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PubMed2003

Ceramide accumulation precedes caspase-3 activation during apoptosis of A549 human lung adenocarcinoma cells.

Ceramide, the basic structural unit of sphingolipids, controls the balance between cell growth and death by inducing apoptosis. We have previously shown that accumulation of ceramide, triggered by hydrogen peroxide (H(2)O(2)) or by short-chain ceramide analogs, induces apoptosis of lung epithelial cells. Here we elucidate the link between caspase-3 activation, at the execution phase, and ceramide accumulation, at the commitment phase of apoptosis in A549 human lung adenocarcinoma cells. The induction of ceramide accumulation by various triggers of ceramide generation, such as H(2)O(2), C(6)-ceramide, or UDP-glucose-ceramide glucosyltransferase inhibitor dl-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, triggered the activation of caspase-3. This ceramide elevation also induced the cleavage of the death substrate poly(ADP-ribose) polymerase and was followed by apoptotic cell death. Ceramide-mediated apoptosis was blocked by a general caspase inhibitor, Boc-d-fluoromethylketone, and by overexpression of the antiapoptotic protein Bcl-2. Notably, overexpression of Bcl-2 reduced the basal cellular levels of ceramide and prevented the induction of ceramide generation by C(6)-ceramide, which implies ceramide generation as a possible target for the antiapoptotic effects of Bcl-2.

Adenocarcinoma↗
PubMed1999

Modification of ceramide metabolism increases cancer cell sensitivity to cytotoxics.

In the preceding report we demonstrated that MCF-7-AdrR cells (adriamycin resistant) were insensitive to ceramide, whereas MCF-7 wild-type cells were sensitive. It was also shown that the drug resistant cells had an increased capacity to convert ceramide to glucosylceramide. Here we demonstrate that blocking the conversion of ceramide to glucosylceramide increases MCF-7-AdrR cell sensitivity to ceramide as well as to antitumor agents. Treatment of MCF-7 cells with adriamycin elicited a 5-fold increase in ceramide, and caused oligonucleosomal fragmentation, characteristic to apoptosis. Under similar treatment conditions, ceramide was not generated in MCF-7-AdrR cells. In MCF-7-AdrR cells neither C6-ceramide nor tamoxifen was cytotoxic; however, the addition of tamoxifen to the C6-ceramide treatment regimen reduced cell viability to 42% and elicited apoptosis. Treatment of MCF-7-AdrR cells with Adriamycin promoted an increase in ceramide only if tamoxifen was present, in which case ceramide increased 7-fold, and cell viability decreased to 50%. The employment of another agent, RU486 (Mifepristone), which blocks ceramide glycosylation, increased MCF-7-AdrR cell sensitivity to adriamycin in a dose-dependent manner. Our data show that agents that block ceramide glycosylation potentiate cellular sensitivity to ceramide and to chemotherapeutic drugs, and suggest that the ceramide metabolic pathway is an important target for anticancer drug development.

Antineoplastic Agents↗
PubMed1992

Ceramide stimulates a cytosolic protein phosphatase.

A sphingomyelin cycle has been identified whereby the action of certain extracellular agents results in reversible sphingomyelin hydrolysis and the concomitant generation of ceramide. Moreover, a cell-permeable ceramide, C2-ceramide (N-acetylsphingosine), is a potent modulator of cell proliferation and differentiation. We report herein that C2-ceramide, C6-ceramide, and natural ceramides activate a cytosolic serine/threonine protein phosphatase in a dose-dependent manner. Initial activation is observed at concentrations of ceramide as low as 0.1 microM with peak response occurring at 5-10 microM. However, other closely related sphingolipids, sphingosine and sphingomyelin, were largely inactive. Ceramide-stimulated phosphatase was inhibited by okadaic acid, an inhibitor of protein phosphatases, with an IC50 of 0.1-1 nM, depending on the concentration of ceramide. Ceramide-stimulated phosphatase was insensitive to Mg2+ and Mn2+ cations. Using sequential anion exchange chromatography, ceramide-stimulated phosphatase activity could be resolved from ceramide-nonresponsive phosphatases. The activity of partially purified enzyme was stimulated 3.5-fold by ceramide. The identification of a phosphatase as a molecular target for the action of ceramide defines a novel intracellular signaling pathway with potential roles in the regulation of cell proliferation and differentiation.

