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PubMed2001

FAS activation induces dephosphorylation of SR proteins; dependence on the de novo generation of ceramide and activation of protein phosphatase 1.

The search for potential targets for ceramide action led to the identification of ceramide-activated protein phosphatases (CAPP). To date, two serine/threonine protein phosphatases, protein phosphatase 2A (PP2A) and protein phosphatase 1 (PP1), have been demonstrated to function as ceramide-activated protein phosphatases. In this study, we show that treatment with either anti-FAS IgM (CH-11) (150 ng/ml) or exogenous d-(e)-C(6-)ceramide (20 microm) induces the dephosphorylation of the PP1 substrates, serine/arginine-rich (SR) proteins, in Jurkat acute leukemia T-cells. The serine/threonine protein phosphatase inhibitor, calyculin A, but not the PP2A-specific inhibitor, okadaic acid, inhibited both FAS- and ceramide-induced dephosphorylation of SR proteins. Anti-FAS IgM treatment of Jurkat cells led to a significant increase in levels of endogenous ceramide beginning at 2 h with a maximal increase of 10-fold after 7 h. A 2-h pretreatment of Jurkat cells with fumonisin B(1) (100 microm), a specific inhibitor of CoA-dependent ceramide synthase, blocked 80% of the ceramide generated and completely inhibited the dephosphorylation of SR proteins in response to anti-FAS IgM. Moreover, pretreatment of Jurkat cells with myriocin, a specific inhibitor of serine-palmitoyl transferase (the first step in de novo synthesis of ceramide), also blocked FAS-induced SR protein dephosphorylation, thus demonstrating a role for de novo ceramide. These results were further supported using A549 lung adenocarcinoma cells treated with d-(e)-C(6-)ceramide. Dephosphorylation of SR proteins was inhibited by fumonisin B(1) and by overexpression of glucosylceramide synthase; again implicating endogenous ceramide generated de novo in regulating the dephosphorylation of SR proteins in response to FAS activation. These results establish a specific intracellular pathway involving both de novo ceramide generation and activation of PP1 to mediate the effects of FAS activation on SR proteins.

Blotting, Western↗
PubMed2002

Membrane restructuring via ceramide results in enhanced solute efflux.

The capacity of ceramides to modify the permeability barrier of cell membranes has been explored. Membrane efflux induced either by in situ generated ceramides (through enzymatic cleavage of sphingomyelin) or by addition of ceramides to preformed membranes has been studied. Large unilamellar vesicles composed of different phospholipids and cholesterol, and containing entrapped fluorescent molecules, have been used as a system to assay ceramide-dependent efflux. Small proportions of ceramide (10 mol % of total lipid) that may exist under physiological conditions of ceramide-dependent signaling have been used in most experiments. When long chain (egg-derived) ceramides are used, both externally added or enzymatically produced ceramides induce release of vesicle contents. However, the same proportion of ceramides generated by sphingomyelinase induce faster and more extensive efflux than when added in organic solution to the preformed vesicles. Under our conditions 10 mol % of N-acetylsphingosine (C(2)-ceramide) did not induce any efflux. On the other hand, sphingomyelinase treatment of bilayers containing 50 mol % sphingomyelin gave rise to release of fluorescein-derivatised dextrans of molecular mass approximately 20 kDa, i.e. larger than cytochrome c. These results have been discussed in the light of our own previous data (Ruiz-Argüello, M. B., Basañez, G., Goñi, F. M., and Alonso, A. (1996) J. Biol. Chem. 271, 26616-26621) and of the observations by Siskind and Colombini (Siskind, L. J., and Colombini, M. (2000) J. Biol. Chem. 275, 38640-38644). Our spectroscopic observations appear to be in good agreement with the electrophysiological studies of the latter authors. Furthermore, some experiments in this paper have been designed to explore the mechanism of ceramide-induced efflux. Two properties of ceramide, namely its capacity to induce negative monolayer curvature and its tendency to segregate into ceramide-rich domains, appear to be important in the membrane restructuring process.

Bacillus cereus↗
PubMed2002

Ceramide signaling in fenretinide-induced endothelial cell apoptosis.

