Involvement of neutral ceramidase in ceramide metabolism at the plasma membrane and in extracellular milieu

Neutral ceramidase is a type II integral membrane protein, which is occasionally secreted into the extracellular milieu after the processing of its N-terminal anchor. We found that when overexpressed in CHOP cells, neutral ceramidase hydrolyzed cell surface ceramide, which increased in amount after the treatment of cells with bacterial sphingomyelinase, leading to an increase in the cellular level of sphingosine and sphingosine 1-phosphate. On the other hand, knockdown of the endogenous enzyme by siRNA decreased the cellular level of both sphingolipid metabolites. The treatment of cells with bovine serum albumin significantly reduced the cellular level of sphingosine, but not sphingosine 1-phosphate, generated by overexpression of the enzyme. The cellular level of sphingosine 1-phosphate increased with overexpression of the cytosolic sphingosine kinase. These results suggest that sphingosine 1-phosphate is mainly produced inside of the cell after the incorporation of sphingosine generated on the plasma membranes. The enzyme also seems to participate in the hydrolysis of serum-derived ceramide in the vascular system. Significant amounts of sphingosine as well as sphingosine 1-phosphate were generated in the cell-free conditioned medium of ceramidase transfectants, compared with mock transfectants. No increase in these metabolites was observed if serum or bacterial sphingomyelinase was omitted from the conditioned medium, suggesting that the major source of ceramide is the serum-derived sphingomyelin. A sphingosine 1-phosphate receptor, S1P(1), was internalized much faster by the treatment of S1P(1)-overexpressing cells with conditioned medium of ceramidase transfectants than that of mock transfectants. Collectively, these results clearly indicate that the enzyme is involved in the metabolism of ceramide at the plasma membrane and in the extracellular milieu, which could regulate sphingosine 1-phosphate-mediated signaling through the generation of sphingosine.     

Sphingolipids in apoptosis, survival and regeneration in the nervous system

Simple sphingolipids such as ceramide, sphingosine and sphingosine 1-phosphate are key regulators of diverse cellular functions. Their roles in the nervous system are supported by extensive evidence derived primarily from studies in cultured cells. More recently animal studies and studies with human samples have revealed the importance of ceramide and its metabolites in the development and progression of neurodegenerative disorders. The roles of sphingolipids in neurons and glial cells are complex, cell dependent, and many times contradictory. In this review I will summarize the effects elicited by ceramide and ceramide metabolites in cells of the nervous system, in particular those effects related to cell survival and death, emphasizing the molecular mechanisms involved. I also discuss recent evidence for the implication of sphingolipids in the development and progression of certain dementias.     

Sphingolipids in apoptosis, survival and regeneration in the nervous system

Simple sphingolipids such as ceramide, sphingosine and sphingosine 1-phosphate are key regulators of diverse cellular functions. Their roles in the nervous system are supported by extensive evidence derived primarily from studies in cultured cells. More recently animal studies and studies with human samples have revealed the importance of ceramide and its metabolites in the development and progression of neurodegenerative disorders. The roles of sphingolipids in neurons and glial cells are complex, cell dependent, and many times contradictory. In this review I will summarize the effects elicited by ceramide and ceramide metabolites in cells of the nervous system, in particular those effects related to cell survival and death, emphasizing the molecular mechanisms involved. I also discuss recent evidence for the implication of sphingolipids in the development and progression of certain dementias.     

Insertional mutagenesis of the mouse acid ceramidase gene leads to early embryonic lethality in homozygotes and progressive lipid storage disease in heterozygotes

