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Sphingoid bioregulators in the differentiation of cells of neural origin
Lipl "°' ELSEVIER
J. Lipid Mediators Cell Signalling 14 (1996) 263-275
Sphingoid bioregulators in the differentiation of cells of neural origin Guido Tettamanti*, Alessandro Prinetti, Rosaria Bassi, Paola Viani, Paola Giussani, Laura Riboni Department of Medical Chemistry and Biochemistry, University o[" Milan, via Saldini 50, 20133, Milan, Italy
Abstract
The involvement of ceramide in the differentiation of two neuroblastoma cell lines, Neuro2a and SH-SY5Y, and cerebellar granule cells in primary culture was investigated. The following results were obtained: (a) the cellular content of ceramide markedly increased with induced differentiation of Neuro2a cells (inducers: RA, FCS deprivation), SH-SY5Y cells (inducers: RA, PMA), and spontaneous differentiation of cerebellar granule cells; (b) all the investigated cells in the differentiated form displayed a higher ability to produce ceramide from exogenously administered [3H]Sph-SM and expressed a higher content of neutral sphingomyelinase and, in the case of cerebellar granule cells, also of acidic sphingomyelinase; (c) inhibition of ceramide biosynthesis by Fumonisin B1 blocked the process of differentiation in Neuro2a and cerebellar granule cells; and (d) treatments capable of enhancing ceramide level (administration of sphingosine or C2-Ceramide) induced differentiation in both Neuro2a and SH-SY5Y cells. The data obtained support the notion that ceramide plays a general biomodulatory role in neural cell differentiation. Keywords: Neural differentiation; Neuroblastoma cells; Cultured neurons; Sphingoid bioregulators; Ceramide
Abbreviations: RA, retinoic acid; PMA, phorbol-12 myristate, 13-acetate; Cer, ceramide; C2-Cer, N-acetyl-erythro-sphingosine; Sph, D-erythro-sphingosine; SM, sphingomyelin; SM-ase, sphingomyelinase; N-SM-ase, neutral sphingomyelinase; A-SM-ase, acidic sphingomyelinase; FB, Fumonisin B1; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum. * Corresponding author. Tel.: + 39 2 70645247; fax: + 39 2 2363584; email: [email protected],unimi.it 0929-7855/96/'$15.00 © 1996 Elsevier Science B.V. All rights reserved PlI S0929-7855(96)00535-4
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1. Introduction
Increasing evidence indicate that molecules of sphingoid nature are involved in the modulation of cell response to different extracellular signals (Kolesnick, 1992; Hannun and Bell, 1993; Merrill, 1994). Particularly, ceramide (N-acyl-erythro-sphingosine) acts as a key molecule in a new signal transduction pathway, the 'sphingomyelin pathway or cycle' (Kolesnick, 1992; Hannun, 1994). In fact, in several cell lines the activation of specific receptors by vitamin D3 and cytokines (tumor necrosis factor e, interleukin-lfl and ~-interferon) induces sphingomyelin hydrolysis by sphingomyelinase, leading to increased levels of ceramide. This sphingoid molecule modulates the activity of specific protein kinases and phosphatases, as well as transcriptional factors that, in turn, control cell proliferation, oncogenesis, differentiation and apoptosis (Hakomori, 1990; Kolesnick and Golde, 1994; Merrill, 1994; Hannun, 1994; Hannun and Obeid, 1995). Several studies support a regulatory role of sphingolipids and sphingoid molecules in neuronal development (Tettamanti and Riboni, 1994). In cultured hippocampal neurons sphingolipid biosynthesis is necessary for axonal outgrowth (Harel and Futerman, 1993) and branching (Schwarz et al., 1995). Furthermore, in T9 glioma cells, the addition of a cell-permeable ceramide analog (C2-Cer) mimics nerve growth factor in causing growth inhibition and neurite outgrowth, with concomitant increase of the cellular level of ceramide (Dobrowsky et al., 1994). Evidence was also provided for the involvement of ceramide in retinoic acid induced differentiation of Neuro2a neuroblastoma cells (Riboni et al., 1995). In this experimental model Cer levels are strictly related to the process of cell differentiation and the increase of cellular Cer, obtained by different treatments, leads to neurite outgrowth and inhibition of cell growth. On these premises, we faced the question whether ceramide is a general biomodulator of neuronal differentiation. To this purpose, we employed either neurotumoral cells, the murine (Neuro2a) and human (SH-SY5Y) neuroblastoma clones, or primary cultures of cerebellar granule cells. The results obtained strongly support that Cer represents a crucial mediator of differentiation in all these cells.
