Involvement of sphingomyelinases in TNF signaling pathways

Sphingomyelin (N-acylsphingosin-1-phosphorylcholine) is a phospholipid preferentially found in the plasma membrane of mammalian cells. Signaling through the sphingomyelin pathway is associated with generation of ceramide, which acts as a second messenger in activating a variety of cellular functions. Ceramide belongs to the group of sphingosine-based lipid second messenger molecules that are critically involved in the regulation of signal transduction of diverse cell surface membrane receptors. The emerging picture suggests that coupling of ceramide to specific signaling cascades is both stimulus- and cell type-specific and depends on the subcellular topology of its production. Following membrane receptor triggering, neutral and acid isoforms of sphingomyelinases are rapidly activated generating ceramide through sphingomyelin hydrolysis. Here the molecular mechanisms of TNF-induced activation of sphingomyelinases and the functional consequences of ceramide generation will be discussed.     

The Gbetagamma dimer drives the interaction of heterotrimeric Gi proteins with nonlamellar membrane structures

Heterotrimeric G proteins are peripheral membrane proteins that propagate signals from membrane receptors to regulatory proteins localized in distinct cellular compartments. To facilitate signal amplification, G proteins are in molar excess with respect to G protein-coupled receptors. Because G proteins are capable of translocating from membrane to cytosol, protein-lipid interactions play a crucial role in signal transduction. Here, we studied the binding of heterotrimeric G proteins (Galphabetagamma) to model membranes (liposomes) and that of the entities formed upon receptor-mediated activation (Galpha and Gbetagamma). The model membranes used were composed of defined membrane lipids capable of organizing into either lamellar or nonlamellar (hexagonal H(II)) membrane structures. We demonstrated that although heterotrimeric G(i) proteins and Gbetagamma dimers can bind to lipid bilayers of phosphatidylcholine, their binding to membranes was markedly and significantly enhanced by the presence of nonlamellar phases of phosphatidylethanolamine. Conversely, activated G protein alpha subunits showed an opposite membrane binding behavior with a marked preference for lamellar membranes. These results have important consequences in cell signaling. First, the binding characteristics of the Gbetagamma dimer account for the lipid binding behavior and the cellular localization of heterotrimeric G proteins. Second, the distinct protein-lipid interactions of heterotrimeric G proteins, Gbetagamma dimers, and Galpha subunits with membrane lipids explain, in part, their different cellular mobilizations during signaling upon receptor activation. Finally, their differential interactions with lipids suggest an active role of the membrane lipid secondary structure in the propagation of signals through G protein-coupled receptors.     

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.     

Ras signaling from plasma membrane and endomembrane microdomains

Ras proteins are compartmentalized by dynamic interactions with both plasma membrane microdomains and intracellular membranes. The mechanisms underlying Ras compartmentalization involve a series of protein/lipid, lipid/lipid and cytoskeleton interactions, resulting in the generation of discrete microdomains from which Ras operates. Segregation of Ras proteins to these different platforms regulates the formation of Ras signaling complexes and the generation of discrete signal outputs. This temporal and spatial modulation of Ras signal transduction provides a mechanism for the generation of different biological outcomes from different Ras isoforms, as well as flexibility in the signal output from a single activated isoform.     

Role of sphingomyelinase and ceramide in modulating rafts: do biophysical properties determine biologic outcome?

Recent biophysical data suggest that the properties of ceramide observed in model membranes may apply to biological systems. In particular, the ability of ceramide to form microdomains, which coalesce into larger platforms or macrodomains, appears to be important for some cellular signaling processes. Several laboratories have now demonstrated similar reorganization of plasma membrane sphingolipid rafts, via ceramide generation, into macrodomains. This event appeared necessary for signaling upon activation of a specific set of cell surface receptors. In this article, we review the properties and functions of rafts, and the role of sphingomyelinase and ceramide in the biogenesis and re-modeling of these rafts. As clustering of some cell surface receptors in these domains may be critical for signal transduction, we propose a new model for transmembrane signal transmission.     

