sphingosine-phosphorylcholine and psychosine-3--sulfate-ester

Sphingolipids act as signaling mediators that regulate a diverse range of cellular events. Although numerous sphingolipid functions have been studied, little is known about the effect of sphingolipids on monocyte differentiation into macrophages. Here, we report that two lysosphingolipids, sphingosylphosphorylcholine (SPC) and lysosulfatide (LSF), inversely affect macrophagic differentiation of monocytic cell lines, U937 and THP-1. Molecular analyses revealed that SPC enhances, whereas LSF suppresses, phorbol ester-induced classical (M1-polarized) differentiation to macrophages. The expression of CD11b, a macrophage marker, was induced in accordance with the activation status of the Raf/MEK/ERK signaling pathway in which SPC and LSF had opposite effects. Pharmacological inhibition of this pathway aborted the differentiation, indicating that this signaling pathway is required. Consistently, SPC promoted, while LSF inhibited, monocyte adhesion to fibronectin, through the phosphatidylinositol-3-kinase (PI3K)/Akt signaling pathway. The effects of SPC on Raf/MEK/ERK and PI3K/Akt signaling were dependent on G(i/o), whereas the SPC-induced calcium influx was dependent on G(q). Thus SPC utilizes G-protein coupled receptor. In contrast, the effects of LSF were independent of G(i/o) and G(q). These results suggest that SPC enhances, whereas LSF suppresses, monocyte differentiation into macrophages through regulating the Raf/MEK/ERK and PI3K/Akt signaling pathways via distinct mechanisms.

Topics: Base Sequence; Calcium; Cell Differentiation; Cell Line; DNA Primers; GTP-Binding Protein alpha Subunits, Gi-Go; GTP-Binding Protein alpha Subunits, Gq-G11; Humans; Macrophages; MAP Kinase Signaling System; Monocytes; Phosphatidylinositol 3-Kinases; Phosphorylcholine; Proto-Oncogene Proteins c-akt; Psychosine; raf Kinases; Receptors, Lysosphingolipid; Signal Transduction; Sphingosine; Tetradecanoylphorbol Acetate; U937 Cells

Apoptotic cell death following injury of vascular endothelium is assumed to play an important role in the pathogenesis of atherosclerosis. In this report, we demonstrate that high density lipoproteins (HDL), a major anti-atherogenic lipoprotein fraction, protect endothelial cells against growth factor deprivation-induced apoptosis. HDL blocked the mitochondrial pathway of apoptosis by inhibiting dissipation of mitochondrial potential (Deltapsi(m)), generation of reactive oxygen species, and release of cytochrome c into the cytoplasm. As a consequence, HDL prevented activation of caspases 9 and 3 and apoptotic alterations of the plasma membrane such as increase of permeability and translocation of phosphatidylserine. Treatment of endothelial cells with HDL induced activation of the protein kinase Akt, an ubiquitous transducer of anti-apoptotic signals, and led to phosphorylation of BAD, a major Akt substrate. Suppression of Akt activity both by wortmannin and LY-294002 or by a dominant negative Akt mutant abolished the anti-apoptotic effect of HDL. Two bioactive lysosphingolipids present in HDL particles, sphingosylphosphorylcholine and lysosulfatide, fully mimicked the survival effect of HDL by blocking the mitochondrial pathway of apoptosis and potently activating Akt. In conclusion, the present study identifies HDL as a carrier of endogenous endothelial survival factors and suggests that inhibition of endothelial apoptosis by HDL-associated lysosphingolipids may represent an important and novel aspect of the anti-atherogenic activity of HDL.

Topics: Apoptosis; Arteriosclerosis; Cell Survival; Cells, Cultured; Endothelium, Vascular; Humans; Lipoproteins, HDL; Mitochondria; Phosphatidylinositol 3-Kinases; Phosphorylcholine; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Psychosine; Sphingosine

Although a number of cellular components of cytokinesis have been identified, little is known about the detailed mechanisms underlying this process. Here, we report that the lipid metabolite psychosine (galactosylsphingosine), derived from galactosylceramide, induced formation of multinuclear cells from a variety of nonadherent and adherent cells due to inhibition of cytokinesis. When psychosine was added to the human myelomonocyte cell line U937, which was the most sensitive among the cell lines tested, cleavage furrow formed either incompletely or almost completely. However, abnormal contractile movement was detected in which the cellular contents of one of the hemispheres of the contracting cell were transferred into its counterpart. Finally, the cleavage furrow disappeared and cytokinesis was reversed. Psychosine treatment also induced giant clots of actin filaments in the cells that probably consisted of small vacuoles with filamentous structures, suggesting that psychosine affected actin reorganization. These observations could account for the formation of multinuclear globoid cells in the brains of patients with globoid cell leukodystrophy, a neurological disorder characterized by the accumulation of psychosine due to galactosylceramidase deficiency.

