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INTRODUCTION |
Ceramide, a key sphingolipid metabolite in both the biosynthesis
and degradation of complex sphingolipids, is involved in the signal
transduction of different extracellular stimuli that lead to cell
proliferation, cell differentiation, cell cycle arrest, and apoptotic
cell death (reviewed in Refs. 1-3). In all these events, the ceramide
concentration, possibly at specific subcellular sites, is crucial.
There is evidence that different activators such as cytokines, growth
factors, and hormones elicit their biological effect by modulating the
activity of sphingomyelinases, ceramide synthase, or neutral ceramidase
(reviewed in Refs. 1 and 3-5) and thus affect ceramide levels.
A role of ceramide as an intracellular mediator of specific
extracellular agents has been recognized also in cells from the central
nervous system (reviewed in Refs. 6-8). In neuronal and glial cells,
the administration of differentiating or apoptotic agents results
in increased cellular levels of ceramide, which, in turn, participate
in the cascade of events producing the final effects (9-12). In a
recent study (13), we demonstrated that ceramide plays a role in the
growth control of glial cells by basic fibroblast growth factor
(bFGF),1 a factor stimulating
astrocyte proliferation during neuronal development, as a response to
injury, and in tumorigenesis (14-17). In particular, we found that, in
quiescent primary astrocytes, the induction of proliferation by bFGF is
paralleled by an early and marked decrease in the cellular content of
ceramide, an event associated with the signaling pathways responsible
for the mitogenic activity of bFGF (13). However, the metabolic pathway
responsible for the reduction of the ceramide levels in astrocytes
after bFGF treatment still remained to be discovered. In fact, the
bFGF-stimulated astrocytes showed no variation in either sphingomyelin
(SM) degradation or ceramide cleavage (13). Note that these variations
were observed in a glioma cell line stimulated by neurotrophins (9) and
in bFGF-treated extraneural cells (18), respectively. Thus, this study focused on the metabolic pathway underlying the decrease in
cellular ceramide, an early event triggered by bFGF in stimulating astrocyte proliferation. Our results strongly support the involvement of SM biosynthesis in the response of astrocytes to mitogenic doses of
bFGF.
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EXPERIMENTAL PROCEDURES |
Materials--
Basal modified Eagle's medium (BMEM), fetal calf
serum,
N-hexanoyl-D-erythro-sphingosine
(C6-ceramide), actinomycin D, brefeldin A (BFA), and bovine
serum albumin were from Sigma. High performance thin-layer
chromatography (HPTLC) silica gel plates were from Merck (Darmstadt,
Germany). Cycloheximide and D609 (tricyclodecan-9-yl xanthogenate) were
from BIOMOL Research Laboratories Inc. (Plymouth Meeting, PA). bFGF was
from PeproTech EC Ltd. (London, United Kingdom).
L-[3H]Serine (Ser; 19.7 Ci/mmol),
C6-[3H]ceramide (labeled at C-3 of the
long chain base; 19.6 Ci/mmol), and
[methyl-3H]thymidine (20 Ci/mmol) were from
PerkinElmer Life Sciences. Ganglioside GM1 (ganglioside nomenclature is
based on that of Svennerholm (73)) and
D-erythro-sphingosine (Sph), isotopically tritiated at C-3, were prepared and purified as previously described (19); their specific radioactivity was 1.9 Ci/mmol, and the radiochemical purity, as assessed by HPTLC and autoradioscanning, was
better than 98%. Standard radioactive sphingolipids were obtained as
previously reported (20).
Cell Cultures--
Primary astrocyte cultures were prepared from
the cerebella of 8-day-old neonatal rats as previously described (21,
22). Cells were plated at a density of 105
cells/cm2 on dishes (35 mm for proliferation assays and 60 mm for metabolic studies) coated with poly-L-lysine. The
growth medium consisted of BMEM supplemented with 2 mM
glutamine, 180 µM gentamycin, 30 mM glucose,
and 10% heat-inactivated fetal calf serum at 37 °C in a humidified
atmosphere of 5% CO2 and 95% air. The medium was changed
after 24 h and every 2nd day thereafter. On the 10th day in
culture, when the type 1 astrocytes accounted for >90% of the cell
population (22), the plates were washed twice with supplemented BMEM
containing 0.5% fetal calf serum, and the cells were incubated for
48-72 h in the same medium prior to use as quiescent cells. At the
time of experiments, 20 ng/ml bFGF was added, and the cells were
incubated for different times. Cell viability was assessed by
the trypan blue exclusion test.
