JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M005151200 on August 4, 2000

J. Biol. Chem., Vol. 275, Issue 44, 34028-34034, November 3, 2000
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
275/44/34028    most recent
M005151200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Fukasawa, M.
Right arrow Articles by Hanada, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fukasawa, M.
Right arrow Articles by Hanada, K.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

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-beta -cyclodextrin*

Masayoshi FukasawaDagger , Masahiro NishijimaDagger , Hiroyuki Itabe§, Tatsuya Takano§, and Kentaro HanadaDagger

From the Dagger  Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162-8640, Japan and the § Department of Microbiology and Molecular Pathology, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Tsukui-gun, Kanagawa 199-0195, Japan

Received for publication, June 14, 2000, and in revised form, August 4, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-beta -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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Both cholesterol and sphingomyelin (SM)1 are preferentially distributed in the plasma membrane of cells (1). 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 (2, 3). 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 (3). 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 (4-7). 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 (8, 9).

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 (2, 3, 10). 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 (11) and also enhances solubility of GPI-anchored proteins in the non-ionic detergent Triton X-100 (12). 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 (13), and to enhance conversion of prion protein, a GPI-anchored protein, to its scrapie isoform (14). 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 (15). 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 (16-18). 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 (19). One is LY-A strain defective in ATP-dependent endoplasmic reticulum to Golgi apparatus trafficking of ceramide directed to SM biosynthesis (20). 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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- 3-(4,5-Dimethyl-thiazoyl-2-yl)-2,5-diphenyltetrazolium bromide (MTT), amphotericin B, and methyl-beta -cyclodextrin (Mbeta CD) were purchased from Sigma; recombinant Bacillus cereus SMase was from Higeta Shoyu (Japan); B. cereus phosphatidylcholine-specific phospholipase C (PC-PLC) was from Roche Molecular Biochemicals (Germany); Streptomyces sp. cholesterol oxidase was from Calbiochem. [1a,2a(n)-3H]Cholesterol (42 Ci/mmol) was obtained from Amersham Pharmacia Biotech, and [methyl-14C]choline chloride (55 mCi/mmol) was from American Radiolabeled Chemicals Inc.

Cells and Cell Cultures-- The CHO-K1 cell line was obtained from the American Type Culture Collection (ATCC CCL). CHO-K1-derived mutant cell lines, LY-A and LY-B strains, have been previously established by us (19). LY-B/cLCB1, a corrected revertant of LY-B strain, was previously obtained by stable transfection of LY-B cells with the cDNA encoding hamster LCB1 subunit of serine palmitoyltransferase (19). Ham's F-12 medium supplemented with 10% newborn calf serum, penicillin G (100 units/ml), and streptomycin sulfate (100 µg/ml) was used as a normal culture medium. Nutridoma-BO medium (F-12 medium containing 1% Nutridoma-SP (Roche Molecular Biochemicals), 0.1% fetal bovine serum, and 10 µM sodium oleate-bovine serum albumin complex, and gentamicin (10 µg/ml)) was used as a sphingolipid-deficient culture medium (19). CHO cells were maintained in the normal culture medium in the routine manner in a 5% CO2 atmosphere in 100% humidity at 33 °C. For cultivation in a sphingolipid-deficient medium, cells were seeded, incubated in the normal culture medium at 37 °C for 1 day, and, after washing twice with serum-free Ham's F-12 medium, cultured in Nutridoma-BO medium for 2 days. In some experiments, Nutridoma-BO medium was supplemented with 1 µM D-erythro-sphingosine (Matreya, Inc., Pleasant Gap, PA) as described previously (19).

Assay for Viability of Cells Exposed to Amphotericin B or Mbeta CD-- Cells were seeded on day 0 at 2 × 104 cells/well (in 12-well plates) in 2 ml of the normal culture medium. On day 1, each well of cells received 2 ml of Nutridoma-BO medium. On day 3, cell monolayers were incubated in 0.5 ml of Ham's F-12 medium containing amphotericin B or Mbeta CD at 37 °C. After washing with phosphate-buffered saline (PBS), cell monolayers were incubated in 200 µl of MTT (5 mg/ml in PBS) at 37 °C for 1 h, and the MTT solution was removed. Formazan produced in the cells was dissolved in 400 µl of 40 mM HCl in propan-2-ol, and the absorbance at 570 nm of the solution was measured with a spectrophotometer.