Ceramide is an important cellular lipid involved in signal transduction and the biosynthesis of complex sphingolipids. It can be hydrolyzed into sphingosine, another important signaling lipid, by the activity of ceramidases. Point mutations in the gene (Asah1) encoding one ceramidase, acid ceramidase (AC), lead to the lysosomal storage disorder Farber disease (FD). To investigate the role of AC in mammalian development, we disrupted the mouse gene Asah1 in embryonic stem cells by homologous recombination mediated insertion of an AC targeting vector into the wild-type sequence. Genotype analysis of over 150 offspring or embryos from heterozygous intercrosses revealed an absence of Asah1(-/-) individuals at embryonic day (E) 8.5 or later, although the ratio of wild-type to Asah1(+/-) individuals from these intercrosses was 1:2. Northern blot analysis showed that AC expression was turned on early in development, by E7.0, and continued through at least E17. In contrast, expression of the related lipid hydrolase, acid sphingomyelinase, was shut down by E11. Asah1(+/-) mice survived and lived a normal lifespan, but developed a progressive lipid storage disease in several of their organs, particularly the liver. These histopathological findings in Asah1(+/-) animals correlated with an up to twofold increase in the ceramide content of these tissues and a reduction n AC activity, confirming that the gene insertion event disrupted AC activity and ceramide metabolism. These results provide direct in vivo evidence that normal ceramide metabolism, and AC activity in particular, is essential for mammalian development. The animals and embryos described here should be a valuable resource for investigators studying the role of ceramide in cell growth and development, as well as those interested in the pathogenesis of FD and other sphingolipid storage disorders.     

Role of sphingosine 1-phosphate in the mitogenesis induced by oxidized low density lipoprotein in smooth muscle cells via activation of sphingomyelinase, ceramidase, and sphingosine kinase.

Oxidized LDL (oxLDL) have been implicated in diverse biological events leading to the development of atherosclerotic lesions. We previously demonstrated that the proliferation of cultured vascular smooth muscle cells (SMC) induced by oxLDL is preceded by an increase in neutral sphingomyelinase activity, sphingomyelin turnover to ceramide, and stimulation of mitogen-activated protein kinases (Augé, N., Escargueil-Blanc, I., Lajoie-Mazenc, I., Suc, I., Andrieu-Abadie, N., Pieraggi, M. T., Chatelut, M., Thiers, J. C., Jaffrézou, J. P., Laurent, G., Levade, T., Nègre-Salvayre, A., and Salvayre, R. (1998) J. Biol. Chem. 273, 12893-12900). Since ceramide can be converted to other bioactive metabolites, such as the well established mitogen sphingosine 1-phosphate (S1P), we investigated whether additional ceramide metabolites are involved in the oxLDL-induced SMC proliferation. We report here that incubation of SMC with oxLDL increased the activities of both acidic and alkaline ceramidases as well as sphingosine kinase, and elevated cellular sphingosine and S1P. Furthermore, the mitogenic effect of oxLDL was inhibited by D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol and N,N-dimethylsphingosine which are inhibitors of ceramidase and sphingosine kinase, respectively. These findings suggest that S1P is a key mediator of the mitogenic effect of oxLDL. In agreement with this conclusion, exogenous addition of sphingosine stimulated the proliferation of cultured SMC, and this effect was abrogated by dimethylsphingosine but not by fumonisin B1, an inhibitor of the acylation of sphingosine to ceramide. Exogenous S1P also promoted SMC proliferation. Altogether, these results strongly suggest that the mitogenic effect of oxLDL in SMC involves the combined activation of sphingomyelinase(s), ceramidase(s), and sphingosine kinase, resulting in the turnover of sphingomyelin to a number of sphingolipid metabolites, of which at least S1P is critical for mitogenesis.     

Cell-nonautonomous function of ceramidase in photoreceptor homeostasis

Neutral ceramidase, a key enzyme of sphingolipid metabolism, hydrolyzes ceramide to sphingosine. These sphingolipids are critical structural components of cell membranes and act as second messengers in diverse signal transduction cascades. Here, we have isolated and characterized functional null mutants of Drosophila ceramidase. We show that secreted ceramidase functions in a cell-nonautonomous manner to maintain photoreceptor homeostasis. In the absence of ceramidase, photoreceptors degenerate in a light-dependent manner, are defective in normal endocytic turnover of rhodopsin, and do not respond to light stimulus. Consistent with a cell-nonautonomous function, overexpression of ceramidase in tissues distant from photoreceptors suppresses photoreceptor degeneration in an arrestin mutant and facilitates membrane turnover in a rhodopsin null mutant. Furthermore, our results show that secreted ceramidase is internalized and localizes to endosomes. Our findings establish a role for a secreted sphingolipid enzyme in the regulation of photoreceptor structure and function.     

