20). Briefly, the cells were maintained at 37 °C in
100-mm Petri dishes and bathed in RPMI 1640 medium supplemented with 2 mM glutamine, 15% fetal calf serum, and 50 µg/ml
gentamycin under a 5% CO2 atmosphere. Hygromycin-resistant
clones, including the BEp75AR clone, were grown in complete medium
supplemented with the antibiotic (150 µg/ml).
Apoptosis: Induction and Protection--
Apoptosis of both
parental and p75NTR-overexpressing clone cells was induced
by overnight administration of exogenous C2-ceramide (50 µM) at 37 °C. Inhibition of apoptosis was induced
in p75NTR-overexpressing clone cells by preincubation with
the caspase inhibitors Ac-YVAD-CMK, Z-DEVD-FMK and DEVD-CHO (100 µM) for 24 or 48 h at 37 °C. NGF protection
against p75NTR-induced apoptosis was studied by
preincubating the p75NTR-overexpressing clone cells for 24 or 48 h with NGF (6 nM). To study the role of NO, the
wild-type SK-N-BE cells were preincubated either with the NO donor SNAP
(50, 100, and 300 µM) in the presence or absence of the
NO scavenger hemoglobin (100 µM) or with the membrane-permeable cGMP analogue 8-Br-cGMP (500 µM) for
15 min at 37 °C before the overnight C2-ceramide
treatment. The cells of the p75NTR-overexpressing clones
that undergo spontaneous apoptosis were incubated for a total of
48 h in the presence of SNAP (300 µM), with changes
of the medium occurring every 12 h.
Apoptosis Detection--
Apoptotic cells were identified by flow
cytometry following two different protocols (21, 22). The first is
based on the identification of a typical sub-G1 peak in
single parameter DNA histograms. DNA was stained in unfixed cells
incubated for 60 min at 37 °C in sodium citrate (0.1%), propidium
iodide (50 mg/ml), RNase A (100 µg/ml), and Nonidet P-40 (0.01%). In
the second method, useful for evaluating the fraction of cells
undergoing apoptosis, phosphatidylserine exposure was monitored by
staining for 15 min at room temperature with fluorescein
isothiocyanate-annexin V (0.5 µg/ml) in the presence of divalent
cations. The loss of the ability to exclude vital dyes was assessed
using propidium iodide in an isotonic buffer (10 µg/ml in PBS). Early
apoptotic cells exclude the dye, but are stained with annexin V,
whereas non-apoptotic cells are negative for both. Cells were then
analyzed either for DNA content or for annexin V staining using a
fluorescence-activated cell sorter (FACStar Plus, Becton Dickinson,
Sunnyvale, CA). Quantification of apoptotic cells was also assessed by
monitoring internucleosomal fragmentation of genomic DNA using an
immunoassay kit based on the recognition of released nucleosomes by
mouse monoclonal antibodies directed against DNA and histones (Cell
Death Detection Elisa Plus kit, Roche Molecular Biochemicals).
Ceramide Measurements--
Ceramide levels were assayed as
described (16). Samples of 1 × 106 cells were
processed for lipid extraction; dried under nitrogen; resuspended in a
mixture containing cardiolipin (5 mM),
diethylenetriaminepentaacetic acid (1 mM), and octyl
Electrophoresis and Western Blot Analysis--
Wild-type SK-N-BE
cells and clones stably transfected with p75NTR were
cultured, harvested using a Pasteur pipette, washed in PBS, and lysed
in cell lysis buffer (150 mM NaCl, 15 mM
MgCl2, 1 mM EGTA, 50 mM Hepes-KOH,
10% glycerol, and 1% Triton X-100, pH 7.5) supplemented with a
protease inhibitor mixture (0.2 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, and 1 µg/ml
o-phenanthroline). After 30 min at 4 °C on a slowly
rotating device, the cell lysates were centrifuged (for 3 min at 5000 rpm in an Eppendorf microcentrifuge), and the supernatants were assayed
for protein content by the bicinchoninic acid procedure.
