Role of sphingolipids in arachidonic acid metabolism.

The arachidonic acid (AA) cascade is regulated mainly by the actions of two rate-limiting enzymes, phospholipase A2 (PLA2) and inducible cyclooxygenase-2 (COX-2). PLA2 acts to generate AA, which serves as the precursor substrate for COX-2 in the metabolic pathway leading to prostaglandin production. Amongst more than 30 members of the PLA2 family, cytosolic PLA2α (cPLA2α, group IVA) plays a major role in releasing AA from cellular membranes. Sphingolipids are a novel class of bioactive lipids that play key roles in the regulation of several cellular processes including growth, differentiation, inflammatory responses, and apoptosis. Recent studies implicated a regulatory function of sphingolipids in prostaglandin production. Whereas ceramide-1-phosphate and lactosylceramide activate cPLA2α directly, sphingosine-1-phosphate induces COX-2 expression. Sphingomyelin has been shown to inhibit the activity of cPLA2α. In addition, several sphingolipid analogs including a therapeutic agent currently used clinically are also reported to be inhibitors of cPLA2α. This review explores the role of sphingolipids in the regulation of cPLA2α and COX-2.

Membrane microdomains in immunity: glycosphingolipid-enriched domain-mediated innate immune responses.

Over the last 30 years, many studies have indicated that glycosphingolipids (GSLs) expressed on the cell surface may act as binding sites for microorganisms. Based on their physicochemical characteristics, GSLs form membrane microdomains with cholesterol, sphingomyelin, glycosylphosphatidylinositol (GPI)-anchored proteins, and various signaling molecules, and GSL-enriched domains have been shown to be involved in these defense responses. Among the GSLs, lactosylceramide (LacCer, CDw17) can bind to various microorganisms. LacCer is expressed at high levels on the plasma membrane of human neutrophils, and forms membrane microdomains associated with the Src family tyrosine kinase Lyn. LacCer-enriched membrane microdomains mediate superoxide generation, chemotaxis, and non-opsonic phagocytosis. Therefore, LacCer-enriched membrane microdomains are thought to function as pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs) expressed on microorganisms. In contrast, several pathogens have developed infection mechanisms using membrane microdomains. In addition, some pathogens have the ability to avoid degradation by escaping from the vacuolar compartment or preventing phagosome maturation, utilizing membrane microdomains, such as LacCer-enriched domains, of host cells. The detailed molecular mechanisms of these membrane microdomain-associated host-pathogen interactions remain to be elucidated.

The hypothesis on function of glycosphingolipids and ABO blood groups revisited.

Twenty-five years ago the author proposed new ideas of glycoprotein (GPs) and glycosphingolipid (GSLs) functions at the cell membrane. The GPs, apart from their glycan carrying capacity, were assumed to have specific, protein associated, functions. In contrast, GSLs such as those of globo and neolacto/lacto series, were considered to be energetically cheap membrane packing substances, filling in membrane spaces not covered with functional GPs. The terminal carbohydrate structures of the neolacto/lacto GSLs, i.e., sialic acid residues and ABH glycotopes, were postulated to have either regulatory or protective functions, respectively. A special active role was ascribed to terminal ß-galactosyl residues of GSLs and GPs. Gangliosides were considered to be functional GSLs. In the present review the author discusses these old ideas in context of the contemporary knowledge and comes to the conclusion that they have not aged.

Glycosphingolipids regulate ameloblastin expression in dental epithelial cells.

Neurotrophin 4 (NT-4) and its receptors regulate the differentiation of ameloblasts in tooth development. Gangliosides, sialic acids that contain glycosphingolipids (GSLs), are involved in a variety of membrane-associated cell physiological functions such as ligand-receptor signal transmission. However, the expression patterns and functions of GSLs during tooth development remain unclear. In this study, we identified strong expressions of GM3 and LacCer in dental epithelium, which give rise to differentiation into enamel-secreting ameloblasts. Exogenous GM3 and LacCer in dental epithelial cells induced the expression of ameloblastin (Ambn), while it was also interesting that GM3 synergistically exerted enhancement of NT-4-mediated Ambn expression. In addition, consistently exogenous GM3 and LacCer in dental epithelial cells induced distinct activation of extracellular signal-regulated kinase 1/2 (ERK1/2), an event upstream of the expression of Ambn. Furthermore, depletion of GSLs from dental epithelial cells by D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) inhibited Ambn expression as well as phosphorylation of ERK1/2. In contrast, exogenous addition of GM3 or LacCer rescued the phosphorylation of ERK1/2 repressed by pre-treatment with D-PDMP. Taken together, these results suggest that GM3 and LacCer are essential for NT-4-mediated Ambn expression, and contribute to dental epithelial cell differentiation into ameloblasts.