Cell Line↗
PubMed1995

N-acetylsphingosine (C2-ceramide) inhibited neutrophil superoxide formation and calcium influx.

Ceramide, a product arising from sphingomyelinase activity, has been shown to act as an intracellular second messenger in effecting growth inhibition, cellular differentiation, and apoptosis. In the present study, the relative effects of cell-permeable ceramides, N-acetylsphingosine (C2-ceramide) and N-hexanoylsphingosine (C6-ceramide), on neutrophil responses were measured. When cells were activated with fMet-Leu-Phe, C2-ceramide both potentiated (< 1 microM) and inhibited (> 1 microM) superoxide generation. C2- and C6-ceramide inhibited phorbol ester-induced superoxide release from neutrophils at IC50 values of 5 and 120 microM, respectively. C2-ceramide had no effect on semipurified protein kinase C activity. Neither ceramide affected significantly the general level of phosphorylated proteins in phorbol ester-treated cells. C2-ceramide (1-20 microM) alone did not change cytosolic free Ca2+ levels but inhibited Ca2+ and Mn2+ influx in fMet-Leu-Phe-activated neutrophils. In contrast, sphingosine enhanced Ca2+ entry; thus, ceramide conversion to sphingosine was not significant. Unlike C2-ceramide, C2-dihydroceramide failed to block superoxide generation or Ca2+ influx. Preincubation of cells with 10 nM okadaic acid reversed slightly the effects of C2-ceramide. Calyculin A, tautomycin, and much higher concentrations of okadaic acid inhibited agonist-induced Ca2+ influx. We postulate that C2-ceramide may inhibit neutrophil superoxide release by activation of type 2A protein phosphatases. Results suggest that protein phosphatase type 1 up-regulates Ca2+ entry, whereas type 2A (or a ceramide-activated subtype) forestalls Ca2+ entry by inactivating a calcium influx factor.

Alkaloids↗
PubMed2004

Opposite regulation of the mitochondrial apoptotic pathway by C2-ceramide and PACAP through a MAP-kinase-dependent mechanism in cerebellar granule cells.

The sphingomyelin-derived messenger ceramides provoke neuronal apoptosis through caspase-3 activation, while the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) promotes neuronal survival and inhibits caspase-3 activity. However, the mechanisms leading to the opposite regulation of caspase-3 by C2-ceramide and PACAP are currently unknown. Here, we show that PACAP prevents C2-ceramide-induced inhibition of mitochondrial potential and C2-ceramide-evoked cytochrome c release. C2-ceramide stimulated Bax expression, but had no effect on Bcl-2, while PACAP abrogated the action of C2-ceramide on Bax and stimulated Bcl-2 expression. The effects of C2-ceramide and PACAP on Bax and Bcl-2 were blocked, respectively, by the JNK inhibitor L-JNKI1 and the MEK inhibitor U0126. L-JNKI1 prevented the alteration of mitochondria induced by C2-ceramide while U0126 suppressed the protective effect of PACAP against the deleterious action of C2-ceramide on mitochondrial potential. Moreover, L-JNKI1 inhibited the stimulatory effect of C2-ceramide on caspase-9 and -3 and prevented C2-ceramide-induced cell death. U0126 blocked PACAP-induced Bcl-2 expression, abrogated the inhibitory effect of PACAP on ceramide-induced caspase-9 activity, and promoted granule cell death. The present study reveals that C2-ceramide and PACAP exert opposite effects on Bax and Bcl-2 through, respectively, JNK- and ERK-dependent mechanisms. These data indicate that the mitochondrial pathway plays a pivotal role in the pro- and anti-apoptotic effects of C2-ceramide and PACAP.

Animals↗
PubMed1999

Multidrug resistance modulators and doxorubicin synergize to elevate ceramide levels and elicit apoptosis in drug-resistant cancer cells.

