Neutral sphingomyelinases and nSMase2: Bridging the gaps

There is strong evidence indicating a role for ceramide as a second messenger in processes such as apoptosis, cell growth and differentiation, and cellular responses to stress. Ceramide formation from the hydrolysis of sphingomyelin is considered to be a major pathway of stress-induced ceramide production with magnesium-dependent neutral sphingomyelinase (N-SMase) identified as a prime candidate in this pathway. The recent cloning of a mammalian N-SMase-nSMase2- and generation of nSMase2 knockout/mutant mice have now provided vital tools with which to further study the regulation and roles of this enzyme in both a physiological and pathological context. In the present review, we summarize current knowledge on N-SMase relating this to what is known about nSMase2. We also discuss the future areas of nSMase2 research important for molecular understanding of this enzyme and its physiological roles.     

Role for neutral sphingomyelinase-2 in tumor necrosis factor alpha-stimulated expression of vascular cell adhesion molecule-1 (VCAM) and intercellular adhesion molecule-1 (ICAM) in lung epithelial cells: p38 MAPK is an upstream regulator of nSMase2

Neutral sphingomyelinases (N-SMases) are major candidates for stress-induced ceramide production. However, there is little information on the physiological regulation and roles of the cloned N-SMase enzyme, nSMase2. In this study, nSMase2 was found to translocate acutely to the plasma membrane of A549 epithelial cells in response to tumor necrosis factor alpha (TNF-alpha) in a time- and dose-dependent manner. Additionally, TNF-alpha increased N-SMase activity rapidly and transiently both endogenously and in cells overexpressing nSMase2. Furthermore, the translocation of nSMase2 was regulated by p38-alpha MAPK, but not ERK or JNK, and the increase in endogenous N-SMase activity was abrogated by p38 MAPK inhibition. In addition, both p38-alpha MAPK and nSMase2 were implicated in the TNF-alpha-stimulated up-regulation of the adhesion proteins vascular cell adhesion molecule-1 (VCAM) and intercellular adhesion molecule-1 (ICAM), but this was largely independent of NF-kappaB activation. These data reveal p38 MAPK as an upstream regulator of nSMase2 and indicate a role for nSMase2 in pro-inflammatory responses induced by TNF-alpha as a regulator of adhesion proteins.     

Biochemical properties of mammalian neutral sphingomyelinase 2 and its role in sphingolipid metabolism

Neutral sphingomyelinase (N-SMase) is one of the key enzymes involved in the generation of ceramide; however, the gene(s) encoding for the mammalian N-SMase is still not well defined. Previous studies on the cloned nSMase1 had shown that the protein acts primarily as lyso-platelet-activating factor-phospholipase C. Recently the cloning of another putative N-SMase, nSMase2, was reported. In this study, biochemical characterization of the mouse nSMase2 was carried out using the overexpressed protein in yeast cells in which the inositol phosphosphingolipid phospholipase C (Isc1p) was deleted. N-SMase activity was dependent on Mg(2+) and was activated by phosphatidylserine and inhibited by GW4869. The ability of nSMase2 to recognize endogenous sphingomyelin (SM) as substrate was investigated by overexpressing nSMase2 in MCF7 cells. Mass measurements showed a 40% decrease in the SM levels in the overexpressor cells, and labeling studies demonstrated that nSMase2 accelerated SM catabolism. Accordingly, ceramide measurement showed a 60 +/- 15% increase in nSMase2-overexpressing cells compared with the vector-transfected MCF7. The role of nSMase2 in cell growth was next investigated. Stable overexpression of nSMase2 resulted in a 30-40% decrease in the rate of growth at the late exponential phase. Moreover, tumor necrosis factor induced approximately 50% activation of nSMase2 in MCF7 cells overexpressing the enzyme, demonstrating that nSMase2 is a tumor necrosis factor-responsive enzyme. In conclusion, these results 1) show that nSMase2 is a structural gene for nSMase, 2) suggest that nSMase2 acts as a bona fide N-SMase in cells, and 3) implicate nSMase2 in the regulation of cell growth and cell signaling.     

