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W2092187419 abstract "We examined the effects of reduction of sphingomyelin level on cholesterol behavior in cells using 2 types of Chinese hamster ovary cell mutants deficient in sphingomyelin synthesis: LY-A strain defective in intracellular trafficking of ceramide for sphingomyelin synthesis, and LY-B strain defective in the enzyme catalyzing the initial step of sphingolipid biosynthesis. Although the sphingomyelin content in LY-A and LY-B cells was ∼40 and ∼15%, respectively, of the wild-type level without accumulation of ceramide, these mutant cells were almost identical in cholesterol content and also in plasma membrane cholesterol level to the wild-type cells. However, density gradient fractionation analysis of Triton X-100-treated lysates of cells prelabeled with [3H]cholesterol showed that the [3H]cholesterol level in the low-density floating fraction was lower in sphingomyelin-deficient cells than in wild-type cells. When cells were exposed to methyl-β-cyclodextrin, cholesterol was more efficiently fluxed from sphingomyelin-deficient cells than wild-type cells. These results suggest that the steady state level of cholesterol at the plasma membrane is little affected by the sphingomyelin levels in Chinese hamster ovary cells, but that sphingomyelin levels play an important role in the retention of cholesterol in the plasma membrane against efflux to extracellular cholesterol-acceptors, due to interaction between sphingomyelin and cholesterol in detergent-resistant membrane domains. We examined the effects of reduction of sphingomyelin level on cholesterol behavior in cells using 2 types of Chinese hamster ovary cell mutants deficient in sphingomyelin synthesis: LY-A strain defective in intracellular trafficking of ceramide for sphingomyelin synthesis, and LY-B strain defective in the enzyme catalyzing the initial step of sphingolipid biosynthesis. Although the sphingomyelin content in LY-A and LY-B cells was ∼40 and ∼15%, respectively, of the wild-type level without accumulation of ceramide, these mutant cells were almost identical in cholesterol content and also in plasma membrane cholesterol level to the wild-type cells. However, density gradient fractionation analysis of Triton X-100-treated lysates of cells prelabeled with [3H]cholesterol showed that the [3H]cholesterol level in the low-density floating fraction was lower in sphingomyelin-deficient cells than in wild-type cells. When cells were exposed to methyl-β-cyclodextrin, cholesterol was more efficiently fluxed from sphingomyelin-deficient cells than wild-type cells. These results suggest that the steady state level of cholesterol at the plasma membrane is little affected by the sphingomyelin levels in Chinese hamster ovary cells, but that sphingomyelin levels play an important role in the retention of cholesterol in the plasma membrane against efflux to extracellular cholesterol-acceptors, due to interaction between sphingomyelin and cholesterol in detergent-resistant membrane domains. sphingomyelin detergent-resistant membrane glycosylphosphatidylinositol sphingomyelinase Chinese hamster ovary 3-(4,5-dimethyl-thiazoyl-2-yl)-2,5-diphenyltetrazolium bromide methyl-β-cyclodextrin phosphatidylcholine-specific phospholipase C phosphate-buffered saline N-acetylneuraminyl lactosylceramide glucosylceramide 50% lethal dose 4-morpholinoethanesulfonic acid Both cholesterol and sphingomyelin (SM)1 are preferentially distributed in the plasma membrane of cells (1van Meer G. Annu. Rev. Cell Biol. 1989; 5: 247-275Crossref PubMed Scopus (345) Google Scholar). Recent developments in membrane biology have demonstrated various lines of evidence that the plasma membrane has microdomains, termed detergent-resistant membrane (DRM) domains or lipid rafts, which are involved in various cellular events, including signal transduction and membrane trafficking (2Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8015) Google Scholar, 3Brown D.A. London E. J. Biol. Chem. 2000; 275: 17221-17224Abstract Full Text Full Text PDF PubMed Scopus (2043) Google Scholar). DRM domains are highly enriched in cholesterol and sphingolipids, and probably exist as liquid-ordered phase, characterized by a conformationally ordered state of the acyl chains of phospholipids that are laterally diffusible (3Brown D.A. London E. J. Biol. Chem. 2000; 275: 17221-17224Abstract Full Text Full Text PDF PubMed Scopus (2043) Google Scholar). Formation of the liquid-ordered phase reflects the fact that cholesterol interacts favorably with phospholipid acyl chains in an extended conformation. SM and saturated glycerophospholipids readily form the extended acyl chain conformation, while unsaturated phospholipids having cis-configuration of the double bond do not. Many studies with model membranes have indicated that cholesterol interacts with SM more strongly than with unsaturated glycerophospholipids, the predominant forms of natural glycerophospholipids (4Demel R.A. Jansen J.W.C.M. van Dijck P.W.M. van Deenen L.L.M. Biochim. Biophys. Acta. 1977; 465: 1-10Crossref PubMed Scopus (300) Google Scholar, 5Yeagle P.L. Young J.E. J. Biol. Chem. 1986; 261: 8175-8181Abstract Full Text PDF PubMed Google Scholar, 6Lund-Katz S. Laboda H.M. McLean L.R. Phillips M.C. Biochemistry. 1988; 27: 3323-3416Crossref Scopus (206) Google Scholar, 7Ramstedt B. Slotte J.P. Biophys. J. 1999; 76: 908-915Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). In addition, previous studies with model membrane vesicles consisting of pure lipids have shown that cholesterol enhances detergent insolubility of SM, and that sphingolipids or saturated glycerophospholipids tending to form the lipid-ordered phase are also important for detergent insolubility of cholesterol (8Schroeder R. London E. Brown D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12130-12134Crossref PubMed Scopus (635) Google Scholar,9Schroeder R.J. Ahmed S.N. Zhu Y. London E. Brown D.A. J. Biol. Chem. 1998; 273: 1150-1157Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar).Only a few reports, however, have addressed the issue of the participation of SM in the formation of DRM domains in cells, whereas many studies have demonstrated various lines of evidence that cholesterol is responsible for formation and functions of DRM domains in cells using specific biological and chemical tools to inhibit cholesterol synthesis and to remove cholesterol from cells (2Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8015) Google Scholar, 3Brown D.A. London E. J. Biol. Chem. 2000; 275: 17221-17224Abstract Full Text Full Text PDF PubMed Scopus (2043) Google Scholar, 10Anderson R.G. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1714) Google Scholar). We previously showed evidence that reduction of the cellular sphingolipid level renders glycosyl phosphatidylinositol (GPI)-anchored proteins more sensitive to bacterial phosphatidylinositol-specific phospholipase C (11Hanada K. Izawa K. Nishijima M. Akamatsu Y. J. Biol. Chem. 1993; 268: 13820-13823Abstract Full Text PDF PubMed Google Scholar) and also enhances solubility of GPI-anchored proteins in the non-ionic detergent Triton X-100 (12Hanada K. Nishijima M. Akamatsu Y. Pagano R.E. J. Biol. Chem. 1995; 270: 6254-6260Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). The blockage of ceramide synthesis by fumonisin B1, a potent inhibitor of dihydrosphingosine-N-acyltransferase, has been shown to inhibit folate uptake via the GPI-anchored receptors (13Stevens V.L. Tang J. J. Biol. Chem. 1997; 272: 18020-18025Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar), and to enhance conversion of prion protein, a GPI-anchored protein, to its scrapie isoform (14Naslavsky N. Shmeeda H. Friedlander G. Yanai A. Futerman A.H. Barenholz Y. Taraboulos A. J. Biol. Chem. 1999; 274: 20763-20771Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). These previous studies have indicated that sphingolipids are important components of DRM domains. However, a mouse melanoma cell mutant defective in glycosphingolipid synthesis has very recently been shown to retain almost normal DRM domains (15Ostermeyer A.G. Beckrich B.T. Ivarson K.A. Grove K.E. Brown D.A. J. Biol. Chem. 1999; 274: 34459-34466Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). It is therefore imperative that we determine whether SM is a key component of DRM domains.Analysis of SM-deficient cells may yield insight into the biological role of SM. Although treatment of cells with bacterial sphingomyelinase (SMase) is a convenient method for depletion of SM from cell surfaces, this method has the unavoidable flaw that degradation of SM by SMase results in accumulation of ceramide, which serves as a modulator of various cellular functions containing the metabolism and dynamics of cholesterol (16Gupta A.K. Rudney H. J. Lipid Res. 1991; 32: 125-136Abstract Full Text PDF PubMed Google Scholar, 17Härmälä A.-S. Pörn M.I. Slotte J.P. Biochim. Biophys. Acta. 1993; 1210: 97-104Crossref PubMed Scopus (25) Google Scholar, 18Ridgway N.D. Biochim. Biophys. Acta. 1995; 1256: 39-46Crossref PubMed Scopus (26) Google Scholar). Thus, when using this method, one must carefully eliminate the possibility that effects of SMase treatment of cells are due to accumulation of ceramide, rather than depletion of SM. Unfortunately, this possibility cannot be eliminated by showing that externally supplied ceramides do not mimic the SMase effects, because long-chain natural ceramide in culture medium is hard to incorporate into the plasma membrane of cells, and short-chain unnatural ceramides that are readily incorporated into cells are quite different in physicochemical properties from natural ceramide produced by treatment of cells with SMase. One way of overcoming this problem is to use cell mutants defective in production of SM without accumulation of ceramide. Recently, we have isolated 2 types of Chinese hamster ovary (CHO) cell mutants defective in sphingolipid biosynthesis (19Hanada K. Hara T. Fukasawa M. Yamaji A. Umeda M. Nishijima M. J. Biol. Chem. 1998; 273: 33787-33794Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). One is LY-A strain defective in ATP-dependent endoplasmic reticulum to Golgi apparatus trafficking of ceramide directed to SM biosynthesis (20Fukasawa M. Nishijima M. Hanada K. J. Cell Biol. 1999; 144: 673-685Crossref PubMed Scopus (148) Google Scholar). Thus, the level of SM, but not of glycosphingolipids, in LY-A cells is lower than the wild-type level. Another mutant, LY-B strain, has a defect in the LCB1 subunit of serine palmitoyltransferase, the enzyme catalyzing the first step in sphingolipid biosynthesis, thereby being incapable of de novo synthesis of any sphingolipid species.In the present study, by analysis with CHO cell mutants, we demonstrated that reduction of cellular SM to ∼15% of the wild-type level without accumulation of ceramide causes no change in the plasma membrane cholesterol level, but that SM plays a role in the retention of plasma membrane cholesterol against efflux to extracellular acceptors of cholesterol. In addition, this study showed for the first time that SM is implicated in the formation of DRM domains in cells.DISCUSSIONIn this study, we investigated the effects of reduction of cellular SM on cholesterol behavior in the plasma membrane using CHO cell mutants defective in SM production. There were no differences in the content of cellular cholesterol and its distribution to the cholesterol oxidase-sensitive pool between these mutant and wild-type cells, even when the SM content in the mutant cells was 15–40% of the wild-type level (Tables I and II, and Fig. 2). LY-B cells were also deficient in ceramide and glycosphingolipids, while the ceramide and glycosphingolipid content in LY-A cells was similar to the wild-type levels (Table I, and see also Ref. 19Hanada K. Hara T. Fukasawa M. Yamaji A. Umeda M. Nishijima M. J. Biol. Chem. 1998; 273: 33787-33794Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Hence, the possibility was eliminated that the failure to affect the cholesterol levels under SM-deficient conditions was due to functional compensation by an increase of ceramide or glycosphingolipid content. These results indicated that the reduction of cellular SM to ∼15% of the wild-type level has no effect on the steady-state level of cellular cholesterol and its distribution to the plasma membrane. Enrichment of SM in the plasma membrane has been hypothesized to force cholesterol to be also enriched in the plasma membrane, based on the observations that addition of SM to cultured human skin fibroblasts results in an increase in cellular cholesterol content according to the increase in cellular SM level (35Kudchodkar B.J. Albers J.J. Bierman E.L. Atherosclerosis. 1983; 46: 353-367Abstract Full Text PDF PubMed Scopus (19) Google Scholar), and that cholesterol interacts with SM more strongly than with glycerophospholipids in model membranes (6Lund-Katz S. Laboda H.M. McLean L.R. Phillips M.C. Biochemistry. 1988; 27: 3323-3416Crossref Scopus (206) Google Scholar, 36Wattenberg B.W. Silbert D.F. J. Biol. Chem. 1983; 258: 2284-2289Abstract Full Text PDF PubMed Google Scholar, 37van Blitterswijk W.J. van der Meer B.W. Hilkmann H. Biochemistry. 