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Lipid rafts are small, dynamic membrane areas characterized by the clustering of selected membrane lipids as the result of the spontaneous separation of glycolipids, sphingolipids, and cholesterol in a liquid-ordered phase. The exact dynamics underlying phase separation of membrane lipids in the complex biological membranes are still not fully understood. Nevertheless, alterations in the membrane lipid composition affect the lateral organization of molecules belonging to lipid rafts. Neural lipid rafts are found in brain cells, including neurons, astrocytes, and microglia, and are characterized by a high enrichment of specific lipids depending on the cell type. These lipid rafts seem to organize and determine the function of multiprotein complexes involved in several aspects of signal transduction, thus regulating the homeostasis of the brain. The progressive decline of brain performance along with physiological aging is at least in part associated with alterations in the composition and structure of neural lipid rafts. In addition, neurodegenerative conditions, such as lysosomal storage disorders, multiple sclerosis, and Parkinson's, Huntington's, and Alzheimer's diseases, are frequently characterized by dysregulated lipid metabolism, which in turn affects the structure of lipid rafts. Several events underlying the pathogenesis of these diseases appear to depend on the altered composition of lipid rafts. Thus, the structure and function of lipid rafts play a central role in the pathogenesis of many common neurodegenerative diseases.

Lactosylceramide [LacCer; β-Gal-(1-4)-β-Glc-(1-1)-Cer] has been shown to contain very long fatty acids that specifically modulate neutrophil properties. The interactions between LacCer and proteins and their role in cell signaling processes were assessed by synthesizing two molecular species of azide-photoactivable tritium-labeled LacCer having acyl chains of different lengths. The lengths of the two acyl chains corresponded to those of a short/medium and very long fatty acid, comparable to the lengths of stearic and lignoceric acids, respectively. These derivatives, designated C18-[3H]LacCer-(N3) and C24-[3H]LacCer-(N3), were incorporated into the lipid rafts of plasma membranes of neutrophilic differentiated HL-60 (D-HL-60) cells. C24-[3H]LacCer-(N3), but not C18-[3H]LacCer-(N3), induced the phosphorylation of Lyn and promoted phagocytosis. Incorporation of C24-[3H]LacCer-(N3) into plasma membranes, followed by illumination, resulted in the formation of several tritium-labeled LacCer-protein complexes, including the LacCer-Lyn complex, into plasma membrane lipid rafts. Administration of C18-[3H]LacCer-(N3) to cells, however, did not result in the formation of the LacCer-Lyn complex. These results suggest that LacCer derivatives mimic the biological properties of natural LacCer species and can be utilized as tools to study LacCer-protein interactions, and confirm a specific direct interaction between LacCer species containing very long fatty acids, and Lyn protein, associated with the cytoplasmic layer via myristic/palmitic chains.

Tumor-infiltrating immune cells can be conditioned by molecules released within the microenvironment to thwart antitumor immune responses, thereby facilitating tumor growth. Among immune cells, neutrophils play an important protumorigenic role by favoring neoangiogenesis and/or by suppressing antitumor immune responses. Tumor-derived oxysterols have recently been shown to favor tumor growth by inhibiting dendritic cell migration toward lymphoid organs. We report that tumor-derived oxysterols recruit protumor neutrophils in a liver X receptor (LXR)–independent, CXCR2-dependent manner, thus favoring tumor growth by promoting neoangiogenesis and immunosuppression. We demonstrate that interfering with the oxysterol–CXCR2 axis delays tumor growth and prolongs the overall survival of tumor-bearing mice. These results identify an unanticipated protumor function of the oxysterol–CXCR2 axis and a possible target for cancer therapy.

The bulk structure of biological membranes consists of a bilayer of amphipathic lipids. According to the fluid mosaic model proposed by Singer and Nicholson, the glycerophospholipid bilayer is a two-dimensional fluid construct that allows the lateral movement of membrane components. Different types of lateral interactions among membrane components can take place, giving rise to multiple levels of lateral order that lead to highly organized structures. Early observations suggested that some of the lipid components of biological membranes may play active roles in the creation of these levels of order. In the late 1980s, a diverse series of experimental findings collectively gave rise to the lipid raft hypothesis. Lipid rafts were originally defined as membrane domains, i.e., ordered structures created as a consequence of the lateral segregation of sphingolipids and differing from the surrounding membrane in their molecular composition and properties. This definition was subsequently modified to introduce the notion that lipid rafts correspond to membrane areas stabilized by the presence of cholesterol within a liquid-ordered phase. During the past two decades, the concept of lipid rafts has become extremely popular among cell biologists, and these structures have been suggested to be involved in a great variety of cellular functions and biological events. During the same period, however, some groups presented experimental evidence that appeared to contradict the basic tenets that underlie the lipid raft concept. The concept is currently being re-defined, with greater consistency regarding the true nature and role of lipid rafts. In this article we will review the concepts, criticisms, and the novel confirmatory findings relating to the lipid raft hypothesis.

The dihydroceramide, ceramide, sphingomyelin, lactosylceramide, and ganglioside species of A2780 human ovarian carcinoma cells treated with the synthetic retinoids N-(4-hydroxyphenyl)retinamide (fenretinide, 4-HPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR) in culture were characterized by ESI-MS. We characterized 32 species of ceramide and dihydroceramide, 15 of sphingomyelin, 12 of lactosylceramide, 9 of ganglioside GM2, and 6 of ganglioside GM3 differing for the long-chain base and fatty acid structures. Our results indicated that treatment with both 4-HPR and 4-oxo-4-HPR led to a marked increase in dihydroceramide species, while only 4-oxo-4-HPR led to a minor increase of ceramide species. Dihydroceramides generated in A2780 cells in response to 4-HPR or 4-oxo-4-HPR differed for their fatty acid content, suggesting that the two drugs differentially affect the early steps of sphingolipid synthesis. Dihydroceramides produced upon treatments with the drugs were further used for the synthesis of complex dihydrosphingolipids, whose levels dramatically increased in drug-treated cells.

