Ceramide-monohexosides and ceramide-dihexosides in lipoproteins of the membranes of fat globules in bovine milk.
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Amino Alcohols↗
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Membrane-destabilizing properties of C2-ceramide may be responsible for its ability to inhibit platelet aggregation.
We have studied the effects of short-chain ceramides on platelet structure and function. N-Acetylsphingosine (C2-ceramide), a cell-permeable short-chain analogue, and N-acetyldihydrosphingosine (C2-dihydroceramide), which lacks the 4-5 double bond, have been investigated. C2-Ceramide (15 microM) inhibited ADP-induced aggregation by 50% at a platelet concentration of 1.25 x 10(8)/mL, while it took twice that concentration to inhibit aggregation by 50% when the platelet concentration was doubled. This indicates that the effect of C2-ceramide on ADP-induced platelet aggregation depends on the ratio of ceramide to total platelet lipid, with a ratio of 0.2 giving significant inhibition. C2-Ceramide at a ceramide: lipid ratio of 0.2 caused platelets to form fenestrations and pseudopodia which were longer and thinner than those caused by agonists such as ADP or thrombin. C2-Dihydroceramide had no effect on ADP-induced aggregation or platelet morphology at any ceramide:lipid ratio. Platelet lysis was induced by C2-ceramide at higher ceramide:lipid ratios (0.5), whereas C2-dihydroceramide did not induce lysis, suggesting that C2-ceramide is able to destabilize membranes. This was tested directly by assessing whether the ceramides induced leakage of 6-carboxyfluorescein from lipid vesicles. C2-Ceramide caused nearly total leakage of dye from the vesicles at a ceramide:lipid ratio of 10. The leakage caused by C2-dihydroceramide at a ceramide:lipid ratio of 10 was equal to that induced by C2-ceramide at a ratio of 0.2 (approximately 3%). The ability of the ceramides to destabilize membranes was also examined by measuring changes in fluorescence anisotropy of the fluorescent dye 1,6-diphenyl-1,3,5-hexatriene (DPH) incorporated into lipid vesicles. C2-Ceramide induced a larger decrease in anisotropy than a detergent (Triton X-100) which is known to lyse membranes. C2-Dihydroceramide did not alter membrane fluidity. The ability of C2-ceramide to cause platelet fenestrations, formation of irregular platelet pseudopodia, platelet lysis, lipid vesicle leakage, and increases in the fluidity of lipid vesicles all suggest that C2-ceramide inhibits platelet aggregation because it destabilizes the platelet membrane. C2-Dihydroceramide did not inhibit platelet aggregation and lacked the nonspecific effects on membranes that C2-ceramide possessed, suggesting that C2-dihydroceramide is not an appropriate control for the nonspecific effects of C2-ceramide.
Adenine↗
Ceramide signalling and the immune response.
Ceramide, produced through either the induction of SM hydrolysis or synthesized de novo transduces signals mediating differentiation, growth, growth arrest, apoptosis, cytokine biosynthesis and secretion, and a variety of other cellular functions. A generalized ceramide signal transduction scheme is shown in Fig. 2 in which ceramide is generated through the activation of distinct SMases residing in separate subcellular compartments in response to specific stimuli. Clearly, specificity of cellular responses to ceramide depends upon many factors which include the nature of the stimulus, co-stimulatory signals and the cell type involved. Ceramide derived from neutral SMase activation is thought to be involved in modulating CAPK and MAP kinases, PLA2 (arachidonic acid mobilization), and CAPP while ceramide generated through acid SMase activation appears to be primarily involved in NF-kappa B activation. While there is no apparent cross-talk between these two ceramide-mediated signalling pathways, there is likely to be significant cross-talk between ceramide signalling and other signal transduction pathways (e.g., the PKC and MAP kinase pathways). Other downstream targets for ceramide action include Cox, IL-6 and IL-2 gene expression, PKC zeta, Vav, Rb, c-Myc, c-Fos, c-Jun and other transcriptional regulators. Many, if not all, of these ceramide-mediated signalling events have been identified in the various cells comprising the immune system and are integral to the optimal functioning of the immune system. Although the role of the SM pathway and the generation of ceramide in T and B lymphocytes have only recently been recognized, it is clear from these studies that signal transduction through SM and ceramide can strongly affect the immune response, either directly through cell signalling events, or indirectly through cytokines produced by other cells as the result of signalling through the SM pathway. An overview of the signalling mechanisms coupling ceramide to the modulation of the immune response is depicted in Fig. 3 and shows how ceramide may play pivotal roles in regulating a number of complex processes. The SM pathway represents a potentially valuable focal point for therapeutic control of immune responses, perhaps for either enhancement of the activity of T cells in the elimination of tumors, or the down-regulation of lymphocyte function in instances of autoimmune disease. The recent explosion of knowledge regarding ceramide signalling notwithstanding, a number of critical questions need to be answered before a comprehensive, mechanistic understanding can be formulated relative to the incredibly varied effects of ceramide on cell function. For example, (i) how is a structurally simple molecule like ceramide able to mediate so many different, and sometimes paradoxical, physiological responses ranging from cell proliferation and differentiation to inhibition of cell growth and apoptosis, (ii) what are the molecular identities and modes of activation of the various SMase isoforms, (iii) what determines the distribution of the unique isoforms of SMase in cells of different lineages or at different stages of differentiation, (iv) what is the relative contribution of ceramide generated through SM hydrolysis versus de novo synthesis, and (v) by what means does ceramide interact with specific intracellular targets? Although a number of ceramide-activatable kinases, phosphatases, and their protein substrates have been identified, a more extensive search for additional cellular targets will be indispensable in determining the phosphorylation cascades linking the activation of the SM pathway to the regulation of nuclear events. Clearly, cross-talk between ceramide-induced signal transduction cascades and other signalling pathways adds to the inherent difficulty in distinguishing the specific effects of complex, intertwining signalling pathways.
