SMS overexpression and knockdown: impact on cellular sphingomyelin and diacylglycerol metabolism, and cell apoptosis

Sphingomyelin synthase (SMS), the last enzyme in the sphingomyelin (SM) biosynthetic pathway, uses ceramide and phosphatidylcholine as substrates to produce SM and diacylglycerol (DAG). To evaluate the role of SMS in apoptosis, we generated Chinese hamster ovary cells that stably express human SMS1 or SMS2. We found that SMS1 or SMS2 overexpression results in a significant increase in cellular levels of SM (24% or 20%) and DAG (35% or 31%), respectively, compared with controls. Cells overexpressing SMS1 or SMS2 were more likely to undergo lysis mediated by lysenin (a protein that causes lysis through its affinity with SM-rich microdomains in the plasma membrane) than were controls, indicating SM enrichment of the plasma membrane. SMS1 and SMS2 overexpression also led to higher retention of DiIC16 fluorescence compared with wild-type cells, indicating an increased number of detergent-insoluble microdomains and significantly increased tumor necrosis factor-alpha-mediated apoptosis. To further evaluate the relationship between SMS activity and cell apoptosis, we used SMS1 and SMS2 small interfering RNA (siRNA) to knock down their mRNA in THP-1-derived macrophages. We found that SMS1 or SMS2 siRNA significantly reduces intracellular SM (by 20% or 23%), plasma membrane SM (as indicated by the rate of lysenin-mediated cell lysis), and DAG levels (24% or 20%), respectively, while significantly reducing lipopolysaccharide-mediated apoptosis compared with controls. These results indicate that SMS1 and SMS2 are key factors in the control of SM and DAG levels within the cell and thus influence apoptosis.     

Both sphingomyelin synthases SMS1 and SMS2 are required for sphingomyelin homeostasis and growth in human HeLa cells

Sphingomyelin (SM) is a vital component of cellular membranes in organisms ranging from mammals to protozoa. Its production involves the transfer of phosphocholine from phosphatidylcholine to ceramide, yielding diacylglycerol in the process. The mammalian genome encodes two known SM synthase (SMS) isoforms, SMS1 and SMS2. However, the relative contributions of these enzymes to SM production in mammalian cells remained to be established. Here we show that SMS1 and SMS2 are co-expressed in a variety of cell types and function as the key Golgi- and plasma membrane-associated SM synthases in human cervical carcinoma HeLa cells, respectively. RNA interference-mediated depletion of either SMS1 or SMS2 caused a substantial decrease in SM production levels, an accumulation of ceramides, and a block in cell growth. Although SMS-depleted cells displayed a reduced SM content, external addition of SM did not restore growth. These results indicate that the biological role of SM synthases goes beyond formation of SM.     

Dynamic modification of sphingomyelin in lipid microdomains controls development of obesity, fatty liver, and type 2 diabetes

Lipid microdomains or caveolae, small invaginations of plasma membrane, have emerged as important elements for lipid uptake and glucose homeostasis. Sphingomyelin (SM) is one of the major phospholipids of the lipid microdomains. In this study, we investigated the physiological function of sphingomyelin synthase 2 (SMS2) using SMS2 knock-out mice, and we found that SMS2 deficiency prevents high fat diet-induced obesity and insulin resistance. Interestingly, in the liver of SMS2 knock-out mice, large and mature lipid droplets were scarcely observed. Treatment with siRNA for SMS2 also decreased the large lipid droplets in HepG2 cells. Additionally, the siRNA of SMS2 decreased the accumulation of triglyceride in liver of leptin-deficient (ob/ob) mice, strongly suggesting that SMS2 is involved in lipid droplet formation. Furthermore, we found that SMS2 exists in lipid microdomains and partially associates with the fatty acid transporter CD36/FAT and with caveolin 1, a scaffolding protein of caveolae. Because CD36/FAT and caveolin 1 exist in lipid microdomains and are coordinately involved in lipid droplet formation, SMS2 is implicated in the modulation of the SM in lipid microdomains, resulting in the regulation of CD36/FAT and caveolae. Here, we established new cell lines, in which we can completely distinguish SMS2 activity from SMS1 activity, and we demonstrated that SMS2 could convert ceramide produced in the outer leaflet of the plasma membrane into SM. Our findings demonstrate the novel and dynamic regulation of lipid microdomains via conformational changes in lipids on the plasma membrane by SMS2, which is responsible for obesity and type 2 diabetes.     

