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Volume 17, Number 18,
Issue of September 15, 1997
pp. 6952-6960
Copyright ©1997 Society for Neuroscience
Involvement of Sphingosine 1-Phosphate in Nerve Growth
Factor-Mediated Neuronal Survival and Differentiation
Lisa Cseh Edsall,
Grisha G. Pirianov, and
Sarah Spiegel
Department of Biochemistry and Molecular Biology, Georgetown
University Medical Center, Washington, DC 20007
 ABSTRACT
- INTRODUCTION
- MATERIALS AND METHODS
- RESULTS
- DISCUSSION
- FOOTNOTES
- REFERENCES
ABSTRACT 
Sphingolipid metabolites, such as ceramide and
sphingosine-1-phosphate (SPP), are emerging as a new class of second
messengers involved in cellular proliferation, differentiation, and
apoptosis. Nerve growth factor (NGF), a neurotrophic factor for
pheochromocytoma PC12 cells, induced a biphasic increase in the
activity of sphingosine kinase, the enzyme that catalyzes the formation
of SPP. This activation was blocked by K252a, an inhibitor of tyrosine
kinase A (trkA). A rapid 1.7-fold increase was followed by a marked
prolonged increase reaching a maximum of fourfold to fivefold
stimulation with a concomitant increase in SPP levels and a
corresponding decrease in endogenous sphingosine levels. Levels of
ceramide, the precursor of sphingosine, were only slightly decreased by
NGF in serum-containing medium. However, NGF decreased the elevation of
ceramide induced by serum withdrawal. Treatment of PC12 cells with SPP
did not induce neurite outgrowth or neurofilament expression, yet it
enhanced neurofilament expression elicited by suboptimal doses of NGF. Moreover, SPP also protected PC12 cells from apoptosis induced by serum
withdrawal. To further substantiate a role for SPP in the
cytoprotective actions of NGF, we found that
N,N-dimethylsphingosine, a competitive inhibitor of
sphingosine kinase, also induced apoptosis and interfered with the
survival effect of NGF. These effects were counteracted by exogenous
SPP. Moreover, other structurally related compounds, such as
dihydrosphingosine 1-phosphate and lysophosphatidic acid, had no
significant protective effects. Our results suggest that activation of
sphingosine kinase and subsequent formation of SPP may play an
important role in the differentiation and survival effects induced by
NGF.
Key words:
NGF;
sphingolipid metabolites;
sphingosine 1-phosphate;
apoptosis;
neuronal differentiation;
trkA;
signal transduction
INTRODUCTION
Sphingolipid metabolites are
emerging as an important new class of lipid second messengers (Hannun,
1994 ; Kolesnick and Golde, 1994; Spiegel and Milstien, 1995). Several
cytokines, including tumor necrosis factor  (TNF-), vitamin
D3,  -interferon, and interleukin 1, stimulate
sphingomyelinase, leading to increased levels of ceramide. Ceramide, in
turn, can arrest cell growth and induce differentiation and more
recently has been implicated as a key component of programmed cell
death, also known as apoptosis (Kolesnick and Golde, 1994; Hannun and
Obeid, 1995). In contrast, further metabolites of ceramide, such as
sphingosine produced from ceramide by ceramidase and
sphingosine-1-phosphate (SPP) produced by sphingosine kinase-dependent
phosphorylation of sphingosine, have been implicated as second
messengers in the mitogenic actions of certain growth factors (Olivera
and Spiegel, 1993). Thus, dissociation of growth factor-induced
mitogenesis from cytokine-mediated apoptosis may be attributable to
distinct sphingolipid-derived second messengers.
The role of sphingolipid metabolites in the regulation of
neuronal survival, development, and death is now beginning to be appreciated (Riboni et al., 1995). Nerve growth factor (NGF) plays an
important role in the survival and development of neurons in the
central and peripheral nervous systems (Levi-Montalcini, 1987). The
effects of NGF are predominantly mediated by tyrosine kinase A (trkA),
the high-affinity NGF receptor that initiates complex signal
transduction cascades, ultimately enhancing differentiation and
promoting survival of neurons (Greene and Kaplan, 1995). The low-affinity neurotrophin receptor p75NGFR, a member
of the tumor necrosis factor receptor superfamily, binds all
NGF-related neurotrophins (Kaplan and Stephens, 1994; McDonald and
Chao, 1995). The functional significance of p75NGFR
in signal transduction is still not completely understood, but it has
been suggested to mediate apoptosis of developing neurons in the
absence of trkA (Van der Zee et al., 1996). Recently, it has been
demonstrated that NGF activates sphingomyelinase in rat T9 anaplastic
glioblastoma cells through p75NGFR (Dobrowsky et
al., 1994), indicating that ceramide may play a role in
p75NGFR signaling. Interestingly, sphingomyelinase
activation was diminished in cells coexpressing the
p75NGFR receptor and trkA but was reinstated when
trkA function was blocked (Dobrowsky et al., 1995). This is in
agreement with recent observations that the trkA receptor may
negatively regulate the functional activity of
p75NGFR (Bothwell, 1996; Carter et al., 1996; Van
der Zee et al., 1996). Because the relative balance of sphingolipid
metabolites ceramide and SPP has recently been shown to influence
opposing pathways of apoptosis and cell survival in HL-60 and U937
cells (Cuvillier et al., 1996), it was of interest to determine whether
SPP production might play a role in the neurotrophic actions of NGF.
For this purpose, we used rat pheochromocytoma PC12 cells that
coexpress both p75NGFR and trkA (Greene and Kaplan,
1995). Our results indicate that activation of sphingosine kinase and
subsequent production of SPP may play an important biological role in
cell survival signal transduction pathways activated by the binding of
NGF to trkA.
