De novo ceramide accumulation due to inhibition of its conversion to complex sphingolipids in apoptotic photosensitized cells

The oxidative stress induced by photodynamic therapy (PDT) with the photosensitizer phthalocyanine 4 is accompanied by increases in ceramide mass. To assess the regulation of de novo sphingolipid metabolism during PDT-induced apoptosis, Jurkat human T lymphoma and Chinese hamster ovary cells were labeled with [14C]serine, a substrate of serine palmitoyltransferase (SPT), the enzyme catalyzing the initial step in the sphingolipid biosynthesis. A substantial elevation in [14C]ceramide with a concomitant decrease in [14C]sphingomyelin was detected. The labeling of [14C]ceramide was completely abrogated by the SPT inhibitor ISP-1. In addition, ISP-1 partly suppressed PDT-induced apoptosis. Pulse-chase experiments showed that the contribution of sphingomyelin degradation to PDT-initiated increase in de novo ceramide was absent or minor. PDT had no effect on either mRNA amounts of the SPT subunits LCB1 and LCB2, LCB1 protein expression, or SPT activity in Jurkat cells. Moreover in Chinese hamster ovary cells LCB1 protein underwent substantial photodestruction, and SPT activity was profoundly inhibited after treatment. We next examined whether PDT affects conversion of ceramide to complex sphingolipids. Sphingomyelin synthase, as well as glucosylceramide synthase, was inactivated by PDT in both cell lines in a dose-dependent manner. These results are the first to show that in the absence of SPT up-regulation PDT induces accumulation of de novo ceramide by inhibiting its conversion to complex sphingolipids.     

Endotoxin activates de novo sphingolipid biosynthesis via nuclear factor kappa B-mediated upregulation of Sptlc2

Sphingolipids are membrane components and are involved in cell proliferation, apoptosis and metabolic regulation. In this study we investigated whether de novo sphingolipid biosynthesis in macrophages is regulated by inflammatory stimuli. Lipopolysaccharide (LPS) treatment upregulated Sptlc2, a subunit of serine palmitoyltransferase (SPT), mRNA and protein in Raw264.7 and mouse peritoneal macrophages, but Sptlc1, another subunit of SPT, was not altered. SPT activation by LPS elevated cellular levels of ceramides and sphingomyelin (SM). Pharmacological inhibition of nuclear factor kappa B (NFκB) prevented LPS-induced upregulation of Sptlc2 while transfection of p65 subunit of NFκB upregulated Sptlc2 and increased cellular ceramide levels. In contrast, MAP kinases were not involved in regulation of sphingolipid biosynthesis. Analysis of Sptlc2 promoter and chromatin immunoprecipitation (ChIP) assay showed that NFκB binding sites are located in Sptlc2 promoter region. Our results demonstrate that inflammatory stimuli activate de novo sphingolipid biosynthesis via NFκB and may play a critical role in lipid metabolism in macrophages.     

Sphingolipid-free Leishmania are defective in membrane trafficking, differentiation and infectivity

Sphingolipids are structural components of the eukaryotic plasma membrane that are involved, together with cholesterol, in the formation of lipid microdomains (rafts). Additionally, sphingolipid metabolites have been shown to modulate a wide variety of cellular events, including differentiation and apoptosis. To investigate the role of de novo sphingolipid biosynthesis in Leishmania, we have focused on serine palmitoyltransferase (SPT), which catalyses the first, rate-limiting step in the synthetic pathway. Genetic ablation of one SPT subunit, LmLCB2, yields viable null parasites that can no longer synthesize ceramide and sphingolipids de novo. Unexpectedly, LmLCB2 expression (and sphingolipid biosynthesis) is stage regulated in Leishmania, being undetectable in intramacrophage parasites. As expected from this observation, the LmLCB2 null mutants maintain infectivity in vivo. However, they are compromised in their ability to form infective extracellular parasites, correlating with a defect in association of the virulence factor, leishmanolysin or GP63, with lipid rafts during exocytosis and an observed relocalization of a second virulence factor, lipophosphogycan, during differentiation. Thus, de novo sphingolipid biosynthesis is critical for membrane trafficking events in extracellular Leishmania but has at best a minor role in intracellular pathogenesis.     

