Basic Fibroblast Growth Factor-induced Proliferation of Primary Astrocytes

EVIDENCE FOR THE INVOLVEMENT OF SPHINGOMYELIN BIOSYNTHESIS*

Laura RiboniDagger, Paola Viani, Rosaria Bassi, Paola Giussani, and Guido Tettamanti

From the Department of Medical Chemistry and Biochemistry, Study Center for the Functional Biochemistry of Brain Lipids, University of Milan, via Fratelli Cervi 93, LITA-Segrate, Segrate, 20090 Milan, Italy

Received for publication, December 21, 2000, and in revised form, January 11, 2001



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We recently reported that the marked decrease in cellular ceramide in primary astrocytes is an early event associated with the mitogenic activity of basic fibroblast growth factor (bFGF) (Riboni, L., Viani, P., Bassi, R., Stabieini, A., and Tettamanti, G. (2000) GLIA 32, 137-145). Here we show that a rapid activation of sphingomyelin biosynthesis appears to be the major mechanism responsible for the fall in ceramide levels induced by bFGF. When quiescent astrocytes were treated with bFGF, an increased amount of newly synthesized ceramide (from either L-[3H]serine or [3H]sphingosine) was directed toward the biosynthesis of sphingomyelin. Conversely, bFGF did not appear to affect ceramide levels by other metabolic pathways involved in ceramide turnover such as sphingomyelin degradation and ceramide biosynthesis, degradation, and glucosylation. Enzymatic studies demonstrating a relevant and rapid increase in sphingomyelin synthase activity after bFGF treatment have provided a convincing explanation for the activation of sphingomyelin biosynthesis. The bFGF-induced increase in sphingomyelin synthase appears to depend on a post-translational activation mechanism. Moreover, in the presence of brefeldin A, the activation of sphingomyelin biosynthesis was abolished, suggesting that the enzyme is located in a compartment other than the Golgi apparatus. Also the phosphatidylcholine-specific phospholipase C inhibitor D609 exerted a potent inhibitory effect on sphingomyelin biosynthesis. Finally, we demonstrate that inhibition of sphingomyelin biosynthesis by brefeldin A or D609 led to a significant inhibition of bFGF-stimulated mitogenesis. All this supports that, in primary astrocytes, the early activation of sphingomyelin synthase is involved in the bFGF signaling pathway leading to proliferation.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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).

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).

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).

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.

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).

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    FOOTNOTES

* This work was supported by Grants MURST PRIN 1998 and 2000 and MURST ex 60% (to L. R.) and Grant MURST PRIN 1999 (to P. V.) from the Italian Ministry of University and Research and by Target Project on Biotechnology Contract 99.00500.PF49 from the Italian Research Council (to G. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 39-02-26423201; Fax: 39-02-26423209; E-mail: laura.riboni@unimi.it.

Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M011570200

    ABBREVIATIONS

The abbreviations used are: bFGF, basic fibroblast growth factor; SM, sphingomyelin; BMEM, basal modified Eagle's medium; C6-ceramide, N-hexanoyl-D-erythro-sphingosine; BFA, brefeldin A; HPTLC, high performance thin-layer chromatography; Ser, L-serine; Sph, D-erythro-sphingosine; Glc-Cer, glucosylceramide; PC, phosphatidylcholine.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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