Cell Physiological Phenomena↗
Differential binding of ceramide to MEKK1 in glomerular endothelial and mesangial cells.
Previously, we have shown that ceramide is able to directly bind to and activate c-Raf and to trigger the downstream classical mitogen-activated protein kinase (MAPK/ERK) cascade in glomerular mesangial cells [Proc. Natl. Acad. Sci. USA 93 (1996) 6959]. In this study, we show that ceramide acts differently in glomerular endothelial cells in that treatment of endothelial cells with exogenous ceramide leads to a potent activation of the stress-activated protein kinase (SAPK/JNK) cascade but not to an activation of the classical ERK cascade. A similar effect was observed with the inflammatory cytokines TNFalpha and IL-1beta, which activate a sphingomyelinase and thereby increase intracellular ceramide levels. The activation of JNKs as shown by c-Jun phosphorylation assays was paralleled by increased phosphorylation of the two JNK isoforms, p45 and p54. In addition, also the activator of JNKs, SEK1, was found to be increasingly phosphorylated by exogenous ceramide as well as by TNFalpha. In contrast, dihydroceramide had no effect on JNK or SEK1 phosphorylation. To see whether ceramide directly binds to MEKK1, which is the c-Raf analog in the SAPK cascade, a radioiodinated photoaffinity labeling analogue of ceramide, (N-[3-[[[2-(125I)iodo-4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzyl]oxy]-carbonyl] propanoyl]-D-erythro-sphingosine) ([125I]TID-ceramide) was used. Stimulation of endothelial cells with this [125I]TID-ceramide for 5 min followed by a short photolysis defined MEKK1 as a direct target of ceramide. With the same method, protein kinase C-alpha (PKC-alpha) was identified as a ceramide target. In contrast, no binding to c-Raf or the MEKK1 activator p65-PAK could be detected. A direct binding of ceramide to MEKK1 was also confirmed by affinity chromatography using a ceramide-coupled sepharose column. Furthermore, the ceramide-activated SAPK/JNK cascade is clearly involved in the mechanism of apoptosis, since in the presence of a JNK inhibitor, ceramide-induced DNA fragmentation is significantly reduced. In summary, we have shown that ceramide potently activates the SAPK cascade but not the ERK cascade in endothelial cells, which contrasts to mesangial cells where ceramide activates the ERK pathway and has only a minor effect on the SAPK cascade. Regarding the direct target of ceramide binding and action in endothelial cells, we identified MEKK1 as a further member of the growing family of ceramide-activated protein kinases.
Animals↗
The structural requirements for ceramide activation of serine-threonine protein phosphatases.
