Key points are not available for this paper at this time.
Sphingomyelin (SM) is a vital component of cellular membranes in organisms ranging from mammals to protozoa. Its production involves the transfer of phosphocholine from phosphatidylcholine to ceramide, yielding diacylglycerol in the process. The mammalian genome encodes two known SM synthase (SMS) isoforms, SMS1 and SMS2. However, the relative contributions of these enzymes to SM production in mammalian cells remained to be established. Here we show that SMS1 and SMS2 are co-expressed in a variety of cell types and function as the key Golgi- and plasma membrane-associated SM synthases in human cervical carcinoma HeLa cells, respectively. RNA interference-mediated depletion of either SMS1 or SMS2 caused a substantial decrease in SM production levels, an accumulation of ceramides, and a block in cell growth. Although SMS-depleted cells displayed a reduced SM content, external addition of SM did not restore growth. These results indicate that the biological role of SM synthases goes beyond formation of SM. Sphingomyelin (SM) is a vital component of cellular membranes in organisms ranging from mammals to protozoa. Its production involves the transfer of phosphocholine from phosphatidylcholine to ceramide, yielding diacylglycerol in the process. The mammalian genome encodes two known SM synthase (SMS) isoforms, SMS1 and SMS2. However, the relative contributions of these enzymes to SM production in mammalian cells remained to be established. Here we show that SMS1 and SMS2 are co-expressed in a variety of cell types and function as the key Golgi- and plasma membrane-associated SM synthases in human cervical carcinoma HeLa cells, respectively. RNA interference-mediated depletion of either SMS1 or SMS2 caused a substantial decrease in SM production levels, an accumulation of ceramides, and a block in cell growth. Although SMS-depleted cells displayed a reduced SM content, external addition of SM did not restore growth. These results indicate that the biological role of SM synthases goes beyond formation of SM. Sphingolipids are ubiquitous components of biomembranes in eukaryotic cells. The most abundant sphingolipid species in mammalian cells is sphingomyelin (SM), 3The abbreviations used are: SM, sphingomyelin; DAG, diacylglycerol; GlcCer, glucosylceramide; PC, phosphatidylcholine; PE, phosphatidylethanolamine; SMS, sphingomyelin synthase; siRNA, small interfering RNA duplex; NBD, 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)); NS, non-silencing; LA, lamin A; HBSS, Hanks' buffered saline solution; BSA, bovine serum albumin; EPC, ethanolamine phosphorylceramide; MS, mass spectrometry. which comprises 5–15% of total phospholipids. SM synthesis involves the transfer of a phosphocholine head group from phosphatidylcholine (PC) to ceramide with concomitant production of diacylglycerol (DAG) (1Ullman M.D. Radin N.S. J. Biol. Chem. 1974; 249: 1506-1512Abstract Full Text PDF PubMed Google Scholar, 2Voelker D.R. Kennedy E.P. Biochemistry. 1982; 21: 2753-2759Crossref PubMed Scopus (160) Google Scholar). The enzyme catalyzing this reaction, SM synthase, thus occupies a central position at a crossroad of sphingolipid and glycerolipid metabolism and has considerable biological potential as a regulator of the proapoptotic factor ceramide and mitogenic factor DAG. Indeed several lines of evidence indicate that SM synthesis plays a critical role in cell growth and survival. Chinese hamster ovary mutant cells with a thermolabile serine palmitoyltransferase, the rate-limiting enzyme in sphingolipid synthesis, die in the absence of externally added sphingoid base when shifted to the non-permissive temperature. The mutant cells could be rescued by added SM but not by glucosylceramide (GlcCer), the precursor of higher glycosphingolipids (3Hanada K. Nishijima M. Kiso M. Hasegawa A. Fujita S. Ogawa T. Akamatsu Y. J. Biol. Chem. 1992; 267: 23527-23533Abstract Full Text PDF PubMed Google Scholar). 