Differential Effects of Ceramide and Sphingosine 1-Phosphate on ERM Phosphorylation: PROBING SPHINGOLIPID SIGNALING AT THE OUTER PLASMA MEMBRANE*

ERM proteins are regulated by phosphorylation of the most C-terminal threonine residue, switching them from an activated to an inactivated form. However, little is known about the control of this regulation. Previous work in our group demonstrated that secretion of acid sphingomyelinase acts upstream of ERM dephosphorylation, suggesting the involvement of sphingomyelin (SM) hydrolysis in ERM regulation. To define the role of specific lipids, we employed recombinant bacterial sphingomyelinase (bSMase) as a direct probe of SM metabolism at the plasma membrane. bSMase induced a rapid dose- and time-dependent decrease in ERM dephosphorylation. ERM dephosphorylation was driven by ceramide generation and not by sphingomyelin depletion, as shown using recombinant sphingomyelinase D. The generation of ceramide at the plasma membrane was sufficient for ERM regulation, and no intracellular SM hydrolysis was required, as was visualized using Venus-tagged lysenin probe, which specifically binds SM. Interestingly, hydrolysis of plasma membrane bSMase-induced ceramide using bacterial ceramidase caused ERM hyperphosphorylation and formation of cell surface protrusions. The effects of plasma membrane ceramide hydrolysis were due to sphingosine 1-phosphate formation, as ERM phosphorylation was blocked by an inhibitor of sphingosine kinase and induced by sphingosine 1-phosphate. Taken together, these results demonstrate a new regulatory mechanism of ERM phosphorylation by sphingolipids with opposing actions of ceramide and sphingosine 1-phosphate. The approach also defines a tool kit to probe sphingolipid signaling at the plasma membrane.     

Accumulation of sphingomyelin in Niemann-Pick disease type C cells disrupts Rab9-dependent vesicular trafficking of cholesterol

Niemann-Pick disease type C (NPC) is a genetic disorder in which patient cells have endosomal/lysosomal accumulation of cholesterol and sphingolipids. However, the relationship between sphingolipids and cholesterol accumulation in NPC cells has not been established. Here, we investigated the role of sphingomyelin (SM) on the accumulation of cholesterol in NPC cells. Reduction of SM by inhibition of the ceramide transfer protein CERT decreased the cholesterol accumulation in NPC cells. The accumulation of SM in NPC cells inhibited the transport of cholesterol to the endoplasmic reticulum. Overexpression of Rab9 in NPC cells reduced the cholesterol accumulation, which was recovered by treatment with SM. In NPC cells that overexpressed a Rab9 constitutively active mutant, SM treatment did not lead to the cholesterol accumulation. These results indicate that SM negatively regulates the Rab9-dependent vesicular trafficking of cholesterol, and a reduction in SM levels in NPC cells recovers the Rab9-dependent vesicular trafficking defect.     

Sphingolipids and gangliosides of the nervous system in membrane function and dysfunction

Simple sphingolipids such as ceramide and sphingomyelin (SM) as well as more complex glycosphingolipids play very important roles in cell function under physiological conditions and during disease development and progression. Sphingolipids are particularly abundant in the nervous system. Due to their amphiphilic nature they localize to cellular membranes and many of their roles in health and disease result from membrane reorganization and from lipid interaction with proteins within cellular membranes. In this review we discuss some of the functions of sphingolipids in processes that entail cellular membranes and their role in neurodegenerative diseases, with an emphasis on SM, ceramide and gangliosides.     

Oleic acid modulates the post-translational glycosylation of macrophage ApoE to increase its secretion