The protein phosphatases1 (PP1) and 2A (PP2A) serve as ceramide-activated protein phosphatases (CAPP). In this study, the structural requirements for interaction between ceramide and CAPP were determined. D-erythro-C(6) ceramide activated the catalytic subunit of PP2A (PP2Ac) approximately 3-fold in a stereospecific manner. In contrast, saturation of the 4-5 double bond, producing D-erythro-dihydro C(6) ceramide, inhibited PP2Ac (IC(50) = 8.5 microM). Furthermore, phyto C(6) ceramide, D-erythro-dehydro C(6) ceramide, and D-erythro-cis-C(6) ceramide had no effect on PP2Ac activity. Modification of the sphingoid chain also abolished the ability of ceramide to activate PP2Ac. Further studies demonstrated the requirement for the amide group, the primary hydroxyl group, and the secondary hydroxyl group of the sphingoid backbone for activation of PP2Ac through the synthesis and evaluation of D-erythro-urea C(6) ceramide, L-erythro-urea C(6) ceramide, D-erythro-N-methyl C(6) ceramide, D-erythro-L-O-methyl C(6) ceramide, D-erythro-3-O-methyl C(6) ceramide, and (2S) 3-keto C(6) ceramide. None of these compounds induced significant activation of PP2Ac. Liposome binding studies were also conducted using analogs of D-erythro-C C(6) ceramide, and the results showed that the ability of ceramide analogs to influence CAPP (activation or inhibition) was associated with the ability of the analogs to bind to CAPP. This study demonstrates strict structural requirements for interaction of ceramide with CAPP, and disclose ceramide as a very specific regulator of CAPP. The studies also begin to define features that transform ceramide analogs into inhibitors of CAPP.
Amides↗
Ceramide activates heterotrimeric protein phosphatase 2A.
Ceramide activates a cytosolic protein phosphatase present in rat T9 glioma cells and rat brain. Ceramide-activated protein phosphatase (CAPP) was found to share several properties with protein phosphatase 2A (PP2A) leading to the hypothesis that ceramide may directly activate PP2A. PP2A was isolated as a heterotrimer (AB'C, AB alpha C), heterodimer (AC), or free C subunit, and the effect of ceramide on the catalytic activity was assessed. C2-ceramide, 5-20 microM, activated heterotrimeric PP2A up to 3.5-fold but had no effect on the activity of AC or C. Ceramides possessing hexanoyl, decanoyl, and myristoyl but not stearoyl acyl chains also activated heterotrimeric PP2A. Ceramide activation of heterotrimeric PP2A required the presence of a B subunit since trypsinization or heparin treatment abolished ceramide activation. Activation of heterotrimeric PP2A was specific for ceramide because related sphingolipids had no effect. Moreover, dihydro-C2-ceramide, which lacks the trans double bond in the sphingoid base, inhibited AB'C activity by > 90% at 10 microM. The specificity of activation of AB'C and AB alpha C by stereoisomers of C2-ceramide was found to differ. Whereas activation of AB'C by either DL-erythro- or threo-C2-ceramide was similar, AB alpha C was activated by either D- or L-erythro-C2-ceramide but not by the threo isomers. CAPP isolated from T9 cells was most effectively activated by D-erythro-C2-ceramide. CAPP was found to possess two peaks of ceramide activated phosphatase activity. The initial peak of activity was coincident with the elution of AB'C and was stimulated 1.8-fold by 20 microM C2-ceramide. A second peak of phosphatase activity was negligible in the absence of ceramide but was stimulated 5.5-fold by 20 microM C2-ceramide. These results support the hypothesis that ceramide is a specific lipid second messenger modulating heterotrimeric PP2A activity.
Animals↗
Astroglial expression of ceramide in Alzheimer's disease brains: a role during neuronal apoptosis.
Accumulating evidences indicate that ceramide is closely involved in apoptotic cell death in neurodegenerative disorders and aging. We examined ceramide levels in the cerebrospinal fluid (CSF) or brain tissues from patients with neurodegenerative disorders and the mechanism of how intra- and extracellular ceramide was regulated during neuronal apoptosis. We screened the ceramide levels in the CSF of patients with neurodegenerative disorders, and found that ceramide was significantly increased in patients with Alzheimer's disease (AD) than in patients with age-matched amyotrophic lateral sclerosis (ALS) and other neurological controls. With immunohistochemistry in AD brains, ceramide was aberrantly expressed in astroglia in the frontal cortices, but not detected in ALS and control brains. To explore for the regulation of ceramide in astroglia in Alzheimer's disease brains, we examined the metabolism of ceramide during neuronal apoptosis. In retinoic acid (RA)-induced neuronal apoptosis, RA slightly increased de novo synthesis of ceramide, but interestingly, RA dramatically inhibited conversion of [14C] ceramide to glucosylceramide (GlcCer), suggesting that the increase of ceramide mass is mainly due to inhibition of the ceramide-metabolizing enzyme GlcCer synthase. In addition, a significant increase of the [14C] ceramide level in the culture medium was detected by chasing and turnover experiments without alteration of extracellular [14C] sphingomyelin levels. A 2.5-fold increase of ceramide mass in the supernatant was also detected after 48 h of treatment with RA. These results suggest a regulatory mechanism of intracellular ceramide through inhibition of GlcCer synthase and a possible role of ceramide as an extracellular/intercellular mediator for neuronal apoptosis. The increased ceramide level in the CSF from AD patients, which may be derived from astroglia, raises a possibility of neuronal apoptosis by the response to intercellular ceramide in AD.
