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Originally published In Press as doi:10.1074/jbc.M401205200 on February 19, 2004

J. Biol. Chem., Vol. 279, Issue 18, 18688-18693, April 30, 2004
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Expression Cloning of a Human cDNA Restoring Sphingomyelin Synthesis and Cell Growth in Sphingomyelin Synthase-defective Lymphoid Cells*

Shohei Yamaoka, Michihiko Miyaji, Toshiyuki Kitano{ddagger}, Hisanori Umehara, and Toshiro Okazaki§

From the Departments of Hematology/Oncology and Clinical Immunology and the {ddagger}Outpatient Oncology Unit, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawaharacho, Sakyo-ku, Kyoto 6068507, Japan

Received for publication, February 3, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Sphingomyelin (SM) synthase has been assumed to be involved in both cell death and survival by regulating pro-apoptotic mediator ceramide and pro-survival mediator diacylglycerol. However, its precise functions are ambiguous due to the lack of molecular cloning of SM synthase gene(s). We isolated WR19L/Fas-SM(-) mouse lymphoid cells, which show a defect of SM at the plasma membrane due to the lack of SM synthase activity and resistance to cell death induced by an SM-directed cytolytic protein lysenin. WR19L/Fas-SM(-) cells were also highly susceptible to methyl-{beta}-cyclodextrin (M{beta}CD) as compared with the WR19L/Fas-SM(+) cells, which are capable of SM synthesis. By expression cloning method using WR19L/Fas-SM(-) cells and M{beta}CD-based selection, we have succeeded in cloning of a human cDNA responsible for SM synthase activity. The cDNA encodes a peptide of 413 amino acids named SMS1 (putative molecular mass, 48.6 kDa), which contains a sterile {alpha} motif domain near the N-terminal region and four predicted transmembrane domains. WR19L/Fas-SM(-) cells expressing SMS1 cDNA (WR19L/Fas-SMS1) restored the resistance against M{beta}CD, the accumulation of SM at the plasma membrane, and SM synthesis by transferring phosphocholine from phosphatidylcholine to ceramide. Furthermore, WR19L/Fas-SMS1 cells, as well as WR19L/Fas-SM(-) cells supplemented with exogenous SM, restored cell growth ability in serum-free conditions, where the growth of WR19L/Fas-SM(-) cells was severely inhibited. The results suggest that SMS1 is responsible for SM synthase activity in mammalian cells and plays a critical role in cell growth of mouse lymphoid cells.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Diverse kinds of phospho- and glycerolipids such as diacylglycerol (DAG),1 inositol phosphatides, and phosphatidic acid are recognized as bioactive molecules in cell growth and survival (1, 2). Sphingolipid ceramide has recently emerged as a signal mediator of cell functions including apoptosis, differentiation, and secretion (3). Various stresses such as ultraviolet, irradiation, heat shock, hypoxia, and biological factors such as tumor necrosis factor-{alpha}, interferon-{gamma}, and Fas antibody require ceramide generation to execute apoptosis, suggesting the implications of SM as a source of ceramide generation in the induction of cell death (4, 5). It was reported that SM dose-dependently inhibits both deoxycholate-induced apoptosis and subsequent hyper-proliferation in colon epithelial cells (6) and decreases the number of aberrant crypts of colon (7), suggesting the implications of SM in cell death and growth.

