sphingolipids

Kidney International, Vol. 58 (2000), pp. 11–26

PERSPECTIVES IN BASIC SCIENCE

Sphingolipids

JAMES A. SHAYMAN

Nephrology Division, Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, Michigan, USA

Sphingolipids. A significant corpus of work over the last decade cated in many of the most critical and fundamental as-has firmly established an important role for sphingolipids in a pects of cellular biology, including growth, differentia-variety of important biological processes. Such processes in- tion, apoptosis, and oncogenesis. On the other hand,clude signaling events related to cell growth, differentiation,

many investigators have been skeptical that there existsprogrammed cell death, and stress responses. These processesany significant role for sphingolipids in several of thesenot only involve those sphingolipids that accumulate as a result

of a variety of inherited lysosomal storage disorders, but, in critical processes, suggesting that proof of concept re-addition, sphingolipids associated with long-chain base metab- mains to be established.olism. This article reviews the chemical properties, pathways, The kidney has historically served as an importantregulated metabolism, and signaling function of sphingolipids.

tissue for the study of sphingolipids. Many glycosphingo-In addition, the potential roles of sphingolipids in renal-specificlipids are uniquely expressed in renal tissue. Renal epi-processes are considered. While a variety of cellular functions

have been ascribed to sphingolipids, in many cases proof of thelia also serves as a paradigm for the elucidation ofthe concept has yet to be well established. Thus, a number of many of the proposed cellular functions of sphingolipids.critical questions can be posed in interpreting these studies. More recently, sphingolipids have received the attentionSeveral of these questions are considered.

of the renal academic community, serving as the subjectfor a growing number of studies in renal physiology andpathophysiology. It is therefore timely to consider howThe identification of cellular lipids as signaling mole-recent advances in sphingolipid studies may impact oncules was first suggested almost one half of a centuryour understanding of renal physiology, cell biology, andago when it was observed that amylase secretion waspathophysiology.closely related to the phospholipid content of pigeon

I have previously reviewed the renal biochemistry andpancreas [1]. In subsequent years, several cellular phos-signaling function of sphingolipids [2, 3]. In the presentpholipids have been identified as substrates for the gen-review, emerging concepts in these areas are highlighted,eration of signaling molecules, including phophatidylino-and areas in which proof of concept has not been fullysitol 4,5-bisphosphate and phosphatidylcholine. Theestablished are emphasized. However, significant em-identification of sphingomyelin as a substrate for thephasis is also placed on how these concepts may help usgeneration of receptor-elicited signaling molecules is ato better understand those processes that have histori-more recent occurrence. Only since 1989 have investiga-cally been viewed as sphingolipid dependent and thosetors seriously pursued the concept of sphingolipids asprocesses that we may wish to re-evaluate in the contextpotentially important cell-signaling mediators. As a re-of recent advances in sphingolipid biology.sult, the number of published studies in sphingolipid

biology has grown exponentially in the last 10 years.The corpus of work in the cell biology of sphingolipids CHEMICAL PROPERTIES

has, on the one hand, been met with considerable excite-Significant work has been focused on understandingment. Not only ceramide, but a variety of additional

the chemical features of sphingolipids based on the beliefsphingolipids are now argued to have second-messengerthat the functional biochemistry of these compounds canfunction. Additionally, sphingolipids have been impli-be better understood. Sphingolipids are, by definition,those compounds that contain a long-chain sphingoidbase. The most common long-chain bases are C-18 andKey words: lipids, cell signaling, renal physiology, apoptosis, oncogene-

sis, epithelia. C-20 sphingosines [4]. These bases contain a C4-C5 dou-ble bond in the trans D-erythro conformation. C-18 andReceived for publication November 17, 1999C-20 sphinganines (dihydrosphingosines) are the nextand in revised form February 9, 2000

Accepted for publication February 21, 2000 most common long-chain bases. These lack the doublebond. 2000 by the International Society of Nephrology

11

Shayman: Sphingolipids12

When a fatty acid is linked to the amide group of golipids. Factors that further promote clustering includethe content of sialic acid [11], the presence of Ca21 ionsthe long-chain base, the resultant lipid is referred to as

ceramide. The fatty acids vary in composition, but they [12], the ceramide content [13], and proteins such asVIP21 (caveolin) [14]. The theoretical ability of sphingo-are typically long. The most common fatty acids are

palmitic (C16:0) and stearic (C18:0). a-Hydoxylated fatty lipids to create distinct microenvironments within the cellmembrane has led investigators to focus on these sitesacids are often present as well.

The primary alcoholic function at carbon one of cera- as “hot spots” for transmembrane signaling sites, as wellas sites of vesciculation and membrane transport [15].mide is the site of attachment for a variety of additional

moieties. These most commonly include phosphocho- Several acronyms have been applied to these theoreti-cal membrane domains. These include “detergent-resistantline-producing sphingomyelins and saccharides produc-

ing a multitude of glycosphingolipids. When a single mo- membranes,” “detergent insoluble glycolipid-enrichedmembrane domains,” and “lipid rafts” [16]. Small tubo-nosaccharide is present (glucose or galactose), the lipid

is referred to as a cerebroside. There may be as many as vesicular invaginations within the plasma membrane,termed caveolae, are included within detergent-resistant15 to 20 sugars in the oligosaccharide chain. When these

sugars are uncharged, the products are referred to as neu- fractions. In addition, density gradient methods can beused to isolate caveolin-containing membrane fractionstral glycosphingolipids. Acidic glycosphingolipids result

when one or more sialic acids are present (gangliosides) that are enriched in glycosphingolipids [17]. On the otherhand, glycosphingolipid-enriched fractions can be iso-or when a sulfate group is present (sulfatides) [5]. Less

commonly, a phosphate may be placed at the C-1 carbon- lated from cells not containing caveolin or having mor-phologic evidence of caveolae [18].producing ceramide-1-phosphate [6]. Recently, it has been

shown that an additional acyl group may be present at Some investigators have argued, however, that suchdomains are unlikely to be present. The interactionsthe C-1 position as well producing 1-O-acylceramide [7].

The ceramide portion of a sphingolipid is highly hy- required to form lateral domains between lipid speciesdrophobic. When a polar head group is present, such as are not sufficiently strong or long lived to be biologicallya phosphorylcholine or oligosaccharide, the sphingolipid significant [19]. In addition, the energy required to demixbecomes amphipathic in character. The presence of the lipid species once aggregated into domains is small.phosphorylcholine head group on both phospatidylcho- While microscopy-based experimental evidence exists inline and sphingomyelin would lead one to conjecture support of such lipid rafts, a number of critical questionsthat physicochemical properties are similar because of need to be addressed to provide a more cogent modela similar molecular conformation. However, there are of their biological significance [20]. These questions in-pronounced differences between the two lipids. First, clude a determination of the size, composition, and dy-differences in the number of methylene groups (para- namics of these structures, as well as the relationship offinnic tail length) are more pronounced between the such rafts to the cytoskeleton and understanding howlong-chain base and fatty acids in ceramide than in the the outer membrane leaflet that is enriched in putativediglyceride function of phosphatidylcholine. These para- raft components (glycosphingolipids, cholesterol, sphin-finnic tails are believed to interdigitate with like mole- gomyelin, and GPI-linked proteins) interacts with thecules on either side of the lipid bilayer [8]. In addition, inner leaflet of the membrane.the interfacial region at the juncture of the lipid bilayerand the polar head group of sphingomyelin is believed

SPHINGOLIPID PATHWAYSto be more polar than the comparable region in phospha-De novo formation of long-chain sphingoid bases be-tidylcholine. This creates more potential sites of interac-

gins with the condensation of palmitoyl-CoA and serine,tion between water and other compartments of the lipidforming 3-ketodihydrosphingosine (Fig. 1) [21]. This pyr-bilayer, including the phosphate, free hydroxyl at C-3,idoxal 59-phosphate–dependent enzyme demonstratesand the trans double bond at C4 and C5 [9]. The conse-distinct specificities for various acyl-CoAs, with palmi-quences of these chemical interactions include a highertoyl-CoA preferred over stearoyl-CoA by a ratio of 5:1melting point, increased microviscosity, enhanced stabil-[22]. This specificity is reflected in the relative abundanceity, and decreased permeability to electrolytes than is ob-of 18 and 20 carbon long-chain bases found in mamma-served with glycerolipids of homologous structures [10].lian sphingolipids. The reduction to sphinganine (dihy-The compositional diversity of membrane lipids isdrosphingosine) is then catalyzed by an oxidoreductase,thought to facilitate a lateral phase separation of differ-which is NADPH dependent [23]. Dihydroceramide (N-ent lipid components. In the case of sphingolipids, theacylsphinganine) is formed by an acylation reaction thatdecrement in membrane fluidity may cause microdo-uses acyl-CoAs as substrates. The enzyme catalyzing themains or clusters of lipids rich in ceramide to form. Thisacylation of sphinganine is acyl-CoA:sphingoid base N-separation may be facilitated by the composition of the

polar head groups on both neutral and acidic glycosphin- acyltransferase. This enzyme is probably present in many

Shayman: Sphingolipids 13

Fig. 1. Long-chain base synthesis resulting inceramide formation.

different forms, differing in substrate preference and enzyme that acylates dihydrosphinganine has not beenestablished. There does not appear to exist alternativetissue distribution. The fatty acyl-CoA preference as

characterized to date is stearoyl-CoA . lignoceryl- routes of de novo sphingosine synthesis other than viathe acylation of sphinganine, unsaturation of dihydrocer-CoA . palmitoyl-CoA . oleyl-CoA [24].

Ceramide is formed through the de novo synthetic amide, and deacylation of ceramide by one of severalceramidases. However, long-chain base acylation may,route by the unsaturation at the 4,5 positions of the

sphingoid amine by another oxidoreductase [25]. This in theory, occur through two alternative routes. First,an acyl-CoA independent, ATP-dependent mechanismreaction is nicotinamide adenine dinucleotide (NAD)

dependent. Ceramide may alternatively be synthesized may occur through the reversal of ceramidase [26]. Sec-ond, platelet-activating factor may serve as the substrateby the acylation of sphingosine by an acyl-CoA:sphin-

goid base N-acyltransferase. Whether this is the same for N-acetylation [27].


Fig. 2. Alternative pathways for ceramide metabolism.

Ceramide is a key intermediate in the formation of gomyelin is thought to synthesized by the transfer ofphosphocholine from phosphatidylcholine to ceramidemost of the biologically important sphingolipids (Fig. 2).

Aside from its deacylation to form free sphingosine, [29]. The responsible enzyme is sphingomyelin synthase.This enzyme has been localized to the cis and medialthese products use the C1 hydroxyl group on ceramide

as the site for additional groups. These products include Golgi membranes [30]. However, a second enzyme withsimilar activity has been detected in the plasma mem-sphingomyelin, cerebrosides and other glycosphingo-

lipids, ceramide-1-phosphate, ceramide-phosphoethano- brane [31]. Because the sphingomyelin synthase pathwaymay regulate levels of the precursor ceramide and of alamine, and 1-O-acylceramide.