Aged↗
Ceramides modulate protein kinase C activity and perturb the structure of Phosphatidylcholine/Phosphatidylserine bilayers.
We studied the effects of natural ceramide and a series of ceramide analogs with different acyl chain lengths on the activity of rat brain protein kinase C (PKC) and on the structure of bovine liver phosphatidylcholine (BLPC)/dipalmitoylphosphatidylcholine (DPPC)/dipalmitoylphosphatidylserine (DPPS) (3:1:1 molar ratio) bilayers using (2)H-NMR and specific enzymatic assays in the absence or presence of 7.5 mol % diolein (DO). Only a slight activation of PKC was observed upon addition of the short-chain ceramide analogs (C(2)-, C(6)-, or C(8)-ceramide); natural ceramide or C(16)-ceramide had no effect. In the presence of 7.5 mol % DO, natural ceramide and C(16)-ceramide analog slightly attenuated DO-enhanced PKC activity. (2)H-NMR results demonstrated that natural ceramide and C(16)-ceramide induced lateral phase separation of gel-like and liquid crystalline domains in the bilayers; however, this type of membrane perturbation has no direct effect on PKC activity. The addition of both short-chain ceramide analogs and DO had a synergistic effect in activating PKC, with maximum activity observed with 20 mol % C(6)-ceramide and 15 mol % DO. Further increases in C(6)-ceramide and/or DO concentrations led to decreased PKC activity. A detailed (2)H-NMR investigation of the combined effects of C(6)-ceramide and DO on lipid bilayer structure showed a synergistic effect of these two reagents to increase membrane tendency to adopt nonbilayer structures, resulting in the actual presence of such structures in samples exceeding 20 mol % ceramide and 15 mol % DO. Thus, the increased tendency to form nonbilayer lipid phases correlates with increased PKC activity, whereas the actual presence of such phases reduced the activity of the enzyme. Moreover, the results show that short-chain ceramide analogs, widely used to study cellular effects of ceramide, have biological effects that are not exhibited by natural ceramide.
1,2-Dipalmitoylphosphatidylcholine↗
Ceramide inactivates cellular protein kinase Calpha.
Ceramide mediates the effects of extracellular agents on cellular growth, differentiation and apoptosis. In this study, we explored the mechanisms by which ceramide induces its cellular effects. In Molt-4 cells, phorbol 12-myristate 13-acetate (PMA) induced retinoblastoma gene product (Rb) phosphorylation, and ceramide inhibited this effect, suggesting an inhibitory effect of ceramide on the protein kinase C (PKC) pathway, the primary target of PMA. Molt-4 cells contained primarily PKCalpha and betaII isoforms of PKC. To determine the effects of ceramide on PKC, we developed an immunoprecipitation assay for PKCalpha activity. Exposure of Molt-4 cells to C6-ceramide resulted in a concentration and time-dependent inhibition of immunoprecipitated protein kinase Calpha (PKCalpha). Initial inhibition was observed as early as 4.5 h after treatment of cells with C6-ceramide, and the activity was completely lost by 13 h. Inhibition of PKCalpha activity was seen at concentrations of ceramide as low as 5 microM with maximal effects occurring at a concentration of 15 microM. Both C2 and C6-ceramide were inhibitory, but C2 and C6 dihydroceramides were not. Ceramide did not directly inhibit PKCalpha in vitro or modulate the levels of PKCalpha protein, suggesting that ceramide acted indirectly. Moreover, ceramide did not inhibit PMA-induced translocation of PKCalpha. Taken together, these results suggested that ceramide caused inactivation of PKCalpha. Since PKC requires phosphorylation for activity, we determined the effects of ceramide on phosphorylation of PKCalpha. C6-ceramide inhibited basal and PMA-induced phosphorylation of PKCalpha. In addition, okadaic acid, a potent phosphatase inhibitor, slightly stimulated PKC activity and blocked the effects of ceramide on PKCalpha inhibition. These results demonstrate that ceramide causes inhibition/inactivation of PKCalpha and suggest these effects of ceramide may be mediated by a protein phosphatase.
Biological Transport↗
Heat-induced elevation of ceramide in Saccharomyces cerevisiae via de novo synthesis.