The dihydroceramide, ceramide, sphingomyelin, lactosylceramide, and ganglioside species of A2780 human ovarian carcinoma cells treated with the synthetic retinoids N-(4-hydroxyphenyl)retinamide (fenretinide, 4-HPR) and 4-oxo-N-(4-hydroxyphenyl)retinamide (4-oxo-4-HPR) in culture were characterized by ESI-MS. We characterized 32 species of ceramide and dihydroceramide, 15 of sphingomyelin, 12 of lactosylceramide, 9 of ganglioside GM2, and 6 of ganglioside GM3 differing for the long-chain base and fatty acid structures. Our results indicated that treatment with both 4-HPR and 4-oxo-4-HPR led to a marked increase in dihydroceramide species, while only 4-oxo-4-HPR led to a minor increase of ceramide species. Dihydroceramides generated in A2780 cells in response to 4-HPR or 4-oxo-4-HPR differed for their fatty acid content, suggesting that the two drugs differentially affect the early steps of sphingolipid synthesis. Dihydroceramides produced upon treatments with the drugs were further used for the synthesis of complex dihydrosphingolipids, whose levels dramatically increased in drug-treated cells.

Human fibroblasts from normal subjects and Niemann-Pick A (NPA) disease patients were fed with two labeled metabolic precursors of sphingomyelin (SM), [3H]choline and photoactivable sphingosine, that entered into the biosynthetic pathway allowing the synthesis of radioactive phosphatidylcholine and SM, and of radioactive and photoactivable SM ([3H]SM-N3). Detergent resistant membrane (DRM) fractions prepared from normal and NPA fibroblasts resulted as highly enriched in [3H]SM-N3. However, lipid and protein analysis showed strong differences between the two cell types. After cross-linking, different patterns of SM-protein complexes were found, mainly associated with the detergent soluble fraction of the gradient containing most cell proteins. After cell surface biotinylation, DRMs were immunoprecipitated using streptavidin. In conditions that maintain the integrity of domain, SM-protein complexes were detectable only in normal fibroblasts, whereas disrupting the membrane organization, these complexes were not recovered in the immunoprecipitate, suggesting that they involve proteins belonging to the inner membrane layer. These data suggest that differences in lipid and protein compositions of these cell lines determine specific lipid-protein interactions and different clustering within plasma membrane. In addition, our experiments show that photoactivable sphingolipids metabolically synthesized in cells can be used to study sphingolipid protein environments and sphingolipid-protein interactions.

Human fibroblasts from normal subjects and Niemann-Pick A (NPA) disease patients were fed with two labeled metabolic precursors of sphingomyelin (SM), [3H]choline and photoactivable sphingosine, that entered into the biosynthetic pathway allowing the synthesis of radioactive phosphatidylcholine and SM, and of radioactive and photoactivable SM ([3H]SM-N3). Detergent resistant membrane (DRM) fractions prepared from normal and NPA fibroblasts resulted as highly enriched in [3H]SM-N3. However, lipid and protein analysis showed strong differences between the two cell types. After cross-linking, different patterns of SM-protein complexes were found, mainly associated with the detergent soluble fraction of the gradient containing most cell proteins. After cell surface biotinylation, DRMs were immunoprecipitated using streptavidin. In conditions that maintain the integrity of domain, SM-protein complexes were detectable only in normal fibroblasts, whereas disrupting the membrane organization, these complexes were not recovered in the immunoprecipitate, suggesting that they involve proteins belonging to the inner membrane layer. These data suggest that differences in lipid and protein compositions of these cell lines determine specific lipid-protein interactions and different clustering within plasma membrane. In addition, our experiments show that photoactivable sphingolipids metabolically synthesized in cells can be used to study sphingolipid protein environments and sphingolipid-protein interactions.

Dendritic cell (DC) migration to secondary lymphoid organs is a crucial step to initiate adaptive immune responses. This step requires the expression of a functional CCR7 chemokine receptor on DC undergoing maturation. Here, we show that the natural retinoid 9‐cisretinoic acid (9cRA) and the synthetic retinoid fenretinide (4‐HPR) specifically inhibit the functional up‐regulation of CCR7 on maturing human DCs, without affecting early steps of DC maturation. As a consequence, mature DCs do not migrate in vitro toward the chemokine CCL19. Importantly, 4‐HPR and 9cRA by inhibiting the expression of CCR7 on bone marrow‐derived murine DCs dampen their in vivo migration to draining lymph nodes. 4‐HPR also inhibits the expression of the chemokine receptors CXCR4, therefore, impairing in vitro migration of human DCs to CXCL12 and inhibiting in vivo the CXCR4‐dependent migration of the posterior lateral line primordium (PLLp) in zebrafish embryos. Taken together, these data highlight a novel function of retinoids and suggest the possibility of using retinoids to treat inflammatory or autoimmune diseases.