MATERIALS AND METHODS
Materials. Mouse 2.5 S nerve growth factor was
obtained from Upstate Biotechnology Inc. (Lake Placid, NY). Type I rat
tail collagen was purchased from Collaborative Research (Lexington, MA). 12-O-Tetradecanoylphorbol-13-acetate (TPA),
dihydrosphingosine, diethylenetriaminepentaacetic acid (DETPAC),
monoclonal anti-neurofilament M, essentially fatty acid-free bovine
serum albumin (BSA), and bovine brain type IV ceramides were obtained
from Sigma (St. Louis, MO). O-Phthalaldehyde was obtained
from Aldrich (Milwaukee, WI). SPP, sphingosine, and
N,N-dimethylsphingosine were purchased from Biomol Research
Laboratory Inc. (Plymouth Meeting, PA). Lysophosphatidic acid and
cardiolipin were purchased from Avanti Polar Lipids (Birmingham, AL).
Dihydrosphingosine-1-phosphate was kindly provided by Dr. Alan P. Kozikowski (Georgetown University Institute for Cognitive and
Computational Sciences). [methyl-3H]Thymidine (83 Ci/mmol), [-32P]ATP (3000 Ci/mmol), and
[3H]acetic anhydride (50 mCi/mmol) were purchased
from Amersham (Arlington Heights, IL). Serum and medium were obtained
from Biofluids (Rockville, MD). Octyl- -D-glucopyranoside
and Escherichia coli diacylglycerol kinase were purchased
from Calbiochem (La Jolla, CA).
Cell culture. Rat pheochromocytoma PC12 cells were kindly
provided by Dr. Gordon Guroff (National Institute of Child Health and
Human Development, National Institutes of Health, Bethesda, MD). Cells
were maintained in RPMI medium supplemented with 10% heat-inactivated
horse serum, 5% fetal bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin (Batistatou and Greene, 1991). Cells were plated
at a density of 5 × 104/cm2. In some experiments,
cells were seeded onto plates coated with rat tail collagen (50 µg/ml).
Sphingosine kinase activity. After various treatments, cells
were washed twice with PBS and harvested by scraping in buffer A [0.1
M Tris-HCl, pH 7.4, containing 20% (v/v) glycerol, 1 mM mercaptoethanol, 1 mM EDTA, 1 mM
Na3VO4, 15 mM NaF, 10 µg/ml leupeptin and aprotinin, 1 mM PMSF, and 0.5 mM 4-deoxypyridoxine]. Cells were lysed by freeze-thawing
three times, and the cytosolic fraction was prepared by centrifugation
at 13,000 × g for 20 min. Incubated together were 100 µl of cytosolic fraction (20-50 µg) with 10 µl of sphingosine (1 mM), delivered as a sphingosine-BSA complex (4 mg/ml BSA).
Buffer A was added to a final volume of 190 µl, and reactions were
started by addition of 10 µl of [-32P]ATP (10-20
µCi, 20 mM) containing 100 mM
MgCl2. Samples were incubated for 30 min at 37°C. Lipids
were then extracted with chloroform/methanol/concentrated HCl
(100:200:1, v/v), and phases were separated as described previously
(Olivera et al.,1994). Lipids from the organic phase were resolved by
TLC on Silica Gel 60 plates using 1-butanol/methanol/acetic acid/water
(80:20:10:20, v/v) as the solvent system. Labeled SPP was visualized by
autoradiography, scraped from the plate, and counted with a
scintillation counter.
Measurement of SPP. SPP levels were determined as described
previously (Yatomi et al., 1995) with minor modifications. Briefly, after treatment with NGF for various times, cells were washed two times
with 1 M Tris-HCl, pH 7.4, harvested in 3.5 ml of
methanol/chloroform/1 M KCl (2:1:0.5, v/v), and sonicated
on ice. Alkaline extraction was performed by the addition of 4.1 ml of
chloroform/1 M KCl/concentrated NH4OH (2:2:0.1,
v/v). The aqueous phase containing SPP was collected and re-extracted
with 3.2 ml of chloroform/concentrated HCl (3:0.2, v/v). Organic phases
containing SPP were then evaporated under N2 and
solubilized in 20 µl of 0.008N NaOH/methanol and 20 µl of 10 mM [3H]acetic anhydride (10 µCi).
Acetylation reactions proceeded at 37°C for 2 hr. Acetylated SPP was
extracted with 2.77 ml of methanol/chloroform/1 M
KCl/concentrated HCl (0.75:1:1:0.02, v/v). The chloroform phases were
washed twice with 1 ml of chloroform/methanol/water (3:48:47, v/v) and
then evaporated under N2. Samples were dissolved in 200 µl of chloroform/methanol (2:1, v/v) and applied to Silica Gel 60 TLC
plates. After development with butanol/acetic acid/water (3:1:1, v/v),
acetylated SPP was visualized by autoradiography using
3H-enhancer spray (DuPont, Billerica, MA), scraped from the
plates, and counted with a scintillation counter.