Significance of glycosphingolipid fatty acid chain length on membrane microdomain-mediated signal transduction.

Lactosylceramide (LacCer), a neutral glycosphingolipid, is abundantly expressed on human neutrophils, and specifically recognizes several pathogenic microorganisms. LacCer forms membrane microdomains coupled with the Src family kinase Lyn on the plasma membrane, and ligand binding to LacCer activates Lyn, resulting in neutrophil functions. In contrast, neutrophilic differentiated HL-60 cells do not have Lyn-associated LacCer-enriched microdomains and lack LacCer-mediated functions. In neutrophil plasma membranes, the very long fatty acid C24:0 and C24:1 chains are the main components of LacCer, whereas plasma membrane of D-HL-60 cells mainly includes C16-LacCer species. Here, we suggest that LacCer species containing very long fatty acid chains are indispensable for the association of Lyn with LacCer-enriched microdomains and LacCer-mediated functions.

Functions of sphingolipid metabolism in mammals--lessons from genetic defects.

Much is known about the pathways that control the biosynthesis, transport and degradation of sphingolipids. During the last two decades, considerable progress has been made regarding the roles this complex group of lipids play in maintaining membrane integrity and modulating responses to numerous signals. Further novel insights have been provided by the analysis of newly discovered genetic diseases in humans as well as in animal models harboring mutations in the genes whose products control sphingolipid metabolism and action. Through the description of the phenotypic consequences of genetic defects resulting in the loss of activity of the many proteins that synthesize, transport, bind, or degrade sphingolipids, this review summarizes the (patho)physiological functions of these lipids.

Platelet derived growth factor recruits lactosylceramide to induce cell proliferation in UDP Gal:GlcCer: beta1 --> 4Galactosyltransferase (GalT-V) mutant Chinese hamster ovary cells.

Recent molecular cloning studies have suggested the presence of at least two beta4Gal transferase genes (beta4GalT-V and beta4GalT-VI) that may encode lactosylceramide synthase but whether they are functional in vivo and whether they mediate growth factor induced phenotypic change such as cell proliferation is not known. Our previous studies lead to the suggestion that various risk factors in atherosclerosis such as oxidized LDL, shear stress, nicotine, tumor necrosis factor-alpha converge upon LacCer synthase to induce critical phenotypic changes such as cell proliferation and cell adhesion. However, whether platelet-derived growth factor also recruits LacCer synthase in mediating cell proliferation is not known. Here we have employed a Chinese hamster ovary mutant cell line Pro(-)5Lec20 to determine whether this enzyme physiologically functions to mediate cell proliferation. We show that PDGF stimulates the activity of UDP galactose:glucosylceramide, beta1,4galactosyltransferase. The activity of LacCer synthase increased about 2.5 fold within 2.5-5 min of incubation with PDGF in both wild type and Pro(-)5Lec20 cells. Concomitantly, there was an increase in the generation of superoxide radicals, p44MAPK phosphorylation and cell proliferation in CHO cells. D-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), a potent inhibitor of GlcCer synthase/LacCer synthase impaired PDGF mediated induction of LacCer synthase activity, superoxide generation, p44 MAPK activation and cell proliferation in Pro(-)5Lec20 cells. PDGF-induced superoxide generation was also mitigated by the use of diphenylene iodonium; an inhibitor of NADPH oxidase activity that is required for superoxide generation. This inhibition was bypassed by the addition of lactosylceramide. Thus, beta4GalT-V gene produces a bona fide LacCer synthase that can function in vivo to generate LacCer. Moreover, this enzyme alone can mediate PDGF induced activation of a signal transduction cascade involving superoxide generation, p44MAPK activation, phosphorylation of Akt and cell proliferation.

Novel role of lactosylceramide in vascular endothelial growth factor-mediated angiogenesis in human endothelial cells.