1987; 26: 1746-1756Crossref PubMed Scopus (176) Google Scholar). However, our analyses with CHO cells suggest that neither SM, glycosphingolipids, nor ceramide is essential for the preferential distribution of cholesterol to the plasma membrane. Consistent with these results, a recent study has shown that most cellular cholesterol is present in the plasma membrane even after depletion of cell surface SM by treatment of human skin fibroblasts with bacterial SMase (38Pörn M.I. Slotte J.P. Biochem. J. 1995; 308: 269-274Crossref PubMed Scopus (46) Google Scholar). It is unlikely that the reduced level of SM in the CHO mutant cells is still sufficient for a stoichiometric association with cholesterol at the plasma membrane, because the estimated amount of SM in LY-B cells (∼20 nmol/μmol cellular phospholipid) is far less than that of cholesterol (∼300 nmol/μmol cellular phospholipid) (Tables I andII), and 70–80% of cholesterol in both LY-B and wild-type cells appears to be distributed in the plasma membrane (Fig. 2). Energy-dependent transport of de novosynthesized cholesterol from the endoplasmic reticulum to the plasma membrane may be primarily responsible for enrichment of cholesterol in the plasma membrane (39DeGrella R.F. Simoni R.D. J. Biol. Chem. 1982; 257: 14256-14262Abstract Full Text PDF PubMed Google Scholar, 40Smart E.J. Ying Y. Donzell W.C. Anderson R.G. J. Biol. Chem. 1996; 271: 29427-29435Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar).We previously showed that sphingolipids are involved in the detergent insolubility of a GPI-anchored protein in CHO cells (12Hanada K. Nishijima M. Akamatsu Y. Pagano R.E. J. Biol. Chem. 1995; 270: 6254-6260Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). However, another research group has recently shown that glycosphingolipids are dispensable for formation of DRM domains in murine melanoma cells (15Ostermeyer A.G. Beckrich B.T. Ivarson K.A. Grove K.E. Brown D.A. J. Biol. Chem. 1999; 274: 34459-34466Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar), although the original concept of lipid-raft is based on the assumption that the hydrogen bond interaction between the sugar head groups of glycosphingolipids and GPI-anchored proteins is important for formation of detergent-resistant rafts (41Simons K. van Meer G. Biochemistry. 1988; 27: 6197-6202Crossref PubMed Scopus (1079) Google Scholar). Ostermeyer et al. (15Ostermeyer A.G. Beckrich B.T. Ivarson K.A. Grove K.E. Brown D.A. J. Biol. Chem. 1999; 274: 34459-34466Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar) also demonstrated that there is no difference in MβCD-mediated efflux of cholesterol between glycosphingolipid-depleted cells and control cells. We herein demonstrated that CHO mutant LY-A cells having a lower level of SM, but not of glycosphingolipids, had a lower level of DRM-associated cholesterol than wild-type cells (Fig. 3). These results indicate that SM plays a role in the formation of DRM domains in CHO cells. This conclusion is consistent with the observation that the liquid-ordered phase of lipid bilayers forms in model membranes containing cholesterol and SM (42Ahmed S.N. Brown D.A. London E. Biochemistry. 1997; 36: 10944-10953Crossref PubMed Scopus (609) Google Scholar). In model membranes, total mol % of lipids tending to form the liquid-ordered phase has been shown to be important for the formation of DRM domains (8Schroeder R. London E. Brown D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12130-12134Crossref PubMed Scopus (635) Google Scholar, 9Schroeder R.J. Ahmed S.N. Zhu Y. London E. Brown D.A. J. Biol. Chem. 1998; 273: 1150-1157Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, 42Ahmed S.N. Brown D.A. London E. Biochemistry. 1997; 36: 10944-10953Crossref PubMed Scopus (609) Google Scholar). Considering that levels of total sphingolipids in SM-deficient CHO mutant cells are lower than the wild-type level (Table I), the total level of SM and glycosphingolipids, but not the level of SM itself, might be a key factor for the formation of DRM domains in cells (see also below).Various investigators have demonstrated that treatment of cells with exogenous SMase enhances efflux of cellular cholesterol to the extracellular cholesterol acceptors, such as high density lipoproteins and cyclodextrins (31Neufeld E.B. Cooney A.M. Pitha J. Dawidowicz E.A. Dwyer N.K. Pentchev P.G. Blanchette-Mackie E.J. J. Biol. Chem. 1996; 271: 21604-21613Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 43Slotte J.P. Härmälä A.S. Jansson C. Pörn M.I. Biochim. Biophys. Acta. 1990; 1030: 251-257Crossref PubMed Scopus (56) Google Scholar, 44Stein O. Ben-Naim M. Dabach Y. Hollander G. Stein Y. Biochim. Biophys. Acta. 1992; 1126: 291-297Crossref PubMed Scopus (21) Google Scholar, 45Pörn M.I. Ares M.P. Slotte J.P. J. Lipid Res. 1993; 34: 1385-1392Abstract Full Text PDF PubMed Google Scholar, 46Ito J. Nagayasu Y. Yokoyama S. J. Lipid Res. 2000; 41: 894-904Abstract Full Text Full Text PDF PubMed Google Scholar). Nevertheless, these previous studies did not eliminate the possibility that the enhanced efflux of cholesterol after SMase treatment was due to accumulation of ceramide. We herein demonstrated that the reduction of cellular SM content without any increase in ceramide level renders plasma membrane cholesterol prone to flux to the extracellular acceptor MβCD (Fig.4). Thus, we conclude that SM play a role in the retention of cholesterol in the plasma membrane against efflux to the extracellular cholesterol-acceptor, probably due to favorable van der Waals and hydrogen-bonding interactions between cholesterol and SM (47Kan C.C. Ruan Z.S. Bittman R. Biochemistry. 1991; 30: 7759-7766Crossref PubMed Scopus (79) Google Scholar, 48Bittman R. Kasireddy C.R. Mattjus P. Slotte J.P. Biochemistry. 