Measurement of sphingosine. Sphingosine levels were
determined by an enzymatic method as described previously (Olivera et al., 1994) or by HPLC analysis (Wilson et al., 1988) with slight modifications. For both assays, after treatment with NGF cells were
washed in 1 M Tris-HCl, pH 7.4, and scraped in 0.5 ml of methanol. Samples were extracted with chloroform/methanol/1
M KCl (1:0.5:1, v/v). For HPLC analysis, aliquots of the
organic layer (~50-100 nmol of total cellular phospholipid),
containing dihydrosphingosine as an internal standard, were saponified
by addition of 0.5 ml of 0.1N KOH/methanol and incubation at 37°C for
60 min. The reactions were terminated by the addition of 10 µl of
concentrated HCl, and samples were extracted with 1 ml of chloroform/1
M KCl (1:1, v/v). Organic extracts were dried under N2, resuspended in 50 µl methanol, and derivatized
with o-phthalaldehyde for subsequent HPLC analysis (Wilson
et al., 1988). Sphingosine and dihydrosphingosine were separated
isocratically on a Cosmosil 5C18-AR column (4.6 × 250 mm; Nacalai
Tesque, Kyoto, Japan) using a Waters (Milford, MA) 600E pump with
methanol/5 mM NaH2PO4, pH 7.0 (60:40, v/v), at a flow rate of 1 ml/min. A Waters 420-AC fluorescence detector was used to detect fluorescent derivatives with
an excitation filter transmitting at a maximum of 340 nM and a cut-off emission filter at 400 nM. Sphingosine levels
were quantified using the Rainin (Woburn, MA) Dynamax HPLC software package.
Measurement of ceramide. Lipids were extracted, and
mass amounts of ceramide in cellular extracts were measured by the DAG kinase enzymatic method (Olivera and Spiegel, 1992). Briefly, an
aliquot (10-50 nmol of total phospholipid) of the chloroform phase
from cellular lipid extracts was dried under a nitrogen stream. The
lipids or standard bovine brain type IV ceramides were resuspended in
40 µl of 7.5% (w/v) octyl--D-glucopyranoside/5 mM cardiolipin in 1 mM DETPAC/10 mM
imidazole, pH 6.6, and solubilized by freeze-thawing and subsequent
sonication. The enzymatic reaction was started by the addition of 20 µl of DTT (20 mM), 10 µl of E. coli
diacylglycerol kinase (0.88 U/ml), 20 µl of
[-32P]ATP (10-20 µCi, 10 mM), and 100 µl of reaction buffer (in mM: 100 imidazole, pH 6.6, 100 NaCl, 25 MgCl2, and 2 EGTA). After incubation for 1 hr at room temperature, lipids were extracted with 1 ml of
chloroform/methanol/concentrated HCl (100:200:1, v/v) and 0.17 ml of 1 M KCl. Labeled phosphatidic acid and ceramide-1-phosphate were resolved by TLC with chloroform/acetone/methanol/acetic acid/water (10:4:3:2:1, v/v). Bands corresponding to ceramide were scraped from
the plates and counted with a scintillation counter or, alternatively, quantified with a Molecular Dynamics (Sunnyvale, CA) Storm
PhosphorImager.
Measurement of total cellular phospholipids. Total
phospholipids present in cellular lipid extracts used for sphingolipid analysis were quantified as described previously (Van Veldhoven and
Mannaerts, 1987) with minor modifications. Briefly, to dried aliquots
of cellular lipid extracts, 40 µl of a mixture of 10N H2SO4/70% perchloric acid (3:1, v/v)
was added, and samples were incubated for 30 min at 210°C. After
cooling, 75 µl of water and 400 µl of 4.2% ammonium molybdate in
4N HCl/0.045% (w/v) malachite green (1:3 v/v) was added. Samples were
incubated at 37°C for 15 min, and absorbances were measured at 660 nm.
Quantitation of DNA fragmentation. DNA fragmentation was
determined in cells prelabeled with [3H]thymidine
(1 µCi/ml) for 24 hr (Duke and Cohen, 1992). Cells were washed gently
twice with serum-free medium and then incubated in the same medium with
the indicated agents for 4-24 hr at 37°C. Cells were harvested,
lysed in TTE (10 mM Tris, pH 7.4, 10 mM EDTA,
and 0.2% Triton X-100), and fragmented DNA was separated from intact
chromatin by centrifugation. Pellets were suspended in TTE, and TCA was
added to a final concentration of 12.5%.
[3H]Thymidine incorporated into both intact and
fragmented DNA was determined by liquid scintillation counting (Duke
and Cohen, 1992). Percent fragmented DNA = 100 × (fragmented/(fragmented + intact chromatin)).
Staining of apoptotic nuclei. Cells were cultured in
serum-free medium in the absence or presence of the indicated agents for 24 hr, washed in PBS, and fixed in 3.7% formaldehyde for 10 min.
After washing with PBS, fixed cells were then incubated with bisbenzimide trihydrochloride (24 µg/ml in 30% glycerol/PBS; Hoechst 33258, Calbiochem) for 10 min. Cells were then examined with a Zeiss
(Petersburg, VA) Photoscope II fluorescent microscope. At a minimum,
500 cells were scored. Apoptotic cells were distinguished by condensed,
fragmented nuclear regions.
Determination of cell survival. Intact nuclei were
determined as described previously (Batistatou and Greene, 1991).
Briefly, PC12 cells cultured in 24-well collagen-coated plates were
treated for 24 hr with various agents in serum-free medium. Medium was then aspirated, and cells were lysed in 200 µl of 10% Zapoglobin (Fisher Scientific, Houston, TX) in 0.5× PBS containing 0.5% Triton X-100, 2 mM MgCl2, and 15 mM
NaCl. This solution provides a uniform suspension of nuclei, which were
counted with a hemocytometer. Four separate fields were counted for
each sample.
Neurofilament assay. PC12 cells were seeded onto
collagen-coated plates, and after 24 hr, cells were washed two times
with serum-free medium and then treated with the indicated agents. Cells were scraped in lysis buffer [20 mM HEPES, pH 7.2, 1% Nonidet P-40, 10% (v/v) glycerol, 50 mM NaF, 1 mM PMSF, 1 mM
Na3VO4, and 10 µg/ml leupeptin].