The integrin CD11b/CD18 plays a central role in neutrophil phagocytosis. Although CD11b/CD18 binds a wide range of ligands, including C3bi and β‐glucan, and transmits outside‐in signaling, the mechanism of this signaling responsible for phagocytosis remains obscure. Here, we report that lactosylceramide (LacCer)‐enriched lipid rafts are required for CD11b/CD18‐mediated phagocytosis of nonopsonized zymosans (NOZs) by human neutrophils. Anti‐CD11b and anti‐LacCer antibodies inhibited the binding of NOZs to neutrophils and the phagocytosis of NOZs. During phagocytosis of NOZ, CD11b and LacCer were accumulated and colocalized in the actin‐enriched phagocytic cup regions. Immunoprecipitation experiments suggested that CD11b/CD18 was mobilized into the LacCer‐enriched lipid rafts during phagocytosis of NOZs. DMSO‐treated, neutrophil‐like HL‐60 cells (D‐HL‐60 cells) lacking Lyn‐coupled, LacCer‐mediated signaling showed little phagocytosis of NOZs. However, loading of D‐HL‐60 cells with C24 fatty acid chain‐containing LacCer (C24‐LacCer) reconstructed functional Lyn‐associated, LacCer‐enriched lipid rafts, and restored D‐HL‐60 cell NOZ phagocytic activity, which was inhibited by anti‐LacCer and anti‐CD11b antibodies. Lyn knockdown by small interfering RNA blocked the effect of C24:1‐LacCer loading on D‐HL‐60 cell phagocytosis of NOZs. CD11b/CD18 activation experiments indicated phosphorylation of LacCer‐associated Lyn by activation of CD11b. Taken together, these observations suggest that CD11b activation causes translocation of CD11b/CD18 into Lyn‐coupled, LacCer‐enriched lipid rafts, allowing neutrophils to phagocytose NOZs via CD11b/CD18.

The ceramide (Cer) and sphingomyelin (SM) species of cultured differentiated rat cerebellar granule cells and human fibroblasts were characterized by electrospray ionization-mass spectrometry. We identified 35 different species of Cer and 18 species of SM in human fibroblasts, and 35 different species of Cer and 9 species of SM were characterized in rat neurons. The main Cer species of rat cerebellar granule cells contained d18:1 sphingosine linked with palmitic, stearic, or nervonic fatty acid, and the two main SM species were d18:1,16:0 and d18:1,18:0. Both sphingolipids were enriched in detergent-resistant membranes (DRMs; or lipid rafts), and significant differences were found in the sphingolipid patterns of DRMs and of detergent-soluble fractions (DSF) from these cells. In human fibroblasts, the main Cer species were d18:1,16:0, d18:2,16:0, d18:1,24:0, d18:2,24:0, d18:1,24:1, and d18:2,24:1; the most represented species of SM were d18:1,16:0, d18:1,24:0, and d18:1,24:1. In these cells, SM was highly enriched in DRMs and Cer was mainly associated with DSF, and the species found in DRMs were markedly different from those found in DSF.

The ceramide (Cer) and sphingomyelin (SM) species of cultured differentiated rat cerebellar granule cells and human fibroblasts were characterized by electrospray ionization-mass spectrometry. We identified 35 different species of Cer and 18 species of SM in human fibroblasts, and 35 different species of Cer and 9 species of SM were characterized in rat neurons. The main Cer species of rat cerebellar granule cells contained d18:1 sphingosine linked with palmitic, stearic, or nervonic fatty acid, and the two main SM species were d18:1,16:0 and d18:1,18:0. Both sphingolipids were enriched in detergent-resistant membranes (DRMs; or lipid rafts), and significant differences were found in the sphingolipid patterns of DRMs and of detergent-soluble fractions (DSF) from these cells. In human fibroblasts, the main Cer species were d18:1,16:0, d18:2,16:0, d18:1,24:0, d18:2,24:0, d18:1,24:1, and d18:2,24:1; the most represented species of SM were d18:1,16:0, d18:1,24:0, and d18:1,24:1. In these cells, SM was highly enriched in DRMs and Cer was mainly associated with DSF, and the species found in DRMs were markedly different from those found in DSF.

PTX (Paclitaxel®) is an antimitotic agent used in the treatment of a number of major solid tumours, particularly in breast and ovarian cancer. This study was undertaken to gain insight into the molecular alterations producing PTX resistance in ovarian cancer. PTX treatment is able to induce apoptosis in the human ovarian carcinoma cell line, CABA I. PTX-induced apoptosis in CABA I cells was accompanied by an increase in the cellular Cer (ceramide) levels and a decrease in the sphingomyelin levels, due to the activation of sphingomyelinases. The inhibition of acid sphingomyelinase decreased PTX-induced apoptosis. Under the same experimental conditions, PTX had no effect on Cer and sphingomyelin levels in the stable PTX-resistant ovarian carcinoma cell line, CABA-PTX. The acquisition of the PTX-resistant phenotype is accompanied by unique alterations in the complex sphingolipid pattern found on lipid extraction. In the drug-resistant cell line, the levels of sphingomyelin and neutral glycosphingolipids were unchanged compared with the drug-sensitive cell line. The ganglioside pattern in CABA I cells is more complex compared with that of CABA-PTX cells. Specifically, we found that the total ganglioside content in CABA-PTX cells was approximately half of that in CABA I cells, and GM3 ganglioside content was remarkably higher in the drug-resistant cell line. Taken together our findings indicate that: i) Cer generated by acid sphingomyelinase is involved in PTX-induced apoptosis in ovarian carcinoma cells, and PTX-resistant cells are characterized by their lack of increased Cer upon drug treatment, ii) PTX resistance might be correlated with an alteration in metabolic Cer patterns specifically affecting cellular ganglioside composition.

Treatment with methyl-β-cyclodextrin (MCD) induced a time- and dose-dependent efflux of cholesterol, sphingolipids, and phosphatidylcholine (PC) from cerebellar neurons differentiated in culture. With a “mild” treatment, the loss of cell lipids induced a deep reorganization of the remaining membrane lipids. In fact, the amount of PC associated with a Triton X-100-insoluble membrane fraction (highly enriched in sphingolipids and cholesterol in nontreated cells) was lowered by the treatment. This suggested a reduction of the lipid domain area. However, the cholesterol and sphingolipid enrichment of this fraction remained substantially unchanged, suggesting the existence of dynamic processes aimed at preserving the segregation of cholesterol and sphingolipids in membrane domains. Under these conditions, the lipid membrane domains retained the ability to sort signaling proteins, such as Lyn and c-Src, but cells displayed deep alterations in their membrane permeability. However, normal membrane permeability was restored by loading cells with cholesterol. When MCD treatment was more stringent, a large loss of cell lipids occurred, and the lipid domains were much less enriched in cholesterol and lost the ability to sort specific proteins.