Reducing plasma membrane sphingomyelin increases insulin sensitivity

It has been shown that inhibition of de novo sphingolipid synthesis increases insulin sensitivity. For further exploration of the mechanism involved, we utilized two models: heterozygous serine palmitoyltransferase (SPT) subunit 2 (Sptlc2) gene knockout mice and sphingomyelin synthase 2 (Sms2) gene knockout mice. SPT is the key enzyme in sphingolipid biosynthesis, and Sptlc2 is one of its subunits. Homozygous Sptlc2-deficient mice are embryonic lethal. However, heterozygous Sptlc2-deficient mice that were viable and without major developmental defects demonstrated decreased ceramide and sphingomyelin levels in the cell plasma membranes, as well as heightened sensitivity to insulin. Moreover, these mutant mice were protected from high-fat diet-induced obesity and insulin resistance. SMS is the last enzyme for sphingomyelin biosynthesis, and SMS2 is one of its isoforms. Sms2 deficiency increased cell membrane ceramide but decreased SM levels. Sms2 deficiency also increased insulin sensitivity and ameliorated high-fat diet-induced obesity. We have concluded that Sptlc2 heterozygous deficiency- or Sms2 deficiency-mediated reduction of SM in the plasma membranes leads to an improvement in tissue and whole-body insulin sensitivity.     

Biosynthesis of sphinganine-analog mycotoxins

Sphinganine-analog mycotoxins (SAMT) are polyketide-derived natural products produced by a number of plant pathogenic fungi and are among the most economically important mycotoxins. The toxins are structurally similar to sphinganine, a key intermediate in the biosynthesis of ceramides and sphingolipids, and competitive inhibitors for ceramide synthase. The inhibition of ceramide and sphingolipid biosynthesis is associated with several fatal diseases in domestic animals and esophageal cancer and neural tube defects in humans. SAMT contains a highly reduced, acyclic polyketide carbon backbone, which is assembled by a single module polyketide synthase. The biosynthesis of SAMT involves a unique polyketide chain-releasing mechanism, in which a pyridoxal 5'-phosphate-dependent enzyme catalyzes the termination, offloading and elongation of the polyketide chain. This leads to the introduction of a new carbon-carbon bond and an amino group to the polyketide chain. The mechanism is fundamentally different from the thioesterase/cyclase-catalyzed polyketide chain releasing found in bacterial and other fungal polyketide biosynthesis. Genetic data suggest that the ketosynthase domain of the polyketide synthase and the chain-releasing enzyme are important for controlling the final product structure. In addition, several post-polyketide modifications have to take place before SAMT become mature toxins.     

Involvement of de novo ceramide biosynthesis in macrophage death induced by activation of ATP-sensitive P2X7 receptor

Macrophage ionotropic P2X7 receptors regulate cell-death through ill-defined signaling pathways. Here, we investigated the role of ceramide, an apoptogenic sphingolipid and showed that ATP stimulated ceramide accumulation in macrophages. Benzoylbenzoyl-ATP, a potent P2X7 agonist, was able to mimic the effects of ATP on ceramide accumulation while oxidized ATP had the opposite effect. Ceramide accumulation was blocked by de novo ceramide biosynthesis inhibitors. Interestingly, ATP-induced caspase-3/7 activation was dependent on ceramide generation. Finally, we showed that de novo ceramide biosynthesis is involved in ATP-induced macrophage death in a caspase-dependent manner. Our results indicate a novel role of ceramide in P2X7-regulated cell-death.     