BACKGROUND: To provide insight for the development of more effective clinical agents, the authors attempted to elucidate the mechanisms of action of multidrug resistance (MDR) modulators. Previously, the authors found that MDR modulators blocked the conversion of ceramide to glucosylceramide in MDR cells, thereby enhancing cytotoxicity. Because ceramide is a critical component of the apoptosis signaling cascade, the current study examined the impact of therapy using agents that elicit ceramide formation combined with agents that block ceramide glycosylation. METHODS: Doxorubicin-resistant human breast carcinoma cells (MCF-7-AdrR) were treated with either doxorubicin, tamoxifen, cyclosporine A, or the cyclosporine A analog SDZ PSC 833 (PSC 833) or with combinations thereof, and ceramide and glucosylceramide metabolisms were measured by cell radiolabeling. Cell viability was quantitated spectrophotometrically and apoptosis was evaluated analyzing DNA integrity by gel electrophoresis. RESULTS: Whereas cyclosporine A blocked the generation of glucosylceramide in MCF-7-AdrR cells, a chemical cousin, PSC 833, elicited a 3-fold increase in glucosylceramide and a 5-fold increase in ceramide levels at 24 hours. The PSC 833 response was time-dependent(as early as 30 minutes) and dose-dependent (as low as 0.1 microM). The appearance of ceramide foreran the generation of glucosylceramide. Sphingomyelin levels were not decreased in response to PSC 833; however, Fumonisin B1, a ceramide synthase inhibitor, blocked PSC 833-induced ceramide generation. Adding tamoxifen, which blocks ceramide glycosylation, to the PSC 833 regimen boosted ceramide levels 11-fold over controls and caused DNA fragmentation. A 3-component regimen comprised of tamoxifen, doxorubicin, and PSC 833 increased ceramide levels 26-fold and brought cell viability to zero. CONCLUSIONS: These results demonstrate that MDR modulators can be used separately, in combination, or in conjunction with chemotherapy at clinically relevant concentrations to manipulate cellular ceramide levels and restore sensitivity in the drug resistant setting. As such, this represents a new direction in the treatment of cancer.

Antineoplastic Agents↗
PubMed2001

Taxol-induced ceramide generation and apoptosis in human breast cancer cells.

PURPOSE: Taxol has emerged as a valuable antimitotic chemotherapeutic agent, particularly in advanced breast and ovarian cancers. Although much is known about cytotoxic mechanisms, the effectiveness of Taxol cannot be solely explained by microtubular interaction. This study was undertaken to determine whether ceramide generation plays a role in Taxol-induced apoptosis. METHODS: Hormone-independent MDA-MB-468 and hormone-dependent MCF-7 breast cancer cell lines were employed, and ceramide metabolism was characterized using [3H]palmitic acid as lipid precursor. RESULTS: Exposure of cells to Taxol resulted in enhanced formation of [3H]ceramide. Ceramide increased nearly 2-fold in MDA-MB-468 cells exposed to 50 nM Taxol, and more than 2.5-fold in MCF-7 cells exposed to 1.0 microM Taxol. These concentrations mirrored the EC50 (amount of drug eliciting 50% cell kill) for Taxol in the two cell lines. Use of cell-permeable C6-ceramide as a medium supplement revealed that MDA-MB-468 cells were 20-fold more sensitive to ceramide than MCF-7 cells (P < 0.001). Ceramide was generated as early as 6 h after exposure to Taxol in MDA-MB-468 cells, whereas the earliest signs of apoptosis were detected 12 h after treatment, and by 24 h the apoptotic index was six times that of untreated cells. Both fumonisin B1, a ceramide synthase inhibitor, and L-cycloserine, a serine palmitoyltransferase inhibitor, blocked Taxol-induced ceramide generation, whilst sphingomyelin levels remained unchanged, indicating a de novo pathway of ceramide formation. L-Cycloserine reduced Taxol-induced apoptosis by 30% in MDA-MB-468 cells and totally blocked Taxol-induced apoptosis in MCF-7 cells. CONCLUSIONS: These results suggest that Taxol-induced apoptosis is, in part, attributable to ceramide and sphingoid bases. This is of relevance to drug mechanism studies, as ceramide is a known messenger of apoptosis. Clinical use of Taxol with ceramide-enhancing agents may maximize cytotoxic potential.

Antineoplastic Agents, Phytogenic↗
PubMedDate not supplied

Production and metabolism of ceramide in normal and ischemic-reperfused myocardium of rats.