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 (4Yamaoka S. Miyaji M. Kitano T. Umehara H. Okazaki T. J. Biol. Chem. 2004; 279: 18688-18693Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). Finally fluctuations in SM synthase activity have been linked to mitogenic and proapoptotic signaling in a variety of mammalian cell types (5Luberto C. Hannun Y.A. J. Biol. Chem. 1998; 273: 14550-14559Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 6Riboni L. Viani P. Bassi R. Giussani P. Tettamanti G. J. Biol. Chem. 2001; 276: 12797-12804Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 7Itoh M. Kitano T. Watanabe M. Kondo T. Yabu T. Taguchi Y. Iwai K. Tashima M. Uchiyama T. Okazaki T. Clin. Cancer Res. 2003; 9: 415-423PubMed Google Scholar). The molecular basis for the requirement of SM synthesis in cell growth is not clear, and any of the following scenarios may apply. To begin with, SM accumulates in the outer leaflet of the plasma membrane, and its high packing density and affinity for cholesterol likely contribute to the barrier function of this organelle. In addition, SM at the plasma membrane provides a reservoir of lipid signaling molecules that are liberated by acidic or neutral SMases in response to a variety of biological stimuli (8Andrieu-Abadie N. Levade T. Biochim. Biophys. Acta. 2002; 1585: 126-134Crossref PubMed Scopus (159) Google Scholar); these molecules include ceramide, sphingosine, and sphingosine 1-phosphate, and have been implicated in the regulation of cell proliferation, differentiation, and apoptosis (9Ogretmen B. Hannun Y.A. Nat. Rev. Cancer. 2004; 4: 604-616Crossref PubMed Scopus (1009) Google Scholar, 10Spiegel S. Milstien S. Nat. Rev. Mol. Cell. Biol. 2003; 4: 397-407Crossref PubMed Scopus (1758) Google Scholar). As SM has a strong capacity to form microdomains, its production in the trans Golgi may affect the lateral organization of other membrane components and hence provide a physical basis for sorting events that help establish the compositional and functional differences between the endoplasmic reticulum, plasma membrane, and Golgi itself (11Holthuis J.C. Pomorski T. Raggers R.J. Sprong H. Van Meer G. Physiol. Rev. 2001; 81: 1689-1723Crossref PubMed Scopus (255) Google Scholar). Moreover SM synthesis in the trans Golgi may create a local pool of DAG, which provides a cue for protein kinase D recruitment and the formation of secretory vesicles (12Baron C.L. Malhotra V. Science. 2002; 295: 325-328Crossref PubMed Scopus (341) Google Scholar). Finally by regulating the cellular levels of the proapoptotic factor ceramide and mitogenic factor DAG in opposite directions, SM synthesis may have a direct impact on cell and Science. PubMed Scopus Google Scholar, Y.A. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). an in we a of SM synthases are the from mammals and to the K. J. J.C. J. 2004; PubMed Scopus Google Scholar, P. J.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In of SM production a of SM synthase (SMS) The human genome two of these SMS1 and SMS2. The enzymes to SM synthase and in SM synthesis is known to the Golgi and plasma membrane K. J. J.C. J. 2004; PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar). In line with these (4Yamaoka S. Miyaji M. Kitano T. Umehara H. Okazaki T. J. Biol. Chem. 2004; 279: 18688-18693Abstract Full Text Full Text PDF PubMed Scopus (192) Google reported the of human SMS1 a mouse lymphoid cell line with reduced SM synthase activity and to cell that SMS1 a SM synthase activity in mammalian cells with a critical role in cell growth. However, the molecular basis of the SM synthase defect in the lymphoid cell line has not been a study that of mouse cells to apoptosis is to of SMS1 M. S. M. Van J. PubMed Scopus Google Scholar). cells are in growth when cultured under serum-free exogenous is the of SMS1 and SMS2 to SM production and growth in mammalian cells to be In the study we used RNA to SMS1 or SMS2 from human cervical carcinoma HeLa cells and the of these on the Golgi- and plasma membrane-associated SM synthase SM production