For electrophoresis and Western blot analyses, protein suspensions were
solubilized (10:7 ratio) in SDS sample buffer (48 mM
Tris-HCl, pH 6.8, 0.8 M sucrose, 4% SDS, 8%
Caspase Activity Measurements--
Samples of 1 × 106 cells were rinsed in cold PBS and lysed in a buffer
containing 25 mM Hepes, pH 7.5, 5 mM EDTA, 1 mM EGTA, 5 mM MgCl2, 5 mM dithiothreitol, 1% CHAPS, 10 µg/ml each pepstatin and
leupeptin, and 1 mM phenylmethylsulfonyl fluoride. The cell lysates were centrifuged (for 3 min at 5000 rpm in an Eppendorf microcentrifuge), and the supernatants were stored at
Lysates (25 µg of protein) were incubated at 37 °C in a buffer
containing 25 mM Hepes, pH 7.5, 10% sucrose, 0.1% CHAPS,
and 1 mM dithiothreitol supplemented with Ac-DEVD-AMC (50 µM). The increase in fluorescence following the cleavage
of the fluorogenic AMC moiety was monitored and then quantified in a
Perkin-Elmer LS50 fluorometer (excitation, 380 nm; and emission, 460 nm). For quantitation, standard curves using increasing concentrations of AMC moiety were performed in parallel.
NO Synthase Activity Assay--
Neuronal NO synthase activity
was quantified by measuring the conversion of
L-[3H]arginine to
L-[3H]citrulline (24). Cells (maintained for
48 h in medium without L-arginine) were harvested and
washed with cold PBS, and the pellet was resuspended in homogenization
buffer containing 20 mM Hepes, pH 7.2, supplemented with
320 mM sucrose, 1 mM dithiothreitol, 1 mM EDTA, and a mixture of protease inhibitors (0.2 mM phenylmethylsulfonyl fluoride, 2 mM
benzamidine, and 10 µg/ml o-phenanthroline). The samples
were immediately processed by adding 25 µl of cell extract (300-400
µg of protein) to 90 µl of reaction buffer (25 mM Tris, pH 7.4, supplemented with 3 µM tetrahydrobiopterin, 1 µM FAD, 1 µM FMN, 100 nM
calmodulin, 1 mM NADPH, and 50 µM unlabeled
L-arginine) containing
L-[3H]arginine (0.5 µCi/sample) in the
presence of 10 µl of 6 mM CaCl2. The reaction
was carried out for 10 min at 37 °C and then stopped by addition of
2 ml of 50 mM Hepes, pH 5.5, supplemented with 5 mM EDTA. The solution was then applied to Dowex 50-X8-400
columns converted to the basic form. [3H]Citrulline not
retained by the ion-exchange resin was immediately eluted and
quantified in a Beckman p75NTR Signaling and Cell Death in
p75NTR-overexpressing Cell Clones--
Receptor-induced
apoptosis develops through a series of intracellular events. In
addition to (or concomitantly with) the establishment of the
death-inducing signaling complex by the recruitment to the plasma
membrane receptors of specific cytosolic proteins (see Ref. 7),
activation of lipid-specific enzyme(s) occurs, with ensuing generation
of metabolites that appear to play a role in the development of the
program. In particular, this is the case of ceramide (25-27), a
sphingosine metabolite known to induce apoptosis even when administered
to the cells as a synthetic, membrane-permeable analogue. Fig.
1 demonstrates the sensitivity of SK-N-BE
cells to one of these analogues, C2-ceramide. The compound
was administered in a range of concentrations (1-50 µM)
(Fig. 1B), including the range of the endogenous metabolite
estimated in our cell clones under resting conditions (5-12
µM) (see also Fig. 2). At all concentrations tested, the
lipid metabolite was found to be distinctly more efficacious in the
clones stably transfected with p75NTR cDNA than in the
wild-type parental neuroblastoma cells (Fig. 1, A and
B). Such an increased effect occurred in at least rough proportion to receptor expression: moderate in the cells of the BEp75Hy
Subsequent experiments were carried out to investigate whether the role
of ceramide is only pharmacological or is connected with the regulation
of p75NTR-induced apoptosis. Fig.