Sphingolipid-related metabolites have been implicated as potential signaling molecules in many studies with mammalian cells as well as in some studies with yeast. Our previous work showed that sphingolipid-deficient strains of Saccharomyces cerevisiae are unable to resist a heat shock, indicating that sphingolipids are necessary for surviving heat stress. Recent evidence suggests that one role for the sphingolipid intermediate ceramide may be to act as a second messenger to signal accumulation of the thermoprotectant trehalose. We examine here the mechanism for generating the severalfold increase in ceramide observed during heat shock. As judged by compositional analysis and mass spectrometry, the major ceramides produced during heat shock are similar to those found in complex sphingolipids, a mixture of N-hydroxyhexacosanoyl C18 and C20 phytosphingosines. Since the most studied mechanism for ceramide generation in animal cells is via a phospholipase C-type sphingomyelin hydrolysis, we examined S. cerevisiae for an analogous enzyme. Using [3H]phytosphingosine and [3H]inositol-labeled yeast sphingolipids, a novel membrane-associated phospholipase C-type activity that generated ceramide from inositol-P-ceramide, mannosylinositol-P-ceramide, and mannose(inositol-P)2-ceramide was demonstrated. The sphingolipid head groups were concomitantly liberated with the expected stoichiometry. However, other data demonstrate that the ceramide generated during heat shock is not likely to be derived by breakdown of complex sphingolipids. For example, the water-soluble fraction of heat-shocked cells showed no increase in any of the sphingolipid head groups, which is inconsistent with complex sphingolipid hydrolysis. Rather, we find that de novo ceramide synthesis involving ceramide synthase appears to be responsible for heat-induced ceramide elevation. In support of this hypothesis, we find that the potent ceramide synthase inhibitor, australifungin, completely inhibits both the heat-induced increase in incorporation of [3H]sphinganine into ceramide as well as the heat-induced increase in ceramide as measured by mass. Thus, heat-induced ceramide most likely arises by temperature activation of the enzymes that generate ceramide precursors, activation of ceramide synthase itself, or both.
Antifungal Agents↗
Renal cortical ceramide patterns during ischemic and toxic injury: assessments by HPLC-mass spectrometry.
Ceramides are a class of signaling molecules that can acutely accumulate in tissues as part of a "stress response." They are classically measured by the diacylglycerol kinase assay, which, in general, measures total ceramide rather than individual moieties within the diverse ceramide family. The present study was undertaken to 1) adapt current HPLC-mass spectrometry technology for measuring individual renal ceramides, and 2) use this technique to more fully characterize the nature of the renal ceramide "stress" reaction. Renal cortical tissues were obtained from CD-1 mice under control conditions and 2 or 18 h after renal injury (ischemia-reperfusion and glycerol-mediated myohemoglobinuria). C24, C22, and C16 ceramides were identified in normal renal cortex, constituting 70, 10, and 20% of the total ceramide pool, respectively. Within each of these families, heterogeneity was apparent because of differing degrees of unsaturation (0-3 double bonds) in the constituent fatty acid of ceramide. Renal injury dramatically changed ceramide profiles: 1) total ceramide increased by approximately 300%; 2) although all ceramides participated in this reaction, they did so to differing degrees; 3) this caused pronounced changes in ceramide distribution patterns; 4) injury induced a striking shift toward unsaturated (vs. saturated) fatty acids within the C22 and C24 (but not the C16) ceramide pools; and 5) the extent of these qualitative changes differed according to the etiology of the initiating renal damage. Thus we conclude that ceramide stress response involves major qualitative (and not simply quantitative) changes in ceramide expression that are partially disease dependent. These findings underscore the fact that simply measuring total renal ceramide content (e.g., by diacylglycerol kinase assay) substantially oversimplifies the nature and, hence, the potential implications of the ceramide stress reaction.
Animals↗
Ceramide-mediated growth inhibition and CAPP are conserved in Saccharomyces cerevisiae.