Vascular endothelial growth factor (VEGF) has been implicated in angiogenesis associated with coronary heart disease, vascular complications in diabetes, inflammatory vascular diseases, and tumor metastasis. The mechanism of VEGF-driven angiogenesis involving glycosphingolipids such as lactosylceramide (LacCer), however, is not known. To demonstrate the involvement of LacCer in VEGF-induced angiogenesis, we used small interfering RNA (siRNA)-mediated silencing of LacCer synthase expression (GalT-V) in human umbilical vein endothelial cells. This gene silencing markedly inhibited VEGF-induced platelet endothelial cell adhesion molecule-1 (PECAM-1) expression and angiogenesis. Second, we used D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), an inhibitor of LacCer synthase and glucosylceramide synthase, that significantly mitigated VEGF-induced PECAM-1 expression and angiogenesis. Interestingly, these phenotypic changes were reversed by LacCer but not by structurally related compounds such as glucosylceramide, digalactosylceramide, and ceramide. In a human mesothelioma cell line (REN) that lacks the endogenous expression of PECAM-1, VEGF/LacCer failed to stimulate PECAM-1 expression and tube formation/angiogenesis. In REN cells expressing human PECAM-1 gene/protein, however, both VEGF and LacCer-induced PECAM-1 protein expression and tube formation/angiogenesis. In fact, VEGF-induced but not LacCer-induced angiogenesis was mitigated by SU-1498, a VEGF receptor tyrosine kinase inhibitor. Also, VEGF/LacCer-induced PECAM-1 expression and angiogenesis was mitigated by protein kinase C and phospholipase A2 inhibitors. These results indicate that LacCer generated in VEGF-treated endothelial cells may serve as an important signaling molecule for PECAM-1 expression and in angiogenesis. This finding and the reagents developed in our report may be useful as anti-angiogenic drugs for further studies in vitro and in vivo.

A novel role of lactosylceramide in the regulation of lipopolysaccharide/interferon-gamma-mediated inducible nitric oxide synthase gene expression: implications for neuroinflammatory diseases.

In the present study a possible role of glycosphingolipids (GSLs) in inducible nitric oxide synthase (iNOS) gene expression and nitric oxide (NO) production after spinal cord injury (SCI) in rats has been established. In primary rat astrocytes lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) treatment increased the intracellular levels of lactosylceramide (LacCer) and induced iNOS gene expression. d-Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol.HCI (PDMP), a glucosylceramide synthase and LacCer synthase (galactosyltransferase, GalT-2) inhibitor, inhibited LPS/IFN-gamma induced iNOS expression, which was reversed by exogenously supplied LacCer, but not by other glycosphingolipids. LPS/IFN-gamma caused a rapid increase in the activity of GalT-2 and synthesis of LacCer. Silencing of GalT-2 gene with the use of antisense oligonucleotides resulted in decreased LPS/IFN-gamma-induced iNOS, TNF-alpha, and IL-1beta gene expression. The PDMP-mediated reduction in LacCer production and inhibition of iNOS expression correlated with decreased Ras and ERK1/2 activation along with decreased IkappaB phosphorylation, NF-kappaB DNA binding activity, and NF-kappaB-luciferase reporter activity. LacCer-mediated Ras activation was redox-mediated and was attenuated by antioxidants N-acetyl cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC). In vivo administration of PDMP after SCI resulted in improved functional outcome (Basso, Beattie, Bresnahan score); inhibition of iNOS, TNF-alpha, and IL-1beta expression; decreased neuronal apoptosis; and decreased tissue necrosis and demyelination. The in vivo studies supported the conclusions drawn from cell culture studies and provided evidence for the possible role of GalT-2 and LacCer in SCI-induced inflammation and pathology. To our knowledge this is the first report of a role of LacCer in iNOS expression and the advantage of GSL depletion in attenuating post-SCI inflammation to improve the outcome of SCI.

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

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

Lactosylceramide is essential for the osteoclastogenesis mediated by macrophage-colony-stimulating factor and receptor activator of nuclear factor-kappa B ligand.

Glycosphingolipids and their metabolites play important roles in a variety of biological processes. Several signal molecules are localized in a glycolipid-enriched microdomain on the cell surface, and their signals are regulated by the glycolipid composition. However, the function of glycolipids in osteoclastogenesis has not been clearly understood. We found that D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), a glucosylceramide synthase inhibitor, completely inhibits the osteoclast formation induced by macrophage-colony-stimulating factor and receptor activator of nuclear factor-kappa B ligand (RANKL) in a dose-dependent manner. Expression of RANK, the receptor of RANKL, induced by macrophage colony-stimulating factor, was reduced markedly in D-PDMP-treated cells. d-PDMP also inhibited the phosphorylation of the inhibitor of nuclear factor-kappa B and extracellular signal-regulated kinase 1/2 induced by RANKL. In several experiments with the addition of glycolipids to D-PDMP-treated purified bone marrow cells, lactosylceramide (LacCer) strongly affected the differentiation into tartrate-resistant acid phosphatase mononucleated cells, but not positive multinucleated cells. GM3 and GM1 also recovered, but less effectively compared with LacCer. Moreover, exogenous LacCer recovered the reduced expression of RANK and the phosphorylation of inhibitor of NF-kappa B and extracellular signal-regulated kinase 1/2 after stimulation by RANKL at the same level of cells without D-PDMP treatment. Our data suggest that glycosphingolipids, especially LacCer, are necessary for the initiation step of RANKL-induced osteoclastogenesis.

