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Dipartimento di Medicina Sperimentale e Patologia, Università ‘La Sapienza’ Roma, Rome, Italy;
* Dipartimento di Medicina Sperimentale, Università di L’Aquila, Italy; and
Department of Neurosciences, University of California, San Diego, School of Medicine, Center for Molecular Genetics, La Jolla, California, USA
1Correspondence: Dipartimento di Medicina Sperimentale e Patologia. Università ‘La Sapienza’ Roma, Policlinico Umberto I, Viale Regina Elena 324, Rome, 00161 Italy. E-mail: roberta.misasi{at}uniroma1.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: signal-regulated protein kinase • BrdU • S-1-P • PC12 cells
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Saposin D stimulates acid ceramidase activity
primarily by interaction with the enzyme (2)
. The
metabolic significance of saposins was made obvious by the discovery of
their role in human lysosomal sphingolipid storage disorders
(3)
and with mutations in the prosaposin gene.
Prosaposin is located in the plasma membranes of neural cells; it
is secreted in body fluids (1
, 4
, 5)
and functions as a
neurotrophic factor (6
, 7)
. The neurotrophic factor
activity of prosaposin resides in a 12 amino acid residue stretch
localized in the NH2-terminal portion of the
saposin C domain (8)
. Several synthetic peptides of 14–22
amino acids (ProsaptidesTM) derived from this region are as bioactive
as prosaposin itself (8
, 9)
. ProsaptideTM treatment
prevented sensory loss in paclitaxel induced neuropathy in rats and
enhanced rat cerebellar granule neuron survival (10
, 11)
.
Prosaposin and ProsaptideTM treatment increased sulfatide
concentrations in Schwann cells and oligodendrocytes, and rescued
Schwann cells from cell death (12)
.
A putative G-protein-coupled receptor for prosaposin has been partially
purified and characterized (13)
. Prosaposin as well as
ProsaptideTM (22 mer) bound to PC12 pheochromocytoma cells and
activated extracellular signal-regulated protein kinases (ERKs)
(14)
. Both ERK-1 and ERK-2 (p44 MAPK, ERK-1; p42 MAPK,
ERK-2) are signaling factors that activate proliferation or
differentiation (15
, 16)
. Either proliferation or
differentiation may occur in PC12 cells after effector treatment. For
example, nerve growth factor binding receptor TrkA activates a program
of rapid cellular changes, followed by neuronal differentiation
(17)
. In contrast, epidermal growth factor (EGF) treatment
enhances cell proliferation and does not initiate neurite formation
(18)
. It has been suggested that, in neurons,
discrimination between prolonged and transient activation of the ERK
signaling pathway may be the cause of these opposite effects
(19)
. Others have suggested the mechanism might be more
complex (20
, 21)
.
Evidence has suggested that branching pathways of sphingolipid
metabolism may mediate either mitogenic or apoptotic effects
(22
23
24
25
26
27
28)
. Ceramide induced apoptosis in several cell lines
(24)
, whereas sphingosine and sphingosine 1-phosphate
(S-1-P) were mitogenic (25
, 26)
. Thus, the intracellular
ratio of ceramide to S-1-P may be a critical factor in determining the
fate of cells (29)
.
Prosaposin activated acid ceramidase (30)
; consequent
lipid derivative second messengers could affect the signaling
pathway(s). In this report we demonstrate that prosaposin treatment
induced G0/G1-arrested PC12 cells to enter the S phase of the cell
cycle and activated ERK phosphorylation; this treatment also prevented
apoptosis. We propose that prosaposin is an important regulator of the
death cascade, leading to sphingosine kinase activation and S-1-P
generation. This leads us to propose that such a mechanism may play a
key role in the regulation of S-1-P as an intracellular second
messenger important for cell proliferation and survival.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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.
Approximately1.0 x 106 cells per 25
cm2 tissue culture flasks were placed in
Dulbecco’s modified Eagle’s medium (DMEM; high glucose) containing
10% fetal calf serum (FCS), 5% horse serum (HS), 100 U/ml penicillin,
100 mg/ml streptomycin, and 250 pg/ml fungizone at 37°C, 6%
CO2, and grown to 60–70% confluency.