In this paper, we show that caveolin-1 is abundantly present in a cell line of immortalized gonadotropin-releasing hormone-expressing neurons (GN11). In contrast to GN11, caveolin is undetectable in a cognate cell line of immortalized gonadotropin-releasing hormone-secreting neurons (GT1-7). These two cell lines are characterized by a radically different sphingolipid metabolism. After incubation in the presence of tracer amount of [1-3H]sphingosine, GN11 and GT1-7 neurons incorporated similar amounts of radioactivity. In GT1-7 neurons, [1-3H]sphingosine metabolism was markedly oriented toward the biosynthesis of complex sphingolipids. In fact, almost all the radioactivity in the lipid extracts from GT1-7 cells was associated with biosynthetic products (ceramide, sphingomyelin, and glycosphingolipids). In particular glycosphingolipids represented more than 65% of total lipid radioactivity in these cells, and the main glycosphingolipid was GM3 ganglioside (about 47% of total lipid radioactivity). In the case of GN11 neurons, a high portion of [1-3H]sphingosine underwent complete degradation, as indicated by the formation of high levels of radioactive phosphatidylethanolamine (about 23% of lipid radioactivity). Moreover, the main complex sphingolipid in GN11 neurons was not a glycolipid, but sphingomyelin (its level in these cells, about 54% of lipid radioactivity, was two-fold higher than in GT1-7). Glycolipids, gangliosides in particular, were present in low amount (9.5% of lipid radioactivity) if compared with the cognate GT1-7 cell line, and GM3 was almost absent in GN11 neurons. Despite the radical differences in ganglioside and caveolin content, from both cell types a membrane fraction similarly enriched in sphingolipids was prepared. In the case of GN11 cells, this fraction was also enriched in caveolin. The presence of caveolin or GM3 may correlate with different functional properties linked to the stage of neuronal maturation, since GN11 and GT1-7 are representative, respectively, of immature, migrating, and differentiated, postmigratory gonadotropin-releasing hormone-positive neurons.

In the present paper, we report on the properties of sphingolipid-enriched domains of rat cerebellar granule cells in culture at different stages of neuronal development. The major lipid components of these domains were glycerophospholipids and cholesterol. Glycerophospholipids were 45–75‥ and cholesterol 15–45‥ of total lipids of the domains. This corresponded to 5–17‥ of total cell glycerophospholipids and 15–45‥ of total cell cholesterol. Phosphatidylcholine, mainly dipalmitoylphosphatidylcholine, was 66–85‥ of all the glycerophospholipids associated with these domains. Consequently, the palmitoyl residue was significantly enriched in the domains. The surface occupied by these structures increased during development. 40–70‥ of cell sphingolipids segregated in sphingolipid-enriched membrane domains, with the maximum ganglioside density in fully differentiated neurons. A high content of ceramide was found in the domains of aging neurons. Then, the sphingolipid/glycerophospholipid molar ratio was more than doubled during the initial stage of development, whereas the cholesterol/glycerophospholipid molar ratio gradually decreased during in vitrodifferentiation. Phosphorylated phosphoinositides, which were scant in the domains of undifferentiated cells, dramatically increased during differentiation and aging in culture. Proteins were minor components of the domains (0.1–2.8‥ of all domain components). Phosphotyrosine-containing proteins were selectively recovered in the sphingolipid-enriched domain. Among these, Src family protein-tyrosine kinases, known to participate to the process of neuronal differentiation, were associated with the sphingolipid-enriched domains in a way specific for the type of kinase and for the developmental stage of the cell. Proteins belonging to other signaling pathways, such as phosphoinositide 3-kinase and its downstream target, Akt, were not associated with the domains.

Sphingolipid-enriched membrane domains, characterized by a particular protein and lipid composition, have been detected in a variety of cells. However, limited data are available concerning these domains in neuronal cells. We analyzed the lipid and protein composition of a sphingolipid-enriched membrane fraction prepared from primary rat cerebellar granule cells differentiated in culture. Although the protein content of this fraction was only 1.4% of total cellular protein, 60% of the gangliosides, 67% of the sphingomyelin, 50% of the ceramide, and 40% of the cholesterol were located in this fraction. The protein pattern of the sphingolipid-enriched domain fraction was dramatically different from that associated with the cell homogenate. This fraction contained 25% of the tyrosine-phosphorylated proteins and was enriched in two proteins with apparent molecular masses of 135 and 15 kDa. 12% of cellular glycerophospholipids were located in the fraction, with phosphatidylcholine having the highest enrichment. The molar ratio between proteins, glycerophospholipids, cholesterol, sphingomyelin,ceramide and gangliosides in cerebellar granule cells was 1.6:41.6:6.1:1.3:0.3:1 in the cell homogenate and 0.04:8.3:4.0:1.4:0.2:1 in the sphingolipid-enriched membrane fraction. These data indicate that selected proteins segregate with sphingolipids in specialized domains in the membrane of cultured neurons.