Labeling of Cells with [3H]Cholesterol and [14C]Choline Chloride-- Cells were seeded on day 0 at 5 × 104 cells/well (in 12-well plates) in 2 ml of the normal culture medium. On day 1, each well of cells received 2 ml of Nutridoma-BO medium. On day 2, cell monolayers were incubated for 16 h in 2 ml of Nutridoma-BO medium containing 1 µCi of [3H]cholesterol or 0.5 µCi of [14C]choline chloride. After washing with serum-free Ham's F-12, the cells were further incubated for 4 h in 2 ml of Nutridoma-BO medium before the following experiments were performed. There were no apparent differences in the levels of cell-associated radioactivity among cell types used. Most of the radioactivity (>95%) was unesterified cholesterol type in the [3H]cholesterol labeling.

Measurement of Cholesterol Oxidase-sensitive Cholesterol Pool-- Treatment of cells with cholesterol oxidase was performed essentially as described previously (21). In brief, cells labeled with [3H]cholesterol were washed with ice-cold PBS and fixed for 10 min on ice with 0.125% glutaraldehyde in PBS. After washing with PBS, fixed cells were incubated in 500 µl of PBS containing 2 units/ml cholesterol oxidase with or without 0.1 unit/ml SMase for 30 min at 37 °C. Then cells were chilled on ice and washed with PBS. After extraction of cellular lipids from the cells, labeled sterols were separated on TLC plates with a solvent of hexane/diethylether/acetic acid (260/60/3, v/v). The radioactivity in cholesterol and cholestenone separated by TLC was determined by liquid scintillation counting.

Efflux of [3H]Cholesterol and [14C]Choline-containing Phospholipids from Cells to Mbeta CD-- After being rinsed with serum-free Ham's F-12, [3H]cholesterol or [14C]choline-labeled cell monolayers were incubated in 0.5 ml of Ham's F-12 medium containing Mbeta CD at 37 °C, as described in figure legends. Then the medium incubated with cells was retrieved and centrifuged (5,000 × g, 5 min), and the supernatant was regarded as the medium fraction. Cell monolayers were washed with PBS and lysed with 500 µl of 0.1% sodium dodecyl sulfate. The radioactivity of each 100 µl of the medium fraction and the cell lysate fraction was counted by liquid scintillation counting.

Flotation of DRM Fractions-- DRM fractions were separated as described previously with a few modifications (22). In brief, subconfluent cells labeled with [3H]cholesterol in a 10 cm-dish were extracted for 30 min on ice with 100 µl of 1% Triton X-100 in MN buffer (25 mM MES-NaOH (pH 6.5) containing 0.15 M NaCl and a protease inhibitor mixture (CompleteTM EDTA-free, Roche Molecular Biochemicals)). The extracts were diluted into 500 µl of MN buffer, mixed with 500 µl of 85% (w/v) sucrose in MN buffer, and layered under 7 ml of a 10-30% sucrose gradient in MN buffer. After centrifugation at 75,000 × g for 20 h at 4 °C, fractions (0.5 ml) were collected from the bottom of the resulting gradient.

Determination of Content of Various Lipids in Cells-- Cells were seeded at 7.5 × 105 cells in 30 ml of normal culture medium per 150-mm dish, incubated for 1 day, and then cultivated in 30 ml of Nutridoma-BO medium for 2 days. For supplementation with sphingosine, 150 µl of 200 µM D-erythro-sphingosine/bovine serum albumin complex was added to the medium at a final concentration of 1 µM every day during culture in Nutridoma-BO medium (19). The cells were harvested by scraping, and lipids were extracted from the cells (23). Then phospholipids were separated on TLC plates with a solvent of chloroform/methanol/acetic acid (65/25/10, v/v), and the content of the individual phospholipid type was determined by the method of Rouser et al. (24), using NaH2PO4 as a standard. The amount of ceramide was determined by a bacterial diacylglycerol kinase method with an assay kit (Amersham Pharmacia Biotech), according to the manufacturer's instructions. The amounts of N-acetylneuraminyl lactosylceramide (GM3) and glucosylceramide (GlcCer) were determined by densitometric analysis, as described previously (19). Average molecular weight values of natural GM3, GlcCer, and ceramide were assumed to be 1198, 727, and 565, respectively. The amount of cholesterol was determined by the cholesterol oxidase method, as described previously (25).