Sphingomyelin was originally proposed by Sribney product, diacylglycerol, the regulation of these sphingo-myelin synthases and sites of action may have importantand Kennedy to be formed by the transfer of CDP-

choline to ceramide [28]. This pathway has not been implications for cell-signaling phenomena, as detailedlater in this article.subsequently confirmed. Instead, the majority of sphin-


Two additional pathways for sphingomyelin synthesis kinase phosphorylates the C-1 position of either sphingo-sine or sphinganine. It has been characterized as both amay exist. First, a two-step pathway involving the forma-

tion of ceramide phosphoethanolamine followed by its cytosolic enzyme and as membrane bound.The terminal catabolism of sphingosine involves themethylation has been described [32]. This appears to be

a relatively minor pathway. Second, lyso-sphingomyelin action of sphingosine-1-phosphate lyase, which degradessphingosine-1-phosphate to form ethanolamine phos-(sphingosylphosphorylcholine) may be acylated directly

[33]. Sphingosylphosphorylcholine has been demon- phate and a fatty aldehdye. C-18 sphingosine is degradedto trans-2-hexadecenal, and C-18 sphinganine is de-strated to have cellular actions akin to sphingosine-1-

phosphate [34]. However, there is limited evidence in graded to palmitaldehdye [43]. Alternatively, sphingo-sine-1-phosphate may be dephosphorylated by the cera-support of the existence of this lysolipid in mammalian

cells. Recently, a bacterial N-deacylase with activity mide-1-phosphate phosphatase [44].Another pathway for sphingosine metabolism is N-against sphingomyelin has been reported [35]. A compa-

rable activity in mammalian tissues has not been identi- methylation by sphingosine N-methyltransferase [45].Both monomethyl and dimethyl sphingosines may befied.

Glucosylceramide is formed from ceramide and UDP- formed by this pathway.glucose by UDP-glucose:ceramide glycosyltransferase[36]. Glucosylceramide is the precursor for most of the

REGULATION OF METABOLISMmammalian glycosphingolipids. The reaction occurs on

The subcellular sites of metabolism and the subcellularthe cytoplasmic side of the cis Golgi [37]. Subsequentdistribution of sphingolipids are important determinantsglycosylation steps occur within the luminal side of thein understanding the cellular biology of these lipids. Thecis Golgi. How ceramide is translocated from the endo-role of various sphingolipids in cell-signaling events hasplasmic reticulum to the Golgi and how glucosylceramidebeen difficult at times to reconcile with some of theis flipped into the luminal side of the Golgi is unknown.known properties and with the subcellular localization ofGlucosylceramide can then be further glycosylatedthe relevant enzymes. In addition, it is unknown whetherwithin the Golgi or can be directly transported to the plas-many of these enzymes that colocalize to specific cellularma membrane. The latter pathway may occur throughorganelles are present in multienzyme complexes.the action of a glycolipid transfer protein.

In general, all of the enzymes associated with de novoCeramide may also undergo phosphorylation at thelong-chain base synthesis and ceramide formation areC-1 position, forming ceramide-1-phosphate [38]. Thispresent in the smooth endoplasmic reticulum [46]. Fur-pathway is catalyzed by a distinct calcium-dependentthermore, the active sites for these enzymes appear toceramide kinase. Ceramide-1-phosphate may also bebe on the cytosolic face of the endoplasmic reticulum.formed by the action of bacterial diglyceride kinase [39].The localization and site of the dihydroceramide oxido-This latter reaction serves as the basis for the analyticalreductase have not been established. Acyl-CoA:sphin-determination of ceramide levels. To date, ceramide ki-goid base acyltransferase has also been shown to localizenase activity has been identified in synaptic vesicles [6],to mitochondria [47]. Whether this mitochondrial activ-leukemia cells [39], and neutrophils [40].ity may have some role in ceramide-induced apoptosisAnother recently described pathway for ceramide(discussed later in this article) is an intriguing yet unan-involves its acylation at the C-1 position forming 1-O-swered question.acylceramide. The substrate for this acylation is phospha-

Sphingomyelin biosynthesis appears to occur primar-tidylethanolamine or phosphatidylcholine [7]. This reac-ily within the cis and medial Golgi membranes, with thetion is catalyzed by a microsomal phospholipase A2 thatactive site within the luminal leaflet. This implies thattransacylates the fatty acid [41]. The phospholipase isde novo synthesized ceramide must be transported fromunique in that it is calcium independent and has an acidicthe endoplamic reticulum to the Golgi. In addition, therepH optimum.must be a translocation of ceramide across the lipid bi-The main catabolic pathways for sphingolipids in gen-layer. In addition, a similar enzymatic activity has beeneral and for ceramide in particular appear to be throughidentified in the plasma membrane [48]. This latter find-endosomal/lysosomal pathways. Glycosphingolipids areing implies that ceramide formed from sphingomyelindegraded via one or more glycosidases, and ceramide ishydrolysis may be resynthesized at the site of its degrada-degraded by an acidic ceramidase. The latter reactiontion within the plasma membrane without intracellularresults in the formation of sphingosine and a fatty acid.transport to the Golgi.Free sphingosine is released from the lysosome by an

The degradation of sphingomyelin is catalyzed byuncharacterized pathway. Free sphingosine is further ca-a phosphodiesterase-producing phosphorylcholine andtabolized outside of the lysosome. It may be reacylatedceramide. This sphingomyelinase activity is separableforming ceramide or phosphorylated by sphingosine ki-

nase to form sphingosine phosphate [42]. Sphingosine into at least seven distinct activities. These include (1)


an acidic, lysosomal sphingomyelinase [49], (2) a zinc- in both its complexity and, at times, inconsistencies. Insome cases, the implication that sphingolipids such asdependent serum sphingomyelinase [50], (3) a neutral,

magnesium-dependent, membrane associate sphingomy- ceramide may play a primary role in cell signaling hasbeen highly controversial. This has been particularly trueelinase [51], (4) a neutral Mg21-independent sphingo-

myelinase identified in the myelin sheath [52], (5) a for hypotheses stating that ceramide regulates nuclearfactor-kB (NF-kB) signaling and apoptosis [67, 68].magnesium and dithiothreitol-stimulated neutral sphin-

gomyelinase found in the nuclei of ascites cells [53], (6) In general, those agonists that increase cell ceramidelevels cause cell growth arrest, differentiation, or apopto-a neutral sphingomyelinase localized to the chromatin

and nuclear envelop of liver cells [54], and (7) an alkaline sis. These include the cytokines [69], environmentalstressors [70], and pharmacologic agents [71]. The down-sphingomyelinase in the bile and intestine [55].

Many of these sphingomyelinase activities have been stream cellular responses are most often consistent withthese effects and include NF-kB activation [72], cyclo-implicated in cellular sphingolipid-signaling events. Thus,

considerable attention has been focused on the biochem- oxygenase transcription [73], heat-shock protein tran-scription [74], selectin expression [75], and cytokine se-istry of these lipases in recent years. Structurally, the best

studied of these sphingomyelinases is the acid, lysosomal cretion [76]. Conversely, the growth factors are oftenassociated with an increase in sphingosine and sphingo-sphingomyelinase [56]. This lipase is deficient in patients

with Niemann-Pick disease, a lysosomal storage disor- sine-1-phosphate [77]. These opposing activities have ledCuvillier to propose a “yin yang” model of intracellularder. The zinc-dependent serum sphingomyelinase has

been determined to be a product of the same gene [57]. A sphingolipids with ceramide and sphingosine-1-phos-phate playing opposing roles [78]. In this model, a sphin-neutral, magnesium-dependent, glutathione-stimulated

enzyme has recently been sequenced [58]. Whether this golipid “rheostat” may exist within cells, the setting orbalance of which determines whether cells undergo alipase is a signaling sphingomyelinase is uncertain.

Both sphingomyelin and glycosphingolipids are highly proliferative or differentiation of apoptotic response.Four primary observations in support of a role forenriched in the outer leaflet of the plasma membrane.

sphingomyelinase-stimulated ceramide formation as aThe mechanism whereby sphingolipids flip from thetransduction event: First, several extracellular stimuliouter to the inner leaflet is unknown. One hypothesis istrigger ceramide generation and sphingomyelin degrada-that the multidrug resistance protein serves as a mem-tion. Multiple activators have been associated with thebrane “flipase” for placement of glycosphingolipids suchgeneration of cellular sphingolipids. An incomplete list-as glucosylceramide in the outer leaflet [59]. It is note-ing would include cytokines [tumor necrosis factor-aworthy that tumor cells that express high levels of multi-(TNF-a) [79], interleukin (IL)-1b [80], IL-2 [81], anddrug resistance protein are characterized by high con-g-interferon [82]], nerve growth factor [83], hormonestents of glucosylceramide [60].(glucocorticoids [84], progesterone [85], 1,25 (OH)2 vita-min D3 [86], bradykinin [87], retinoic acid [88], endo-

A SIGNALING ROLE FOR SPHINGOLIPIDS thelin 1 [89]), antigens (FAS ligand [90], anti-CD28 [91]),Interest in the role of sphingolipids as signaling mole- environmental stressors (ionizing radiation [92], heat

cules began after the discovery that sphingosine could shock [74], ischemia [93], hypoxia [94], peroxide [70]),act as an inhibitor of protein kinase C [61]. In 1989, pharmacologicals (daunorubicin [95], vincristine [96],Okazaki, Bell, and Hannun reported the first example Ara-C [97], phorbol esters [98], calcium channel blockersof agonist-coupled degradation of sphingomyelin [62]. [99], protein kinase C inhibitors [100]), and infectiveSince that time, the number of published studies on agents (viruses [101], bacteria [102], and bacterial toxinssphingolipid-based signaling has increased exponen- [103]).tially. While the majority of attention has been focused Second, sphingomyelinase activity increases in a kinet-on ceramide, the product of sphingomyelin hydrolysis, ically consistent manner. The degree of increase in cera-as the principle sphingolipid mediator of intracellular mide is variably reported, ranging from a 20% increasesignaling events, several sphingolipid metabolites gener- to a 2000% increase [90, 94]. Concurrent measurementsated both as a result of sphingomyelinase activity and of sphingomyelin and ceramide have been made in manythrough de novo synthesis have been implicated in sig- studies. In general, sphingomyelin levels or radiolabelingnaling events. Sphingolipids implicated in signaling decrease over time and at a magnitude that is consistentevents include ceramide [63], sphingosine [64], sphingo- with the appearance of ceramide. Often, the sphingomy-sine-1-phosphate [65], and ceramide-1-phosphate [66]. elin levels return to control values as the ceramide con-The primary proposed signaling functions for these tent decreases, implying that the ceramide is recycled tosphingolipids are outlined in Figure 3. sphingomyelin, presumably via the sphingomyelin syn-