levels, lipid and cell growth. from from and from other from and RNA cells cultured at and in medium with with human human K. J. J.C. J. 2004; PubMed Scopus Google or mouse from L. of and in medium RNA on HeLa cells with small interfering RNA as J. A. K. T. 2001; PubMed Scopus Google Scholar). with in medium with or serum as serum by to and J. Res. Full Text PDF PubMed Google Scholar). and on and HeLa cells. SMS1 and SMS2 that a strong and in of the protein of and the protein lamin as RNA cells of with and for or as and cells and SMS1 or SMS2 for as K. J. J.C. J. 2004; PubMed Scopus Google Scholar). to the of human SMS2 to and used to of the for this study and used at a of for a from J. for from and mouse from the and protein K. J. J.C. J. 2004; PubMed Scopus Google Scholar). in saline and with a a with as K. J. J.C. J. 2004; PubMed Scopus Google Scholar). and on density as J. Biol. Chem. Full Text PDF PubMed Google with HeLa cells on and in added K. J. J.C. J. 2004; PubMed Scopus Google with of a at for and in and added to a of at in a for at and from the to and for and SM synthase To this for at in Hanks' buffered saline and in a total of by addition of of under and to from the by for in on a and with cells with or for or in the of for in serum at in saline and to lipid the of and J. Physiol. PubMed Scopus Google Scholar). The under by base in at with and by in or in the of by to on a and with cell lipid to the of J. M. J. Biol. Chem. Full Text PDF PubMed Google but the lipid under in and with for SM, PC, ceramide, and as M. R. M. P. J. PubMed Scopus Google Scholar). by PubMed Scopus Google sphingolipid to M. S. 1974; PubMed Scopus Google and cholesterol as in M. J. J. Res. Full Text PDF PubMed Google Scholar). The lipid molecular species and by as in M. R. M. P. J. PubMed Scopus Google an on line to a mass of the of HeLa cells as M. Nishijima M. K. J. Biol. PubMed Scopus Google with cells on for in with at in for at and with in for at a SM SM synthase activity as A. T. A. Meer G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). In HeLa cells with and for at in and by a in The medium and by the of and J. Physiol. PubMed Scopus Google and to from the by in and as cells at with in as of the medium for or serum with exogenous SM or as (3Hanada K. Nishijima M. Kiso M. Hasegawa A. Fujita S. Ogawa T. Akamatsu Y. J. Biol. Chem. 1992; 267: 23527-23533Abstract Full Text PDF PubMed Google Scholar). In in a small of under and in at of with a for at temperature. the cells with and in and the of and cells on a To cells in and with V. of and the cells by as SMS1 and SMS2 in that SMS1 and SMS2 are in human K. J. J.C. J. 2004; PubMed Scopus Google Scholar). may that most cell types SM synthase To we mammalian cell lines for SMS1 and SMS2 RNA from human cervical carcinoma HeLa cells, human cells, human carcinoma cells, mouse cells, and mouse cells. As in the of SMS1 and SMS2 in cell lines These results indicate that SMS1 and SMS2 are by of and HeLa and SMS2 in SM synthesis is known to in the Golgi and at the plasma membrane, K. J. J.C. J. 2004; PubMed Scopus Google Scholar). However, SMS1 and SMS2 are for SM synthesis at these is not to the impact of SMS1 and SMS2 and depletion on Golgi- and plasma membrane-associated SM synthase To this HeLa cells with SMS1 or SMS2 with K. J. J.C. 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M. and SM species to a Moreover by a in ceramide and a in with SMS1 cells, cells with SMS2 a in SM levels a in ceramide levels, and a in levels and The molecular species of the that in and cells that SMS1 and SMS2 have with SMS1 and SMS2 displayed a lipid to that of cells. depletion on the of the PE, and or on the levels of cholesterol or DAG K. M. S. J. A. P. and J. C. M. of and SMS-depleted HeLa of of of of in a The impact of depletion on cellular SM levels in of the reduced enzyme activity However, cells with under serum-free a in SM K. M. S. J. A. P. and J. C. M. the impact of depletion on cellular SM levels be to of SM from SMS1 and SMS2 for in