Determination of Protein Concentration-- Protein concentrations were determined by the method of Lowry et al. (26) using bovine serum albumin as a standard.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reduction of the SM Level without Accumulation of Ceramide in CHO Mutant Cells-- The CHO cell mutant LY-B strain defective in the LCB1 subunit of serine palmitoyltransferase is unable to synthesize any sphingolipid species de novo, and another mutant LY-A strain defective in ATP-dependent trafficking of ceramide from the endoplasmic reticulum to the Golgi apparatus for SM synthesis is impaired in SM synthesis but not ceramide or glycosphingolipid synthesis (19, 20). As shown in Table I, when cells were cultured in a sphingolipid-deficient medium for 2 days, the SM levels in LY-B and LY-A cells were ~15 and ~40%, respectively, of the level (25.1 ± 1.0 nmol/mg of protein) in wild-type CHO-K1 cells, being in agreement with our previous study (19). The contents of GM3, the sole ganglioside in CHO cells, in LY-B and LY-A cells were ~20 and ~90%, respectively, of the wild-type level (3.2 ± 0.3 nmol/mg of protein), and GlcCer contents in LY-B and LY-A cells were ~20 and ~110%, respectively, of the wild-type level (1.1 ± 0.1 nmol/mg of protein). Other glycosphingolipids were undetectable even in CHO-K1 cells. The contents of ceramide in LY-B and LY-A cells were ~5 and ~100%, respectively, of the wild-type level (0.5 nmol/mg of protein). The reduced contents of SM and GM3 in LY-B cells were reversed to the wild-type levels by genetic complementation of the LY-B strain with the hamster LCB1 cDNA (this revertant was designated the LY-B/cLCB1 strain) or by supplementation of the culture medium with D-erythro-sphingosine, which is metabolically converted to complex sphingolipids in cells. Therefore, these CHO cell strains represented an appropriate model cell system for examining whether reduction of SM level without any increase in ceramide level affected behavior of cholesterol in intact cells.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Phospholipid and sphingolipid levels in CHO cells
CHO cells were cultivated in Nutridoma-BO medium at 37 °C for 2 days. Lipids were extracted from the cells, and after separation of lipids by TLC, the amounts of phospholipids, GM3, GlcCer, and ceramide were determined. The data shown are the mean values ± S.D. from triplicate experiments. The amounts of total sphingolipids were expressed as the sum of SM, GM3, GlcCer, and ceramide contents. PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; PI, phosphatidylinositol; SL, sphingolipids.

Cholesterol Content in SM-deficient CHO Cells-- After culture of cells in the sphingolipid-deficient medium for 2 days, the content of cholesterol in cells was determined. There was no appreciable difference in the content of unesterified cholesterol among LY-A, LY-B, LY-B/cLCB1, and wild-type cells (Table II). Cultivation of cells under sphingosine-supplied conditions did not significantly affect the cholesterol level. The content of esterified cholesterol was too low to be accurately determined in any of the cell types used (data not shown), because cells were cultured under lipoprotein-deficient conditions. These results were consistent with those of our previous study showing that reduction in cellular sphingolipid level through inactivation of SPT did not affect cholesterol level in CHO cells (12, 19).

                              
View this table:
[in this window]
[in a new window]
 
Table II
Cholesterol content in CHO cells
Cells were cultivated in Nutridoma-BO medium supplemented with (+) or without (-) 1 µM D-erythro-sphingosine at 37 °C for 2 days. Then, after extraction of lipids from the cells, the amounts of cholesterol and phospholipids were determined as described under "Experimental Procedures." The cholesterol level was corrected for the phospholipid level. Total phospholipid levels in CHO-K1, LY-A, LY-B, LY-B (+sphingosine), LY-B/cLCB1, LY-B/cLCB1 (+sphingosine) were 168.2 ± 2.4, 156.6 ± 4.9, 186.6 ± 5.2, 173.6 ± 5.9, 177.2 ± 10.4, and 185.0 ± 7.0 nmol/mg protein, respectively. The data shown are the mean values ± S.D. from triplicate experiments.

Effect of SM Deficiency in CHO Cells on Amphotericin B Susceptibility-- To address the question of whether reduction in cellular SM level affected the level of plasma membrane-associated cholesterol, we first compared amphotericin B susceptibility between SM-deficient and control cells. Cytolytic susceptibility of cells to the polyene macrolide amphotericin B has been suggested to represent a semi-quantitative measure of plasma membrane cholesterol level in intact cells because reduction of the plasma membrane cholesterol level in CHO cells renders the cells resistant to amphotericin B, which exhibits a cytolytic activity after specific binding to sterols at the cell surface (27, 28). After cultivation in the sphingolipid-deficient medium for 2 days, cells were incubated with amphotericin B at various concentrations for 1 h at 37 °C, and then the viability of the cells was determined. Wild-type cells lost viability after exposure to amphotericin B in a dose-dependent manner with a LD50 (LD50) of ~7 µg/ml, and the LD50 values of amphotericin B susceptibility in LY-A, LY-B, and LY-B/cLCB1 cells were also found to be 4-7 µg/ml (Fig. 1). The observation that LY-A and LY-B cells did not display any higher resistance to amphotericin B than wild-type cells suggested that there was no reduction in plasma membrane cholesterol level in the SM-deficient mutant cells, compared with the control cells. On the other hand, analysis of the binding capability of cells to the cytolysin lysenin, the specific membrane receptor of which is SM, has shown that SM levels in the plasma membranes of LY-A and LY-B cells are lower than the wild-type level (19).