2A suggests that this may
indeed be the case. In fact, compared with wild-type cells, the resting
levels of endogenous ceramide were higher in the
p75NTR-expressing clones: 50% in the Hy
The dependence of ceramide levels and caspase activity on
p75NTR was further investigated in the transfected clones
by treatment with a natural ligand of the receptor, NGF. For both
parameters, this neurotrophin (6 nM) induced significant
decreases, which, however, required long periods (tenths of hours) to
develop (see Fig. 3 for clone B). Since
the employed cells express neither TrkA nor any other Trk receptor, as
revealed by Western blots (Fig.
4B) (20), the protective
effect of NGF can only be attributed to the expressed
p75NTR.
An Apoptosis-resistant p75NTR-transfected Clone--
A
new clone transfected with the p75NTR cDNA (BEp75AR,
indicated below as clone AR) differs profoundly from those described so far (20). The level of p75NTR expression was high in clone
AR, comparable to that in clones B and H (Fig. 4A), and Trk
receptors were not detectable (Fig. 4B). Yet the AR clone
tendency to apoptosis was low, not only under resting conditions, but
also after ceramide administration (Fig. 4C, left
and middle panels). In contrast, clone AR was as susceptible
as the other clones to treatments known to induce apoptosis by
non-receptor mechanisms, such as exposure to the Ca2+
ionophore ionomycin (5 µM) (Fig. 4C,
right panel; and data not shown). In terms of endogenous
ceramide, clone AR resembled the p75NTR-overexpressing
clones, B and H, with high resting levels (~11.5 µM),
which decreased with time following exposure to NGF (Fig. 4D).
A number of mechanisms that could account for the resistance to
apoptosis of the AR clone were investigated. The levels of the
apoptosis controller oncogene product, Bcl-2, and of caspases were
similar to those found in the other clones (Western blotting) (data not
shown). Yet when caspase activity was assayed by the use of a
fluorescent substrate, clone AR exhibited very low levels, thus
resembling not the other p75NTR-overexpressing clones, but
the wild-type parental cells (Fig. 5).
Moreover, the caspase activity remained unchanged when clone AR was
exposed to exogenous ceramide, a treatment that, even in wild-type
cells, induces large increases, exceeding with time the levels observed
in the unstimulated p75NTR-overexpressing clones (Fig. 5).
From these data, we conclude that, in the AR clone, the tendency to
apoptosis induced by p75NTR overexpression is balanced
specifically by one (or more) additional event(s) by which the death
signaling cascade is blocked upstream of caspase activation.
Role of NO--
A series of experiments was carried out to explore
the possibility that NO has a role in the regulation of apoptosis in
SK-N-BE clones. NO synthase activity was found to vary profoundly in
the various clones. Compared with controls, it was clearly increased in
the resistant AR clone and greatly reduced in the sensitive clones,
especially clones H and B (Fig. 6). In
general terms, therefore, this variability correlates inversely to the
sensitivity to exogenous ceramide. Exposure to the blocker
L-NAME induced large drops in the NO synthase activity,
which were parallel in all the preparations investigated (Fig. 6).
Additional evidence for a role of NO was obtained by experiments in
which spontaneous apoptosis was significantly reduced in the
p75NTR-overexpressing B and H clones by pretreatment with
the NO donor SNAP as revealed by various tests, including fluorescein
isothiocyanate-annexin V staining and nucleosome accumulation (Table
II). The same donor, administered to
wild-type cells, was able to prevent (in a dose-dependent fashion) the apoptosis induced by exogenous ceramide (50 µM) (Fig. 7A)
and to inhibit the activity of caspases (Fig. 7B). Such a protection was due to NO generation from the donor, and not to other
effects of the compound, because it faded out completely when the
messenger was trapped by addition of hemoglobin (100 µM)
to the cell incubation mixture (Fig. 7A). Likewise, the high caspase resting levels were reduced in the
p75NTR-overexpressing cells by the NO donor (Fig.