Cells were arrested in G0/G1 phase of the cell
cycle by serum starvation (32)
.
Proteins and antibodies
Milk prosaposin was prepared as described (1)
.
5-Bromo-2'-deoxyuridine (BrdU) was purchased from Sigma Chemical Co.
(St. Louis, Mo.).
A monoclonal antibody anti-BrdU was purchased from Calbiochem (La Jolla, Calif.). Fluorescein isothiocyanate (FITC) -conjugated rabbit anti-mouse immunoglobulin G (IgG) was purchased from DAKO (Dakopatts, Glostrup, Denmark).
Evaluation of DNA synthesis
Cells were grown to 60–70% confluency and then placed in
serum-free media. To arrest the cells in G0/G1
phase of the cell cycle (32)
, after serum starvation cells
were stimulated with either serum (10% FCS, 5% HS) or prosaposin (10
nM) for different times (0, 5, 10, 15, or 30 min or 3, 6, 18, or
24 h). In parallel experiments, cells were alternatively
preincubated (30 min) in the presence of 50 µM MEK inhibitor PD98059
(2'-amino-3'-methoxyflavone; Calbiochem) (33)
, pertussis
toxin (Recombinant holotoxin, 100 ng/ml; Calbiochem), or 50 µM
N-oleoyl-ethanolamine (Matreya Inc., Pleasant Gap, Pa.), a ceramidase
inhibitor (34)
. 5-Bromo-2'-deoxyuridine (120 µM) was
then added to the cell culture and incubated for 3 h at 37°C.
Monolayers were rinsed with phosphate-buffered saline (PBS), scraped,
resuspended in PBS, and pelleted by centrifugation. The cell pellet was
resuspended in 0.5 ml ice-cold PBS and fixed in ice-cold
acetone/methanol 1:5 for 1 h at 4°C. Cells were washed twice
with PBS, then with PBS containing Tween 20 (0.5%), followed by 2 ml
of 2N HCl (Merck, Darmstadt, Germany), and incubated for 45 min at
20°C. The cells were washed twice and the pellet resuspended in 1 ml
of Na-tetraborate 0.1M; 1 ml of PBS/Tween was then added and
centrifuged. Cells were resuspended in PBS/Tween containing 1% bovine
serum albumin (BSA) and incubated with anti-BrdU diluted 1:10 for 45
min at 20°C. After two washes in PBS/Tween, FITC-conjugated rabbit
anti-mouse IgG diluted 1:40 was added to the cells and incubated for 30
min at 4°C. Cells were then washed twice in PBS, resuspended in 1 ml
of PBS, and treated with 50 µl RNase (Sigma: 10 mg/ml in PBS). A
solution of propidium iodide (PI; Sigma) was added to give a final
concentration of 100 µg/ml. The cells were incubated for 15 min in
the dark at 20°C and analyzed by an EPICS profile cytometer (Coulter
Electronics, Hialeah, Fla.).
Confluent and quiescent cultures were incubated in DMEM with or without
10% FCS-5% HS with or without prosaposin (10 nM). After 48 h the
cells were removed from the dishes and counted (Cell Dyn 3500, Abbott
Diagnostic, Chicago, Ill.). A trypan blue exclusion test
(35)
was performed to evaluate the viability of the
cultures.
Evaluation of cell apoptosis
Subconfluent PC12 cell cultures, washed in serum free DMEM and
incubated in the presence or absence of 10 nM prosaposin for 30', were
treated with 1 µM 77 staurosporine or 20 µM C6 ceramide for 24 h at 37°C. In parallel experiments, cells were alternatively
preincubated for 30 min in the presence of either 50 µM MEK inhibitor
PD98059 (2'-amino-3'-methoxyflavone; Calbiochem) (33)
or
50 µM N-oleoyl-ethanolamine (Matreya Inc.), a ceramidase inhibitor
(34)
.