Equal amounts of protein from the cell lysates were electrophoresed on
a 7.5% SDS-polyacrylamide gel and subsequently transblotted to
nitrocellulose (0.45 µM). Membranes were probed with
monoclonal anti-neurofilament, M and immunoreactive bands were detected
by chemiluminescence using horseradish peroxidase-conjugated anti-mouse
IgG and quantitated by densitometric analysis.
RESULTS
NGF activates sphingosine kinase and increases SPP levels
via the trkA receptor
We previously reported that PDGF, but not epidermal growth
factor, stimulated sphingosine kinase activity and increased SPP levels
in Swiss 3T3 fibroblasts (Olivera and Spiegel, 1993). It was of
interest to examine whether NGF, a well known pleiotropic growth factor
for PC12 cells, could also regulate levels of sphingolipid metabolites.
Treatment of PC12 cells with NGF markedly stimulated sphingosine kinase
activity. A rapid 75% increase in kinase activity was observed within
15 min that was then followed by a gradual and prolonged elevation of
fourfold to fivefold by 72 hr (Fig. 1A). Similar results
were obtained in the absence or presence of serum. After treatment for
24 hr, significant activation of sphingosine kinase was observed at a
concentration of NGF as low as 1 ng/ml, with maximum stimulation
plateauing at a concentration of 10 ng/ml (Fig. 1B).
Because most of the actions of NGF are known to be mediated by binding
to the trkA receptor (Greene and Kaplan, 1995), we investigated whether
trkA function was required for activation of sphingosine kinase by NGF.
K252a, a relatively specific inhibitor of trkA (Berg et al., 1992),
completely blocked both phases of activation of sphingosine kinase
induced by NGF (Fig. 1C), indicating that the trkA receptor
is essential for NGF-induced sphingosine kinase activation. In many
cell types, activation of protein kinase C (PKC) by the phorbol ester
TPA results in stimulation of sphingosine kinase activity (Mazurek et
al., 1994; Spiegel and Milstien, 1995; Cuvillier et al., 1996). In
agreement, TPA transiently stimulated sphingosine kinase activity in
PC12 cells (Fig. 1D). However, in contrast to the
prolonged activation of sphingosine kinase induced by NGF, the effects
of TPA rapidly diminished after 1 hr, and further treatment with TPA
did not have any significant effects, probably because of downregulation of PKC.
Fig. 1.
NGF stimulates sphingosine kinase in rat
pheochromocytoma PC12 cells. A, Time course. PC12 cells
were treated with NGF (100 ng/ml) for the indicated times, and
cytosolic sphingosine kinase activity was measured as described in
Materials and Methods. Data are the mean ± SD of triplicate
cultures expressed as fold increase over control values. Similar
results were obtained in three independent experiments.
B, Dose response. PC12 cells were treated with or without various concentrations of NGF for 24 hr, and cytosolic sphingosine kinase activity was measured. Data are the mean ± SD
of triplicate cultures expressed as fold increase over control values.
C, Inhibition by the trkA receptor inhibitor K252a. PC12 cells were treated with NGF (100 ng/ml) in the absence (open
bars) or presence (solid bars) of K252a (200 nM), and sphingosine kinase activity was measured after 15 min and 4 hr. Results are expressed as fold increase over control
values and are the mean ± SD of triplicate cultures. Similar
results were obtained in three separate experiments. D,
TPA stimulates sphingosine kinase activity. PC12 cells were treated
with TPA (200 nM) for the indicated times, and sphingosine
kinase activity was measured. Sphingosine kinase activity is expressed
as fold increase over control values and is the mean ± SD of
triplicate cultures.
[View Larger Version of this Image (39K GIF file)]
Activation of sphingosine kinase by NGF was accompanied by an increase
in cellular levels of SPP (Fig.
2A). A small but
significant increase was observed after 1 hr, which increased to
2.5-fold after 24 hr treatment with NGF in the presence of serum.
Treatment with NGF for up to 1 hr had no significant effect on
sphingosine levels as measured by HPLC (Wilson et al., 1988). However,
after 6 and 24 hr, there were small but significant decreases in
sphingosine (Fig. 2B), approximately corresponding to
the mass increase in SPP after NGF treatment. Identical results were
found when levels of sphingosine were measured by the enzymatic assay
method recently described by our laboratory (Olivera et al., 1994). It
should be noted that the ratio of sphingosine to sphinganine
(dihydrosphingosine) is ~20:1, and the levels of
dihydrosphingosine-1-phosphate in PC12 cells were below our detection
limits. Levels of ceramide, the precursor of sphingosine, which are 10- to 20-fold greater than sphingosine or SPP levels in PC12 cells,
decreased ~23% after NGF treatment for 24 hr in serum-containing
medium. In agreement with previous studies (Jayadev et al., 1995;
Hannun, 1996), serum deprivation induced a significant increase in
ceramide levels (Fig. 2C). Interestingly, NGF decreased the
elevation of ceramide induced by serum withdrawal (Fig.
2C).
Fig. 2.