The general picture that has emerged of sphingolipid thase. There is some evidence in support of a membrane-associated sphingomyelin synthase on the inner leafletsignaling is both exciting in its implications and confusing


Fig. 3. Some proposed signaling functions of ceramide and its metabolites.

of the plasma membrane. There are three implications potential problems confounding the interpretation of theexperimental data.for this model. First, sphingomyelin hydrolysis, ceramide

formation, and sphingomyelin resynthesis would neces- Short-chain ceramides, usually containing an acetylgroup or C6 or C8 fatty acid at the amide linkage, havesarily occur at this site. Second, the ceramide generated

would act at this site on a local target or be transported been extremely popular in signaling studies. These ana-logues retain the D-erythro stereospecificity of naturalfrom this site to one where such targets exist. The cera-

mide would then be reutilized, or ceramide synthesized ceramides as well as the critical C4-C5 double bond. Inaddition, in early studies, these compounds were be-de novo would serve as substrate for resynthesis. Third,

a significant amount of diacylglycerol, the product of lieved to be relatively inert with regard to cellular metab-olism. Thus, any biological activities observed with thesesphingomyelin synthesis, would be generated and thus

may have independent biological activity. compounds were interpreted as mimics for the activitiesof endogenous ceramides.Not only is the degree of ceramide elevation highly

variable, but the time course for ceramide accumulation Several difficulties exist with this interpretation. First,these ceramides are quite avidly metabolized by cellsis as well. Four patterns of ceramide accumulation have

been described. These include a rapid increase occurring [114]. Original studies often used analogues radiolabeledwithin the fatty acid. The liberation of this fatty acid bywithin a few minutes and declining to baseline within 15

to 20 minutes [104], a gradual increase occurring within a ceramidase would result in a long-chain base, which wasunlabeled and in which the metabolism was thereforean hour and decreasing within two to three hours [105],

a rapid increase with a persistent elevation lasting several impossible to follow. More recent studies have demon-strated that following the addition of these compounds,hours [76], and a gradual increase occurring after several

hours and persisting for 24 hours or longer [106]. Surpris- both short- and long-chain sphingomyelins and glyco-sphingolipids are synthesized as well as free sphingosine,ingly, these patterns can be observed with the same ago-

nist (IL-1b) [76, 104–107] and are not associated with long-chain ceramides, and long-chain sphingomyelins.Another problem is that the concentrations of ceramidesthe degree of rise in ceramide.

A third observation in support of a role for sphingomy- added to cells (typically in the micromolar range) resultin intracellular levels of ceramides that are approxi-elin hydrolysis is that the biological consequences of

sphingomyelin degradation and ceramide generation can mately 100 times those found normally. Additionally, insome cases, comparable biological effects are observed,often be replicated by maneuvers that augment or inhibit

ceramide levels. These methods include the addition of irrespective of the stereoselectivity of the analogue em-ployed. Finally, because of distinct physiochemical dif-short chain, cell-permeant ceramides [108], the addition

of bacterial sphingomyelinase [109], the use of inhibitors ferences in these analogues, short-chain ceramides maydestabilize membranes through detergent-like effectsof long-chain base acylation [110], the use of inhibitors

that decrease ceramide utilization through glycosylation that are independent of their endogenous biological ac-tivities [115].pathways [111], the use of inhibitors of ceramide degra-

dation [112], or the addition of natural long-chain cera- The use of exogenous sphingomyelinase has also beenproblematic, because the majority of sphingomyelin ismides [113]. Each of these methods is associated with


in the outer leaflet of the plasma membrane where it dered some discussion because in other systems sphin-gomyelin hydrolysis and diacylglycerol formation occurwould be accessible to the bacterial enzyme. However,

the pool of sphingomyelin that is susceptible to agonist- simultaneously, D609 may not be specific for phospholi-pase C, and protein kinase C activation can, at times,stimulated sphingomyelinase activity is believed to exist

on the inner membrane leaflet [116]. Thus, ceramide abrogate biological effects thought to be secondary toceramide generation, such as apoptosis.produced with the bacterial enzyme would be present

at a site that may have little to do with the endogenous Another lipid mediator of sphingomyelinase may bearachidonic acid, the product of phospholipase A2 [79].signaling mechanisms. It is therefore not surprising that

the use of this approach often does not replicate the In HL60 cells, arachidonate formation precedes sphingo-myelin hydrolysis. The effect was reproducible by mel-biology under investigation.

Inhibitors of glucosylceramide formation (1-phenyl- litin, an activator of phospholipase A2, and is absent ina cell line defective in cytosolic phospholipase A2. The2-decanoylamino-3-morpholino-propanol [PDMP] and

1-phenyl-2-palmitoylamino-3-pyrrolidino-propenol [P4]) specificity of this effect for arachidonate remains to bedetermined, since other polyunsaturated fatty acids may[117, 118], of ceramide synthase (fumonisin B1) [119], or

ceramidase (IS, 2R)-D-erythro-2-(N-myristoylamino)-1- activate neutral sphingomyelinase. In addition, whetherthis effect is specific for the cytosolic phospholipase A2phenyl-propanol [MAPP]) [120] have also found popu-

larity. These are also potentially problematic. The ability or whether other phospholipases may mediate this effectis unknown.of PDMP and its related homologues to raise ceramide

has recently been found to reside at a second site of Glutathione, a tripeptide, serves a major role in pro-tecting cells against oxidative stress. Reduced glutathi-action, the inhibition of 1-O-acylceramide synthase [41].

De novo ceramide formation, although operative in one is an antioxidant that is depleted in cell death. Bothreduced and oxidized glutathione have been identifiedsome systems [71], is generally not the basis for ceramide

accumulation. Thus, even if highly specific, this fungal as inhibitors of the neutral magnesium-dependent sphin-toxin may not alter endogenous ceramide levels. In gen- gomyelinase [127, 128]. Support for a role for glutathioneeral, there is little assurance that, even if ceramide levels in the regulation of ceramide formation is based on ex-can be manipulated by these agents, the ceramide accu- periments demonstrating that prevention of sphingomy-mulates at a site within the cell at which it would be elin hydrolysis and apoptosis can be prevented by reple-most biologically active. This consideration would also tion of intracellular glutathione.apply for the use of the bacterial sphingomyelinase or The interaction of specific membrane proteins cou-the cell permeant ceramides. pling receptors that activate sphingomyelinases with the

The fourth and final evidence in support of ceramide lipases has been proposed. One such protein is factoras an important signaling molecule is that some of the associated with neutral sphingomyelinase (FAN) [129].same responses can be replicated in yeast systems. Yeasts This is a novel WD (tryptophase-aspartic acid) repeathave been particularly valuable models for understand- protein suggested to link the neutral sphingomyelinaseing sphingolipid biology. A signaling role for ceramides with the mitogen-activated protein kinase pathway.as activators of protein phosphatase 2A [121] and fortheir role as mediators of the yeast heat shock response

CELLULAR TARGETS FOR SPHINGOLIPIDS[122] has been garnered from the use of this model. MoreSeveral effectors have been demonstrated for sphin-recently, an important role for sphingosine-1-phosphates

golipid activities. Perhaps the best characterized effectorhas been identified by use of S. cervesiae, first as theis the set of orphan receptors termed Edg-1, Edg-3, andbasis for the identification of the sphingosine kinase [123]H218/Edg-5 [130]. These comprise a family of structur-and sphingosine-1-phosphate lyase genes [124], and sec-ally homologous receptors with high affinity and speci-ond through manipulation of long-chain base phosphateficity for sphingosine-1-phosphate. Edg-3 and Edg-5 arelevels within yeast. These latter studies have demon-44% similar to each other and 50% similar to Edg-1. Asstrated a marked protective effect of these metabolitesseven transmembrane-spanning proteins, these recep-in models of heat shock and nutrient deprivation [125].tors are G-protein coupled. Two related receptors, Edg-2Several mediators have been implicated in the regula-and Edg-4, appear to couple to lysophosphatidic acidtion of signaling sphingomyelinases. Diacylglycerol was[131]. Edg-1 was named as the “endothelial differentia-perhaps the first lipid to be suggested to regulate TNF-tion gene” and was identified as an early response genea–induced ceramide formation. In this model, the hydro-in endothelial cell differentiation. These receptors arelysis of phosphatidylcholine was thought to be upstreamubiquitously expressed in multiple tissues.from an acidic sphingomyelinase [126]. Support for this

Several signaling events are observed following sphin-was based largely on the use of D609, believed at thegosine-1-phosphate binding to cells. These include thetime to be a specific inhibitor of the phosphatidylcholine-

specific phospholipase C. This observation has engen- activation of mitogen-activated protein kinases [132],


phospholipase C [133], phospholipase D [134], and po- has been reported to be the kinase suppressor of ras(KSR) in Drosophila [149]. It is suggested to directlytassium channels [135]. In addition, there is inhibition

of adenylyl cyclase [133] and mobilization of intracellular activate Raf and mitogen-activated protein kinase activa-tion in response to TNF-a.calcium [136]. Of potential importance is the observation

that both P-cadherin transcription and serum response A third potential effector for ceramide is protein ki-nase C z [150]. Ceramide has been reported to directlyactivation can be induced with sphingosine-1-phosphate

[137]. The latter response is rho dependent and mediates bind to this protein kinase C. A common feature for allof these putative targets is the presence of a cysteine-richstress-fiber formation. Several other responses to sphin-

gosine-1-phosphate have been observed, including mito- domain that may constitute a portion of the ceramide-binding site. Additional suggested ceramide targets in-genesis, inhibition of ceramide-induced apoptosis, inhi-

bition of cell motility, neurite retraction, and platelet clude the CPP32, a caspase 3-like apoptotic protease andthe stress activated/c-jun N-terminal protein kinase.activation.