Apoptosis was measured by both morphological analysis and flow
cytometry. DNA fragmentation was studied by PI staining
(36)
, followed by flow cytometric analysis (EPICS Profile,
Coulter Electronics). Cells were fixed with cold 70% ethanol in PBS
for 1 h at 4°C. After centrifugation at 200 g for 10
min at 4°C, cells were washed once in PBS. The pellet was resuspended
in 0.5 ml PBS; 50 µl of RNase (Type I-A, Sigma: 10 mg/ml in PBS) was
added, followed by 1 ml PI (Sigma: 100 µg/ml in PBS) solution. The
cells were incubated in the dark at room temperature for 15 min and
kept at 4°C until measured. A trypan blue exclusion test was
performed to evaluate the viability of the cultures. The labeling of
DNA strand breaks via the TUNEL reaction was performed by a Peroxidase
ApopTag kit (ONCOR, Gaithersburg, Md.) (37)
. The staining
was performed following the manufacturer’s instructions and cells were
examined by an optical microscope. Morphological analysis was also
performed by labeling the cells with Hoechst 33258 (Sigma) (5 µg/ml)
and cells were examined in an inverted fluorescence microscope (320 nm
UV excitation). Viable cells were identified by their intact nuclei,
and fragmented or condensed nuclei were scored as apoptotic.
Sphingosine kinase assay
PC12 cells (2x107) were resuspended in
300 µl of homogenization buffer: 20 mM MOPS (Sigma) pH 7.2, 200 mM
sucrose, 10 mM EDTA (Sigma), 10 mM EGTA (Sigma), 10 mM
ß-mercaptoethanol (Sigma), 1 mM phenylmethylsulfonyl fluoride
(Sigma), 0.0125% leupeptin (Sigma), and 0.5 mM 4-deoxypyridoxine
(Sigma). Cells were then disrupted by freeze-thawing and centrifuged at
105,000 g for 60 min.
The protein concentration of supernatants was determined using Bio-Rad protein assay (Hercules, Calif.) with BSA as the standard.
PC12 cytosolic extracts (15–120 µg) were incubated in 254 µl
reaction mixture containing 100 mM MOPS, pH 7.2, 60 mM
MgCl2, 5% glycerol, 5 mM ß-mercaptoethanol, 51
mM ß-octyl-glucoside (Sigma), 50 µM D-erythro-sphingosine
(Sigma). D-Erythro-sphingosine was dried under a stream of nitrogen
from an ethanol solution and dissolved by sonication in buffer (255 mM
ß-octyl glucoside, 100 mM MOPS, pH 7.2, 5% glycerol, and 5 mM
ß-mercaptoethanol). The reaction was started by the addition of 10
µl of 5 mM 
-[32P]-ATP (Amersham, Bucks,
U.K.), added to give a specific activity of 100,000 cpm/nmol. Assay
tubes were incubated at room temperature for the times indicated and
the reaction was stopped by the addition of 1 ml of methanol/chloroform
(2:1, v:v) containing 5% triethylamine (Sigma). S-1-P was converted to
N-caproyl-sphingosine-phosphate by addition of 20 µl of caproic
anhydride (Sigma), followed by incubation for 30 min at room
temperature. Excess caproic anhydride was removed by addition of 1 ml
of 0.2N methanolic NaOH for 30 min at room temperature. After
incubation, lipids were extracted by addition of 330 µl of methanol,
1.66 ml of chloroform, 1 ml of a 1% perchloric acid solution, and 150
µl of 70% perchloric acid and the tubes were vortexed. After
centrifuging, the lower phase was washed twice with 2 ml of the 1%
perchloric acid solution.
The organic phases were dried under nitrogen, resuspended in chloroform for thin-layer chromatography analysis (TLC). Sphingosine was resolved using Silica gel 60 F254 plates (Merck, Darmstadt, Germany) and butanol:H2O:acetic acid (3:1:1, v:v:v) as a solvent system. N-Caproyl-sphingosine-1-phosphate migrated with an Rf = 0.47 and the corresponding radioactive spot was visualized by autoradiography, scraped from the plate, and counted by liquid scintillation.
Radioactive measurements were converted to pmol product by using the specific activity of ATP.
Metabolic labeling of sphingolipids
Metabolic labeling of sphingolipids was performed
according to Xia et al. (38)
with slight modifications.