Stress stimuli can mediate apoptosis by generation of the lipid second messenger, ceramide. Herein we investigate the molecular mechanism of ceramide signaling in endothelial apoptosis induced by fenretinide (N-(4-hydroxyphenyl)retinamide (4-HPR)). 4-HPR, a synthetic derivative of retinoic acid that induces ceramide in tumor cell lines, has been shown to have antiangiogenic effects, but the molecular mechanism of these is largely unknown. We report that 4-HPR was cytotoxic to endothelial cells (50% cytotoxicity at 2.4 microm, 90% at 5.36 microm) and induced a caspase-dependent endothelial apoptosis. 4-HPR (5 microm) increased ceramide levels in endothelial cells 5.3-fold, and the increase in ceramide was required to achieve the apoptotic effect of 4-HPR. The 4-HPR-induced increase in ceramide was suppressed by inhibitors of ceramide synthesis, fumonisin B(1), myriocin, and l-cycloserine, and 4-HPR transiently activated serine palmitoyltransferase, demonstrating that 4-HPR induced de novo ceramide synthesis. Sphingomyelin levels were not altered by 4-HPR, and desipramine had no effect on ceramide level, suggesting that sphingomyelinase did not contribute to the 4-HPR-induced ceramide increase. Finally, the pancaspase inhibitor, t-butyloxycarbonyl-aspartyl[O-methyl]-fluoromethyl ketone, suppressed 4-HPR-mediated apoptosis but not ceramide accumulation, suggesting that ceramide is upstream of caspases. Our results provide the first evidence that increased ceramide biosynthesis is required for 4-HPR-induced endothelial apoptosis and present a molecular mechanism for its antiangiogenic effects.

Acyltransferases↗
PubMed2001

Ceramide conversion to sphingosine-1-phosphate is essential for survival in C3H10T1/2 cells.

Ceramide and sphingosine-1-phosphate (S1P) are important dietary lipids involved in cell growth, differentiation, apoptosis and cell survival. Treatment of C3H10T1/2 murine fibroblast cells (10T1/2) with ceramide did not induce apoptosis, a commonly observed effect of ceramide treatment. To determine whether the metabolism of ceramide played a role in this resistance to apoptosis, inhibitors of ceramidase and sphingosine kinase, two important enzymes in sphingolipid metabolism, were used. Treatment of 10T1/2 cells both without or with ceramide plus N-oleoyl-ethanolamine (NOE) and (1S,2R)-D-erythro-s-(N-myristoylamino)-1-phenol-1-propanol (MAPP), two ceramidase inhibitors, resulted in fourfold and eightfold increases, respectively, in apoptosis. Cells treated without or with ceramide plus N,N-dimethylsphingosine (DMS), a potent sphingosine kinase inhibitor, resulted in fourfold and sixfold increases, respectively, in apoptosis. In all treatments the induction of apoptosis was prevented by the addition of S1P. With the addition of S1P with NOE and MAPP as well as with ceramide, treatments reduced the apoptotic response by 30 and 35%, respectively; whereas the addition of S1P with the DMS only and ceramide with DMS treatments reduced the apoptotic response by 60 and 70%, respectively. Studies using labeled ceramide demonstrated ceramide was metabolized to S1P. In addition, a 14-fold increase in apoptosis occurred in cells treated with a nonhydrolyzable analog of ceramide, ceramine, compared with vehicle control. Because inhibiting the conversion of ceramide to S1P resulted in apoptosis, the lack of an apoptotic response to ceramide alone for C3H10T1/2 cells is attributable to the conversion of this pro-apoptotic sphingolipid to the anti-apoptotic metabolite S1P, which is essential for cell survival.

Amidohydrolases↗
PubMed2003

Bacterial ceramides and sphingophospholipids induce apoptosis of human leukaemic cells.