2. Materials and methods
2.1. Materials
Culture media, fetal calf serum (FCS), crystalline bovine serum albumin, bovine brain sphingomyelin (SM), D-erythro-sphingosine (C-18) (Sph), retinoic acid (RA), Fumonisin B1 (FB), phorbol, 12-myristate, 13-acetate (PMA) were from Sigma (St. Louis, MO); N-acetyl-erythro-sphingosine (C2-Cer) and DLthreo-sphinganine were from Matreya (Pleasant Gap, PA); HPTLC silica gel plates were from Merck (Darmstadt, Germany). Sphingomyelin, tritiated at the
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C-3 of the long chain base ([3H]Sph-SM, 2.9 Ci/mmol), as well as standard [3H]sphingolipids were prepared as previously described (Riboni et al., 1992).
2.2. CeH Cultures The murine neuroblastoma cell line, clone NB2a, (Neuro2a, CCL-131, American Cell Type Culture Collection, Bethesda, MD) and the cloned human neuroblastoma cell line, SH-SY5Y (kindly provided by Dr. D. Fornasari, Center of Cellular and Molecular Pharmacology, C.N.R., Milan, Italy) were employed. These cells were cultured in Falcon dishes in D M E M (Neuro2a) or RPMI 1640 (SH-SY5Y) supplemented with 10% FCS, 4 m M l.-glutamine, 1 m M sodium pyruvate, 100 U/ml of penicillin and 100 /Lg/ml of streptomycin sulfate in 5% CO2-95% air humidified atmosphere at 37°C. Neural differentiation was induced as follows: (a) Neuro2a cells. Cells were plated at a 1.8 x 104/cm 2 cell density and 48 h after plating the medium was replaced with 2% F C S - D M E M containing 20 /tM RA, or with serum free D M E M , the incubation being continued up to 48 h (Shea et al., 1985); differentiation was complete after 24 48 h; (b) SH-SY5Y cells. Cells were plated at a 4.0 x 104 c m 2 cell density and 48 h after plating the medium was replaced with 10% FCS-RPMI 1640 containing 10 /tM RA or 20 nM PMA and the incubation continued up to 72 h (Pahlman et al., 1984); differentiation was complete after 4 8 - 7 2 h. Cerebellar granule cells were prepared and cultured as described (Gallo et al., 1982); differentiation started about 2 days after plating and was completed after 7 8 days in culture.
2.3. Metabolism of exogenously added [3H]Sph-SM The ability of neuroblastoma cells and cerebellar granule cells to metabolically process SM added to the culture medium was assessed on undifferentiated and differentiated cells. At the time of experiment, cells were fed with 1 /~M [3H]Sph-SM (2.9 /zCi/ml) up to 2 h (Riboni et al., 1994; Riboni et al., 1995). At the end of the pulse period, cells were washed, harvested by scraping and lyophilized (Riboni et al., 1994). The influence of endocytosis and lysosomal activity on the metabolic processing was assessed by performing experiments at 4°C (a condition that blocks endocytosis), or in the presence of 50 /~M chloroquine (a drug which inhibits the activity of lysosomal enzymes) (Riboni et al., 1991).
2.4. Lipid extraction and quantification Extraction and purification of identification and quantification formed as described (Riboni et was determined by the method respectively.
total cell lipids, as well as H P T L C separation, of the individual radiolabeled lipids, were peral., 1992; 1995). The content of Cer and Sph of Preiss et al. (1986) and Ohta et al. (1994),
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2.5. Treatment o f cultured cells with exogenous sphingosine and C2-Cer
In order to increase the cellular levels of Cer, SH-SY5Y cells were treated with Sph or C2-Cer in 10% FCS RPMI 1640, according to Riboni et al. (1995). 2. 6. Morphological differentiation
The degree of morphological differentiation was assessed by phase-contrast microscopy, counting 200 300 cells in 4 - 5 random fields in each dish. Cells bearing neurite-like processes were scored as differentiated by the criteria previously reported (Riboni et al., 1995). Data were expressed as percentage of neurite-bearing cells over total cells counted. 2. 7. Other methods
Radioactivity was determined by liquid scintillation counting, fluorography, and radiochromatoscanning (Digital Autoradiograph Berthold, Germany) (Riboni and Tettamanti, 1991). Total protein were assayed (Lowry et al., 1951) using bovine serum albumin as the standard. Mg2+-stimulated neutral (N-SM-ase) and acidic (A-SM-ase) sphingomyelinases were assayed on the cell homogenate, using [3H]SphSM as substrate, according to Mooibroek et al. (1985) and Gatt et al. (1981), respectively.