7C), which, in contrast, failed to modify the high levels of
endogenous ceramide (data not shown). As far as the mechanisms by which
the protective effects of NO are induced, experiments were carried out
in which both wild-type and clone B cells were treated with the
membrane-permeable cGMP analogue 8-Br-cGMP (0.5 mM) before
exogenous ceramide administration or under resting conditions. Since
this treatment did not affect the caspase activity (Fig. 7,
B and C) or apoptosis (data not shown), we
conclude that one of the classical mediators of NO action, the
activation of guanylate cyclase, is most likely not involved in its
anti-apoptotic effect.
Interest about p75NTR has been greatly stimulated by
the conflicting results reported about its function. In cells in which
its expression occurs together with that of the other NGF receptor, the
tyrosine kinase TrkA, p75NTR has been shown to contribute
to the trophic effects induced by ligand binding, playing either a
tuning (4, 5, 28, 29) or a true synergistic (30) role. Recently,
however, p75NTR has been recognized as a death receptor,
belonging to the same molecular family as the tumor necrosis factor- To reconcile these results, a detailed understanding of the
p75NTR transduction system would be needed. Activation of
other death receptors, such as the tumor necrosis factor- New and profoundly different results were obtained with clone AR, the
p75NTR-overexpressing SK-N-BE clone that diverges from the
others because of its resistance to apoptosis. Resting clone AR cells
are characterized by high ceramide levels and by caspase expression
levels comparable to those of the other clones. The signaling
difference of clone AR emerged when its low caspase activity failed to
rise and its cells failed to enter apoptosis when exposed to exogenous
ceramide. Apparently, therefore, the resistance of clone AR is due to a transduction blockade localized between p75NTR signaling
and ceramide generation on the one hand and caspase activation on the
other. Such a blockade appears specific for the receptor-initiated
pathway since, when other inducing treatments were employed, no
difference in apoptosis development was observed between clone AR and
the other clones. Of the cell properties that could be possibly
responsible for clone AR resistance, some (expression of Trk receptors
and increased levels of Bcl-2) could be excluded. A difference detected
in clone AR with respect to the other overexpressing clones and also
the wild-type SK-N-BE cells was, in contrast, its high NO synthase
activity. Thus, NO could have a part in the lack of caspase activation
and apoptosis in clone AR cells; whether other, so far unidentified
protective factor(s) are also involved remains to be established.
In previous studies, the conclusions about the role of NO in nerve cell
apoptosis had been conflicting. In many cases, the messenger was
reported to induce (38-40) and in others to protect from (41, 42)
programmed cell death. In our work, the protective role of NO was
documented by (i) the inverse correlation between NO synthase activity
and apoptosis (both spontaneous and induced by ceramide) and (ii) the
effects of an NO donor (SNAP) that was able to prevent the ceramide
activation of caspases and induced marked decreases in programmed death
rates in both the wild-type cells and p75NTR-overexpressing
B and H clones. The latter effects were specific because they
disappeared when the cells were incubated in the presence of the NO
scavenger hemoglobin. Thus, endogenous NO appears to be among the
controllers modulating the efficacy of the p75NTR-triggered
death signal. This finding is of importance for at least two reasons.