Suppression of integrin expression and tumorigenicity by sulfation of lactosylceramide in 3LL Lewis lung carcinoma cells.

To investigate the cellular functions of sulfated glycosphingolipids, we introduced the cerebroside sulfotransferase (CST) gene into J5 cells, a subclone of 3LL Lewis lung carcinoma cells. The J5 cells lack acidic glycosphingolipids but accumulate their common biosynthetic precursor, lactosylceramide. We established the stable CST transfectants, J5/CST-1 and J5/CST-2 clones, highly expressing sulfated lactosylceramide (SM3). Both clones exhibited more spherical morphology in comparison to mock transfectant, and their adhesiveness to fibronectin and laminin was significantly lower. The loss of cell-substratum interactions in these SM3-expressing cells could be attributed to decreased expression of integrins (alpha(5), alpha(6), and beta(1)) on the cell surface and their whole cellular levels. However, the levels of H-2K(b) and H-2D(b) antigens remained unchanged. Reverse transcriptase-polymerase chain reaction and Northern blot analyses for these integrins exhibited significant decrease of beta(1) gene expression in J5/CST-1 and 2, but there was no change in the levels of alpha(5) and alpha(6) transcripts. Deglycosylation by endoglycosidase H treatment clearly demonstrated that the precursor form of beta(1) integrin, possessing high mannose oligosaccharide chains, was preferentially decreased in the CST transfectants. These results demonstrate that endogenous SM3 negatively regulates beta(1) integrin expression at the transcriptional level, and the decrease of alpha integrin proteins in the CST transfectants was due to the post-transcriptional modification. We suggest the putative importance of the intracellular pre-beta(1) integrin pool for normal integrin maturation and subsequent function. Although the rates of cell proliferation in vitro for mock and CST transfectants were similar, tumorigenicity of J5/CST-1 and -2 cells inoculated into syngeneic C57/BL6 mice was greatly decreased or even absent. This was probably due to global loss of the efficient cell-matrix interactions, which are essential for the development of malignant tumors in vivo. Thus, we showed the evidence that cellular SM3 negatively regulates the cell-substratum interaction, resulting in the loss of tumorigenicity.

N-Oleoylethanolamine inhibits glucosylation of natural ceramides in CHP-100 neuroepithelioma cells: possible implications for apoptosis.

We report that N-oleoylethanolamine (NOE), widely employed as a ceramidase inhibitor, also inhibits glucosylation of naturally occurring ceramides. When CHP-100 neuroepithelioma cells were exposed for 18h to non-toxic NOE concentrations (i.e. up to 70 microM), basal incorporation of labelled hexose into glucosylceramide (GlcCer) and higher order neutral glycosphingolipids was significantly inhibited. In cells treated with 30 microM N-hexanoylsphingosine (C6-Cer), NOE affected only marginally short-chain glucocerebroside accumulation, but markedly decreased accumulation of glucocerebrosides originating from glucosylation of a long-chain ceramide (Lc-Cer) pool produced upon C6-Cer treatment. Evidence is provided that NOE effects neither are mediated by their effects on ceramidase nor are due to enhanced long-chain GlcCer (Lc-GlcCer) conversion to higher order glycosylated derivatives. NOE inhibition of Lc-GlcCer generation was accompanied by enhanced accumulation of Lc-Cer and by potentiation of apoptosis induced by C6-Cer; the possible causal relationships between these two phenomena are discussed.

Lactosylceramide mediates tumor necrosis factor-alpha-induced intercellular adhesion molecule-1 (ICAM-1) expression and the adhesion of neutrophil in human umbilical vein endothelial cells.