Sphingolipid-enriched membrane domains, characterized by a particular protein and lipid composition, have been detected in a variety of cells. However, limited data are available concerning these domains in neuronal cells. We analyzed the lipid and protein composition of a sphingolipid-enriched membrane fraction prepared from primary rat cerebellar granule cells differentiated in culture. Although the protein content of this fraction was only 1.4% of total cellular protein, 60% of the gangliosides, 67% of the sphingomyelin, 50% of the ceramide, and 40% of the cholesterol were located in this fraction. The protein pattern of the sphingolipid-enriched domain fraction was dramatically different from that associated with the cell homogenate. This fraction contained 25% of the tyrosine-phosphorylated proteins and was enriched in two proteins with apparent molecular masses of 135 and 15 kDa. 12% of cellular glycerophospholipids were located in the fraction, with phosphatidylcholine having the highest enrichment. The molar ratio between proteins, glycerophospholipids, cholesterol, sphingomyelin, ceramide and gangliosides in cerebellar granule cells was 1.6:41.6:6. 1:1.3:0.3:1 in the cell homogenate and 0.04:8.3:4.0:1.4:0.2:1 in the sphingolipid-enriched membrane fraction. These data indicate that selected proteins segregate with sphingolipids in specialized domains in the membrane of cultured neurons.

Differentiation and neuritogenesis of mouse neuroblastoma Neuro2a cells are induced by exogenous ganglioside but are not induced by nerve growth factor because its receptor is absent in these cells. In view of the emerging concept of the "glycosphingolipid-enriched domain" (GEM), we studied the mechanism of the ganglioside effect, focusing on the structure and function of such a domain. GEM in Neuro2a cells, separated as a low density membrane fraction, contains essentially all glycosphingolipids and sphingomyelin, together with five signal transducer molecules (c-Src, Lyn, Csk, Rho A, Ha-Ras). (3)H-Labeled Il(3)NeuAc-LacCer (GM3), Gb4Cer (globoside), and Il(3)NeuAc-Gg4Cer (GM1) added exogenously to cells were incorporated and concentrated in the low density GEM fraction. In contrast, more than 50% of glycerophospholipids and 30% of cholesterol were found in the high density fraction. (3)H-Labeled phosphatidylcholine added exogenously to cells was incorporated exclusively in the high density fraction. c-Src, the predominant signal transducer in the microdomain, was coimmunoprecipitated with anti-GM3 antibody DH2 or with anti-Csk; reciprocally, Csk was coimmunoprecipitated with anti-c-Src, indicating a close association of GM3, c-Src, and Csk. Brief stimulation of an isolated GEM fraction by the exogenous addition of GM3, but not lactosylceramide, caused enhanced c-Src phosphorylation with a concomitant decrease of Csk level in GEM. A decreased Csk/c-Src ratio in GEM may cause activation of c-Src because Csk is a negative regulator of c-Src. The effect of exogenous GM3 on c-Src activity was also observed in intact Neuro2a cells. Activation of c-Src was followed by rapid and prolonged (60 min) enhancement of mitogen-activated protein kinase activity leading to neuritogenesis. Thus, the ganglioside induction of neuritogenesis in Neuro2a cells is mediated by GEM structure and function.

Differentiation and neuritogenesis of mouse neuroblastoma Neuro2a cells are induced by exogenous ganglioside but are not induced by nerve growth factor because its receptor is absent in these cells. In view of the emerging concept of the “glycosphingolipid-enriched domain” (GEM), we studied the mechanism of the ganglioside effect, focusing on the structure and function of such a domain. GEM in Neuro2a cells, separated as a low density membrane fraction, contains essentially all glycosphingolipids and sphingomyelin, together with five signal transducer molecules (c-Src, Lyn, Csk, Rho A, Ha-Ras). 3H-Labeled Il3NeuAc-LacCer (GM3), Gb4Cer (globoside), and Il3NeuAc-Gg4Cer (GM1) added exogenously to cells were incorporated and concentrated in the low density GEM fraction. In contrast, more than 50% of glycerophospholipids and 30% of cholesterol were found in the high density fraction.3H-Labeled phosphatidylcholine added exogenously to cells was incorporated exclusively in the high density fraction. c-Src, the predominant signal transducer in the microdomain, was coimmunoprecipitated with anti-GM3 antibody DH2 or with anti-Csk; reciprocally, Csk was coimmunoprecipitated with anti-c-Src, indicating a close association of GM3, c-Src, and Csk. Brief stimulation of an isolated GEM fraction by the exogenous addition of GM3, but not lactosylceramide, caused enhanced c-Src phosphorylation with a concomitant decrease of Csk level in GEM. A decreased Csk/c-Src ratio in GEM may cause activation of c-Src because Csk is a negative regulator of c-Src. The effect of exogenous GM3 on c-Src activity was also observed in intact Neuro2a cells. Activation of c-Src was followed by rapid and prolonged (60 min) enhancement of mitogen-activated protein kinase activity leading to neuritogenesis. Thus, the ganglioside induction of neuritogenesis in Neuro2a cells is mediated by GEM structure and function.

The metabolic fate of exogenous [3H]sphingosine was investigated in five types of cultured cells: primary cultures of neurons and astrocytes, murine and human neuroblastoma cells and human skin fibroblasts. After administration of 40 nM [3-3H]sphingosine into a cell-conditioned medium containing fetal calf serum, all cell types rapidly and efficiently incorporated the long-chain base in a time-dependent fashion. In all cases, after a 120 min pulse, the amount of radioactivity taken up was in the range of the endogenous sphingosine content. However, unchanged [3H]sphingosine represented only a very minor portion of the label incorporated into cells throughout the pulse period (10–120 min), indicating rapid and efficient sphingosine metabolism in these cells. Most of the [3H]sphingosine taken up was metabolically processed, either by degradation (assessed as 3H2O release into the culture medium) or by N-acylation (mainly to radioactive ceramide, sphingomyelin, neutral glycolipids and gangliosides). [3H]Sphingosine 1-phosphate accounted for less than 2% of the total radioactivity incorporated in all cases. Throughout the pulse period and in all cell types, 3H-labelled organic metabolites largely prevailed over 3H2O, indicating that N-acylation is the major metabolic fate of sphingosine in these cells under apparently physiological conditions. These results are consistent with the notion that sphingosine has a rapid turnover in the cells studied, and indicate that regulation of the basal level of this bioactive molecule occurs mainly through N-acylation.