(i) It shows that the protective role of endogenously generated NO
against receptor-triggered apoptosis is not confined to individual
receptors and cell types (CD95 in T lymphocytes (16, 17) and the tumor
necrosis factor- In conclusion, the panel of stably transfected SK-N-BE neuroblastoma
clones lacking Trk have shown to be interesting models for the study of
the signaling and physiological role of the p75NTR
receptor. The higher efficacy of the latter in inducing apoptosis in
the absence rather than in the presence of its ligand might account for
the neurotrophic role attributed in previous studies (30) to the NGF
activation of p75NTR working synergistically with TrkA. In
other cell types, however, classical apoptotic responses have been
triggered following ligand activation of p75NTR (12, 13),
as expected for a bona fide death receptor. These apparently
conflicting results call attention to the mechanisms that regulate
intracellularly the development of the apoptotic program. In this study
on p75NTR-expressing SK-N-BE clones, the event that seems
to play a major role is the generation of NO. In fact, high levels of
the gaseous messenger inhibit the activation of caspases by a mechanism
that appears to be cGMP-independent. Therefore, in the case of
p75NTR-induced apoptosis, at least two levels of control
appear to be functioning: at the receptor, with its alternative
free/bound signaling; and within the cytoplasm, where multiple signals,
including those triggered by NO, appear to participate in the
regulation of the life-or-death decision (11).
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptor and CD95 (Fas,
APO-1) (see Ref. 6). The latter are known to induce apoptosis via
complex signal cascades including the generation of a lipid messenger,
ceramide, and the proteolytic activation of cysteine proteases, the
caspases (7, 8). At the moment, a pro-apoptotic role of
p75NTR is supported by results in a variety of systems,
including cultured cells as well as knockout and transgenic mice
(9-11).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
clone
expressed ~16% of protein in comparison with overexpressing clones.
The resistant BEp75AR clone was obtained from one of the clones
overexpressing the transfected receptor by isolating a rapidly growing
colony that exhibited no sign of spontaneous apoptosis from a
preparation kept in the selection medium.
-glucopyranoside (7.5%); and labeled using the diacylglycerol
kinase assay. Ceramide phosphate was isolated by thin-layer
chromatography (Silica Gel 60) using
CHCl3/CH3OH/CH3COOH (65:15:5,
v/v/v) as solvent and quantified by densitometric analysis of the
radioactive spots. Authentic ceramide 1-phosphate was identified by
autoradiography at RF = 0.25.
-mercaptoethanol, and 0.008% bromphenol blue) and heated on a
boiling water bath for 5 min. The solubilized proteins were loaded and
separated on Laemmli-type SDS-polyacrylamide gels run as described
(23). Polyacrylamide concentrations were typically 4% in the stacking gel and either 7 or 15% in the running gel for Trk or
p75NTR, Bcl-2, and caspase-1 and -3, respectively. The
separated proteins were electrotransferred in a cold room onto a 0.2-mm
pore nitrocellulose membrane at a constant 70 mA for 16 h using a
buffer containing 25 mM Tris, 192 mM glycine,
and 20% methanol, pH 8.3. After brief staining with 0.2% (w/v)
Ponceau Red in 3% trichloroacetic acid to reveal the standard
molecular mass markers, the blots were blocked with 5% (w/v) low-fat
dried milk in PBS supplemented with 0.01% (v/v) Tween 20 (PBS/Tween)
and then exposed for 1 h at room temperature to the primary
antibody. Blots were washed in 2.5% (w/v) milk in PBS/Tween (5 × 10 min). Immunostaining was then developed by the enhanced
chemiluminescence kit, and the relevant bands were revealed were
quantitated using a Molecular Dynamics Imagequant apparatus.
80 °C until used. Protein content was assayed by the bicinchoninic acid procedure.
-counter after addition of scintillation
mixture. Samples containing either L-NAME (500 µM) or EDTA (2 mM) were processed in parallel
as controls.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
clone (Hy
clone) and much
stronger in those of the BEp75B clone (B clone), where expression is
~7-fold higher.
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Fig. 1.
Spontaneous and ceramide-induced apoptosis in
wild-type SK-N-BE cells and clones expressing different levels of the
p75NTR receptor. A, apoptosis in wild-type
cells (panel a) and two transfected clones with different
levels of p75NTR expression (low levels in clone
Hy (panel b) and high levels in clone B
(panel c)) is shown before (insets) and after
overnight exposure to exogenous ceramide (50 µM).
