sphingosine-1-phosphate and dihydroceramide

Atherosclerosis-a systemic inflammatory disease-is the number one cause of mortality and morbidity worldwide. As such, the prevention of disease progression is of global interest in order to reduce annual deaths at a significant scale. Atherosclerosis is characterized by plaque formation in the arteries, resulting in vascular events such as ischemic stroke or myocardial infarction. A better understanding of the underlying pathophysiological processes at the cellular and molecular level is indispensable to identify novel therapeutic targets that may alleviate disease initiation or progression. Sphingolipids-a lipid class named after the chimeric creature sphinx-are considered to play a critical and, metaphorically, equally chimeric regulatory role in atherogenesis. Previous studies identified six common sphingolipids, namely dihydroceramide (DhCer), ceramide (Cer), sphingosine-1-phosphate (S1P), sphingomyelin (SM), lactosylceramide (LacCer), and glucosylceramide (GluCer) in carotid plaques, and demonstrated their potential as inducers of plaque inflammation. In this review, we point out their specific roles in atherosclerosis by focusing on different cell types, carrier molecules, enzymes, and receptors involved in atherogenesis. Whereas we assume mainly atheroprotective effects for GluCer and LacCer, the sphingolipids DhCer, Cer, SM and S1P mediate chimeric functions. Initial studies demonstrate the successful use of interventions in the sphingolipid pathway to prevent atherosclerosis. However, as atherosclerosis is a multifactorial disease with a variety of underlying cellular processes, it is imperative for future research to emphasize the circumstances in which sphingolipids exert protective or progressive functions and to evaluate their therapeutic benefits in a spatiotemporal manner.

Topics: Antigens, CD; Atherosclerosis; Ceramides; Chimera; Glucosylceramides; Humans; Lactosylceramides; Lysophospholipids; Plaque, Atherosclerotic; Sphingolipids; Sphingomyelins; Sphingosine

Sphingolipidoses are monogenic lipid storage diseases caused by variants in enzymes of lipid synthesis and metabolism. We describe an autosomal recessive complex neurological disorder affecting consanguineous kindred. All four affected individuals, born at term following normal pregnancies, had mild to severe intellectual disability, spastic quadriplegia, scoliosis and epilepsy in most, with no dysmorphic features. Brain MRI findings were suggestive of leukodystrophy, with abnormal hyperintense signal in the periventricular perioccipital region and thinning of the body of corpus callosum. Notably, all affected individuals were asymptomatic at early infancy and developed normally until the age of 8-18 months, when deterioration ensued. Homozygosity mapping identified a single 8.7 Mb disease-associated locus on chromosome 1q41-1q42.13 between rs1511695 and rs537250 (two-point LOD score 2.1). Whole exome sequencing, validated through Sanger sequencing, identified within this locus a single disease-associated homozygous variant in DEGS1, encoding C4-dihydroceramide desaturase, an enzyme of the ceramide synthesis pathway. The missense variant, segregating within the family as expected for recessive heredity, affects an evolutionary-conserved amino acid of all isoforms of DEGS1 (c.656A>G, c.764A>G; p.(N219S), p.(N255S)) and was not found in a homozygous state in ExAC and gnomAD databases or in 300 ethnically matched individuals. Lipidomcs analysis of whole blood of affected individuals demonstrated augmented levels of dihydroceramides, dihydrosphingosine, dihydrosphingosine-1-phosphate and dihydrosphingomyelins with reduced levels of ceramide, sphingosine, sphingosine-1-phosphate and monohexosylceramides, as expected in malfunction of C4-dihydroceramide desaturase. Thus, we describe a sphingolipidosis causing a severe regressive neurological disease.

Topics: Adolescent; Adult; Brain; Ceramides; Cerebrosides; Child; Child, Preschool; Exome Sequencing; Fatty Acid Desaturases; Female; Genetic Predisposition to Disease; Genetic Variation; Homozygote; Humans; Infant; Intellectual Disability; Lysophospholipids; Male; Mutation, Missense; Nervous System Diseases; Pedigree; Phenotype; Sequence Analysis, DNA; Sphingosine; Young Adult