The qualitative and quantitative pattern of endogenous gangliosides and the routes of metabolic processing of exogenous GM1,3H labeled in the sphingosine moiety (Sph-3H GM1) were studied in cerebellar granule cells during differentiation in vitro. During the first 7–8 days in culture the ganglioside content markedly increased, and the qualitative pattern showed, in percentage terms, a drastic decrease of GD3 and a marked increase of GD2, O-Ac-GT1b, O-Ac-GQ1b and GQ1b. After pulse with (Sph-3H) GM1, at all the investigated days in culture, different radiolabelled lipids were formed indicating that taken up exogenous GM1 was degraded and that its catabolic fragments, and partly GM1 itself, were used for biosynthetic purposes; moreover radioactive water was measured in the culture medium during chase indicating that labelled sphingosine underwent also degradation. The uptake of exogenous GM1 and the extent of its metabolic processing per cell unit increased during differentiation: a) GM2 was the major metabolic product and was relatively more abundant at 2 than 7 days in culture; b) the percentage of metabolites of biosynthetic origin over total metabolites increased during differentiation, especially at the short pulse times; c) among the metabolites of anabolic origin sphingomyelin equalled gangliosides at 2 days, whereas it was largely overcome by gangliosides at 7 days in culture; d) at 4 and 7 days in culture a radioactive substance, not yet identified, was present, whereas no trace of it was found at 2 days. In conclusion, cerebellar granule cells in culture feature a different pattern of endogenous gangliosides and display different ability to metabolically process exogenous GM1 ganglioside in the undifferentiated and fully differentiated stage.

Cerebellar granule cells differentiated in culture were incubated with ganglioside [ 3H-Sph]GDla in order to have it inserted into the plasma membrane, internalized by endocytosis, and metabolized. The metabolites formed included GMI. product of GDla desialosylation. No GMI or other metabolites were present in the incubation medium, whereas with the lysosomal apparatus blocked by chloroquine. or GDI a endocytosis prevented at 4° C, the only metabolite formed was GMI. These results suggest that GDI a desialosylation did not occur either extracellularly or intracellularly but likely, at the membrane level. Similar results were obtained with [ 3H-Gal]GDlb. whereas no degradation of [ 3H-NeuAc]GMI took place in the presence of chloroquine or at 4°C. In conclusion, cerebellar granule cells express in vivoa sialidasc, presumably located on the cell surface. that affects GDIa and GDIb but not GMI.

The possible relationship between metabolism and biological effects of sphingosine was investigated in Neuro2a cells. [C3-3H]-sphingosine, administered at different doses (80 pmol-80 nmolmg cell protein). Amounts up to hundredfold were rapidly taken up and metabolized, the intracellular content of sphingosine being processed within 2 h. At low doses, [3H]-sphingosine represented a minor portion of the cellular radiolabel, and N-acylated metabolites, particularly ceramide, prevailed over degradation products. Neuro2a cell differentiation took place in conjunction with ceramide increase. At increasing exogenous sphingosinecell ratio, the acylation process became saturated while sphingosine degradation increased proportionally. From this point on [3H]-sphingosine accumulated and cell toxicity occurred. In conclusion, in Neuro2a cells the biological effects exerted by exogenous sphingosine are strictly connected to the exogenous sphingosinecell ratio and to the capacity of the cell to metabolize sphingosine.

Mouse melanoma B16 cells are characterized by the predominant presence of ganglioside GM3 and adhere to lactosylceramide- or Gg3-coated plates through interaction of GM3 with lactosylceramide or Gg3, whereby not only adhesion but also spreading and enhancement of cell motility occur (Kojima, N., Hakomori, S. (1991) J. Biol. Chem. 266, 17552–17558). We now report that the adhesion process is based essentially on a glycosphingolipid-enriched microdomain (GEM) at the B16 cell surface, since >90% of GM3 present in the original cells is found in GEM, and GEM is also enriched in several signal transducer molecules, e.g.c-Src, Ras, Rho, and focal adhesion kinase (FAK). GEM was isolated as a low density membranous fraction by homogenization of B16 cells in lysis buffer under two different conditions (i.e.buffer containing 1% Triton X-100, or hypertonic sodium carbonate without detergent), followed by sucrose density gradient centrifugation. A close association of GM3 with c-Src, Rho, and FAK was indicated by co-immunoprecipitation of GM3 present in GEM by anti-GM3 monoclonal antibody DH2, followed by Western blotting with antibodies directed to these transducer molecules. The following data indicate that GEM is a structural and functional unit for initiation of GM3-dependent cell adhesion coupled with signal transduction. 1) Tyrosine phosphorylation in FAK was greatly enhanced in B16 cells adhered to Gg3-coated plates but was minimal in cells adhered to GM3-coated, GlcCer-coated, or noncoated plates. 2) GTP loading on Ras and Rho increased significantly when cells were adhered to Gg3-coated plates, compared with GM3-coated, GlcCer-coated, or noncoated plates. Since Ras and Rho are closely associated with GM3 in GEM, cell adhesion/stimulation through GM3 in GEM may induce activation of Ras and Rho through enhanced GTP binding.