Identification of a family of animal sphingomyelin synthases

Sphingomyelin (SM) is a major component of animal plasma membranes. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, yielding diacylglycerol as a side product. This reaction is catalysed by SM synthase, an enzyme whose biological potential can be judged from the roles of diacylglycerol and ceramide as anti- and proapoptotic stimuli, respectively. SM synthesis occurs in the lumen of the Golgi as well as on the cell surface. As no gene for SM synthase has been cloned so far, it is unclear whether different enzymes are present at these locations. Using a functional cloning strategy in yeast, we identified a novel family of integral membrane proteins exhibiting all enzymatic features previously attributed to animal SM synthase. Strikingly, human, mouse and Caenorhabditis elegans genomes each contain at least two different SM synthase (SMS) genes. Whereas human SMS1 is localised to the Golgi, SMS2 resides primarily at the plasma membrane. Collectively, these findings open up important new avenues for studying sphingolipid function in animals.     

Expression cloning of a human cDNA restoring sphingomyelin synthesis and cell growth in sphingomyelin synthase-defective lymphoid cells

Sphingomyelin (SM) synthase has been assumed to be involved in both cell death and survival by regulating pro-apoptotic mediator ceramide and pro-survival mediator diacylglycerol. However, its precise functions are ambiguous due to the lack of molecular cloning of SM synthase gene(s). We isolated WR19L/Fas-SM(-) mouse lymphoid cells, which show a defect of SM at the plasma membrane due to the lack of SM synthase activity and resistance to cell death induced by an SM-directed cytolytic protein lysenin. WR19L/Fas-SM(-) cells were also highly susceptible to methyl-beta-cyclodextrin (MbetaCD) as compared with the WR19L/Fas-SM(+) cells, which are capable of SM synthesis. By expression cloning method using WR19L/Fas-SM(-) cells and MbetaCD-based selection, we have succeeded in cloning of a human cDNA responsible for SM synthase activity. The cDNA encodes a peptide of 413 amino acids named SMS1 (putative molecular mass, 48.6 kDa), which contains a sterile alpha motif domain near the N-terminal region and four predicted transmembrane domains. WR19L/Fas-SM(-) cells expressing SMS1 cDNA (WR19L/Fas-SMS1) restored the resistance against MbetaCD, the accumulation of SM at the plasma membrane, and SM synthesis by transferring phosphocholine from phosphatidylcholine to ceramide. Furthermore, WR19L/Fas-SMS1 cells, as well as WR19L/Fas-SM(-) cells supplemented with exogenous SM, restored cell growth ability in serum-free conditions, where the growth of WR19L/Fas-SM(-) cells was severely inhibited. The results suggest that SMS1 is responsible for SM synthase activity in mammalian cells and plays a critical role in cell growth of mouse lymphoid cells.     

The domain responsible for sphingomyelin synthase (SMS) activity

Sphingomyelin synthase (SMS) sits at the crossroads of sphingomyelin (SM), ceramide, diacylglycerol (DAG) metabolism. It utilizes ceramide and phosphatidylcholine as substrates to produce SM and DAG, thereby regulating lipid messengers which play a role in cell survival and apoptosis. There are two isoforms of the enzyme, SMS1 and SMS2. Both SMS1 and SMS2 contain two histidines and one aspartic acid which are evolutionary conserved within the lipid phosphate phosphatase superfamily. In this study, we systematically mutated these amino acids using site-directed mutagenesis and found that each point mutation abolished SMS activity without altering cellular distribution. We also explored the domains which are responsible for cellular distribution of both enzymes. Given their role as a potential regulator of diseases, these findings, coupled with homology modeling of SMS1 and SMS2, will be useful for drug development targeting SMS.     