Recent work has attempted to determine which cellu- Historically, it has been difficult to reconcile the roleof ceramide as an effector with potential sites of forma-lar responses are coupled to which receptor and a sub-

units. At present, it appears that Edg-1 couples to mem- tion in the plasma membrane, cytosol, and acidic lyso-somal or endosomal compartments. Recently, using abers of the Gi family. Edg-3 and Edg-5 couple to Gi in

addition to Gq and G13 [138]. The diversity of receptors photo-crosslinking to a ceramide analogue, cathepsin D,an acidic aspartate protease was identified as a potentialand G-protein interactions not only explains the relative

sensitivity of some but not all sphingosine-1-phosphate target of ceramide generated by the acidic sphingomyeli-nase [151].signaling to inhibitors such as pertussis toxin, but may

clarify the differing cellular responses to this agonist.Sphingosine-1-phosphate is formed intracellularly and

APOPTOSIS AND SPHINGOLIPID SIGNALINGwas originally identified as a putative lipid second mes-

Of the proposed roles for sphingolipids in cell signal-senger. It can mobilize calcium from intracellular storesing, none has engendered greater interest and generated[139]. Sphingosine phosphate kinase activity is stimu-greater controversy than the concept that ceramide is alated by factors such as phorbol ester [140], platelet-signal for programmed cell death. Two primary observa-derived growth factor [141], and Fc receptor ligationtions have been critical in the development of this hy-[142]. Inhibition of its activity by N,N-dimethylsphingo-pothesis. First, agonists that induce apoptosis (for exam-sine inhibits cell growth and promotes apoptosis [143].ple, TNF-a, irradiation, chemotherapeutic drugs, andThe kinase has been cloned in yeast and mammalianCD95) activate cellular sphingomyelinases and raise cellcells. There is a high degree of conservation of its primaryceramide levels [90, 92, 96, 97]. Second, the exogenoussequence between yeast, worms, and mammals. Thereaddition of cell-permeant ceramides or of bacterialis also a significant degree of similarity with diacylglyc-sphingomyelinase induces an apoptotic response [152].erol z. The lyase and phosphatases have also been clonedUnder this hypothesis, agonists in the form of cell stres-from yeast. Deletion of these genes by homologous re-sors or of peptide hormones serve as cell death signals bycombination creates an enhanced resistance to heatactivating one or more sphingomyelinases and increasingshock and nutrient deprivation.ceramide, which then acts as a critical effector for theSeveral potential cytosolic effectors of ceramide activ-apoptotic pathway.ity have also been identified. One target is one or more

Under the current model for apoptosis, death recep-cytosolic phosphatases termed “ceramide-activated pro-tors such as the TNF and CD95 receptors activate deathtein phosphatase” (CAPP) [144]. This enzyme is relatedeffector domain-containing enzymes termed caspasesto the 2A class of heterotrimeric protein phosphatases.(“cysteine protease that cleaves after aspartic acid”).Support for this target is observed in yeast in whichActivation is mediated by binding to the cytoplasmicceramide-induced growth arrest can be mitigated in yeastportions of these receptors through adaptor proteinslacking the PP2A subunits [145]. Protein phosphatase 1such as TRADD, TRF-2, and FADD. Death receptor-may also be stimulated by ceramide [146].independent stimuli such as etoposide and irradiationAn important substrate for ceramide-stimulated phos-induce caspase activation at other sites. “Inducer” cas-phatase activity may be the retinoblastoma protein. Cer-pases (caspase 8) appear to act upstream of the mito-amide and sphingosine stimulate cellular phosphatasechondrial release of cytochrome c into the cytosol, whichactivity against Rb protein and induce cell cycle arrestin association with Apaf-1 and caspase-9 subsequentlyat the G1/S transition [147].process and activate executioner caspases (for example,A second activity that has been identified as a cera-caspase-3, -6, and -7).mide effector has been termed ceramide-activated pro-

The challenge for proponents of ceramide as a media-tein kinase (CAP kinase) [148]. This represents a mem-ber of the family of proline-directed protein kinases. It tor of programmed cell death has been to define the


sphingolipid signaling events in the context of well-estab- similar strategy has been employed for Fabry disease andis currently under clinical investigation with promisinglished pathways for apoptosis. Unfortunately, no consis-

tent view for the role or the site of action of ceramide in preliminary results [159]. Recombinant enzyme replace-ment, however, is an exceptionally expensive pharmaco-either the cell death effector-dependent or -independent

pathways. The published data are inconsistent in this logic modality caused by the small potential market andsignificant manufacturing costs. b-Glucosidase currentlyregard. Many of the critical questions surrounding the

properties and biological role of sphingomyelin signaling represents the most expensive drug on the market, withyearly costs in excess of $200,000.detailed previously in this article impact on the evalua-

tion of the strength of the ceramide hypothesis. These An alternative approach is the use of “substrate deple-tion,” first proposed by Radin [160]. Here, glycosphingo-questions include identifying the critical sphingomyeli-

nase involved in ceramide formation (for example, acidic lipid synthesis is blocked by inhibition of a precursorsuch as glucosylceramide. Support for this approach hasvs. neutral sphingomyelinase), determining the cellular

site for ceramide accumulation, defining the critical ef- been reported using a form of “experimental epistasis.”By crossing knockout mice with the Sandhoff phenotypefectors of ceramide action, and demonstrating that sphin-

gomyelin hydrolysis and ceramide accumulation occur (due to GM2 accumulation) with those defective in Gal-NAc transferase, Liu et al were able to prolong lifein a kinetically consistent manner.

Thus, both neutral and acidic sphingomyelinases have and postpone the neurological phenotype in the doublehomozygous-affected offspring [161]. Support for sub-been implicated in cell death pathways [153, 154]. Cera-

mides formed secondary to exogenous sphingomyelinase strate depletion has also been reported for mouse modelsof Tay-Sachs [162] and Sandhoff disease [163] using N-(presumably in the outer membrane leaflet), within

acidic cellular compartments, and via activation of a neu- butyldeoxynojirimycin, a relatively inactive inhibitor ofthe cerebroside synthase. In these studies, mice weretral sphingomyelinase (presumably within the inner

membrane leaflet) have been implicated in the cell death treated with between 1.2 and 4.8 g/kg/day orally withthe inhibitor. The mice exhibited some prolongation ofresponse. Ceramide has been shown to activate effectorlife and reversal of the accumulation of ganglioside[155] and executioner caspases [156]. Effector caspasewithin their brains. Given the high dose of drug requiredactivation, however, may only result from the exposureand the secondary toxicities of N-butyldeoxynojirimycin,of cells to short-chain ceramides. Finally, apoptotic stim-the development of more active and less toxic inhibitorsuli have been associated both with very rapid and slowlyis required. Nevertheless, N-butyldeoxynojirimycin isrising ceramide levels. Perhaps when the role of ceramidecurrently under evaluation in phase I and phase II trialsin the cell death response is fully delineated will morefor the treatment of Gaucher and Fabry diseases. At thefundamental questions regarding sphingolipid signalingtime of submission, the results of these trials had yet tobe answered.be reported.

In vitro support for the treatment of Fabry disease isSPHINGOLIPIDS AND THE KIDNEY based on the ability of two different PDMP homologues

With the discovery of new insights into sphingolipid to deplete Gb3 levels in lymphoblasts from a patientbiochemistry, cellular function, and pharmacology, in- with Fabry disease [164]. Concentrations as low as 10vestigators have begun to consider what role sphingoli- nmol/L of either 49-hydroxyphenyl- or ethylenedioxy-pids may play in renal physiology and pathophysiology. phenyl-2-decanoylamino-3-pyrrolidino-propanol result-

Gb3 levels accumulate in Anderson-Fabry disease, a ed in greater than 70% depletion of Gb3. By contrastlysosomal storage disorder affecting the kidney. In this even 1000-fold higher concentrations of N-butyldeoxy-X-linked disorder, affected males accumulate Gb3 and nojirimycin were unable to significantly lower Gb3 con-globobiosylceramide in their kidneys, vascular, hearts, tent. Preliminary in vivo studies using a-glactosidase Aand peripheral nerve secondary to a loss of a-galac- knockout mice suggest that renal, hepatic, and cardiactosidase A activity, resulting in renal failure, cardiovas- Gb3 levels can be significantly lowered with prolongedcular disease, and neuropathy. At least 30,000 individuals treatment with the ethylenedioxy P4 homologue.are believed to suffer from this sphingolipidosis world- The verocytotoxin elaborated by pathogenic strains ofwide and 6000 within the United States [157]. However, Escherichia coli (O157:H7) associated with hemolyticfewer than 100 patients are reported in the U.S. Renal uremic syndrome and hemorrhagic colitis binds to globo-Data System to suffer end-stage renal disease as a result triaosylceramide. The epidemiological basis for hemo-of Fabry disease. This disparity suggests that many pa- lytic uremic syndrome was established by Karmali et altients go undiagnosed. [165]. Gb3 is expressed on platelets; however, verotoxin

Gaucher disease, caused by a defect in b-glucosidase, is does not affect platelet aggregation. It is therefore thecurrently treated with enzyme replacement therapy using cellular distribution of Gb3 within kidney and endothe-

lium of this glycosphingolipid receptor that correlatesmannose-terminated recombinant glucosidase [158]. A


best with the pathophysiology of this disorder [166]. In ceramide in mediating renal injury is established, thiswill provide important and obvious strategic approachessupport of this view is the observation that adult glomer-

uli poorly express Gb3, whereas Gb3 is highly expressed to intervention.in the glomeruli of pediatric patients.

Hemolytic uremic syndrome is also prevalent in theCONCLUDING THOUGHTSolder population, and yet glomeruli from normal older

Recent studies in the biochemistry and cellular biologypatients are verotoxin receptor negative. This may beof sphingolipids have provided several potentially excit-explained by the observation that Gb3 levels can being opportunities for providing new insights into normalinduced in response to multiple cytokines (particularlyand aberrant renal function. In many cases, there existsTNF, IL-1b, and lipopolysaccharide), suggesting a rolesubstantial evidence for a critical role of the unique lip-for glycolipids in the mediation of inflammatory re-ids, such as receptors for bacterial toxin binding andsponses [167]. Gb3 can also be induced by viral infectionligands for the Edg family of receptors. In other cases,such as with Epstein-Barr virus and HIV. It is a Burkittdebate persists, and further work is required to establishlymphoma-associated antigen [168]. It is thus possibleproof of concept. Such proof will undoubtedly progressthat viral prodromes may render adult patients suscepti-as additional enzymes in the metabolic pathways ofble to hemolytic uremic syndromes. Such a syndromesphingolipids are sequenced and characterized, as extra-has been reported for HIV [169].cellular and intracellular targets of sphingolipid messen-If sphingosine-1-phosphate is produced and releasedgers are unequivocally identified, and as new inhibitorsfrom activated platelets, then one might speculate thatof these enzymes and glycolipid receptors become avail-thrombotic disorders such as hemolytic uremic syndromeable.may be mediated in part by sphingosine-1-phosphate