Ceramide has been shown to be a key signaling molecule involved in the apoptotic effect of tumor necrosis factor alpha (TNF-alpha) and other cytokines. Given the importance of cytokines such as TNF-alpha in myocardial ischemia-reperfusion injury, we hypothesize that ceramide is increased during ischemia or reperfusion, and that the activity of enzymes responsible for its production or breakdown should be increased and/or decreased, respectively. Therefore, in the present study, we characterized the enzymatic activities responsible for ceramide production and metabolism in the myocardium of rats, and determined the contribution of these enzymes to altered ceramide levels during myocardial ischemia and reperfusion. The basal ceramide concentration in the myocardium of rats was 34.0 pmol/mg tissue. As determined by the conversion of 14C-sphingomyelin into ceramide and 14C-choline phosphate, both neutral (N-) and acidic (A-) SMase were detected in the myocardium, with a conversion rate of 0.09 +/- 0.008 and 0.32 +/- 0.05 nmol/min per mg protein, respectively. The activity of A-SMase (78 % of total cellular activity) was significantly higher in microsomes than in cytosol, while the activity of N-SMase was similar in both fractions. Ceramidase, a ceramide-metabolizing enzyme, was also detected in the myocardium of rats. It metabolized ceramide into sphingosine at a rate of 9.94 +/- 0.42 pmol/min per mg protein. In anesthetized rats, 30 min of ischemia had no apparent effect on ceramide concentrations in the myocardium, while 30 min of ischemia followed by 3 h of reperfusion resulted in a significant increase in ceramide by 48 %. The activities of both N- and A-SMase were reduced by 44 % and 32 %, respectively, in the myocardium subjected to ischemia followed by reperfusion, but unaltered in the ischemic myocardium. It was also found that myocardial ischemia followed by reperfusion produced a marked inhibition of ceramidase (by 29 %). These results demonstrate that the myocardium of rats expresses N- and A-SMase and ceramidase, which contribute to the production and metabolism of ceramide, respectively. Tissue ceramide concentrations increased in reperfused myocardium. These increases in ceramide were not associated with enhanced SMase activity, but rather with reduced ceramidase activity.

Animals↗
PubMed2003

Modulation by C2 ceramide of the nicotinic transmission within the coeliac ganglion in the rabbit.

We have investigated the modulation by ceramide of the nicotinic activation of the prevertebral sympathetic neurons. Our study was performed in vitro in rabbit isolated coeliac ganglion, using intracellular recording techniques. We have used C(2) ceramide, a permeant analog of ceramide. The effects of C(2) ceramide were first assessed when nicotinic activation was elicited without modulatory mechanisms (fast excitatory postsynaptic potentials triggered by stimulation of the thoracic splanchnic nerves with a single pulse). In all the neurons tested, C(2) ceramide triggered an increase in the amplitude of the fast excitatory postsynaptic potentials demonstrating a direct facilitatory effect on the nicotinic activation. We then investigated the effects of C(2) ceramide on modulatory mechanisms of this activation. These mechanisms occur when a train of pulses of supramaximum intensity is applied on the splanchnic nerves. During the train, a gradual depression of fast nicotinic activation occurred: the pulses failed to systematically elicit action potentials. We have previously demonstrated that this regulatory phenomenon is partly modulated by nitric oxide which exerts a dual effect: facilitation or inhibition of the nicotinic activation. In all the neurons tested, C(2) ceramide decreased the number of action potentials fired during a train of pulses, demonstrating an indirect inhibitory effect on the nicotinic activation. The use of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (nitric oxide scavenger) suppressed the inhibitory effect of C(2) ceramide, demonstrating that this effect is mediated through the nitric oxide pathway. C(2) dihydro-ceramide, an inactive analog of ceramide, was without effect on the nicotinic activation of the ganglionic neurons. These results demonstrate that ceramide exerts a complex modulation of the nicotinic activation of the prevertebral neurons: direct facilitation and indirect inhibition involving the nitric oxide pathway. In fact, C(2) ceramide plays a key gating role in the dual effect of the nitric oxide pathway by activating the inhibitory effect. The existence of this gating mechanism involving ceramide and nitric oxide opens new perspectives in terms of our understanding of the modulation of synaptic transmission within the prevertebral ganglia. Our study demonstrates that sphingolipids are involved in complex modulations of the synaptic activation within the prevertebral ganglia, and thus contribute to their integrative properties.