Induction of apoptosis through B-cell receptor cross-linking occurs via de novo generated C16-ceramide and involves mitochondria

B-cells, triggered via their surface B-cell receptor (BcR), start an apoptotic program known as activation-induced cell death (AICD), and it is widely believed that this phenomenon plays a role in the restriction and focusing of the immune response. Although both ceramide and caspases have been proposed to be involved in AICD, the contribution of either and the exact molecular events through which AICD commences are still unknown. Here we show that in Ramos B-cells, BcR-triggered cell death is associated with an early rise of C16 ceramide that derives from activation of the de novo pathway, as demonstrated using a specific inhibitor of ceramide synthase, fumonisin B1 (FB1), and using pulse labeling with the metabolic sphingolipid precursor, palmitate. There was no evidence for activation of sphingomyelinases or hydrolysis of sphingomyelin. Importantly, FB1 inhibited several specific apoptotic hallmarks such as poly(A)DP-ribose polymerase cleavage and DNA fragmentation. Electron microscopy revealed morphological evidence of mitochondrial damage, suggesting the involvement of mitochondria in BcR-triggered apoptosis, and this was inhibited by FB1. Moreover, a loss of mitochondrial membrane potential was observed in Ramos cells after BcR cross-linking, which was inhibited by the addition of FB1. Interestingly, benzyloxycarbonyl-Val-Ala-dl-Asp, a broad spectrum caspase inhibitor did not inhibit BcR-induced mitochondrial membrane permeability transition but did block DNA fragmentation. These results suggest a crucial role for de novo generated C16 ceramide in the execution of AICD, and they further suggest an ordered and more specific sequence of biochemical events in which de novo generated C16 ceramide is involved in mitochondrial damage resulting in a downstream activation of caspases and apoptosis.     

Acute activation of de novo sphingolipid biosynthesis upon heat shock causes an accumulation of ceramide and subsequent dephosphorylation of SR proteins

Recent studies are beginning to implicate sphingolipids in the heat stress response. In the yeast Saccharomyces cerevisiae, heat stress has been shown to activate de novo biosynthesis of sphingolipids, whereas in mammalian cells the sphingolipid ceramide has been implicated in the heat shock responses. In the current study, we found an increase in the ceramide mass of Molt-4 cells in response to heat shock, corroborating findings in HL-60 cells. Increased ceramide was determined to be from de novo biosynthesis by two major lines of evidence. First, the accumulation of ceramide was dependent upon the activities of both ceramide synthase and serine palmitoyltransferase. Second, pulse labeling studies demonstrated increased production of ceramide through the de novo biosynthetic pathway. Significantly, the de novo sphingolipid biosynthetic pathway was acutely induced upon heat shock, which resulted in a 2-fold increased flux in newly made ceramides within 1-2 min of exposure to 42.5 degrees C. Functionally, heat shock induced the dephosphorylation of the SR proteins, and this effect was demonstrated to be dependent upon the accumulation of de novo-produced ceramides. Thus, these studies disclose an evolutionary conserved activation of the de novo pathway in response to heat shock. Moreover, SR dephosphorylation is emerging as a specific downstream target of accumulation of newly made ceramides in mammalian cells.     

JNK3 signaling pathway activates ceramide synthase leading to mitochondrial dysfunction

A cardinal feature of brain tissue injury in stroke is mitochondrial dysfunction leading to cell death, yet remarkably little is known about the mechanisms underlying mitochondrial injury in cerebral ischemia/reperfusion (IR). Ceramide, a naturally occurring membrane sphingolipid, functions as an important second messenger in apoptosis signaling and is generated by de novo synthesis, sphingomyelin hydrolysis, or recycling of sphingolipids. In this study, cerebral IR-induced ceramide elevation resulted from ceramide biosynthesis rather than from hydrolysis of sphingomyelin. Investigation of intracellular sites of ceramide accumulation revealed the elevation of ceramide in mitochondria because of activation of mitochondrial ceramide synthase via post-translational mechanisms. Furthermore, ceramide accumulation appears to cause mitochondrial respiratory chain damage that could be mimicked by exogenously added natural ceramide to mitochondria. The effect of ceramide on mitochondria was somewhat specific; dihydroceramide, a structure closely related to ceramide, did not inflict damage. Stimulation of ceramide biosynthesis seems to be under control of JNK3 signaling: IR-induced ceramide generation and respiratory chain damage was abolished in mitochondria of JNK3-deficient mice, which exhibited reduced infarct volume after IR. These studies suggest that the hallmark of mitochondrial injury in cerebral IR, respiratory chain dysfunction, is caused by the accumulation of ceramide via stimulation of ceramide synthase activity in mitochondria, and that JNK3 has a pivotal role in regulation of ceramide biosynthesis in cerebral IR.     