Identification and Characterization of Murine Mitochondria-associated Neutral Sphingomyelinase (MA-nSMase), the Mammalian Sphingomyelin Phosphodiesterase 5

Sphingolipids play important roles in regulating cellular responses. Although mitochondria contain sphingolipids, direct regulation of their levels in mitochondria or mitochondria-associated membranes is mostly unclear. Neutral SMase (N-SMase) isoforms, which catalyze hydrolysis of sphingomyelin (SM) to ceramide and phosphocholine, have been found in the mitochondria of yeast and zebrafish, yet their existence in mammalian mitochondria remains unknown. Here, we have identified and cloned a cDNA based on nSMase homologous sequences. This cDNA encodes a novel protein of 483 amino acids that displays significant homology to nSMase2 and possesses the same catalytic core residues as members of the extended N-SMase family. A transiently expressed V5-tagged protein co-localized with both mitochondria and endoplasmic reticulum markers in MCF-7 and HEK293 cells; accordingly, the enzyme is referred to as mitochondria-associated nSMase (MA-nSMase). MA-nSMase was highly expressed in testis, pancreas, epididymis, and brain. MA-nSMase had an absolute requirement for cations such as Mg²⁺ and Mn²⁺ and activation by the anionic phospholipids, especially phosphatidylserine and the mitochondrial cardiolipin. Importantly, overexpression of MA-nSMase in HEK293 cells significantly increased in vitro N-SMase activity and also modulated the levels of SM and ceramide, indicating that the identified cDNA encodes a functional SMase. Thus, these studies identify and characterize, for the first time, a mammalian MA-nSMase. The characterization of MA-nSMase described here will contribute to our understanding of pathways regulated by sphingolipid metabolites, particularly with reference to the mitochondria and associated organelles.     

Identification of Heat Shock Protein 60 as a Regulator of Neutral Sphingomyelinase 2 and Its Role in Dopamine Uptake

Activation of sphingomyelinase (SMase) by extracellular stimuli is the major pathway for cellular production of ceramide, a bioactive lipid mediator acting through sphingomyelin (SM) hydrolysis. Previously, we reported the existence of six forms of neutral pH–optimum and Mg²⁺-dependent SMase (N-SMase) in the membrane fractions of bovine brain. Here, we focus on N-SMase ε from salt-extracted membranes. After extensive purification by 12,780-fold with a yield of 1.3%, this enzyme was eventually characterized as N-SMase2. The major single band of 60-kDa molecular mass in the active fractions of the final purification step was identified as heat shock protein 60 (Hsp60) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. Proximity ligation assay and immunoprecipitation study showed that Hsp60 interacted with N-SMase2, prompting us to examine the effect of Hsp60 on N-SMase2 and ceramide production. Interestingly, Hsp60 siRNA treatment significantly increased the protein level of N-SMase2 in N-SMase2-overexpressed HEK293 cells. Furthermore, transfection of Hsp60 siRNA into PC12 cells effectively increased both N-SMase activity and ceramide production and increased dopamine re-uptake with paralleled increase. Taken together, these results show that Hsp60 may serve as a negative regulator in N-SMase2-induced dopamine re-uptake by decreasing the protein level of N-SMase2.     

Neutral sphingomyelinase: past, present and future

Sphingomyelin and its metabolic products are now known to have second messenger functions in a variety of cellular signaling pathways. At the epicenter of the sphingomyelin--cell signaling pathway is a family of phospholipases called sphingomyelinases. These enzymes cleave sphingomyelin to produce ceramide and phosphocholine. Ceramide in turn serves as a lipid second messenger that induces a variety of cell regulatory phenomenon such as programmed cell death (apoptosis), cell differentiation, cell proliferation, and sterol homeostasis. Neutral sphingomyelinase (N-SMase) is a Mg2+ sensitive enzyme that can be activated by a host of physiologically relevant and structurally diverse molecules like tumor necrosis factor-alpha (TNF-alpha), oxidized human low density lipoproteins (Ox-LDL), and several growth factors. Large amounts of ceramide accumulate in human fatty streaks and plaques along with Ox-LDL, growth factors, and proinflammatory cytokines in human atherosclerosis. A further role of ceramide and N-SMase in atherosclerosis was uncovered by the finding that Ox-LDL and TNF-alpha stimulated N-SMase activity. In turn, ceramide and/or a homolog serves as an important stress signaling molecule in signal transduction, which leads to apoptosis. Interestingly, an antibody against N-SMase can abrogate Ox-LDL and TNF-alpha induced apoptosis, and therefore may be useful for additional studies of apoptosis in experimental animals. Overexpression of recombinant human N-SMase in human aortic smooth muscle cells markedly stimulate apoptosis, presumably via the multioligomerization of the 'death domain'. Since plaque stability is an integral aspect of atherosclerosis management, activation of N-SMase and subsequent apoptosis may be vital events in the onset of plaque rupture, stroke and heart failure. In contrast to these observations in human hepatocytes, TNF-alpha mediated N-SMase activation did not induce apoptosis. Rather it stimulated the maturation of sterol regulatory element (SRE) binding protein (SREBP-1). Moreover, a cell permeable ceramide was found to reconstitute the phenomenon above in a sterol-independent fashion. These findings provide alternate avenues for therapy of patients with hypercholesterolemia and atherosclerosis. The findings reported here suggests that N-SMase plays important cell regulatory roles and provide an exciting opportunity to further these findings to understand the pathophysiology of human disease states.     