3. Results
3.1. Cer and sphingosine levels during differentiation o f neuroblastoma and cerebellar granule cells'
The content of endogenous Cer significantly increased during the process of differentiation in neuroblastoma cells and cerebellar granule cells (Fig. 1). The increase was: (a) 65% and 56% in Neuro2a cells, after 24 h treatment with RA, or FCS deprivation, respectively; (b) 52% and 45% in SH-SY5Y cells, after 48 h treatment with RA and PMA, respectively; (c) 113°/,, from the 2 ° to the 8 ° day in culture in cerebellar granule cells. The sphingosine content, that did not change during differentiation of Neuro2a and SH-SY5Y cells, underwent a 40')'0 increase in differentiated cerebellar granule cells. As shown in Fig. 2 in Neuro2a cells, induced to differentiate by serum withdrawal, the increase of Cer was prompt, and reached the maximal value after 30 min. This level was maintained constant as long as cells were cultivated in the absence of serum, but returned to the starting value upon FCS addition, with concomitant reversal of differentiation. The same results were obtained with Neuro2a cells induced to differentiate with RA (Riboni et al., 1995) and SH-SY5Y cells differentiating under the stimulation of RA. Also in the case of cerebellar granule cells (Fig. 2) the Cer level sharply increased after the 2nd day in culture and maintained costantly high values in differentiated cells (7-10 days in culture).
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3.2. Metabolic origin of Cer in differentiated neuroblastoma and cerebellar granule cells The ability to produce Cer from SM by differentiated and undifferentiated Neuro2a cells and cerebellar granule cells was inspected by pulsing both cell types with [3H]Sph-SM and following the formation of [3H]-metabolites. In the used experimental conditions [3H]Cer was the major radiolabeled metabolite of exogenous [3H]Sph-SM, accounting for more than half of total metabolites in both differentiated and undifferentiated cells. It was produced (Fig. 3) more rapidly and to a higher extent in the differentiated form of both Neuro2a cells (induced to differentiate by RA treatment) and cerebellar granule cells. When [3H]Sph-SM was administered under conditions which block endocytosis or lysosomal degradation, [3H]Cer formation was only partially reduced in all cells (Fig. 4). This indicates that only a portion of [3H]Sph-SM is internalized into cells and processed in the lysosomes, the remainder being produced at an extralysosomal level (possibly the
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plasma membrane). Anyway, the amount of [3H]Cer produced in the presence of chloroquine or at 4°C was higher in the differentiated than undifferentiated forms of both cell types. As shown in Fig. 5, the activity of Mg 2 +-stimulated N-SM-ase (extralysosomal) was substantially higher in differentiated (by RA treatment or FCS deprivation) than undifferentiated Neuro2a cells, whereas A-SM-ase did not change during differentiation. Instead, in the case of cerebellar granule cells, both N- and A-SM-ases underwent a marked (about 4-fold) increase during differentiation. The contribution of Cer biosynthesis to elevate Cer levels in differentiated cells was inspected by administering FB, the known inhibitor of ceramide synthase (Harel and Futerman, 1993; Merrill et al., 1993). In the presence of 25 ¢tM FB, neural differentiation was reduced in both Neuro 2a (Fig. 6) and cerebellar granule cells (data not shown).
3.3. EJ)'ect of induced increase of Cer content on the differentiation of neuroblastoma cells Undifferentiated Neuro2a and SH-SY5Y cells were treated with Sph, a condition that is known to increase the cellular Cer level (Riboni et al., 1995), and the differentiation process was morphologically assessed. The addition of exogenous Sph resulted in a dose (1-5 /~M) and time-dependent stimulation of neurite outgrowth in both SH-SY5Y (Fig. 7) and Neuro2a cells. After 24 h (Neuro2a cells)
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or 48 h (SH-SY5Y cells) of treatment the morphological differentiation appeared to be complete in both cell lines• The stimulation of neurite outgrowth after Sph administration was concomitant to the increase of Cer content but it was not detectable in the presence of 25 ¢tM FB (data not shown). As shown in Fig. 8, also the addition of a cell-permeable Cer analogue (C2-Cer, 1 - 2 p M) stimulates neurite outgrowth in both Neuro2a and SH-SY5Y cells, while equimolar concentrations of M DL-threo-sphinganine were ineffective.