I have attempted to provide a balanced and criticalactivity. Sphingosine-1-phosphate stimulates mesangialappraisal of the current status of sphingolipid biochemistrycell proliferation. Additionally, the Edg-1 and Edg-3 re-and function. In many cases, the importance of sphingo-ceptors can be detected in developing mouse kidneylipids is well established and unequivocal. In other cases,(abstract; Arend et al, J Am Soc Nephrol (Suppl)although many proposed functions for sphingolipids are10:466A, 1999). Given the ability of sphingosine-1-phos-very exciting in their implications in cell biology andphate to stimulate smooth muscle contraction, one mightpathophysiology, they remain unproven since they arespeculate that this lipid may mediate much of the renalfundamentally descriptive. The potential role of sphin-pathology and hypertension observed in this syndrome.golipids in protein sorting through lipid rafts, as media-Conversely, ceramide has been implicated as a pot-tors of injury and repair and as receptors for pathogens,ential mediator of vascular relaxation. Cell-permeantprovides fertile ground for work by the renal investiga-ceramides have been demonstrated to attenuate agonist-tive community. Classic models of renal function andstimulated vasoconstriction [170]. One potential mecha-disease not only provide particularly useful opportunitiesnism for this effect may be the induction of endothelialfor clarifying the role of sphingolipids in the kidney andnitric oxide synthase (eNOS) by ceramide. Exogenousvasculature, but also provide the opportunity to establishceramides have been reported to increase NO synthasefundamentally and unequivocally a role for sphingolipidsactivity as well as the translocation of eNOS from the

endothelial membrane to intracellular sites. Bradykinin, in these vital processes.a known inducer of eNOS, also raises cell ceramide levels

Reprint requests to James A. Shayman, M.D., Nephrology Division,[171]. Department of Internal Medicine, University of Michigan, Box 0676,Finally, ceramide levels have been demonstrated to Room 1560 MSRBII, 1150 West Medical Center Drive, Ann Arbor,

Michigan 48109-0676, USA.rise in ischemic renal injury. These changes reportedlyE-mail: [email protected] due to both sphingomyelinase activation [172] and

de novo synthesis by ceramide synthase [173]. Establish-REFERENCESing whether the increased ceramide in fact is a critical

1. Hokin LE, Hokin MR: Evidence for phosphatidic acid as thesignal in the ischemic injury or more trivially whether itsodium carrier. Nature 184:1068–1069, 1959increases secondary to generalized lipid degradation will

2. Shayman JA, Radin NS: The structure and function of renalrequire additional studies. The delayed kinetics of cera- glycosphingolipids. Am J Physiol 260:F291–F302, 1991

3. Shayman JA: Sphingolipids: Their role in intracellular signalingmide formation in many models of apoptosis and itsand renal growth. J Am Soc Nephrol 7:171–182, 1996more likely role in regulating executioner caspases will

4. Karlsson KA: On the chemistry and occurrence of sphingolipidneed to be confirmed in models of renal ischemia. In long-chain bases. Chem Phys Lipids 5:6–43, 1970

5. Radin NS, Martin FB, Brown JR: Galactolipide metabolism.addition, better characterization of the multiple isoformsJ Biol Chem 224:499–507, 1957of the neutral sphingomyelinase and the development

6. Bajjalieh SM, Martin TFJ, Floor E: Synaptic vesicle ceramideof sphingomyelinase-specific inhibitors will be critical to kinase: A calcium-stimulated lipid kinase that co-purifies with

brain synaptic vesicles. J Biol Chem 264:14917–14921, 1990proving this concept. However, if and when a role for


7. Abe A, Shayman JA, Radin NS: A novel enzyme that catalyzes 31. Van Den Hill A, Van Heusden GP, Wirtz KWA: The synthesisthe esterification of N-acetylsphingosine. J Biol Chem 271:14383– of sphingomyelin in the Morris hepatomas 7777 and 5123D is14389, 1996 restricted to the plasma membrane. Biochim Biophys Acta 833:

8. Bar LK, Barenholz Y, Thompson TE: Effect of sphingomyelin 354–357, 1985composition on the phase structure of phosphatidylcholine-sphin- 32. Vos JP, Dehaas CGM, Can Golde LM, Lopes-Gardozo MJ:gomyelin bilayers. Biochemistry 36:2507–2516, 1997 Relationships between phosphatidylcholine, phosphatidylethano-

9. White D: Form and Function of Phospholipids. Amsterdam, Else- lamine, and sphingomyelin metabolism in cultured oligodendro-vier Publications, 1973, pp 441–481 cytes. J Neurochem 68:1252–1260, 1997

10. Barenholz Y: Physiology of Membrane Fluidity, edited by Shi- 33. Brady RO, Bradley RM, Young OM, Kaller HJ: An alterna-nitzky M, Sphingomyelin-lecithin balance in membranes: Com- tive pathway for the enzymatic synthesis of sphingomyelin. J Biolposition, structure, and junction relationships, Boca Raton, CRC Chem 240:3693–3694, 1965Press, 1984, pp 131–173 34. Ghosh TK, Bian J, Gill DL: Intracellular calcium release medi-

11. Myers M, Wortman C, Freire E: Modulation of neuraminidase ated by sphingosine derivatives generated in cells. Scienceactivity by the physical state of phospholipid bilayers containing 248:1653–1656, 1990gangliosides Gd1a and Gt1b. Biochemistry 23:1442–1448, 1984 35. Ito M, Kurita T, Kita K: A novel enzyme that cleaves the N-

12. Masserini M, Freire E: Thermotropic characterization of phos- acyl linkage of ceramides in various glycosphingolipids as well asphatidylcholine vesicles containing ganglioside GM1 with homo- sphingomyelin to produce their lyso forms. J Biol Chem 270:geneous ceramide chain length. Biochemistry 25:1043–1049, 1986 24370–24374, 1995

13. Masserini M, Palestini P, Freire E: Influence of glycolipid oligo- 36. Shayman JA, Abe A: Glucosylceramide synthase: Assay andsaccharide and long-chain base composition on the thermotropic properties. Methods Enzymol 311:42–49, 1999properties of dipalmitoylphosphatidylcholine large unilamellar 37. Coste H, Martel MB, Got R: Topology of glucosylceramidevesicles containing gangliosides. Biochemistry 28:5029–5032, 1989 synthesis in Golgi membranes from porcine submaxillary glands.

14. Fra A, Masserini M, Palestini P, Sonnino S, Simons K: A photo- Biochim Biophys Acta 858:6–12, 1986reactive derivative of ganglioside GM1 specifically cross-links 38. Dressler KA, Kolesnick RN: Ceramide 1-phosphate, a novelVIP21-caveolin on the cell surface. FEBS Lett 375:11–14, 1995 phospholipid in human leukemia (HL-60) cells: Synthesis via cera-

15. Okamoto T, Schlegel AM, Scherer PE, Lisanti MP: Caveolins, mide from sphingomyelin. J Biol Chem 265:14917–14921, 1990a family of scaffolding proteins for organizing “preassembled 39. Bohnenberger E, Sandermann H Jr: Diglyceride kinase fromsignaling complexes” at the plasma membrane. J Biol Chem Escherichia coli: Purification in organic solvent and some proper-273:5419–5422, 1998 ties of the enzyme. Eur J Biochem 94:401–407, 1979

16. Brown DA, London E: Functions of lipid rafts in biological 40. Hinkovska-Galcheva VT, Boxer LA, Mansfield PJ, Harsh D:membranes. Annu Rev Cell Dev Biol 14:111–136, 1998 The formation of ceramide-1-phosphate during neutrophil phago-

17. Smart EJ, Ying YS, Mineo C, Anderson RG: A detergent-free cytosis and its role in liposome fusion. J Biol Chem 273:33203–method for purifying caveolae membrane from tissue culture cells. 33209, 1998Proc Natl Acad Sci USA 92:10104–10108, 1995 41. Abe A, Shayman JA: Purification and characterization of 1-O-18. Iwabuchi K, Handa K, Hakomori S: Separation of “glycosphin- acylceramide synthase, a novel phospholipase A2. J Biol Chemgolipid signaling domain” from caveolin-containing membrane

11:8467–8474, 1998fraction in mouse melanoma B16 cells and its role in cell adhesion42. Stoffel W, Heimann G, Hellenbroich B: Sphingosine kinasecoupled with signaling. J Biol Chem 273:33766–33773, 1998

in blood platelets. Hoppe Seylers Z Physiol Chem 354:562–565,19. Edidin M: Lipid microdomains in cell surface membranes. Curr1973Opin Struct Biol 7:528–532, 1997

43. Stoffel W: Studies on the biosynthesis and degradation of sphin-20. Jacobson K, Dietrich C: Looking at lipid rafts? Trends Cell Biolgosine bases. Chem Phys Lipids 5:139–158, 19709:87–91, 1999

44. Waggoner DW, Gomez-Munoz A, Dewald J, Brindley DN:21. Brady RO, Koval GJ: The enzyme synthesis of sphingosine.Phosphatidate phosphohydrolase catalyzes the hydrolysis of cera-J Biol Chem 233:26–31, 1958mide 1-phosphate, lysophosphatidate, and sphingosine 1-phos-22. Williams RD, Wang E, Merrill AHJ: Enzymology of long-phate. J Biol Chem 271:16506–16509, 1997chain base synthesis by liver: Characterization of serine palmitoyl-

45. Igarashi Y, Hakomori S: Enzymatic synthesis of N,N-dimethyl-transferase in rat liver microsomes. Arch Biochem Biophyssphingosine: Demonstration of the sphingosine: N-methyltrans-228:282–291, 1984ferase in mouse brain. Biochem Biophys Res Commun 164:1411–23. Stoffel W, Le Kim D, Sticht G: Synthesis and degradation of1416, 1989spingosine bases in Ansenula ciferrii. Hoppe Seyler’s Z Physiol

46. Mandon EC, Ehses I, Rother J, Van Echten G, Sandhoff K:Chem 349:1637–1644, 1968Subcellular localization and membrane topology of serine palmi-24. Sribney M: Enzymatic synthesis of ceramide. Biochim Biophystoyltransferase, 3-dehydrosphinganine reductase and sphinganineActa 125:242–247, 1966N-acyltransferase in mouse liver. J Biol Chem 267:11144–11148,25. Rother J, Van Echten G, Schwarzmann G, Sandhoff K: Bio-1992synthesis of sphingolipids: Dihydroceramide and not sphinganine

47. Morell P, Radin NS: Specificity in ceramide biosynthesis fromis desaturated by cultured cells. Biochem Biophys Res Communlong chain bases and various fatty acyl coenzyme As by brain89:14–20, 1992microsomes. J Biol Chem 245:342–350, 197026. Gatt S: Enzymatic hydrolysis of sphingolipids. I. Hydrolysis and

48. Van Helvoort A, Van’t Hof W, Ritsema T, Sandra A, Vansynthesis of ceramides by an enzyme from rat brain. J Biol ChemMeer G: Conversion of diacylglycerol to phosphatidylcholine on241:3724–3730, 1966the basolateral surface of epithelial (Madin-Darby canine kidney)27. Lee T, Ou M, Shinozaki K, Malone B, Snyder F: Biosynthesiscells: Evidence for the reverse action of a sphingomyelin synthase.of N-acetylsphingosine by platelet-activating factor: SphingosineJ Biol Chem 269:1763–1769, 1994CoA-independent transacetylase in HL-60 cells. J Biol Chem