Cooperative prosurvival activity by ERK and Akt in human alveolar macrophages is dependent on high levels of acid ceramidase activity

Human alveolar macrophages are unique in that they have an extended life span in contrast to precursor monocytes. In evaluating the role of sphingolipids in alveolar macrophage survival, we found high levels of sphingosine, but not sphingosine-1-phosphate. Sphingosine is generated by the action of ceramidase(s) on ceramide, and alveolar macrophages have high constitutive levels of acid ceramidase mRNA, protein, and activity. The high levels of acid ceramidase were specific to alveolar macrophages, because there was little ceramidase protein or activity (or sphingosine) in monocytes from matching donors. In evaluating prolonged survival of alveolar macrophages, we observed a requirement for constitutive activity of ERK MAPK and the PI3K downstream effector Akt. Blocking acid ceramidase but not sphingosine kinase activity in alveolar macrophages led to decreased ERK and Akt activity and induction of cell death. These studies suggest an important role for sphingolipids in prolonging survival of human alveolar macrophages via distinct survival pathways.     

Ceramide/sphingosine/sphingosine 1-phosphate metabolism on the cell surface and in the extracellular space

Sphingolipid metabolites, ceramide, sphingosine, and sphingosine 1-phosphate, have emerged as a new class of lipid biomodulators of various cell functions. These metabolites are known to function not only as intracellular second messengers, but also in the extracellular space. Sphingosine 1-phosphate especially has numerous functions as an important extracellular mediator that binds to cell surface S1P receptors. Recent studies have also shown that sphingolipid-metabolizing enzymes function not only in intracellular organelles but also in the extracellular spaces, including the outer leaflet of the plasma membrane. This review focuses on the metabolic enzymes (acid and alkaline sphingomyelinases, neutral ceramidase, and sphingosine kinase) that are involved in the production of the sphingolipid metabolites in these extracellular spaces, and on the metabolic pathway itself.     

Nitric oxide induces neutral ceramidase degradation by the ubiquitin/proteasome complex in renal mesangial cell cultures

The neutral ceramidase is a key enzyme in the regulation of cellular ceramide levels. Previously we have reported that stimulation of rat renal mesangial cells with nitric oxide (NO) donors leads to an inhibition of neutral ceramidase activity which is due to increased degradation of the enzyme. This and the concomitant activation of the sphingomyelinase results in an amplification of ceramide levels. Here, we show that the NO-triggered degradation of neutral ceramidase involves activation of the ubiquitin/proteasome complex. The specific proteasome inhibitor lactacystin completely reverses the NO-induced degradation of ceramidase protein and neutral ceramidase activity. As a consequence, the cellular amount of ceramide, which drastically increases by NO stimulation, is reduced in the presence of lactacystin. Furthermore, ubiquitinated neutral ceramidase accumulates after NO stimulation. In summary, our data clearly show that the ubiquitin/proteasome complex is an important determinant of neutral ceramidase activity and thereby regulates the availability of ceramide.     

Bimodal effect of advanced glycation end products on mesangial cell proliferation is mediated by neutral ceramidase regulation and endogenous sphingolipids

Advanced glycation end-products (AGE) are generated by chronic hyperglycaemia and may cause diabetic microvascular complications such as diabetic nephropathy. Many factors influence the development of diabetic nephropathy; however, dysregulation of mesangial cell (MC) proliferation appears to play an early and crucial role. In this study, we investigated the effects of AGE on rat MC proliferation and the involvement of sphingolipids in the AGE response. Results show a bimodal effect of AGE on MC proliferation. Thus, low AGE concentrations (<1 microm) induced a significant increase (+26%) of MC proliferation, whereas higher concentrations (10 microm) markedly reduced it (-24%). In parallel, AGE exerted biphasic effects on neutral ceramidase expression and activity. Low AGE concentrations increased neutral ceramidase activity and expression, whereas high AGE concentrations showed opposite effects. Surprisingly, neutral ceramidase modulation did not result in changes of ceramide levels. However, the AGE (10 microm)-inhibitory effect on MC proliferation was associated with accumulation of sphingosine and was specifically prevented by blocking glucosylceramide synthesis, suggesting that the high AGE concentration effects are mediated by sphingosine and/or glycolipids. On the other hand, treatment of cells with low AGE concentrations led to an increase of sphingosine kinase activity and sphingosine-1-phosphate production that drove the increase of MC proliferation. Interestingly, in glomeruli isolated from streptozotocin-diabetic rats, a time-dependent modulation of ceramidase activity was observed as compared with controls. These results suggest that AGE regulate MC growth by modulating neutral ceramidase and endogenous sphingolipids.     