4. Discussion A previous paper (Riboni et al., 1995) showed that in Neuro2a cells RA-induced differentiation is characterized by an early increase of endogenous Cer level, and that treatments capable of increasing cellular ceramide are followed by neuronal differentiation. On the other hand, removal of RA results in the decrease of Cer and reversal of differentiation. It was also established that the increased formation of Cer in RA treated cells is due to activation of both SM degradation and de novo Cer biosynthesis•
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G. Tettamanti et al. / J. Lipid Mediators Cell Signalling 14 (1996) 263-275
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This paper provides clear evidence that the involvement of Cer in neuronal differentiation is a more generalized phenomenon. In fact, the increase of endogenous Cer occurs (a) in Neuro2a cells not only stimulated by RA, but also by FCS deprivation, a condition that is known (Shea et al., 1985) to induce differentiation of these cells; (b) in human neuroblastoma cells SH-SY5Y, concomitantly with differentiation promoted by RA or PMA; and (c) in primary cultures of cerebellar granule cells that undergo spontaneous differentiation in vitro from the 2nd to the 7-8th day in culture. Secondly, SM degradation contributes to enhance the Cer level in all the examined cell types, either neurotumoral cell lines or primary cultures of neurons. Particularly, Cer formation in differentiated cells remains elevated also when endocytosis or lysosomal degradation are inhibited, indicating that an extralysosomal, possibly the plasma membrane-bound N-SM-ase, is mainly responsible of the increased SM degradation in differentiated cells. Consistent with this interpretation is the evidence, provided here, that the activity of the Mg 2+-dependent N-SM-ase, an enzyme especially concentrated in neural tissues (Gatt, 1976) and in cells of neuronal origin (Spence et al., 1982; Das et al., 1984) increases during neuronal differentiation. However, in cerebellar granule cells, besides N-SMase, A-SM-ase appears to be much more active in the differentiated than undifferentiated form, suggesting that in these cells also the lysosomal enzyme is implicated
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in the differentiation process. This latter finding could be important, since the sphingomyelin pathway, in the tumor necrosis factor biosignaling system, has been described as involving both N- and A-SM-ases, and Cer action depends on the topology of its production (Wiegmann et al., 1994). Moreover, Cer increase, concomitant to neural differentiation, appears to be due not only to stimulation of SM degradation. In fact, treatment with Fumonisin B1, which inhibits Cer synthase, but does not affect the activity of N-SMase (Riboni et al., 1995), reduced, but not suppressed, neurite outgrowth in both Neuro2a and cerebellar granule cells, indicating that Cer biosynthesis also concurs to enhance Cer level with differentiation. An additional piece of evidence supporting a general biomodulatory role of Cer in neural differentiation is that conditions leading to enhanced intracellular Cer content stimulate neurite outgrowth, not only in Neuro2a but also in SH-SY5Y neuroblastoma cells. In fact, supplying of exogenous Sph or C2-Cer is followed by promotion of neurite formation in both neuroblastoma cell lines, and co-treatment with FB inhibits the differentiation process elicited by Sph, but not that due to Cz-Cer. Remarkably, the stimulation of neuroblastoma cells neuritogenesis by exogenous Sph appears to be rather specific since the DL-threo-dihydroderivative of Sph does not exhert such an effect. All these observations strongly suggest that Cer represents a bioactive molecule instrumental to neuronal cells differentiation. Within the events governing neuronal differentiation, it would be very interesting to ascertain the downstream targets of Cer as well as the crosstalk between Cer and other biosignaling molecules.
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Acknowledgements This w o r k was s u p p o r t e d in p a r t b y g r a n t s f r o m the Consiglio N a z i o n a l e delle Ricerche, C . N . R . , R o m a , I t a l y ( T a r g e t P r o j e c t " B i o t e c h n o l o g y a n d B i o i n s t r u m e n t a t i o n " , g r a n t N ° 93.01094.PF70) a n d f r o m the M i n i s t r y o f U n i v e r s i t y a n d Research, M . U . R . S . T . , R o m e , I t a l y (40% Project).
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J. Lipid Mediators Cell Signalling 14 (1996) 263-275
Sphingoid bioregulators in the differentiation of cells of neural origin Guido Tettamanti*, Alessandro Prinetti, Rosaria Bassi, Paola Viani, Paola Giussani, Laura Riboni Department of Medical Chemistry and Biochemistry, University o[" Milan, via Saldini 50, 20133, Milan, Italy
Abstract
The involvement of ceramide in the differentiation of two neuroblastoma cell lines, Neuro2a and SH-SY5Y, and cerebellar granule cells in primary culture was investigated. The following results were obtained: (a) the cellular content of ceramide markedly increased with induced differentiation of Neuro2a cells (inducers: RA, FCS deprivation), SH-SY5Y cells (inducers: RA, PMA), and spontaneous differentiation of cerebellar granule cells; (b) all the investigated cells in the differentiated form displayed a higher ability to produce ceramide from exogenously administered [3H]Sph-SM and expressed a higher content of neutral sphingomyelinase and, in the case of cerebellar granule cells, also of acidic sphingomyelinase; (c) inhibition of ceramide biosynthesis by Fumonisin B1 blocked the process of differentiation in Neuro2a and cerebellar granule cells; and (d) treatments capable of enhancing ceramide level (administration of sphingosine or C2-Ceramide) induced differentiation in both Neuro2a and SH-SY5Y cells. The data obtained support the notion that ceramide plays a general biomodulatory role in neural cell differentiation. Keywords: Neural differentiation; Neuroblastoma cells; Cultured neurons; Sphingoid bioregulators; Ceramide