1994; 33: 11776-11781Crossref PubMed Scopus (150) Google Scholar). This conclusion does not exclude the possibility that total sphingolipids (SM, ceramide, and glycosphingolipids) or complex sphingolipids (SM and glycosphingolipids) are responsible for the retention of cholesterol. However, treatment of cells with bacterial SMase not only enhances cholesterol efflux from cells (Refs. 31Neufeld E.B. Cooney A.M. Pitha J. Dawidowicz E.A. Dwyer N.K. Pentchev P.G. Blanchette-Mackie E.J. J. Biol. Chem. 1996; 271: 21604-21613Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar and 43Slotte J.P. Härmälä A.S. Jansson C. Pörn M.I. Biochim. Biophys. Acta. 1990; 1030: 251-257Crossref PubMed Scopus (56) Google Scholar, 44Stein O. Ben-Naim M. Dabach Y. Hollander G. Stein Y. Biochim. Biophys. Acta. 1992; 1126: 291-297Crossref PubMed Scopus (21) Google Scholar, 45Pörn M.I. Ares M.P. Slotte J.P. J. Lipid Res. 1993; 34: 1385-1392Abstract Full Text PDF PubMed Google Scholar, 46Ito J. Nagayasu Y. Yokoyama S. J. Lipid Res. 2000; 41: 894-904Abstract Full Text Full Text PDF PubMed Google Scholar, and Fig. 6) and also reduces cholesterol distribution to DRM domains (46Ito J. Nagayasu Y. Yokoyama S. J. Lipid Res. 2000; 41: 894-904Abstract Full Text Full Text PDF PubMed Google Scholar), although total sphingolipid levels most likely remain unchanged after SMase treatment. Therefore, levels of SM itself or total complex sphingolipids in cells are likely to be important for the formation of DRM domains and the retention of cholesterol against efflux.Interestingly, MβCD-mediated efflux of cholesterol from cells results in reduction of cholesterol level in DRM, but not non-DRM domains (Fig.3). The simplest explanation for this observation is that MβCD-mediated efflux of cholesterol preferentially occurs at the DRM domains in cells. Fielding and Fielding (22Fielding P.E. Fielding C.J. Biochemistry. 1995; 34: 14288-14292Crossref PubMed Scopus (256) Google Scholar, 49Fielding C.J. Fielding P.E. J. Lipid Res. 1997; 38: 1503-1521Abstract Full Text PDF PubMed Google Scholar) have suggested that cholesterol efflux from the cell surface to high density lipoproteins occurs largely at DRM domains of the plasma membrane. Alternatively, MβCD-mediated efflux of cholesterol may occur at non-DRM domains of the plasma membrane, but cellular cholesterol is rapidly redistributed from DRM to non-DRM domains for homeostasis of the cholesterol level of non-DRM domains. A previous study has shown that de novosynthesized cholesterol at the endoplasmic reticulum is delivered to DRM domains of the plasma membrane by a caveolin-dependent mechanism, and that the cholesterol delivered to DRM domains then rapidly flows to non-DRM domains at the plasma membrane (40Smart E.J. Ying Y. Donzell W.C. Anderson R.G. J. Biol. Chem. 1996; 271: 29427-29435Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar).It is also noteworthy that cytotoxic susceptibility of CHO cells to MβCD is enhanced when cellular SM level is reduced (Figs. 5 and 6). Because MβCD-mediated efflux of cholesterol is enhanced in SM-deficient cells (Fig. 4), it is likely that cholesterol is depleted below the level necessary for sustaining viability more easily from SM-deficient cells than from control cells. The higher toxicity of MβCD to SM-deficient cells than control cells may allow the use of this drug for a positive selection of revertants of SM-deficient mutant cells. Both cholesterol and sphingomyelin (SM)1 are preferentially distributed in the plasma membrane of cells (1van Meer G. Annu. Rev. Cell Biol. 1989; 5: 247-275Crossref PubMed Scopus (345) Google Scholar). Recent developments in membrane biology have demonstrated various lines of evidence that the plasma membrane has microdomains, termed detergent-resistant membrane (DRM) domains or lipid rafts, which are involved in various cellular events, including signal transduction and membrane trafficking (2Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8015) Google Scholar, 3Brown D.A. London E. J. Biol. Chem. 2000; 275: 17221-17224Abstract Full Text Full Text PDF PubMed Scopus (2043) Google Scholar). DRM domains are highly enriched in cholesterol and sphingolipids, and probably exist as liquid-ordered phase, characterized by a conformationally ordered state of the acyl chains of phospholipids that are laterally diffusible (3Brown D.A. London E. J. Biol. Chem. 2000; 275: 17221-17224Abstract Full Text Full Text PDF PubMed Scopus (2043) Google Scholar). Formation of the liquid-ordered phase reflects the fact that cholesterol interacts favorably with phospholipid acyl chains in an extended conformation. SM and saturated glycerophospholipids readily form the extended acyl chain conformation, while unsaturated phospholipids having cis-configuration of the double bond do not. Many studies with model membranes have indicated that cholesterol interacts with SM more strongly than with unsaturated glycerophospholipids, the predominant forms of natural glycerophospholipids (4Demel R.A. Jansen J.W.C.M. van Dijck P.W.M. van Deenen L.L.M. Biochim. Biophys. Acta. 1977; 465: 1-10Crossref PubMed Scopus (300) Google Scholar, 5Yeagle P.L. Young J.E. J. Biol. Chem. 1986; 261: 8175-8181Abstract Full Text PDF PubMed Google Scholar, 6Lund-Katz S. Laboda H.M. McLean L.R. Phillips M.C. Biochemistry. 1988; 27: 3323-3416Crossref Scopus (206) Google Scholar, 7Ramstedt B. Slotte J.P. Biophys. J. 1999; 76: 908-915Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). In addition, previous studies with model membrane vesicles consisting of pure lipids have shown that cholesterol enhances detergent insolubility of SM, and that sphingolipids or saturated glycerophospholipids tending to form the lipid-ordered phase are also important for detergent insolubility of cholesterol (8Schroeder R. London E. Brown D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12130-12134Crossref PubMed Scopus (635) Google Scholar,9Schroeder R.J. Ahmed S.N. Zhu Y. London E. Brown D.A. J. Biol. Chem. 1998; 273: 1150-1157Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar). Only a few reports, however, have addressed the issue of the participation of SM in the formation of DRM domains in cells, whereas many studies have demonstrated various lines of evidence that cholesterol is responsible for formation and functions of DRM domains in cells using specific biological and chemical tools to inhibit cholesterol synthesis and to remove cholesterol from cells (2Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8015) Google Scholar, 3Brown D.A. London E. J. Biol. Chem. 2000; 275: 17221-17224Abstract Full Text Full Text PDF PubMed Scopus (2043) Google Scholar, 10Anderson R.G. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1714) Google Scholar). We previously showed evidence that reduction of the cellular sphingolipid level renders glycosyl phosphatidylinositol (GPI)-anchored proteins more sensitive to bacterial phosphatidylinositol-specific phospholipase C (11Hanada K. Izawa K. Nishijima M. Akamatsu Y. J. Biol. Chem. 1993; 268: 13820-13823Abstract Full Text PDF PubMed Google Scholar) and also enhances solubility of GPI-anchored proteins in the non-ionic detergent Triton X-100 (12Hanada K. Nishijima M. Akamatsu Y. Pagano R.E. J. Biol. Chem. 1995; 270: 6254-6260Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). The blockage of ceramide synthesis by fumonisin B1, a potent inhibitor of dihydrosphingosine-N-acyltransferase, has been shown to inhibit folate uptake via the GPI-anchored receptors (13Stevens V.L. Tang J. J. Biol. Chem. 1997; 272: 18020-18025Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar), and to enhance conversion of prion protein, a GPI-anchored protein, to its scrapie isoform (14Naslavsky N. Shmeeda H. Friedlander G. Yanai A. Futerman A.H. Barenholz Y. Taraboulos A. J. Biol. Chem. 1999; 274: 20763-20771Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). These previous studies have indicated that sphingolipids are important components of DRM domains. However, a mouse melanoma cell mutant defective in glycosphingolipid synthesis has very recently been shown to retain almost normal DRM domains (15Ostermeyer A.G. Beckrich B.T. Ivarson K.A. Grove K.E. Brown D.A. J. Biol. Chem. 1999; 274: 34459-34466Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). It is therefore imperative that we determine whether SM is a key component of DRM domains. Analysis of SM-deficient cells may yield insight into the biological role of SM. Although treatment of cells with bacterial sphingomyelinase (SMase) is a convenient method for depletion of SM from cell surfaces, this method has the unavoidable flaw that degradation of SM by SMase results in accumulation of ceramide, which serves as a modulator of various cellular functions containing the metabolism and dynamics of cholesterol (16Gupta A.K. Rudney H. J. Lipid Res. 1991; 32: 125-136Abstract Full Text PDF PubMed Google Scholar, 17Härmälä A.-S. Pörn M.I. Slotte J.P. Biochim. Biophys. Acta. 1993; 1210: 97-104Crossref PubMed Scopus (25) Google Scholar, 18Ridgway N.D. Biochim. Biophys. Acta. 1995; 1256: 39-46Crossref PubMed Scopus (26) Google Scholar). Thus, when using this method, one must carefully eliminate the possibility that effects of SMase treatment of cells are due to accumulation of ceramide, rather than depletion of SM. Unfortunately, this possibility cannot be eliminated by showing that externally supplied ceramides do not mimic the SMase effects, because long-chain natural ceramide in culture medium is hard to incorporate into the plasma membrane of cells, and short-chain unnatural ceramides that are readily incorporated into cells are quite different in physicochemical properties from natural ceramide produced by treatment of cells with SMase. One way of overcoming this problem is to use cell mutants defective in production of SM without accumulation of ceramide. Recently, we have isolated 2 types of Chinese hamster ovary (CHO) cell mutants defective in sphingolipid biosynthesis (19Hanada K. Hara T. Fukasawa M. Yamaji A. Umeda M. Nishijima M. J. Biol. Chem. 1998; 273: 33787-33794Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). One is LY-A strain defective in ATP-dependent endoplasmic reticulum to Golgi apparatus trafficking of ceramide directed to SM biosynthesis (20Fukasawa M. Nishijima M. Hanada K. J. Cell Biol. 1999; 144: 673-685Crossref PubMed Scopus (148) Google Scholar). Thus, the level of SM, but not of glycosphingolipids, in LY-A cells is lower than the wild-type level. Another mutant, LY-B strain, has a defect in the LCB1 subunit of serine palmitoyltransferase, the enzyme catalyzing the first step in sphingolipid biosynthesis, thereby being incapable of de novo synthesis of any sphingolipid species. In the present study, by analysis with CHO cell mutants, we demonstrated that reduction of cellular SM to ∼15% of the wild-type level without accumulation of ceramide causes no change in the plasma membrane cholesterol level, but that SM plays a role in the retention of plasma membrane cholesterol against efflux to extracellular acceptors of cholesterol. In addition, this study showed for the first time that SM is implicated in the formation of DRM domains in cells. DISCUSSIONIn this study, we investigated the effects of reduction of cellular SM on cholesterol behavior in the plasma membrane using CHO cell mutants defective in SM production. There were no differences in the content of cellular cholesterol and its distribution to the cholesterol oxidase-sensitive pool between these mutant and wild-type cells, even when the SM content in the mutant cells was 15–40% of the wild-type level (Tables I and II, and Fig. 2). LY-B cells were also deficient in ceramide and glycosphingolipids, while the ceramide and glycosphingolipid content in LY-A cells was similar to the wild-type levels (Table I, and see also Ref. 19Hanada K. Hara T. Fukasawa M. Yamaji A. Umeda M. Nishijima M. J. Biol. Chem. 1998; 273: 33787-33794Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Hence, the possibility was eliminated that the failure to affect the cholesterol levels under SM-deficient conditions was due to functional compensation by an increase of ceramide or glycosphingolipid content. These results indicated that the reduction of cellular SM to ∼15% of the wild-type level has no effect on the steady-state level of cellular cholesterol and its distribution to the plasma membrane. Enrichment of SM in the plasma membrane has been hypothesized to force cholesterol to be also enriched in the plasma membrane, based on the observations that addition of SM to cultured human skin fibroblasts results in an increase in cellular cholesterol content according to the increase in cellular SM level (35Kudchodkar B.J. Albers J.J. Bierman E.L. Atherosclerosis. 1983; 46: 353-367Abstract Full Text PDF PubMed Scopus (19) Google Scholar), and that cholesterol interacts with SM more strongly than with glycerophospholipids in model membranes (6Lund-Katz S. Laboda H.M. McLean L.R. Phillips M.C. Biochemistry. 1988; 27: 3323-3416Crossref Scopus (206) Google Scholar, 36Wattenberg B.W. Silbert D.F. J. Biol. Chem. 1983; 258: 2284-2289Abstract Full Text PDF PubMed Google Scholar, 37van Blitterswijk W.J. van der Meer B.W. Hilkmann H. Biochemistry. 