Dopamine transporter trafficking is regulated by neutral sphingomyelinase 2/ceramide kinase

Dopamine (DA) reuptake is the primary mechanism to terminate dopaminergic transmission in the synaptic cleft. The dopamine transporter (DAT) has an important role in the regulation of DA reuptake. This study provides anatomical and physiological evidence that DAT recycling is regulated by ceramide kinase via the sphingomyelin pathway. First, the results show that DAT and neutral sphingomyelinase 2 (nSMase2) were successfully co-precipitated from striatal samples and were colocalized in the mouse striatum or PC12 cells. We also identified a protein-protein interaction between nSMase2 and DAT through in situ proximity ligation assay experiments in the mouse striatum. Second, dopamine (DA) stimulated the formation of ceramide and increased nSMase activity in PC12 cells, while treatment with a cell-permeable ceramide-1-phosphate (C1P) increased DA uptake. Third, we used inhibitors and siRNA to inhibit nSMase2 and ceramide kinase and observed the effects on DAT recycling in PC12 cells. Treatment with ceramide kinase inhibitor K1, or nSMase inhibitor GW4869, decreased DA uptake in PC12 cells, although the application of FB₁, a ceramide synthase inhibitor, did not affect DA uptake. Transfection of nSMase2 and CERK siRNA decreased DAT surface level in PC12 cells. These results suggested that SM-derived C1P affects cell surface levels of DAT.     

Molecular modeling of human neutral sphingomyelinase provides insight into its molecular interactions

The neutral sphingomyelinase (N-SMase) is considered a major candidate for mediating the stress-induced production of ceramide, and it plays an important role in cell-cycle arrest, apoptosis, inflammation, and eukaryotic stress responses. Recent studies have identified a small region at the very N-terminus of the 55 kDa tumour necrosis factor receptor (TNF-R55), designated the neutral sphingomyelinase activating domain (NSD) that is responsible for the TNF-induced activation of N-SMase. There is no direct association between TNF-R55 NSD and N-SMase; instead, a protein named factor associated with N-SMase activation (FAN) has been reported to couple the TNF-R55 NSD to N-SMase. Since the three-dimensional fold of N-SMase is still unknown, we have modeled the structure using the protein fold recognition and threading method. Moreover, we propose models for the TNF-R55 NSD as well as the FAN protein in order to study the structural basis of N-SMase activation and regulation. Protein-protein interaction studies suggest that FAN is crucially involved in mediating TNF-induced activation of the N-SMase pathway, which in turn regulates mitogenic and proinflammatory responses. Inhibition of N-SMase may lead to reduction of ceramide levels and hence may provide a novel therapeutic strategy for inflammation and autoimmune diseases. Molecular dynamics (MD) simulations were performed to check the stability of the predicted model and protein-protein complex; indeed, stable RMS deviations were obtained throughout the simulation. Furthermore, in silico docking of low molecular mass ligands into the active site of N-SMase suggests that His135, Glu48, Asp177, and Asn179 residues play crucial roles in this interaction. Based on our results, these ligands are proposed to be potent and selective N-SMase inhibitors, which may ultimately prove useful as lead compounds for drug development.     