Abbreviations: RA, retinoic acid; PMA, phorbol-12 myristate, 13-acetate; Cer, ceramide; C2-Cer, N-acetyl-erythro-sphingosine; Sph, D-erythro-sphingosine; SM, sphingomyelin; SM-ase, sphingomyelinase; N-SM-ase, neutral sphingomyelinase; A-SM-ase, acidic sphingomyelinase; FB, Fumonisin B1; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum. * Corresponding author. Tel.: + 39 2 70645247; fax: + 39 2 2363584; email: [email protected],unimi.it 0929-7855/96/'$15.00 © 1996 Elsevier Science B.V. All rights reserved PlI S0929-7855(96)00535-4
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1. Introduction
Increasing evidence indicate that molecules of sphingoid nature are involved in the modulation of cell response to different extracellular signals (Kolesnick, 1992; Hannun and Bell, 1993; Merrill, 1994). Particularly, ceramide (N-acyl-erythro-sphingosine) acts as a key molecule in a new signal transduction pathway, the 'sphingomyelin pathway or cycle' (Kolesnick, 1992; Hannun, 1994). In fact, in several cell lines the activation of specific receptors by vitamin D3 and cytokines (tumor necrosis factor e, interleukin-lfl and ~-interferon) induces sphingomyelin hydrolysis by sphingomyelinase, leading to increased levels of ceramide. This sphingoid molecule modulates the activity of specific protein kinases and phosphatases, as well as transcriptional factors that, in turn, control cell proliferation, oncogenesis, differentiation and apoptosis (Hakomori, 1990; Kolesnick and Golde, 1994; Merrill, 1994; Hannun, 1994; Hannun and Obeid, 1995). Several studies support a regulatory role of sphingolipids and sphingoid molecules in neuronal development (Tettamanti and Riboni, 1994). In cultured hippocampal neurons sphingolipid biosynthesis is necessary for axonal outgrowth (Harel and Futerman, 1993) and branching (Schwarz et al., 1995). Furthermore, in T9 glioma cells, the addition of a cell-permeable ceramide analog (C2-Cer) mimics nerve growth factor in causing growth inhibition and neurite outgrowth, with concomitant increase of the cellular level of ceramide (Dobrowsky et al., 1994). Evidence was also provided for the involvement of ceramide in retinoic acid induced differentiation of Neuro2a neuroblastoma cells (Riboni et al., 1995). In this experimental model Cer levels are strictly related to the process of cell differentiation and the increase of cellular Cer, obtained by different treatments, leads to neurite outgrowth and inhibition of cell growth. On these premises, we faced the question whether ceramide is a general biomodulator of neuronal differentiation. To this purpose, we employed either neurotumoral cells, the murine (Neuro2a) and human (SH-SY5Y) neuroblastoma clones, or primary cultures of cerebellar granule cells. The results obtained strongly support that Cer represents a crucial mediator of differentiation in all these cells.
2. Materials and methods
2.1. Materials
Culture media, fetal calf serum (FCS), crystalline bovine serum albumin, bovine brain sphingomyelin (SM), D-erythro-sphingosine (C-18) (Sph), retinoic acid (RA), Fumonisin B1 (FB), phorbol, 12-myristate, 13-acetate (PMA) were from Sigma (St. Louis, MO); N-acetyl-erythro-sphingosine (C2-Cer) and DLthreo-sphinganine were from Matreya (Pleasant Gap, PA); HPTLC silica gel plates were from Merck (Darmstadt, Germany). Sphingomyelin, tritiated at the
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C-3 of the long chain base ([3H]Sph-SM, 2.9 Ci/mmol), as well as standard [3H]sphingolipids were prepared as previously described (Riboni et al., 1992).
2.2. CeH Cultures The murine neuroblastoma cell line, clone NB2a, (Neuro2a, CCL-131, American Cell Type Culture Collection, Bethesda, MD) and the cloned human neuroblastoma cell line, SH-SY5Y (kindly provided by Dr. D. Fornasari, Center of Cellular and Molecular Pharmacology, C.N.R., Milan, Italy) were employed. These cells were cultured in Falcon dishes in D M E M (Neuro2a) or RPMI 1640 (SH-SY5Y) supplemented with 10% FCS, 4 m M l.-glutamine, 1 m M sodium pyruvate, 100 U/ml of penicillin and 100 /Lg/ml of streptomycin sulfate in 5% CO2-95% air humidified atmosphere at 37°C. Neural differentiation was induced as follows: (a) Neuro2a cells. Cells were plated at a 1.8 x 104/cm 2 cell density and 48 h after plating the medium was replaced with 2% F C S - D M E M containing 20 /tM RA, or with serum free D M E M , the incubation being continued up to 48 h (Shea et al., 1985); differentiation was complete after 24 48 h; (b) SH-SY5Y cells. Cells were plated at a 4.0 x 104 c m 2 cell density and 48 h after plating the medium was replaced with 10% FCS-RPMI 1640 containing 10 /tM RA or 20 nM PMA and the incubation continued up to 72 h (Pahlman et al., 1984); differentiation was complete after 4 8 - 7 2 h. Cerebellar granule cells were prepared and cultured as described (Gallo et al., 1982); differentiation started about 2 days after plating and was completed after 7 8 days in culture.