SM is produced by SM synthase, which is thought to be the only enzyme to synthesize SM in mammalian cells (8). The enzyme catalyzes the reaction in which phosphocholine moiety is transferred from phosphatidylcholine (PC) to ceramide. Thus, the activation of SM synthase subsequently increases the levels of DAG and decreases ceramide at the same time (8). DAG is an important signaling molecule for cell growth through protein kinase C activation (9–12) and acts competitively against ceramide-induced apoptosis (4, 13). It has been reported that after thioacetamide-induced injury, the SM/PC ratio significantly increased in microsomal fraction from liver, suggesting the involvement of SM synthase in tissue recovery (14). In cerebellar astrocytes, the level of ceramide is rapidly down-regulated by basic fibroblast growth factor via activating SM synthase (15). In SV40-transformed lung fibroblasts, SM synthase regulates the levels of ceramide and DAG in an opposite direction (16). We recently reported that SM synthase was activated to inhibit ceramide generation in IL-2-induced proliferation of natural killer cells,2 whereas the activity in nucleus was inhibited with ceramide generation in Fas-induced T cell apoptosis (17). We also showed its in vivo implication that the level of ceramide was decreased via activation of SM synthase in chemotherapy-resistant blast cells obtained from refractory leukemia patients than in chemotherapy-sensitive leukemic blasts (18). Thus, SM synthase is assumed to play an important role in cell death and survival, in vitro as well as in vivo.

We previously proposed the "SM cycle," a pathway that consisted of SM synthase and sphingomyelinase as a novel biological system to regulate the cellular level of ceramide for cell death and differentiation (19). In contrast to the studies of the acid and neutral sphingomyelinases in cell death (20, 21), the biological implication of SM synthase has not been elucidated due to the lack of molecular cloning of its responsible gene(s). We recently found mouse lymphoid cell variants designated WR19L/Fas-SM(-), which are defective of SM synthesis and susceptible to methyl-{beta}-cyclodextrin (M{beta}CD)-induced cell death (30). By an expression cloning method using WR19L/Fas-SM(-) cells and M{beta}CD-based cell selection, we isolated a human cDNA responsible for SM synthase activity. The cDNA clone encodes a peptide of 413 amino acids, named SMS1, which contains a sterile {alpha} motif (SAM) domain and four putative transmembrane domains. SMS1 was identical to the peptide that was recently identified as a human SM synthase by Huitema et al. (24). In serum-free condition, where the cell growth of WR19L/Fas-SM(-) was inhibited, the cells expressing SMS1 cDNA (WR19L/Fas-SMS1) restored the growth ability and accumulation of SM at the surface of the plasma membrane. The restoration of cell growth was also observed when WR19L/Fas-SM(-) cells were maintained in the serum-free medium supplemented with exogenous SM. Here, we show the critical role of SM synthesized through SM synthase in mammalian cell growth, and the localization, active site and biological function of SMS1 are also discussed.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Materials—Lysenin, M{beta}CD, and ceramide from bovine brain were purchased from Sigma; PC from egg yolk, SM from bovine brain, and a cell viability assay kit with 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium (WST-8) were from Nacalai tesque (Kyoto, Japan); GP2–293 packaging cell, pLIB retroviral expression vector, and human HeLa cDNA retroviral expression library were from Clontech; D-erythro-C6-NBD-ceramide and C6-NBD-sphingomyelin were from Matreya (Pleasant Gap, PA); L-[U-14C]serine, cytidine 5'-diphospho [methyl-14C]choline, L-3-phosphatidyl [N-methyl-14C]choline, 1,2-dipalmitoyl, and [N-methyl-14C]sphingomyelin were from Amersham Biosciences.

Cell Culture—WR19L/Fas cells were kindly gifted from Dr. Yonehara (Institute for Virus Research, Kyoto University). The SM-defective WR19L/Fas-SM(-) cells and the SM-containing WR19L/Fas-SM(+) cells were isolated from the original WR19L/Fas cells by a dilution cloning method. The cells were routinely maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 50 µM 2-mercaptoethanol, and 75 µg/ml kanamycin in 5% CO2 and 100% humidity at 37 °C. For culture in serum-free medium, the cells were washed, reseeded at 1 x 105 cells/ml, and incubated in the RPMI 1640 medium with 5 µg/ml human insulin and bovine holo transferrin in the presence or absence of 50 µM SM in 5% CO2 at 37 °C. After 48 h incubation, the cell numbers were counted with dye exclusion method using 0.25% trypan blue (Nakalai tesque, Kyoto, Japan).