49. Barnholz Y, Roitman A, Gatt S: Enzymatic hydrolysis of sphin-271:209–217, 1996golipids. II. Hydrolysis of sphingomyelin by an enzyme from rat28. Sribney M, Kennedy EP: The enzymatic synthesis of sphingomye-brain. J Biol Chem 241:3731–3737, 1966lin. J Biol Chem 233:1315–1322, 1958

50. Spence MW, Byers DM, Palmer FB, Cook HW: A new Zn21-29. Ullman MD, Radin NS: The enzymatic formation of sphingomy-stimulated sphingomyelinase in fetal bovine serum. J Biol Chemelin from ceramide and lecithin in mouse liver. J Biol Chem264:5358–5363, 1989249:1506–1513, 1974

51. Rao BG, Spence MW: Sphingomyelinase activity at pH 7.4 in30. Lipsky N, Pagano RE: Sphingolipid metabolism in cultured fi-human brain and a comparison to activity at pH 5.0. J Lipid Resbroblasts: Microscopic and biochemical studies employing a fluo-17:506–515, 1976rescent ceramide analogue. Proc Natl Acad Sci USA 80:2608–

2612, 1983 52. Chakraborty G, Ziemba S, Drivas A, Ledeen RW: Myelin con-


tains neutral sphingomyelinase activity that is stimulated by tumor of the sphingomyelin pathway but inhibit nuclear factor-kappaB activation. J Biol Chem 270:24518–24524, 1995necrosis factor-alpha. J Neurosci Res 50:466–476, 1997

53. Levade T, Vidal F, Vermeersch S, Andrieu N, Gatt S, Salvayre 73. Subbaramaiah K, Chung WJ, Dannenberg AJ: Ceramide regu-lates the transcription of cyclooxygenase-2: Evidence for involve-R: Degradation of fluorescent and radiolabelled sphingomyelins

in intact cells by a non-lysosomal pathway. Biochim Biophys Acta ment of extracellular signal-regulated kinase/c-Jun N-terminalkinase and p38 mitogen-activated protein kinase pathways. J Biol12358:277–287, 1995

54. Nilsson A: The presence of spingomyelin and ceramide-cleaving Chem 273:32943–32949, 199874. Chang Y, Abe A, Shayman JA: Ceramide formation during heatenzymes in the small intestinal tract. Biochim Biophys Acta

176:339–347, 1969 shock: A potential mediator of alpha B-crystallin transcription.Proc Natl Acad Sci USA 92:12275–12279, 199555. Duan RD, Nilsson A: Purification of a newly identified alkaline

sphingomyelinase in human bile and effects of bile salts and phos- 75. Masamune A, Igarashi Y, Hakomori S: Regulatory role of cera-mide in interleukin (IL)-1-beta-induced E-selectin expression inphatidylcholine on enzyme activity. Hepatology 26:823–830, 1997

56. Schuchman EH, Suchi M, Takahashi T, Sandhoff K, Desnick human umbilical vein endothelial cells: Ceramide enhances IL-1beta action, but is not sufficient for E-selectin expression. J BiolRJ: Human acid sphingomyelinase: Isolation, nucleotide sequence

and expression of the full-length and alternatively spliced cDNAs. Chem 271:9368–9375, 199676. Mathias S, Younes A, Kan CC, Orlow I, Joseph C, KolesnickJ Biol Chem 266:8531–8539, 1991

57. Schissel SL, Keesler GA, Schuchman EH, Williams KJ, Tabas RN: Activation of the sphingomyelin signaling pathway in intactEL4 cells and in a cell-free system by IL-1 beta. Science 259:519–I: The cellular trafficking and zinc dependence of secretory and

lysosomal sphingomyelinase, two products of the acid sphingomy- 522, 199377. Olivera A, Spiegel S: Sphingosine-1-phosphate as second mes-elinase gene. J Biol Chem 273:18240–18259, 1998

58. Tomiuk S, Hofmann K, Nix M, Zumbansen M, Stoffel W: senger in cell proliferation induced by PDGF and FCS mitogens.Nature 365:557–560, 1993Cloned mammalian neutral sphingomyelinase: Functions in sphin-

golipid signaling? Proc Natl Acad Sci USA 95:3638–3643, 1998 78. Cuvillier O, Pirianov G, Kleuser B, Vanek PG, Coso OA,Gutkind S, Spiegel S: Suppression of ceramide-mediated pro-59. Van Helvoort A, Smith AJ, Sprong H, Fritzsche I, Schinkel

AH, Borst P, Van Meer G: MDR1 P-glycoprotein specifically grammed cell death by sphingosine-1-phosphate. Nature 381:800–803, 1996translocates phosphatidylcholine. Cell 87:507–517, 1996

60. Lavie Y, Cao HT, Bursten SL, Giuliano AE, Cabot MC: Accu- 79. Jayadev S, Linardic CM, Hannun YA: Identification of arachi-donic acid as a mediator of sphingomyelin hydrolysis in responsemulation of glucosylceramides in multidrug-resistant cancer cells.

J Biol Chem 271:19530–19536, 1996 to tumor necrosis factor alpha. J Biol Chem 269:5757–5763, 199480. Jacobs LS, Kester M: Sphingolipids as mediators of effects of61. Hannun YA, Loomis CR, Merrill AH, Bell RM: Sphingosine

inhibition of protein kinase C activity and of phorbol dibutyrate platelet-derived growth factor in vascular smooth muscle cells.Am J Physiol 265:C740–C747, 1993binding in vitro and in human platelets. J Biol Chem 261:12604–

12609, 1986 81. Borchardt RA, Lee WT, Kalen A, Buckley RH, Peters C,Schiff S, Bell RM: Growth-dependent regulation of cellular62. Okazaki T, Bell RM, Hannun YA: Sphingomyelin turnover

induced by vitamin D3 in HL-60 cells: Role in cell differentiation. ceramides in human T-cells. Biochim Biophys Acta 1212:327–336,1994J Biol Chem 264:19076–19080, 1989

63. Hannun YA: The sphingomyelin cycle and the second messenger 82. Kim MY, Linardic C, Obeid L, Hannun Y: Identification ofsphingomyelin turnover as an effector mechanism for the actionfunction of ceramide. J Biol Chem 269:3125–3128, 1994

64. Hannun YA, Bell RM: Lysosphingolipids inhibit protein kinase of tumor necrosis factor alpha and gamma-interferon: Specificrole in cell differentiation. J Biol Chem 266:484–489, 1991C: Implications for the sphingolipidoses. Science 235:670–674,

1987 83. Dobrowsky RT, Jenkins GM, Hannun YA: Neurotrophins in-duce sphingomyelin hydrolysis. Modulation by co-expression of65. Spiegel S, Cuvillier O, Edsall LC, Kohama T, Menzeleev

R, Olah Z, Olivera A, Pirianov G, Thomas DM, Tu Z, Van p75NTR with Trk receptors. J Biol Chem 270:22135–22142, 199584. Quintans J, Kilkus J, McShan CL, Gottschalk AR, DawsonBrocklyn JR, Wang F: Sphingosine-1-phosphate in cell growth

and cell death. Ann NY Acad Sci 845:11–18, 1998 G: Ceramide mediates the apoptotic response of WEHI 231 cellsto anti-immunoglobulin, corticosteroids and irradiation. Biochem66. Gomez-Munoz A, Duffy PA, Martin A, O’Brien L, Byun HS,

Bittman R, Brindley DN: Short-chain ceramide-1-phosphates Biophys Res Commun 202:710–714, 199485. Strum JC, Swenson KI, Turner JE, Bell RM: Ceramide triggersare novel stimulators of DNA synthesis and cell division: Antago-

nism by cell-permeable ceramides. Mol Pharmacol 47:833–839, meoitic cell cycle progression in Xenopus oocytes: A potentialmediator of progesterone-induced maturation. J Biol Chem 270:1995

67. Wiegmann K, Schutze S, Machleidt T, Witte D, Kronke M: 13541–13547, 199586. Geilen CC, Bektas M, Wieder T, Orfanos CE: The vitaminFunctional dichotomy of neutral and acidic sphingomyelinases in

tumor necrosis factor signaling. Cell 78:1005–1015, 1994 D3 analogue, calcipotriol, induces sphingomyelin hydrolysis inhuman keratinocytes. FEBS Lett 378:88–92, 199668. Betts JC, Agranoff AB, Nabel GJ, Shayman JA: Dissociation

of endogenous cellular ceramide from NK-kappa B activation. 87. Meacci E, Vasta V, Farnararo M, Bruni P: Bradykinin increasesceramide and sphingosine content in human fibroblasts: PossibleJ Biol Chem 269:8455–8458, 1994

69. Ballou LR, Chao CP, Holness MA, Barker SC, Raghow R: involvement of glycosphingolipids. Biochem Biophys Res Com-mun 221:1–7, 1996Interleukin-1-mediated PGE2 production and sphingomyelin me-

tabolism: Evidence for the regulation of cyclooxzygenase gene 88. Riboni L, Prinetti A, Bassi R, Caminiti A, Tettamanti G: Amediator role of ceramide in the regulation of neuroblastomaexpression by sphingosine and ceramide. J Biol Chem 267:20044–

20050, 1992 Neuro2a cell differentiation. J Biol Chem 270:26868–26875, 199589. Catalan RE, Aragones MD, Martinez AM, Fernandez I:70. Verheij M, Bose R, Lin XH, Yao B, Jarvis WD, Grant S, Birrer

MJ, Szabo E, Zon LI, Kyriakis JM, Haimovitz-Friedman A, Involvement of sphingolipids in the endothelin-1 signal transduc-tion mechanism in rat brain. Neurosci Lett 220:121–124, 1996Fuks Z, Kolesnick RN: Requirement for ceramide-initiated

SAPK/JNK signaling in stress-induced apoptosis. Nature 380:75– 90. Cifone MG, Maria R, Roncaioli P, Rippo MR, Azuma M, LanierLL, Santoni A, Testi R: Apoptotic signaling through CD95 (Fas/79, 1996

71. Bose R, Verheij M, Haimovitz-Friedman A, Scotto K, Fuks Z, Apo-1) activates an acidic sphingomyelinase. J Exp Med 180:1547–1552, 1994Kolesnick R: Ceramide synthase mediates daunorubicin-induced

apoptosis: an alternative mechanism for generating death signals. 91. Boucher LM, Wiegmann K, Futterer A, Pfeffer K, MachleidtT, Schutze S, Mak TW, Kronke M: CD28 signals through acidicCell 82:405–414, 1995

72. Andrieu N, Salvayre R, Jaffrezou JP, Levade T: Low tempera- sphingomyelinase. J Exp Med 181:2059–2068, 199592. Wright SC, Zheng H, Zhong J: Tumor cell resistance to apopto-tures and hypertonicity do not block cytokine-induced stimulation


sis due to a defect in the activation of sphingomyelinase and the killing of multidrug- resistant KB cells by inhibitors of glucosylcer-amide synthase. Br J Cancer 81:423–430, 199924 kDa apoptotic protease (AP24). FASEB J 10:325–332, 1996