2.3. Metabolism of exogenously added [3H]Sph-SM The ability of neuroblastoma cells and cerebellar granule cells to metabolically process SM added to the culture medium was assessed on undifferentiated and differentiated cells. At the time of experiment, cells were fed with 1 /~M [3H]Sph-SM (2.9 /zCi/ml) up to 2 h (Riboni et al., 1994; Riboni et al., 1995). At the end of the pulse period, cells were washed, harvested by scraping and lyophilized (Riboni et al., 1994). The influence of endocytosis and lysosomal activity on the metabolic processing was assessed by performing experiments at 4°C (a condition that blocks endocytosis), or in the presence of 50 /~M chloroquine (a drug which inhibits the activity of lysosomal enzymes) (Riboni et al., 1991).
2.4. Lipid extraction and quantification Extraction and purification of identification and quantification formed as described (Riboni et was determined by the method respectively.
total cell lipids, as well as H P T L C separation, of the individual radiolabeled lipids, were peral., 1992; 1995). The content of Cer and Sph of Preiss et al. (1986) and Ohta et al. (1994),
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2.5. Treatment o f cultured cells with exogenous sphingosine and C2-Cer
In order to increase the cellular levels of Cer, SH-SY5Y cells were treated with Sph or C2-Cer in 10% FCS RPMI 1640, according to Riboni et al. (1995). 2. 6. Morphological differentiation
The degree of morphological differentiation was assessed by phase-contrast microscopy, counting 200 300 cells in 4 - 5 random fields in each dish. Cells bearing neurite-like processes were scored as differentiated by the criteria previously reported (Riboni et al., 1995). Data were expressed as percentage of neurite-bearing cells over total cells counted. 2. 7. Other methods
Radioactivity was determined by liquid scintillation counting, fluorography, and radiochromatoscanning (Digital Autoradiograph Berthold, Germany) (Riboni and Tettamanti, 1991). Total protein were assayed (Lowry et al., 1951) using bovine serum albumin as the standard. Mg2+-stimulated neutral (N-SM-ase) and acidic (A-SM-ase) sphingomyelinases were assayed on the cell homogenate, using [3H]SphSM as substrate, according to Mooibroek et al. (1985) and Gatt et al. (1981), respectively.
3. Results
3.1. Cer and sphingosine levels during differentiation o f neuroblastoma and cerebellar granule cells'
The content of endogenous Cer significantly increased during the process of differentiation in neuroblastoma cells and cerebellar granule cells (Fig. 1). The increase was: (a) 65% and 56% in Neuro2a cells, after 24 h treatment with RA, or FCS deprivation, respectively; (b) 52% and 45% in SH-SY5Y cells, after 48 h treatment with RA and PMA, respectively; (c) 113°/,, from the 2 ° to the 8 ° day in culture in cerebellar granule cells. The sphingosine content, that did not change during differentiation of Neuro2a and SH-SY5Y cells, underwent a 40')'0 increase in differentiated cerebellar granule cells. As shown in Fig. 2 in Neuro2a cells, induced to differentiate by serum withdrawal, the increase of Cer was prompt, and reached the maximal value after 30 min. This level was maintained constant as long as cells were cultivated in the absence of serum, but returned to the starting value upon FCS addition, with concomitant reversal of differentiation. The same results were obtained with Neuro2a cells induced to differentiate with RA (Riboni et al., 1995) and SH-SY5Y cells differentiating under the stimulation of RA. Also in the case of cerebellar granule cells (Fig. 2) the Cer level sharply increased after the 2nd day in culture and maintained costantly high values in differentiated cells (7-10 days in culture).