Differential effects of sphingomyelin hydrolysis and resynthesis on the activation of NF-kappa B in normal and SV40-transformed human fibroblasts

The precise role of ceramide in NF-kappaB signaling remains unclear. The recent observation of differential sphingomyelin synthase (SMS) activity in normal (low SMS) versus SV40-transformed (high SMS) WI38 human lung fibroblasts provides an opportunity to assess the involvement of ceramide and SMS in NF-kappaB activation. Treatment of normal WI38 fibroblasts with bacterial sphingomyelinase resulted in a 4-fold elevation of ceramide and blocked NF-kappaB activation by serum stimulation. Such inhibition was not observed in SV40-transformed fibroblasts. Under regular growth conditions, after sphingomyelinase was washed out, normal WI38 did not show SM re-synthesis nor NF-kappaB activation. In SV40-WI38, on the other hand, sphingomyelinase washout induced resynthesis of SM due to the action of SMS on ceramide generated at the plasma membrane. NF-kappaB activation correlated with SM resynthesis. This activation was abrogated by D609, which inhibited SM resynthesis but not the initial formation of ceramide. The differential activity of SMS may explain the effects of ceramide in NF-kappaB signaling: in the absence of significant SMS activity, ceramide inhibits NF-kappaB, whereas with high SMS, the conversion of the ceramide signal to a diacylglycerol signal by the action of SMS stimulates NF-kappaB. These results also suggest a role for SMS in regulating NF-kappaB.     

Regulation of cell migration by sphingomyelin synthases: sphingomyelin in lipid rafts decreases responsiveness to signaling by the CXCL12/CXCR4 pathway

Sphingomyelin synthase (SMS) catalyzes the formation of sphingomyelin, a major component of the plasma membrane and lipid rafts. To investigate the role of SMS in cell signaling and migration induced by binding of the chemokine CXCL12 to CXCR4, we used mouse embryonic fibroblasts deficient in SMS1 and/or SMS2 and examined the effects of SMS deficiency on cell migration. SMS deficiency promoted cell migration through a CXCL12/CXCR4-dependent signaling pathway involving extracellular signal-regulated kinase (ERK) activation. In addition, SMS1/SMS2 double-knockout cells had heightened sensitivity to CXCL12, which was significantly suppressed upon transfection with the SMS1 or SMS2 gene or when they were treated with exogenous sphingomyelin but not when they were treated with the SMS substrate ceramide. Notably, SMS deficiency facilitated relocalization of CXCR4 to lipid rafts, which form platforms for the regulation and transduction of receptor-mediated signaling. Furthermore, we found that SMS deficiency potentiated CXCR4 dimerization, which is required for signal transduction. This dimerization was significantly repressed by sphingomyelin treatment. Collectively, our data indicate that SMS-derived sphingomyelin lowers responsiveness to CXCL12, thereby reducing migration induced by this chemokine. Our findings provide the first direct evidence for an involvement of SMS-generated sphingomyelin in the regulation of cell migration.     

Role of ceramide/sphingomyelin (SM) balance regulated through “SM cycle” in cancer

Sphingomyelin synthase (SMS), which comprises of two isozymes, SMS1 and SMS2, is the only enzyme that generates sphingomyelin (SM) by transferring phosphocholine of phosphatidylcholine to ceramide in mammals. Conversely, ceramide is generated from SM hydrolysis via sphingomyelinases (SMases), ceramide de novo synthesis, and the salvage pathway. The biosynthetic pathway for SM and ceramide content by SMS and SMase, respectively, is called “SM cycle.” SM forms a SM-rich microdomain on the cell membrane to regulate signal transduction, such as proliferation/survival, migration, and inflammation. On the other hand, ceramide acts as a lipid mediator by forming a ceramide-rich platform on the membrane, and ceramide exhibits physiological actions such as cell death, cell cycle arrest, and autophagy induction. Therefore, the regulation of ceramide/SM balance by SMS and SMase is responsible for diverse cell functions not only in physiological cells but also in cancer cells. This review outlines the implications of ceramide/SM balance through “SM cycle” in cancer progression and prevention. In addition, the possible involvement of “SM cycle” is introduced in anti-cancer tumor immunity, which has become a hot topic to innovate a more effective and safer way to conquer cancer in recent years.     