Animals↗
PubMed2000

Ceramide-induced cell death is independent of the Fas/Fas ligand pathway and is prevented by Nur77 overexpression in A20 B cells.

The role of ceramide in triggering apoptosis is still a matter of debate. While in some experimental systems, ceramide was shown to mediate Fas-induced cell death, in other instances it was claimed to induce the expression of Fas ligand (FasL), killing cells in a caspase-dependent fashion. We found that, in mature A20 B cells, ceramide-induced apoptosis is independent of the caspase pathway, since we observed no ICE-like, CPP32-like and Mch2 activities and no PARP proteolysis. Moreover, we were unable to protect these cells from ceramide-induced apoptosis using caspase inhibitors, while they blocked Fas-induced apoptosis and no FasL induction could be detected following ceramide treatment. These results suggest that ceramide does not induce apoptosis through the Fas/FasL pathway. We also found that overexpression of Nur77, a zinc-finger transcription factor described to upregulate FasL, antagonizes ceramide-induced apoptosis, but not Fas-induced apoptosis. This further supports the hypothesis that Fas and ceramide death pathways are independent in A20 cells. Ceramide-induced cell death was associated with increased c-myc, p53, Bax and p27kip1 levels; in contrast, cells transfected with Nur77 (A20Nur77), resistant to ceramide-induced apoptosis, showed a marked downregulation of p53 after ceramide treatment, with neither Bax nor p27kip1 induction. In conclusion, our results suggest that, in the A20 B cell line, Fas and ceramide trigger two distinct pathways and that Nur77 overexpression confers protection against ceramide-mediated apoptosis which correlates with inhibition of p53, Bax and p27kip1 induction.

Animals↗
PubMed2005

Human T-cell lymphotropic virus type I-transformed T-cells have a partial defect in ceramide synthesis in response to N-(4-hydroxyphenyl)retinamide.

Treatment with the synthetic retinoid HPR [N-(4-hydroxyphenyl)-retinamide] causes growth arrest and apoptosis in HTLV-I (human T-cell lymphotropic virus type-I)-positive and HTLV-I-negative malignant T-cells. It was observed that HPR-mediated growth inhibition was associated with ceramide accumulation only in HTLV-I-negative cells. The aim of the present study was to investigate the mechanism by which HPR differentially regulates ceramide metabolism in HTLV-I-negative and HTLV-I-positive malignant T-cells. Clinically achievable concentrations of HPR caused early dose-dependent increases in ceramide levels only in HTLV-I-negative cells and preceded HPR-induced growth suppression. HPR induced de novo synthesis of ceramide in HTLV-I-negative, but not in HTLV-I-positive, cells. Blocking ceramide glucosylation in HTLV-I-positive cells, which leads to accumulation of endogenous ceramide, rendered these cells more sensitive to HPR. Exogenous cell-permeant ceramides that function partially by generating endogenous ceramide induced growth suppression in all tested malignant lymphocytes, were consistently found to be less effective in HTLV-I-positive cells confirming their defect in de novo ceramide synthesis. Owing to its multipotent activities, the HTLV-I-encoded Tax protein was suspected to inhibit ceramide synthesis. Tax-transfected Molt-4 and HELA cells were less sensitive to HPR and C6-ceramide mediated growth inhibition respectively and produced lower levels of endogenous ceramide. Together, these results indicate that HTLV-I-positive cells are defective in de novo synthesis of ceramide and that therapeutic modalities that bypass this defect are more likely to be successful.

CD4-Positive T-Lymphocytes↗
PubMed1998

Cell-cycle-dependent changes in ceramide levels preceding retinoblastoma protein dephosphorylation in G2/M.