Metabolic Studies--
In pulse experiments, quiescent cells
were fed 200 nM [3H]Ser, 40 nM
[3H]Sph, or 1 µM
[D-erythro-sphingosine-3H]GM1
in serum-free supplemented BMEM (20) in the absence or presence of 20 ng/ml bFGF. In chase experiments, after a pulse with the above
radiolabeled compounds in the absence of bFGF, the cells were submitted
to a period of chase in serum-free supplemented BMEM containing 20 ng/ml bFGF. After appropriate pulse or chase times (see below), the
medium was carefully collected, and the cells were rapidly washed with
cold phosphate-buffered saline, harvested by a rubber scraper, and
submitted to lipid extraction. The total lipids were extracted and
partitioned from cells at 4 °C as recently described (20). After
partitioning, the organic phase was subjected to a mild alkaline
methanolysis (treatment with methanolic 0.1 N KOH at
37 °C for 1 h). After counting for radioactivity, the obtained
methanolyzed organic phase and aqueous phase were analyzed by HPTLC
using the following solvent systems (by volume): A,
chloroform/methanol/water (55:20:3); B, chloroform, methanol, and 32%
NH4OH (40:10:1); C, chloroform, methanol, and 0.2%
CaCl2 (55:45:10); and D, chloroform, methanol, and 0.5%
CaCl2 (55:45:10). After HPTLC, the plates were radioscanned
with a digital autoradiograph (Berthold, Bad Wiedbad, Germany)
and then submitted to fluorography. Recognition and identification of
[3H]ceramide, [3H]SM,
[3H]glucosylceramide (Glc-Cer), and other
3H-labeled metabolites were performed as previously
described (19, 20). In some cases, the medium was centrifuged
(1000 × g for 10 min at 4 °C), and the supernatant
was processed for volatile radioactivity (20).
Treatment of Cultured Astrocytes with Metabolic
Inhibitors--
Quiescent cultures were incubated for different times
in supplemented BMEM containing 20 ng/ml bFGF in the absence or
presence of 1 µM actinomycin D, 10 µg/ml cycloheximide,
1-2 µg/ml BFA, or 10-20 µg/ml D609. Stock solutions of
cycloheximide and BFA were prepared in distilled methanol and ethanol,
respectively, and added to the medium to the desired final
concentration (the final solvent concentration never exceeded 0.1%).
D609 was dissolved in sterile phosphate-buffered saline on the day of
the experiment. In the presence of all inhibitors at the concentrations
used, no sign of cell toxicity was detected for up to 24 h of treatment.
Sphingomyelin Synthase Assay--
SM synthase was assayed as
previously described (23), with some modifications. The astrocytes were
washed with phosphate-buffered saline and collected by scraping in
ice-cold lysis buffer containing 25 mM Tris-HCl (pH 7.4), 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml each
aprotinin, leupeptin, and pepstatin. Cell homogenates, obtained by
sonication in lysis buffer (3 × 10 s at 4 °C), were used
fresh as the enzyme source. In the enzyme assay, the reaction mixture
contained 50 mM Tris-HCl (pH 7.4), 25 mM KCl, 1 mM EDTA, and 5-20 µg of cell proteins in a final volume
of 50 µl. The reaction was started by the addition of 2 nmol of
C6-[3H]ceramide as an equimolar complex with
fatty acid-free bovine serum albumin (complex specific activity of 300 nCi/nmol). After incubation for 10-30 min at 37 °C, the reaction
was terminated by the addition of 200 µl of chloroform/methanol (1:2,
by volume) at 4 °C; the tubes were centrifuged, and aliquots of the
supernatant were applied to an HPTLC plate that was developed in
solvent system D (see above). Background values were obtained by
blocking the reaction at time 0, with incubation at 37 °C being omitted.
Proliferation Assays--
Quiescent cultures were incubated with
bFGF for 24 h, and 1 µCi/ml [3H]thymidine was
added to each dish 4 h before cell harvesting. The
[3H]thymidine incorporation into trichloroacetic
acid-insoluble materials was then determined. Each independent
experiment was performed at least in triplicate, using independent cultures.
