Expression Cloning of a Human cDNA Restoring Sphingomyelin Synthesis and Cell Growth in Sphingomyelin Synthase-defective Lymphoid Cells

Abstract

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-␤-cyclodextrin (M␤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␤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 ␣ 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␤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.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-␣, interferon-␥, 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 a...

Both Sphingomyelin Synthases SMS1 and SMS2 Are Required for Sphingomyelin Homeostasis and Growth in Human HeLa Cells

“…Remarkably cells treated with SMS1 siRNA for 7 days displayed only a minor (up to 20%) reduction in SM levels compared with NS or LA siRNA-treated cells, namely from 7.7 to 6.3 mol % of total phospholipid (Table 1). This reduction was reached within 3 days of siRNA treatment 4 and affected all major SM species to a similar extent (Fig. 9).…”

“…Remarkably cells treated with SMS1 or SMS2 siRNAs for 3 days displayed a 4-and 2-fold decrease in [ 14 C]PC levels compared with NS or LA siRNA-treated cells, respectively. 4 This was likely due to a general down-regulation of phospholipid biosynthesis in SMS-depleted cells because incorporation of 3-L-[ 14 C]serine into phosphatidylserine was affected to a similar extent (3-and 2-fold decrease in SMS1 and SMS2 siRNAtreated cells, respectively). Nevertheless after normalization against [ 14 C]PC levels, SMS1-depleted cells displayed a marked reduction in the amount of [ 14 C]SM compared with NS or LA siRNA-treated cells, namely from 45% after 3 days to 70% after 7 days of depletion ( Fig.…”

“…The mutant cells could be rescued by added SM but not by glucosylceramide (GlcCer), the precursor of higher glycosphingolipids (3). Another study reported the isolation of a mouse lymphoid cell line with diminished SM synthase activity and a defect in cell growth when cultured under serum-free conditions; growth could be restored by supplementing the medium with exogenous SM (4). Finally fluctuations in SM synthase activity have been linked to mitogenic and proapoptotic signaling in a variety of mammalian cell types (5-7).…”

Tales and Mysteries of the Enigmatic Sphingomyelin Synthase Family

“…59 Due to a decreased SM content of the plasma membrane, these lymphoid cells are highly susceptible to the toxic effects of methyl-`-cyclodextrin (M`CD), a compound used to deplete cholesterol from cellular membranes. SMS1 was recovered from a human cDNA expression library on the basis of its ability to restore M`CD resistance.…”

Caspase-mediated inhibition of sphingomyelin synthesis is involved in FasL-triggered cell death

“…17 Two different mammalian genes encoding SMS have been cloned so far. 18,19 The corresponding proteins, SMS1 and SMS2, are both localized at the Golgi, SMS2 being localized at the plasma membrane as well. 18 Both SMS1 and 2 transfer the phosphocholine moiety from phosphatidylcholine (PC) to ceramide to generate SM and diacylglycerol (DAG).…”