Exogenous sphingomyelin, radiolabelled at the sphingosine moiety, was administered to primary cultures of cerebellar granule cells and astrocytes for different pulse times (20 min–2 h) and the fate of the radioactivity was followed. Ceramide was the main metabolic product in both cells, whereas sphingosine, glucosyl-ceramide and gangliosides GM3 and GD3 were produced only in astrocytes. When endocytosis was prevented and the lysosomal apparatus inactivated, ceramide formation was reduced slightly in granule cells and almost completely blocked in astrocytes, with disappearance of sphingosine, glucosyl-ceramide, GM3 and GD3. These data indicate that (a) ceramide is rapidly produced in cerebellar granule cells and astrocytes, presumably at the level of the plasma membrane in the first cell type, and of the lysosomes in the second one; (b) sphingosine is produced in cerebellar astrocytes by lysosomal sphingomyelin degradation and is partly reused for glucosyl-ceramide and ganglioside biosynthesis.

Cerebellar granule cells in culture were subjected to a pulse (0.5–4 h)‐chase (0–4 h) of 10−6M [3H]ganglioside GM1 carrying the radioactive label at the level of NeuAc ([3H‐NeuAc]GM1), Sph ([3H‐Sph]GM1) or Gal ([3H‐GallGMl) and the formed [3H]metabolites were determined. With all forms of [3H]GM1, there was formation of [3H]catabolites, including [3H]H2O and [3H]biosynthetic products obtained by recycling of [3H]NeuAc, [3H]Sph and [3H]Gal released during intralysosomal ganglioside degradation (salvage processes). Much higher amounts of [3MH2O were produced from [3H‐Gal]GM1 than [3H‐Sph]GM1 and [3H‐NeuAc]GM1; conversely, more products from salvage processes (polysialogangliosides GD1a, GDlb, GTlb, O‐acetylated GT1b, protein‐bound radioactivity) were obtained with [3H‐NeuAc]GM1; than the two other forms of [3H]GM1, Liberated [3H]NeuAc produced 10‐fold less tritiated water and 10‐fold higher salvage products than [3H]Gal. Using [3H‐NeuAc]GM1; granule cells appeared to metabolize 7.7% of membrane‐incorporated exogenous GM1 per hour with a high degree of NeuAc recycling and the calculated metabolic half‐life was 6.5 h.

Exogenous sphingomyelin, radiolabelled at the sphingosine moiety, was administered to primary cultures of cerebellar granule cells and astrocytes for different pulse times (20 min–2 h) and the fate of the radioactivity was followed. Ceramide was the main metabolic product in both cells, whereas sphingosine, glucosyl‐ceramide and gangliosides GM3 and GD3 were produced only in astrocytes. When endocytosis was prevented and the lysosomal apparatus inactivated, ceramide formation was reduced slightly in granule cells and almost completely blocked in astrocytes, with disappearance of sphingosine, glucosyl‐ceramide, GM3 and GD3. These data indicate that (a) ceramide is rapidly produced in cerebellar granule cells and astrocytes, presumably at the level of the plasma membrane in the first cell type, and of the lysosomes in the second one; (b) sphingosine is produced in cerebellar astrocytes by lysosomal sphingomyelin degradation and is partly reused for glucosyl‐ceramide and ganglioside biosynthesis.

The possible involvement of protein phosphatase in ceramide-mediated neural cell differentiation was investigated. Neuroblastoma Neuro2a cell differentiation induced by retinoic acid, or conditions causing an increase in cellular ceramide, was significantly inhibited by the serine/threonine phosphatase inhibitor okadaic acid, at concentrations as low as 2.5 nM. A crude cytosolic preparation from Neuro2a cells was found to have a cation-independent protein phosphatase activity that was stimulated by ceramide in a dose-dependent manner. Short- and long-chain ceramides, but not sphingosine and related dihydroderivatives, were active. Ceramide-activated protein phosphatase activity from Neuro2a cells was inhibited by 5 nM okadaic acid. The data indicate that a type 2A protein phosphatase is involved in ceramide-mediated differentiation of Neuro2a cells.

Mouse melanoma B16 cells are characterized by the predominant presence of ganglioside GM3 and adhere to lactosylceramide- or Gg3-coated plates through interaction of GM3 with lactosylceramide or Gg3, whereby not only adhesion but also spreading and enhancement of cell motility occur (Kojima, N., Hakomori, S. (1991) J. Biol. Chem. 266, 17552-17558). We now report that the adhesion process is based essentially on a glycosphingolipid-enriched microdomain (GEM) at the B16 cell surface, since >90% of GM3 present in the original cells is found in GEM, and GEM is also enriched in several signal transducer molecules, e.g. c-Src, Ras, Rho, and focal adhesion kinase (FAK). GEM was isolated as a low density membranous fraction by homogenization of B16 cells in lysis buffer under two different conditions (i.e. buffer containing 1% Triton X-100, or hypertonic sodium carbonate without detergent), followed by sucrose density gradient centrifugation. A close association of GM3 with c-Src, Rho, and FAK was indicated by co-immunoprecipitation of GM3 present in GEM by anti-GM3 monoclonal antibody DH2, followed by Western blotting with antibodies directed to these transducer molecules. The following data indicate that GEM is a structural and functional unit for initiation of GM3-dependent cell adhesion coupled with signal transduction. 1) Tyrosine phosphorylation in FAK was greatly enhanced in B16 cells adhered to Gg3-coated plates but was minimal in cells adhered to GM3-coated, GlcCer-coated, or noncoated plates. 2) GTP loading on Ras and Rho increased significantly when cells were adhered to Gg3-coated plates, compared with GM3-coated, GlcCer-coated, or noncoated plates. Since Ras and Rho are closely associated with GM3 in GEM, cell adhesion/stimulation through GM3 in GEM may induce activation of Ras and Rho through enhanced GTP binding.