Other Methods--
Total protein was assayed (24) using bovine
serum albumin (fraction V) as the standard. SM was determined, after
perchloric acid digestion, as reported (25, 26). Radioactivity was
determined by liquid scintillation counting, fluorography, or
radiochromatoscanning using the digital autoradiograph. Statistical
significance of differences was determined by Student's t test.
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RESULTS |
Effect of bFGF on Ceramide Metabolism from
[3H]Ser--
To investigate the biochemical mechanisms
involved in the rapid decrease in ceramide upon bFGF treatment, the
initial experiments were focused on the possible effects of bFGF on
ceramide biosynthesis. For this purpose, [3H]Ser,
the precursor of the de novo biosynthetic pathway of
ceramide, was administered to quiescent and bFGF-treated astrocytes,
and its metabolic fate was followed in pulse and pulse-chase
experiments. In pulse experiments, 45 and 90 min after
[3H]Ser administration, quiescent and bFGF-treated cells
incorporated similar amounts of radioactivity, which increased with
time (Fig. 1, upper left
panel). Under these experimental conditions, also the amount of
3H-labeled sphingolipids, measured after partitioning and
mild alkaline methanolysis, increased with pulse time, but remained unaffected by bFGF treatment (Fig. 1, upper middle panel).
Within the 3H-labeled sphingolipids, the radiolabeled
ceramide also increased during the pulse, with no difference between
the control and bFGF-stimulated astrocytes (Fig. 1, upper right
panel). After a 90-min pulse with [3H]Ser,
radioactive ceramide decreased with chase time, and
[3H]SM was detected as the major 3H-labeled
sphingolipid (Fig. 1, lower panels). Under such conditions, the bFGF administration was followed by a marked, significant reduction
of [3H]ceramide (Fig. 1, lower left panel);
the amount of this sphingolipid was ~40 and 50% lower than that in
quiescent cells after 2 and 4 h of chase, respectively. At the
same times, in bFGF-treated cells, the radioactivity incorporated into
SM was increased (Fig. 1, lower middle panel), whereas the
amount of radioactive Glc-Cer was similar to that in quiescent cells
(Fig. 1, lower right panel). These data suggest that
ceramide utilization as a precursor of SM biosynthesis could be
stimulated by bFGF.

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Fig. 1.
Effect of bFGF on [3H]Ser
incorporation into sphingolipids. Upper panels, 200 nM [3H]Ser was administered to quiescent
astrocytes in the absence or presence of 20 ng/ml bFGF. Incorporation
of radioactivity into the total lipid extract (left panel),
sphingolipids (middle panel), and ceramide (right
panel) was measured after 45 and 90 min of pulse. Lower
panels, quiescent cells were incubated with 200 nM
[3H]Ser for 90 min and then submitted to a chase in the
absence or presence of 20 ng/ml bFGF. Incorporation of radioactivity
into ceramide (left panel), SM (middle panel),
and Glc-Cer (right panel) was measured after 2 and 4 h
of chase. Data are the means ± S.D. of three experiments
performed in duplicate on independent cultures. *, p < 0.05; **, p < 0.01 (bFGF-treated versus
quiescent cells at the same chase time).
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Effect of bFGF on Ceramide Metabolism from Exogenous and Catabolic
[3H]Sph--
Taking into account that exogenous Sph is
rapidly incorporated first into ceramide and then into SM in astrocytes
(27, 28), an additional study using [3H]Sph was
performed. Quiescent cells were submitted to a 20-min pulse with this
molecule (sufficient to obtain [3H]ceramide as the major
3H-labeled metabolite) in the absence or presence of bFGF.
The results demonstrate that, in quiescent and bFGF-treated cells, the
amount of [3H]ceramide synthesized from
[3H]Sph was similar (132 ± 10 and 125 ± 12 nCi/mg of protein, respectively). In a second set of experiments, after
a 20-min pulse of quiescent cells with [3H]Sph, the fate
of newly synthesized [3H]ceramide was followed in a chase
in the absence or presence of bFGF. In bFGF-treated cells, a
significant decrease in radiolabeled ceramide (Fig.