Ceramide-enriched membrane domains--structure and function

Membrane lipids seem to be organized and not randomly distributed in the cell membrane. In particular, sphingolipids seem to interact with cholesterol in the outer leaflet of the cell membrane resulting in the formation of distinct membrane domains, i.e. rafts. The generation of ceramide within rafts alters their biophysical properties and results in the formation of large ceramide-enriched membrane platforms. These platforms serve to cluster receptor molecules and to organize intracellular signalling molecules to facilitate signal transduction via a receptor upon stimulation. Thus, ceramide-enriched membrane domains amplify not only receptor-, but also stress-mediated signalling events. Although many receptors cluster, the molecular mechanisms mediating this important and general event in signal transduction need to be identified.     

Ceramide-enriched membrane domains—Structure and function

Membrane lipids seem to be organized and not randomly distributed in the cell membrane. In particular, sphingolipids seem to interact with cholesterol in the outer leaflet of the cell membrane resulting in the formation of distinct membrane domains, i.e. rafts. The generation of ceramide within rafts alters their biophysical properties and results in the formation of large ceramide-enriched membrane platforms. These platforms serve to cluster receptor molecules and to organize intracellular signalling molecules to facilitate signal transduction via a receptor upon stimulation. Thus, ceramide-enriched membrane domains amplify not only receptor-, but also stress-mediated signalling events. Although many receptors cluster, the molecular mechanisms mediating this important and general event in signal transduction need to be identified.     

Asymmetric addition of ceramides but not dihydroceramides promotes transbilayer (flip-flop) lipid motion in membranes

Transbilayer lipid motion in membranes may be important in certain physiological events, such as ceramide signaling. In this study, the transbilayer redistribution of lipids induced either by ceramide addition or by enzymatic ceramide generation at one side of the membrane has been monitored using pyrene-labeled phospholipid analogs. When added in organic solution to preformed liposomes, egg ceramide induced transbilayer lipid motion in a dose-dependent way. Short-chain (C6 and C2) ceramides were less active than egg ceramide, whereas dihydroceramides or dioleoylglycerol were virtually inactive in promoting flip-flop. The same results (either positive or negative) were obtained when ceramides, dihydroceramides, or diacylglycerols were generated in situ through the action of a sphingomyelinase or of a phospholipase C. The phenomenon was dependent on the bilayer lipid composition, being faster in the presence of lipids that promote inverted phase formation, e.g., phosphatidylethanolamine and cholesterol; and, conversely, slower in the presence of lysophosphatidylcholine, which inhibits inverted phase formation. Transbilayer motion was almost undetectable in bilayers composed of pure phosphatidylcholine or pure sphingomyelin. The use of pyrene-phosphatidylserine allowed detection of flip-flop movement induced by egg ceramide in human red blood cell membranes at a rate comparable to that observed in model membranes. The data suggest that when one membrane leaflet becomes enriched in ceramides, they diffuse toward the other leaflet. This is counterbalanced by lipid movement in the opposite direction, so that net mass transfer between monolayers is avoided. These observations may be relevant to the physiological mechanism of transmembrane signaling via ceramides.     

神经酰胺与细胞凋亡

在多细胞有机体中,凋亡对正常组织的发育和稳态是必需的。神经酰胺（ceramide,Cer）作为一种脂类分子,不仅是细胞膜的重要组成部分,而且是重要的凋亡参与者。在细胞膜上,它能够自发地聚集,形成招募死亡受体和配体的结构域从而促进信号的传导和放大;在细胞质中,它作为信号通路的中介者,通过许多不同的分子机制去调控机体的凋亡通路;在细胞核膜和核内,通过其代谢变化,决定细胞的增殖或凋亡;而且它的浓度和位置以及与其它分子的比例及其相互作用都会影响到细胞和组织的命运。综述了近来关于Cer及其代谢物的凋亡作用。     

Vectorial budding of vesicles by asymmetrical enzymatic formation of ceramide in giant liposomes