Inhibition of ceramide de novo synthesis by myriocin produces the double effect of reducing pathological inflammation and exerting antifungal activity against A. fumigatus airways infection

BackgroundFungal infections develop in pulmonary chronic inflammatory diseases such as asthma, Chronic Obstructive Pulmonary Disease (COPD) and Cystic Fibrosis (CF). The available antifungal drugs may fail to eradicate fungal pathogens, that can invade the lungs and vessels and spread by systemic circulation taking advantage of defective lung immunity. An increased rate of sphingolipid de novo synthesis, leading to ceramide accumulation, was demonstrated in CF and COPD inflamed lungs. The inhibitor of sphingolipid synthesis myriocin reduces inflammation and ameliorates the response against bacterial airway infection in CF mice. Myriocin also inhibits sphingolipid synthesis in fungi and exerts a powerful fungistatic effect.MethodsWe treated Aspergillus fumigatus infected airway epithelial cells with myriocin and we administered myriocin-loaded nanocarriers to A. fumigatus infected mice lung.ResultsWe demonstrate here that de novo synthesized ceramide mediates the inflammatory response induced by A. fumigatus infection in airway epithelia. CF epithelial cells are chronically inflamed and defective in killing internalized conidia. Myriocin treatment reduced ceramide increase and inflammatory mediator release whereas it upregulated HO1 and NOD2, allowing the recovery of a functional killing of conidia in these cells. Myriocin-loaded nanocarriers, intratracheally administered to mice, significantly reduced both the inflammatory response induced by A. fumigatus pulmonary challenge and fungal lung invasion.ConclusionsWe conclude that inhibition of sphingolipid synthesis can be envisaged as a dual anti-inflammatory and anti-fungal therapy in patients suffering from chronic lung inflammation with compromised immunity.General significanceMyriocin represents a powerful agent for inflammatory diseases and fungal infection.     

Screening of fungal species for fumonisin production andfumonisin-like disruption of sphingolipid biosynthesis

Fumonisins are mycotoxins produced by several species of Fusaria. They are found on corn and in corn-based products, can cause fatal illnesses in some animals and are suspected human esophageal carcinogens. Fumonisins are believed to cause toxicity by blocking ceramide synthase, a key enzyme in sphingolipid biochemistry which converts sphinganine (or sphingosine) and fatty acyl CoA to ceramide. Relatively fewfungal species have been evaluated for their ability to produce fumonisins. Fewer have been studied to determine if they produce ceramide synthase inhibitors, whether fumonisin-like structures or not, therefore potentially having toxicity similar to fumonisins. We analyzed corn cultures of 49 isolates representing 32 diversespecies of fungi for their ability to produce fumonisins. We also evaluated the culture extracts for ceramide synthase activity. Only cultures prepared with species reported previously to produce fumonisins – Fusarium moniliforme and F. proliferatum – tested positive for fumonisins. Extracts of these cultures inhibited ceramide synthase, as expected. None of the other fungal isolates we examined produced fumonisins or other compounds capable of inhibiting ceramide synthase. Although the fungi we selected for these studies represent only a few ofthe thousands of species that exist, they share the commonality that they are frequently associated with cereal grasses, including corn, either as pathogens or as asymptomatic endophytes. Thus,these results should be encouraging to those attempting to find ways to genetically manipulate fumonisin-producing fungi, tomake corn more resistant, or to develop biocontrol measures because it appears that only a relatively few fungal contaminants of corn can produce fumonisins. This revised version was published online in June 2006 with corrections to the Cover Date.     