Cell Labeling—The cells were reseeded at 5 x 105 cells/ml in the RPMI 1640 medium with 2% FBS and L-[14C]serine (specific activity; 155 mCi/mmol) and incubated at 37 °C in 5% CO2 for 36 h. The labeled cells were incubated at 37 °C in 5% CO2 for 2 h. The cell lipids were extracted by the method of Bligh and Dyer (19), applied on a silica Gel 60 TLC plate (Merck), and developed with solvent containing methyl acetate/propanol/chloroform/methanol/0.25% KCl (25:25:25:10:9). The radioactive spots were visualized and quantified by using a BAS 2000 Image Analyzer (Fuji Film).

FACS Analyses—The cells were incubated with 500 ng/ml lysenin in the presence of 20 µg/ml propidium iodide (Molecular Probes) at room temperature for 15 min and analyzed with FACS Calibur (BD Biosciences). For detection of SM localized at the plasma membrane, the cells were stained on ice for 30 min with non-toxic lysenin fused to maltose-binding protein (MBP-lysenin) (25), kindly provided by Dr. T. Kobayashi (The Institute of Physical and Chemical Research (RIKEN), Japan). The cells were washed with ice-cold phosphate-buffered saline supplemented with 1% FCS and 0.1% NaN3 and incubated with rabbit anti-MBP antiserum (New England BioLabs, Beverly, MA) on ice for 30 min. After being washed again, the cells were incubated for 30 min with phycoerythrin-conjugated anti-rabbit IgG (Sigma) and subjected to fluorescence-activating cell sorter (FACS) analysis using FACS Calibur. The data analysis was performed by Cell Quest software (BD Biosciences).

Confocal Microscopy—For visualization of SM localized at the plasma membrane, the cells settled onto slides coated with poly-L-lysine were fixed in 4% formaldehyde and stained with lysenine-MBP at 4 °C for 45 min followed with anti-MBP. After being stained with a phycoerythrin-conjugated anti-rabbit IgG monoclonal antibody, the cells were examined using confocal microscopy using a Zeiss LSM 310 laser scan confocal microscope (Carl Zeiss, Oberkochen, Germany).

Expression Cloning of SMS1 cDNA—The expression cloning method performed in this study was based on the study of Hanada et al. (26). Pantropic retroviral particles containing the G glycoprotein of vesicular stomatitis virus (VSV-G) were prepared using a human HeLa cDNA retroviral expression library kit and GP2–293 packaging cells (Clontech). After infection for 24 h, the WR19L/Fas-SM(-) cells were cultured in the RPMI 1640 medium containing 2% FBS overnight. After being washed with serum-free RPMI 1640 medium, the cells were incubated in 1.5 mM M{beta}CD in RPMI 1640 medium for 5 min at 37 °C, replenished with the normal culture medium to a final concentration of FBS at 5%, and then cultured at 37 °C for 60 h. The cells were reseeded, cultured in the RPMI 1640 medium containing 2% FBS overnight, and subjected again to the treatment with appropriate concentrations of M{beta}CD. After a total of two cycles of 1.5 mM M{beta}CD treatment followed by two cycles of 3 mM and two subsequent cycles of 5 mM, an M{beta}CD-resistant variant of WR19L/SM(-) was isolated by a limiting dilution.

By genomic PCR using primers specific to the pLIB expression vector (5' and 3' pLIB Primer, Clontech), the 2.0-kb cDNA integrated in the genome of the M{beta}CD-resistant cell was amplified and cloned into pGEM-T Easy vector (Promega, Madison, WI). After sequencing and computer analysis, the cDNA was subcloned into the pLIB expression vector and transfected into the WR19L/Fas-SM(-) cells via the VSV-G retroviral particles. A resultant cell was isolated by a limiting dilution method, which was designated WR19L/Fas-SMS1 cells, and subjected to various assays. Integration of the cDNA into the genome of WR19L/Fas-SMS1 cells was confirmed with PCR.