The genus Sphingobacterium, whose members are Gram-negative non-fermentative rods, possesses ceramides and related sphingophospholipids (SPLs) with isoheptadecasphinganine and 2-hydroxy or non-hydroxy isopentadecanoic acid. This paper reports evidence that ceramides isolated from Sphingobacterium spiritivorum ATCC 33861 induce endonucleolytic DNA cleavage in human myeloid leukaemia HL-60 cells in vitro, which is the primary characteristic biochemical marker for apoptosis or programmed cell death. Ceramides and SPLs also induced DNA fragmentation and caspase-3 activation, followed by changes in morphology, such as alterations in the size of nuclei and cells, and cell cycle shortening. Apoptotic activity correlated with the ceramide structure. Ceramide with a 2-hydroxy fatty acid showed stronger apoptotic activity than ceramide with a non-hydroxy fatty acid. Furthermore, the major five SPLs (ceramide phosphorylethanolamine-1 and -2, ceramide phosphorylinositol-1 and -2, and ceramide phosphorylmannose-1) showed apoptosis-inducing activity in HL-60 cells, indicating that the ceramide moiety of the SPLs plays a crucial role as the intracellular second messenger but that their hydrophilicity is less important in this regard. The hydrophilic part of SPLs may play a role in other cellular response systems. The involvement of Fas antigen was implicated in the apoptotic event since Fas antigen expression was observed after 3 or 4 h stimulation of HL-60 cells with bacterial ceramides. However, a time-course study for caspase-3 activation indicated maximal activity at 1 h after stimulation with bacterial ceramides, suggesting that two (or possibly more) mechanisms of signal transduction, Fas-dependent and Fas-independent, may be involved. Fas antigen expression and caspase-3 activation by five kinds of SPLs were observed after 3 or 4 h. These results indicate that there is a difference in the response of HL-60 cells to bacterial ceramides and SPLs.

Apoptosis↗
PubMed2002

Ceramide, a putative second messenger for nerve growth factor, modulates the TTX-resistant Na(+) current and delayed rectifier K(+) current in rat sensory neurons.

Because nerve growth factor (NGF) is elevated during inflammation and is known to activate the sphingomyelin signalling pathway, we examined whether NGF and its putative second messenger, ceramide, could modulate the excitability of capsaicin-sensitive adult and embryonic sensory neurons. Using the whole-cell patch-clamp recording technique, exposure of isolated sensory neurons to either 100 ng ml(-1) NGF or 1 microM N-acetyl sphingosine (C2-ceramide) produced a 3- to 4-fold increase in the number of action potentials (APs) evoked by a ramp of depolarizing current in a time-dependent manner. Intracellular perfusion with bacterial sphingomyelinase (SMase) also increased the number of APs suggesting that the release of native ceramide enhanced neuronal excitability. Glutathione, an inhibitor of neutral SMase, completely blocked the NGF-induced augmentation of AP firing, whereas dithiothreitol, an inhibitor of acidic SMase, was without effect. In the presence of glutathione and NGF, exogenous ceramide still enhanced the number of evoked APs, indicating that the sensitizing action of ceramide was downstream of NGF. To investigate the mechanisms of action for NGF and ceramide, isolated membrane currents were examined. Both NGF and ceramide facilitated the peak amplitude of the TTX-resistant sodium current (TTX-R I(Na)) by approximately 1.5-fold and shifted the activation to more hyperpolarized voltages. In addition, NGF and ceramide suppressed an outward potassium current (I(K)) by approximately 35 %. Ceramide reduced I(K) in a concentration-dependent manner. Isolation of the NGF- and ceramide-sensitive currents indicates that they were delayed rectifier types of I(K). The inflammatory prostaglandin, PGE(2), produced an additional suppression of I(K) after exposure to ceramide (approximately 35 %), suggesting that these agents might act on different targets. Thus, our findings indicate that the pro-inflammatory agent, NGF, can rapidly enhance the excitability of sensory neurons. This NGF-induced sensitization is probably mediated by activation of the sphingomyelin signalling pathway to liberate ceramide(s), wherein ceramide appears to be the second messenger involved in modulating neuronal excitability.

Animals↗
PubMed2003

Liposomal delivery enhances short-chain ceramide-induced apoptosis of breast cancer cells.