NGF induces changes in levels of sphingolipid
metabolites. A, Mass levels of SPP. PC12 cells were
treated with NGF (100 ng/ml) for the indicated times, and SPP was
quantified as described in Materials and Methods. SPP levels are
expressed as picomoles per milligram of protein (dotted
bars) and as femtomoles per nanomoles of phospholipid
(hatched bars) and are the mean ± SD of triplicate cultures. B, Sphingosine levels. After treatment with
NGF (100 ng/ml) for the indicated times, cellular lipids were
extracted, and sphingosine levels were measured by HPLC as described in
Materials and Methods. Sphingosine levels are expressed as picomoles
normalized to total phospholipids and are the mean ± SD of
triplicate cultures. C, Ceramide levels. PC12 cells were
cultured in serum-free medium in the absence (open bars)
or presence (hatched bars) of NGF (100 ng/ml) for the
indicated times. Ceramide levels were determined as described in
Materials and Methods. For controls, ceramide levels were determined in
untreated PC12 cells cultured in serum-containing medium for 24 hr and
were found to be 5.3 ± 0.2 pmol/nmol of phospholipids. Ceramide
levels were normalized to total phospholipids and are expressed as the
mean ± SD of fold increases over control values. Similar results
were obtained in three independent experiments.
[View Larger Version of this Image (28K GIF file)]
Effect of SPP on NGF-induced neuritogenesis
NGF induces neuritogenesis and enhances the survival of PC12
cells (Greene, 1978; Kaplan and Stephens, 1994). Because NGF markedly
stimulates sphingosine kinase activity, leading to an increase in SPP
levels, it was important to determine whether SPP plays a role in the
pleiotropic effects of NGF. NGF induced dose-dependent expression of
neurofilament, a protein exclusively found in neuronal cells (Tsuneishi
et al., 1993), the expression of which correlates with neurite
outgrowth. SPP alone did not significantly induce neurite outgrowth,
nor did it increase neurofilament expression (data not shown). However,
SPP enhanced the ability of suboptimal concentrations of NGF to induce
neurofilament protein expression (Fig.
3A). To examine the possible
role of endogenous SPP in the actions of NGF, we used
N,N-dimethylsphingosine, a potent inhibitor of sphingosine
kinase (Cuvillier et al., 1996). N,N-Dimethylsphingosine not
only inhibited NGF-induced neurite outgrowth, it markedly reduced
neurofilament expression in response to optimal concentrations of NGF
(Fig. 3B), an effect that was partially reversed by the
addition of SPP (data not shown). Because relatively high
concentrations of N,N-dimethylsphingosine have also been
shown to inhibit PKC activity (Khan et al., 1990), it was important to
determine whether the inhibitory effects of
N,N-dimethylsphingosine were attributable specifically to
inhibition of sphingosine kinase. Treatment of PC12 cells with
N,N-dimethylsphingosine, at concentrations that block the
neurotrophic effects of NGF and inhibit sphingosine kinase activity and
concomitant SPP formation, had no significant effect on TPA-induced
activation of PKC (data not shown). Thus, the effect of
N,N-dimethylsphingosine on differentiation does not appear
to be mediated via inhibition of PKC.
Fig. 3.
Involvement of SPP in NGF-induced expression of
neurofilament protein. A, SPP enhances the effect of
suboptimal concentrations of NGF on levels of neurofilament protein.
After treatment with NGF (0.1-100 ng/ml), SPP (10 µM),
or a combination of SPP (10 µM) and NGF (0.1 or 1 ng/ml),
neurofilament expression was determined as described in Materials and
Methods. Twenty-five micrograms of total cellular protein was loaded in
each lane of an SDS-PAGE gel. After transblotting to nitrocellulose,
blots were probed with monoclonal anti-neurofilament M antibody.
Results shown are from a representative experiment. Similar results
were obtained in five independent experiments. B,
N,N-Dimethylsphingosine (DMS), an
inhibitor of sphingosine kinase, inhibits neurofilament expression induced by NGF. PC12 cells were treated with the indicated
concentrations NGF in the absence or presence of
N,N-dimethylsphingosine (10 µM), and
neurofilament levels were analyzed by Western blotting. Similar results
were obtained in four independent experiments.
[View Larger Version of this Image (31K GIF file)]
Effect of SPP on neuronal cell death
In addition to promoting neuronal differentiation, NGF is
essential for the survival of neurons in the central and peripheral nervous systems (Levi-Montalcini, 1987). We have recently shown that
SPP can reverse apoptosis induced by TNF-, Fas ligation, and
ceramide elevation, suggesting that the intracellular balance between
levels of ceramide and SPP is a critical factor that determines the
fate of cells (Cuvillier et al., 1996). Moreover, ceramide has been
implicated in apoptosis induced by growth factor withdrawal (Hannun,
1996), and exogenous ceramide induces apoptosis in PC12 cells
(Hartfield et al., 1997). In this study, we found that there was a
significant increase in ceramide levels on removal of trophic support
(Fig. 2C), which may contribute to the apoptotic response. In agreement with previous reports (Batistatou and Greene, 1991; Xia et
al., 1995), serum deprivation induced apoptosis in PC12 cells within 4 hr, as determined by quantitative DNA fragmentation (Fig.
4A), and shrinkage of
cell bodies and condensation of nuclei were clearly evident after 24 hr
(Fig. 4B). NGF eliminated DNA fragmentation and
nuclei condensation and enhanced cell survival (Fig.
4A, Tables 1, 2). Exogenous SPP also prevented DNA
fragmentation because of serum deprivation, albeit to a lesser extent
than NGF (Fig. 4A). In addition, SPP partially
prevented the appearance of nuclei with apoptotic features determined
by staining with the DNA-specific fluorochrome bisbenzimide (Fig.
4C), whereas, as expected, NGF almost completely prevented
the apoptotic response (Fig. 4D). The protective
effect of SPP was specific, because other structurally related lipid
analogs, such as lysophosphatidic acid or
dihydrosphingosine-1-phosphate, which lacks the trans double
bond present in SPP, did not significantly prevent apoptosis resulting
from serum withdrawal after 9 hr (Fig.
5A) or 18 hr (Fig.