View larger version (25K):
[in this window]
[in a new window]
  		Fig. 1.   Effect of amphotericin B on viability of CHO cells. Cells were incubated in Ham's F-12 containing various concentrations of amphotericin B for 1 h at 37 °C, and the viability of cells was determined using MTT as described under "Experimental Procedures." The data shown are the mean values ± S.D. from triplicate experiments. Open circles, CHO-K1; closed circles, LY-A; open squares, LY-B; open triangles, LY-B/cLCB1.

Effect of SM deficiency in CHO Cells on the Pool Size of Cholesterol Oxidase-sensitive Cholesterol-- To examine the effect of SM deficiency on the distribution of cholesterol between the plasma membrane and the intracellular membranes, we measured the pool size of cholesterol sensitive to extracellular cholesterol oxidase in CHO cells. Cells were labeled with [3H]cholesterol in the sphingolipid-deficient medium for 16 h, fixed with glutaraldehyde, and then treated with bacterial SMase and cholesterol oxidase. Pretreatment of the fixed cells with SMase is necessary to correct for the influence of membrane SM on accessibility of cholesterol oxidase to membrane cholesterol (29). LY-A, LY-B, LY-B/cLCB1, and wild-type cells were quite similar in the pool size of cholesterol oxidase-sensitive [3H]cholesterol, which amounted to ~70% of total cell-associated [3H]cholesterol (Fig. 2). Cultivation of LY-B or LY-B/cLCB1 cells under sphingosine-supplied conditions did not affect the level of cholesterol oxidase-sensitive [3H]cholesterol. These results indicated that the reduction of cellular SM level to ~15% of the wild-type level does not appreciably affect the distribution of cholesterol between the plasma membrane and the intracellular membranes.


View larger version (67K):
[in this window]
[in a new window]
  		Fig. 2.   Cholesterol oxidase sensitivity of [3H]cholesterol in various sphingolipid-deficient cells in the presence or absence of exogenous SMase. [3H]Cholesterol-labeled cells fixed by glutaraldehyde were incubated with PBS containing 2 units/ml cholesterol oxidase with (+) or without (-) 0.1 unit/ml SMase for 30 min at 37 °C. Then the percent conversion of [3H]cholesterol to [3H]cholestenone was determined as described under "Experimental Procedures." 1, CHO-K1; 2, LY-A; 3, LY-B; 4, LY-B cultured under 1 µM sphingosine; 5, LY-B/cLCB1; 6, LY-B/cLCB1 cultured under 1 µM sphingosine.

When the fixed cells were not treated with SMase, the apparent pool size of cholesterol oxidase-sensitive [3H]cholesterol was inversely correlated to the SM content in cells (Fig. 2). This observation is explained by the previously elucidated mechanism that the accessibility of cholesterol oxidase to membrane cholesterol is restricted by SM coexisting in the membrane (21). Thus, this result demonstrated another line of evidence that plasma membrane SM levels are lower in LY-A and LY-B cells than in wild-type cells.

Reduced Distribution of Cholesterol to DRM Domains in SM-deficient CHO Cells-- We next examined whether the distribution of cholesterol to DRM domains was affected by the reduction in cellular SM level. Lipid and protein components associated with DRM are thought to be fractionated into a Triton X-100-insoluble floating fraction by sucrose density gradient centrifugation (22). Thus, lysates of the [3H]cholesterol-labeled cells treated with Triton X-100 were separated in a sucrose density gradient, and the radioactivity of [3H]cholesterol in the fractions enriched in caveolin, a protein marker of DRM domains, was determined. As shown in Fig. 3, the low-density floating fraction (fraction number 7-13) contained ~60% of total [3H]cholesterol radioactivity in the extract from wild-type cells, whereas the low-density floating fraction in LY-A and LY-B had less than 40 and 30%, respectively, of total [3H]cholesterol radioactivity in the cell extracts (Fig. 3). Western blot analysis showed that the expression levels of caveolin were nearly identical among these 3 cell types (data not shown). In addition, SM and caveolin in these 3 cell types were highly enriched in low-density floating fractions (data not shown). These results indicated that the reduction of cellular SM level affects distribution of cholesterol to DRM domains.


View larger version (21K):
[in this window]
[in a new window]
  		Fig. 3.   Effect of Mbeta CD-treatment of CHO cells on the cholesterol level distributed to DRM domains. [3H]Cholesterol-labeled wild-type CHO-K1 (A), LY-A (B), and LY-B (C) cells were incubated with Ham's F-12 with (closed circles) or without (open circles) 5 mM Mbeta CD for 5 min at 37 °C. Then, after lysis of cells in 1% Triton X-100, the lysate was fractionated by sucrose density gradient centrifugation, and the radioactivity of the resulting fractions was measured.