2, upper left panel)
concomitant with an increase in [3H]SM (upper right
panel) was detected after both 30 and 60 min of chase. At the same
times, quite similar amounts of radioactive Glc-Cer and GM3 were
produced in quiescent and bFGF-treated cells (Fig. 2, lower
panels). Under these conditions, also the complete degradation of
[3H]ceramide did not appear to be modified by bFGF. In
fact, the amount of 3H2O (detectable as the
final degradation product) released in the medium after a 60-min chase
accounted for 2.5 ± 0.27 and 2.3 ± 0.29 nCi/mg of protein
in quiescent and bFGF-treated cells, respectively.

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Fig. 2.
Effect of bFGF on [3H]Sph
incorporation into sphingolipids. Quiescent astrocytes were
incubated with 40 nM [3H]Sph for 20 min and
then submitted to a chase for 30 or 60 min in the absence or presence
of 20 ng/ml bFGF. The radioactivity incorporated into ceramide, SM,
Glc-Cer, and GM3 is reported. Data are the means ± S.D. of three
experiments performed in triplicate on independent cultures. *,
p < 0.05; **, p < 0.01 (bFGF-treated
versus quiescent cells at the same chase time).
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Since Sph in cells is derived essentially from sphingolipid degradation
and is mainly recycled for biosynthetic purposes (1), further
experiments were performed to confirm that ceramide, synthesized from
the salvage of intracellularly produced Sph, was affected in
bFGF-stimulated cells. Thus, we followed the turnover of radiolabeled ceramide and the formation of SM during a chase, after a pulse with
[D-erythro-sphingosine-3H]GM1.
The experimental conditions used allowed us to follow the recycling of
Sph produced from ganglioside degradation and to determine the fate of
ceramide produced from the salvage of catabolically produced Sph (29).
The results demonstrate that bFGF did not affect the total amount of
radioactivity measured in the organic phase (Fig.
3, upper left panel), but did
affect the distribution within the major components. In fact, a marked
reduction of [3H]ceramide paralleled by a relevant
increase in [3H]SM occurred at both chase times (Fig. 3,
lower panels). Under such conditions, no significant
variation of [3H]Glc-Cer was detected (Fig. 3,
upper right panel).

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Fig. 3.
Effect of bFGF on [3H]Sph
salvage pathways. Quiescent astrocytes were incubated with 1 µM
[D-erythro-sphingosine-3H]GM1
for 2 h at 4 °C and then submitted to a chase for 2 or 3 h
in the absence or presence of 20 ng/ml bFGF. The radioactivity
incorporated into the organic phase, Glc-Cer, ceramide, and SM is
reported. Data are the means ± S.D. of two experiments performed
in duplicate on independent cultures. *, p < 0.05; **,
p < 0.01 (bFGF-treated versus quiescent
cells at the same chase time).
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In Vitro Assay of SM Synthase--
On the basis of this evidence,
we next assessed the in vitro activity of SM synthase. In
these experiments, quiescent astrocytes were incubated with bFGF for
different times, subsequently scraped off the dishes, and assayed for
enzyme activity using C6-[3H]ceramide as
substrate and optimized conditions. As shown in Fig.
4, SM synthase activity was found to be
enhanced by bFGF treatment. When the reaction rate was linear with
incubation time and enzyme protein (Fig. 4, upper panels),
the formation rate of the reaction product
C6-[3H]SM was noticeably higher
(1.8-2-fold) in the bFGF-treated cells than in the quiescent cells
after a 1-h treatment with bFGF. In this in vitro assay, the
activity of Glc-Cer synthase was 0.38 ± 0.04 and 0.42 ± 0.05 nmol/mg/min in quiescent and bFGF-treated astrocytes,
respectively.

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Fig. 4.