5B). Similar results were obtained using a quantitative DNA
fragmentation assay (data not shown). To substantiate a role for
endogenous SPP in the cytoprotective effect of NGF further, we again
used the competitive inhibitor of sphingosine kinase, N,N-dimethylsphingosine. At a concentration that markedly
inhibited sphingosine kinase activity and decreased SPP levels,
N,N-dimethylsphingosine increased DNA fragmentation (Fig.
4A). Moreover, N,N-dimethylsphingosine also inhibited the antiapoptotic effect of NGF (Fig.
4A). If SPP is a critical component in the
cytoprotective effect of NGF, as suggested by these results, then
addition of SPP should overcome the effects of
N,N-dimethylsphingosine. Indeed, SPP was able to reverse the
effect of N,N-dimethylsphingosine on induced cell death
effectively and also to restore the cytoprotective activity of NGF
(Fig. 4A). Similar results were obtained with two
additional independent assays of apoptosis. Quantitation of condensed,
fragmented apoptotic nuclei (Table 1) as well as determination of cell
survival (Table 2) showed that SPP exerted the same cytoprotective
effects as found in the DNA fragmentation assays and also counteracted the effects of N,N-dimethylsphingosine.
Fig. 4.
Effects of SPP and
N,N-dimethylsphingosine on apoptosis in PC12 cells.
A, PC12 cells were prelabeled with
[3H]thymidine and then treated in serum-free
medium for 4 hr without or with NGF (100 ng/ml), SPP (5 µM), dimethylsphingosine (DMS, 10 µM), or the indicated combinations, and DNA fragmentation
was determined. Data are expressed as percent DNA fragmentation and are
the mean ± SD of triplicate cultures. Similar results were obtained in three independent experiments. PC12 cells were incubated for 24 hr in serum-free medium without (B) or
with 5 µM SPP (C) or 100 ng/ml NGF
(D) and then stained with the DNA-specific
fluorochrome bisbenzimide.
[View Larger Version of this Image (44K GIF file)]
Table 1.
Effects of NGF and SPP on Apoptosis Induced by Serum
Withdrawal
| Treatment |
Apoptotic cells (%)
|
|
| None |
73.5 ± 0.9 |
| SPP |
53.9
± 1.3 |
| NGF |
9.5 ± 1.6 |
| DMS |
83.4 ± 0.2 |
| DMS + NGF |
18.5 ± 1.9 |
| DMS + SPP |
52.6 ± 0.9 |
| DMS + NGF + SPP |
10.5 ± 1.0 |
|
|
PC12 cells were incubated for 24 hr in serum-free medium in the
absence or presence of NGF (100 ng/ml), SPP (5 µM),
N,N-dimethylsphingosine (DMS, 10 µM), or the
indicated combinations. Changes of nuclear morphology characteristic of
apoptosis were quantified after staining with the DNA-specific
fluorochrome bisbenzimide. Cells with chromatin condensation or
segmentation of nuclei into three or more fragmentations were
considered apoptotic. At a minimum, 500 cells in each field were
scored. The data represent two independent determinations from three
separate experiments.
|
|
Table 2.
SPP enhances short-term survival of PC12 cells in
serum-free medium
| Treatment |
Surviving cells (%)
|
|
| None |
52.2 ± 1.2 |
| SPP |
72.2
± 0.2 |
| NGF |
90.9 ± 1.0 |
| DMS |
50.5 ± 0.6 |
| DMS + NGF |
59.8 ± 1.3 |
| DMS + SPP |
75.1 ± 0.9 |
| DMS + NGF + SPP |
85.5 ± 1.2 |
|
|
PC12 cells were incubated for 24 hr in serum-free medium in the
absence or presence of NGF (100 ng/ml), SPP (5 µM),
N,N-dimethylsphingosine (DMS, 10 µM), or the
indicated combinations. After 24 hr in culture, viable cells were
quantified by determining the number of intact nuclei, as described in
Materials and Methods. The numbers of surviving cells are presented
relative to the number of cells initially plated (12.5 × 104, designated as 100%). Comparable results were
obtained in two separate experiments.
|
|
Fig. 5.
Effects of SPP analogs on apoptosis induced
by trophic factor withdrawal. PC12 cells were incubated in serum-free
medium in the absence or presence of the indicated concentrations of
SPP, dihydrosphingosine-1-phosphate (DHSPP) or
lysophosphatidic acid (LPA) for 9 hr
(A) or 18 hr (B) and then
stained with bisbenzimide. Cells with chromatin condensation or
segmentation of nuclei into three or more fragmentations were
considered apoptotic. At a minimum, 500 cells in each field were
scored. Similar results were obtained in three independent
experiments.
[View Larger Version of this Image (35K GIF file)]
DISCUSSION
NGF is generally considered to be important for survival and
differentiation of neurons (Greene, 1978; Kaplan and Stephens, 1994;
Xia et al., 1995). These effects are mainly mediated by binding of NGF
to the high-affinity trkA tyrosine kinase receptor, which initiates
several parallel signaling cascades, including activation of
phospholipase C, phosphatidylinositol-3 kinase, and the
extracellular signal-regulated kinase (ERK) pathway (Greene and Kaplan,
1995). More recently, attention has also been focused on the
low-affinity NGF receptor p75NGFR, which not only
regulates trkA signaling and NGF binding affinity to trkA (Benedetti et
al., 1993; Hantzopoulos et al., 1994; Verdi et al., 1994), but
additionally is instrumental in engaging death pathways of NGF
(Rabizadeh et al., 1993; Cassacia-Bonnefil et al., 1996; Frade et al.,
1996; Van der Zee et al., 1996). Early in development, binding of NGF
to p75NGFR causes the elimination of excess retinal
neurons of chick embryos, which do not contain trkA (Frade et al.,
1996). Similarly, p75NGFR mediates apoptosis
in vivo of developing neostriatum and cholinergic forebrain
neurons only in the absence of trkA (Van der Zee et al., 1996).