Mbeta CD serves as a high-affinity acceptor for cholesterol (30-34). We further investigated the distribution of cholesterol to DRM domains after the Mbeta CD-mediated efflux of plasma membrane cholesterol. The [3H]cholesterol-labeled cells were exposed to 5 mM Mbeta CD for 5 min at 37 °C and treated with cold 1% Triton X-100. After fractionation by sucrose density gradient centrifugation, the radioactivity of each fraction was measured. In all cell types examined, significantly reduced distribution of [3H]cholesterol to the DRM fractions was observed after the Mbeta CD-mediated efflux of cellular cholesterol, whereas the amounts of [3H]cholesterol in non-DRM fractions were little affected (Fig. 3). The reduction in [3H]cholesterol distribution in the DRM domains was more marked in LY-A and LY-B cells than in wild-type cells (Fig. 3). Note that the 3 cell types sustained ~100% viability after exposure to 5 mM Mbeta CD for 5 min (data not shown, and see also Fig. 5B). These results suggested that efflux of cellular cholesterol to Mbeta CD mainly reduced the level of cholesterol associated with DRM but not non-DRM domains, and that the reduction ratio was inversely correlated with cellular SM content.

Efflux of Cellular Cholesterol and Phospholipids from SM-deficient CHO Cells in the Presence of Mbeta CD-- To confirm that the reduction in cellular SM level affected Mbeta CD-mediated efflux of cholesterol from cells, we determined time courses of cholesterol efflux from wild-type and SM-deficient mutant cells to a serum-free medium containing Mbeta CD. After labeling of cells with [3H]cholesterol in the sphingolipid-deficient medium for 16 h, the labeled cells were exposed to 1 mM Mbeta CD for various times up to 60 min, and the radioactivity of [3H]cholesterol released from the cells to the medium was determined. The Mbeta CD-mediated [3H]cholesterol efflux rates from LY-A and LY-B cells were ~2- and ~3-fold, respectively, of the wild-type level throughout the time window examined, while there was no significant difference in the efflux rate between wild-type cells and LY-B/cLCB1 cells (Fig. 4A). The enhanced [3H]cholesterol efflux from LY-B cells was suppressed to the wild-type level when cells were cultured under sphingosine-supplied conditions. The enhanced [3H]cholesterol efflux from SM-deficient cells was not due to cell lysis during exposure to Mbeta CD because almost all cells sustained trypan blue extrusion ability under the conditions used.


View larger version (17K):
[in this window]
[in a new window]
  		Fig. 4.   Efflux of cholesterol and choline-containing phospholipids from CHO cells by Mbeta CD. [3H]Cholesterol-labeled (A) or [14C]choline-labeled (B) cells were incubated with Ham's F-12 containing 1 mM Mbeta CD for the indicated periods at 37 °C. The radioactivity fluxed to the medium and the cell associated radioactivity were measured. The fluxed radioactivity (the mean values ± S.D. from triplicate experiments) is shown as the percentage of total radioactivity recovered. Open circles, CHO-K1; closed circles, LY-A; open squares, LY-B; closed squares, LY-B cultured under 1 µM sphingosine; open triangles, LY-B/cLCB1.

To determine the lipid specificity of Mbeta CD-mediated efflux, the efflux of cellular phospholipids was examined. After metabolic labeling of phospholipids with [14C]choline for 16 h, cells were exposed to 1 mM Mbeta CD for various periods, and the radioactivity of [14C]choline-labeled phospholipids released to the medium was measured. The fraction of [14C]choline-labeled phospholipids released during 1-h exposure to 1 mM Mbeta CD was less than 5% of the initial cell-associated level in all cell types examined (Fig. 4B). These control experiments confirmed the specific enhancement of cholesterol efflux via Mbeta CD through SM deficiency in cells and also the lack of appreciable lysis of cells.

SM-deficient CHO Cells Are Hyper-susceptible to Mbeta CD-mediated Cell Lysis-- We observed that exposure of CHO cells to Mbeta CD at higher concentrations caused greater lysis of cells. This observation and the adverse correlation between Mbeta CD-mediated cholesterol efflux and cellular SM content raised the possibility that the reduction in cellular SM level enhanced the cytolytic susceptibility of the cells to Mbeta CD. This was the case. When wild-type and LY-B/cLCB1 cells were exposed to various concentrations of Mbeta CD for 1 h at 37 °C, the cells maintained ~100% viability at 10 mM Mbeta CD, but lost viability almost completely at 20 mM Mbeta CD (Fig. 5A). In contrast, SM-deficient cells were much more susceptible to this drug, and almost complete loss of the viability of LY-A and LY-B cells was observed at 5 and 2.5 mM, respectively, of Mbeta CD (Fig. 5A). When cells were exposed to 5 mM Mbeta CD for various periods, LY-A and LY-B cells almost completely lost their viability by 90 min, whereas wild-type and LY-B/cLCB1 cells sustained ~100% viability at least up to 90 min (Fig. 5B). The enhanced Mbeta CD susceptibility of LY-B cells was suppressed to near the wild-type level by addition of D-erythro-sphingosine to the culture medium (Fig. 5B).