There has been increasing interest in a potential role for fatty acids in adversely affecting organismal substrate utilization and contributing to the cardiovascular complications in insulin resistance. Fatty acids have already been implicated in regulating the expression of a number of genes in resident cells of the vessel wall. In the current studies, we evaluated a potential role for fatty acids in the regulation of macrophage apoE expression. Incubation in oleic acid increased the synthesis and secretion of apoE by human monocyte-derived macrophages. Part of this stimulation was mediated at a post-translational locus. Oleic acid increased the secretion of apoE from macrophages that constitutively expressed a human apoE3 cDNA. Incubation in palmitic acid decreased apoE secretion from these cells. The effect of oleic acid on apoE secretion could not be accounted for by the known effect of fatty acid on cellular sterol, because incubation in oleic acid did not suppress the degradation of nascent apoE. Incubation in oleic acid for at least 6 h was required to observe an effect on apoE secretion. Oleic acid altered the glycosylation pattern of cellular and secreted apoE, with a loss of the most heavily sialylated isoform. Oleic acid had no effect on the glycosylation of interleukin 6 secreted from macrophages. Elimination of apoE glycosylation, by substitution of threonine 194 with alanine, eliminated oleic acid-mediated stimulation of apoE secretion. These results indicate that oleic acid increases apoE secretion from macrophages at a locus involving post-translational glycosylation.     

ApoE-dependent sterol efflux from macrophages is modulated by scavenger receptor class B type I expression

Macrophages express a number of proteins involved in sterol efflux pathways, including apolipoprotein E (apoE) and scavenger receptor class B type I (SR-BI). We have investigated a potential interaction between these two sterol efflux pathways in modulating overall macrophage sterol flux. We utilized an experimental system in which we increased expression of each of these proteins to a high physiologic range in order to perform our evaluation. We show that in apoE-expressing cells, a 4-fold increase in SR-BI expression leads to reduction of sterol and phospholipid efflux. SR-BI-mediated reduction in sterol efflux was only observed in cells that expressed endogenous apoE. In J774 cells that did not express apoE, a similar increase in SR-BI level led to increased sterol efflux. The divergent response of sterol efflux after increased SR-BI was maintained in the presence of a number of structurally diverse extracellular sterol acceptors. Increased SR-BI expression also enhanced sterol efflux to exogenously added apoE. Investigation of a potential mechanism for reduced efflux in apoE-expressing cells indicated that SR-BI expression reduced macrophage apoE by accelerating the degradation of newly synthesized apoE. This led to decreased secretion of apoE and reduced the fraction of apoE sequestered on the cell surface. Thus, enhanced SR-BI expression in macrophages can reduce the cellular level and secretion of apoE by accelerating degradation of the newly synthesized protein. This reduction of endogenous apoE is accompanied by reduced sterol efflux from macrophages.     

Synthetic, non-natural sphingolipid analogs inhibit the biosynthesis of cellular sphingolipids, elevate ceramide and induce apoptotic cell death

Numerous studies have demonstrated the participation of sphingolipids in signal transduction and regulation of cell growth. Several cellular stress agents have been shown to elevate ceramide, the basic precursor of all sphingolipids, initiating a cascade of events leading to arrest of the cell cycle, apoptosis and cell death. Aiming at inhibiting metabolic pathways of sphingolipid metabolism that might lead to an increase of cellular ceramide, we have synthesized non-natural analogs of ceramide, sphingosine and trimethylsphingosine. When the respective analogs were applied to HL60 human myeloid leukemic cells they inhibited the biosynthesis of sphingomyelin (SPM) and glycosphingolipids and induced apoptosis that led to cell death. A fluorescent procedure which has been developed for quantifying the biosynthesis of cellular ceramide indicated an increase in the ceramide content following an incubation with the synthetic analogs. These results suggest that the newly synthesized sphingolipid analogs might be valuable for potential application as a therapeutic modality in leukemia and other malignancies.     

Molecular mechanisms and regulation of ceramide transport

De novo biosynthesis of sphingolipids begins in the endoplasmic reticulum (ER) and continues in the Golgi apparatus and plasma membrane. A crucial step in sphingolipid biosynthesis is the transport of ceramide by vesicular and non-vesicular mechanisms from its site of synthesis in the ER to the Golgi apparatus. The recent discovery of the ceramide transport protein CERT has revealed a novel pathway for the delivery of ceramide to the Golgi apparatus for sphingomyelin (SM) synthesis. In addition to a ceramide-binding START domain, CERT has FFAT (referring to two phenylalanines [FF] in an acidic tract) and pleckstrin homology (PH) domains that recognize the ER integral membrane protein VAMP-associated protein (VAP) and Golgi-associated PtdIns 4-phosphate, respectively. Mechanisms for vectorial transport involving dual-organellar targeting and sites of deposition of ceramide in the Golgi apparatus are proposed. Similar Golgi-ER targeting motifs are also present in the oxysterol-binding protein (OSBP), which regulates ceramide transport and SM synthesis in an oxysterol-dependent manner. Consequently, this emerges as a potential mechanism for integration of sphingolipid and cholesterol metabolism. The identification of organellar targeting motifs in other related lipid-binding/transport proteins indicate that concepts learned from the study of ceramide transport can be applied to other lipid transport processes.     