Ceramide is emerging as a potential physiologic regulator of growth and differentiation in mammalian cells. This regulation may be mediated through the action of a serine/threonine ceramide-activated protein phosphatase (CAPP). In this study, the existence of a ceramide-mediated pathway of cell regulation in Saccharomyces cerevisiae was investigated. Incubating exponentially growing S. cerevisiae cells with 1-20 microM cell-permeable ceramide (C2-ceramide) produced a dose-dependent inhibition of proliferation. A number of other lipids and detergents, such as arachidonate, oleate, Triton X-100, dioctanoylglycerol, and phenylaminoalcohol ceramide analogs, were largely ineffective, demonstrating the specificity of the response. Stereospecificity was demonstrated, in that the D enantiomer of erythro-C2-ceramide was more potent than the L enantiomer. More dramatically, a highly specific structural requirement for C2-ceramide was demonstrated, in that 1-12 microM C2-dihydroceramide was completely ineffective at inhibiting growth. Since C2-dihydroceramide lacks the 4-5 trans double bond present in C2-ceramide, this suggests that the antiproliferative properties of C2-ceramide depend upon the presence of the double bond. This raises an interesting possibility; the dehydrogenase responsible for introduction of the double bond during endogenous ceramide synthesis may regulate cell growth by controlling the cellular concentrations of dihydroceramide and ceramide. The oxygenase responsible for introduction of the final hydroxyl group in phytoceramide could provide a similar regulatory function in yeast. The potential role of CAPP in ceramide action in yeast was investigated next. Crude extracts of S. cerevisiae also contained a ceramide-dependent serine/threonine phosphatase activity, which was sensitive to inhibition by okadaic acid. This enzyme exhibited stereospecificity and structural requirements identical to that of the ceramide-induced growth inhibition. We conclude that both the growth-inhibitory response to ceramide and CAPP activity are conserved in S. cerevisiae. The identical stereochemical and structural requirements in both the biological and phosphatase assays suggest that the anti-proliferative effects of ceramide in yeast may be mediated in part through the action of CAPP.
Ceramides↗
Sphingolipid metabolic changes during chiral C2-ceramides induced apoptosis in human leukemia cells.
N-acetylsphingosine (C2-ceramide) is a synthetic water-soluble ceramide mimicking the activity of natural ceramides. By fixing chiral conformation on carbon numbers 2 and 3 in the ceramide structure, four chiral C2-ceramides naming d-erythro-, l-erythro-, d-threo- and l-threo C2-ceramide were synthesized. We have investigated the chiral effects of these C2-ceramides on the sphingolipid metabolism, particularly on both the sphingolipid biosynthetic pathway and on the degradation pathway. In both HL-60 and U937 cells, the chiral C2-ceramide (10 microM) showed sphingosine accumulation monitored fluoromatrically by a high performance liquid chromatographic separation of the sphingoid bases. Most importantly, in HL-60 cells, l-erythro C2-ceramide induced a 50 fold increase in sphingosine as compared to the control, while l-threo C2-ceramide exhibited a minimal 7-fold increase. In contrast, sphinganine, another sphingoid base, showed less accumulation by any chiral C2-ceramide tested under the same conditions. These results suggested that chiral C2-ceramide primarily acts on the sphingolipid degradation pathway rather than on the sphingolipid biosynthetic route. The strong G0/G1 phase arrest in the cell cycle by treatment of l-erythro C2-ceramide indicates that the blockade of the sphingolipid degradation pathway might be concomitantly involved in the dysfunction of the cell cycle. On the other hand, the fact that all chiral C2-ceramides tested failed to inhibit the activity of sphingosine kinase acting on the removal of sphingosine by producing sphingosine-l-phosphate demonstrates that chiral C2- ceramides may increase sphingosine by activating various ceramidases by which natural ceramides are divided into sphingosine and free fatty acids. However, the precise steps involved in this interaction are still unknown.
Apoptosis↗
C2-ceramide primes specifically for the superoxide anion production induced by N-formylmethionylleucyl phenylalanine (fMLP) in human neutrophils.
Activated sphingomyelinases release ceramide molecules believed to be involved in intracellular signalling. The present study investigated whether soluble C2-ceramide modulates some of the effects of N-formylmethionylleucyl phenylalanine (fMLP) and other agonists on human neutrophils (or polymorphonuclear leukocytes-PMN); principally superoxide anion (O2-) production. The preincubation of PMN for 15 min with C2-ceramide increased by up to almost 3-fold the amounts of O2- generated in response to 0.1 and 1 microM fMLP. Priming was detected at C2-ceramide concentrations of 2 microM to 4 microM per million PMN. Though less potent than C2-ceramide, C6-ceramide (N-hexanoylsphingosine) could prime for O2- generated in response to 0.1 microM fMLP, with maximal effects obtained at 10-20 microM. In contrast, micromolar concentrations of sphingosine, dihydroceramide, and ceramide-phosphate, failed to exert any potentiating effect on fMLP-induced O2- generation. As expected, TNF-alpha (1000 U/ml), also primed for fMLP-induced O2- production; however, the combination of TNF-alpha and C2-ceramide showed no additive effect. Moreover, S. aureus sphingomyelinase (0.1 U/ml), was unable to reproduce the priming effects of C2-ceramide and TNF-alpha. C2-ceramide at 2 microM did not enhance the production of O2- induced by 100 nM recombinant human interleukin-8 (IL-8), leukotriene B4 (LTB4), platelet-activating factor (PAF) or 20 mM sodium fluoride (NaF). Furthermore, C2-ceramide (2 microM) did not enhance the mobilization of calcium, the release of arachidonic acid or the accumulation of phosphatidylethanol, induced by 100 nM fMLP. This suggests that probably neither phospholipases C, A2 or D (PLC, PLA2, PLD) were involved in the priming effect by C2-ceramide. However, C2-ceramide inhibited in a dose-related manner the production of O2- induced by phorbol 12-myristate 13-acetate (PMA) and mezerein. Furthermore, PMA-stimulated PLD activity was also significantly reduced by a preincubation of PMN with C2-ceramide. The priming of O2- production by C2-ceramide could involve yet unidentified mechanisms specific for fMLP, or it might imply that cytokines such as TNF-alpha have different mechanisms than C2-ceramide.