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3.2. Metabolic origin of Cer in differentiated neuroblastoma and cerebellar granule cells The ability to produce Cer from SM by differentiated and undifferentiated Neuro2a cells and cerebellar granule cells was inspected by pulsing both cell types with [3H]Sph-SM and following the formation of [3H]-metabolites. In the used experimental conditions [3H]Cer was the major radiolabeled metabolite of exogenous [3H]Sph-SM, accounting for more than half of total metabolites in both differentiated and undifferentiated cells. It was produced (Fig. 3) more rapidly and to a higher extent in the differentiated form of both Neuro2a cells (induced to differentiate by RA treatment) and cerebellar granule cells. When [3H]Sph-SM was administered under conditions which block endocytosis or lysosomal degradation, [3H]Cer formation was only partially reduced in all cells (Fig. 4). This indicates that only a portion of [3H]Sph-SM is internalized into cells and processed in the lysosomes, the remainder being produced at an extralysosomal level (possibly the
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plasma membrane). Anyway, the amount of [3H]Cer produced in the presence of chloroquine or at 4°C was higher in the differentiated than undifferentiated forms of both cell types. As shown in Fig. 5, the activity of Mg 2 +-stimulated N-SM-ase (extralysosomal) was substantially higher in differentiated (by RA treatment or FCS deprivation) than undifferentiated Neuro2a cells, whereas A-SM-ase did not change during differentiation. Instead, in the case of cerebellar granule cells, both N- and A-SM-ases underwent a marked (about 4-fold) increase during differentiation. The contribution of Cer biosynthesis to elevate Cer levels in differentiated cells was inspected by administering FB, the known inhibitor of ceramide synthase (Harel and Futerman, 1993; Merrill et al., 1993). In the presence of 25 ¢tM FB, neural differentiation was reduced in both Neuro 2a (Fig. 6) and cerebellar granule cells (data not shown).
3.3. EJ)'ect of induced increase of Cer content on the differentiation of neuroblastoma cells Undifferentiated Neuro2a and SH-SY5Y cells were treated with Sph, a condition that is known to increase the cellular Cer level (Riboni et al., 1995), and the differentiation process was morphologically assessed. The addition of exogenous Sph resulted in a dose (1-5 /~M) and time-dependent stimulation of neurite outgrowth in both SH-SY5Y (Fig. 7) and Neuro2a cells. After 24 h (Neuro2a cells)
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or 48 h (SH-SY5Y cells) of treatment the morphological differentiation appeared to be complete in both cell lines• The stimulation of neurite outgrowth after Sph administration was concomitant to the increase of Cer content but it was not detectable in the presence of 25 ¢tM FB (data not shown). As shown in Fig. 8, also the addition of a cell-permeable Cer analogue (C2-Cer, 1 - 2 p M) stimulates neurite outgrowth in both Neuro2a and SH-SY5Y cells, while equimolar concentrations of M DL-threo-sphinganine were ineffective.
4. Discussion A previous paper (Riboni et al., 1995) showed that in Neuro2a cells RA-induced differentiation is characterized by an early increase of endogenous Cer level, and that treatments capable of increasing cellular ceramide are followed by neuronal differentiation. On the other hand, removal of RA results in the decrease of Cer and reversal of differentiation. It was also established that the increased formation of Cer in RA treated cells is due to activation of both SM degradation and de novo Cer biosynthesis•
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This paper provides clear evidence that the involvement of Cer in neuronal differentiation is a more generalized phenomenon. In fact, the increase of endogenous Cer occurs (a) in Neuro2a cells not only stimulated by RA, but also by FCS deprivation, a condition that is known (Shea et al., 1985) to induce differentiation of these cells; (b) in human neuroblastoma cells SH-SY5Y, concomitantly with differentiation promoted by RA or PMA; and (c) in primary cultures of cerebellar granule cells that undergo spontaneous differentiation in vitro from the 2nd to the 7-8th day in culture. Secondly, SM degradation contributes to enhance the Cer level in all the examined cell types, either neurotumoral cell lines or primary cultures of neurons. Particularly, Cer formation in differentiated cells remains elevated also when endocytosis or lysosomal degradation are inhibited, indicating that an extralysosomal, possibly the plasma membrane-bound N-SM-ase, is mainly responsible of the increased SM degradation in differentiated cells. Consistent with this interpretation is the evidence, provided here, that the activity of the Mg 2+-dependent N-SM-ase, an enzyme especially concentrated in neural tissues (Gatt, 1976) and in cells of neuronal origin (Spence et al., 1982; Das et al., 1984) increases during neuronal differentiation. However, in cerebellar granule cells, besides N-SMase, A-SM-ase appears to be much more active in the differentiated than undifferentiated form, suggesting that in these cells also the lysosomal enzyme is implicated
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in the differentiation process. This latter finding could be important, since the sphingomyelin pathway, in the tumor necrosis factor biosignaling system, has been described as involving both N- and A-SM-ases, and Cer action depends on the topology of its production (Wiegmann et al., 1994). Moreover, Cer increase, concomitant to neural differentiation, appears to be due not only to stimulation of SM degradation. In fact, treatment with Fumonisin B1, which inhibits Cer synthase, but does not affect the activity of N-SMase (Riboni et al., 1995), reduced, but not suppressed, neurite outgrowth in both Neuro2a and cerebellar granule cells, indicating that Cer biosynthesis also concurs to enhance Cer level with differentiation. An additional piece of evidence supporting a general biomodulatory role of Cer in neural differentiation is that conditions leading to enhanced intracellular Cer content stimulate neurite outgrowth, not only in Neuro2a but also in SH-SY5Y neuroblastoma cells. In fact, supplying of exogenous Sph or C2-Cer is followed by promotion of neurite formation in both neuroblastoma cell lines, and co-treatment with FB inhibits the differentiation process elicited by Sph, but not that due to Cz-Cer. Remarkably, the stimulation of neuroblastoma cells neuritogenesis by exogenous Sph appears to be rather specific since the DL-threo-dihydroderivative of Sph does not exhert such an effect. All these observations strongly suggest that Cer represents a bioactive molecule instrumental to neuronal cells differentiation. Within the events governing neuronal differentiation, it would be very interesting to ascertain the downstream targets of Cer as well as the crosstalk between Cer and other biosignaling molecules.