Microsomal Triglyceride Transfer Protein Transfers and Determines Plasma Concentrations of Ceramide and Sphingomyelin but Not Glycosylceramide

Sphingolipids, a large family of bioactive lipids, are implicated in stress responses, differentiation, proliferation, apoptosis, and other physiological processes. Aberrant plasma levels of sphingolipids contribute to metabolic disease, atherosclerosis, and insulin resistance. They are fairly evenly distributed in high density and apoB-containing lipoproteins (B-lps). Mechanisms involved in the transport of sphingolipids to the plasma are unknown. Here, we investigated the role of microsomal triglyceride transfer protein (MTP), required for B-lp assembly and secretion, in sphingolipid transport to the plasma. Abetalipoproteinemia patients with deleterious mutations in MTP and absence of B-lps had significantly lower plasma ceramide and sphingomyelin but normal hexosylceramide, lactosylceramide, and different sphingosines compared with unaffected controls. Furthermore, similar differential effects on plasma sphingolipids were seen in liver- and intestine-specific MTP knock-out (L,I-Mttp^−/−) mice, suggesting that MTP specifically plays a role in the regulation of plasma ceramide and sphingomyelin. We hypothesized that MTP deficiency may affect either their synthesis or secretion. MTP deficiency had no effect on ceramide and sphingomyelin synthesis but reduced secretion from primary hepatocytes and hepatoma cells. Therefore, MTP is involved in ceramide and sphingomyelin secretion but not in their synthesis. We also found that MTP transferred these lipids between vesicles in vitro. Therefore, we propose that MTP might regulate plasma ceramide and sphingomyelin levels by transferring these lipids to B-lps in the liver and intestine and facilitating their secretion.     

Regulation of macrophage apoE secretion and sterol efflux by the LDL receptor

Factors that regulate apolipoprotein E (apoE) secretion by macrophages will have important effects on vessel wall lipid flux and atherosclerosis. Macrophages express the LDL receptor, which binds apoE with high affinity and could thereby affect the net secretion of apoE from macrophages. In these studies, we demonstrate that treatment of J774 macrophages transfected to constitutively express a human apoE3 cDNA with simvastatin, to increase LDL receptor activity, reduces the secretion of apoE. To further examine the relationship between LDL receptor expression and apoE secretion from macrophages, mouse peritoneal macrophages (MPMs) were isolated from mice with constitutively high expression of human LDL receptor to increase overall LDL receptor expression by 2- to 3-fold. Cells with increased LDL receptor expression also showed reduced apoE secretion compared with MPMs with basal LDL receptor expression. The effect of changes in LDL receptor expression on apoE secretion was isoform-specific, with greater reduction of apoE4 compared with apoE3 secretion and no reduction of apoE2 secretion, paralleling the known affinity of each isoform for LDL receptor binding. The effect of the LDL receptor on apoE secretion for each isoform was further reflected in LDL receptor-dependent changes in apoE-mediated cholesterol efflux. These results establish a regulatory interaction between two branches of macrophage sterol homeostatic pathways that could facilitate a rapid response to changes in macrophage sterol content relative to need.     

Apolipoprotein A-I-stimulated apolipoprotein E secretion from human macrophages is independent of cholesterol efflux

