The yins and yangs of ceramide

Since their discovery over 100 years ago,sphingolipids have caught the eyes and the imagination of scientists.Modern science has made many new insights on the cell biology and day-to-day functions of many integral sphingolipids,especially those of ceramids.Ceramide is recognized as a vital second messenger in the signal transduction process mediated by receptors of many cytokines and growth factors.A great part of our current understanding of ceramide has been achieved from apoptosis-related studies,however recent data in the fields of immunology,endocrinology and neurobiology,also suggest a fundamental involvement of ceramide in the onset of diseases.Therefore,understanding the diology of ceramide could be a key to unraveling many biological mechanisms and provide information for the treatment of some common diseases.     

Synthesis and biological activity of α-galactosyl ceramide KRN7000 and galactosyl (α1→2) galactosyl ceramide

We herein report a faster and less cumbersome synthesis of the biologically attractive, α-galactosyl ceramide (α-GalCer), known as KRN7000, and its analogues. More importantly, the use of a silicon tethered intramolecular glycosylation reaction gave easy access to the diglycosyl ceramide Gal(α1→2)GalCer, which has been shown to require uptake and processing to the biologically active α-GalCer derivative.     

Effects of ceramide,ceramidase inhibition and expression of ceramide kinase on cytosolic phospholipase A2α; additional role of ceramide-1-phosphate in phosphorylation and Ca2+ signaling

Ceramide and the metabolites including ceramide-1-phosphate (C1P) and sphingosine are reported to regulate the release of arachidonic acid (AA) and/or phospholipase A₂ (PLA₂) activity in many cell types including lymphocytes. Recent studies established that C1P, a product of ceramide kinase, interacts directly with Ca²⁺ binding regions in the C2 domain of α type cytosolic PLA₂ (cPLA₂α), leading to translocation of the enzyme from the cytosol to the perinuclear region in cells. However, a precise mechanism for C1P-induced activation of cPLA₂α has not been well elucidated; such as the phosphorylation signal caused by the extracellular signal-regulated kinases (ERK1/2) pathway, a downstream of the protein kinase C activation with 4β-phorbol myristate acetate (PMA), is required or not. In the present study, we showed that the increase in intracellular ceramide levels (exogenously added cell permeable ceramides and an inhibition of ceramidase by (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol and the increase in C1P formation by transfection with the vector for human ceramide kinase significantly enhanced the Ca²⁺ ionophore (A23187) -induced release of AA via cPLA₂α's activation in CHO cells. Ceramides did not show additional effects on the release from the cells treated with the inhibitor of ceramidase. Ceramides and C2-C1P neither had effect on the intracellular mobilization of Ca²⁺ nor the phosphorylation of cPLA₂α in cells. A23187/PMA-induced release of AA was enhanced by ceramides and C2-C1P and by expression of ceramide kinase. Our findings suggest that C1P is a stimulatory factor on cPLA₂α that is independent of the Ca²⁺ signal and the PKC-ERK-mediated phosphorylation signal.     

The many faces (and phases) of ceramide and sphingomyelin I – single lipids

Ceramides, the simplest kind of two-chained sphingolipids, contain a single hydroxyl group in position 1 of the sphingoid base. Sphingomyelins further contain a phosphocholine group at the OH of position 1 of ceramide. Ceramides and sphingomyelins show a variety of species depending on the fatty acyl chain length, hydroxylation, and unsaturation. Because of the relatively high transition temperature of sphingomyelin compared to lecithin and, particularly, of ceramides with 16:0–18:0 saturated chains, a widespread idea on their functional importance refers to formation of rather solid domains enriched in sphingomyelin and ceramide. Frequently, and especially in the cell biology field, these are generally (and erroneously) assumed to occur irrespective on the type of N-acyl chain in these lipids. This is because most studies indicating such condensed ordered domains employed sphingolipids with acyl chains with 16 carbons while scarce attention has been focused on the influence of the N-acyl chain on their surface properties. However, abundant evidence has shown that variations of the N-acyl chain length in ceramides and sphingomyelins markedly affect their phase state, interfacial elasticity, surface topography, electrostatics and miscibility and that, even the usually conceived “condensed” sphingolipids and many of their mixtures, may exhibit liquid-like expanded states. This review is a summarized overview of our work and of related others on some facts regarding membranes composed of single molecular species of ceramide and sphingomyelin. A second part is dedicated to discuss the miscibility properties between species of sphingolipids that differ in N-acyl and oligosaccharide chains.     

