The role of sphingomyelin and sphingomyelin synthases in cell death,proliferation and migration—from cell and animal models to human disorders

Sphingomyelin constitutes membrane microdomains such as lipid raft, caveolae, and clathrin-coated pits and implicates in the regulation of trans-membrane signaling. On the other hand, sphingomyelin emerges as an important molecule to generate bioactive sphingolipids through ceramide. Sphingomyelin synthase is an enzyme that generates sphingomyelin and diacylglycerol from phosphatidylcholine and ceramide. Although ceramide has a well-known role as a lipid mediator to regulate cell death and survival, the only known biological role of sphingomyelin regulated by sphingomyelin synthases was limited to being a source of bioactive lipids. Here, we describe the basic characters of sphingomyelin synthases and discuss additional roles for sphingomyelin and sphingomyelin synthase in biological functions including cell migration, apoptosis, autophagy, and cell survival/proliferation as well as in human disorders such as cancer and cardiovascular disorders. It is expected that a better understanding of the role of sphingomyelin regulated by sphingomyelin synthase will shed light on new mechanisms in cell biology, physiology and pathology. In the future, novel therapeutic procedures for currently incurable diseases could be developed through modifying the function of not only sphingolipids, such as sphingomyelin and ceramide, but also of their regulatory enzymes. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.     

Lipidomic analysis of Toxoplasma gondii reveals unusual polar lipids

Analysis of the polar lipids of Toxoplasma gondii by electrospray ionization tandem mass spectrometry provides a detailed picture of the lipid molecular species of this parasitic protozoan. Most notably, T. gondii contains a relatively high level, estimated to about 2% of the total polar lipid, of ceramide phosphoethanolamine. The ceramide phosphoethanolamine has a fatty amide profile with only 16- and 18-carbon species. Compared with the host fibroblasts in which it was grown, T. gondii also has higher levels of phosphatidylcholine but lower levels of sphingomyelin and phosphatidylserine. Analysis at the molecular species level indicated that T. gondii has greater amounts of shorter-chain fatty acid in its polar lipid molecular species than the host fibroblasts. Shorter-chain fatty acids with a combined total of 30 or fewer acyl carbons make up 21% of Toxoplasma's, but only 3% of the host's, diacyl phosphatidylcholine. Furthermore, diacyl phosphatidylcholine with two saturated acyl chains with 12, 14, or 16 carbons make up over 11% of parasite phosphatidylcholine but less than 3% of the host phosphatidylcholine molecular species. The distinctive T. gondii tachyzoite lipid profile may be particularly suited to the function of parasitic membranes and the interaction of the parasite with the host cell and the host's immune system. Combined with T. gondii genomic data, these lipidomic data will assist in elucidation of metabolic pathways for lipid biosynthesis in this important human pathogen.     

Effect of Ceramide on Nonraft Proteins

The currently accepted model of biological membranes involves a heterogeneous, highly dynamic organization, where certain lipids and proteins associate to form cooperative platforms (“rafts”) for cellular signaling or transport processes. Ceramides, a lipid species occurring under conditions of cellular stress and apoptosis, are considered to stabilize these platforms, thus modulating cellular function. The present study focuses on a previously unrecognized effect of ceramide generation. In agreement with previous studies, we find that ceramide leads to a depletion of sphingomyelin from mixtures with palmitoyl oleoyl phosphatidylcholine bilayers, forming a ceramide–sphingomyelin-rich gel phase that coexists with a fluid phase rich in palmitoyl oleoyl phosphatidylcholine. Interestingly, however, this latter phase has an almost fourfold smaller bending rigidity compared to a sphingomyelin–palmitoyl oleoyl phosphatidylcholine mixture lacking ceramide. The significant change of membrane bulk properties can have severe consequences for conformational equilibria of membrane proteins. We discuss these effects in terms of the lateral pressure profile concept for a simple geometric model of an ion channel and find a significant inhibition of its activity.     