In the present study, the roles of telomerase and prostaglandin E(2) (PGE(2)) in platelet-derived growth factor (PDGF's) and fibroblast growth factor-2 (FGF-2's) effects against C(2)-ceramide-induced cell death were investigated. C(2)-ceramide reduced the viability of NIH3T3 cells in a condition without calf serum (CS) in accordance with decreasing telomerase activity according to the TRAP assay. The addition of CS significantly protected cells from C(2)-ceramide-induced apoptosis through increased telomerase activity, and the phosphorylations of PDGF and the FGF-2-like receptor in NIH3T3 cells were detected. Adding PDGF and FGF-2 decreased the cytotoxic effect elicited by C(2)-ceramide through stimulating telomerase activity, which was blocked by adding a telomerase inhibitor (TI). Activations of ERKs and JNKs were detected in PDGF- and FGF-2-treated NIH3T3 cells, and the telomerase activities induced by PDGF and FGF were respectively inhibited by the addition of the ERK inhibitor, PD98059, and the JNK inhibitor, SP600125. Accordingly, induction of cyclooxygenase-2 (COX-2) protein expression and PGE(2) production was detected in PDGF- and FGF-2-treated NIH3T3 cells, and the telomerase activities stimulated by PDGF and FGF were reduced by adding a specific COX-2 inhibitor, NS398, through a decrease in PGE(2) production. Incubation of cells with PGE(2) or the EP1 agonist, 17-PT, but not the EP2 agonist, sulprostone, the EP3 agonist, butaprost, or the EP4 agonist, PGE(1) alcohol, significantly enhanced the telomerase activity of NIH3T3 cells. PGE(2) protection of NIH3T3 cells against C(2)-ceramide-induced cell death was identified by the MTT and LDH-release assays, and it was inhibited by adding the EP1 antagonist, SC-19220. Ceramide metabolites including ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P), and a standard control of exogenous ceramide C(2)-dihydroceramide show no effect on the telomerase activity and viability of NIH3T3 cells. The involvement of COX-2/PGE(2)-mediated telomerase activation by PDGF and FGF-2 against C(2)-ceramide-induced cell death is first demonstrated herein.

Topics: Animals; Apoptosis; Cell Survival; Ceramides; Cyclooxygenase 2; Cytoprotection; Dinoprostone; Enzyme Activation; Enzyme Inhibitors; Extracellular Signal-Regulated MAP Kinases; Fibroblast Growth Factor 2; JNK Mitogen-Activated Protein Kinases; Lysophospholipids; Mice; NIH 3T3 Cells; Phosphorylation; Platelet-Derived Growth Factor; Sphingosine; Telomerase

Novel immunomodulatory molecule FTY720 is a synthetic analog of myriocin, but unlike myriocin FTY720 does not inhibit serine palmitoyltransferase. Although many of the effects of FTY720 are ascribed to its phosphorylation and subsequent sphingosine 1-phosphate (S1P)-like action through S1P(1,3-5) receptors, studies on modulation of intracellular balance of signaling sphingolipids by FTY720 are limited. In this study, we used stable isotope pulse labeling of human pulmonary artery endothelial cells with l-[U-(13)C, (15)N]serine as well as in vitro enzymatic assays and liquid chromatography-tandem mass spectrometry methodology to characterize FTY720 interference with sphingolipid de novo biosynthesis. In human pulmonary artery endothelial cells, FTY720 inhibited ceramide synthases, resulting in decreased cellular levels of dihydroceramides, ceramides, sphingosine, and S1P but increased levels of dihydrosphingosine and dihydrosphingosine 1-phosphate (DHS1P). The FTY720-induced modulation of sphingolipid de novo biosynthesis was similar to that of fumonisin B1, a classical inhibitor of ceramide synthases, but differed in the efficiency to inhibit biosynthesis of short-chain versus long-chain ceramides. In vitro kinetic studies revealed that FTY720 is a competitive inhibitor of ceramide synthase 2 toward dihydrosphingosine with an apparent K(i) of 2.15 microm. FTY720-induced up-regulation of DHS1P level was mediated by sphingosine kinase (SphK) 1, but not SphK2, as confirmed by experiments using SphK1/2 silencing with small interfering RNA. Our data demonstrate for the first time the ability of FTY720 to inhibit ceramide synthases and modulate the intracellular balance of signaling sphingolipids. These findings open a novel direction for therapeutic applications of FTY720 that focuses on inhibition of ceramide biosynthesis, ceramide-dependent signaling, and the up-regulation of DHS1P generation in cells.

Topics: Cells, Cultured; Ceramides; Chromatography, Liquid; Endothelium, Vascular; Fingolimod Hydrochloride; Humans; Immunosuppressive Agents; Lung; Lysophospholipids; Oxidoreductases; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Propylene Glycols; Pulmonary Artery; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Serine C-Palmitoyltransferase; Sphingosine; Tandem Mass Spectrometry; Up-Regulation

Resistance to chemotherapeutic drugs often limits their clinical efficacy. Previous studies have implicated the bioactive sphingolipid sphingosine-1-phosphate (S-1-P) in regulating sensitivity to cisplatin [cis-diamminedichloroplatinum(II)] and showed that modulating the S-1-P lyase can alter cisplatin sensitivity. Here, we show that the members of the sphingosine kinase (SphK1 and SphK2) and dihydroceramide synthase (LASS1/CerS1, LASS4/CerS4, and LASS5/CerS5) enzyme families each have a unique role in regulating sensitivity to cisplatin and other drugs. Thus, expression of SphK1 decreases sensitivity to cisplatin, carboplatin, doxorubicin, and vincristine, whereas expression of SphK2 increases sensitivity. Expression of LASS1/CerS1 increases the sensitivity to all the drugs tested, whereas LASS5/CerS5 only increases sensitivity to doxorubicin and vincristine. LASS4/CerS4 expression has no effect on the sensitivity to any drug tested. Reflecting this, we show that the activation of the p38 mitogen-activated protein (MAP) kinase is increased only by LASS1/CerS1, and not by LASS4/CerS4 or LASS5/CerS5. Cisplatin was shown to cause a specific translocation of LASS1/CerS1, but not LASS4/CerS4 or LASS5/CerS5, from the endoplasmic reticulum (ER) to the Golgi apparatus. Supporting the hypothesis that this translocation is mechanistically involved in the response to cisplatin, we showed that expression of SphK1, but not SphK2, abrogates both the increased cisplatin sensitivity in cells stably expressing LASS1/CerS and the translocation of the LASS1/CerS1. The data suggest that the enzymes of the sphingolipid metabolic pathway can be manipulated to improve sensitivity to the widely used drug cisplatin.