Topics: Actins; Cell Division; Humans; Leukodystrophy, Globoid Cell; Phagocytosis; Phosphorylcholine; Psychosine; Sphingosine; Tumor Cells, Cultured; U937 Cells

Our earlier studies demonstrated that high-density lipoproteins (HDLs) stimulate multiple signaling pathways, including activation of phosphatidylcholine-specific phospholipases C and D (PC-PLs) and phosphatidylinositol-specific phospholipase C (PI-PLC). However, only activation of PC-PLs was linked to the HDL-induced cholesterol efflux. In the study presented here, the role of HDL-induced PI-PLC activation was studied. In human skin fibroblasts, HDL potently induced PI-PLC as inferred from enhanced phosphatidylinositol bisphosphate (PtdInsP(2)) turnover and Ca(2+) mobilization. The major protein component of HDL, apo A-I, did not induce PtdInsP(2) turnover or Ca(2+) mobilization in these cells. Both HDL and apo A-I promoted cellular cholesterol efflux, whereas only HDL induced fibroblast proliferation. Inhibition of PI-PLC with U73122 or blocking intracellular Ca(2+) elevation with Ni(2+) or EGTA markedly reduced the extent of HDL-induced cell proliferation but had no effect on cholesterol efflux. In fibroblasts from patients with Tangier disease which are characterized by defective cholesterol efflux, neither HDL-induced PtdInsP(2) breakdown and Ca(2+) mobilization nor cell proliferation was impaired. HDL-induced fibroblast proliferation, PtdInsP(2) turnover, and Ca(2+) mobilization were fully mimicked by the lipid fraction isolated from HDL. Analysis of this fraction with high-performance liquid chromatography (HPLC) and time-of-flight secondary ion mass spectroscopy (TOF-SIMS) revealed that the PI-PLC-inducing activity is identical with two bioactive lysosphingolipids, namely, lysosulfatide (LSF) and sphingosylphosphorylcholine (SPC). Like native HDL, LSF and SPC induced PtdInsP(2) turnover, Ca(2+) mobilization, and fibroblast proliferation. However, both compounds did not promote cholesterol efflux. In conclusion, two agonist activities are carried by HDL. Apo A-I stimulates phosphatidylcholine breakdown and thereby facilitates cholesterol efflux, whereas LSF and SPC trigger PI-PLC activation and thereby stimulate cell proliferation.

Topics: Apolipoprotein A-I; Calcium Signaling; Cells, Cultured; Cholesterol; DNA; Egtazic Acid; Enzyme Activation; Estrenes; Fibroblasts; Humans; Lipoproteins, HDL; Lysophospholipids; Mitogens; Nickel; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositol Diacylglycerol-Lyase; Phosphoinositide Phospholipase C; Phosphorylcholine; Psychosine; Pyrrolidinones; Sphingolipids; Sphingosine; Tangier Disease; Type C Phospholipases

The cell numbers of three mouse neuroblastoma cell lines were decreased upon incubation with lysosphingolipids in the following order of effectiveness: lysosulfatide (lysoCS) greater than psychosine (Ps) greater than sphingosylphosphocholine (SPC). The different cell lines showed characteristic sensitivities to various concentrations of lysolipids less than 150 microM. Interestingly, only SPC induced neurite outgrowth and changed the lipid composition, modifying the amounts of cholesterol, sphingomyelin (SM) and ganglioside GM3 in all cell lines. The effect of SPC on these cell lines was comparable to the effect of N-acetyl SPC (NAcSPC) rather than that of SM.

Topics: Animals; Cell Division; Cell Line; Choline; Gangliosides; Kinetics; Lipid Metabolism; Mice; Neuroblastoma; Phospholipids; Phosphorylcholine; Psychosine; Sphingosine; Tumor Cells, Cultured