Assay for Sphingomyelin Synthase Activity—The cells were homogenized in an ice-cold buffer containing 20 mM Tris-HCl, pH 7.4, 2 mM EDTA, 10 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, and 2.5 µg/ml leupeptin. The lysates containing 500 µg of cell protein were added to a reaction solution containing 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 20 µM C6-NBD-ceramide, 120 µM PC and incubated at 37 °C for 30 min. The lipids were extracted by the method of Bligh and Dyer (19), applied on the TLC plates, and developed with solvent containing chloroform/methanol/12 mM MgCl2 in H2O (65:25:4). The fluorescent lipids were visualized by FluorImager SI system (Amersham Biosciences). For the assay for transferase activity, 20 µM ceramide and 120 µM [N-methyl-14C]PC (specific activity; 57 mCi/mmol) or [methyl-14C]CDP-choline (specific activity; 54 mCi/mmol) were used in the reaction solution instead of the NBD-ceramide and PC. The radioactive spots were visualized using the BAS 2000 system.

Assay for Viability and Growth Rate of Cells Exposed to M{beta}CD and Lysenin—For the assay using M{beta}CD, 1 x 106 of the cells were washed and resuspended in 1 ml of the serum-free RPMI 1640 medium, treated with appropriate concentrations of M{beta}CD, and incubated in 5% CO2 at 37 °C for 5 min. After the addition of 1 ml of the normal culture medium, the cells were further incubated for 12 h. The viability of the cells was measured using a cell viability kit with WST-8 (Nakalai tesque). For the assay using lysenin, 7 x 105 of the cells were washed and resuspended in 1 ml of prewarmed phosphate-buffered saline, treated with the appropriate concentrations of lysenin and incubated in 5% CO2 at 37 °C for 1 h. After the addition of FBS, the cell number was counted with the 0.25% trypan blue dye exclusion method.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Mouse Lymphoid Cells Defective of Sphingomyelin Synthase Activity—During investigation of the sphingolipid metabolism in mouse lymphoid cells named WR19L/Fas, which overexpress the human Fas antigen, the variant clones altering SM synthase activity (from 150 to nearly 0 pmol/mg protein/h) have been isolated. One of the variants (clone 6) severely diminished the SM synthase activity (Fig. 1A). Conversion of C6-NBD-ceramide to C6-NBD-SM in the cell lysate of the clone 6, named WR19L/Fas-SM(-), was not detected on a TLC plate, in contrast to the clone 2 showing the highest SM synthase activity, named WR19L/Fas-SM(+) (Fig. 1B). This finding was supported by the fact that WR19L/Fas-SM(-) cells did not synthesize [14C]serine-labeled SM (Fig. 1C).



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  		FIG. 1.
Deficiency of SM synthesis, SM synthase activity, and SM localized at the plasma membrane in the WR19L/Fas-SM(-) cells. A, SM synthase activity in the cell lysates of the variant WR19L/Fas cells was assessed by the generation of C6-NBD-SM. The NBD-labeled products were quantified by a fluorospectro-photometer. B, SM synthase activity of WR19L/Fas-SM(-) and -SM(+). The NBD-labeled products developed on the TLC plate were visualized by a fluorospectro-photometer. The reaction was performed in the presence of 0.5 mM UDP-glucose. Cer, ceramide; GC, glucosylceramide; C, the cellular lipids were labeled with [14C]serine, extracted by the Bligh and Dyer method, and assessed by TLC. The radiolabeled products developed on the TLC plate were visualized by BAS 2000 system. PE, phosphatidylethanolamine; PS, phosphatidylserine. D, SM localized at the plasma membrane was assessed by FACS analysis and confocal microscopy. FACS analysis was performed for the cells treated with the MBP-conjugated modified lysenin (shaded with dark blue) and the control cells (unshaded). For the results of the confocal microscopy, the fluorescence of phycoerythrin (PE) was pseudo-colored with red. E, The cells stained with 500 ng/ml lysenin in the presense of 20 µg/ml propidium iodide were assessed by FACS analysis. The data were the average and 1 S.D. obtained from three independent experiments (A) and were the representative of three independent experiments (B–E).