Apolipoprotein A-I (apoA-I)-mediated cholesterol efflux involves the binding of apoA-I to the plasma membrane via its C terminus and requires cellular ATP-binding cassette transporter (ABCA1) activity. ApoA-I also stimulates secretion of apolipoprotein E (apoE) from macrophage foam cells, although the mechanism of this process is not understood. In this study, we demonstrate that apoA-I stimulates secretion of apoE independently of both ABCA1-mediated cholesterol efflux and of lipid binding by its C terminus. Pulse-chase experiments using (35)S-labeled cellular apoE demonstrate that macrophage apoE exists in both relatively mobile (E(m)) and stable (E(s)) pools, that apoA-I diverts apoE from degradation to secretion, and that only a small proportion of apoA-I-mobilized apoE is derived from the cell surface. The structural requirements for induction of apoE secretion and cholesterol efflux are clearly dissociated, as C-terminal deletions in recombinant apoA-I reduce cholesterol efflux but increase apoE secretion, and deletion of central helices 5 and 6 decreases apoE secretion without perturbing cholesterol efflux. Moreover, a range of 11- and 22-mer alpha-helical peptides representing amphipathic alpha-helical segments of apoA-I stimulate apoE secretion whereas only the C-terminal alpha-helix (domains 220-241) stimulates cholesterol efflux. Other alpha-helix-containing apolipoproteins (apoA-II, apoA-IV, apoE2, apoE3, apoE4) also stimulate apoE secretion, implying a positive feedback autocrine loop for apoE secretion, although apoE4 is less effective. Finally, apoA-I stimulates apoE secretion normally from macrophages of two unrelated subjects with genetically confirmed Tangier Disease (mutations C733R and c.5220-5222delTCT; and mutations A1046D and c.4629-4630insA), despite severely inhibited cholesterol efflux. We conclude that apoA-I stimulates secretion of apoE independently of cholesterol efflux, and that this represents a novel, ABCA-1-independent, positive feedback pathway for stimulation of potentially anti-atherogenic apoE secretion by alpha-helix-containing molecules including apoA-I and apoE.     

Inactivation of ceramide transfer protein during pro-apoptotic stress by Golgi disassembly and caspase cleavage

The mammalian Golgi apparatus is composed of multiple stacks of cisternal membranes organized laterally into a polarized ribbon. Furthermore, trans-Golgi membranes come in close apposition with ER (endoplasmic reticulum) membranes to form ER-trans-Golgi contact sites, which may facilitate transfer of newly synthesized ceramide from the ER to SM (sphingomyelin) synthase at the trans-Golgi via CERT (ceramide transfer protein). CERT interacts with both ER and Golgi membranes, and together with Golgi morphology contributes to efficient SM synthesis. In the present study, we show that Golgi disassembly during pro-apoptotic stress induced by TNFα (tumour necrosis factor α) and anisomycin results in decreased levels of CERT at the Golgi region. This is accompanied by a caspase-dependent loss of full-length CERT and reduction in de novo SM synthesis. In vitro, CERT is cleaved by caspases 2, 3 and 9. Truncated versions of CERT corresponding to fragments generated by caspase 2 cleavage at Asp213 were mislocalized and did not promote efficient de novo SM synthesis. Thus it is likely that during cellular stress, disassembly of Golgi structure together with inactivation of CERT by caspases causes a reduction in ceramide trafficking and SM synthesis, and could contribute to the cellular response to pro-apoptotic stress.     

Formation of ceramide-enriched domains in lipid particles enhances the binding of apolipoprotein E

We investigated the interaction between apolipoprotein E (apoE) and ceramide (CER)-enriched domains on the particles, by using lipid emulsions containing sphingomyelin (SM) or CER as model particles of lipoproteins. The sphingomyelinase (SMase)-induced aggregation of emulsion particles was prevented by apoE. CER increased the amount of apoE bound to emulsion particles. The confocal images of CER-containing large emulsions with two fluorescent probes showed three-dimensional microdomains enriched in CER. SMase also induced the formation of CER-enriched domains. We propose apoE prefers to bind on CER-enriched domains exposed on particle surface, and thus inhibits the aggregation or fusion of the particles.     