Activity of SM synthase in quiescent and
bFGF-treated astrocytes. Quiescent astrocytes were incubated with
20 ng/ml bFGF for 1 h (upper panels) or 2 h
(lower panel) in culture. The cells were then scraped off
the plates and assayed for SM synthase using
C6-[3H] ceramide as substrate, as described
under "Experimental Procedures." Upper panels, SM
synthase was measured at different incubation times (left
panel; 20 µg of protein/50 µl) or at different protein
concentrations (right panel; 20-min incubation, 50-µl
final volume) in quiescent and bFGF-treated cells. Lower
panel, shown is the effect of inhibition of RNA or protein
synthesis on SM synthase activity. Quiescent astrocytes were incubated
with 10 µg/ml cycloheximide (CYC) for 10 min or with 1 µM actinomycin D (ACT) or 20 µg/ml D609 for
30 min prior to incubation with bFGF for 2 h. The cells were then
scraped off the plates, homogenized, and assayed for SM synthase. All
values are the means ± S.D. of at least three individual
experiments.
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Preincubation with the RNA synthesis inhibitor actinomycin or the
protein synthesis inhibitor cycloheximide had no effect on the
bFGF-stimulated increase in SM synthase activity (Fig. 4, lower
panel). Recent evidence demonstrating that D609, a
phosphatidylcholine (PC)-specific phospholipase C inhibitor (30, 31),
dramatically inhibits SM synthase in SV40-transformed fibroblasts (23)
prompted us to investigate the effect of this compound on
bFGF-stimulated SM synthase. The addition of 20 µg/ml D609 to
astrocytes during bFGF stimulation resulted in ~85% inhibition of SM
synthase activity (Fig. 4, lower panel). A similar effect
(~80% inhibition) was obtained at 10 µg/ml.
Effect of bFGF on Cellular SM Content--
The activation of SM
biosynthesis after bFGF treatment led us to evaluate whether the
cellular level of SM was also affected by this growth factor. The
results demonstrate that after 2 and 4 h of bFGF treatment, the
amount of total SM in quiescent cells (13.67 ± 1.18 and
13.91 ± 1.16 nmol/mg of protein, respectively) was very similar
to that in bFGF-treated cells (13.97 ± 1.26 and 14.31 ± 1.42 nmol/mg of protein, respectively).
Effect of BFA on SM Biosynthesis--
To obtain evidence
concerning the subcellular location of the bFGF-stimulated SM synthase,
a study was made of the effect of BFA, an inhibitor of the anterograde
vesicular transport between Golgi compartments (32), on
[3H]Sph incorporation into SM and other sphingolipids. In
both quiescent and bFGF-treated astrocytes, the presence of BFA during
a pulse with [3H]Sph resulted in an increase in
[3H]ceramide and, above all, [3H]Glc-Cer,
paralleled by a marked reduction of [3H]SM (Fig.
5). Interestingly, the observed
bFGF-induced decrease in [3H]ceramide and concomitant
elevation of [3H]SM did not occur with BFA.

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Fig. 5.
Effect of BFA on SM biosynthesis in
bFGF-treated astrocytes. Quiescent astrocytes were incubated with
40 nM [3H]Sph for 2 h in the absence or
presence of 20 ng/ml bFGF. When present, 1 µg/ml BFA was added 30 min
prior to the pulse and maintained thereafter. The radioactivity
incorporated into ceramide (left panel), Glc-Cer
(middle panel), and SM (right panel) is reported.
Data are the means ± S.D. of three experiments performed in
duplicate on independent cultures. **, p < 0.01 (bFGF-treated versus quiescent cells).
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Effect of Drugs Affecting SM Synthase on the Mitogenic Activity of
bFGF--
The possible connection between stimulation of SM synthase
and the mitogenic effect of bFGF was investigated. For this purpose, [3H]thymidine incorporation into DNA was assessed in
cells treated with BFA or D609 in the early phase of bFGF stimulation.
As shown in Fig. 6, in astrocytes treated
with bFGF, both BFA and D609 exerted a potent inhibitory effect on the
incorporation of [3H]thymidine into DNA.

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Fig. 6.
Effects of inhibitors of SM biosynthesis on
bFGF-induced astrocyte proliferation. Quiescent cells were
incubated with 20 ng/ml bFGF in the absence (bar 1) or
presence of 1 or 2 µg/ml BFA (bars 2 and 3,
respectively) or 10 or 20 µg/ml D609 (bars 4 and
5, respectively) for 4 h. The medium containing
inhibitors was then removed, and all cells were further incubated with
bFGF for up to 24 h. Cells were pulsed for the last 4 h with
[3H]thymidine and processed as described under
"Experimental Procedures." Data are expressed as percent of the
radioactivity incorporated in bFGF-stimulated astrocytes in the absence
of inhibitors (control, taken as 100%). Each bar is the mean ± S.D. of three independent experiments performed in triplicate.