Dual role for phosphoinositides in regulation of yeast and mammalian phospholipase D enzymes

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that regulates diverse biological processes by binding to a family of G protein-coupled receptors or as an intracellular second messenger. Mammalian S1P phosphatase (SPP-1), which degrades S1P to terminate its actions, was recently cloned based on homology to a lipid phosphohydrolase that regulates the levels of phosphorylated sphingoid bases in yeast. Confocal microscopy surprisingly revealed that epitope-tagged SPP-1 is intracellular and colocalized with the ER marker calnexin. Moreover, SPP-1 activity and protein appeared to be mainly enriched in the intracellular membranes with lower expression in the plasma membrane. Treatment of SPP-1 transfectants with S1P markedly increased ceramide levels, predominantly in the intracellular membranes, diminished survival, and enhanced apoptosis. Remarkably, dihydro-S1P, although a good substrate for SPP-1 in situ, did not cause significant ceramide accumulation or increase apoptosis. Ceramide accumulation induced by S1P was completely blocked by fumonisin B1, an inhibitor of ceramide synthase, but only partially reduced by myriocin, an inhibitor of serine palmitoyltransferase, the first committed step in de novo synthesis of ceramide. Furthermore, S1P, but not dihydro-S1P, stimulated incorporation of [3H]palmitate, a substrate for both serine palmitoyltransferase and ceramide synthase, into C16-ceramide. Collectively, our results suggest that SPP-1 functions in an unprecedented manner to regulate sphingolipid biosynthesis and is poised to influence cell fate.     

Identification of fumonisin B1 as an inhibitor of argininosuccinate synthetase using fumonisin affinity chromatography and in vitro kinetic studies

Fumonisin B1, a fungal mycotoxin that grows on corn and other agricultural products, alters sphingolipid metabolism by inhibiting ceramide synthase. The precise mechanism of fumonisin B1 toxicity has not been completely elucidated; however, a central feature in the cytotoxicity is alteration of sphingolipid metabolism through interruption of de novo ceramide synthesis. An affinity column consisting of fumonisin B1 covalently bound to an HPLC column matrix was used to isolate a rat liver protein that consistently bound to the column. The protein was identified as argininosuccinate synthetase by protein sequencing. The enzyme-catalyzed formation of argininosuccinic acid from citrulline and aspartate by recombinant human and rat liver argininosuccinate synthetase was inhibited by fumonisin B1. Fumonisin B1 showed mixed inhibition against citrulline, aspartate, and ATP to the enzyme. Fumonisin B1 had a Ki' of approximately 6 mM with the recombinant human argininosuccinate synthase and a Ki' of 35 mM with a crude preparation of enzyme prepared from rat liver. Neither tricarballylic acid nor hydrolyzed fumonisin B1 inhibited recombinant human argininosuccinate synthetase. This is the first demonstration of fumonisin B1 inhibition of argininosuccinate synthethase, a urea cycle enzyme, which adds to the list of enzymes that are inhibited in vitro by fumonisin B1 (ceramide synthase, protein serine/threonine phosphatase). The extent of the inhibition of argininosuccinate synthetase in cells, and the possible role of this enzyme inhibition in the cellular toxicity of FB1, remains to be established.     

Selective substrate supply in the regulation of yeast de novo sphingolipid synthesis

The heat stress response of Saccharomyces cerevisiae is characterized by transient cell cycle arrest, altered gene expression, degradation of nutrient permeases, trehalose accumulation, and translation initiation of heat shock proteins. Importantly heat stress also induces de novo sphingolipid synthesis upon which many of these subprograms of the heat stress response depend. Despite extensive data addressing the roles for sphingolipids in heat stress, the mechanism(s) by which heat induces sphingolipid synthesis remains unknown. This study was undertaken to determine the events and/or factors required for heat stress-induced sphingolipid synthesis. Data presented indicate that heat does not directly alter the in vitro activity of serine palmitoyltransferase (SPT), the enzyme responsible for initiating de novo sphingolipid synthesis. Moreover deletion of the small peptide Tsc3p, which is thought to maximize SPT activity, specifically reduced production of C(20) sphingolipid species by over 70% but did not significantly decrease overall sphingoid base production. In contrast, the fatty-acid synthase inhibitor cerulenin nearly completely blocked sphingoid base production after heat, indicating a requirement for endogenous fatty acids for heat-mediated sphingoid base synthesis. Consistent with this, genetic studies show that fatty acid import does not contribute to heat-induced de novo synthesis under normal conditions. Interestingly the absence of medium serine also ameliorated heat-induced sphingoid base production, indicating a requirement for exogenous serine for the response, and consistent with this finding, disruption of synthesis of endogenous serine did not affect heat-induced sphingolipid synthesis. Serine uptake assays indicated that heat increased serine uptake from medium by 100% during the first 10 min of heat stress. Moreover treatments that increase serine uptake in the absence of heat including acute medium acidification and glucose treatment also enhanced de novo sphingoid base synthesis equivalent to that induced by heat stress. These data agree with findings from mammalian systems that availability of substrates is a key determinant of flux through sphingolipid synthesis. Moreover data presented here indicate that SPT activity can be driven by several factors that increase serine uptake in the absence of heat. These findings may provide insights into the many systems in which de novo synthesis is increased in the absence of elevated in vitro SPT activity.     