Control of inflammatory responses by ceramide,sphingosine 1-phosphate and ceramide 1-phosphate

Inflammation is a network of complex processes involving a variety of metabolic and signaling pathways aiming at healing and repairing damage tissue, or fighting infection. However, inflammation can be detrimental when it becomes out of control. Inflammatory mediators involve cytokines, bioactive lipids and lipid-derived metabolites. In particular, the simple sphingolipids ceramides, sphingosine 1-phosphate, and ceramide 1-phosphate have been widely implicated in inflammation. However, although ceramide 1-phosphate was first described as pro-inflammatory, recent studies show that it has anti-inflammatory properties when produced in specific cell types or tissues. The biological functions of ceramides and sphingosine 1-phosphate have been extensively studied. These sphingolipids have opposing effects with ceramides being potent inducers of cell cycle arrest and apoptosis, and sphingosine 1-phosphate promoting cell growth and survival. However, the biological actions of ceramide 1-phosphate have only been partially described. Ceramide 1-phosphate is mitogenic and anti-apoptotic, and more recently, it has been demonstrated to be key regulator of cell migration. Both sphingosine 1-phosphate and ceramide 1-phosphate are also implicated in tumor growth and dissemination. The present review highlights new aspects on the control of inflammation and cell migration by simple sphingolipids, with special emphasis to the role played by ceramide 1-phosphate in controlling these actions.     

Purification and characterization of human intestinal neutral ceramidase

Sphingolipids are degraded by sphingomyelinase and ceramidase in the gut to ceramide and sphingosine, which may inhibit cell proliferation and induce apoptosis, and thus have anti-tumour effects in the gut. Although previous rodent studies including experiments on knockout mice indicate a role of neutral ceramidase in ceramide digestion, the human enzyme has never been purified and characterized in its purified form. We here report the purification and characterization of neutral ceramidase from human ileostomy content, using octanoyl-[(14)C]sphingosine as substrate. After four chromatographic steps, a homogeneous protein band with 116kDa was obtained. MALDI mass spectrometry identified 16 peptide masses similar to human ceramidase previously cloned by El Bawab et al. [Molecular cloning and characterization of a human mitochondrial ceramidase, J. Biol. Chem. 275 (2000) 21508-21513] and Hwang et al. [Subcellular localization of human neutral ceramidase expressed in HEK293 cells, Biochem. Biophys. Res. Commun. 331 (2005) 37-42]. By RT-PCR and 5'-RACE methods, a predicted partial nucleotide sequence of neutral ceramidase was obtained from a human duodenum biopsy sample, which was homologous to that of known neutral/alkaline ceramidases. The enzyme has neutral pH optimum and catalyses both hydrolysis and formation of ceramide without distinct bile salt dependence. It is inhibited by Cu(2+) and Zn(2+) ions and by low concentrations of cholesterol. The enzyme is a glycoprotein but deglycosylation does not affect its activity. Our study indicates that neutral ceramidase is expressed in human intestine, released in the intestinal lumen and plays a major role in ceramide metabolism in the human gut.     

A simple,highly sensitive,and facile method to quantify ceramide at the plasma membrane

The role of ceramide in biological functions is typically based on the elevation of cellular ceramide, measured by LC-MS in the total cell lysate. However, it has become increasingly appreciated that ceramide in different subcellular organelles regulates specific functions. In the plasma membrane, changes in ceramide levels might represent a small percentage of the total cellular ceramide, evading MS detection but playing a critical role in cell signaling. Importantly, there are currently no efficient techniques to quantify ceramide in the plasma membrane. Here, we developed a method to measure the mass of ceramide in the plasma membrane using a short protocol that is based on the hydrolysis of plasma membrane ceramide into sphingosine by the action of exogenously applied bacterial recombinant neutral ceramidase. Plasma membrane ceramide content can then be determined by measuring the newly generated sphingosine at a stoichiometry of 1:1. A key step of this protocol is the chemical fixation of cells to block cellular sphingolipid metabolism, especially of sphingosine to sphingosine 1-phosphate. We confirmed that chemical fixation does not disrupt the lipid composition at the plasma membrane, which remains intact during the time of the assay. We illustrate the power of the approach by applying this protocol to interrogate the effects of the chemotherapeutic compound doxorubicin. Here we distinguished two pools of ceramide, depending on the doxorubicin concentration, consolidating different reports. In summary, we have developed the first approach to quantify ceramide in the plasma membrane, allowing the study of new avenues in sphingolipid compartmentalization and function.     