It is therapeutically desirable to effectively deliver ceramide, an antimitogenic and proapoptotic lipid second messenger, to transformed cell types. However, the targeted delivery of cell-permeable ceramide analogs, including C6-ceramide, to cells may be impeded by the hydrophobicity of these bioactive lipids, resulting in reduced efficacy. The objective of this study is to develop and optimize liposomal vehicles to augment ceramide delivery to a breast adenocarcinoma cell line. We designed conventional, cationic, and pegylated drug release vesicles to efficaciously deliver ceramide to MDA-MB-231 breast adenocarcinoma cells. In vitro pharmacokinetic analysis demonstrated that liposomal ceramide delivery resulted in significantly greater accumulation of ceramide in MDA-MB-231 cells. Ceramide-formulated liposomes significantly inhibited MDA-MB-231 cell proliferation as compared with nonliposomal administration of ceramide. Ceramide-induced apoptosis correlated with the pharmacokinetic profile and the diminished proliferation in this highly aggressive, metastatic cell line. Liposomal ceramide formulations inhibited phosphorylated Akt levels and stimulated caspase-3/7 activity more effectively than nonliposomal ceramide, events consistent with apoptosis. Together, these results indicate that bioactive ceramide analogs can be incorporated into conventional, cationic, or pegylated liposomal vehicles for improved drug delivery and release.

Apoptosis↗
PubMed1996

Tumor necrosis factor-alpha and its second messenger, ceramide, stimulate apoptosis in cultured ovarian follicles.

In the mammalian ovary, only a small fraction of follicles fully mature and ovulate, while most of them die via apoptosis. Multiple factors promoting follicle survival have been identified, but intraovarian mediators of apoptosis are poorly known. Tumor necrosis factor-alpha (TNF alpha) is a cytokine capable of inducing apoptosis in diverse cell types, and the apoptotic effect of TNF alpha is, partially, coupled to the sphingomyelin signaling pathway with ceramide as a second messenger. Because TNF alpha has been localized in the rat ovary, and TNF alpha treatment increases granulosa cell ceramide production, we studied the effect of treatment with TNF alpha and ceramide on follicle apoptosis. Immature rats were implanted with diethylstilbestrol to stimulate the development of early antral follicles. Follicles were isolated and cultured in a serum-free medium for 24 h with or without hormone treatments. During culture, spontaneous follicle apoptosis occurred (10-fold increase in DNA fragmentation), which was partially blocked by 100 ng/ml FSH (60% suppression). The effect of FSH was counteracted by TNF alpha in a dose-dependent manner, with the maximal effect at 100 ng/ml TNF alpha (90% reversal of FSH action). In situ analysis indicated that the granulosa cell is the follicle cell type undergoing DNA fragmentation. A membrane-permeable ceramide analog, C2-ceramide N-acetyl sphingosine, mimicked the effect of TNF alpha and was able to completely abolish the action of FSH at 50 microM. In contrast, another ceramide analog, C2-dihydroceramide N-acetyl dihydrosphingosine, did not alter the effect of FSH, verifying the specificity of ceramide action. To study the mechanism of TNF alpha and ceramide action, the effect of sodium aurathiomalate (ATM), an inhibitor of interleukin-1 beta-converting enzyme/ced-3-related cystine proteases known to be essential in the execution of mammalian cell apoptosis, was studied. Treatment with ATM (1 mM) prevented the apoptosis-inducing effect of both TNF alpha and ceramide, suggesting a role for cysteine proteases in mediating follicle apoptosis. Treatment with either TNF alpha or ceramide increased both basal and FSH-stimulated progesterone production by cultured follicles. Concomitant treatment by ATM did not alter the stimulatory effect of TNF alpha or ceramide on progesterone production, ruling out nonspecific toxic effect of the inhibitor and indicating that the apoptotic and steroidogenic pathways are independent. In summary, treatment with TNF alpha or its second messenger, ceramide, stimulates apoptosis of early antral follicles in culture, suggesting a potential role for TNF alpha as an intraovarian regulator of follicle atresia by acting through the ceramide signaling pathway.

Animals↗
PubMed2001

Ceramide inhibits the inwardly rectifying potassium current in GH(3) lactotrophs.