Briefly, cells (4x106) were incubated with
3H-serine (4 µCi/ml, specific activity 33
Ci/mmol, Amersham) in DMEM containing 10% FCS and 5% horse serum for
48 h. The cells were washed once with PBS, incubated for 24 h
in serum-free DMEM, and then incubated with or without prosaposin, 10
nM, for 5 min. Where indicated, 50 µM N-oleoyl-ethanolamine (Matreya
Inc.), a ceramidase inhibitor (34)
, was added 30 min
before prosaposin treatment. After incubation, the cells were washed
with PBS, scraped from the flasks in 1 ml trypsin/EDTA, and sedimented
by centrifugation (300 g for 10 min at 4°C). Cell pellets
were suspended in 0.3 ml of TRIS/HCl 20 mM pH 7.4 containing 0.2%
Triton X-100 and homogenized by sonication (2x10 s, using a probe
sonicator). Lipids were extracted by 0.9 ml chloroform/methanol (1:1).
The lipid phase was evaporated under nitrogen, dissolved in chloroform,
and lipids were separated by bidimensional TLC on Silica Gel C60 F254
analytical plates using chloroform/methanol/acetic acid/water
(50/30/8/5, by vol) as first solvent and 1-butanol/acetic acid/water
(3/1/1, by vol) as second solvent. Radioactive spots were visualized by
autoradiography after spraying ENHANCER (NENTM Life Science Products,
Boston, Mass.) and identified based on comigration with authentic
standards revealed by exposure to iodine vapors. All radioactive spots
were scraped off, counted by liquid scintillation, and normalized by
radioactivity recovered in total cellular lipids.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Flow cytometric analysis was carried out using BrdU as specific marker
for cells in the S phase, in addition to PI; exponentially growing PC12
cells incorporate BrdU, reflecting active DNA synthesis. As already
reported (32)
, a very low level of DNA synthesis was
observed after 3 days of serum starvation (Fig. 1a
, 4.7% of BrdU-positive cells). When the cells were treated
with prosaposin in the absence of serum, BrdU incorporation after
3 h was significantly higher than untreated cells value
(P<0.001, as revealed by Kolmogorov-Smirnov test)
(39)
(Fig. 1c
, 48.6% of BrdU-positive cells).
When the cells were treated with prosaposin 10 nM for 18 h, the
BrdU incorporation was still high (Fig. 1d
, 28.4% of
BrdU-positive cells) and consistent with that observed in
serum-stimulated cells (Fig. 1b
, 33.9% of BrdU-positive
cells), indicating that prosaposin treatment induced PC12 entry in the
S phase of the cell cycle.
|
To investigate the kinetics of this stimulation, cells were treated
with prosaposin at different incubation times (Fig. 2
). The analysis of BrdU incorporation showed an initial wave at 30 min,
reaching a peak at 3 h (Fig. 2)
.
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DNA synthesis after prosaposin addition was correlated with an increase
in cell division. Cell number increase was clearly evident after
24 h (43±5) x 103
cell/cm2 and maximal after 48 h of exposure
to prosaposin (50±5) x 103
prosaposin-treated cell/cm2 vs. (36±4) x
103 control cell/cm2; this
increase in cell number and the space of time between the two related
phenomena was comparable to that shown after treatment with S-1-P or
EGF (25)
. Thus, the kinetics of the response of PC12 cells
to prosaposin was similar to other known mitogens.
ERK involvement in prosaposin effect on DNA synthesis
To investigate whether the prosaposin effect on DNA synthesis was
under the control of ERK, we used the synthetic MEK inhibitor PD98059,
which is known to specifically prevent MEK-1 activation without
affecting the activity of other kinases (35)
and viability
of the cells (not shown). Analysis of the cells stimulated with
prosaposin in either the presence or absence of PD98059 showed that
this molecule significantly inhibited the prosaposin effect on DNA
synthesis (P<0.001) (Fig. 3
). We have previously reported that this dose of PD98059 blocked
prosaposin-induced ERK phosphorylation within 5 min in primary Schwann
cells (40)
. These results suggest that prosaposin-induced
transient phosphorylation of ERK is required for prosaposin-induced
stimulation of DNA synthesis.