The many faces (and phases) of ceramide and sphingomyelin II – binary mixtures

A rather widespread idea on the functional importance of sphingolipids in cell membranes refers to the occurrence of ordered domains enriched in sphingomyelin and ceramide that are largely assumed to exist irrespective of the type of N-acyl chain in the sphingolipid. Ceramides and sphingomyelins are the simplest kind of two-chained sphingolipids and show a variety of species, depending on the fatty acyl chain length, hydroxylation, and unsaturation. Abundant evidences have shown that variations of the N-acyl chain length in ceramides and sphingomyelins markedly affect their phase state, interfacial elasticity, surface topography, electrostatics, and miscibility, and that even the usually conceived “condensed” sphingolipids and many of their mixtures may exhibit liquid-like expanded states. Their lateral miscibility properties are subtlety regulated by those chemical differences. Even between ceramides with different acyl chain length, their partial miscibility is responsible for a rich two-dimensional structural variety that impacts on the membrane properties at the mesoscale level. In this review, we will discuss the miscibility properties of ceramide, sphingomyelin, and glycosphingolipids that differ in their N-acyl or oligosaccharide chains. This work is a second part that accompanies a previous overview of the properties of membranes formed by pure ceramides or sphingomyelins, which is also included in this Special Issue.     

Fatty acid synthase causes drug resistance by inhibiting TNF-α and ceramide production

Fatty acid synthase (FASN) is a key enzyme in the synthesis of palmitate, the precursor of major nutritional, energetic, and signaling lipids. FASN expression is upregulated in many human cancers and appears to be important for cancer cell survival. Overexpression of FASN has also been found to associate with poor prognosis and higher risk of recurrence of human cancers. Indeed, elevated FASN expression has been shown to contribute to drug resistance. However, the mechanism of FASN-mediated drug resistance is currently unknown. In this study, we show that FASN overexpression causes resistance to multiple anticancer drugs via inhibiting drug-induced ceramide production, caspase 8 activation, and apoptosis. We also show that FASN overexpression suppresses tumor necrosis factor-α production and nuclear factor-κB activation as well as drug-induced activation of neutral sphingomyelinase. Thus, TNF-α may play an important role in mediating FASN function in drug resistance.     

A signaling cascade mediated by ceramide,src and PDGFRβ coordinates the activation of the redox-sensitive neutral sphingomyelinase-2 and sphingosine kinase-1

Stress-inducing agents, including oxidative stress, generate the sphingolipid mediators ceramide (Cer) and sphingosine-1-phosphate (S1P) that are involved in stress-induced cellular responses. The two redox-sensitive neutral sphingomyelinase-2 (nSMase2) and sphingosine kinase-1 (SK1) participate in transducing stress signaling to ceramide and S1P, respectively; however, whether these key enzymes are coordinately regulated is not known. We investigated whether a signaling link coordinates nSMase2 and SK1 activation by H₂O₂. In mesenchymal cells, H₂O₂ elicits a dose-dependent biphasic effect, mitogenic at low concentration (5 μM), and anti-proliferative and toxic at high concentration (100 μM).     

神经酰胺(ceramide)信使途径

生物体内神经酰胺有多种生成途径。作为重要的第二信使,神经酰胺在多种信号转导过程中发挥广泛作用,从而影响细胞生长、增殖、分化、凋亡及损伤等多种生理、病理过程。此机制的发现被认为是近年来信号转导研究的重要进展之一。     

Synthesis of truncated analogues of the iNKT cell agonist, α-galactosyl ceramide (KRN7000), and their biological evaluation

Stimulation of iNKT cells by ??-galactosyl ceramide (??-GalCer), also known as KRN7000, and its truncated analogue OCH induces both Th1- and Th2-cytokines, with OCH inducing a Th2-cytokine bias. Skewing of the iNKT cells’ response towards either a Th1- or Th2-cytokine profile offers potential therapeutic benefits. The length of both the acyl and the sphingosine chains in ??-galactosyl ceramides is known to influence the cytokine release profile. We have synthesized analogues of ??-GalCer with truncated sphingosine chains for biological evaluation, with particular emphasis on the Th1/Th2 distribution. Starting from a common precursor, d-lyxose, the sphingosine derivatives were synthesised via a straightforward Wittig condensation.     