Ceramide functions as a growth-inhibitory lipid-signalling molecule and might have a role in mediating the effects of extracellular agents on cell growth, differentiation and senescence. Here we investigate the roles of ceramide in cell cycle progression. With the use of the model of serum withdrawal, we were able to synchronize Wi-38 human diploid fibroblasts at different stages of cell cycle. Serum stimulation resulted in G0 to G1/S progression as determined by flow cytometric analysis and [3H]thymidine incorporation. Analyses of endogenous ceramide levels demonstrated that ceramide levels remained relatively constant on serum stimulation, indicating that ceramide might not be critical during G1/S transition. Treating exponentially growing Wi-38 human diploid fibroblasts with nocodazole led to cell cycle arrest at the G2/M phase of the cell cycle; 2 h after the removal of nocodazole, retinoblastoma (Rb) protein became dephosphorylated and the cells exited from G2/M and moved to the G1 phase of the new cycle. When cells were released from G2/M block by nocodazole, and before Rb protein dephosphorylation, endogenous ceramide levels transiently increased up to 2-fold at 0.5 h after the removal of nocodazole. Fumonisin B1, an inhibitor of ceramide synthase, inhibited the elevation of ceramide levels. Desipramine and SR33557, both acid sphingomyelinase inhibitors, did not have an appreciable effect on the elevation of ceramide levels. Furthermore, fumonisin B1 inhibited Rb protein dephosphorylation induced by endogenous ceramide but not by exogenous ceramide. These results demonstrate for the first time changes in ceramide during cell cycle progression and suggest that ceramide synthesized de novo might function as an endogenous modulator of Rb protein and cell cycle progression.

Carboxylic Acids↗
PubMed1998

Role of enhanced ceramide generation in DNA damage and cell death in chemical hypoxic injury to LLC-PK1 cells.

BACKGROUND: Ceramide has been implicated to be a second messenger in the cell signaling pathway involved in cell growth, proliferation, and apoptotic cell death. However, there is little information of a role of ceramide in DNA damage and cell death in hypoxic injury known to induce necrotic cell death. METHODS: Ceramide generation was measured in LLC-PK1 cells exposed to chemical hypoxia with a mitochondrial electron transport inhibitor, antimycin A and glucose deprivation. The effect of inhibition of ceramide generation on chemical hypoxia-induced DNA damage and cell death and the effect of exogenous ceramide on cellular injury were also determined. RESULTS: Chemical hypoxia resulted in a rapid increase in ceramide production prior to any evidence of DNA damage and cell death in LLC-PK1 cells. The inhibitor of ceramide synthase, fumonisin B1, provided a marked protection against chemical hypoxia-induced DNA strand breaks, DNA fragmentation and cell death. Fumonisin B1 did not affect adenosine triphosphate (ATP) depletion induced by antimycin A, suggesting that fumonisin B1 does not alter cellular uptake of antimycin A. We confirmed the ability of ceramide synthase inhibitor, fumonisin B1, to suppress chemical hypoxia-induced ceramide generation. Exposure of LLC-PK1 cells to synthetic ceramide, C2- and C6-ceramide, but not C2-dihydroceramide, the structural analog of C2-ceramide, resulted in DNA strand breaks, DNA fragmentation and cell death in a dose- and time-dependent manner similar to the effect of chemical hypoxia. CONCLUSIONS: Our data indicate that ceramide is a key modulator for DNA damage and cell death in chemical hypoxia to renal tubular epithelial cells.

Animals↗
PubMed1998

Altered sphingomyelinase and ceramide expression in the setting of ischemic and nephrotoxic acute renal failure.

Diverse physical and chemical stimuli can activate sphingomyelinases (SMases), resulting in sphingomyelin (SM) hydrolysis with ceramide release. Since ceramide can profoundly impact a host of homeostatic mechanisms, the concept of a "SM (or SMase) signaling pathway" has emerged. We recently documented that ceramide levels fall abruptly during renal ischemia, and then rebound to twice normal values during early reperfusion (30 to 90 min) Therefore, the present study assessed whether these ceramide changes are paralleled, and hence potentially mediated, by comparable changes in SMase activity. Mice were subjected to 45 minutes of renal ischemia +/- 30 minutes, 90 minutes, or 24 hours of reperfusion. Renal cortices (or isolated proximal tubules) were then assayed for SMase activity (acidic, neutral forms). To characterize whether early post-ischemic ceramide increments are a relatively persistent event, ceramide was assayed following a 24-hour reperfusion period. Finally, to assess whether the observed perturbations were unique to post-ischemic injury, SMase and ceramide were quantified in the setting of glycerol-induced myohemoglobinuria and anti-glomerular basement membrane (alpha GBM) antibody-induced acute renal failure (ARF). Ischemia induced abrupt declines (approximately 50%) in both acidic and neutral SMase activities, and these persisted in an unremitting fashion throughout 24 hours of reperfusion. Nevertheless, increased ceramide expression (2x normal) resulted. Myohemoglobinuria also suppressed acidic/neutral SMases, and again, "paradoxical" ceramide increments were observed. Finally, alpha GBM nephritis increased ceramide levels, but in this instance, a correlate was increased SMase activity. These results suggest that: (1) ceramide is an acute renal "stress rectant" increasing in response to diverse renal insults; (2) this response may occur independently of the classic SM pathway, since the ceramide increments can seemingly be dissociated from increased SMase activity; and (3) given the well documented impact of ceramide and the SM(ase) pathway on apoptosis, cell proliferation, differentiation, and tissue inflammation, the present results have potentially broad ranging implications for the induction and evolution of diverse forms of ARF.