HeLa that SM synthesis is for cell growth in mammalian cells (3Hanada K. Nishijima M. Kiso M. Hasegawa A. Fujita S. Ogawa T. Akamatsu Y. J. Biol. Chem. 1992; 267: 23527-23533Abstract Full Text PDF PubMed Google Scholar, S. Miyaji M. Kitano T. Umehara H. Okazaki T. J. Biol. Chem. 2004; 279: 18688-18693Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar). or enzymes a role in this is To this we the of NS, LA, and SMS2 on the growth and of HeLa cells cultured in or with or on cell growth or with In growth of cells with SMS1 of the and that depletion of SMS1 caused a in cell with cells to in by a in the of cells, from to The of cells did not with SMS1 with SMS2 displayed a growth a in to and a in cells apoptosis to The growth in cells of of and could be by any of SMS2 that in cell SM synthase activity and and these results we that HeLa cells SMS1 and SMS2 to growth. (4Yamaoka S. Miyaji M. Kitano T. Umehara H. Okazaki T. J. Biol. Chem. 2004; 279: 18688-18693Abstract Full Text Full Text PDF PubMed Scopus (192) Google reported the isolation of a mouse lymphoid cell line with diminished SM synthase activity and a defect in cell growth when cultured in serum-free of these cells could be restored by external addition of SM. As in supplementing the medium with exogenous SM at to the growth in SM mouse lymphoid cells did not restore growth in or HeLa cells. These results that a in SM levels is not the or of the growth defect in HeLa cells. reported that the mammalian genome two SMS1 and SMS2 K. J. J.C. J. 2004; PubMed Scopus Google Scholar). In this study we that SMS1 and SMS2 function as the Golgi- and plasma membrane-associated SM synthases in human cervical carcinoma HeLa cells, respectively. Moreover SMS1 and SMS2 to SM production in and to be for growth in HeLa cells. Although and cells displayed a reduced SM content, external addition of SM did not restore growth. These results indicate that the requirement of SM synthases for growth in HeLa cells goes beyond formation of SM. SMS1 is to the Golgi and lines of evidence that this enzyme is for the SM synthase activity in HeLa cells. of SMS1 to a substantial in an SM synthase activity that with the enzyme and from the plasma membrane on a density in to the in cells, which a in plasma membrane-associated SM synthase HeLa cells with to SMS1 of the SM synthase activity that with on a the activity in cells. Finally SMS1 depletion vital of the Golgi with this in cells. the Golgi of a of cultured cells J. Biol. PubMed Scopus Google Scholar, A. Cell. Full Text PDF PubMed Scopus Google but the by which this lipid accumulates in the Golgi is indicate that molecular of at the Golgi that the Golgi is to formation of to of its and absence of a head cellular membranes and the in the Golgi be The is to and be from cells with addition of the head group on the leaflet of the Golgi membrane P. Meer G. J. Biol. PubMed Scopus Google Scholar). is with the that of the Golgi in is by S. Mol. Biol. Cell. PubMed Scopus Google a of the enzyme for synthesis in the Golgi J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). However, not an that SMS1 at the Golgi by a lipid that its to other cellular that of the Golgi to be this involves production of or between and levels in the Golgi are by SMS1 DAG, In any that of the Golgi provides a to SM synthase activity at this in As reported for B. J. Biol. Chem. Full Text PDF PubMed Google and cells A. T. A. Meer G. J. Biol. Chem. Full Text PDF PubMed Google we that HeLa cells a plasma membrane-associated SM synthase activity in cells, reduced in cells, and did not with in SMS1 with the that SMS2 at the plasma membrane, these indicate that SMS2 as the not cell SM synthase in HeLa cells. SMS2 is in K. J. J.C. J. 2004; PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, N. J.C. C. M. J. PubMed Scopus Google and in a variety of mammalian cell lines we that the of a cell SM synthase activity is a of mammalian cells. In enzyme on of cells that SMS1 and SMS2 are for the of SM synthase activity in HeLa cells with