The neutral sphingomyelinase family: identifying biochemical connections

Neutral sphingomyelinases (N-SMases) are considered to be key mediators of stress-induced ceramide production. The extended family of N-SMases is a subset of the DNaseI superfamily and comprises members from bacteria, yeast and mammals. In recent years, the identification and cloning of mammalian N-SMase family members has led to significant advances in understanding their physiological roles and regulation. However, there is still limited information on their regulation at the biochemical and molecular level. In this review, we summarize current knowledge about the biochemical regulation of the eukaryotic N-SMases and identify the major areas where knowledge is lacking. In recent years, research into the roles and regulation of N-SMases has moved in great strides with the cloning and characterization of multiple N-SMase isoforms and the development of knockout mice. However, as researchers continue to move forward in understanding the physiological functions of these various N-SMase isoforms, it has become exceedingly important to define howthese isoforms are regulated at the biochemical and molecular level. This is crucial for the development of future tools to study N-SMase signaling such as, for example, phospho-specific antibodies designating activation states. This is also an important part of identifying novel roles of N-SMases in physiological and pathological states. Finally, only by obtaining a more complete understanding of the workings of these enzymes at the molecular level, will investigators be able to design appropriate compounds that can target and inhibit their activity both efficiently and specifically. Certainly, the last of these is crucial when considering the potential of N-SMases as therapeutic targets. With this in mind, we sincerely hope that the next decade of research will even surpass the last ten years in advancing our understanding of the eukaryotic N-SMase family.     

Neutral sphingomyelinase inhibitor scyphostatin prevents and ceramide mimics mechanotransduction in vascular endothelium

Recently, we showed that neutral sphingomyelinase (N-SMase) is concentrated at the endothelial cell surface in caveolae and is activated to produce ceramide in an acute and transient manner by increase in flow rate and pressure in rat lung vasculature (Czarny M, Liu J, Oh P, and Schnitzer JE, J Biol Chem 278: 4424-4430, 2003). Here, we report further on our investigations of this new acute mechanotransduction pathway. We employed three experimental models to explore the role of N-SMase and ceramides in mechanosignaling: 1) a cell-free, in vitro model using isolated luminal plasma membranes of rat lung endothelium; 2) a fluid shear stress model using monolayers of intact bovine aorta endothelial cell in culture; and 3) an in situ model using controlled perfusion of the rat lung vasculature. Scyphostatin, which specifically inhibited N-SMase but not acid SMase activity, prevented mechanoactivation of N-SMase as well as downstream tyrosine and mitogen-activated protein kinases. Cell-permeable ceramide analogs (N-acetylsphingosine, C2-ceramide, and N-hexanoylsphingosine, C6-ceramide) but not the inactive dihydroderivatives D2-ceramide and D6-ceramide (N-acetylsphinganine and N-hexanoylsphinganine, respectively) mimic rapid mechano-induced tyrosine phosphorylation of cell surface proteins as well as mechanoactivation of Src-like kinases and the extracellular regulated kinase pathway. The responses common to ceramide and mechanical stress were inhibited by genistein, herbamycin A, and PP2, but not PP3, which suggests an obligate role of Src-like kinases in ceramide-mediated mechanotransduction. Ceramides also induced serine/threonine phosphorylation to activate the Akt/endothelial nitric oxide synthase pathway. Thus N-SMase at the plasma membrane in caveolae may be an upstream initiating mechanosensor, which acutely triggers mechanotransduction by generation of the lipid second messenger ceramide.     

The extended family of neutral sphingomyelinases

The neutral sphingomyelinases (N-SMases) are considered major candidates for mediating the stress-induced production of ceramide, and N-SMase activity has been identified, characterized, and cloned from bacteria, yeast, and mammalian cells. Although the level of identity between these enzymes is low, a number of key residues thought to be involved in metal binding and catalysis are conserved. This has led to the suggestion of a common catalytic mechanism, and thus, these enzymes are considered to form an extended family of N-SMases. Despite considerable research into N-SMase activity in cell culture and various tissues, the lack, until recently, of molecular identification of specific N-SMase enzymes had precluded specific insights into the regulation, physiological, and pathological roles of these proteins. In this review, we summarize, for the first time, current knowledge of the N-SMase family, focusing on cloned members from bacteria, yeast, and mammalian cells. We also briefly consider the major future directions for N-SMase research which promises highly significant and specific insight into sphingolipid-mediated functions.     

Neutral sphingomyelinase 2 is palmitoylated on multiple cysteine residues. Role of palmitoylation in subcellular localization

The neutral sphingomyelinases (nSMases) are considered major candidates for mediating the stress-induced production of ceramide. nSMase2, which has two hydrophobic segments near the NH(2)-terminal region, has been reported to be located at the plasma membrane and play important roles in ceramide-mediated signaling. In this study, we found that nSMase2 is palmitoylated on multiple cysteine residues via thioester bonds. Site-directed mutagenesis of cysteine residues to alanine indicated that two cysteine clusters of the enzyme are multiply palmitoylated; one cluster is located between the two hydrophobic segments, and the second one is located in the middle of the catalytic region of the protein. When overexpressed in the confluent phase of MCF-7 cells, wild-type nSMase2 was strictly localized in the plasma membranes, and the cysteine mutants of each palmitoylated cysteine cluster were seen not only at the plasma membrane but also in some punctate structures. Furthermore, mutation of all potential palmitoylation sites resulted in a dramatic reduction in the plasma membrane distribution and an increase in the punctate structures. The palmitoylation-deficient mutant was directed to lysosomes and rapidly degraded. Palmitoylation had no effect on enzyme activity but affected membrane-association properties of the protein. Finally, the catalytic region of nSMase2 where palmitoylation occurs was found to be localized at the inner leaflet of the plasma membrane. In summary, the results from this study reveal for the first time the palmitoylation of nSMase2 via thioester bonds and its importance in the subcellular localization and stability of this protein.     