The effects of ceramide on ion currents in rat pituitary GH(3) cells were investigated. Hyperpolarization-elicited K(+) currents present in GH(3) cells were studied to determine the effect of ceramide and other related compounds on the inwardly rectifying K(+) current (I(K(IR))). Ceramide (C(2)-ceramide) suppressed the amplitude of I(K(IR)) in a concentration-dependent manner, with an IC(50) value of 5 microM. Ceramide caused a rightward shift in the midpoint for the activation curve of I(K(IR)). Pretreatment with PD-98059 (30 microM) or U-0126 (30 microM) did not prevent ceramide-mediated inhibition of I(K(IR)). However, the magnitude of ceramide-induced inhibition of I(K(IR)) was attenuated in GH(3) cells preincubated with dithiothreitol (10 microM). TNF alpha (100 ng/g) also suppressed I(K(IR)). In the inside-out configuration, application of ceramide (30 microM) to the bath slightly suppressed the activity of large conductance Ca(2+)-activated K(+) channels. Under the current clamp mode, ceramide (10 microM) increased the firing of action potentials. Cells that exhibited an irregular firing pattern were converted to those displaying a regular firing pattern after application of ceramide (10 microM). Ceramide also suppressed I(K(IR)) in neuroblastoma IMR-32 cells. Therefore, ceramide can produce a depressant effect on I(K(IR)). The blockade of this current by ceramide may affect cell function.

Action Potentials↗
PubMed2001

Role of ceramide synthase in oxidant injury to renal tubular epithelial cells.

Ceramide has been implicated to play an important role in the cell signaling pathway involved in apoptosis. Most studies that have used the apoptotic model of cellular injury have suggested that enhanced ceramide generation is the result of the breakdown of sphingomyelin by sphingomyelinases. However, the role of ceramide synthase in enhanced ceramide generation in response to oxidant stress has not been previously examined in any tissue. Hydrogen peroxide (H(2)O(2)) (1 mM) resulted in a rapid increase in ceramide generation (as measured by in vitro diacylglycerol kinase assay) in LLC-PK1 cells. The intracellular ceramide level was significantly increased at 5 min after exposure of cells to H(2)O(2) and thereafter continuously increased up to 60 min. H(2)O(2) also resulted in a rapid increase (within 5 min) in ceramide synthase activity (as measured by incorporation of [(14)C] from the labeled palmytoyl-CoA into dihydroceramide) in microsomes. In contrast, the exposure of cells to H(2)O(2) did not result in any significant change in sphingomyelin content or acid or neutral sphingomyelinase activity. An increase in ceramide production induced by H(2)O(2) preceded any evidence of DNA damage and cell death. The specific inhibitor of ceramide synthase, fumonisin B1 (50 microM), was able to suppress H(2)O(2)-induced ceramide generation and provided a marked protection against H(2)O(2)-induced DNA strand breaks, DNA fragmentation, and cell death. Taken together, these data provide the first evidence that H(2)O(2) is a regulator of ceramide synthase rather than sphingomyelinases and that ceramide synthase-dependent ceramide generation plays a key role in DNA damage and cell death in oxidant stress to renal tubular epithelial cells.

Animals↗
PubMed1999

Ceramide toxicity and metabolism differ in wild-type and multidrug-resistant cancer cells.

Previously we demonstrated that multidrug-resistant (MDR) cancer cells have elevated levels of a glycosylated form of ceramide, glucosylceramide. Here we compared ceramide metabolism and ceramide toxicity in MCF-7 and in adriamycin-resistant (MCF-7-AdrR) human breast cancer cells. MCF-7-AdrR cells were resistant to C6-ceramide (1-10 microM); however, in MCF-7 cells treated with C6-ceramide, viability dropped sharply. Ceramide, when supplemented, was not metabolized by MCF-7 cells. In contrast, ceramide was efficiently converted to glucosylceramide by MCF-7-AdrR cells. Analysis of extracellular [3H]ceramide in radiolabeled cells showed that MCF-7-AdrR cells do not have an enhanced capacity to efflux ceramide compared with MCF-7 cells. Triphenylethylene anti-estrogens, known modulators of drug resistance, were effective inhibitors of ceramide conversion to glucosylceramide, suggesting that blocking ceramide metabolism plays a role in chemosensitization. The anti-progestine, RU486, also blocked glucosylceramide synthesis in cells; however, LY117018, a raloxifene analog, was without influence. We propose that an enhanced capacity to glycosylate ceramide as evidenced in MCF-7-AdrR cells, is a molecular determinant of drug resistance, particularly as regards resistance to ceramide-enhancing agents such as anthracyclines, ionizing radiation, and tumor necrosis factor-alpha.