1987; 26: 1746-1756Crossref PubMed Scopus (176) Google Scholar). However, our analyses with CHO cells suggest that neither SM, glycosphingolipids, nor ceramide is essential for the preferential distribution of cholesterol to the plasma membrane. Consistent with these results, a recent study has shown that most cellular cholesterol is present in the plasma membrane even after depletion of cell surface SM by treatment of human skin fibroblasts with bacterial SMase (38Pörn M.I. Slotte J.P. Biochem. J. 1995; 308: 269-274Crossref PubMed Scopus (46) Google Scholar). It is unlikely that the reduced level of SM in the CHO mutant cells is still sufficient for a stoichiometric association with cholesterol at the plasma membrane, because the estimated amount of SM in LY-B cells (∼20 nmol/μmol cellular phospholipid) is far less than that of cholesterol (∼300 nmol/μmol cellular phospholipid) (Tables I andII), and 70–80% of cholesterol in both LY-B and wild-type cells appears to be distributed in the plasma membrane (Fig. 2). Energy-dependent transport of de novosynthesized cholesterol from the endoplasmic reticulum to the plasma membrane may be primarily responsible for enrichment of cholesterol in the plasma membrane (39DeGrella R.F. Simoni R.D. J. Biol. Chem. 1982; 257: 14256-14262Abstract Full Text PDF PubMed Google Scholar, 40Smart E.J. Ying Y. Donzell W.C. Anderson R.G. J. Biol. Chem. 1996; 271: 29427-29435Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar).We previously showed that sphingolipids are involved in the detergent insolubility of a GPI-anchored protein in CHO cells (12Hanada K. Nishijima M. Akamatsu Y. Pagano R.E. J. Biol. Chem. 1995; 270: 6254-6260Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). However, another research group has recently shown that glycosphingolipids are dispensable for formation of DRM domains in murine melanoma cells (15Ostermeyer A.G. Beckrich B.T. Ivarson K.A. Grove K.E. Brown D.A. J. Biol. Chem. 1999; 274: 34459-34466Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar), although the original concept of lipid-raft is based on the assumption that the hydrogen bond interaction between the sugar head groups of glycosphingolipids and GPI-anchored proteins is important for formation of detergent-resistant rafts (41Simons K. van Meer G. Biochemistry. 1988; 27: 6197-6202Crossref PubMed Scopus (1079) Google Scholar). Ostermeyer et al. (15Ostermeyer A.G. Beckrich B.T. Ivarson K.A. Grove K.E. Brown D.A. J. Biol. Chem. 1999; 274: 34459-34466Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar) also demonstrated that there is no difference in MβCD-mediated efflux of cholesterol between glycosphingolipid-depleted cells and control cells. We herein demonstrated that CHO mutant LY-A cells having a lower level of SM, but not of glycosphingolipids, had a lower level of DRM-associated cholesterol than wild-type cells (Fig. 3). These results indicate that SM plays a role in the formation of DRM domains in CHO cells. This conclusion is consistent with the observation that the liquid-ordered phase of lipid bilayers forms in model membranes containing cholesterol and SM (42Ahmed S.N. Brown D.A. London E. Biochemistry. 1997; 36: 10944-10953Crossref PubMed Scopus (609) Google Scholar). In model membranes, total mol % of lipids tending to form the liquid-ordered phase has been shown to be important for the formation of DRM domains (8Schroeder R. London E. Brown D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12130-12134Crossref PubMed Scopus (635) Google Scholar, 9Schroeder R.J. Ahmed S.N. Zhu Y. London E. Brown D.A. J. Biol. Chem. 1998; 273: 1150-1157Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, 42Ahmed S.N. Brown D.A. London E. Biochemistry. 1997; 36: 10944-10953Crossref PubMed Scopus (609) Google Scholar). Considering that levels of total sphingolipids in SM-deficient CHO mutant cells are lower than the wild-type level (Table I), the total level of SM and glycosphingolipids, but not the level of SM itself, might be a key factor for the formation of DRM domains in cells (see also below).Various investigators have demonstrated that treatment of cells with exogenous SMase enhances efflux of cellular cholesterol to the extracellular cholesterol acceptors, such as high density lipoproteins and cyclodextrins (31Neufeld E.B. Cooney A.M. Pitha J. Dawidowicz E.A. Dwyer N.K. Pentchev P.G. Blanchette-Mackie E.J. J. Biol. Chem. 1996; 271: 21604-21613Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 43Slotte J.P. Härmälä A.S. Jansson C. Pörn M.I. Biochim. Biophys. Acta. 1990; 1030: 251-257Crossref PubMed Scopus (56) Google Scholar, 44Stein O. Ben-Naim M. Dabach Y. Hollander G. Stein Y. Biochim. Biophys. Acta. 1992; 1126: 291-297Crossref PubMed Scopus (21) Google Scholar, 45Pörn M.I. Ares M.P. Slotte J.P. J. Lipid Res. 1993; 34: 1385-1392Abstract Full Text PDF PubMed Google Scholar, 46Ito J. Nagayasu Y. Yokoyama S. J. Lipid Res. 2000; 41: 894-904Abstract Full Text Full Text PDF PubMed Google Scholar). Nevertheless, these previous studies did not eliminate the possibility that the enhanced efflux of cholesterol after SMase treatment was due to accumulation of ceramide. We herein demonstrated that the reduction of cellular SM content without any increase in ceramide level renders plasma membrane cholesterol prone to flux to the extracellular acceptor MβCD (Fig.4). Thus, we conclude that SM play a role in the retention of cholesterol in the plasma membrane against efflux to the extracellular cholesterol-acceptor, probably due to favorable van der Waals and hydrogen-bonding interactions between cholesterol and SM (47Kan C.C. Ruan Z.S. Bittman R. Biochemistry. 1991; 30: 7759-7766Crossref PubMed Scopus (79) Google Scholar, 48Bittman R. Kasireddy C.R. Mattjus P. Slotte J.P. Biochemistry. 1994; 33: 11776-11781Crossref PubMed Scopus (150) Google Scholar). This conclusion does not exclude the possibility that total sphingolipids (SM, ceramide, and glycosphingolipids) or complex sphingolipids (SM and glycosphingolipids) are responsible for the retention of cholesterol. However, treatment of cells with bacterial SMase not only enhances cholesterol efflux from cells (Refs. 31Neufeld E.B. Cooney A.M. Pitha J. Dawidowicz E.A. Dwyer N.K. Pentchev P.G. Blanchette-Mackie E.J. J. Biol. Chem. 1996; 271: 21604-21613Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar and 43Slotte J.P. Härmälä A.S. Jansson C. Pörn M.I. Biochim. Biophys. Acta. 1990; 1030: 251-257Crossref PubMed Scopus (56) Google Scholar, 44Stein O. Ben-Naim M. Dabach Y. Hollander G. Stein Y. Biochim. Biophys. Acta. 1992; 1126: 291-297Crossref PubMed Scopus (21) Google Scholar, 45Pörn M.I. Ares M.P. Slotte J.P. J. Lipid Res. 1993; 34: 1385-1392Abstract Full Text PDF PubMed Google Scholar, 46Ito J. Nagayasu Y. Yokoyama S. J. Lipid Res. 2000; 41: 894-904Abstract Full Text Full Text PDF PubMed Google Scholar, and Fig. 6) and also reduces cholesterol distribution to DRM domains (46Ito J. Nagayasu Y. Yokoyama S. J. Lipid Res. 2000; 41: 894-904Abstract Full Text Full Text PDF PubMed Google Scholar), although total sphingolipid levels most likely remain unchanged after SMase treatment. Therefore, levels of SM itself or total complex sphingolipids in cells are likely to be important for the formation of DRM domains and the retention of cholesterol against efflux.Interestingly, MβCD-mediated efflux of cholesterol from cells results in reduction of cholesterol level in DRM, but not non-DRM domains (Fig.3). The simplest explanation for this observation is that MβCD-mediated efflux of cholesterol preferentially occurs at the DRM domains in cells. Fielding and Fielding (22Fielding P.E. Fielding C.J. Biochemistry. 