Ceramide and Its Interconvertible Metabolite Sphingosine Function as Indispensable Lipid Factors Involved in Survival and Dendritic Differentiation of Cerebellar Purkinje Cells

Abstract: Ceramide generated from sphingomyelin has emerged as a new but conserved type of biologically active lipid. We previously found that endogenous sphingolipids are required for the normal growth of cultured cerebellar Purkinje neurons and that sphingomyelin is present abundantly in the somatodendritic region of these cells. To gain further insight into a potential role of the sphingomyelin/ceramide pathway, we investigated the effects of depletion of sphingolipids on the phenotypic growth and survival of immature Purkinje cells and the ability of ceramide or other sphingolipids to antagonize these effects. Inhibition of ceramide synthesis by ISP-1, a specific inhibitor of serine palmitoyltransferase, decreased cellular levels of sphingolipids. This treatment resulted in a decrease in cell survival accompanied by an induction of apoptotic cell death and aberrant dendritic differentiation of Purkinje cells with no detectable changes in other cerebellar neurons. Cell-permeable ceramides, sphingosine, or sphingomyelin overcame these abnormalities more effectively than other sphingolipids when added simultaneously with ISP-1. Exposure to bacterial sphingomyelinase in turn enhanced cell survival and dendritic branching complexity of Purkinje cells at different optimal concentrations. Furthermore, cell-permeable ceramide acted synergistically with the neurotrophin family, which has been previously shown to support Purkinje cell survival. These observations suggest that ceramide is a requisite for the survival and the dendritic differentiation of Purkinje cells.     

The C-terminal lipid-binding domain of apolipoprotein E is a highly efficient mediator of ABCA1-dependent cholesterol efflux that promotes the assembly of high-density lipoproteins

This study was undertaken to identify the alpha-helical domains of human apoE that mediate cellular cholesterol efflux and HDL assembly via ATP-binding cassette transporter A1 (ABCA1). The C-terminal (CT) domain (residues 222-299) of apoE was found to stimulate ABCA1-dependent cholesterol efflux in a manner similar to that of intact apoE2, -E3, and -E4 in studies using J774 macrophages and HeLa cells. The N-terminal (NT) four-helix bundle domain (residues 1-191) was a relatively poor mediator of cholesterol efflux. On a per molecule basis, the CT domain stimulated cholesterol efflux with the same efficiency (Km approximately 0.2 microM) as intact apoA-I and apoE. Gel filtration chromatography of conditioned medium from ABCA1-expressing J774 cells revealed that, like the intact apoE isoforms, the CT domain promoted the assembly of HDL particles with diameters of 8 and 13 nm. Removal of the CT domain abolished the formation of HDL-sized particles, and only larger particles eluting in the void volume were formed. Studies with CT truncation mutants of apoE3 and peptides indicated that hydrophobic helical segments governed the efficiency of cellular cholesterol efflux and that conjoined class A and G amphipathic alpha-helices were required for optimal efflux activity. Collectively, the data suggest that the CT lipid-binding domain of apoE encompassing amino acids 222-299 is necessary and sufficient for mediating ABCA1 lipid efflux and HDL particle assembly.     

Entamoeba invadens: Sphingolipids metabolic regulation is the main component of a PKC signaling pathway in controlling cell growth and proliferation

The sphingolipids biosynthesis pathway generates bioactive molecules crucial to the regulation of physiological processes. We have recently reported that DAG (diacylglycerol) generated during sphingomyelin synthesis, plays an important role in PKC (protein kinase C) activation, necessary for the transit through the cell cycle (G1 to S transition) and cell proliferation (Cerbon and Lopez-Sanchez, 2003. Diacylglycerol generated during sphingomyelin synthesis is involved in protein kinase C activation and cell proliferation in Madin-Darby canine kidney cells. Biochem. J. 373, 917-924). Since pathogenic Entamoeba invadens synthesize the sphingolipids inositol-phosphate ceramide (IPC) and ethanolamine-phosphate ceramide (EPC) as well as sphingomyelin (SM), we decided to investigate when during growth initiation, the synthesis of sphingolipids takes place, DAG is generated and PKC is activated. We found that during the first 6 h of incubation there was a significant increase in the synthesis of all three sphingolipids, accompanied by a progressive increment (up to 4-fold) in the level of DAG, and particulate PKC activity was increased 4-8 times. The enhanced DAG levels coincided with decrements in the levels of sphingoid bases, conditions adequate for the activation of PKC. Moreover, we found that inhibition of sphingolipid synthesis with myriocin, specific inhibitor of the synthesis of sphinganine, reduce DAG generation, PKC activation and cell proliferation. All these inhibitory processes were restored by metabolic complementation with exogenous d-erythrosphingosine, indicating that the DAG generated during sphingolipid synthesis was necessary for PKC activation and cell proliferation. Also, we show that PI (phosphatidylinositol), PE (phosphatidylethanolamine) and PC (phosphatidylcholine) are the precursors of their respective sphingolipids (IPC, EPC and SM), and therefore sources of DAG to activate PKC.     