Sphingomyelin synthase as a potential target for D609-induced apoptosis in U937 human monocytic leukemia cells

Tricyclodecan-9-yl-xanthogenate (D609) is a selective tumor cytotoxic agent. However, the mechanisms of action of D609 against tumor cells have not been well established. Using U937 human monocytic leukemia cells, we examined the ability of D609 to inhibit sphingomyelin synthase (SMS), since inhibition of SMS may contribute to D609-induced tumor cell cytotoxicity via modulating the cellular levels of ceramide and diacylglycerol (DAG). The results showed that D609 is capable of inducing U937 cell death by apoptosis in a dose- and time-dependent manner. The induction of U937 cell apoptosis was associated with an inhibition of SMS activity and a significant increase in the intracellular level of ceramide and decrease in that of sphingomyelin (SM) and DAG, which resulted in an elevation of the ratio between ceramide and DAG favoring the induction of apoptosis. In addition, incubation of U937 cells with C(6)-ceramide and/or H7 (a selective PKC inhibitor) reduced U937 cell viability; whereas pretreatment of the cells with a PKC activator, PMA or 1-oleoyl-2-acetylglycerol (OAG), attenuated D609-induced U937 cell apoptosis. These results suggest that SMS is a potential target of D609 and inhibition of SMS may contribute to D609-induced tumor cell death via modulation of the cellular levels of ceramide and DAG.     

Reducing plasma membrane sphingomyelin increases insulin sensitivity

It has been shown that inhibition of de novo sphingolipid synthesis increases insulin sensitivity. For further exploration of the mechanism involved, we utilized two models: heterozygous serine palmitoyltransferase (SPT) subunit 2 (Sptlc2) gene knockout mice and sphingomyelin synthase 2 (Sms2) gene knockout mice. SPT is the key enzyme in sphingolipid biosynthesis, and Sptlc2 is one of its subunits. Homozygous Sptlc2-deficient mice are embryonic lethal. However, heterozygous Sptlc2-deficient mice that were viable and without major developmental defects demonstrated decreased ceramide and sphingomyelin levels in the cell plasma membranes, as well as heightened sensitivity to insulin. Moreover, these mutant mice were protected from high-fat diet-induced obesity and insulin resistance. SMS is the last enzyme for sphingomyelin biosynthesis, and SMS2 is one of its isoforms. Sms2 deficiency increased cell membrane ceramide but decreased SM levels. Sms2 deficiency also increased insulin sensitivity and ameliorated high-fat diet-induced obesity. We have concluded that Sptlc2 heterozygous deficiency- or Sms2 deficiency-mediated reduction of SM in the plasma membranes leads to an improvement in tissue and whole-body insulin sensitivity.     

Sphingomyelin synthases regulate production of diacylglycerol at the Golgi

SMS [SM (sphingomyelin) synthase] is a class of enzymes that produces SM by transferring a phosphocholine moiety on to ceramide. PC (phosphatidylcholine) is believed to be the phosphocholine donor of the reaction with consequent production of DAG (diacylglycerol), an important bioactive lipid. In the present study, by modulating SMS1 and SMS2 expression, the role of these enzymes on the elusive regulation of DAG was investigated. Because we found that modulation of SMS1 or SMS2 did not affect total levels of endogenous DAG in resting cells, whereas they produce DAG in vitro, the possibility that SMSs could modulate subcellular pools of DAG, once acute activation of the enzymes is triggered, was investigated. Stimulation of SM synthesis was induced by either treatment with short-chain ceramide analogues or by increasing endogenous ceramide at the plasma membrane, and a fluorescently labelled conventional C1 domain [from PKC (protein kinase C)] enhanced in its DAG binding activity was used to probe subcellular pools of DAG in the cell. With this approach, we found, using confocal microscopy and subcellular fractionation, that modulation of SMS1 and, to a lesser extent, SMS2 affected the formation of DAG at the Golgi apparatus. Similarly, down-regulation of SMS1 and SMS2 reduced the localization of the DAG-binding protein PKD (protein kinase D) to the Golgi. These results provide direct evidence that both enzymes are capable of regulating the formation of DAG in cells, that this pool of DAG is biologically active, and for the first time directly implicate SMS1 and SMS2 as regulators of DAG-binding proteins in the Golgi apparatus.     