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DISCUSSION |
The major finding of this study is that an increase in SM
biosynthesis appears to be the major mechanism responsible for the rapid decrease in ceramide levels induced by bFGF in primary
astrocytes, an event associated with bFGF-stimulated astrocyte
proliferation (13). In fact, when quiescent cells were treated with
bFGF, an increased amount of newly synthesized ceramide (either from [3H]Ser or [3H]Sph) was directed toward the
biosynthesis of SM. On the other hand, under the same conditions, the
biosynthesis of ceramide through either the de novo pathway
or Sph recycling was not affected by bFGF. The activation of SM
biosynthesis was further confirmed by studies demonstrating a
significant increase in SM synthase activity after 1-2 h of
stimulation with bFGF. Conversely, no effect was detected on ceramide
biosynthesis, SM degradation, or ceramide removal by ceramidase in
the early phases of bFGF stimulation (this study and Ref. 13). Under
our experimental conditions, also the biosynthesis of Glc-Cer,
particularly efficient in astrocytes (28) and necessary for bFGF
stimulation of axonal growth in hippocampal neurons (33, 34), was not
influenced by the growth factor.
To the best of our knowledge, this is the first evidence of an
association between activation of SM biosynthesis and growth stimulation of cells. In this respect, it is worth noting that SM
synthase recently emerged as a negative regulator of endogenous ceramide levels involved in signaling processes (35-37). Moreover, it
has been demonstrated that SV40-transformed lung fibroblasts contain a
significantly higher SM synthase activity than normal lung fibroblasts,
and a role for SM synthase in the regulation of cell growth has been
suggested (23). Our results appear to corroborate this hypothesis.
We also observed that the stimulation of SM synthase activity by bFGF
was not paralleled by significant changes in cellular SM content. This
apparent contradiction could be explained by the evidence that the
cellular concentration of SM in rat cerebellar astrocytes is 20-fold
higher than that of ceramide (13, 27).
Evidence from the literature supports that SM can be synthesized at
more than one subcellular site. Although most studies indicate that SM
synthase is mainly located in the cis- and medial-Golgi (38-44), a different enzyme catalyzing the same reaction has been detected in the plasma membrane (38, 42, 45-50), possibly exposed to
the cytosolic leaflet (49). Recently, evidence for a form of SM
synthase in the trans-Golgi network (51) and at the nuclear level (52) has also come to light. Using BFA as a tool to inspect the
subcellular localization of SM biosynthesis, we demonstrated that this
compound exerts a dual action on ceramide metabolic processing,
consisting of a striking increase in Glc-Cer and a marked reduction of
SM biosynthesis in both quiescent and bFGF-treated astrocytes. Since
BFA disrupts the Golgi apparatus, resulting in the redistribution of
the cis,trans-Golgi stacks in the endoplasmic reticulum (32), the increase in Glc-Cer upon BFA treatment is in
agreement with the cis,medial-Golgi stack location of the
glucosyltransferase involved in its biosynthesis (53-55). On the other
hand, the inhibition of SM biosynthesis by BFA in astrocytes is in
contrast with the cis-Golgi as the major location of SM
synthase (38-44). A possible explanation of the different effect
exerted by BFA on Glc-Cer and SM biosynthesis could reside in the
different topology of Glc-Cer synthase and SM synthase (cytosolic and
luminal sides of the Golgi membrane, respectively). This hypothesis
does not appear to be likely since ceramide can "flip-flop" across
membranes very rapidly (t1/2 of seconds) (56), and
BFA does not appear to affect this spontaneous flipping. In fact, this
drug is able to induce the increase in SM synthesis, besides that of
Glc-Cer, in different non-neuronal cells (57-60). On the other hand,
the BFA-induced inhibition of SM biosynthesis in astrocytes is in
agreement with the inhibitory effect exerted by BFA on SM synthesis in
other cell types, including neuronal cells (50, 61, 62). This evidence
is in favor of a location of this metabolic step distal to the Golgi
apparatus. It thus appears that, in the central nervous system, both
neurons and astrocytes share an extra-Golgi location as the major site
of SM biosynthesis. This might be of functional relevance considering the SM involvement in the signaling mechanisms of the cells of the
nervous system. It is worth noting that BFA treatment also precludes
the stimulating effect of bFGF on SM biosynthesis, suggesting that the
SM synthase activated by this growth factor is located in a compartment
other than the Golgi apparatus. Although the results with BFA do not
allow the full identification of the subcellular site of
SM synthase, evidence from the literature suggests that it could
reside in the plasma membrane or a related compartment (trans-Golgi network) connected to signaling mechanisms.