Implication of Sphingomyelin/Ceramide Molar Ratio on the Biological Activity of Sphingomyelinase

Sphingolipid signaling plays an important, yet not fully understood, role in diverse aspects of cellular life. Sphingomyelinase is a major enzyme in these signaling pathways, catalyzing hydrolysis of sphingomyelin to ceramide and phosphocholine. To address the related membrane dynamical structural changes and their feedback to enzyme activity, we have studied the effect of enzymatically generated ceramide in situ on the properties of a well-defined lipid model system. We found a gel-phase formation that was about four times faster than ceramide generation due to ceramide-sphingomyelin pairing. The gel-phase formation slowed down when the ceramide molar ratios exceeded those of sphingomyelin and stopped just at the solubility limit of ceramide, due to unfavorable pairwise interactions of ceramide with itself and with monounsaturated phosphatidylcholine. A remarkable correlation to in vitro experiments suggests a regulation of sphingomyelinase activity based on the sphingomyelin/ceramide molar ratio.     

Changes in the balance between mitogenic and antimitogenic lipid second messengers during proliferation, cell arrest, and apoptosis in T-lymphocytes.

Control of lymphocyte cell survival and proliferation is critical for both the immune response and for the prevention of autoimmune and infectious diseases. The actions of interleukin-2, the major T-cell regulatory cytokine, are mediated by the complex network of divergent signalling pathways controlled by its high-affinity receptor. Various studies have indicated that the generation of certain lipid second messengers is an important mechanism in the control of proliferation and cell death. We have examined the relationship between diacylglycerol and ceramide and the levels of the lipids phosphatidylcholine and sphingomyelin, their potential precursors, in the human T-cell line Kit 225 cultured in three distinct conditions to favor apoptosis, cell arrest, and proliferation. Our data show that, in proliferating cells, the ratios of diacylglycerol/ceramide and phosphatidylcholine/sphingomyelin are higher than those found in arrested cells and increase with time in culture. These ratios are rapidly reversed in apoptotic cells. Further experiments reveal that de novo synthesis of both diacylglycerol and phosphatidylcholine is greatest in proliferating cells, whereas sphingomyelin synthase activity is increased in cells undergoing apoptosis. In summary, our results demonstrate for the first time that the ratio of mitogenic/antimitogenic lipids changes dramatically during T-cell proliferation and cell death. These results indicate that lipid second messengers and the enzymes that are responsible for their generation may provide targets for novel therapeutic interventions in the clinical management of immunosuppression and autoimmune disease.     

Uptake and intracellular transport pathways of fluorescent lipid analogues inAmoeba proteus (Rhizopoda: Amoebida)

Summary The uptake and intracellular transport of 5 different lipid analogues derived from phosphatidylethanolamine, phosphatidylcholine, sphingomyelin, ceramide, and cholesterol have been studied in livingAmoeba proteus using fluorescence microscopy. Phosphatidylcholine and sphingomyelin are predominantly transported from the external environment to the cell interior in a manner consistent with induced macropinocytosis. On the other hand, phosphatidylethanolamine, ceramide, and cholesterol mainly enter the cellular matrix by carrier-mediated, ATP-dependent transmembrane transport. In general, all lipid analogues are first imported to a large pool of endosomal or lysosomal vacuoles, and then partitioned to numerous tiny cisternal elements; only sphingomyelin remains in the lysosomes and is not exported to other membrane compartments. The ultrastructural localization of ceramide indicates that the cisternal elements result from the decay of the Golgi apparatus into single cisternae during lipid accumulation. As a whole, the transport pathway of lipid analogues inA. proteus from the cell surface to different cell organelles shows many similarities to respective processes in a variety of metazoan cells.Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Characterization and comparison of lipids in different squid nervous tissues

We have studied the lipid composition of brain (optic and cerebral lobes), stellate ganglia and fin nerves of the squid. Cholesterol, phosphatidylethanolamine and phosphatidylcholine were the major lipids in these nervous tissues. Phosphatidylethanolamine contained about 3% of its amount in [corrected] plasmalogen form. Phosphatidylserine and -inositol, sphingomyelin and ceramide 2-aminoethylphosphonate were also present in significant amounts. In addition, cardiolipin and free fatty acids were detected in brain (each 2-3% of total lipids) and stellate ganglia (about 1% each), but not in fin nerves. Phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol from brain contained large amounts of polyunsaturated fatty acids, namely 20:4, 20:5 and 22:6 in the n-3 family. On the other hand, phosphatidylcholine, cardiolipin, and sphingomyelin, and ceramide 2-aminoethylphosphonate contained only saturated or monounsaturated C16-C18 fatty acids. The aldehyde moieties of ethanolamine plasmalogen were also C16-C18 saturated or monounsaturated. These lipid compositions are compared with those in other invertebrate nervous systems.     