Purification and characterization of a magnesium-dependent neutral sphingomyelinase from bovine brain

The magnesium-dependent, plasma membrane-associated neutral sphingomyelinase (N-SMase) catalyzes hydrolysis of membrane sphingomyelin to form ceramide, a lipid signaling molecule implied in intracellular signaling. We report here the biochemical purification to apparent homogeneity of N-SMase from bovine brain. Proteins from Nonidet P-40 extracts of brain membranes were subjected to four purification steps yielding a N-SMase preparation that exhibited a specific enzymatic activity 23,330-fold increased over the brain homogenate. When analyzed by two-dimensional gel electrophoresis, the purified enzyme presented as two major protein species of 46 and 97 kDa, respectively. Matrix-assisted laser desorption/ionization-mass spectrometry analysis of tryptic peptides revealed at least partial identity of these two proteins. Amino acid sequencing of tryptic peptides showed no apparent homologies of bovine N-SMase to any known protein. Peptide-specific antibodies recognized a single 97-kDa protein in Western blot analysis of cell lysates. The purified enzyme displayed a K(m) of 40 microM for sphingomyelin with an optimal activity at pH 7-8. Bovine brain N-SMase was strictly dependent on Mg(2+), whereas Zn(2+) and Ca(2+) proved inhibitory. The highly purified bovine N-SMase was effectively blocked by glutathione and scyphostatin. Scyphostatin proved to be a potent inhibitor of N-SMase with 95% inhibition observed at 20 microM scyphostatin. The results of this study define a N-SMase that fulfills the biochemical and functional criteria characteristic of the tumor necrosis factor-responsive membrane-bound N-SMase.     

Crocin prevents the death of PC-12 cells through sphingomyelinase-ceramide signaling by increasing glutathione synthesis

Crocin is a pharmacologically active component of Crocus sativus L. (saffron) that has been used in traditional Chinese medicine. In a previous study, we demonstrated that crocin inhibits apoptosis in PC-12 cells by affecting the function of tumor necrosis factor-alpha. In this study, we found that depriving cultured PC-12 cells of serum/glucose causes a rapid increase in cellular ceramide levels, followed by an increase in the phosphorylation of c-jun kinase (JNK). The accumulation of ceramide was found to depend on the activation of magnesium-dependent neutral sphingomyelinase (N-SMase), but not on de novo synthesis. The serum/glucose-deprived PC-12 cells also decreased the cellular levels of glutathione (GSH), which is the potent inhibitor of N-SMase. Treating the PC-12 cells with crocin prevented N-SMase activation, ceramide production, and JNK phosphorylation. We also found that the chemical can enhance the activities of GSH reductase and gamma-glutamylcysteinyl synthase (gamma-GCS), contributing to a stable GSH supply that blocks the activation of N-SMase. Thus our data suggest that crocin combats the serum/glucose deprivation-induced ceramide formation in PC-12 cells by increasing GSH levels and prevents the activation of JNK pathway, which is reported to have a role of the signaling cascade downstream ceramide for neuronal cell death.     

Inhibition of neutral sphingomyelinase decreases elevated levels of nitrative and oxidative stress markers in liver ischemia–reperfusion injury