 
Lysenin is reported as an SM-direct cytolysin purified from the earthworm (27), for which binding to SM causes poring of the plasma membrane and subsequent cell death (22, 23, 25). Hanada et al. (22) previously showed that Chinese hamster ovary cells, which express SM in the outer surface of the plasma membrane, were sensitive to lysenin-induced cell death. They also showed that reduced accumulation of SM in the variant Chinese hamster ovary cells, LY-A and LY-B, causes the significant resistance against lysenin (22). In LY-A cells, the reduction of SM is caused by the lack of non-vesicular transporter for ceramide between endoplasmic reticulum (ER) and Golgi apparatus (CERT) (26), whereas in LY-B cells, it is due to the lack of LCB1, a component of serine palmitoyltransferase (22). CERT is involved in SM synthesis by transferring ceramide from the endoplasmic reticulum to the cytoplasmic surface of Golgi apparatus (26, 31). Recently, Kobayashi and co-workers (25) reported a modified lysenin, which specifically binds to SM without the induction of cell death. By using the modified lysenin conjugated with MBP, we examined the accumulation of SM on the cellular surface of WR19L/Fas-SM(-) cells. Binding of the modified lysenin was positively detected in WR19L/Fas-SM(+) cells but not in WR19L/Fas-SM(-) cells by FACS analysis and confocal microscopy using anti-MBP antibody (Fig. 1D), indicating that the accumulation of SM on the outer surface of the WR19L/Fas-SM(-) cells was severely reduced. The results were supported by the fact that WR19L/Fas-SM(+) cells underwent cell death by treatment with the cytotoxic lysenin, whereas WR19L/Fas-SM(-) cells did not, when we examined cell viability by staining with propidium iodide and subsequent FACS analysis (Fig. 1E). These facts suggest that the severe reduction of SM at the cellular surface of WR19L/Fas-SM(-) cells is due to the lack of enzymatic activity of SM synthase.

Expression Cloning of a Human cDNA Responsible for Resistance to Methyl-{beta}-cyclodextrin-induced Cell Death—It has been reported that SM strongly interacts with cholesterol in biological and artificial membranes (28) and that SM is required to form the membrane microdomains (lipid rafts) related to cell functions such as cell death and growth (29, 30). LY-A cells were sensitive to M{beta}CD-induced cell death due to the decrease of the SM level in plasma membrane (30). We similarly observed that WR19L/Fas-SM(-) cells were highly sensitive to M{beta}CD-induced cell death, whereas WR19L/Fas-SM(+) cells were not (Fig. 2D). This finding allowed us to screen WR19L/Fas-SM(-) cells complemented with the ability of SM synthesis using M{beta}CD as a selective agent. Using a pantropic retroviral transfection system, WR19L/SM(-) cells were transfected with a cDNA expression library of the human HeLa cell, and the variant cells, which were resistant to M{beta}CD due to the expression of SM in the plasma membrane, were selected. The variant cells were isolated to a single clone by a limiting dilution method. The purified cells integrated a human cDNA of 1967 bp in the genome, which encodes a peptide of 413 amino acids with 48.6 kDa of a predicted molecular mass (Fig. 2A). The BLAST algorithm (32) and the SOSUI program (33) suggested that this peptide carries a SAM domain in the N-terminal region and four transmembrane helices, respectively (Fig. 2B). The SAM domain is suggested to be involved in signal transduction, development, and transcriptional regulation (34, 35). A variety of proteins such as ephrin-related receptor tyrosine kinase, a variant of p53 (p73), and DAG kinase {delta} contain the SAM domain(s), which may play a role in protein-protein or protein-lipid interaction (35, 36). Recently, Huitema et al. (24) reported a family of SM synthases using a bioinformatics and functional cloning strategy in yeast. They identified the human cDNAs encoding the peptides that shared a sequence motif with the lipid phosphate phosphatases and Aur1p proteins required for inositolphosphorylceramide production in yeast (24). One of the human peptides, SMS1, was identical to our peptide. They further demonstrated that SMS1 was localized at Golgi apparatus and predicted the six transmembrane domains and an exoplasmic catalytic site, which is consistent with the characteristics of SM synthase suggested previously (37–39). Molecular structure of SMS1, including the transmembrane domains, should be clarified by further detailed analysis.