Plasma lipid profiling in a large population-based cohort

We have performed plasma lipid profiling using liquid chromatography electrospray ionization tandem mass spectrometry on a population cohort of more than 1,000 individuals. From 10 μl of plasma we were able to acquire comparative measures of 312 lipids across 23 lipid classes and subclasses including sphingolipids, phospholipids, glycerolipids, and cholesterol esters (CEs) in 20 min. Using linear and logistic regression, we identified statistically significant associations of lipid classes, subclasses, and individual lipid species with anthropometric and physiological measures. In addition to the expected associations of CEs and triacylglycerol with age, sex, and body mass index (BMI), ceramide was significantly higher in males and was independently associated with age and BMI. Associations were also observed for sphingomyelin with age but this lipid subclass was lower in males. Lysophospholipids were associated with age and higher in males, but showed a strong negative association with BMI. Many of these lipids have previously been associated with chronic diseases including cardiovascular disease and may mediate the interactions of age, sex, and obesity with disease risk.     

The role of endogenous phosphatidylcholine and ceramide in the biosynthesis of sphingomyelin in mouse fibroblasts

The intracellular location of sphingomyelin formation via the cholinephosphotransferase reaction from both endogenous an exogenous phosphatidylcholine and ceramide substrates has been studied in the subcellular membrane fractions prepared from mouse fibroblasts. The enzyme was found to be located in both the plasma membrane and the Golgi fractions. Activity in the Golgi fraction was stimulated to a greater extent by the addition of exogenous ceramide than was the activity in the plasma membrane fraction. It is concluded that endogenous phosphatidylcholine is available to the cholinephosphotransferase at saturating concentration and, therefore, is not rate-limiting. In contrast, the very low concentration of endogenous ceramide seems to limit the reaction rate, necessitating supplementation with exogenous material Both endogenous substrates are shown to be utilized in an intramembranous rather than an intermembranous reaction. The capacity of the plasma membrane fraction to synthesize sphingomyelin from endogenous phosphatidylcholine and ceramide was found to be sufficiently high to account for the rate of net synthesis of plasma membrane-bound sphingomyelin observed in the logarithmically multiplying cell culture. In contrast, the Golgi fraction displayed only 26% of the expected capacity, but it was stimulated 6-fold by the addition of exogenous ceramide. These results demonstrate that the total cellular sphingomyelin of the mouse fibroblasts can be provided via the cholinephosphotransferase pathways and that the plasma membrane and the Golgi fraction are most probably the intracellular sites of sphingomyelin biosynthesis.     

Cholera toxin B-subunit prevents activation and proliferation of human CD4+ T cells by activation of a neutral sphingomyelinase in lipid rafts

The inhibition of human CD4+ T lymphocyte activation and proliferation by cholera toxin B-subunit (CTB) is a well-established phenomenon; nevertheless, the exact mechanism remained unclear. In the present study, we propose an explanation for the rCTB-induced inhibition of CD4+ T lymphocytes. rCTB specifically binds to GM1, a raft marker, and strongly modifies the lipid composition of rafts. First, rCTB inhibits sphingomyelin synthesis; second, it enhances phosphatidylcholine synthesis; and third, it activates a raft-resident neutral sphingomyelinase resembling to neutral sphingomyelinase type 1, thus generating a transient ceramide production. We demonstrated that these ceramides inhibit protein kinase Calpha phosphorylation and its translocation into the modified lipid rafts. Furthermore, we show that rCTB-induced ceramide production activate NF-kappaB. Combined all together: raft modification in terms of lipids, ceramide production, protein kinase Calpha inhibition, and NF-kappaB activation lead to CD4+ T cell inhibition.     