Sphingomyelin is an abundant component of eukaryotic membranes. A specific enzyme, sphingomyelinase can convert this lipid to ceramide, a central second messenger in cellular signaling for apoptosis (programmed cell death), differentiation, and senescence. We used microinjection and either Hoffman modulation contrast or fluorescence microscopy of giant liposomes composed of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), N-palmitoyl-sphingomyelin (C16:0-SM), and Bodipy-sphingomyelin as a fluorescent tracer (molar ratio 0.75:0.20:0.05, respectively) to observe changes in lipid lateral distribution and membrane morphology upon formation of ceramide. Notably, in addition to rapid domain formation (capping), vectorial budding of vesicles, i.e., endocytosis and shedding, can be induced by the asymmetrical sphingomyelinase-catalyzed generation of ceramide in either the outer or the inner leaflet, respectively, of giant phosphatidylcholine/sphingomyelin liposomes. These results are readily explained by 1) the lateral phase separation of ceramide enriched domains, 2) the area difference between the adjacent monolayers, 3) the negative spontaneous curvature, and 4) the augmented bending rigidity of the ceramide-containing domains, leading to membrane invagination and vesiculation of the bilayer.     

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.     

Shape transitions and lattice structuring of ceramide-enriched domains generated by sphingomyelinase in lipid monolayers

Sphingomyelinases (SMases) hydrolyze the membrane constituent sphingomyelin (SM) to phosphocholine and ceramide (Cer). Growing evidence supports that SMase-induced SM-->Cer conversion leads to the formation of lateral Cer-enriched domains which drive structural reorganization in lipid membranes. We previously provided visual evidence in real-time for the formation of Cer-enriched domains in SM monolayers through the action of the neutral Bacillus cereus SMase. In this work, we disclose a succession of discrete morphologic transitions and lateral organization of Cer-enriched domains that underlay the SMase-generated surface topography. We further reveal how these structural parameters couple to the generation of two-dimensional electrostatic fields, based upon the specific orientation of the lipid dipole moments in the Cer-enriched domains. Advanced image processing routines in combination with time-resolved epifluorescence microscopy on Langmuir monolayers revealed: 1), spontaneous nucleation and circular growth of Cer-enriched domains after injection of SMase into the subphase of the SM monolayer; 2), domain-intrinsic discrete transitions from circular to periodically undulating shapes followed by a second transition toward increasingly branched morphologies; 3), lateral superstructure organization into predominantly hexagonal domain lattices; 4), formation of super-superstructures by the hexagonal lattices; and 5), rotationally and laterally coupled domain movement before domain border contact. All patterns proved to be specific for the SMase-driven system since they could not be observed with Cer-enriched domains generated by defined mixtures of SM/Cer in enzyme-free monolayers at the same surface pressure (pi = 10 mN/m). Following the theories of lateral shape transitions, dipolar electrostatic interactions of lipid domains, and direct determinations of the monolayer dipole potential, our data show that SMase induces a domain-specific packing and orientation of the molecular dipole moments perpendicular to the air/water interface. In consequence, protein-driven generation of specific out-of-equilibrium states, an accepted concept for maintenance of transmembrane lipid asymmetry, must also be considered on the lateral level. Lateral enzyme-specific out-of-equilibrium organization of lipid domains represents a new level of signal transduction from local (nm) to long-range (microm) scales. The cross-talk between lateral domain structures and dipolar electrostatic fields adds new perspectives to the mechanisms of SMase-mediated signal transduction in biological membranes.     

Gangliosides as components of lipid membrane domains

Cell membrane components are organized as specialized domains involved in membrane-associated events such as cell signaling, cell adhesion, and protein sorting. These membrane domains are enriched in sphingolipids and cholesterol but display a low protein content. Theoretical considerations and experimental data suggest that some properties of gangliosides play an important role in the formation and stabilization of specific cell lipid membrane domains. Gangliosides are glycolipids with strong amphiphilic character and are particularly abundant in the plasma membranes, where they are inserted into the external leaflet with the hydrophobic ceramide moiety and with the oligosaccharide chain protruding into the extracellular medium. The geometry of the monomer inserted into the membrane, largely determined by the very large surface area occupied by the oligosaccharide chain, the ability of the ceramide amide linkage to form a network of hydrogen bonds at the water-lipid interface of cell membranes, the Delta(4) double bond of sphingosine proximal to the water-lipid interface, the capability of the oligosaccharide chain to interact with water, and the absence of double bonds into the double-tailed hydrophobic moiety are the ganglioside features that will be discussed in this review, to show how gangliosides are responsible for the formation of cell lipid membrane domains characterized by a strong positive curvature.     