Ceramide and ceramide 1-phosphate are negative regulators of TNF-α production induced by lipopolysaccharide

LPS is a constituent of cell walls of Gram-negative bacteria that, acting through the CD14/TLR4 receptor complex, causes strong proinflammatory activation of macrophages. In murine peritoneal macrophages and J774 cells, LPS at 1-2 ng/ml induced maximal TNF-α and MIP-2 release, and higher LPS concentrations were less effective, which suggested a negative control of LPS action. While studying the mechanism of this negative regulation, we found that in J774 cells, LPS activated both acid sphingomyelinase and neutral sphingomyelinase and moderately elevated ceramide, ceramide 1-phosphate, and sphingosine levels. Lowering of the acid sphingomyelinase and neutral sphingomyelinase activities using inhibitors or gene silencing upregulated TNF-α and MIP-2 production in J774 cells and macrophages. Accordingly, treatment of those cells with exogenous C8-ceramide diminished TNF-α and MIP-2 production after LPS stimulation. Exposure of J774 cells to bacterial sphingomyelinase or interference with ceramide hydrolysis using inhibitors of ceramidases also lowered the LPS-induced TNF-α production. The latter result indicates that ceramide rather than sphingosine suppresses TNF-α and MIP-2 production. Of these two cytokines, only TNF-α was negatively regulated by ceramide 1-phosphate as was indicated by upregulated TNF-α production after silencing of ceramide kinase gene expression. None of the above treatments diminished NO or RANTES production induced by LPS. Together the data indicate that ceramide negatively regulates production of TNF-α and MIP-2 in response to LPS with the former being sensitive to ceramide 1-phosphate as well. We hypothesize that the ceramide-mediated anti-inflammatory pathway may play a role in preventing endotoxic shock and in limiting inflammation.     

Role of sphingomyelinase and ceramide in modulating rafts: do biophysical properties determine biologic outcome?

Recent biophysical data suggest that the properties of ceramide observed in model membranes may apply to biological systems. In particular, the ability of ceramide to form microdomains, which coalesce into larger platforms or macrodomains, appears to be important for some cellular signaling processes. Several laboratories have now demonstrated similar reorganization of plasma membrane sphingolipid rafts, via ceramide generation, into macrodomains. This event appeared necessary for signaling upon activation of a specific set of cell surface receptors. In this article, we review the properties and functions of rafts, and the role of sphingomyelinase and ceramide in the biogenesis and re-modeling of these rafts. As clustering of some cell surface receptors in these domains may be critical for signal transduction, we propose a new model for transmembrane signal transmission.     

Globotriaosyl ceramide modulates interferon-α-induced growth inhibition and CD19 expression in Burkitt's lymphoma cells

Previous studies have indicated that globotriaosyl ceramide (Gb3 or CD77) plays a role in -interferon signal transduction and CD19-mediated homotypic adhesion in B cell lines derived from Burkitt's lymphoma. These roles for Gb3 may involve the proteins IFNAR-1 (subunit 1 of the interferon- receptor) and CD19, respectively, both of which have potential Gb3-binding sites in their extracellular domains which resemble those of the verotoxin (Shiga toxin and Shiga-like toxin) B subunit. The majority of this work was performed using wild-type Daudi cells and a single, Gb3-deficient mutant cell line, VT500. In the present investigations, these and additional Daudi-derived cells with varying degrees of sensitivity to interferon- were examined for Gb3 expression, interferon-induced growth inhibition and CD19 expression. The degree of interferon-induced growth inhibition and CD19 expression correlated with Gb3 expression in the various cell lines tested. In addition, reconstitution of the VT500 cell line with Gb3 but not other glycolipids partially restored the sensitivity of cells to IFN-induced growth inhibition. The degree to which reconstitution restored sensitivity to growth inhibition was similar to the results of previous studies in which Gb3 reconstitution restored sensitivity to verotoxin-induced cytotoxicity. These results demonstrate that Gb3 is specifically required for IFN-induced growth inhibition in Daudi cells and provide further evidence of a role for Gb3 in CD19 expression and function in these cells.     

Novel anti-viability ceramide analogs: Design,synthesis, and structure–activity relationship studies of substituted (S)-2-(benzylideneamino)-3-hydroxy-N-tetradecylpropanamides

A group of novel l-serinamides, substituted (S)-2-(benzylideneamino)-3-hydroxy-N-tetradecylpropanamides (3a–o) and substituted (S)-2-(benzylamino)-3-hydroxy-N-tetradecyl propanamides (4c, 4i, 4l, and 4o), were synthesized as potential anti-tumor lead compounds. In vitro cell viability assay results indicate treatment with 3a–o compounds resulted in significant inhibition of cell viability in the chemoresistant breast cancer cell line, MCF-7TN-R. Compounds 3i and 3l, both ortho-substituted analogs, show the greatest efficacy with IC₅₀ values of 10.3 μM and 12.5 μM, respectively. The SAR analysis indicate that the imine functional group of 3a–o is critical for the cellular anti-viability effect, and the partial atomic charge (PAC) value of imine C atom is a valuable structural parameter for predicting the activity of these ceramide analogs.     