112. Meroni SB, Canepa DF, Pellizzari EH, Schteingart HF, Cigor-93. Kubota M, Kitahara S, Shimasaki H, Ueta N: Accumulation ofraga SB: Effect of N-acetylsphingosine (C2) and the ceramidaseceramide in ischemic human brain of an acute case of cerebralinhibitor (1S,2R)-d-erythro-2-(N-myristoylamino)-1-phenyl-1-pro-occlusion. Jpn J Exp Med 59:59–64, 1989panol on the regulation of Sertoli cell function. J Androl 20:619–94. Turinsky J, Bayly BP, O’Sullivan DM: 1,2-Diacylglyerol and625, 1999ceramide levels in rat liver and skeletal muscle in vivo. Am J

113. Ji L, Zhang G, Uematsu S, Akahori Y, Hirabayashi Y: InductionPhysiol 261:E620–E627, 1991of apoptotic DNA fragmentation and cell death by natural cera-95. Jaffrezou JP, Levade T, Bettaieb A, Andrieu N, Bezombes C,mide. FEBS Lett 358:211–214, 1995Maestre N, Vermeersch S, Rousse A, Laurent G: Daunoru-

114. Abe A, Wu D, Shayman JA, Radin NS: Metabolic effects ofbicin-induced apoptosis: Triggering of ceramide generationshort-chain ceramide and glucosolyceramide on sphingolipids andthrough sphingomyelin hydrolysis. EMBO J 15:2416–2427, 1996protein kinase C. Eur J Biochem 210:765–773, 199296. Zhang J, Alter N, Reed JC, Borner C, Obeid LM, Hannun

115. Simon CG Jr, Gear AR: Membrane-destabilizing properties ofYA: Bcl-2 interrupts the ceramide-mediated pathway of cellC2-ceramide may be responsible for its ability to inhibit plateletdeath. Proc Natl Acad Sci USA 93:5325–5328, 1996aggregation. Biochem 37:2059–2069, 199897. Strum JC, Small GW, Pauig SB, Daniel LW: 1-beta-D-Arabino-

116. Zhang P, Liu B, Jenkins GM, Hannun YA, Obeid LM: Expres-furanosylcytosine stimulates ceramide and diglyceride formationsion of neutral sphingomyelinase identifies a distinct pool of sphin-in HL-60 cells. J Biol Chem 269:15493–15497, 1994gomyelin involved in apoptosis. J Biol Chem 272:9609–9612, 199798. Tettamanti G, Prinetti A, Bassi R, Viani P, Giussani P, Riboni

117. Abe A, Radin NS, Shayman JA, Wotring LL, Zipkin RE, Sivaku-L: Sphingoid bioregulators in the differentiation of cells of neuralmar R, Ruggieri JM, Carson KG, Ganem B: Structural andorigin. J Lipid Med Cell Signal 14:263–275, 1996stereochemical studies of potent inhibitors of glucosylceramide99. Jaffrezou JP, Herbert JM, Levade T, Gau MN, Chatelain P,synthase and tumor cell growth. J Lipid Res 36:611–612, 1995Larent G: Reversal of multidrug resistance by calcium channel

118. Shayman JA, Lee L, Abe A, Shu L: Inhibitors of glucosylceramideblocker SR33557 without photoaffinity labeling of P-glycoprotein.synthase. Methods Enzymol 311:373–387, 1999J Biol Chem 266:19858–19864, 1991

119. Merrill AH Jr, Wang E, Vales TR, Smith ER, Schroeder JJ,100. Chmura SJ, Nodzenski E, Weichselbaum RR, Quintans J: Pro-Menaldino DS, Alexander C, Crane HM, Xia J, Liotta DC,tein kinase C inhibition induces apoptosis and ceramide produc-Meredith FI, Riley RT: Fumonisin toxicity and sphingolipidtion through activation of a neutral sphingomyelinase. Cancerbiosynthesis. Adv Exp Med Biol 392:297–306, 1996Res 56:2711–2714, 1996

120. Bielawska A, Greenberg MS, Perry D, Jayadev S, Shayman J,101. Van Veldhoven PP, Matthews TJ, Bolognesi DP, Bell RM:McKay C, Hannun YA: (1S,2R)-d-erythro-2-(N-myristoylam-Changes in bioactive lipids, aklylacylglycerol and ceramide, occurino)-1-phenyl-1-propanol as an inhibitor of ceramidase. J Biolin HIV-infected cells. Biochem Biophys Res Commun 187:209–Chem 271:12646–12654, 1996216, 1992

121. Dobrowsky RT, Kamibayashi C, Mumby MC, Hannun YA: Cer-102. Hedlund M, Svensson M, Nilsson A, Duan RD, Svanborg C: amide activates heterotrimeric protein phosphatase 2A. J BiolRole of the ceramide-signaling pathway in cytokine responses to Chem 268:15523–15530, 1993P-finbriated Escherichia coli. J Exp Med 183:1037–1044, 1996 122. Skrzypek MS, Nagiec MM, Lester RL, Dickson RC: Analysis

103. Taga S, Carlier K, Mishal Z, Capoulade C, Mangeney M, of phosphorylate sphingolipid long-chain bases reveals potentialLecluse Y, Coulaud D, Tetaud C, Pritchard LL, Tursz T, roles in heat stress and growth control in Saccharomyces. J Bacte-Wiels J: Intracellular signaling events in CD77-mediated apopto- riol 181:1134–1140, 1999sis of Burkitt’s lymphoma cells. Blood 90:2757–2767, 1997 123. Lantermann MM, Saba JD: Characterization of sphingosine ki-

104. Welsh N: Interleukin-1 beta-induced ceramide and diacylglycerol nase (SK) activity in Saccharomyces cerevisiae and isolation ofgeneration may lead to activation of the c-Jun NH2-terminal SK-deficient mutants. Biochem J 332:525–531, 1998kinase and the transcription factor ATF2 in the insulin-producing 124. Saba JC, Nara F, Bielawska A, Garrett S, Hannun YA: Thecell line RINm5F. J Biol Chem 271:8307–8312, 1996 BST1 gene of Saccharomyces cerevisiae is the sphingosine-1-phos-

105. Chen J, Nikolova-Karakashian M, Merrill AH, Morgan ET: phate lyase. J Biol Chem 272:26087–26090, 1997Regulation of cytochrome P450 2C11 (CYP2C11) gene expression 125. Mao C, Saba JD, Obeid LM: The dihydrosphingosine-1-phos-by interleukin-1, sphingomyelin hydrolysis, and ceramides in rat phate phosphatases of Saccharomyces cerevisiae are importanthepatocytes. J Biol Chem 270:25233–25238, 1995 regulators of cell proliferation and heat stress responses. Biochem

106. Santana P, Llanes L, Hernandez I, Gallardo G, Quintana J 342:667–675, 1999J, Gonzalez J: Ceramide mediates tumor necrosis factor effects 126. Schutz S, Potthoff K, Machleidt T, Berkovic D, Wiegmann K,on P450-aromatase activity in cultured granulosa cells. Endocri- Kronke M: TNF activates NF-kappa B by phosphatidylcholine-nology 136:2345–2348, 1995 specific phospholipase C-induced “acidic” sphingomyelin break-

107. Ballou LR, Chao CP, Holness MA, Barker SC, Rahow R: down. Cell 71:765–776, 1992Interleukin-1-mediated PGE2 production and sphingomyelin me- 127. Liu B, Hannun YA: Inhibition of the neutral magnesium-depen-tabolism: Evidence for the regulation of cyclooxygenase gene dent sphingomyelinase by glutathione. J Biol Chem 272:16281–expression by sphingosine and ceramide. J Biol Chem 267:20044– 16287, 199720050, 1992 128. Liu B, Andrieu-Abadie N, Levade T, Zhang P, Obeid LM,

108. Goldkorn T, Dressler KA, Muindi J, Radin NS, Mendelsohn Hannun YA: Glutathione regulation of neutral sphingomyelinaseJ, Menaldino D, Liotta D, Kolesnick RN: Ceramide stimulates in tumor necrosis factor-alpha-induced cell death. J Biol Chemepidermal growth factor receptor phosphorylation in A431 human 273:11313–11320, 1998epidermoid carcinoma cells: Evidence that ceramide may mediate 129. Adam-Klages S, Adam D, Wiegmann K, Struve S, Kolanus W,sphingosine action. J Biol Chem 266:16092–16097, 1991 Schneider-Mergener J, Kronke M: FAN, a novel WD-repeat

109. Richard A, Bourgoin S, Naccache PH, L’Heureux GP, Krum protein, couples the p55 TNF-receptor to neutral sphingomyeli-E, McColl SR, Pelletier G: C2-ceramide primes specifically for nase. Cell 86:937–947, 1996the superoxide anion production induced by N-formylmethionyl- 130. Windh RT, Lee MJ, Hla T, An S, Barr AJ, Manning DR:leucyl phenylalanine (fMLP) in human neutrophils. Biochim Bio- Differential coupling of the sphingosine 1-phosphate receptorsphys Acta 1299:259–266, 1996 edg-1, edg-3, and H218/Edg-5 to the g (i), g (q), and G (12) families

110. Xu J, Yeh CH, Chen S, He L, Sensi SL, Canzoniero LM, Choi of heterotrimeric G proteins. J Biol Chem 274:27351–27358, 1999DW, Hsu CY: Involvement of de novo ceramide biosynthesis in 131. Goetzl EJ, An S: Diversity of cellular receptors and functionstumor necrosis factor-alpha/cycloheximide-induced cerebral en- for the lysophospholipid growth factors lysophosphatidic acid anddothelial cell death. J Biol Chem 273:16521–16526, 1998 sphingosine 1-phosphate. FASEB J 12:1589–1598, 1998

132. Wu J, Spiegel S, Sturgill TW: Sphingosine 1-phosphate rapidly111. Nicholson KM, Quinn DM, Kellett GL, Warr JR: Preferential


activates the mitogen-activated protein kinase pathway by a G 150. Muller G, Ayoub M, Storz P, Rennecke J, Fabbro D, Pfizen-maier K: PKC zeta is a molecular switch in signal transduction ofprotein-dependent mechanism. J Biol Chem 270:11484–11488,