The bFGF-induced increase in SM synthase does not seem to depend on
newly synthesized enzyme molecules since inhibitors of RNA and protein
synthesis are without effect. At present, the enzymes involved in SM
biosynthesis have not yet been molecularly defined, and the biochemical
mechanisms responsible for their regulation remain largely unknown.
Since many of the biological activities of bFGF, including its
mitogenic property, have been found to depend on a phosphorylation
cascade initiated by its receptor intrinsic tyrosine kinase (63), it is
tempting to speculate that a phosphorylation mechanism might be the
basis of the bFGF-dependent increase in SM synthase activity.
Further evidence obtained in this work is that BFA and D609, when added
to cells in the initial phases of bFGF stimulation, strongly inhibit
bFGF-stimulated mitogenesis of astrocytes. Both agents exert an
inhibitory effect on SM biosynthesis in primary astrocytes, although
through different mechanisms. Moreover, as far as BFA is concerned, its
morphologic and traffic effects are known to be rapidly and completely
reversed by removing the drug (32). The antimitogenic effect exerted by
this macrocyclic lactone, when administered in the first hours of bFGF
treatment, appears to be the result of an early metabolic impairment
rather than the depletion of cell membrane sphingolipids. Thus, the
data obtained strongly support that the early activation of SM synthase
is involved in the bFGF signaling pathway leading to cell proliferation.
The involvement of SM synthase in bFGF-induced proliferation deserves a
further comment. Since the main pathway of SM biosynthesis in mammalian
cells is catalyzed by SM synthase, and this occurs primarily through
the transfer of the phosphorylcholine group from phosphatidylcholine to
ceramide, the reaction products are both SM and diacylglycerol
(38, 45, 47, 64). Although the signaling pathways that mediate the
proliferating effect of bFGF have not been fully elucidated, there is
evidence that different growth factors, upon activation of their
receptor tyrosine kinases, induce a PC-specific phospholipase C
instrumental to their mitogenic effect (65-67). The evidence that
(a) an extra-Golgi SM synthase is up-regulated by and
necessary for bFGF growth stimulation of astrocytes, (b) the
PC-specific phospholipase C inhibitor D609 exerts an inhibitory effect
on the bFGF-induced stimulation of both SM biosynthesis and astrocyte
proliferation, and (c) neosynthesized phosphatidylcholine is
the major phosphocholine donor for SM synthesis in glial cells (68, 69)
raises the intriguing possibility, recently proposed by Luberto and
Hannun (23), that the PC-specific phospholipase C involved in cell
proliferation may, in part, be a SM synthase. Considering that the
stimulation of a PC-specific phospholipase C in the early phases of the
cell cycle appears to be a critical event in the mammalian cell
division cycle (70) and the recent evidence suggesting that, in primary
astrocytes and astrocytoma cells, a PC-specific phospholipase C is
involved in a receptor-mediated mitogen-activated protein kinase
activation and cell division (71, 72), the definition of the role of PC-specific phospholipase C/SM synthase in astrocyte growth appears to
be of particular interest.
In conclusion, the observations reported in this study suggest that the
biosynthesis of SM, due to an extra-Golgi form of SM synthase, is
up-regulated by bFGF. This activation appears to be functional for this
growth factor for "switching off" the antiproliferative signal of
ceramide and inducing DNA synthesis and astrocyte proliferation. The
uncovering of the mechanisms by which bFGF leads to SM synthase
activation presents a fascinating challenge. The regulation of this or
other enzymes controlling ceramide levels could be a possible tool to
control astrocyte proliferation in diseases such as stroke,
demyelinative disorders, and brain tumors.