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  		FIG. 2.
Expression cloning of a human cDNA responsible for cellular resistance to methyl-{beta}-cyclodextrin. A, nucleotide sequence and predicted amino acid sequence of SMS1. Putative SAM domain sequence and transmembrane (TM) regions are indicated with the thin and thick underline, respectively. SAM domain and transmembrane domains were predicted by the BLAST algorithm and SOSUI program, respectively. B, hydropathy plot of the amino acid sequence of SMS1 analyzed by the method of Kyte and Doolittle (45). Positions of the putative SAM domain and transmembrane regions are indicated with horizontal bars. C, integration of SMS1 cDNA (2 kb) into the genome of WR19L/Fas-SM(-) and -SMS1 cells was examined by PCR. D, the viability of WR19L/Fas-SM(-), -SM(+), and -SMS1 cells exposed to various concentrations of M{beta}CD. The viability of the cells was examined using WST-8. The data were the average and 1 S.D. obtained from three independent experiments.

 
It was recently proposed by Luberto et al. (40) that the Pseudomonas PlcH gene product, which is a secreted protein, is a putative SM synthase. The SMS1 peptide was suggested to be an integral membrane protein and did not show any significant homology with the PlcH product.

The SMS1 cDNA Is Responsible for Sphingomyelin Synthase in Mammalian Cells—Huitema et al. (24) demonstrated the SM synthase activity in the yeast cells expressing SMS1 cDNA. Here, we demonstrated that the loss of SM synthesis in the SM-defective mammalian cells was complemented with SMS1 cDNA. WR19L/Fas-SM(-) cells transfected with SMS1 cDNA, named WR19L/Fas-SMS1 (Fig. 2C), restore the resistance against M{beta}CD-induced cell death (Fig. 2D). Radiolabeling of cellular lipids with [14C]serine revealed that [14C]SM synthesis was also restored in WR19L/Fas-SMS1 cells (Fig. 3A), and whole cell lysate from WR19L/Fas-SMS1 cells generated C6-NBD-SM in the presence of C6-NBD-ceramide and PC (Fig. 3B). These results strongly suggest that the SMS1 cDNA is indispensable for SM synthase activity in mammalian cells. Furthermore, SM synthase activity in WR19L/Fas-SMS1 cells was detected in the presence of [14C]PC but not [14C]CDP-choline (Fig. 3C), suggesting that PC was a phosphocholine donor for SM synthesis by SMS1. These results indicate that the SMS1 protein possesses the characteristics consistent with those of SM synthase reported elsewhere previously (8).



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  		FIG. 3.
Restoration of SM synthesis and SM synthase activity in WR19L/Fas-SMS1. A, the cellular lipids of WR19L/Fas-SM(-) and -SMS1 labeled with [14C]serine were assessed by TLC as described in Fig. 1. Cer, ceramide; GC, glucosylceramide; PE, phosphatidylethanolamine; PS, phosphatidylserine. B, SM synthase activity of WR19L/Fas-SM(-) and -SMS1 cells was assessed in the absence of UDP-glucose as described in the legend for Fig. 1. C, SM synthase activity was assessed using the radiolabeled PC and CDP-choline as the donor of phosphocholine moiety. The radiolabeled products developed on the TLC plate were visualized by BAS 2000 system. The data were the representative of three independent experiments.