Topics: Antineoplastic Agents; Blotting, Western; Cell Survival; Ceramides; Cisplatin; Drug Resistance, Neoplasm; Enzyme Activation; Fluorescent Antibody Technique; Golgi Apparatus; Humans; Immunoprecipitation; Lysophospholipids; Membrane Proteins; Oxidoreductases; p38 Mitogen-Activated Protein Kinases; Phosphotransferases (Alcohol Group Acceptor); Sphingosine; Sphingosine N-Acyltransferase

Previous studies have demonstrated that a number of biochemical actions of ceramide are mediated through protein kinase signalling pathways, such as p42/p44 mitogen-activated protein kinase (p42/p44 MAPK) and c-Jun N-terminal directed protein kinase (JNK). Ceramide-activated protein kinases, such as the kinase suppressor of Ras (KSR) and protein kinase Czeta (PKCzeta), are involved in the regulation of c-Raf, which promotes sequential activation of MEK-1 and p42/p44 MAPK in mammalian cells. However, in cultured airway smooth muscle (ASM) cells, neither KSR nor PKCzeta are involved in the C2-ceramide (C2-Cer)-dependent activation of this kinase cascade. Instead, we found that C2-Cer utilises a novel pathway involving tyrosine kinases, phosphoinositide 3-kinase (PI3K) and conventional PKC isoform(s). We also found that despite its ability to stimulate p42/p44 MAPK, C2-Cer inhibited platelet-derived growth factor (PDGF)-stimulated DNA synthesis. The possibility that growth arrest could be mediated by JNK was discounted on the basis that PDGF, as well as ceramide, stimulated JNK in these cells. Therefore, growth arrest in response to ceramide is mediated by an alternative mechanism.

Topics: Animals; Cell Membrane Permeability; Cell Survival; Cells, Cultured; Ceramides; Chromones; Enzyme Activation; Guinea Pigs; Isoenzymes; JNK Mitogen-Activated Protein Kinases; Lysophospholipids; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Morpholines; Muscle, Smooth; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Platelet-Derived Growth Factor; Precipitin Tests; Protein Kinase C; Protein Serine-Threonine Kinases; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-raf; Quinazolines; Respiratory System; Signal Transduction; Sphingosine; Tyrphostins

In previous studies, we have demonstrated that the inhibitory effects of tumor necrosis factor (TNF) and interleukin (IL)-1 on human erythroid colony formation are indirect and mediated by beta and gamma interferon (IFN), respectively, which act directly upon erythroid colony forming units (CFU-E). The in vitro inhibitory effect of gammaIFN but not betaIFN is reversed by exposure to high concentrations of recombinant human (rh) erythropoietin (EPO). Ceramide, a product of sphingomyelin hydrolysis, is a known mediator of apoptotic effects of TNF, IL-1, and gammaIFN. In this report, the effects of ceramide on CFU-E colony formation and its implication in the model described above are evaluated. Endogenous ceramide produced by exposure to bacterial sphingomyelinase (0.2-2.0 U/mL) and exogenous cell-permeable ceramide (C2-ceramide; 5 and 10 mM) significantly inhibited bone marrow CFU-E colony formation. This effect was reversed by the ceramide antagonist sphingosine-1-phosphate (S-1-P). Inhibition of CFU-E by rhgammaIFN, but not rhbetaIFN, was significantly reversed by S-1-P. rhEPO 10 U/mL reversed CFU-E inhibition by C2-ceramide 10 mM. Exposure of marrow cells to rhgammaIFN led to a 57% increase in ceramide content. The present study demonstrates that colony formation by human CFU-E is inhibited by endogenous and exogenous ceramide, and that inhibition by rhgammaIFN can be reversed by the ceramide antagonist S-1-P. Inhibition of CFU-E colony formation by ceramide and by are both reversed by high concentrations of rhEPO. These findings strongly suggest that ceramide mediates inhibition of human CFU-E colony formation by gammaIFN.

Topics: Apoptosis; Ceramides; Colony-Forming Units Assay; Depression, Chemical; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; fas Receptor; Humans; Interferon-beta; Interferon-gamma; Lysophospholipids; Recombinant Proteins; Sphingomyelin Phosphodiesterase; Sphingosine