Switching head group selectivity in mammalian sphingolipid biosynthesis by active-site-engineering of sphingomyelin synthases

SM is a fundamental component of mammalian cell membranes that contributes to mechanical stability, signaling, and sorting. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, a reaction catalyzed by SM synthase (SMS)1 in the Golgi and SMS2 at the plasma membrane. Mammalian cells also synthesize trace amounts of the SM analog, ceramide phosphoethanolamine (CPE), but the physiological relevance of CPE production is unclear. Previous work revealed that SMS2 is a bifunctional enzyme producing both SM and CPE, whereas a closely related enzyme, SMS-related protein (SMSr)/SAMD8, acts as a monofunctional CPE synthase in the endoplasmic reticulum. Using domain swapping and site-directed mutagenesis on enzymes expressed in defined lipid environments, we here identified structural determinants that mediate the head group selectivity of SMS family members. Notably, a single residue adjacent to the catalytic histidine in the third exoplasmic loop profoundly influenced enzyme specificity, with Glu permitting SMS-catalyzed CPE production and Asp confining the enzyme to produce SM. An exchange of exoplasmic residues with SMSr proved sufficient to convert SMS1 into a bulk CPE synthase. This allowed us to establish mammalian cells that produce CPE rather than SM as the principal phosphosphingolipid and provide a model of the molecular interactions that impart catalytic specificity among SMS enzymes.     

Anti-proliferative Effects of Tricyclodecan-9-yl-xanthogenate (D609) Involve Ceramide and Cell Cycle Inhibition

Tricyclodecan-9-yl-xanthogenate (D609) inhibits phosphatidylcholine (PC)-phospholipase C (PLC) and/or sphingomyelin (SM) synthase (SMS). Inhibiting SMS can increase ceramide levels, which can inhibit cell proliferation. Here, we examined how individual inflammatory and glia cell proliferation is altered by D609. Treatment with 100-μM D609 significantly attenuated the proliferation of RAW 264.7 macrophages, N9 and BV-2 microglia, and DITNC(1) astrocytes, without affecting cell viability. D609 significantly inhibited BrdU incorporation in BV-2 microglia and caused accumulation of cells in G(1) phase with decreased number of cells in the S phase. D609 treatment for 2 h significantly increased ceramide levels in BV-2 microglia, which, following a media change, returned to control levels 22 h later. This suggests that the effect of D609 may be mediated, at least in part, through ceramide increase via SMS inhibition. Western blots demonstrated that 2-h treatment of BV-2 microglia with D609 increased expression of the cyclin-dependent kinase (Cdk) inhibitor p21 and down-regulated phospho-retinoblastoma (Rb), both of which returned to basal levels 22 h after removal of D609. Exogenous C8-ceramide also inhibited BV-2 microglia proliferation without loss of viability and decreased BrdU incorporation, supporting the involvement of ceramide in D609-mediated cell cycle arrest. Our current data suggest that D609 may offer benefit after stroke (Adibhatla and Hatcher, Mol Neurobiol 41:206-217, 2010) through ceramide-mediated cell cycle arrest, thus restricting glial cell proliferation.     

Resistance to alkyl-lysophospholipid-induced apoptosis due to downregulated sphingomyelin synthase 1 expression with consequent sphingomyelin- and cholesterol-deficiency in lipid rafts