1995 TNF-alpha, bifunctionally regulated by ceramide and arachidonicacid. EMBO J 14:1961–1969, 1995133. Okamato H, Takuwa N, Gonda K, Okazaki H, Chang K, Ya-

tomi Y, Shigematsu H, Takuwa Y: EDG1 is a functional sphingo- 151. Heinrich M, Wickel M, Schneider-Brachert W, Sandberg C,Gahr J, Schwandner R, Weber T, Saftig P, Peters C, Brunnersine-1-phosphate receptor that is linked via a Gi/o to multiple

signaling pathways, including phospholipase C activation, Ca21 J, Kronke M, Schutze S: Cathepsin D targeted by acid sphingo-myelinase-derived ceramide. EMBO J 18:5252–5263, 1999mobilization, Ras-mitogen-activated protein kinase activation,

and adenylate cyclase inhibition. J Biol Chem 273:27104–27110, 152. Obeid LM, Linardic CM, Karolak LA, Hannun YA: Pro-grammed cell death induced by ceramide. Science 259:1769–1771,1998

134. Meacci E, Vasta V, Moorman JP, Bobak DA, Bruni P, Moss 1993153. Yoshimura S, Banno Y, Nakashima S, Hayashi K, YamakawaJ, Vaughan M: Effect of Rho and ADP-ribosylation factor

GTPases on phospholipase D activity in intact human adenocarci- H, Sawada M, Sakai N, Nozawa Y: Inhibition of neutral sphingo-myelinase activation and ceramide formation by glutathione innoma A549 cells. J Biol Chem 274:18605–18612, 1999

135. Bunemann M, Brandts B, Zu Heringdorf DM, Van Koppen hypoxic PC12 cell death. J Neurochem 73:675–683, 1999154. Santana P, Pena LA, Haimovitz-Friedman A, Martin S, GreenCJ, Jakobs KH, Pitt L: Activation of muscarinic K1 current in

guinea-pig atrial myocytes by sphingosine-1-phosphate. J Physiol D, McLoughlin M, Cordon-Cardo C, Schuchman EH, Fuks Z,Kolesnick R: Acid sphingomyelinase-deficient human lympho-(Lond) 489:701–707, 1995

136. Meyer Zu Heringdorf D, Lass H, Alemany R, Laser KT, Neu- blasts and mice are defective in radiation-induced apoptosis. Cell86:189–199, 1996mann E, Zhang C, Schmidt M, Rauen U, Jakobs KH, van Kop-

pen CJ: Sphingosine kinase-mediated Ca21 signalling by G-pro- 155. Amarante-Mendes GP, Naekyung Kim C, Liu L, Huang Y,Perkins CL, Green DR, Bhalla K: Bcr-Abl exerts its antiapo-tein-coupled receptors. EMBO J 17:2830–2837, 1998

137. Lee MJ, Van Brocklyn JR, Thangada S, Liu CH, Hand AR, ptotic effect against diverse apoptotic stimuli through blockageof mitochondrial release of cytochrome C and activation ofMenzeleev R, Spiegel S, Hla T: Sphingosine-1-phosphate as a

ligand for the G protein-coupled receptor EDG-1. Science caspase-3. Blood 91:1700–1705, 1998156. Tepper AD, De Vries E, Van Blitterswijk WJ, Borst J: Order-269:1552–1555, 1998

138. Ancillin N, Hla T: Differential pharmacological properties and ing of ceramide formation, caspase activation, and mitochondrialchanges during CD95- and DNA damage-induced apoptosis.signal transduction of the sphingosine 1-phosphate receptors

EDG-1, EDG-3, and EDG-5. J Biol Chem 274:18997–19902, 1999 J Clin Invest 103:971–978, 1999157. Desnick RJ, Ioannou YA, Eng CM: Alpha-galactosidase A defi-139. Ghosh TK, Bian J, Gill DL: Sphingosine 1-phosphate generated

in the endoplasmic reticulum membrane activates release of ciency: Fabry disease, in The Metabolic and Molecular Bases ofInherited Disease (7th ed), New York, McGraw-Hill, pp 2741–stored calcium. J Biol Chem 269:22628–22635, 1994

140. Buehrer BM, Bardes ES, Bell RM: Protein kinase C-dependent 2784, 1995158. Beutler E: Gaucher disease as a paradigm of current issuesregulation of human erythroleukemia (HEL) cell sphingosine

kinase activity. Biochim Biophys Acta 1303:233–242, 1996 regarding single gene mutations of humans. Proc Natl Acad SciUSA 90:5384–5390, 1993141. Rani CS, Wang F, Fuior E, Berger A, Wu J, Sturgill TW,

LeRoith D, Varticovski L, Spiegel S: Divergence in signal trans- 159. Schiffmann R, Murray GJ, Treco D, Daniel P, Sellos-MouraM, Quirk JM, Zirzow GC, Borowski M, Loveday K, Andersonduction pathways of platelet-derived growth factor (PDGF) and

epidermal growth factor (EGF) receptors: Involvement of sphin- T, Gillespie F, Oliver KL, Jeffries NO, Doo E, Liang TJ, KrepsC, Gunter K, Frei K, Crutchfield K, Selden RF, Brady RO:gosine 1-phosphate in PDGF but not EGF signaling. J Biol Chem

272:10777–10783, 1997 Infusion of alpha-galactosidase A reduces tissue globotriaosycera-mide storage in patients with Fabry disease. Proc Natl Acad Sci142. Melendez A, Floto RA, Gillooly DJ, Harnett MM, Allen

JM: FcgammaRI coupling to phospholipase D initiates sphingo- USA 97:365–370, 2000160. Radin NS: Treatment of Gaucher disease with an enzyme inhibi-sine kinase-mediated calcium mobilization and vesicular traffick-

ing. J Biol Chem 273:9393–9403, 1998 tor. Glycoconj J 13:153–157, 1996161. Liu Y, Wada R, Kawai H, Sango K, Deng C, Tai T, McDonald143. Edsall LC, Van Brocklyn JR, Cuvillier O, Kleuser B, Spiegel

SN: N,N-dimethylsphingosine is a potent competitive inhibitor MP, Araujo K, Crawley JN, Bierfreund U, Sandhoff K, Su-zuki K, Proia RL: A genetic model of substrate deprivationof sphingosine kinase but not of protein kinase C: Modulation of

cellular levels of sphingosine 1-kinase and ceramide. Biochemistry therapy for a glycosphingolipid storage disorder. J Clin Invest103:497–505, 199937:12892–12898, 1998

144. Galardi S, Kishikawa K, Kamibayashi C, Mumby MC, Hannum 162. Platt FM, Neises GR, Reinkensmeier G, Townsend MJ, PerryVH, Proia RL, Winchester B, Dwek RA, Butters TD: Preven-YA: Purification and characterization of ceramide-activated pro-

tein phosphatases. Biochemistry 37:11232–11238, 1998 tion of lysosomal storage in Tay-Sachs mice treated with N-butyl-deoxynojirimycin. Science 276:428–431, 1997145. Nickels JT, Broach JR: A ceramide-activated protein phospha-

tase mediates ceramide-induced G1 arrest of Saccharomyces cere- 163. Jeyakumar M, Butters TD, Cortina-Borja M, Hunnam V, ProiaRL, Perry VH, Dwek RA, Platt FM: Delayed symptom onsetvisiae. Genes Dev 10:382–394, 1996

146. Chalfant CE, Kishikawa K, Mumby MC, Kamibayashi C, Bie- and increased life expectancy in Sandhoff disease mice treatedwith N-butyldeoxynojirimycin. Proc Natl Acad Sci USA 96:6388–lawska A, Hannun YA: Long chain ceramides activate protein

phosphatase-1 and protein phosphatase-2A: Activation is stereo- 6393, 1999164. Abe A, Arend LJ, Lee L, Lingwood C, Brady RO, Shaymanspecific and regulated by phopshatidic acid. J Biol Chem 274:

20313–20317, 1999 JA: Glycosphingolipid depletion in Fabry disease lymphoblastswith potent inhibitors of glucosylceramide synthase. Kidney Int147. Dbaibo GS, Pushkareva MY, Jayadev S, Schwarz JK, Horowitz

JM, Obeid LM, Hannun YA: Retinoblastoma gene product as 57:446–454, 2000165. Karmali MA, Petric M, Lim C, Fleming PC, Arbus GS, Liora downstream target for a ceramide-dependent pathway of growth

arrest. Proc Natl Acad Sci USA 92:1347–1351, 1995 H: The association between isopathic hemolytic uremic syndromeand infection by verotoxin-producing Escherichia coli. J Infect148. Zhang Y, Yao B, Delikat S, Bayoumy S, Lin XH, Basu S,

McGinley M, Chan-Hui PY, Lichenstein H, Kolesnick R: Ki- Dis 151:775–782, 1985166. Nyholm PG, Magnusson G, Zheng Z, Norel R, Binnington-nase suppressor of Ras is ceramide-activated protein kinase. Cell

89:63–72, 1997 Boyd B, Lingwood CA: Two distinct binding sites for globotriao-syl ceramide on verotoxins: Identification by molecular modelling149. Mathias S, Younes A, Kan CC, Orlow I, Joseph C, Kolesnick

RN: Activation of the sphingomyelin signaling pathway in intact and confirmation using deoxy analogues and a new glycolipidreceptor for all verotoxins. Chem Biol 3:263–275, 1996EL4 cells and in a cell-free system by IL-1 beta. Science 259:519–

522, 1993 167. Van De Kar NC, Monnens LA, Karmali MA, Van Hinsbergh


VW: Tumor necrosis factor and interleukin-1 induce expression 170. Zheng T, Li W, Wang J, Altura BT, Altura BM: C2-ceramideattenuates phenylephrine-induced vasoconstriction and elevationof the verocytotoxin receptor globotriaosylceramide on human

endothelial cells: Implications for the pathogenesis of the hemo- in [Ca21]i in rat aortic smooth muscle. Lipids 34:689–695, 1999171. Igarashi J, Thatte HS, Prabhakar P, Golan DE, Michel T:lytic uremic syndrome. Blood 80:2755–2764, 1992

168. Wiels J, Holmes EH, Cochran N, Tursz T, Hakomori S: Enzy- Calcium-independent activation of endothelial nitric oxide syn-thase by ceramide. Proc Natl Acad Sci USA 96:12583–12588, 1999matic and organizational difference in expression of a Burkitt

lymphoma-associated antigen (globotriaosylceramide) in Burkitt 172. Zager RA, Conrad DS, Burkhart K: Ceramide accumulationduring oxidant renal tubular injury: Mechanisms and potentiallymphoma and lymphoblastoid lines. J Biol Chem 259:14783–

14787, 1984 consequences. J Am Soc Nephrol 9:1670–1680, 1998173. Ueda N, Kaushal GP, Hong X, Shah SV: Role of enhanced169. Dickie P, Felser J, Eckhaus M, Bryant J, Silver J, Marinos

N, Notkins AL: HIV-associated nephropathy in transgenic mice ceramide generation in DNA damage and cell death in chemicalhypoxic injury to LLC-PK1 cells. Kidney Int 2:399–406, 1998expressing HIV-1 genes. Virology 185:109–119, 1991

sphingolipids

Documents