Different sphingolipids show differential partitioning into sphingolipid/cholesterol-rich domains in lipid bilayers

Two fluorescence-based approaches have been applied to examine the differential partitioning of fluorescent phospho- and sphingolipid molecules into sphingolipid-enriched domains modeling membrane "lipid rafts." Fluorescence-quenching measurements reveal that N-(diphenylhexatrienyl)propionyl- (DPH3:0-)-labeled gluco- and galactocerebroside partition into sphingolipid-enriched domains in sphingolipid/phosphatidylcholine/cholesterol bilayers with substantially higher affinity than do analogous sphingomyelin, ceramide, or phosphatidylcholine molecules. By contrast, the affinity of sphingomyelin and ceramide for such domains is only marginally greater than that of a phosphatidylcholine with similar hydrocarbon chains. By using direct measurements of molecular partitioning between vesicles of different compositions, we show that the relative affinities of different C(6)-NBD- and C(5)-Bodipy-labeled sphingolipids for sphingolipid-enriched domains are quantitatively, and in most circumstances even qualitatively, quite different from those found for species whose N-acyl chains more closely resemble the long saturated chains of cellular sphingolipids. These findings lend support in principle to previous suggestions that differential partitioning of different sphingolipids into "raft" domains could contribute to the differential trafficking of these species in eukaryotic cells. However, our findings also indicate that short-chain sphingolipid probes previously used to examine this phenomenon are in general ill-suited for such applications.     

Functional reconstitution of sphingomyelin synthase in Chinese hamster ovary cell membranes.

Sphingomyelin synthase (phosphatidylcholine:ceramide phosphocholinetransferase) activity in the membranes of Chinese hamster ovary cells was found to be detectable with a fluorescent ceramide analog, containing a short acyl chain, as a substrate. We developed a method for the functional reconstitution of sphingomyelin synthase in detergent-treated membranes. Treatment of membranes with 1.5% octyl glucoside in the absence of exogenous phosphatidylcholine resulted in almost complete loss of sphingomyelin synthase activity, even after removal of the detergent by dialysis. In contrast, membranes treated with the detergent in the presence of exogenous phosphatidylcholine showed partial activity and, after dialysis of this mixture, enzyme activity was restored to almost the same level as the activity in dialyzed intact membranes. The effects of various lipids on enzyme activity in this reconstitution system suggested that L-alpha-phosphatidylcholine was the environmental lipid essential for the functional reconstitution of the enzyme. Furthermore, diacylglycerol was suggested to serve as an inhibitory regulator of sphingomyelin synthesis.     

Epidermal sphingolipids: metabolism, function, and roles in skin disorders

Mammalian epidermis produces and delivers large quantities of glucosylceramide and sphingomyelin precursors to stratum corneum extracellular domains, where they are hydrolyzed to corresponding ceramide species. This cycle of lipid precursor formation and subsequent hydrolysis represents a mechanism that protects the epidermis against potentially harmful effects of ceramide accumulation within nucleated cell layers. Prominent skin disorders, such as psoriasis and atopic dermatitis, have diminished epidermal ceramide levels, reflecting altered sphingolipid metabolism, that may contribute to disease severity/progression. Enzymatic processes in the hydrolysis of glucosylceramide and sphingomyelin, and the roles of sphingolipids in skin diseases, are the focus of this review.     

Intracellular processes associated with glycoprotein transport and processing.