The ALP (alkyl-lysophospholipid) edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine; Et-18-OCH3) induces apoptosis in S49 mouse lymphoma cells. To this end, ALP is internalized by lipid raft-dependent endocytosis and inhibits phosphatidylcholine synthesis. A variant cell-line, S49AR, which is resistant to ALP, was shown previously to be unable to internalize ALP via this lipid raft pathway. The reason for this uptake failure is not understood. In the present study, we show that S49AR cells are unable to synthesize SM (sphingomyelin) due to down-regulated SMS1 (SM synthase 1) expression. In parental S49 cells, resistance to ALP could be mimicked by small interfering RNA-induced SMS1 suppression, resulting in SM deficiency and blockage of raft-dependent internalization of ALP and induction of apoptosis. Similar results were obtained by treatment of the cells with myriocin/ISP-1, an inhibitor of general sphingolipid synthesis, or with U18666A, a cholesterol homoeostasis perturbing agent. U18666A is known to inhibit Niemann-Pick C1 protein-dependent vesicular transport of cholesterol from endosomal compartments to the trans-Golgi network and the plasma membrane. U18666A reduced cholesterol partitioning in detergent-resistant lipid rafts and inhibited SM synthesis in S49 cells, causing ALP resistance similar to that observed in S49AR cells. The results are explained by the strong physical interaction between (newly synthesized) SM and available cholesterol at the Golgi, where they facilitate lipid raft formation. We propose that ALP internalization by lipid-raft-dependent endocytosis represents the retrograde route of a constitutive SMS1- and lipid-raft-dependent membrane vesicular recycling process.     

All members in the sphingomyelin synthase gene family have ceramide phosphoethanolamine synthase activity

Sphingomyelin synthase-related protein (SMSr) synthesizes the sphingomyelin analog ceramide phosphoethanolamine (CPE) in cells. Previous cell studies indicated that SMSr is involved in ceramide homeostasis and is crucial for cell function. To further examine SMSr function in vivo, we generated Smsr KO mice that were fertile and had no obvious phenotypic alterations. Quantitative MS analyses of plasma, liver, and macrophages from the KO mice revealed only marginal changes in CPE and ceramide as well as other sphingolipid levels. Because SMS2 also has CPE synthase activity, we prepared Smsr/Sms2 double KO mice. We found that CPE levels were not significantly changed in macrophages, suggesting that CPE levels are not exclusively dependent on SMSr and SMS2 activities. We then measured CPE levels in Sms1 KO mice and found that Sms1 deficiency also reduced plasma CPE levels. Importantly, we found that expression of Sms1 or Sms2 in SF9 insect cells significantly increased not only SM but also CPE formation, indicating that SMS1 also has CPE synthase activity. Moreover, we measured CPE synthase K_m and V_max for SMS1, SMS2, and SMSr using different NBD ceramides. Our study reveals that all mouse SMS family members (SMSr, SMS1, and SMS2) have CPE synthase activity. However, neither CPE nor SMSr appears to be a critical regulator of ceramide levels in vivo.     

Sphingomyelin synthase 1 activity is regulated by the BCR-ABL oncogene

Sphingomyelin synthase (SMS) produces sphingomyelin while consuming ceramide (a negative regulator of cell proliferation) and forming diacylglycerol (DAG) (a mitogenic factor). Therefore, enhanced SMS activity could favor cell proliferation. To examine if dysregulated SMS contributes to leukemogenesis, we measured SMS activity in several leukemic cell lines and found that it is highly elevated in K562 chronic myelogenous leukemia (CML) cells. The increased SMS in K562 cells was caused by the presence of Bcr-abl, a hallmark of CML; stable expression of Bcr-abl elevated SMS activity in HL-60 cells while inhibition of the tyrosine kinase activity of Bcr-abl with Imatinib mesylate decreased SMS activity in K562 cells. The increased SMS activity was the result of up-regulation of the Sms1 isoform. Inhibition of SMS activity with D609 (a pharmacological SMS inhibitor) or down-regulation of SMS1 expression by siRNA selectively inhibited the proliferation of Bcr-abl-positive cells. The inhibition was associated with an increased production of ceramide and a decreased production of DAG, conditions that antagonize cell proliferation. A similar change in lipid profile was also observed upon pharmacological inhibition of Bcr-abl (K526 cells) and siRNA-mediated down-regulation of BCR-ABL (HL-60/Bcr-abl cells). These findings indicate that Sms1 is a downstream target of Bcr-abl, involved in sustaining cell proliferation of Bcr-abl-positive cells.     