Ceramides↗
PubMed1999

SDZ PSC 833, the cyclosporine A analogue and multidrug resistance modulator, activates ceramide synthesis and increases vinblastine sensitivity in drug-sensitive and drug-resistant cancer cells.

Resistance to chemotherapy is the major cause of cancer treatment failure. Insight into the mechanism of action of agents that modulate multidrug resistance (MDR) is instrumental for the design of more effective treatment modalities. Here we show, using KB-V-1 MDR human epidermoid carcinoma cells and [3H]palmitic acid as metabolic tracer, that the MDR modulator SDZ PSC 833 (PSC 833) activates ceramide synthesis. In a short time course experiment, ceramide was generated as early as 15 min (40% increase) after the addition of PSC 833 (5.0 microM), and by 3 h, [3H]ceramide was >3-fold that of control cells. A 24-h dose-response experiment showed that at 1.0 and 10 microM PSC 833, ceramide levels were 2.5- and 13.6-fold higher, respectively, than in untreated cells. Concomitant with the increase in cellular ceramide was a progressive decrease in cell survival, suggesting that ceramide elicited a cytotoxic response. Analysis of DNA in cells treated with PSC 833 showed oligonucleosomal DNA fragmentation, characteristic of apoptosis. The inclusion of fumonisin B1, a ceramide synthase inhibitor, blocked PSC 833-induced ceramide generation. Assessment of ceramide mass by TLC lipid charring confirmed that PSC 833 markedly enhanced ceramide synthesis, not only in KB-V-1 cells but also in wild-type KB-3-1 cells. The capacity of PSC 833 to reverse drug resistance was demonstrated with vinblastine. Whereas each agent at a concentration of 1.0 microM reduced cell survival by approximately 20%, when PSC 833 and vinblastine were coadministered, cell viability fell to zero. In parallel experiments measuring ceramide metabolism, it was shown that the PSC 833/vinblastine combination synergistically increased cellular ceramide levels. Vinblastine toxicity, also intensified by PSC 833 in wild-type KB-3-1 cells, was as well accompanied by enhanced ceramide formation. These data demonstrate that PSC 833 has mechanisms of action in addition to P-glycoprotein chemotherapy efflux pumping.

Cell Survival↗
PubMed2003

Effect of hypothyreosis on the content of ceramides in rat tissues.

Ceramide is the second messenger in the sphingomyelin signalling pathway. A number of extracellular stimuli increase the content of ceramide in the cell. There are some data indicating that the content of ceramide may also be regulated by hormones. The aim of the present study was to examine the effect of hypothyreosis on the content and composition of ceramide in rat tissues. The rats were thyroidectomized and thereafter they received propylthiouracyl in drinking water. The control rats were sham operated. 30 days after thyroidectomy or sham operation the rats were anaesthetized and samples of the liver, white and red vastus lateralis and left ventricle were taken. One set of samples was frozen in liquid nitrogen for analysis of ceramide. Another set of samples was freshly homogenized in chloroform/methanol for further determination of the content of sphingomyelin phosphorous. The content and composition of ceramide-fatty acids was determined by means of gas-liquid chromatography. Twelve ceramides containing different fatty acid residues were identified in both groups. Hypothyreosis reduced the total content of ceramide in each tissue studied: in the heart by 50.9%, in the red vastus by 28.6%, in the white vastus by 29.4% and in the liver by 22%. Concomitantly, the content of individual ceramides was either reduced, stable or even elevated, depending on the tissue. The content of sphingomyelin was elevated in both sections of the vastus lateralis and remained stable in the heart and the liver. The ratio: total content of sphingomyelin to total content of ceramide was elevated in the muscles and remained stable in the liver. This indicates that the reduction in the content of ceramide in the tissues of hypothyroid rats may be a consequence either of a reduction in the formation of ceramide from sphingomyelin, its increased hydrolysis or both. It is concluded that normal thyroid function is needed to maintain the content and composition of ceramide in the tissues.

Animals↗
PubMed1967

Brain ceramide hexosides in Tay-Sachs disease and generalized gangliosidosis (GM1-gangliosidosis).