Transforming growth factor-β1 suppresses serum deprivation-induced apop-tosis via activation of ERK1/ERK2 and the inhibition of p38 activity and ceramide production

In this report we studied the effects and mechanism of transforming growth factor-β1 (TGF-β1) on serum deprivation-induced cell apoptosis. Serum deprivation induces apoptosis, which is associated with an increase in intracellular ceramide level and with the activation of p38 mitogen-activated protein (MAP) kinase. Inhibition of p38 MAP kinase by SB203580 significantly reduced apoptosis induced by serum-deprivation. Treatment of cells with TGF-β1 stimulated cell proliferation and suppressed the serum deprivation-induced apoptotic response. The anti-apoptotic effect of TGF-β1 is correlated with its ability to inhibit the serum deprivation-induced activation of p38 MAP kinase and the increase in intracellular ceramide level. In     

Do longevity assurance genes containing Hox domains regulate cell development via ceramide synthesis?

A gene family containing a longevity assurance gene (Lag1p) motif is described. Database searches revealed >40 members of this family of transmembrane proteins, two of which have recently been shown to regulate the synthesis of ceramide, a lipid second messenger involved in a variety of cellular processes. We speculate that other family members, some of which contain a Hox domain, may also be involved in the synthesis of specific ceramide pools, perhaps explaining the role of longevity assurance genes in regulating development.     

A fluorometric assay of ceramide glycanase with 4-methylumbelliferyl β-d-lactoside derivatives

4-Methylumbelliferyl 6-O-benzyl--d-lactoside (6Bn-MU-Lac) and some related compounds were synthesizedvia different selective reactions including phase-transfer glycosylation. Their suitability as substrates for a fluorometric assay of ceramide glycanase (CGase) was evaluated. Among others, the 6Bn-MU-Lac, which is resistant to exogalactosidase, was found to be a suitable substrate for routine assay of the CGase activity. For American leech CGase, theK _m value is 0.232 mM at pH 5. Abbreviations: CGase, ceramide glycanase; Gal, galactose; Glc, Glucose; Lac, lactose; MU, 4-methylumbelliferone; MU-Lac, 4-methylumbelliferyl -d-lactoside; bBn-Lac, 6-O-benzyl-lactose; 6Bn-MU-Lac, 4-methylumbelliferyl 6-Obenzyl--d-lactoside; 46Bd-MU-Lac, 4-methylumbelliferyl 4,6-O-benzylidene--d-lactoside; MU-Cel, 4-methylumbellifery -d-cellobioside; 46Bd-MU-Cel, 4-methylumbelliferyl 4,6-O-benzylidene--d-cellobioside; TLC, thin layer chromatography;¹H-NMR, proton nuclear magnetic resonance; GSL, glycosphingolipids; CSA, 10-camphorsulfonic acid. See Scheme 1 for chemical structures.     

An accessory role for ceramide in interleukin-1β induced prostaglandin synthesis

Interleukin-1 (IL-1) is a potent inducer of prostaglandin E₂ (PGE₂) synthesis. We previously showed that ceramide accumulates in fibroblasts treated with IL-1 and that it enhances IL-1-induced PGE₂ production. The present study was undertaken to determine the mechanism(s) by which ceramide and IL-1 interact to enhance PGE₂ production by examining their respective effects on the rate-limiting enzymes in PGE₂ synthesis, cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and cytosolic phospholipase A₂ (cPLA₂). IL-1-induced PGE₂ synthesis required 8 h even though COX-1 was constitutively expressed (both mRNA and protein) and enzymatically active in untreated cells. Conversely, COX-2 mRNA was barely detectable in untreated cells but within 2 h, ceramide or IL-1 alone induced a 5 and 20 fold increase in COX-2 mRNA, respectively. However, IL-1 induced COX-2 protein synthesis was only detectable 6-7 h after maximal COX-2 mRNA induction; COX-2 protein accumulation was not induced by ceramide alone. Ceramide however, reduced the length of time required for IL- 1 to induce COX-2 protein accumulation and increased COX-2 protein accumulation. IL-1 induced a 15 fold increase in COX-1 mRNA including an alternatively spliced form of COX-1. IL-1, but not ceramide induced cPLA₂ mRNA and protein expression which corresponded with the initiation of PGE₂ synthesis. These observations indicate that, (1) while either ceramide or IL-1 rapidly induced COX-2 mRNA, COX-2 protein only accumulated in IL- 1 treated cells after a delay of 6-7 h, (2) IL-1-induced PGE₂ synthesis required both COX-2 and cPLA₂ protein synthesis and, (3) ceramide enhanced (temporally and quantitatively) IL-1-induced COX-2 protein accumulation resulting in enhanced PGE₂ production.