Regulation of the selective uptake of cholesteryl esters from high density lipoproteins by sphingomyelin

Although sphingomyelin (SM) is a major phospholipid in lipoproteins as well as in the membrane rafts where the scavenger receptor class B type I (SR-BI) is localized, its possible role in the selective uptake of cholesteryl ester (CE) by the SR-BI-mediated pathway is unknown. We investigated the effect of SM in lipoproteins and cell membranes on the selective uptake in three different cell lines: SR-BI-transfected CHO cells, hepatocytes (HepG2), and adrenocortical cells (Y1BS1). Incorporation of SM into recombinant high density lipoprotein (rHDL) containing labeled CE resulted in up to 50% inhibition of the selective uptake of CE in all three cell lines. This inhibition was completely reversed by treatment of rHDL with sphingomyelinase (SMase). Selective uptake from plasma HDL was activated by 22-72% after treatment of HDL with SMase. In addition, pretreatment of the cells with SMase resulted in stimulation of CE uptake from rHDL by CHO and Y1BS1, although not by HepG2. Incorporation of ceramide into rHDL resulted in up to 2-fold stimulation of CE uptake, although pretreatment of cells with egg ceramide had no significant effect. These results show that SM and ceramide in the lipoproteins and the cell membranes regulate the SR-BI-mediated selective uptake of CE, possibly by interacting with the sterol ring or with SR-BI itself.     

Ebolavirus requires acid sphingomyelinase activity and plasma membrane sphingomyelin for infection

Acid sphingomyelinase (ASMase) converts the lipid sphingomyelin (SM) to phosphocholine and ceramide and has optimum activity at acidic pH. Normally, ASMase is located in lysosomes and endosomes, but membrane damage or the interaction with some bacterial and viral pathogens can trigger its recruitment to the plasma membrane. Rhinovirus and measles viruses each require ASMase activity during early stages of infection. Both sphingomyelin and ceramide are important components of lipid rafts and are potent signaling molecules. Each plays roles in mediating macropinocytosis, which has been shown to be important for ebolavirus (EBOV) infection. Here, we investigated the role of ASMase and its substrate, SM, in EBOV infection. The work was performed at biosafety level 4 with wild-type virus with specificity and mechanistic analysis performed using virus pseudotypes and virus-like particles. We found that virus particles strongly associate with the SM-rich regions of the cell membrane and depletion of SM reduces EBOV infection. ASM-specific drugs and multiple small interfering RNAs strongly inhibit the infection by EBOV and EBOV glycoprotein pseudotyped viruses but not by the pseudotypes bearing the glycoprotein of vesicular stomatitis virus. Interestingly, the binding of virus-like particles to cells is strongly associated with surface-localized ASMase as well as SM-enriched sites. Our work suggests that ASMase activity and SM presence are necessary for efficient infection of cells by EBOV. The inhibition of this pathway may provide new avenues for drug treatment.     

Conversion of apical plasma membrane sphingomyelin to ceramide attenuates the intoxication of host cells by cholera toxin

Cholera toxin (CT) enters host cells by binding to ganglioside GM1 in the apical plasma membrane (PM). GM1 carries CT retrograde from the PM to the endoplasmic reticulum (ER), where a portion of the toxin, the A1-chain, retro-translocates to the cytosol, causing disease. Trafficking in this pathway appears to depend on the association of CT–GM1 complexes with sphingomyelin (SM)- and cholesterol-rich membrane microdomains termed lipid rafts. Here, we find that in polarized intestinal epithelia, the conversion of apical membrane SM to ceramide by bacterial sphingomyelinase attenuates CT toxicity, consistent with the lipid raft hypothesis. The effect is reversible, specific to toxin entry via the apical membrane, and recapitulated by the addition of exogenous long-chain ceramides. Conversion of apical membrane SM to ceramide inhibits the efficiency of toxin endocytosis, but retrograde trafficking from the apical PM to the Golgi and ER is not affected. This result suggests that the cause for toxin resistance occurs at steps required for retro-translocation of the CT A1-chain to the cytosol.