The carbohydrate composition was determined for ceramide hexosides isolated from brains of patients with Tay-Sachs disease and generalized gangliosidosis (hereby named GM1-gangliosidosis). Gray matter of patients with each disease showed a characteristic abnormal ceramide hexoside pattern. In Tay-Sachs gray matter, ceramide trihexoside is the major component, whereas ceramide tetrahexoside is barely detectable. In GM1-gangliosidosis, ceramide tetrahexoside is the major ceramide hexoside, while ceramide trihexoside is present only in small amount. These two major components have been characterized as the asialo derivatives of, respectively, the "Tay-Sachs ganglioside" (GM2-ganglioside) and the normal major monosialoganglioside (GM1-ganglioside). In both diseases, more than half the ceramide monohexoside of gray matter was glucocerebroside. Gray matter ceramide dihexoside, present in both diseases at higher than normal levels, was mostly ceramide lactoside, with possibly a small amount of ceramide digalactoside. Sulfatide contained only galactose. The abnormal ceramide hexoside pattern is limited to gray matter: white matter showed normal ceramide hexosides, i.e. a preponderance of monohexosides and sulfatide, with no detectable glucocerebroside.

Brain Chemistry↗
PubMed1997

Distinct roles for ceramide and glucosylceramide at different stages of neuronal growth.

Sphingolipids (SLs) are important structural and regulatory components of neuronal plasma membranes. Previous studies using fumonisin B1, an inhibitor of the synthesis of ceramide, the precursor of all SLs, demonstrated that ceramide synthesis is required to sustain axonal growth in hippocampal neurons (; ) and dendritic growth in cerebellar Purkinje cells (). We now show that ceramide plays distinct roles at different stages of neuronal development. (1) During axon growth, ceramide must be metabolized to glucosylceramide (GlcCer) to sustain growth. Thus, whereas D-erythro-ceramide, which is metabolized to GlcCer, is able to antagonize the disruptive effects of fumonisin B1 on axon growth, L-threo-ceramide, which is not metabolized to GlcCer, is ineffective. (2) The formation of minor processes from lamellipodia can be stimulated by incubation with short-acyl chain analogs of ceramide that are active in ceramide-mediated signaling pathways, or by generation of endogenous ceramide by incubation with sphingomyelinase. However, GlcCer synthesis is not required for this initial stage of neuronal development. (3) During minor process formation and during axon growth, incubation with high concentrations of ceramide or sphingomyelinase, but not dihydroceramide, induces apoptosis. Together, these observations are consistent with the possibility that minor process formation and apoptosis can be regulated by ceramide-dependent signaling pathways and that the decision whether to enter these diametrically opposed pathways depends on intracellular ceramide concentrations. In contrast, axonal growth requires the synthesis of GlcCer from ceramide, perhaps to support an intracellular transport pathway.

Animals↗
PubMed1997

Resistance to radiation-induced apoptosis in Burkitt's lymphoma cells is associated with defective ceramide signaling.

Increased sensitivity to ionizing radiation has been shown to be due to defects in double-strand break repair and mutations in the proteins that detect DNA damage. However, it is now recognized that the cellular radiation response is complex and that radioresistance/radiosensitivity may also be regulated at different levels in the radiation signal transduction pathway. Here, we describe a direct relationship between resistance to radiation-induced apoptosis and defective ceramide signaling. Radiation sensitivity in human tumor cells correlated with the immediate accumulation of the second messenger ceramide. In the BL30A Burkitt's lymphoma line, ceramide increased 4-fold by 10 min postirradiation (10 Gy), and in the moderately sensitive HL-60 leukemia cells, ceramide accumulated 2.5-fold above basal levels. In contrast, in all radioresistant tumor cells examined, including several Burkitt's lymphoma lines (BL30K, BL29, and BL36) and the MO59K glioma cell line, ceramide did not accumulate postirradiation. The ability to abrogate ceramide production by pretreatment with the tumor promoter, 12-O-tetradecanoylphorbol 13-acetate, conferred resistance to radiation-induced apoptosis in the sensitive BL30A cells. An isogenic subline of BL30A, BL30K, was resistant to both C8-ceramide (20 microM) and ionizing radiation-induced apoptosis. Bypassing the block in radiation-induced ceramide production by the addition of exogenous ceramide was not sufficient to induce apoptosis; this suggests the existence of a second ceramide-associated signaling defect in these radioresistant cells that confers resistance to ceramide-induced apoptosis. Thus, these results provide compelling evidence that ceramide is an essential mediator of radiation-induced apoptosis and that defective ceramide signaling confers an apoptosis-resistant phenotype in tumor cells.