SMS1 as a of and SMS2 as a of SM synthase of cells with SM that SMS1 and SMS2 contribute to SM production in with a role for Although SMS2 is with the plasma membrane, a of the protein in the Golgi K. J. J.C. J. 2004; PubMed Scopus Google and the of SM production in the at the plasma membrane, or a reduced SM content, cells a in as as a in The that SM production in the Golgi a of ceramide to synthesis, which in the organelle. with cells, cells in SMS2 a in ceramide levels but accumulation of that SM production at the plasma that SMS1 and SMS2 are for the of SM synthase activity in HeLa cells, that depletion caused a in SM be by of SM from the medium cells with under serum-free a in SM could be that mammalian cells are with an of SM M. J. PubMed Scopus Google a of SM synthesis in which ceramide is to ethanolamine transfer of the head group from is to SM by in a to the of to HeLa cells an synthase in we SMS-depleted cells to for a in SM However, of cells with and evidence for an that to SM in we that the impact of depletion on cellular SM is to the growth which in of and which is by a of synthesis and a substantial in SM to decrease in cells as by with we that HeLa cells SMS1 and SMS2 for growth. with a substantial decrease in SM production levels, and cells displayed a reduced SM as as an accumulation of the vital of SM to the barrier function of the plasma membrane and the of ceramide (9Ogretmen B. Hannun Y.A. Nat. Rev. Cancer. 2004; 4: 604-616Crossref PubMed Scopus (1009) Google either may a growth (4Yamaoka S. Miyaji M. Kitano T. Umehara H. Okazaki T. J. Biol. Chem. 2004; 279: 18688-18693Abstract Full Text Full Text PDF PubMed Scopus (192) Google reported that a mouse lymphoid cell line with diminished SM synthase activity in growth when cultured in the absence of serum or externally added SM. These that SM plays a critical role in cell growth. However, we that the growth defect in SMS-depleted HeLa cells of the medium with serum or exogenous SM. Although this not a vital function of SM in cell indicate that the requirement of SMS1 and SMS2 in this is not to a critical of SM. Moreover the of cells two SM synthases for growth. may be that SMS1 and SMS2 molecular species of ceramide to create of SM DAG that an role in growth. However, of SMS1 and SMS2 in thus differences in enzymes as as to as for SM synthesis K. J. J.C. J. 2004; PubMed Scopus Google Scholar). In addition, did not show any in molecular species between the that in and HeLa cells that SMS1 and SMS2 a at indicate that cell growth SM synthases to in the Golgi and at the plasma SM synthesis DAG, and has been reported that the of DAG in the trans Golgi is critical for protein to the plasma membrane, a for cell growth. In protein protein and are to Golgi membranes and these are for formation of secretory vesicles (12Baron C.L. Malhotra V. Science. 2002; 295: 325-328Crossref PubMed Scopus (341) Google Scholar, C. C. A. Y. T. Malhotra V. Nat. Biol. 2004; PubMed Scopus Google Scholar). Moreover an of ceramide synthesis that production of DAG and SM from ceramide and PC, has been to protein kinase D recruitment and to block protein from the trans Golgi to the cell (12Baron C.L. Malhotra V. Science. 2002; 295: 325-328Crossref PubMed Scopus (341) Google Scholar). to its SMS2 may SMS1 by an of SM at the plasma membrane but not with to DAG for secretory formation at the The requirement of SMS2 in cell growth may a role in at the plasma of SM in response to external stimuli ceramide, a regulator of cell growth Y.A. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). ceramide to SM, SMS2 may signaling at the plasma DAG, which is a mitogenic factor N. C. J. 2002; PubMed Scopus Google Scholar). SMS2 may a critical role in cell growth by regulating the between and stimuli at the plasma The of SM synthase in organisms SM that a Here we have that this is for growth in human HeLa cells. The by which SMS1 and SMS2 contribute to cell growth be the of for for on the and and for and with
Tafesse et al. (Mon,) studied this question.