Arachidonic Acid↗
Ceramide interaction with the respiratory chain of heart mitochondria.
A study is presented on the interaction of ceramide with the respiratory chain of rat heart mitochondria, and a comparison is made between the effects elicited by short- and long-chain ceramides. N-Acetylsphingosine (C(2)-ceramide) and N-palmitoylsphingosine (C(16)-ceramide) inhibited to the same extent the pyruvate+malate-dependent oxygen consumption. Succinate-supported respiration was also inhibited by ceramides, but this activity was substantially restored upon the addition of cytochrome c, which, on the contrary, was ineffective toward the ceramide-inhibited NADH-linked substrate oxidation. Direct measurements showed that short- and long-chain ceramides caused a large release of cytochrome c from mitochondria. The ceramide-dependent inhibition of pyruvate+malate and succinate oxidation caused reactive oxygen species to be produced at the level of either complex I or complex III. The activity of the cytochrome c oxidase, measured as ascorbate/TMPD oxidase activity, was significantly stimulated and inhibited by C(2)- and C(16)-ceramide, respectively. Similar effects were observed on the activity of the individual respiratory complexes isolated from bovine heart. Short- and long-chain ceramides had definitely different effects on the mitochondrial membrane potential. C(2)-ceramide caused an almost complete collapse of the respiration-dependent membrane potential, whereas C(16)-ceramide had a negligible effect. Similar results were obtained when the potential was generated in liposome-reconstituted complex III respiring at the steady-state. Furthermore, C(2)-ceramide caused a drop of the membrane potential generated by ATP hydrolysis instead of respiration, whereas C(16)-ceramide did not. Finally, only short-chain ceramides inhibited markedly the reactive oxygen species generation associated with membrane potential-dependent reverse electron flow from succinate to complex I. The emerging indication is that the short-chain ceramide-dependent collapse of membrane potential is a consequence of their ability to perturb the membrane structure, leading to an unspecific increase of its permeability.
Adenosine Triphosphate↗
Ceramide regulates cellular homeostasis via diverse stress signaling pathways.
The sphingolipid ceramide is an important second signal molecule that regulates diverse signaling pathways involving apoptosis, cell senescence, the cell cycle, and differentiation. For the most part, ceramide's effects are antagonistic to growth and survival. Interestingly, ceramide and the pro-growth agonist, diacylglycerol (DAG) appear to be regulated simultaneously but in opposite directions in the sphingomyelin cycle. While ceramide stimulates signal transduction pathways that are associated with cell death or at least are inhibitory to cell growth (eg stress-activated protein kinase, SAPK, pathways), DAG activates the classical and novel isoforms of the protein kinase C (PKC) family. These PKC isoforms are associated with cell growth and cell survival. Furthermore, DAG activation of PKC stimulates other signal transduction pathways that support cell proliferation (eg mitogen-activated protein kinase, MAPK, pathways). Thus, ceramide and DAG generation may serve to monitor cellular homeostasis by inducing pro-death or pro-growth pathways, respectively. The production of ceramide is emerging as a fixture of programmed cell death. Ceramide levels are elevated in response to diverse stress challenges including chemotherapeutic drug treatment, irradiation, or treatment with pro-death ligands such as tumor necrosis factor alpha, TNF alpha. Consistent with this notion, ceramide itself is a potent apoptogenic agent. Ceramide activates stress-activated protein kinases like c-jun N-terminal kinase (JNK) and thus affects transcription pathways involving c-jun. Ceramide activates protein phosphatases such as protein phosphatase 1 (PP1) and protein phosphatase 2 (PP2A). Ceramide activation of protein phosphatases has been shown to promote inactivation of a number of pro-growth cellular regulators including the kinases PKC alpha and Akt, Bcl2 and the retinoblastoma protein. A new role has recently emerged for ceramide in the regulation of protein synthesis. Ceramide-induced activation of double-stranded RNA-dependent protein kinase (PKR), a protein kinase important in anti-viral host defense mechanisms and recently implicated in cellular stress pathways, results in the inhibition of protein synthesis as a prelude to cell death. Taken together, these properties of ceramide suggest that this important second-signal molecule may have useful properties as an anti-neoplastic agent. Thus, strategies to promote ceramide metabolism or use of ceramide analogs directly may one day become useful in the treatment of diseases like leukemia.