Cerebellar granule cells differentiated in culture were incubated with ganglioside [3H‐Sph]GDla in order to have it inserted into the plasma membrane, internalized by endocytosis, and metabolized. The metabolites formed included GMI. product of GDla desialosylation. No GMI or other metabolites were present in the incubation medium, whereas with the lysosomal apparatus blocked by chloroquine. or GDI a endocytosis prevented at 4° C, the only metabolite formed was GMI. These results suggest that GDI a desialosylation did not occur either extracellularly or intracellularly but likely, at the membrane level. Similar results were obtained with [3H‐Gal]GDlb. whereas no degradation of [3H‐NeuAc]GMI took place in the presence of chloroquine or at 4°C. In conclusion, cerebellar granule cells express in vivoa sialidasc, presumably located on the cell surface. that affects GDIa and GDIb but not GMI.

Current studies indicate that ceramide is involved in the regulation of important cell functions, namely cell growth, differentiation, and apoptosis. In the present study, the possible role of ceramide in the differentiation of neuroblastoma Neuro2a cells was investigated. The following results were obtained. (a) Ceramide content of Neuro2a cells, induced to differentiate by retinoic acid (RA) treatment rapidly increased after addition of RA, was maintained at high levels in RA-differentiated cells and returned to the starting levels with removal of RA and reversal of differentiation; under the same conditions, the sphingosine content remained unchanged. (b) After a short pulse with [3H]sphingomyelin or [3H]sphingosine or L-[3H]serine, the metabolic formation of ceramide was markedly higher and more rapid in RA-differentiated than undifferentiated cells. (c) Inhibitors of ceramide biosynthesis (Fumonisin B1, beta-chloroalanine and L-cycloserine) diminished the extent of the differentiating effect of RA and concomitantly Cer content decreased. (d) The activity of neutral sphingomyelinase increased after addition of RA, maintained high levels in RA-differentiated cells, and returned to the initial levels with removal of RA. (e) Experimental conditions that cause an elevation of ceramide content (treatment with sphingosine or ceramide or C2-ceramide or bacterial sphingomyelinase) inhibited cell proliferation and stimulated neurite outgrowth; dihydro-analogues of sphingosine, ceramide, and C2-ceramide had no effect on differentiation. (f) treatment with Fumonisin B1 completely inhibited sphingosine-induced differentiation. These data suggest a specific bioregulatory function of ceramide in the control of Neuro2a cell growth and differentiation and pose the general hypothesis of a mediator role of ceramide in the differentiation of cells of neural origin.

Current studies indicate that ceramide is involved in the regulation of important cell functions, namely cell growth, differentiation, and apoptosis. In the present study, the possible role of ceramide in the differentiation of neuroblastoma Neuro2a cells was investigated. The following results were obtained. (a) Ceramide content of Neuro2a cells, induced to differentiate by retinoic acid (RA) treatment rapidly increased after addition of RA, was maintained at high levels in RA-differentiated cells and returned to the starting levels with removal of RA and reversal of differentiation; under the same conditions, the sphingosine content remained unchanged. (b) After a short pulse with [3H]sphingomyelin or [3H]sphingosine or L-[3H]serine, the metabolic formation of ceramide was markedly higher and more rapid in RA-differentiated than undifferentiated cells. (c) Inhibitors of ceramide biosynthesis (Fumonisin B1, β-chloroalanine and L-cycloserine) diminished the extent of the differentiating effect of RA and concomitantly Cer content decreased. (d) The activity of neutral sphingomyelinase increased after addition of RA, maintained high levels in RA-differentiated cells, and returned to the initial levels with removal of RA. (e) Experimental conditions that cause an elevation of ceramide content (treatment with sphingosine or ceramide or C2-ceramide or bacterial sphingomyelinase) inhibited cell proliferation and stimulated neurite outgrowth; dihydro-analogues of sphingosine, ceramide, and C2-ceramide had no effect on differentiation. (f) treatment with Fumonisin B1 completely inhibited sphingosine-induced differentiation. These data suggest a specific bioregulatory function of ceramide in the control of Neuro2a cell growth and differentiation and pose the general hypothesis of a mediator role of ceramide in the differentiation of cells of neural origin.

N‐(4‐hydroxyphenyl)retinamide (HPR) is a synthetic retinoid of clinical use in breast cancer chemoprevention. Its in vitroeffects on tumor cell lines include growth inhibition and apoptosis and are, to a large extent, independent of the activation of retinoic acid receptors. We have performed a cDNA differential display analysis to identify genes potentially relevant for understanding the mechanism of action of HPR. We report that prosaposin (PSAP), a gene encoding for the common precursor protein of saposins (cofactors involved in the metabolism of various sphingolipids), was significantly up‐regulated, both at transcriptional and protein level, in MCF7 and T47D breast cancer cell lines after treatment with HPR, but not with all trans retinoic acid (ATRA). Remarkably, this response was also induced by oxidants, whereas it was abrogated by antioxidants in cells treated with HPR, which implies a direct role of free radicals generated by HPR in prosaposin up‐regulation. Stable transfectants of T47D cells overexpressing prosaposin (T47D‐PSAP) exhibited morphological changes, reduced proliferation, and impaired attachment to culture plates, which correlated with down‐regulation of α6, β1, and β4 subunits of the integrin family of adhesion receptors. Concordant with a role of prosaposin in ceramide generation, increased amounts of this lipid were found in T47D‐PSAP and in HPR‐treated T47D cells. Moreover, treatment of T47D with C2‐ceramide resulted in the up‐regulation of prosaposin and down‐regulation of integrin receptors. Altogether, these findings establish a link between prosaposin, ceramide, and integrin receptor regulation. In view of the association between integrin down‐regulation and suppression of metastatic capacity, our data raise the possibility that the cancer chemopreventive activity of HPR may rely, at least in part, on its capacity to regulate integrin receptors through prosaposin.

Mycobacterial-specific glycolipids determine pathogenesis after the mycobacteria are engulfed by human neutrophils.

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