1995; 34: 14288-14292Crossref PubMed Scopus (256) Google Scholar, 49Fielding C.J. Fielding P.E. J. Lipid Res. 1997; 38: 1503-1521Abstract Full Text PDF PubMed Google Scholar) have suggested that cholesterol efflux from the cell surface to high density lipoproteins occurs largely at DRM domains of the plasma membrane. Alternatively, MβCD-mediated efflux of cholesterol may occur at non-DRM domains of the plasma membrane, but cellular cholesterol is rapidly redistributed from DRM to non-DRM domains for homeostasis of the cholesterol level of non-DRM domains. A previous study has shown that de novosynthesized cholesterol at the endoplasmic reticulum is delivered to DRM domains of the plasma membrane by a caveolin-dependent mechanism, and that the cholesterol delivered to DRM domains then rapidly flows to non-DRM domains at the plasma membrane (40Smart E.J. Ying Y. Donzell W.C. Anderson R.G. J. Biol. Chem. 1996; 271: 29427-29435Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar).It is also noteworthy that cytotoxic susceptibility of CHO cells to MβCD is enhanced when cellular SM level is reduced (Figs. 5 and 6). Because MβCD-mediated efflux of cholesterol is enhanced in SM-deficient cells (Fig. 4), it is likely that cholesterol is depleted below the level necessary for sustaining viability more easily from SM-deficient cells than from control cells. The higher toxicity of MβCD to SM-deficient cells than control cells may allow the use of this drug for a positive selection of revertants of SM-deficient mutant cells. In this study, we investigated the effects of reduction of cellular SM on cholesterol behavior in the plasma membrane using CHO cell mutants defective in SM production. There were no differences in the content of cellular cholesterol and its distribution to the cholesterol oxidase-sensitive pool between these mutant and wild-type cells, even when the SM content in the mutant cells was 15–40% of the wild-type level (Tables I and II, and Fig. 2). LY-B cells were also deficient in ceramide and glycosphingolipids, while the ceramide and glycosphingolipid content in LY-A cells was similar to the wild-type levels (Table I, and see also Ref. 19Hanada K. Hara T. Fukasawa M. Yamaji A. Umeda M. Nishijima M. J. Biol. Chem. 1998; 273: 33787-33794Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). Hence, the possibility was eliminated that the failure to affect the cholesterol levels under SM-deficient conditions was due to functional compensation by an increase of ceramide or glycosphingolipid content. These results indicated that the reduction of cellular SM to ∼15% of the wild-type level has no effect on the steady-state level of cellular cholesterol and its distribution to the plasma membrane. Enrichment of SM in the plasma membrane has been hypothesized to force cholesterol to be also enriched in the plasma membrane, based on the observations that addition of SM to cultured human skin fibroblasts results in an increase in cellular cholesterol content according to the increase in cellular SM level (35Kudchodkar B.J. Albers J.J. Bierman E.L. Atherosclerosis. 1983; 46: 353-367Abstract Full Text PDF PubMed Scopus (19) Google Scholar), and that cholesterol interacts with SM more strongly than with glycerophospholipids in model membranes (6Lund-Katz S. Laboda H.M. McLean L.R. Phillips M.C. Biochemistry. 1988; 27: 3323-3416Crossref Scopus (206) Google Scholar, 36Wattenberg B.W. Silbert D.F. J. Biol. Chem. 1983; 258: 2284-2289Abstract Full Text PDF PubMed Google Scholar, 37van Blitterswijk W.J. van der Meer B.W. Hilkmann H. Biochemistry. 1987; 26: 1746-1756Crossref PubMed Scopus (176) Google Scholar). However, our analyses with CHO cells suggest that neither SM, glycosphingolipids, nor ceramide is essential for the preferential distribution of cholesterol to the plasma membrane. Consistent with these results, a recent study has shown that most cellular cholesterol is present in the plasma membrane even after depletion of cell surface SM by treatment of human skin fibroblasts with bacterial SMase (38Pörn M.I. Slotte J.P. Biochem. J. 1995; 308: 269-274Crossref PubMed Scopus (46) Google Scholar). It is unlikely that the reduced level of SM in the CHO mutant cells is still sufficient for a stoichiometric association with cholesterol at the plasma membrane, because the estimated amount of SM in LY-B cells (∼20 nmol/μmol cellular phospholipid) is far less than that of cholesterol (∼300 nmol/μmol cellular phospholipid) (Tables I andII), and 70–80% of cholesterol in both LY-B and wild-type cells appears to be distributed in the plasma membrane (Fig. 2). Energy-dependent transport of de novosynthesized cholesterol from the endoplasmic reticulum to the plasma membrane may be primarily responsible for enrichment of cholesterol in the plasma membrane (39DeGrella R.F. Simoni R.D. J. Biol. Chem. 1982; 257: 14256-14262Abstract Full Text PDF PubMed Google Scholar, 40Smart E.J. Ying Y. Donzell W.C. Anderson R.G. J. Biol. Chem. 1996; 271: 29427-29435Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar). We previously showed that sphingolipids are involved in the detergent insolubility of a GPI-anchored protein in CHO cells (12Hanada K. Nishijima M. Akamatsu Y. Pagano R.E. J. Biol. Chem. 1995; 270: 6254-6260Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). However, another research group has recently shown that glycosphingolipids are dispensable for formation of DRM domains in murine melanoma cells (15Ostermeyer A.G. Beckrich B.T. Ivarson K.A. Grove K.E. Brown D.A. J. Biol. Chem. 1999; 274: 34459-34466Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar), although the original concept of lipid-raft is based on the assumption that the hydrogen bond interaction between the sugar head groups of glycosphingolipids and GPI-anchored proteins is important for formation of detergent-resistant rafts (41Simons K. van Meer G. Biochemistry. 1988; 27: 6197-6202Crossref PubMed Scopus (1079) Google Scholar). Ostermeyer et al. (15Ostermeyer A.G. Beckrich B.T. Ivarson K.A. Grove K.E. Brown D.A. J. Biol. Chem. 1999; 274: 34459-34466Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar) also demonstrated that there is no difference in MβCD-mediated efflux of cholesterol between glycosphingolipid-depleted cells and control cells. We herein demonstrated that CHO mutant LY-A cells having a lower level of SM, but not of glycosphingolipids, had a lower level of DRM-associated cholesterol than wild-type cells (Fig. 3). These results indicate that SM plays a role in the formation of DRM domains in CHO cells. This conclusion is consistent with the observation that the liquid-ordered phase of lipid bilayers forms in model membranes containing cholesterol and SM (42Ahmed S.N. Brown D.A. London E. Biochemistry. 