The endothelial expression of adhesion molecules by proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) has been suggested to contribute to the initiation of atherosclerotic plaque formation. Since lactosylceramide (LacCer) accumulates in large quantities in human atherosclerotic plaque, we have explored its role in TNF-alpha-induced expression of intercellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells and their consequent adhesion to polymorphonuclear leukocytes (PMNs). We found that TNF-alpha increased LacCer synthesis by way of stimulating the activity of UDP-galactose:glucosylceramide beta(1-->4)-galactosyltransferase in a time-dependent fashion. The TNF-alpha-induced expression of ICAM-1 was abrogated by D-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), an inhibitor of UDP-galactose:glucosylceramide beta(1-->4)-galactosyltransferase. However, the addition of LacCer reversed the D-PDMP effect on TNF-alpha-induced ICAM-1 expression in human umbilical vein endothelial cells. Northern hybridization analysis of mRNA levels and enzyme-linked immunosorbent assays revealed that LacCer (5 microM) specifically stimulated ICAM-1 at both the transcriptional and translational levels. This was accompanied by the adhesion of PMNs, which was visualized by confocal microscopy. Further studies revealed that LacCer stimulated the endogenous generation of superoxide radicals (O-2) about 5-fold compared with the control by specifically activating plasma membrane-associated NADPH-dependent oxidase. This phenomenon was blocked by the antioxidant N-acetyl-L-cysteine, pyrrolidine dithiocarbamate, and the NADPH oxidase inhibitor, diphenylene iodonium. Overexpression of endogeneous CuZn-superoxide dismutase via an adenoviral vector carrying cDNA for CuZn-superoxide dismutase, also inhibited LacCer-induced ICAM-1 expression in endothelial cells. In sum, our findings suggest that LacCer may play the role of a lipid second messenger in TNF-alpha-induced pathogenesis by activating an oxidant-sensitive transcriptional pathway that leads to the adhesion of PMNs to endothelial cells.

Role of lactosylceramide and MAP kinase in the proliferation of proximal tubular cells in human polycystic kidney disease.

Polycystic kidney disease (PKD) is a common genetic disease characterized by the proliferation of epithelial cells, formation of cysts, and the progression of renal deficiency. We have investigated a possible role of glycosphingolipids in the proliferation of human kidney cells in this disease. The levels of glucosylceramide and lactosylceramide and the activity of glucosylceramide synthase (GlcT-1) and lactosylceramide synthase (GalT-2) were elevated 2-fold and 3-fold, respectively, in the PKD tissue compared to control. Lactosylceramide, but not glucosylceramide (10 microM) derived from PKD exerted a 4-fold stimulation in the proliferation of these cells. However, at a concentration of 40 microM, lactosylceramide and glucosylceramide both stimulated cell proliferation on the order of 10-fold and 2.5-fold, respectively, as compared to control. This phenomenon may be due to the enrichment of lactosylceramide containing shorter chain fatty acids (C16:0-C18:0). Lactosylceramide, but not glucosylceramide exerted a time-dependent stimulation in the phosphorylation of mitogen-activated protein kinase (p44 MAPK) in normal human kidney proximal tubular cells. Moreover, the kidneys and cultured cells from the PKD patients contained higher levels of the p44 MAPK as compared to normal human kidneys. In sum, our studies indicate that lactosylceramide present in the PKD kidney may stimulate cell proliferation via activation of the p44 MAPK, and contribute to the pathophysiology in this disease.

Adhesion of Mycoplasma pneumoniae to sulfated glycolipids and inhibition by dextran sulfate.

A virulent strain of Mycoplasma pneumoniae was metabolically labeled with [3H]palmitate and studied for binding to glycolipids and to WiDr human colon adenocarcinoma cells. The organism binds strongly to sulfatide and other sulfated glycolipids, such as seminolipid and lactosylsulfatide which all contain terminal Gal(3SO4) beta 1-residues and weakly to some neolactoseries neutral glycolipids. M. pneumoniae do not bind gangliosides including the sialylneolacto-series and other neutral glycolipids that were tested. Only metabolically active M. pneumoniae cells bind to sulfatide, as binding is maximal in RPMI medium at 37 degrees C and almost completely abolished in nutrient-deficient medium or by keeping the cells at 4 degrees C. Dextran sulfate but not other sulfated or anionic polysaccharides at 10 micrograms/ml completely inhibits binding of M. pneumoniae to purified sulfatide. Dextran sulfate does not inhibit binding to the neolacto-series neutral glycolipids. Dextran sulfate partially inhibits adhesion of M. pneumoniae to cultured human colon adenocarcinoma cells (WiDr). The biological relevance of these data is suggested by our finding that sulfatide occurs in large amounts in human trachea, lung, and WiDr cells. Thus, there are at least two distinct receptors that mediate binding of M. pneumoniae to cells: glycolipids containing terminal Gal(3SO4) beta 1-residues as reported here, and glycoproteins containing terminal NeuAc alpha 2-3Gal beta 1-4GlcNAc sequences (Roberts, D. D., Olson, L. D., Barile, M. F., Ginsburg, V., and Krivan, H. C. (1989) J. Biol. Chem. 264, 9289-9293).