Acute Kidney Injury↗
PubMed1995

Interaction of ceramides, sphingosine, and sphingosine 1-phosphate in regulating DNA synthesis and phospholipase D activity.

C2- and C6-ceramides (N-acetylsphingosine and N-hexanoylsphingosine, respectively) abolished the stimulation of DNA synthesis by sphingosine 1-phosphate in rat fibroblasts. This inhibition by ceramide was partially prevented by insulin. C2-ceramide did not alter the stimulation of DNA synthesis by insulin and decreased the sphingosine-induced stimulation by only 16%. The ceramides did not significantly modify the actions of sphingosine or sphingosine 1-phosphate in decreasing cAMP concentrations. C2- and C6-ceramides blocked the activation of phospholipase D by sphingosine 1-phosphate, and this inhibition was not affected by insulin. Okadaic acid decreased the activation of phospholipase D by sphingosine 1-phosphate and did not reverse the inhibitory effect of C2-ceramide on this activation. Therefore, this effect of C2-ceramide is unlikely to involve the stimulation of phosphoprotein phosphatase activity. Sphingosine did not activate phospholipase D activity significantly after 10 min. C2-ceramide stimulated the conversion of exogenous [3H]sphingosine 1-phosphate to sphingosine and ceramide in fibroblasts. Ceramides can inhibit some effects of sphingosine 1-phosphate by stimulating its degradation via a phosphohydrolase that also hydrolyzes phosphatidate. Furthermore, C2- and C6-ceramides stimulated ceramide production from endogenous lipids, and this could propagate the intracellular signal. This work demonstrates that controlling the production of ceramide versus sphingosine and sphingosine 1-phosphate after sphingomyelinase activation could have profound effects on signal transduction.

Animals↗
PubMed1998

Sphingomyelinase and ceramide stimulate the expression of inducible nitric-oxide synthase in rat primary astrocytes.

The sphingomyelin signal transduction pathway is known to play a role in mediating the action of various cytokines. Here we examined the possible role of the sphingomyelin signaling pathway on lipopolysaccharide (LPS)- and cytokine-mediated production of NO and the expression of inducible nitric-oxide synthase (iNOS). Sphingomyelinase (SMase) treatment of astrocytes increased the cellular levels of ceramide without the induction of NO production. However, incubation of LPS or cytokine-stimulated astrocytes with SMase or by increasing intracellular ceramide by cell-permeable ceramide analogs (C2- or C6-ceramide) or inhibitor of ceramidase (N-oleoyl ethanolamine) led to a time- and dose-dependent increase in the production of NO. This increase in NO production was accompanied by an increase in iNOS activity, iNOS protein, and iNOS mRNA. Similar to astrocytes, SMase or ceramide analogs also stimulated the LPS- and cytokine-mediated expression of iNOS in the C6 glial cell line. Since activation of NF-kappaB is necessary for the induction of iNOS, we examined the effect of SMase and C2-ceramide on the activation of NF-kappaB. Although SMase or C2-ceramide alone was ineffective in activating NF-kappaB, both stimulated the LPS-mediated activation of NF-kappaB in LPS-activated astrocytes. Inhibition of ceramide and LPS-mediated induction of iNOS by antioxidant inhibitors of NF-kappaB (N-acetylcysteine and pyrrolidine dithiocarbamate) suggest that the stimulatory effect of ceramide on the induction of iNOS is due to the stimulation of NF-kappaB activation and that cellular redox plays a role in the activation of NF-kappaB and induction of iNOS. Inhibition of LPS-mediated as well as LPS and ceramide-mediated induction of iNOS and activation of NF-kappaB by PD98059, a specific inhibitor of activation of mitogen-activated protein (MAP) kinase kinase (MEK), and FPT inhibitor II, a selective inhibitor of Ras farnesyl protein transferase, indicate that the Ras-MAP kinase pathway is involved in LPS-ceramide induced activation of NF-kappaB and induction of iNOS, and that ceramide-mediated signaling events probably converge into the LPS-modulated MAP kinase signaling pathway resulting in greater activation of NF-kappaB and iNOS induction. This study illustrates a novel role of the sphingomyelin-ceramide signaling pathway in stimulating the expression of iNOS via LPS- or cytokine-mediated activation of NF-kappaB in astrocytes.