Sphingolipid signalling domains floating on rafts or buried in caves?

Ceramide is a novel lipid mediator involved in regulating cell growth, cell differentiation and cell death. Many studies have focused on characterizing the stimulus-induced production of ceramide and identifying putative downstream molecular targets. However, little remains known about the localization of the regulated production of ceramide through sphingomyelin metabolism in the plasma membrane. Additionally, it is unclear whether a localized increase in ceramide concentration is necessary to facilitate downstream signalling events initiated by this lipid. Recent studies have suggested that detergent-insoluble plasma membrane domains may be highly localized sites for initiating signal transduction cascades by both tyrosine kinase and sphingolipid signalling pathways. These domains are typically enriched in both sphingolipids and cholesterol and have been proposed to form highly ordered lipid rafts floating in a sea of glycerophospholipids. Alternatively, upon integration of the cholesterol binding protein caveolin, these domains may also form small cave-like structures called caveolae. Emerging evidence suggests that the enhanced sphingomyelin content of these lipid domains make them potential substrate pools for sphingomyelinases to produce a high local concentration of ceramide. The subsequent formation of ceramide microdomains in the plasma membrane may be a critical factor in regulating downstream signalling through this lipid messenger.     

Acid sphingomyelinase amplifies redox signaling in Pseudomonas aeruginosa-induced macrophage apoptosis

Recent studies indicate that distinct membrane microdomains, also named lipid rafts, and ceramide play an important role in infectious biology. Ceramide forms larger ceramide-enriched membrane platforms that are required for diverse signal transduction. In this study, we demonstrate that ceramide-enriched membrane platforms are critically involved in redox signaling that regulates alveolar macrophage apoptosis upon infection with Pseudomonas aeruginosa. In freshly isolated alveolar macrophages, P. aeruginosa infection results in rapid activation of acid sphingomyelinase (Asm), release of ceramide, and formation of ceramide-enriched membrane platforms, which are required for P. aeruginosa-induced activation of NADPH oxidase and production of reactive oxygen species (ROS). Inhibition of NADPH oxidase or removal of intracellular ROS reduced P. aeruginosa-induced activation of the Asm and formation of ceramide-enriched membrane platforms, suggesting that NADPH oxidase-derived ROS regulate Asm-initiated redox signaling in a positive feedback manner. Furthermore, stimulation of JNK and induction of apoptosis upon P. aeruginosa infections are dependent on NADPH oxidase-derived ROS. These findings indicate that ceramide-enriched membrane platforms are essential for amplification of Asm-mediated redox signaling, which mediates JNK activation and thereby apoptosis of alveolar macrophages upon P. aeruginosa infection.     

Lipid second messengers and cell signaling in vascular wall

Agonists of cellular receptors, such as receptor tyrosine kinases, G protein-coupled receptors, cytokine receptors, etc., activate phospholipases (C(gamma), C(beta), A(2), D), sphingomyelinase, and phosphatidylinositol-3-kinase. This produces active lipid metabolites, some of which are second messengers: inositol trisphosphate, diacylglycerides, ceramide, and phosphatidylinositol 3,4,5-trisphosphate. These universal mechanisms are involved in signal transduction to maintain blood vessel functions: regulation of vasodilation and vasoconstriction, mechanical stress resistance, and anticoagulant properties of the vessel lumen surface. Different signaling pathways realized through lipid second messengers interact to one another and modulate intracellular events. In early stages of atherogenesis, namely, accumulation of low density lipoproteins in the vascular wall, cascades of pro-atherogenic signal transduction are triggered through lipid second messengers. This leads to atherosclerosis, the general immuno-inflammatory disease of the vascular system.     