Moreover, death of mature oligodendrocytes is mediated by interaction
of NGF with p75NGFR (Cassacia-Bonnefil et al.,
1996). Although the mechanism by which p75NGFR
initiates the death cascade is not well understood,
p75NGFR displays structural similarity to the TNF
family of receptors that initiate cell death in lymphocytes. Recently,
it has been shown that NGF, similar to TNF-, activates the
sphingomyelinase cycle through p75NGFR (Dobrowsky et
al., 1995; Carter et al., 1996). Moreover, binding of NGF, but not BDNF
or neurotrophin-3, to p75NGFR in mature
oligodendrocytes results in sustained increases in intracellular
ceramide and c-Jun amino-terminal kinase (JNK) activity (Cassacia-Bonnefil et al., 1996). Interestingly, enhanced ceramide production was abolished in the presence of trkA, and inhibition of the
tyrosine kinase activity of trkA with K252a restored the ability of NGF
to induce sphingomyelin hydrolysis (Dobrowsky et al., 1995), suggesting
that there may be crosstalk between these two NGF receptor-dependent
signaling pathways.
In this study, we found that in PC12 cells NGF stimulated the formation
of a further metabolite of ceramide, SPP, by markedly activating
sphingosine kinase, the enzyme that catalyzes the phosphorylation of
sphingosine to SPP. Pretreatment of PC12 cells with K252a blocked both
the increase in sphingosine kinase activity and trkA tyrosine phosphorylation, suggesting that activation of sphingosine kinase by
NGF is mediated by trkA. SPP has been previously implicated as a second
messenger playing an important role in signaling pathways initiated by
the PDGF receptor (Olivera and Spiegel, 1993; Spiegel and Milstien,
1995). We used two approaches to examine the potential role of SPP in
the pleiotropic responses to NGF in PC12 cells. First, addition of
exogenous SPP protected PC12 cells from apoptosis induced by serum
withdrawal. Second, N,N-dimethylsphingosine, a competitive
inhibitor of sphingosine kinase, not only induced apoptosis by itself,
but reduced the cytoprotective effect of NGF, which could then be
restored by readdition of SPP. Thus, it seems that activation of
sphingosine kinase by binding of NGF to trkA and subsequent formation
of SPP may play an important role in the survival effect of NGF.
Elevation of ceramide induced by serum deprivation was also prevented
by NGF treatment, suggesting that exposure of PC12 cells to NGF has a
major impact on the intracellular ratio of ceramide to SPP.
SPP may also play an important role in neuronal differentiation.
Although addition of SPP to PC12 cells did not induce neuritogenesis, it markedly enhanced neurofilament expression induced by NGF. It should
be noted that the effects of exogenous SPP may be complex, because
certain types of neuronal cells may possess cell surface receptors for
SPP. In differentiated N1E-115 neuroblastoma cells, exogenous SPP
induces  -dependent neurite retraction and soma rounding (Postma et
al., 1996), although this effect was not observed in PC12 cells. In
further support of a role for endogenous SPP in PC12 differentiation,
we found that N,N-dimethylsphingosine inhibited NGF-induced
differentiation. Although relatively high concentrations of
N,N-dimethylsphingosine have been shown to also inhibit PKC
activity (Khan et al., 1990), the concentrations used in this study
markedly inhibit sphingosine kinase activity without having a
significant effect on PKC. Previously, it has been shown that
sphingosine can suppress NGF-directed neurite outgrowth in PC12 cells
(Hall et al., 1988). This effect may be mediated by conversion of
sphingosine to ceramide, because elevation of ceramide levels by
treatment of PC12 cells with sphingomyelinase suppresses neurite
outgrowth induced by NGF via a PKC-independent pathway (Tamura et al.,
1994). Moreover, increased ceramide levels within distal neurites
inhibit neurite outgrowth in cultured rat sympathetic neurons,
suggesting that ceramide negatively regulates neurite growth (Posse de
Chaves et al., 1997). In agreement, we observed that NGF decreases the
elevation of ceramide induced by removal of trophic support. As an
added complexity, in Neuro2A neuroblastoma cells, both sphingosine and
ceramide stimulate differentiation (Riboni et al., 1995). Thus, the
role of sphingolipid metabolites in neuronal differentiation may vary
with different cell types.