Apoptotic cells were revealed by FACS analysis. The results shown are
representative of three to five experiments. In this and the following
figures showing FACS analyses, the apoptotic cell peak is indicated by
an arrowhead. B, concentration dependence of
apoptosis induced in the indicated SK-N-BE cells by exogenous ceramide,
administered as described for A. The arbitrary units on the
ordinate refer to absorbance measured at 405 nm.
wt, wild-type.
clone and >100% in the B clone and in the other
p75NTR-overexpressing clone employed, BEp75H (H clone).
Parallel to the results with endogenous ceramide were those with the
cysteine proteases known to participate in the apoptotic operative
cascade, i.e. the caspases (7, 8). The activity of the
latter enzymes, already conspicuous compared with wild-type cells in
the low expression clone, Hy
, reached values 50-85-fold
higher in the overexpressing clones, B and H (Fig. 2B).
Likewise, DNA laddering (a classical sign of apoptosis) was high
(~10-fold of the wild-type value) in clone B, and this value was
significantly decreased when cells were pretreated with
membrane-permeable caspase inhibitors (Ac-YVAD-CMK, Z-DEVD-FMK, and
DEVD-CHO (100 µM), administered either alone or in
combination) (Table I and data not
shown). In contrast, these peptides had no effect on the cell
endogenous ceramide levels (Fig. 2A).
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Fig. 2.
Endogenous ceramide levels
(A) and caspase activity (B) in
wild-type SK-N-BE neuroblastoma cells and in various clones expressing
different levels of the p75NTR receptor. A,
the levels of endogenous ceramide were assayed in the wild-type cells
and in three transfected clones in the absence (stippled
bars) and presence (black bars) of the caspase
inhibitors Ac-YVAD-CMK and Z-DEVD-FMK (100 and 50 µM) as
described under "Experimental Procedures." The results shown
(means ± S.D.) are representative of three consistent
experiments. B, total cell lysates of the wild-type cells
and of various p75NTR-transfected SK-N-BE clones were
incubated at 37 °C with a 50 µM concentration of the
fluorogenic substrate Ac-DEVD-AMC. The fluorescence increase following
the cleavage of the AMC residue was monitored for 10 min at 37 °C.
Data shown (means ± S.D.) are from the results of five
experiments.
Effect of caspase inhibitors and NGF treatment on apoptosis of
clone B cells
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Fig. 3.
Effects of NGF treatment on ceramide levels
(A) and caspase activity (B) in
p75NTR-overexpressing clone B. Clone B cells were
incubated for the indicated time periods in cell culture medium
supplemented with 6 nM NGF. At the beginning of the
incubation as well as 24 and 48 h later, the cells were collected,
washed, and processed for either ceramide assay (A) or
caspase activity (B). The results (means ± S.D. of
five experiments run in duplicate) are expressed as described in the
legend to Fig. 2. In A, the bottom of the columns are the
values in wild-type cells.
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Fig. 4.
Characterization of the
p75NTR-overexpressing clone resistant to spontaneous and
exogenous ceramide-induced apoptosis (AR clone). A,
Western blot analysis of p75NTR expression in wild-type
cells and p75NTR-expressing clones carried out using an
antibody specific for the transfected receptor (lane a,
wild-type cells; lane b, clone B; lane c, clone
H; lane d, clone Hy ; lane e, clone
AR). The two bars on the left indicate the positions of the
two forms (glycosylated and non-glycosylated) of p75NTR (75 and 59 kDa). Thirty µg of protein were loaded on each lane. The
results shown are representative of three to five separate experiments.