LASS5 is the predominant ceramide synthase isoform involved in de novo sphingolipid synthesis in lung epithelia

Ceramide is a key bioactive mediator that inhibits surfactant phosphatidylcholine (PtdCho) synthesis in lung epithelia. Ceramide availability is governed by sphingomyelin (SM) hydrolysis, but less is known regarding its de novo synthesis. In this study, we observed that ceramide synthesis within murine lung epithelia was associated with high-level ceramide synthase (dihydroceramide synthase) activity. Longevity assurance homolog 5 (LASS5) was the predominant ceramide synthase isoform detected in lung epithelia, whereas relatively lower level expression was detected for the other five mammalian homologs. Pulmonary LASS5 was developmentally regulated, but its expression was spatially and gender nonspecific. Exogenously expressed LASS5 in lung epithelia was membrane-associated, triggering increased ceramide synthesis, whereas knockdown studies using fumonisin B1 or LASS5 small, interfering RNA reduced ceramide synthase activity by 78% or 45%, respectively. Overexpression of LASS5 also reduced PtdCho synthesis, but maximal inhibition was achieved when LASS5 was coexpressed with a plasmid encoding a neutral sphingomyelinase involved in SM hydrolysis. These results demonstrate that LASS5 is the major ceramide synthase gene product involved in sphingolipid production that may also regulate PtdCho metabolism in pulmonary epithelia.     

Fumonisins: fungal toxins that shed light on sphingolipid function

Fumonisins are sphinganine analogues produced by Fusarium moniliforme and related fungi. They inhibit ceramide synthase and block the biosynthesis o f complex sphingolipids, promoting accumulation o f sphinganine and sphinganine 1 phosphate. Disruption o f sphingolipid metabolism by fumonisin B(1) alters cell-cell interactions, the behaviour o f cell-surface proteins, the activity o f protein kinases, the metabolism of other lipids, and cell growth and viability. This multitude of effects probably accounts for the toxicity and carcinogenicity of these mycotoxins. Naturally occurring inhibitors o f sphingolipid metabolism such as fumonisins are proving to be powerful tools for studying the diverse roles of sphingolipids in cell regulation and disease.     

Variations of ganglioside biosynthetic pathways in the phenotype conversion from myofibroblasts to lipocytes in murine hepatic stellate cell line

GRX cell line represents hepatic stellate cell and can be transformed from an actively proliferation myofibroblast phenotype into a quiescent fat-storing lipocyte phenotype. Both express the same gangliosides (GM3, GM2, GM1 and GD1a), which are resolved as doublets on HPTLC. Upper/lower band ratio is increased in lipocyte-like cells and the upper band is composed by ceramides with long-chain fatty acids. This study evaluated the contribution of de novo synthesis, sphingosine and Golgi recycling pathways on ganglioside biosynthesis, in both phenotypes. Cells were preincubated with 5 mM β-chloroalanine (SPT: serine palmitoyltransferase inhibitor) or with 25 μM fumonisin B1 (ceramide synthase inhibitor) and then radiolabeled with [U-¹⁴C]galactose in the continued presence of inhibitors. Gangliosides were extracted, purified and analyzed by HPTLC. In myofibroblast-like cells, simple gangliosides use the de novo pathway while complex gangliosides are mainly synthesized by recycling pathways. In lipocyte-like cells, de novo pathway has a lesser contribution and this is in agreement with the lower activity of the committed enzyme of sphingolipid synthesis (SPT) detected in this phenotype. SPT mRNA has an identical expression in both phenotypes. It was also observed that gangliosides doublets from myofibroblast-like cells have the same distribution between triton soluble and insoluble fractions (upper band > lower band) while the gangliosides doublets from lipocyte-like cells show an inversion in the insoluble fraction (lower band > upper band) in comparison to soluble fraction. These results indicate that myofibroblast- and lipocyte-like cells have important differences between the glycosphingolipid biosynthetic pathways, which could contribute with the respective glycosphingolipid-enriched membrane microdomain’s composition.     

