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

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 ϳ40 and ϳ15%, 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 [ 3 H]cholesterol showed that the [ 3 H]cholesterol level in the lowdensity floating fraction was lower in sphingomyelindeficient cells than in wild-type cells. When cells were exposed to methyl-␤-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. 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 liquidordered 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 ...

Dynamics of membrane lipid domains in neuronal cells differentiated in culture

“…* P Ͻ 0.05, ** P Ͻ 0.01 versus control. process has been shown to be dependent on cyclodextrin concentration, time, and cell type (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). The mechanism by which cholesterol is lost by the cells in the presence of cyclodextrins is not clear; this process probably involves different plasma membrane cholesterol pools (22,24) but not the intracellular cholesterol pools (22).…”

“…A very promising experimental approach to a better understanding of the involvement of lipids in the structural and functional properties of membrane domains is represented by the artificial manipulation of lipid composition and/or organization within these structures. To this purpose, a wide variety of experimental approaches allowing the downregulation of the biosynthesis of membrane lipids (15)(16)(17)(18)(19)(20)(21) or the lowering of cholesterol levels in the plasma membrane by external ligands (20)(21)(22)(23)(24)(25)(26)(27)(28) has been introduced. Among these tools, the extraction of cholesterol from cells by treatment with cyclodextrins is certainly the most widely used, as evidenced by the hundreds of papers found combining the two key words "cyclodextrin" and "cholesterol" in a Medline search from 1996 to the present.…”

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

“…We recently found mouse lymphoid cell variants designated WR19L/Fas-SM(Ϫ), which are defective of SM synthesis and susceptible to methyl-␤-cyclodextrin (M␤CD)-induced cell death (30). By an expression cloning method using WR19L/Fas-SM(Ϫ) cells and M␤CD-based cell selection, we isolated a human cDNA responsible for SM synthase activity.…”

Ceramide and raft signaling are linked with each other in UVA radiation-induced gene expression

“…In principle, the UVA radiation-induced decrease in Chol could be the consequence of (i) sphingomyelin hydrolysis and the resulting formation of ceramide, or (ii) vice versa, or (iii) both UVA effects could occur independently from each other. In this regard, in Chinese Hamster Ovary cells a decrease of sphingomyelin levels has been shown to enhance the cellular Chol efflux, indicating that sphingomyelin has a function in retaining Chol in the plasma membrane (Fukasawa et al, 2000). In addition, ceramide has recently been shown to selectively displace Chol from lipid rafts (Megha and London, 2004).…”