 
The SMS1 cDNA Is Essential for Growth in Mammalian Cells—In contrast to the role of ceramide in cell death, the biological implications of SM are still ambiguous. The WR19L/Fas-SMS1 cells were sensitive to lysenin-induced cell death (Fig. 4A), suggesting that overexpression of SMS1 cDNA increases SM at the surface of plasma membrane. In the serumfree condition, the WR19L/Fas-SM(-) cells did not grow well, whereas the WR19L/Fas-SMS1 cells showed normal cell growth as well as the WR19L/Fas-SM(+) cells. The supplement with exogenous SM also restored cell growth of WR19L/Fas-SM(-) cells, although the growth rate was slightly reduced (Fig. 4B). The viable cell number of WR19L/Fas-SM(-) cells after 48 h from supplement with SM (4.4 x 105/ml) seemed to be similar to that of WR19L/Fas-SMS1 cells after 24 h (4.3 x 105/ml; data not shown), suggesting that stimulation for cell growth may be delayed due to the uptake of SM by WR19L/Fas-SM(-) cells. These observations suggest that SM synthesis through SM synthase is essential for cell growth. Huitema et al. (24) suggested that SMS2 is localized at the plasma membrane and may play a role in signal transduction. Here, we demonstrated that the cell growth was closely related with the accumulation of SM at the plasma membrane caused by SMS1, suggesting that SMS1 may be involved in signal transduction for cell growth as well as SMS2. In contrast to our observation, it has been reported that the variant melanoma cells deficient in glucosylceramide synthase showed no significant difference of cell growth as compared with the original cells (41), although glucosylceramide is an essential lipid for the diversity of gly-cosphingolipids. One possible explanation for the difference between SM and glucosylceramide is the significant involvement of SM in the microdomains responsible for various cell signaling events.



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  		FIG. 4.
The SMS1 cDNA is essential for normal growth in mammalian lymphoid cells. A, a viability assay of the WR19L/Fas-SM(-) and -SMS1 cells exposed to lysenin. After treatment with the indicated concentrations of lysenin at 37 °C for 1 h, the cell numbers were assessed by the dye exclusion method. B, growth of WR19L/Fas-SM(-), -SM(+), and -SMS1 cells in serum-free medium and restoration of WR19L/Fas-SM(-) cell growth by supplement of SM. The cells were incubated in serum-free medium for 48 h in the presence or absence of 50 µM SM, and the cell numbers were assessed by the dye exclusion method. The data were the average and 1 S.D. obtained from three independent experiments.

 
SM synthase regulates the levels of pro-apoptotic ceramide and anti-apoptotic DAG in an opposite manner and balances the levels of phospholipid PC and sphingolipid SM (9). SM synthase is closely regulated by the levels of ceramide and DAG, as well as extracellular stresses (15, 18, 31, 42). SM synthase is suggested to localize not only in the plasma membrane (37) and Golgi apparatus (43) but also in the endoplasmic reticulum (38) and nucleus (17, 44). We recently reported that, in Fas-induced Jurkat T cell apoptosis, ceramide increased through inhibition of SM synthase in the nucleus (17). The relationship between the intracellular localization and the regulation of SM synthase activity and the regulatory mechanism for the levels of lipid mediators in cell growth and death through SM synthase should be clarified by detailed analysis of the SMS genes.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AB154421 [GenBank] .

* This work was supported by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ To whom correspondence should be addressed. Tel. and Fax: 81-75-751-3154; E-mail: toshiroo{at}kuhp.kyoto-u.ac.jp.

1 The abbreviations used are: DAG, diacylglycerol; SM, sphingomyelin; PC, phosphatidylcholine; M{beta}CD, methyl-{beta}-cyclodextrin; SAM, sterile {alpha} motif; WST-8, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium; FBS, fetal bovine serum; FACS, fluorescence-activated cell sorter; MBP, maltose-binding protein; NBD, 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)). Back

2 Y. Taguchi, T. Kondo, M. Watanabe, Y. Kozutumi, and T. Okazaki, submitted for publication. Back


   ACKNOWLEDGMENTS
 
We appreciate Dr. K. Hanada (National Institute of Infectious Diseases) for a useful support in terms of retroviral transfection and M{beta}CD screening methods and Drs. Y. Hirabayashi (RIKEN) and Y. Igarashi (Hokkaido University) for careful discussions. We also appreciate Drs. A. Takaori and M. Kobayashi (Kyoto University) for a technical advice for retroviral transfection.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 

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