Activations of three mitogen-activated protein kinases (MAPKs) present
in distinct but related pathways play crucial roles in proliferation,
differentiation, and survival of PC12 cells. The importance of trkA in
NGF-stimulated extracellular signal-regulated kinases ERK-1 and ERK-2
in the Ras cascade leading to proliferation and neuritogenesis has been
well established (Marshall, 1995). Recent studies have implicated
stress-activated protein kinases, also known as JNKs, as well as p38 as
obligatory components of cell death pathways in PC12 cells (Xia et al.,
1995; Park et al., 1996). NGF withdrawal from PC12 cells results in
simultaneous activation of JNK and inhibition of ERK (Xia et al.,
1995). Experiments with dominant-interfering or constitutively
activated forms of various components of the MAPK pathway demonstrate
that activation of JNK and concurrent inhibition of ERK are critical
for promotion of cell death in PC12 cells, and conversely, activation
of ERK signals and suppression of JNK leads to the promotion of cell survival (Xia et al., 1995). It has been proposed that survival and
death of PC12 cells may be determined by the dynamic balance between
ERK and JNK and p38 signaling (Xia et al., 1995). Previously, it has
been shown that transient activation of ERK stimulates cell growth, and
prolonged activation results in differentiation (Marshall, 1995). In a
similar manner, the duration of JNK activation may also be a crucial
factor in mediating the signaling decision. Transient JNK induction
leads to cellular proliferation, whereas sustained activation results
in apoptosis (Chen et al., 1996). Therefore, a single kinase-signaling
pathway may have distinct functions depending on the induction pattern
in the context of other signaling pathways. It is important to note
that ceramide and SPP have opposing effects on these pathways. Ceramide
induces activation of JNK in many cell types, including HepG2 cells
(Kyriakis et al., 1994), HL-60 cells (Westwick et al., 1995), rat
glomerular mesangial cells (Coroneos et al., 1996), airway smooth
muscle cells (Pyne et al., 1996), and U937 monoblastic leukemia cells (Cuvillier et al., 1996). Dominant-negative N-terminal
deletion mutants of c-Jun and dominant-negative mutants of MEK4, the
kinase that phosphorylates and activates JNK, block ceramide-mediated apoptosis (Verheij et al., 1996). However, SPP, in contrast to ceramide, not only stimulates ERK-1 and ERK-2 in many cell types, such
as Swiss 3T3 fibroblasts, airway smooth muscle cells, HL-60 human
promyelocytic cells, and U937 cells (Wu et al., 1995; Cuvillier et al.,
1996; Pyne et al., 1996), it also inhibits JNK activity stimulated by
TNF- or ceramide (Cuvillier et al., 1996). Although growth factors
can increase SPP levels, levels of ceramide are elevated during stress
conditions (Cuvillier et al., 1996). Thus, the relative intracellular
levels of ceramide and SPP and potential consequent activation or
inhibition of distinct members of the MAPK cascades are important
factors in determining the fate of cells (Cuvillier et al., 1996). This
study suggests that such reciprocal situations may also be important
for NGF action. As similarly speculated by Posse de Chaves et al.
(1997), when growth arrest and neurite retraction are required, it is
anticipated that trkA functions would be inhibited, and engagement of
p75NGFR would activate sphingomyelinase activity,
leading to increased ceramide levels, and conversely, when survival and
neurite extensions are required, trkA would not only suppress the
ability of p75NGFR to stimulate sphingomyelinase, it
would also stimulate sphingosine kinase and increase SPP levels. Thus,
crosstalk between p75NGFR and trkA may modulate the
production of ceramide and SPP, affecting pathways involved in
maintenance and differentiation of neuronal cells.
FOOTNOTES
Received March 7, 1997; revised June 12, 1997; accepted June 27, 1997.
This work was supported by Research Grant 1RO1 CA61774 from the
National Institutes of Health. We thank Dr. Gordon Guroff for helpful
suggestions.
Correspondence should be addressed to Sarah Spiegel, Basic Science
Building, Room 353, Georgetown University Medical Center, 3900 Reservoir Road, NW, Washington, DC 20007.
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K. Itagaki and C. J. Hauser
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A. C. Yopp, G. J. Randolph, and J. S. Bromberg
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A. Kihara, M. Ikeda, Y. Kariya, E.-Y. Lee, Y.-M. Lee, and Y. Igarashi
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X. Shu, W. Wu, R. D. Mosteller, and D. Broek
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H. Le Stunff, I. Galve-Roperh, C. Peterson, S. Milstien, and S. Spiegel
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K. R. Johnson, K. P. Becker, M. M. Facchinetti, Y. A. Hannun, and L. M. Obeid
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S. Spiegel and S. Milstien
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T.-Y. Chin, H.-M. Hwang, and S.-H. Chueh
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H. M. ROSENFELDT, J. P. HOBSON, M. MACEYKA, A. OLIVERA, V. E. NAVA, S. MILSTIEN, and S. SPIEGEL
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S. S. Castillo and D. Teegarden
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V. E. Nava, O. Cuvillier, L. C. Edsall, K. Kimura, S. Milstien, E. P. Gelmann, and S. Spiegel
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F. Wang, J. R. Van Brocklyn, L. Edsall, V. E. Nava, and S. Spiegel
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A. Olivera, T. Kohama, L. Edsall, V. Nava, O. Cuvillier, S. Poulton, and S. Spiegel
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A. OLIVERA, L. EDSALL, S. POULTON, A. KAZLAUSKAS, and S. SPIEGEL
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A. Olivera, T. Kohama, Z. Tu, S. Milstien, and S. Spiegel
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H. Liu, M. Sugiura, V. E. Nava, L. C. Edsall, K. Kono, S. Poulton, S. Milstien, T. Kohama, and S. Spiegel
Molecular Cloning and Functional Characterization of a Novel Mammalian Sphingosine Kinase Type 2 Isoform
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S. M. Pitson, P. A. B. Moretti, J. R. Zebol, P. Xia, J. R. Gamble, M. A. Vadas, R. J. D'Andrea, and B. W. Wattenberg
Expression of a Catalytically Inactive Sphingosine Kinase Mutant Blocks Agonist-induced Sphingosine Kinase Activation. A DOMINANT-NEGATIVE SPHINGOSINE KINASE
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E. P. de Chaves, M. Bussiere, B. MacInnis, D. E. Vance, R. B. Campenot, and J. E. Vance
Ceramide Inhibits Axonal Growth and Nerve Growth Factor Uptake without Compromising the Viability of Sympathetic Neurons
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S. M. Mandala, R. Thornton, I. Galve-Roperh, S. Poulton, C. Peterson, A. Olivera, J. Bergstrom, M. B. Kurtz, and S. Spiegel
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