View larger version (22K):
[in this window]
[in a new window]
  		Fig. 5.   SM-deficient CHO cells are hyper-susceptible to Mbeta CD-mediated cell lysis. A, dose-response curve of susceptibility of cells to Mbeta CD. Cells were incubated in Ham's F-12 medium containing various concentrations of Mbeta CD for 1 h at 37 °C. B, time course of susceptibility of cells to Mbeta CD. Cells were incubated in Ham's F-12 containing 5 mM Mbeta CD for the indicated periods at 37 °C. Viability of cells was determined using MTT as described under "Experimental Procedures." The data shown are the mean values ± S.D. from triplicate experiments. Open circles, CHO-K1; closed circles, LY-A; open squares, LY-B; closed squares, LY-B cultured under 1 µM sphingosine; open triangles, LY-B/cLCB1.

We also determined whether depletion of plasma membrane SM by SMase treatment affected Mbeta CD susceptibility. When wild-type cells were treated with 250 milliunits/ml SMase for 1 h at 37 °C, the cells showed hyper-susceptibility to Mbeta CD (Fig. 6), similar to the case with SM-deficient mutant cells. By contrast, treatment of cells with PC-PLC caused no appreciable change in Mbeta CD susceptibility (Fig. 6). Collectively, these results indicated that SM plays a role in the retention of cholesterol in the plasma membrane against Mbeta CD-mediated efflux.


View larger version (18K):
[in this window]
[in a new window]
  		Fig. 6.   Effects of SMase and PC-PLC on viability of CHO-K1 cells. CHO cell monolayers were pretreated with SMase (250 milliunits/ml) and PC-PLC (40 units/ml) for 1 h at 37 °C, and then treated with various concentrations of Mbeta CD for 1 h at 37 °C. Then viability of the cells was determined using MTT as described under "Experimental Procedures." The data shown are the mean values ± S.D. from triplicate experiments. Open circles, no treatment; closed circles, SMase treatment; open squares, PC-PLC treatment.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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. 19). 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 (35), and that cholesterol interacts with SM more strongly than with glycerophospholipids in model membranes (6, 36, 37). 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 (38). 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 and II), 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 novo synthesized cholesterol from the endoplasmic reticulum to the plasma membrane may be primarily responsible for enrichment of cholesterol in the plasma membrane (39, 40).

We previously showed that sphingolipids are involved in the detergent insolubility of a GPI-anchored protein in CHO cells (12). However, another research group has recently shown that glycosphingolipids are dispensable for formation of DRM domains in murine melanoma cells (15), 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 (41). Ostermeyer et al. (15) also demonstrated that there is no difference in Mbeta CD-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 (42). 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 (8, 9, 42). 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 (31, 43-46). 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 Mbeta CD (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 (47, 48). 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. 31 and 43-46, and Fig. 6) and also reduces cholesterol distribution to DRM domains (46), 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, Mbeta CD-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 Mbeta CD-mediated efflux of cholesterol preferentially occurs at the DRM domains in cells. Fielding and Fielding (22, 49) have suggested that cholesterol efflux from the cell surface to high density lipoproteins occurs largely at DRM domains of the plasma membrane. Alternatively, Mbeta CD-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 novo synthesized 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 (40).

It is also noteworthy that cytotoxic susceptibility of CHO cells to Mbeta CD is enhanced when cellular SM level is reduced (Figs. 5 and 6). Because Mbeta CD-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 Mbeta CD 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.

    FOOTNOTES

* This work was supported in part by grants-in-aid from the Ministry of Education, Science and Culture of Japan, CREST of Japan Science and Technology Corporation, and a Special Coordination Fund for Promoting Science and Technology from the Science and Technology Agency of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry and Cell Biology, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo 162-8640, Japan. Fax: 81-3-5285-1157; E-mail: hanak@nih.go.jp.