Acetylcysteine↗
PubMed1998

The formation of ceramide-1-phosphate during neutrophil phagocytosis and its role in liposome fusion.

Ceramide, a product of agonist-stimulated sphingomyelinase activation, is known to be generated during the phagocytosis of antibody-coated erythrocytes by polymorphonuclear leukocytes. Agonist-stimulated formation of ceramide-1-phosphate is now shown to occur in 32PO4-labeled neutrophils. Ceramide-1-phosphate is formed by a calcium-dependent ceramide kinase, found predominately in the neutrophil plasma membrane. The neutrophil kinase is specific for ceramide because, in contrast to the bacterial diglyceride kinase, ceramide is not phosphorylated under conditions specific for diglyceride phosphorylation. Conversely, 1,2-diacylglycerol does not serve as substrate for the neutrophil ceramide kinase. Ceramide kinase activation occurs in a time-dependent fashion, reaching peak activity 10 min after formyl peptide stimulation and challenge with antibody-coated erythrocytes. The lipid kinase activity is optimal at pH 6.8. Because the formation of the phagolysosome is a critical event in phagocytosis, the effect of ceramide-1-phosphate in promoting the fusion of liposomes was determined. Both the addition of increasing concentrations of sphingomyelinase D and ceramide-1-phosphate promoted liposomal fusion. In summary, ceramide-1-phosphate is formed during phagocytosis through activation of ceramide kinase. Ceramide-1-phosphate may promote phagolysosome formation.

Calcium↗
PubMed2000

Suppression of heat shock protein-70 by ceramide in heat shock-induced HL-60 cell apoptosis.

Ceramide has emerged as a mediator of cell growth, differentiation, and apoptosis in many biological systems. Many kinds of stresses are reported to induce apoptosis with an increase of ceramide generation. Here we showed that the intracellular ceramide levels increased in parallel with heat shock (HS)-induced apoptosis in an intensity- and time-dependent manner, and synthetic N-acetylsphingosine (C(2)-ceramide) synergistically enhanced HS-induced apoptosis in HL-60 cells. In order to know the role of ceramide generation in HS-induced apoptosis, we examined the effects of C(2)-ceramide on the levels of mRNA and protein of heat shock proteins (HSPs). The increase of HSP-70 mRNA levels 1-2 h after HS at 42 degrees C for 30 min was suppressed by C(2)-ceramide in a dose-dependent manner. In comparison with HSP-70, the levels of HSP-60 and -90 mRNAs were faintly suppressed by C(2)-ceramide. Similarly, the increase in the protein levels of HSP-70 was significantly suppressed 4-8 h after HS by C(2)-ceramide in a dose-dependent manner. Additionally, in 293 cells, which are constitutively overexpressing HSP-70 gene, the levels of HSP-70 mRNA were suppressed by C(2)-ceramide in parallel with the increase of apoptotic cells. We next examined the mechanisms by which C(2)-ceramide suppressed HS-increased HSP-70 expression. The treatment with C(2)-ceramide did not affect both an activation of a nuclear transcription factor for HSP-70, heat shock factor-1, and an increased transcriptional rate of HSP-70 by HS, but increased the rates of HSP-70 mRNA degradation. In summary, ceramide may efficiently induce HS-induced apoptosis by suppressing anti-apoptotic HSP-70 through a post-transcriptional regulation.

Apoptosis↗