Modulation of THP-1 macrophage and cholesterol-loaded foam cell apolipoprotein E levels by glycosphingolipids

Macrophages synthesize and secrete apolipoprotein E (apoE) constitutively. This process is upregulated under conditions of cholesterol loading. The response to cholesterol is antiatherogenic as it is believed to promote cholesterol efflux from the artery wall. The concentration of lactosyl ceramide (LacCer), a glycosphingolipid recently discovered to regulate cellular signaling, proliferation, and expression of adhesion molecules, is also increased in atherosclerotic tissues. Here we have investigated the effect of exogenous LacCer on macrophage apoE levels. We show that increasing macrophage LacCer levels sevenfold led to reductions in cellular and secreted apoE (15 and 30%, respectively, over a 24-h period) as determined by enzyme-linked immunosorbent assay. A similar effect was also induced by glucosyl ceramide (GlcCer) but not by ganglioside species. When macrophages were converted to cholesterol-loaded foam cells by incubation with acetylated LDL, the resulting increase in cellular apoE levels was inhibited by 26% when the cells were subsequently enriched with LacCer. After metabolic labeling of cellular glycosphingolipids with [14C]palmitate, we also discovered that high-density lipoprotein (HDL) stimulates the efflux of glycosphingolipids from foam cells. These data imply that LacCer and GlcCer may be proatherogenic due to the suppression of macrophage apoE production. Furthermore, the efflux of glycosphingolipids from macrophage foam cells to HDL could indicate a potential pathway for their removal from the artery wall and subsequent delivery to the liver.     

Sphingolipid Turnover in the Hippocampus and Cognitive Dysfunction in Alcoholized Rats: Correction with the Help of Alimentary <Emphasis Type="Italic">n</Emphasis>-3 Fatty Acids

We studied the effect of long-lasting (during 60 days) consumption of 15% ethanol solution by 2- to 4-month-old rats on the turnover of sphingolipids in the hippocampus and cognitive functions in these animals. It was found that chronic action of ethanol led to a decrease in the content of newly synthesized sphingomyelin and to an increase in the level of ceramide and the ceramide/sphingomyelin ratio in the hippocampus; this was accompanied by significant worsening of conditioned-reflex activity in experimental rats (testing of active avoidance in a shuttle chamber). The addition of fish oil containing a considerable amount of n-3 fatty acids to the diet of animals consuming ethanol prevented, to a considerable extent, the development of disorders of sphingolipid turnover and cognitive functions.     

Apolipoprotein-mediated cellular lipid release requires replenishment of sphingomyelin in a phosphatidylcholine-specific phospholipase C-dependent manner

When sphingomyelin is digested by sphingomyelinase in the plasma membrane of rat astrocytes, productions of sphingomyelin, diacylglycerol, and phosphatidylcholine are stimulated. D609, an inhibitor of phosphatidylcholine-specific phospholipase C, suppressed these effects. Similarly, when apolipoprotein A-I removed cellular cholesterol, phosphatidylcholine, and sphingomyelin to generate high density lipoprotein, cholesterol synthesis from acetate subsequently increased, and sphingomyelin synthesis from acetate and serine also increased. D609 inhibited these effects again. D609 also inhibited the cholesterol removal by apoA-I not only from the astrocytes but also from BALB/3T3 and RAW264 cells. D609 decreased cholesterol synthesis, although D609 did not directly inhibit hydroxymethylglutaryl-CoA reductase. ApoA-I-stimulated translocation of newly synthesized cholesterol to cytosol was also decreased by D609. A diacylglycerol analog increased the apoA-I-mediated cholesterol release, whereas ceramide did not influence it. We concluded that removal of cellular sphingomyelin by apolipoproteins is replenished by transfer of phosphorylcholine from phosphatidylcholine to ceramide, and this reaction may limit the removal of cholesterol by apoA-I. This reaction also produces diacylglycerol that potentially triggers subsequent cellular signal cascades and regulates intracellular cholesterol trafficking.