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Alternatively, PC12 cells were treated with pertussis toxin (100 ng/ml) for 30 min before stimulation with prosaposin. Addition of pertussis toxin inhibited the induction of DNA synthesis by prosaposin (P<0.001), indicating that the prosaposin receptor is a pertussis toxin-sensitive G-protein-coupled receptor.
Pretreatment of cells with N-oleoyl-ethanolamine (50 µM) also inhibited the induction of DNA synthesis by prosaposin (P<0.001), suggesting a role for ceramidase activity.
Prosaposin effect on cell apoptosis
To determine whether prosaposin prevented apoptosis in PC12 cells,
cells were incubated with staurosporine (24 h) or C6 ceramide (24 h),
in either the presence or absence of prosaposin, 10 nM. A subdiploid
peak by cytofluorometric analysis indicates DNA fragmentation,
consistent with apoptosis. As reported earlier (41)
, a
much larger hypodiploid peak was observed after 24 h of
staurosporine incubation (35.5±3%) (Fig. 4B
) compared to the control (4.3±0.9%) (Fig. 4A
).
In the presence of prosaposin, a significant (P<0.001)
decrease in hypodiploid cell number was observed (15.6+1.8%) after
staurosporine treatment (Fig. 4C
). In all samples, the
percentage of necrotic cells was less than 2% as revealed by a trypan
blue exclusion test (35)
.
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To confirm these findings, cells were stained with Hoechst 33258 (Fig. 4D
, E
, F
); the nuclei of control PC12 stained
uniformly with this dye, indicating that the nuclei were intact and the
cells were viable (Fig. 4D
). As suspected, treatment of
cells with staurosporine caused nuclear fragmentation and condensation
(Fig. 4E
). In cells treated with staurosporine in the
presence of prosaposin, a decrease in the number of apoptotic cells was
observed (Fig. 4F
).
Similar findings were observed in cells treated with C6 ceramide in the
presence or absence of prosaposin (Fig. 5a
, b
).
|
After C6-ceramide treatment, cells stained with the ‘ApopTag’ kit
showed increased apoptosis (Fig. 5c
, d
); when prosaposin was
added, apoptosis was attenuated (Fig. 5e
). When cells were
pretreated with PD98059 and incubated with prosaposin and C6-ceramide,
the protective effect of prosaposin on apoptosis was attenuated (Fig. 5f
).
Sphingosine kinase activation and sphingosine-1-phosphate
generation after prosaposin treatment
Sphingosine kinase activity has been proposed to be the
rate-limiting step in the metabolism of sphingosine (42)
.
Treatment with prosaposin increased sphingosine kinase activity. When
cells were treated with prosaposin at different incubation times (0, 1,
5, or 30 min), a peak of sphingosine kinase activation occurred by 5
min (and the activity was 450-fold higher than in control cells); the
activity returned to baseline after 30 min (Fig. 6A
). Sphingosine kinase activity at 5 min was proportional
with respect to protein (Fig. 6B
, C
).
|
When cells were labeled with 3H-serine to assay
sphingosine and sphingosine-1-phosphate and prosaposin was added, the
content of sphingosine and S-1-P was increased and a parallel decrease
of ceramide was observed (Fig. 7
).