Sphingolipids and cell signaling: Involvement in apoptosis and atherogenesis

This review considers various functional aspects of cell sphingolipids (sphingomyelin, ceramides) and lysosphingolipids (sphingosine-1-phosphate (S1P) and sphingosine phosphorylcholine). Good evidence now exists that they are actively involved in numerous cell-signaling processes. The enzymes responsible for formation and interconversion of cell sphingolipids (sphingomyelinases, ceramidase, sphingosine kinase, S1P-lyase) exhibit high sensitivity to various stimulating factors. This determines the content of individual cell sphingolipids and therefore the mode of cell response. Special attention is paid to preferential localization of sphingolipids in the rigid plasma membrane domains (rafts) coupled to many signal proteins. The suggestion is discussed that ceramide signaling may be based on the modification of fine molecular interactions in lipid rafts, resulting in its clusterization inducing the signal transduction. The review also highlights involvement of sphingolipids in cell proliferation, apoptosis, and in processes implicated to atherosclerosis.     

CDase is a pan-ceramidase in Drosophila

Ceramidases catalyze the conversion of ceramide to sphingosine. They are acylaminohydrolases that catalyze the deacylation of the amide-linked saturated fatty acid from ceramide to generate sphingosine. They also catalyze the reverse reaction of ceramide biosynthesis using sphingosine and fatty acid. In mammals, different proteins catalyze these reactions while individually exhibiting optimal activity over a narrow pH range and have been accordingly called acid, neutral, and alkaline ceramidases. Several genes encode for variants of alkaline ceramidase in mammals. Brainwashing (Bwa) is the only putative alkaline ceramidase homologue present in Drosophila. In this study we have demonstrated that BWA does not exhibit ceramidase activity and that bwa null mutants display no loss of ceramidase activity. Instead, the neutral ceramidase gene CDase encodes the protein that is responsible for all measurable ceramidase activity in Drosophila. Our studies show strong genetic interaction of Bwa with CDase and the Drosophila ceramide kinase gene (DCERK). We show that, although BWA is unlikely to be a ceramidase, it is a regulator of sphingolipid flux in Drosophila. Bwa exhibits strong genetic interaction with other genes coding for ceramide-metabolizing enzymes. This interaction might partly explain its original identification as a ceramidase.     

Novel pathway of ceramide production in mitochondria: thioesterase and neutral ceramidase produce ceramide from sphingosine and acyl-CoA

Reports suggest that excessive ceramide accumulation in mitochondria is required to initiate the intrinsic apoptotic pathway and subsequent cell death, but how ceramide accumulates is unclear. Here we report that liver mitochondria exhibit ceramide formation from sphingosine and palmitoyl-CoA and from sphingosine and palmitate. Importantly, this activity was markedly decreased in liver from neutral ceramidase (NCDase)-deficient mice. Moreover, the levels of ceramide were dissimilar in liver mitochondria of WT and NCDase KO mice. These results suggest that NCDase is a key participant of ceramide formation in liver mitochondria. We also report that highly purified liver mitochondria have ceramidase, reverse ceramidase, and thioesterase activities. Increased accessibility of palmitoyl-CoA to the mitochondrial matrix with the pore-forming peptide zervamicin IIB resulted in 2-fold increases in palmitoyl-CoA hydrolysis by thioesterase. This increased hydrolysis was accompanied by an increase in ceramide formation, demonstrating that both outer membrane and matrix localized thioesterases can regulate ceramide formation. Also, ceramide formation might occur both in the outer mitochondrial membrane and in the mitochondrial matrix, suggesting the existence of distinct ceramide pools. Taken together, these results suggest that the reverse activity of NCDase contributes to sphingolipid homeostasis in this organelle in vivo.     