B, Western blot analysis carried out with an antibody that
recognizes all the Trk receptors (lane a, PC12 cells,
employed as a positive control; lane b, wild-type SK-N-BE
cells; lane c, AR clone). The two bars on the
left indicate the positions of Trk receptor isoforms in PC12 cells (140 and 110 kDa). The results shown are representative of three to four
separate experiments. C, FACS analysis of the DNA in clone
AR cells while under resting conditions (left panel) and
after overnight treatment with either ceramide (50 µM;
middle panel) or ionomycin (5 µM; right
panel). Representation is as described for Fig. 1A.
Notice that the apoptotic cell peak is appreciable only in
ionomycin-treated cells (arrowhead). The results shown are
representative of three experiments. D, effect of
pretreatment with NGF (6 nM) added for the indicated times
on ceramide levels in clone AR. The values in clone B (see Fig.
2A) are also reported for reference. The results are
means ± S.D. of two experiments run in duplicate.
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Fig. 5.
Caspase activity assayed under resting
conditions and upon exogenous ceramide application in wild-type SK-N-BE
cells and in the various p75NTR-overexpressing clones.
Total cell lysates obtained from either the wild-type (wt)
cells or the various p75NTR-transfected SK-N-BE clones,
under resting conditions or after exogenous ceramide (50 µM) administration for different times (0, 2, 4, 6, and
16 h), were mixed at 37 °C with a 50 µM
concentration of the fluorogenic substrate Ac-DEVD-AMC, and the
fluorescence increase following the cleavage of the AMC residue was
monitored for 10 min in a spectrofluorometer. The results (means ± S.D.) are from three separate experiments run in duplicate.
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Fig. 6.
NO synthase activity in wild-type SK-N-BE
cells and in the various p75NTR-expressing clones. NO
synthase (NOS) activity was assayed in the total homogenates
of wild-type (wt) SK-N-BE cells and of the various
p75NTR-overexpressing clones, prepared both under resting
conditions and in the presence of the NO synthase inhibitor
L-NAME (500 µM), employed as an internal
control. The results shown (means ± S.D.) are from three separate
experiments run in triplicate.
Protective effect of the NO donor SNAP against apoptosis in the
p75NTR-overexpressing clones as revealed by two independent
procedures
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Fig. 7.
Inhibition by the NO donor SNAP of both
apoptosis (A) and caspase activity (B
and C) in wild-type SK-N-BE (A
and B) and clone B (C)
cells. A, wild-type SK-N-BE cells were preincubated for
15 min without (panels a and b) or with
(panels c and d) the NO donor SNAP (300 µM) in the presence (panel d) or absence
(panel c) of the NO scavenger hemoglobin, (100 µM) before receiving, in addition, exogenous ceramide (50 µM overnight) (panels b-d). Apoptotic cells
were revealed by DNA FACS analysis as specified in the legend to Fig.
1A. The results are representative of two consistent
experiments. B, wild-type SK-N-BE cells were preincubated
for 15 min either with the indicated concentrations of SNAP or with the
membrane-permeable cGMP analogue 8-Br-cGMP before receiving, in
addition, exogenous ceramide (50 µM, 4 h).
C, clone B cells were preincubated for 48 h (changes
every 12 h) with either SNAP or 8-Br cGMP at the indicated
concentrations before assaying the caspase activity. In both
B and C, caspase activity measurements were
performed on total cell homogenates by monitoring the cleavage of the
fluorogenic substrate Ac-DEVD-AMC. Values are shown as described in the
legends to Figs. 2B and 5. The results (means ± S.D.)
are from three separate experiments run in duplicate.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
receptor and CD95, and ample evidence of p75NTR-induced
apoptosis has been reported (10, 11). Whether the latter effect is
induced by the receptor when free or after binding of one of its
ligands, such as NGF, remains debated. In two studies in which the
binding mode was reported (in oligodendrocytes (Ref. 12; however, see
also Ref. 31) and in chick retina neurons (Ref. 13)), the pro-apoptotic
activation of p75NTR was shown to trigger the generation of
ceramide. This finding is of importance because the lipid derivative,
no matter whether generated endogenously or administered exogenously,
is known to play a key role in the induction of apoptosis (32, 33). On the other hand, spontaneous apoptosis taking place in
p75NTR-expressing cells in the absence of NGF was
demonstrated in immortalized neural cells and sensory neurons (34, 35)
as well as in the overexpressing SK-N-BE clones employed in this study
(20). The intracellular signaling events operative in these various
cases were not investigated.