Hyperresponsiveness of mice deficient in plasma-secreted sphingomyelinase reveals its pivotal role in early phase of host response

Plasma secretion of acid sphingomyelinase is a hallmark of cellular stress response resulting in the formation of membrane embedded ceramide-enriched lipid rafts and the reorganization of receptor complexes. Consistently, decompartmentalization of ceramide formation from inert sphingomyelin has been associated with signaling events and regulation of the cellular phenotype. Herein, we addressed the question of whether the secretion of acid sphingomyelinase is involved in host response during sepsis. We found an exaggerated clinical course in mice genetically deficient in acid sphingomyelinase characterized by an increased bacterial burden, an increased phagocytotic activity, and a more pronounced cytokine storm. Moreover, on a functional level, leukocyte-endothelial interaction was found diminished in sphingomyelinase-deficient animals corresponding to a distinct leukocytes’ phenotype with respect to rolling and sticking as well as expression of cellular surface proteins. We conclude that hydrolysis of membrane-embedded sphingomyelin, triggered by circulating sphingomyelinase, plays a pivotal role in the first line of defense against invading microorganisms. This function might be essential during the early phase of infection leading to an adaptive response of remote cells and tissues.     

Increased Association of Dynamin Ⅱ with Myosin Ⅱ in Ras Transformed NIH3T3 Cells

Dynamin has been implicated in the formation of nascent vesicles through both endocytic and secretory pathways. However, dynamin has recently been implicated in altering the cell membrane shape during cell migration associated with cytoskeleton-related proteins. Myosin Ⅱ has been implicated in maintaining cell morphology and in cellular movement. Therefore, reciprocal immunoprecipitation was carried out to identify the potential relationship between dynamin Ⅱ and myosin Ⅱ. The dynamin Ⅱ expression level was higher when co-expressed with myosin Ⅱ in Ras transformed NIH3T3 cells than in normal NIH3T3 cells. Confocal microscopy also confirmed the interaction between these two proteins. Interestingly, exposing the NIH3T3 cells to platelet-derived growth factor altered the interaction and localization of these two proteins. The platelet-derived growth factor treatment induced lamellipodia and cell migration, and dynamin Ⅱ inter- acted with myosin Ⅱ. Grb2, a 24 kDa adaptor protein and an essential element of the Ras signaling pathway, was found to be associated with dynamin Ⅱ and myosin Ⅱ gene expression in the Ras transformed NIH3T3 cells. These results suggest that dynamin Ⅱ acts as an intermediate messenger in the Ras signal transduction pathway leading to membrane ruffling and cell migration.     

Exchange of C(16)-ceramide between phospholipid vesicles.

Ceramide is considered to be an important signaling molecule in cellular processes such as cell growth, secretion, differentiation, and apoptosis. This implies that the molecule is able to move between cellular membranes. However, the ability of the molecule to undergo such exchange has been largely ignored despite the profound impact that this ability would have on its mechanism of action in signal-transduction cascades. With this in mind, the ability of a long-chain, radioactive ceramide, (14)C-C(16)-ceramide, to exchange between populations of lipid vesicles was evaluated. The rate of exchange of (14)C-C(16)-ceramide between lipid vesicles at lipid concentrations commonly found in cells (10-110 mM) was on the order of days (t(1/2) of 45-109 h). Simultaneous observations revealed negligible exchange of (3)H-cholesteryl oleate, which was included as a nontransferable marker to control for artifacts such as vesicle fusion and aggregation. In addition, all of the ceramide was exchangeable, and the exchange followed monoexponential kinetics, indicating that the ceramide underwent transbilayer movement at a rate faster than or equal to its rate of intervesicle exchange. Two conclusions can be drawn from these observations: (i) the spontaneous transfer of ceramide between cellular membranes is too slow to play a role in rapid, inter-membrane signaling phenomena and can only be a factor in cell functions that take place over days; and (ii) without the aid of an exchange protein, ceramide can only interact with target molecules that are located at the membrane where the ceramide is formed.