1997; 36: 10944-10953Crossref PubMed Scopus (609) Google Scholar). In model membranes, total mol % of lipids tending to form the liquid-ordered phase has been shown to be important for the formation of DRM domains (8Schroeder R. London E. Brown D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 12130-12134Crossref PubMed Scopus (635) Google Scholar, 9Schroeder R.J. Ahmed S.N. Zhu Y. London E. Brown D.A. J. Biol. Chem. 1998; 273: 1150-1157Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar, 42Ahmed S.N. Brown D.A. London E. Biochemistry. 1997; 36: 10944-10953Crossref PubMed Scopus (609) Google Scholar). Considering that levels of total sphingolipids in SM-deficient CHO mutant cells are lower than the wild-type level (Table I), the total level of SM and glycosphingolipids, but not the level of SM itself, might be a key factor for the formation of DRM domains in cells (see also below). Various investigators have demonstrated that treatment of cells with exogenous SMase enhances efflux of cellular cholesterol to the extracellular cholesterol acceptors, such as high density lipoproteins and cyclodextrins (31Neufeld E.B. Cooney A.M. Pitha J. Dawidowicz E.A. Dwyer N.K. Pentchev P.G. Blanchette-Mackie E.J. J. Biol. Chem. 1996; 271: 21604-21613Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar, 43Slotte J.P. Härmälä A.S. Jansson C. Pörn M.I. Biochim. Biophys. Acta. 1990; 1030: 251-257Crossref PubMed Scopus (56) Google Scholar, 44Stein O. Ben-Naim M. Dabach Y. Hollander G. Stein Y. Biochim. Biophys. Acta. 1992; 1126: 291-297Crossref PubMed Scopus (21) Google Scholar, 45Pörn M.I. Ares M.P. Slotte J.P. J. Lipid Res. 1993; 34: 1385-1392Abstract Full Text PDF PubMed Google Scholar, 46Ito J. Nagayasu Y. Yokoyama S. J. Lipid Res. 2000; 41: 894-904Abstract Full Text Full Text PDF PubMed Google Scholar). Nevertheless, these previous studies did not eliminate the possibility that the enhanced efflux of cholesterol after SMase treatment was due to accumulation of ceramide. We herein demonstrated that the reduction of cellular SM content without any increase in ceramide level renders plasma membrane cholesterol prone to flux to the extracellular acceptor MβCD (Fig.4). Thus, we conclude that SM play a role in the retention of cholesterol in the plasma membrane against efflux to the extracellular cholesterol-acceptor, probably due to favorable van der Waals and hydrogen-bonding interactions between cholesterol and SM (47Kan C.C. Ruan Z.S. Bittman R. Biochemistry. 1991; 30: 7759-7766Crossref PubMed Scopus (79) Google Scholar, 48Bittman R. Kasireddy C.R. Mattjus P. Slotte J.P. Biochemistry. 1994; 33: 11776-11781Crossref PubMed Scopus (150) Google Scholar). This conclusion does not exclude the possibility that total sphingolipids (SM, ceramide, and glycosphingolipids) or complex sphingolipids (SM and glycosphingolipids) are responsible for the retention of cholesterol. However, treatment of cells with bacterial SMase not only enhances cholesterol efflux from cells (Refs. 31Neufeld E.B. Cooney A.M. Pitha J. Dawidowicz E.A. Dwyer N.K. Pentchev P.G. Blanchette-Mackie E.J. J. Biol. Chem. 1996; 271: 21604-21613Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar and 43Slotte J.P. Härmälä A.S. Jansson C. Pörn M.I. Biochim. Biophys. Acta. 1990; 1030: 251-257Crossref PubMed Scopus (56) Google Scholar, 44Stein O. Ben-Naim M. Dabach Y. Hollander G. Stein Y. Biochim. Biophys. Acta. 1992; 1126: 291-297Crossref PubMed Scopus (21) Google Scholar, 45Pörn M.I. Ares M.P. Slotte J.P. J. Lipid Res. 1993; 34: 1385-1392Abstract Full Text PDF PubMed Google Scholar, 46Ito J. Nagayasu Y. Yokoyama S. J. Lipid Res. 2000; 41: 894-904Abstract Full Text Full Text PDF PubMed Google Scholar, and Fig. 6) and also reduces cholesterol distribution to DRM domains (46Ito J. Nagayasu Y. Yokoyama S. J. Lipid Res. 2000; 41: 894-904Abstract Full Text Full Text PDF PubMed Google Scholar), although total sphingolipid levels most likely remain unchanged after SMase treatment. Therefore, levels of SM itself or total complex sphingolipids in cells are likely to be important for the formation of DRM domains and the retention of cholesterol against efflux. Interestingly, MβCD-mediated efflux of cholesterol from cells results in reduction of cholesterol level in DRM, but not non-DRM domains (Fig.3). The simplest explanation for this observation is that MβCD-mediated efflux of cholesterol preferentially occurs at the DRM domains in cells. Fielding and Fielding (22Fielding P.E. Fielding C.J. Biochemistry. 1995; 34: 14288-14292Crossref PubMed Scopus (256) Google Scholar, 49Fielding C.J. Fielding P.E. J. Lipid Res. 1997; 38: 1503-1521Abstract Full Text PDF PubMed Google Scholar) have suggested that cholesterol efflux from the cell surface to high density lipoproteins occurs largely at DRM domains of the plasma membrane. Alternatively, MβCD-mediated efflux of cholesterol may occur at non-DRM domains of the plasma membrane, but cellular cholesterol is rapidly redistributed from DRM to non-DRM domains for homeostasis of the cholesterol level of non-DRM domains. A previous study has shown that de novosynthesized cholesterol at the endoplasmic reticulum is delivered to DRM domains of the plasma membrane by a caveolin-dependent mechanism, and that the cholesterol delivered to DRM domains then rapidly flows to non-DRM domains at the plasma membrane (40Smart E.J. Ying Y. Donzell W.C. Anderson R.G. J. Biol. Chem. 1996; 271: 29427-29435Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar). It is also noteworthy that cytotoxic susceptibility of CHO cells to MβCD is enhanced when cellular SM level is reduced (Figs. 5 and 6). Because MβCD-mediated efflux of cholesterol is enhanced in SM-deficient cells (Fig. 4), it is likely that cholesterol is depleted below the level necessary for sustaining viability more easily from SM-deficient cells than from control cells. The higher toxicity of MβCD to SM-deficient cells than control cells may allow the use of this drug for a positive selection of revertants of SM-deficient mutant cells." @default.
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W2092187419 title "Reduction of Sphingomyelin Level without Accumulation of Ceramide in Chinese Hamster Ovary Cells Affects Detergent-resistant Membrane Domains and Enhances Cellular Cholesterol Efflux to Methyl-β-cyclodextrin" @default.
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