receptor,
is known to trigger not a single, but a number of intracellular
cascades, only some of which lead to apoptosis, whereas others
ultimately induce cell protection and/or proliferation (32, 36, 37). The equilibrium among these various pathways appears to be regulated in
a cell type-specific fashion. A task of this work was to elucidate the
mechanisms by which p75NTR triggers and regulates its
spontaneous apoptotic response in SK-N-BE cells. The results we have
obtained emphasize the role of both ceramide and caspases. The high
levels of endogenous ceramide observed in the
p75NTR-overexpressing cells were shown to decline, although
slowly (24-48 h), after administration of NGF, in parallel with the
strong decrease in the probability of apoptosis (20). The possibility
that high ceramide levels increase the probability of cells to enter
the apoptotic program is confirmed also by their higher sensitivity to the exogenous lipid metabolite. Our ceramide results with NGF are
just the opposite of what was reported for oligodendrocytes (12),
suggesting that the p75NTR transmembrane signaling can
vary, depending on the cell type, and thus explaining the apparently
contradictory results of the literature. In the case of caspases, the
enzymes known to play key operative roles in the cell death program,
our present evidence strongly suggests their involvement in the
p75NTR signaling (7, 8). In the
p75NTR-overexpressing B and H clones, the caspase activity
was found to be very high already under resting conditions. Exposure to exogenous ceramide induced increases not only in the overexpressing cells, but also in the wild-type cells, correlating with the
development of apoptosis. Along the same line, a negative correlation
was found between apoptosis and the effects of caspase inhibitors.
receptor in endothelial cells (19)), but extends to
p75NTR in a nerve cell; and (ii) it provides clues about
the role of NO in the development and plasticity of the nervous system
(see, for example, Ref. 37). Evidence for the involvement of the
gaseous messenger in such processes was already available for the
visual (43, 44) and olfactory (45) systems as well as for peripheral sensory neurons (35, 46); however, the mechanisms involved remained
unclear. Based on the present results, it is conceivable that at least
part of the NO effects, in the above and possibly also in other parts
of the nervous system, are established via its involvement in the
regulation of death signaling programs, such as that activated by
p75NTR.
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FOOTNOTES |
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* This work was supported in part by grants from the Italian Association for Cancer Research; Special Project Apoptosis, University of Bologna; the Co-funding Program of the Italian Ministry of University and Research; the Armenise-Harvard Foundation; and Target Project Biotechnology, Consiglio Nazionale delle Ricerche. The support of Italian Schering Plough is gratefully acknowledged.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These two authors contributed equally to this work.
¶ Supported by a fellowship from INSERM.
§§ To whom correspondence should be addressed: DIBIT, Dept. of Neuroscience, San Raffaele Inst., Via Olgettina, 58, 20132 Milan, Italy. Tel.: 2-2643-2770; Fax: 2-2643-4813; E-mail: meldolesi.jacopo{at}hsr.it.
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ABBREVIATIONS |
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The abbreviations used are:
NGF, nerve growth
factor;
NTR, neurotrophin receptor;
NO, nitric oxide;
AMC, 7-amino-4-methylcoumarin;
CMK, chloromethyl ketone;
FMK, fluoromethyl
ketone;
Z-, benzyloxycarbonyl-;
SNAP, S-nitrosoacetylpenicillamine;
L-NAME, L-N-nitro-L-arginine
methyl ester;
PBS, phosphate-buffered saline;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
FACS, fluorescence-activated cell sorter.
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REFERENCES |
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