|
Pretreatment with the ceramide inhibitor N-oleoyl-ethanolamine
(34)
prevented the increase of sphingosine or
sphingosine-1-P or the decrease of ceramide, after prosaposin addition
(Fig. 7)
. This finding, together with the observation that
N-oleoyl-ethanolamine inhibited the induction of DNA synthesis by
prosaposin (Fig. 3)
, led us to verify the effect of
N-oleoyl-ethanolamine on anti-apoptotic activity of prosaposin. Cells
pretreated with N-oleoyl-ethanolamine (30 min before) and prosaposin
were stimulated with staurosporine (Fig. 8
) or C6 ceramide (not shown). Hoechst 33258 staining revealed that the
addition of N-oleoyl-ethanolamine inhibited the protective effect of
prosaposin, since several cells showed nuclear fragmentation and
condensation (Fig. 8)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Previous data showed that prosaposin binds to PC12 cells and activated
ERK phosphorylation (14)
. Prosaposin’s effect on ERK
activation was inhibited by PD98059 (40)
, a selective MEK
inhibitor. In several cell types, ERK-activated signaling by a
G-protein-dependent mechanism appears to be an absolute requirement for
activating the proliferative response (43)
. We have
demonstrated here that prosaposin-induced PC12 entry in the S phase of
the cell cycle is a transition that requires ERK phosphorylation and
appears to be a consequence of a G-protein-coupled receptor. This
finding is consistent with the observation that the prosaposin receptor
is a pertussis toxin-sensitive G-protein receptor that activated the
ERK pathway, which in Schwann cells is essential for enhanced sulfatide
synthesis (40)
. Taken together, these data support the
view that signaling induced by prosaposin is generated by binding to a
G0-protein-coupled receptor (13)
.
PC12 cells undergo apoptosis after serum deprivation (44)
and after staurosporine (41)
or ceramide (45)
treatment. Prosaposin addition rescues cells from death after serum
deprivation in neuroblastoma cells (12)
, primary
hippocampal neurons (46)
, and Schwann cells
(47)
. In this report, prosaposin addition rescued PC12
cells from apoptosis. One possible molecular mechanism may be that
prosaposin binding blocks calcium channels by activation of a
G-protein-coupled receptor (48)
and thereby modulates
calcium concentrations. Another possible mechanism may be modulation of
the ceramide pathway, which may mediate mitogenic or apoptotic effects.
The effects of prosaposin on voltage-dependent calcium channels is
being investigated. In addition, evidence has suggested that branching
pathways of sphingolipid metabolism may mediate either mitogenic or
apoptotic effects (22
23
24
25
26
27)
. It has been reported that
ceramide induced apoptosis in several cell lines (24)
,
that sphingosine and S-1-P are mitogenic (25
, 26)
, and
that both stimulate the activation of the ERK pathway by a
G-protein-coupled receptor, the S-1-P receptor (28)
. The
S-1-P receptor has been shown to play a distinct role in altering
neuronal cell morphology (27)
and stimulating ERK
signaling pathways in different cell types, reminiscent of effects
elicited on growth factor stimulation. Activation of ERKs by the S-1-P
receptor in quiescent Swiss 3T3 fibroblast is time and concentration
dependent: S-1-P rapidly stimulated ERK activity, reaching a maximal
level within 2.5 min and declining thereafter. This concentration
dependence correlated closely with that for induction of DNA synthesis
(28)
. Prosaposin enhanced sphingosine kinase and led to
intracellular formation of S-1-P; one result may be that the
accumulation of S-1-P in prosaposin-treated PC12 cells is due to
ceramide breakdown. Our hypothesis is that prosaposin modulated
sphingosine kinase activity by increasing intracellular levels of
S-1-P, enhanced proliferation by promoting the G1 to S phase
transition, and suppressed ceramide-induced apoptosis
(49)
. This view is strongly supported by our results
showing that ceramidase inhibitor N-oleoyl-ethanolamine diminishes not
only S-1-P formation, but also the anti-apoptotic and proliferative
effects of prosaposin.
In conclusion, prosaposin may be a key modulator in the ceramide-S-1-P rheostat via the ERK pathway and may represent an important determinant of cell fate. Further studies are in progress to better investigate the kinetics of this pathway.
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ACKNOWLEDGMENTS |
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Received for publication April 7, 2000.
Revision received July 27, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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induces adhesion molecule expression trough the sphingosine kinase pathway. Proc. Natl. Acad. Sci. USA 95,14196-14201
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
450-fold compared to control cells,
as counted by liquid scintillation), thus indicating the activation of
sphingosine kinase, and was back to baseline at 30 min. Representative
of 5 independent experiments. B) Protein dependence of
sphingosine kinase activity expressed as pmol N-caproyl-S-1-P.
Quantitation data from three experiments are shown
(means±SD). C) Protein dependence of
sphingosine kinase activity: one representative of three experiments is
shown.