Sphingomyelinase and cell-permeable ceramide analogs stimulate cellular proliferation in quiescent Swiss 3T3 fibroblasts.

Sphingomyelin or the products derived from its metabolism may constitute a signaling system involved in a variety of cellular processes. The activation of a plasma membrane neutral sphingomyelinase, which catalyzes the first step in sphingomyelin turnover, has been suggested to play an important role in cellular differentiation. We have studied the effect of exogenous staphylococcal sphingomyelinase on DNA synthesis and on the composition of membrane sphingolipids in quiescent Swiss 3T3 fibroblasts. Sphingomyelinase stimulated proliferation of Swiss 3T3 cells and potentiated the mitogenic action of other growth factors, such as insulin, epidermal growth factor, and bombesin. Treatment with sphingomyelinase produced a significant decrease in sphingomyelin accompanied by a corresponding increase in ceramide levels. No significant increases were detected in the levels of products derived from ceramide, i.e. ceramide 1-phosphate, sphingosine, or sphingosine 1-phosphate. To further investigate the role of ceramide in cellular proliferation, we studied the effect of cell-permeable analogs of ceramide on DNA synthesis in quiescent Swiss 3T3 cells. Both N-hexanoylsphingosine and N-acetylsphingosine at low concentrations stimulated [3H]thymidine incorporation and acted synergistically with a wide variety of growth factors known to induce proliferation of quiescent Swiss 3T3 fibroblasts. Similar effects were observed with bovine brain ceramides. These results suggest that ceramide may be involved in the regulation of cellular proliferation.     

Simultaneous quantitative analysis of ceramide and sphingosine in mouse blood by naphthalene-2,3-dicarboxyaldehyde derivatization after hydrolysis with ceramidase

Ceramide and sphingosine are sphingolipids with important functional and structural roles in cells. In this paper we report a new enzyme-based method to simultaneously quantify the levels of ceramide and sphingosine in biological samples. This method utilizes purified human recombinant acid ceramidase to completely hydrolyze ceramide to sphingosine, followed by derivatization of the latter with naphthalene-2,3-dialdehyde (NDA) and quantification by reverse-phase high-performance liquid chromatography. The limits of detection for sphingosine-NDA and ceramidase-derived sphingosine-NDA were 9.6 and 12.3 fmol, respectively, and the limits of quantification were 34.2 and 45.7 fmol, respectively. The recovery of sphingosine and ceramide standards quantified by this assay were between 95.6 and 104.6%. The relative standard deviations for the intra- and interday sphingosine assay were 2.1 and 4.5%, respectively, and those for the ceramide assay were 3.3 and 4.1%, respectively. To validate this procedure, we quantified ceramide and sphingosine in mouse plasma, white blood cells, and hemoglobin, the first reported time that the amounts of these lipids have been documented in individual blood components. We also used this technique to evaluate the ability of a novel ceramide analog, AD2646, to inhibit the hydrolytic activity of acid ceramidase. The results demonstrate that this new procedure can provide sensitive, reproducible, and simultaneous ceramide and sphingosine quantification. The technique also may be used for determining the activity and inhibition of ceramidases and may be adapted for quantifying sphingomyelin and sphingosine-1-phosphate levels. In the future it could be an important tool for investigators studying the role of ceramide/sphingosine metabolism in signal transduction, cell growth and differentiation, and cancer pathogenesis and treatment.     

Acute activation of acid ceramidase affects cytokine-induced cytotoxicity in rat islet β-cells

Ceramidase hydrolyzes ceramide and produces sphingosine as a substrate of sphingosine kinase (SPHK), which transforms sphingosine to sphingosine-1-phosphate. It has been reported that cytokines elicit SPHK activation in rat β-cells. As a sphingosine provider, ceramidase should also be activated. In our previous work, we showed that the increase in mRNA and protein levels in cytokine-treated INS-1 rat β-cells resulted in chronic activation of neutral ceramidase. Here we found that acid ceramidase (AC) is activated by cytokines at an early stage via tyrosine phosphorylation. In addition, basal AC activity was first detected in INS-1 cells and isolated rat islets, and cytokine-induced cell growth was significantly repressed when AC was pharmacologically inhibited.