Apoptosis↗
PubMed1998

Quantitative analysis of ceramide molecular species by high performance liquid chromatography.

A method was developed for quantitative analysis of molecular species of ceramide (N-acyl-sphingosine) and dihydroceramide (N-acyl sphinganine) by high performance liquid chromatography (HPLC). Various N-acyl chain-containing ceramides or dihydroceramides were semi-synthesized as standard materials and allowed to react with anthroyl cyanide, a fluorescent reagent. Anthroyl derivatives of ceramide and dihydroceramide containing C16, C18, C20, C22, and C24 saturated N-acyl chain could be completely separated to each molecular species by reversed-phase HPLC equipped with fluorescence detector, although some ceramide molecular species containing monoenoic acyl chain were eluted together with saturated dihydroceramide species. Ceramide molecular species could be quantified using N-heptadecanoyl or N-tricosanoyl sphingosine as an internal standard, and the lower detection limit was below 1 pmol. This method was applied to the analysis of sphingomyelin and free ceramide in U937 cells. The analysis of the ceramide obtained by hydrolysis of sphingomyelin of U937 cells revealed that the ceramide moiety was mainly composed of N-palmitoyl sphingosine, N-nervonoyl sphingosine, and N-lignoceroyl sphingosine, representing 50.0, 27.4, and 6.7% of sphingomyelin, respectively. The total free ceramide and dihydroceramide of U937 cells was determined to be 254 +/- 5 pmol/10(6) cells. Major molecular species of the free ceramide fraction were N-lignoceroyl, N-palmitoyl, and N-nerovonoyl sphingosine, representing 27.6%, 26.6%, and 13.6% of this fraction, respectively. Different distribution of free ceramide molecular species from sphingomyelin species may suggest that selective metabolism of molecular species occurs in the synthesis or degradation of sphingomyelin. These results indicate that the picomole level of molecular species of ceramide and dihydroceramide is successfully determined by fluorescence HPLC and that this newly developed method may be useful to reveal the metabolism and function of ceramide and related compounds in cultured cells.

Acylation↗
PubMed1999

Ceramide induces caspase-independent apoptosis in rat hepatocytes sensitized by inhibition of RNA synthesis.

Ceramide has been implicated as a second messenger in intracellular signaling pathways leading to apoptosis in nonhepatic cells. To determine whether ceramide can mediate hepatocyte apoptosis, the cytotoxicity of ceramide was determined in rat hepatocytes. The rat hepatocyte cell line, RALA255-10G, and primary rat hepatocytes were completely resistant to toxicity from 10 to 100 micromol/L C2 ceramide. Resistance was not the result of a failure to take up ceramide, because ceramide treatment did cause nuclear factor-kappaB (NF-kappaB) activation. Because ceramide may mediate cell death from tumor necrosis factor alpha (TNF-alpha), the ability of RNA synthesis inhibition and NF-kappaB inactivation to sensitize hepatocytes to ceramide toxicity was examined. RALA hepatocytes were sensitized to ceramide toxicity by coadministration of actinomycin D (ActD). Cell death occurred by apoptosis as determined by the presence of morphological evidence of apoptosis, caspase activation, poly(ADP-ribose) polymerase (PARP) degradation, and DNA hypoploidy. Despite the induction of apoptosis associated with caspase activation, cell death from ActD/ceramide was not blocked by caspase inhibition. Inhibition of NF-kappaB activation also sensitized RALA hepatocytes to ceramide toxicity, but to a lesser extent than for TNF-alpha. Thus, unlike many nonhepatic cell types, rat hepatocytes are resistant to cell death from ceramide because of the transcriptionally dependent up-regulation of a protective gene(s). The ability of ActD and NF-kappaB inactivation to sensitize RALA hepatocytes to ceramide toxicity suggests that ceramide may act as a downstream mediator of TNF-alpha toxicity.

Animals↗