Sphingolipid metabolism is a crucial determinant of cellular fate in nonstimulated proliferating Madin-Darby canine kidney (MDCK) cells

The present report was addressed to study the influence of sphingolipid metabolism in determining cellular fate. In nonstimulated proliferating Madin-Darby canine kidney (MDCK) cells, sphingolipid de novo synthesis is branched mainly to a production of sphingomyelin and ceramide, with a minor production of sphingosylphosphocholine, ceramide 1-phosphate, and sphingosine 1-phosphate. Experiments with (32)P as a radioactive precursor showed that sphingosine 1-phosphate is produced mainly by a de novo independent pathway. Enzymatic inhibition of the de novo pathway and ceramide synthesis affected cell number and viability only slightly, without changing sphingosine 1-phosphate production. By contrast, inhibition of sphingosine kinase-1 activity provoked a significant reduction in both cell number and viability in a dose-dependent manner. When sphingolipid metabolism was studied, an increase in de novo formed ceramide was found, which correlated with the concentration of enzyme inhibitor and the reduction in cell number and viability. Knockdown of sphingosine kinase-1 expression also induced an accumulation of de novo synthesized ceramide, provoking a slight reduction in cell number and viability similar to that induced by a low concentration of the sphingosine kinase inhibitor. Taken together, our results indicate that the level of de novo formed ceramide is controlled by the synthesis of sphingosine 1-phosphate, which appears to occur through a de novo synthesis-independent pathway, most probably the salvage pathway, that is responsible for the MDCK cell fate, suggesting that under proliferating conditions, a dynamic interplay exists between the de novo synthesis and the salvage pathway.     

Enhanced phagocytosis through inhibition of de novo ceramide synthesis

Fcgamma receptors are important mediators of the binding of IgG to and induction of phagocytosis in neutrophils. COS-1 cells provide a potentially useful model for studying these receptors because transfection with the FcgammaRIIA renders these cells phagocytic. During FcgammaRIIA-mediated phagocytosis in COS-1 cells, endogenous ceramide levels increased 52% by 20 min (p < 0.01). Phospholipase D activity increased by 62% (p < 0.01). Correspondingly, the phagocytic index increased by 3.7-fold by 20 min. Two inhibitors of ceramide formation were used to assess the consequences of reduced ceramide generation. l-Cycloserine, an inhibitor that blocks serine palmitoyltransferase activity, lowered both sphingosine and ceramide levels. Under these conditions, the phagocytic index increased 100% in the presence of 2 mm l-cycloserine. The formation of ceramide resulting from the N-acylation of dihydrosphingosine or sphingosine by ceramide synthase is inhibited by the fungal toxin fumonisin B(1). When cells were treated with 5-50 microm fumonisin B(1), the cellular level of ceramide decreased in a concentration-dependent manner, while simultaneously the phagocytic index increased by 52%. Concomitantly, three indirect measures of FcgammaRIIA activity were altered with the fall in ceramide levels. Syk phosphorylation, phospholipase D activity, and mitogen-activated protein (MAP) kinase phosphorylation were increased at 30 min. When Syk phosphorylation was blocked with piceatannol and cells were similarly challenged, phosphatidylinositol 3-kinase activation was blocked, but no changes in either ceramide accumulation or MAP kinase activation were observed. Ceramide formation and MAP kinase activation are therefore not dependent on Syk kinase activity in this system. These results indicate that COS-1 cells provide a useful model for the recapitulation of sphingolipid signaling in the study of phagocytosis. Ceramide formed by de novo synthesis may represent an important mechanism in the regulation of phagocytosis.