Published, JBC Papers in Press, August 4, 2000, DOI 10.1074/jbc.M005151200

    ABBREVIATIONS

The abbreviations used are: SM, sphingomyelin; DRM, detergent-resistant membrane; GPI, glycosylphosphatidylinositol; SMase, sphingomyelinase; CHO, Chinese hamster ovary; MTT, 3-(4,5-dimethyl-thiazoyl-2-yl)-2,5-diphenyltetrazolium bromide; Mbeta CD, methyl-beta -cyclodextrin; PC-PLC, phosphatidylcholine-specific phospholipase C; PBS, phosphate-buffered saline; GM3, N-acetylneuraminyl lactosylceramide; GlcCer, glucosylceramide; LD50, 50% lethal dose; MES, 4-morpholinoethanesulfonic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. van Meer, G. (1989) Annu. Rev. Cell Biol. 5, 247-275
2. Simons, K., and Ikonen, E. (1997) Nature 387, 569-572
3. Brown, D. A., and London, E. (2000) J. Biol. Chem. 275, 17221-17224
4. Demel, R. A., Jansen, J. W. C. M., van Dijck, P. W. M., and van Deenen, L. L. M. (1977) Biochim. Biophys. Acta 465, 1-10
5. Yeagle, P. L., and Young, J. E. (1986) J. Biol. Chem. 261, 8175-8181
6. Lund-Katz, S., Laboda, H. M., McLean, L. R., and Phillips, M. C. (1988) Biochemistry 27, 3416-323
7. Ramstedt, B., and Slotte, J. P. (1999) Biophys. J. 76, 908-915
8. Schroeder, R., London, E., and Brown, D. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 12130-12134
9. Schroeder, R. J., Ahmed, S. N., Zhu, Y., London, E., and Brown, D. A. (1998) J. Biol. Chem. 273, 1150-1157
10. Anderson, R. G. (1998) Annu. Rev. Biochem. 67, 199-225
11. Hanada, K., Izawa, K., Nishijima, M., and Akamatsu, Y. (1993) J. Biol. Chem. 268, 13820-13823
12. Hanada, K., Nishijima, M., Akamatsu, Y., and Pagano, R. E. (1995) J. Biol. Chem. 270, 6254-6260
13. Stevens, V. L., and Tang, J. (1997) J. Biol. Chem. 272, 18020-18025
14. Naslavsky, N., Shmeeda, H., Friedlander, G., Yanai, A., Futerman, A. H., Barenholz, Y., and Taraboulos, A. (1999) J. Biol. Chem. 274, 20763-20771
15. Ostermeyer, A. G., Beckrich, B. T., Ivarson, K. A., Grove, K. E., and Brown, D. A. (1999) J. Biol. Chem. 274, 34459-34466
16. Gupta, A. K., and Rudney, H. (1991) J. Lipid Res. 32, 125-136
17. Härmälä, A.-S., Pörn, M. I., and Slotte, J. P. (1993) Biochim. Biophys. Acta 1210, 97-104
18. Ridgway, N. D. (1995) Biochim. Biophys. Acta 1256, 39-46
19. Hanada, K., Hara, T., Fukasawa, M., Yamaji, A., Umeda, M., and Nishijima, M. (1998) J. Biol. Chem. 273, 33787-33794
20. Fukasawa, M., Nishijima, M., and Hanada, K. (1999) J. Cell Biol. 144, 673-685
21. Slotte, J. P., Hedstrom, G., Rannstrom, S., and Ekman, S. (1989) Biochim. Biophys. Acta 985, 90-96
22. Fielding, P. E., and Fielding, C. J. (1995) Biochemistry 34, 14288-14292
23. Bligh, E. G., and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37, 911-917
24. Rouser, G., Siakotos, A. N., and Fleischer, S. (1966) Lipids 1, 85-86
25. Heider, J. G., and Boyett, R. L. (1978) J. Lipid Res. 19, 514-518
26. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275
27. Krieger, M. (1983) Anal. Biochem. 135, 383-391
28. Dahl, N. K., Reed, K. L., Daunais, M. A., Faust, J. R., and Liscum, L. (1992) J. Biol. Chem. 267, 4889-4896
29. Liscum, L., and Munn, N. J. (1999) Biochim. Biophys. Acta 1438, 19-37
30. Kilsdonk, E. P. C., Yancey, P. G., Stoudt, G. W., Bangerter, F. W., Johnson, W. J., Phillips, M. C., and Rothblat, G. H. (1995) J. Biol. Chem. 270, 17250-17256
31. Neufeld, E. B., Cooney, A. M., Pitha, J., Dawidowicz, E. A., Dwyer, N. K., Pentchev, P. G., and Blanchette-Mackie, E. J. (1996) J. Biol. Chem. 271, 21604-21613
32. Yancey, P. G., Rodrigueza, W. V., Kilsdonk, E. P. C., Stoudt, G. W., Johnson, W. J., Phillips, M. C., and Rothblat, G. H. (1996) J. Biol. Chem. 271, 16026-16034
33. Ohvo, H., Olsio, C., and Slotte, J. P. (1997) Biochim. Biophys. Acta 1349, 131-141
34. Rothblat, G. H., de la Llera-Moya, M., Atger, V., Kellner-Weibel, G., Williams, D. L., and Phillips, M. C. (1999) J. Lipid Res. 40, 781-796
35. Kudchodkar, B. J., Albers, J. J., and Bierman, E. L. (1983) Atherosclerosis 46, 353-367
36. Wattenberg, B. W., and Silbert, D. F. (1983) J. Biol. Chem. 258, 2284-2289
37. van Blitterswijk, W. J., van der Meer, B. W., and Hilkmann, H. (1987) Biochemistry 26, 1746-1756
38. Pörn, M. I., and Slotte, J. P. (1995) Biochem. J. 308, 269-274
39. DeGrella, R. F., and Simoni, R. D. (1982) J. Biol. Chem. 257, 14256-14262
40. Smart, E. J., Ying, Y., Donzell, W. C., and Anderson, R. G. (1996) J. Biol. Chem. 271, 29427-29435
41. Simons, K., and van Meer, G. (1988) Biochemistry 27, 6197-6202
42. Ahmed, S. N., Brown, D. A., and London, E. (1997) Biochemistry 36, 10944-10953
43. Slotte, J. P., Härmälä, A. S., Jansson, C., and Pörn, M. I. (1990) Biochim. Biophys. Acta 1030, 251-257
44. Stein, O., Ben-Naim, M., Dabach, Y., Hollander, G., and Stein, Y. (1992) Biochim. Biophys. Acta 1126, 291-297
45. Pörn, M. I., Ares, M. P., and Slotte, J. P. (1993) J. Lipid Res. 34, 1385-1392
46. Ito, J., Nagayasu, Y., and Yokoyama, S. (2000) J. Lipid Res. 41, 894-904
47. Kan, C. C., Ruan, Z. S., and Bittman, R. (1991) Biochemistry 30, 7759-7766
48. Bittman, R., Kasireddy, C. R., Mattjus, P., and Slotte, J. P. (1994) Biochemistry 33, 11776-11781
49. Fielding, C. J., and Fielding, P. E. (1997) J. Lipid Res. 38, 1503-1521