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Acknowledgements This w o r k was s u p p o r t e d in p a r t b y g r a n t s f r o m the Consiglio N a z i o n a l e delle Ricerche, C . N . R . , R o m a , I t a l y ( T a r g e t P r o j e c t " B i o t e c h n o l o g y a n d B i o i n s t r u m e n t a t i o n " , g r a n t N ° 93.01094.PF70) a n d f r o m the M i n i s t r y o f U n i v e r s i t y a n d Research, M . U . R . S . T . , R o m e , I t a l y (40% Project).
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(sphinganine) N-acyltransferase and de novo sphingolipid biosynthesis in cultured neurons in situ. J. Biol. Chem. 268, 27299 27306. Mooibroek, M.J., Cook, H.W., Clarke, J.T.R. and Spence, M.W. (1985) Catabolism of exogenous and endogenous sphingomyelin and phosphatidylcholine by homogenates and subcellular fractions of cultured neuroblastoma cells. Effects of anesthetics. J. Neurochem. 44, 1551-1558. Ohta, H., Yatomi, Y, Sweeney, E.A., Hakomori, S. I., and Igarashi, Y. (1994) Quantification of free sphingosine in cultured cells by acylation with radioactive acetic anhydride. Anal. Biochem. 222, 489-494. Pahlman, S., Ruusala, A.-I., Abrahamsson, L., Mattson, M.E.K. and Esscher, T. (1984) Retinoic-acid induced differentiation of cultured human neuroblastoma cells: a comparison with phorbolester-induced human differentiation. Cell Diff. 14, 135 144. Preiss, J., Loomis, C.R., Bishop, W.R., Stein, R., Niedel, J.E. and Bell, R.M. (1986) Quantitative measurement of sn-l,2-diacylglycerols present in platelets, hepatocytes, and ras- and sis-transformed normal rat kidney cells. J. Biol. Chem. 261, 8597-8600. Riboni, L. and Tettamanti, G. (1991) Rapid internalization and intracellular metabolic processing of exogenous ganglioside by cerebellar granule cells differentiated in culture. J. Neurochem. 57, 1931 1939. Riboni, L., Prinetti, A., Bassi, R. and Tettamanti, G. (1991) Cerebellar granule cells in culture exhibit a ganglioside sialidase presumably linked to the plasma membrane. FEBS Lett. 287, 42 46. Riboni, L., Bassi, R., Sonnino, S. and Tettamanti, G. (1992) Formation of free sphingosine and ceramide from exogenous ganglioside GM1 by cerebellar granule cells in culture. FEBS Lett. 300, 188 192. Riboni, L., Prinetti, A., Bassi, R. and Tettamanti, G. (1994) Formation of bioactive sphingoid molecules from exogenous sphingomyelin in primary cultures of neurons and astrocytes. FEBS Lett. 352, 323 326. Riboni, L., Prinetti, A., Bassi, R., Caminiti, A. and Tettamanti, G. (1995) A mediator role of ceramide in the regulation of neuroblastoma Neuro2a cell differentiation. J. Biol. Chem. 270, 26868-26875. Schwarz, A., Rapaport, E., Hirschberg, K. and Futerman, A.H. (1995) A regulatory role for sphiongolipids in neuronal growth. J. Biol. Chem. 270, 10990-10998. Shea, T.B., Fischer, I. and Sapirtein, U.S. (1985) Effects of retinoic acid on growth and morphological differentiation of mouse NB2a neuroblastoma cells in culture. Dev. Brain Res. 21, 307 314. Spence, M.W., Wakkary, J., Clarke, J.T.R. and Cook, H.W. (1982) Localization of neutral, magnesiumstimulated sphingomyelinase in plasma membrane of cultured neuroblastoma cells. Biochim. Biophys. Acta 719, 162 164. Tettamanti, G. and Riboni, L. (1994) Gangliosides turnover and neural cell function: a new perspective. Prog. Brain Res. 101, 77 100. Wiegmann, K., Schfitze, S., Machleidt, T., Witte, D. and Kr6nke, M. (1994) Functional dicotomy of neutral and acidic sphingomyelinases in tumor necrosis factor signaling. Cell 78, 1005 1015.