The intracellular transport of mucus glycoprotein precursor (apomucin) from endoplasmic reticulum (ER) to Golgi was quantitated by the immunoprecipitation with 3G12 antimucin monoclonal antibody and by estimation of the apomucin glycosylation using UDP-[3H]galactose. The assembly of the entities carrying apomucin to Golgi was assessed by electron microscopy and by quantitation of the incorporation of [14C]choline, [14C]ethanolamine, and [14C]oleic acid into their lipids. The microscopic image of the isolated transport components revealed a population of 80- to 100-nm vesicles with occasional membranes of the ER used for their synthesis. On the average, the vesicles contained 82 ng apomucin/microgram of protein and 80-90% of the total incorporated lipid precursors. From that, 91% of [14C]choline was detected in phosphatidylcholine, and 9% in phosphatidylethanolamine, lysophosphatidylcholine, and sphingomyelin. With [14C]oleate, 54% of the label was incorporated into ceramide, diglyceride, and phosphatidic acid, 35% to phosphatidylcholine, 7% in phosphatidylethanolamine, and 2% in sphingomyelin. After incubation of the vesicles with Golgi, the apomucin was found glycosylated and the lipids of the transport vesicles incorporated into Golgi membranes. The fusion of the vesicular membranes was accompanied by the synthesis of sphingomyelin. In the Golgi, 39-55% of the radiolabeled phosphatidylcholine of transport vesicles was converted to sphingomyelin. The results indicate that the newly synthesized membranes of apomucin transporting vesicles are enriched in phosphoglycerides and ceramides. Upon fusion with the Golgi, the membranes of the vesicles are replenished with sphingomyelin by exchange reaction between phosphatidylcholine and ceramide.     

The role of sphingomyelin in regulating phase coexistence in complex lipid model membranes: competition between ceramide and cholesterol

The structure, thermotropic phase behavior, dynamic motion and order parameters of bilayer dispersions of egg phosphatidylcholine, egg sphingomyelin, egg ceramide and cholesterol have been determined. The coexistence of gel, liquid-ordered and liquid-disordered structure has been determined by peak fitting analysis of synchrotron X-ray powder patterns. Order parameters and extent of distribution of 16-doxyl-stearic acid spin probe between ordered and disordered environments has been estimated by ESR spectral simulation methods. The presence of ceramide in proportions up to 20 mol% in phosphatidylcholine is characterized by gel-fluid phase coexistence at temperatures up to 46 degrees C depending on the amount of ceramide. Cholesterol tends to destabilize the ceramide-rich domains formed in phosphatidylcholine while sphingomyelin, by formation of stable complexes with ceramide, tends to stabilize these domains. The stability of sphingomyelin-ceramide complexes is evident from the persistence of highly ordered structure probed by ESR spectroscopy and appearance of a sharp wide-angle X-ray reflection at temperatures higher than the gel-fluid transition of ceramide alone in egg phosphatidylcholine bilayers. The competition between ceramide and cholesterol for interaction with sphingomyelin is discussed in terms of control of lipid-mediated signaling pathways by sphingomyelinase and phospholipase A2.     

Phospholipids of Entamoeba invadens.

The major phosphoglycerides present in Entamoeba invadens are phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol. Furthermore, three different sphingolipids could be isolated from the amoeba. In addition to sphingomyelin and a phosphonolipid, ceramide phosphonylethanolamine, a previously unknown sphingolipid was present. This sphingolipid contained a long chain base, inositol, and phosphorus in the ratio of 0.97:0.97: 1.0 and could be identified as ceramide phosphorylinositol. The various individual phospholipids showed different rates of turnover. Phosphatidic acid and phosphatidylinositol had, relative to the other phospholipids, a short half-time of about 12 h. Phosphatidylethanolamine and ceramide phosphorylinositol had a half-time of about 24 and 30 h, respectively. The major phospholipid, phosphatidylcholine and also sphingomyelin and phosphatidylserine showed no turnover. In contrast to the phosphoglycerides, the sphingolipid composition of the amoeba cultivated in different media was rather variable, while the total sphingolipid content remained at 21% of the total amount of phospholipids. The amount of ceramide phosphorylinositol was almost doubled in the cells cultivated on the serum-free medium (T), whereas the amount of sphingomyelin and ceramide phosphonylethanolamine decreased. Evidence is presented that these alterations in the sphingolipid composition of E. invadens are related to the amount of unsaturated fatty acids which were present in the culture medium.     

Association of ethanol with lipid membranes containing cholesterol, sphingomyelin and ganglioside: a titration calorimetry study.