Animals↗
C2-ceramide induces apoptosis in a human squamous cell carcinoma cell line.
BACKGROUND: Previous studies have demonstrated that synthetic cell-permeable analogues of ceramide promote differentiation and inhibit proliferation of keratinocytes, and that the vitamin D3 inducible sphingomyelin cycle generates ceramide in keratinocytes. Although it has been suggested that exogenous ceramide induces apoptosis of keratinocytes, which is similar to their effect on other cell types, such as leukaemia cells, only a few studies have reported ceramide-induced apoptosis of keratinocytes. OBJECTIVE: To determine whether ceramide induces apoptosis of keratinocytes, we used the synthetic ceramide analogue, C2-ceramide (N-acetylsphingosine) and a human squamous cell carcinoma cell line, HSC-I. METHODS: We treated HSC-I cells with C2-ceramide, followed by a viability assay, morphological observations, nick end-labelling (TUNEL), DNA electrophoresis, and electron microscopy. RESULTS: In the viability assay, C2-ceramide was toxic to HSC-I cells in a dose-dependent manner. Manifestations of apoptotic morphology occurred in the ceramide-treated cells, whereas these morphological changes did not occur in cells treated with dihydroceramide (N-acetylsphinganine). TUNEL revealed that many of the ceramide-treated cells showed positive reactivity. DNA electrophoresis demonstrated that C2-ceramide caused internucleosomal fragmentation in a dose- and time-dependent manner. Electron microscopy revealed that the ceramide-treated cells manifested morphological characteristics typical of apoptosis. CONCLUSIONS: The present results demonstrate that C2-ceramide induces apoptosis of transformed human keratinocytes, whereas C2-dihydroceramide does not have such an effect. The fact that ceramide induces apoptosis of keratinocyctes raises the possibility that intracellular ceramide, which is increased with differentiation of the epidermis, might be involved in terminal differentiation, a specialized form of apoptosis of keratinocytes.
Apoptosis↗
Ceramide is a mediator of apoptosis in retina photoreceptors.
PURPOSE: The precise mechanisms involved in photoreceptor apoptosis are still unclear. In the present study, the role of ceramide, a sphingolipid precursor that induces apoptosis on cellular stress, was investigated in relation to the activation of cell death in photoreceptors. METHODS: Rat retina neuronal cultures, with or without docosahexaenoic acid (DHA), were treated with the ceramide analogue acetylsphingosine (C2-ceramide), and with a glucosylceramide synthase inhibitor. Ceramide synthesis in cultures treated with the oxidant paraquat was evaluated with [3H]palmitate. The effect of inhibitors of ceramide de novo synthesis, fumonisin B1 and cycloserine, on photoreceptor apoptosis was investigated. Apoptosis, mitochondrial membrane potential, and Bcl-2 expression were determined. RESULTS: Addition of C2-ceramide induced photoreceptor apoptosis. Paraquat increased formation of [3H]ceramide in photoreceptors, compared with the control, whereas inhibition of ceramide synthesis, immediately before paraquat treatment, prevented paraquat-induced photoreceptor apoptosis. Fumonisin also reduced photoreceptor apoptosis during early development in vitro. DHA, the retina major polyunsaturated fatty acid, which protects photoreceptors from oxidative stress-induced apoptosis, completely blocked C2-ceramide-induced photoreceptor death, simultaneously increasing Bcl-2 expression. Inhibiting glucosylceramide synthase, which catalyzes ceramide glucosylation, before ceramide or paraquat treatment blocked DHA's protective effect. CONCLUSIONS: The results suggest that oxidative stress stimulated an increase in ceramide levels that induced photoreceptor apoptosis. DHA prevented oxidative stress and ceramide damage by upregulating Bcl-2 expression and glucosylating ceramide, thus decreasing its intracellular concentration. This shows for the first time that ceramide is a critical mediator for triggering photoreceptor apoptosis in mammalian retina and suggests that modulating ceramide levels may provide a therapeutic tool for preventing photoreceptor death in neurodegenerative diseases.
Animals↗