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Biol. Chem.Home page
K. Bowden and N. D. Ridgway
OSBP Negatively Regulates ABCA1 Protein Stability
J. Biol. Chem., June 27, 2008; 283(26): 18210 - 18217.
[Abstract] [Full Text] [PDF]

Home page
 J. Virol.Home page
H. Aizaki, K. Morikawa, M. Fukasawa, H. Hara, Y. Inoue, H. Tani, K. Saito, M. Nishijima, K. Hanada, Y. Matsuura, et al.
Critical Role of Virion-Associated Cholesterol and Sphingolipid in Hepatitis C Virus Infection
J. Virol., June 15, 2008; 82(12): 5715 - 5724.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
O. Sano, A. Kobayashi, K. Nagao, K. Kumagai, N. Kioka, K. Hanada, K. Ueda, and M. Matsuo
Sphingomyelin-dependence of cholesterol efflux mediated by ABCG1
J. Lipid Res., November 1, 2007; 48(11): 2377 - 2384.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. Kumagai, M. Kawano, F. Shinkai-Ouchi, M. Nishijima, and K. Hanada
Interorganelle Trafficking of Ceramide Is Regulated by Phosphorylation-dependent Cooperativity between the PH and START Domains of CERT
J. Biol. Chem., June 15, 2007; 282(24): 17758 - 17766.
[Abstract] [Full Text] [PDF]

Home page
 J. Lipid Res.Home page
D. K. Singh and P. V. Subbaiah
Modulation of the activity and arachidonic acid selectivity of group X secretory phospholipase A2 by sphingolipids
J. Lipid Res., March 1, 2007; 48(3): 683 - 692.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. Valeva, N. Hellmann, I. Walev, D. Strand, M. Plate, F. Boukhallouk, A. Brack, K. Hanada, H. Decker, and S. Bhakdi
Evidence That Clustered Phosphocholine Head Groups Serve as Sites for Binding and Assembly of an Oligomeric Protein Pore
J. Biol. Chem., September 8, 2006; 281(36): 26014 - 26021.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. K. Rouquette-Jazdanian, A. Foussat, L. Lamy, C. Pelassy, P. Lagadec, J.-P. Breittmayer, and C. Aussel
Cholera Toxin B-Subunit Prevents Activation and Proliferation of Human CD4+ T Cells by Activation of a Neutral Sphingomyelinase in Lipid Rafts
J. Immunol., November 1, 2005; 175(9): 5637 - 5648.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
Y. Lange, J. Ye, and T. L. Steck
How cholesterol homeostasis is regulated by plasma membrane cholesterol in excess of phospholipids
PNAS, August 10, 2004; 101(32): 11664 - 11667.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.