The association of ethanol at physiologically relevant concentrations with lipid bilayers of different lipid composition has been investigated by use of isothermal titration calorimetry (ITC). The liposomes examined were composed of combinations of lipids commonly found in neural cell membranes: dimyristoyl phosphatidylcholine (DMPC), ganglioside (GM(1)), sphingomyelin and cholesterol. The calorimetric results show that the interaction of ethanol with fluid lipid bilayers is endothermic and strongly dependent on the lipid composition of the liposomes. The data have been used to estimate partitioning coefficients for ethanol into the fluid lipid bilayer phase and the results are discussed in terms of the thermodynamics of partitioning. The presence of 10 mol% sphingomyelin or ganglioside in DMPC liposomes enhances the partitioning coefficient by a factor of 3. Correspondingly, cholesterol (30 mol%) reduces the partitioning coefficient by a factor of 3. This connection between lipid composition and partitioning coefficient correlates with in vivo observations. Comparison of the data with the molecular structure of the lipid molecules suggests that ethanol partitioning is highly sensitive to changes in the lipid backbone (glycerol or ceramide) while it appears much less sensitive to the nature of the head group.     

Stable and unstable lipid domains in ceramide-containing membranes

We applied x-ray diffraction, calorimetry, and infrared spectroscopy to lipid mixtures of palmitoyl-oleoyl phosphatidylcholine, sphingomyelin, and ceramide. This combination of experimental techniques allowed us to probe the stability and structural properties of coexisting lipid domains without resorting to any molecular probes. In particular, we found unstable microscopic domains (compositional/phase fluctuations) in the absence of ceramide, and macroscopically separated fluid and gel phases upon addition of ceramide. We also observed phase fluctuations in the presence of ceramide within the broad phase transition regions. We compare our results with fluorescence spectroscopy data and complement the previously reported phase diagram. We also obtained electron paramagnetic resonance data to assess the possible limitations of techniques employing a single label. Our study demonstrates the necessity of applying a combination of experimental techniques to probe local/global structural and fast/slow motional properties in complex lipid mixtures.     

Biosynthesis of sphingomyelin from erythro-ceramides and phosphatidylcholine by a microsomal cholinephosphotransferase

Mouse liver microsomes were shown to be active in the synthesis of sphingomyelin from ceramide and phosphatidylcholine in a reaction independent of CDPcholine. The conversion was not inhibited by calcium chelating reagents, and no evidence for the involvement of phospholipase C activity in the transformation could be adduced. Activity was also demonstrated in monkey liver and heart microsomes. Mouse brain microsomes produced a sphingomyelin analogue, tentatively identified as ceramide phosphorylethanolamine, but not sphingomyelin. Both [14C]ceramide and [G-14]phosphatidylethanolamine were precursors of the brain product, while phosphatidylcholine was inactive. Progress in the partial characterization of the liver enzyme is also described.     

Comparative analysis of lipid composition of normal and acute-phase high density lipoproteins

In the acute-phase response and in diseases with prolonged acute phases, normal HDL (NHDL) is converted into acute-phase HDL (APHDL) and becomes proinflammatory and unable to protect LDL against oxidative modification. Earlier work had demonstrated that these changes are associated with alterations in apolipoprotein composition and enzymatic activity of APHDL, but the effect of the acute-phase condition on the lipid composition of APHDL had remained obscure. The present study shows marked quantitative differences in lipid composition between NHDL and APHDL. Specifically, APHDL contained 25% less total lipid per milligram of protein. Up to 50% of cholesteryl ester in the lipid core of APHDL was replaced by triacylglycerol; however, the total phospholipid/total neutral lipid ratios were the same as in NHDL, both lipoproteins giving similar calculated lipid core radii. Furthermore, the phosphatidylcholine/sphingomyelin ratio in APHDL was nearly double that in NHDL, indicating a relative loss of sphingomyelin. A decrease was also seen in diacyl and alkenylacyl glycerophosphatidylethanolamine as well as in phosphatidylinositol of APHDL when compared with NHDL. APHDL contained proportionally more saturated and less polyunsaturated and isoprostane-containing species of phosphatidylcholine, as well as more saturated than unsaturated cholesteryl esters. APHDL also contained significantly more free fatty acids, lysophosphatidylcholine, and free cholesterol. These changes in the lipid composition of HDL are consistent with the alterations in the apoprotein composition and enzymatic activity of APHDL and indicate proinflammatory and proatherogenic roles for APHDL.