Oxidative stress and excessive nitric oxide production via induction of inducible nitric oxide synthase (NOS)-2 have been shown in the pathogenesis of liver ischemia–reperfusion (IR) injury. Neutral sphingomyelinase (N-SMase)/ceramide pathway can regulate NOS2 expression therefore this study determined the role of selective N-SMase inhibition on nitrative and oxidative stress markers following liver IR injury. Selective N-SMase inhibitor was administered via intraperitoneal injections. Liver IR injury was created by clamping blood vessels supplying the median and left lateral hepatic lobes for 60 min, followed by 60 min reperfusion. Nitrative and oxidative stress markers were determined by evaluating NOS2 expression, protein nitration, nitrite/nitrate levels, 4-hydroxynonenal (HNE) formation, protein carbonyl levels and xanthine oxidase/xanthine dehydrogenase (XO/XDH) activity. Levels of sphingmyelin and ceramide in liver tissue were determined by an optimized multiple reaction monitoring method using ultra-fast liquid chromatography coupled with tandem mass spectrometry (MS/MS). Spingomyelin levels were significantly increased in all IR groups compared to controls. Treatment with a specific N-SMase inhibitor significantly decreased all measured ceramides in IR injury. NOS2 expression, nitrite/nitrate levels and protein nitration were significantly greater in IR injury and decreased with N-SMase inhibition. Treatment with a selective N-SMase inhibitor significantly decreased HNE formation, protein carbonyl levels and the hepatic conversion of XO. Data confirm the role of nitrative and oxidative injury in IR and highlight the protective effect of selective N-SMase inhibition. Future studies evaluating agents blocking N-SMase activity can facilitate the development of treatment strategies to alleviate oxidative injury in liver I/R injury.     

Neutral sphingomyelinase inhibition alleviates apoptosis,but not ER stress,in liver ischemia–reperfusion injury

Previous studies have revealed the activation of neutral sphingomyelinase (N-SMase)/ceramide pathway in hepatic tissue following warm liver ischemia reperfusion (IR) injury. Excessive ceramide accumulation is known to potentiate apoptotic stimuli and a link between apoptosis and endoplasmic reticulum (ER) stress has been established in hepatic IR injury. Thus, this study determined the role of selective N-SMase inhibition on ER stress and apoptotic markers in a rat model of liver IR injury. Selective N-SMase inhibitor was administered via intraperitoneal injections. Liver IR injury was created by clamping blood vessels supplying the median and left lateral hepatic lobes for 60?min, followed by 60?min reperfusion. Levels of sphingmyelin and ceramide in liver tissue were determined by an optimized multiple reactions monitoring (MRM) method using ultrafast-liquid chromatography (UFLC) coupled with tandem mass spectrometry (MS/MS). Spingomyelin levels were significantly increased in all IR groups compared with controls. Treatment with a specific N-SMase inhibitor significantly decreased all measured ceramides in IR injury. A significant increase was observed in ER stress markers C/EBP-homologous protein (CHOP) and 78?kDa glucose-regulated protein (GRP78) in IR injury, which was not significantly altered by N-SMase inhibition. Inhibition of N-SMase caused a significant reduction in phospho-NF-kB levels, hepatic TUNEL staining, cytosolic cytochrome c, and caspase-3, -8, and -9 activities which were significantly increased in IR injury. Data herein confirm the role of ceramide in increased apoptotic cell death and highlight the protective effect of N-SMase inhibition in down-regulation of apoptotic stimuli responses occurring in hepatic IR injury.     

nSMase2 activation and trafficking are modulated by oxidative stress to induce apoptosis

We have previously shown that accumulation of ceramide, triggered by hydrogen peroxide (H(2)O(2)), induces apoptosis of human airway epithelial (HAE) cells. Under oxidant exposure, a lung sphingomyelinase (SMase) is activated and displays continued ceramide generation and pro-apoptotic signaling, thus leading to the pathological apoptosis that causes lung injury. In a search for a specific SMase that is modulated by oxidative stress, we recently cloned nSMase2 from monkey lung tissue and HAE cells. Here, we show that this nSMase2 is up-regulated by an oxidant (H(2)O(2)) and is inhibited by an antioxidant (glutathione (GSH)). Moreover, nSMase2 subcellular localization is governed by oxidant exposure, which leads to its preferential trafficking to the plasma membrane, where it generates ceramide and induces apoptosis. On the other hand, exposure to GSH results in nSMase2 trafficking to the nucleus, where it neither generates ceramide nor induces apoptosis.     

Bcl-xL interrupts oxidative activation of neutral sphingomyelinase

Recent studies demonstrate a role for intracellular oxidation in the regulation of neutral sphingomyelinase (N-SMase). Glutathione (GSH) has been shown to regulate N-SMase in vitro and in cells. However, it has not been established whether the effects of GSH in cells are due to direct action on N-SMase. In this study, treatment of human mammary carcinoma MCF-7 cells with diamide, a thiol-depleting agent, caused a decrease in intracellular GSH and degradation of sphingomyelin (SM) to ceramide. The SM pool hydrolyzed in response to diamide belonged to the bacterial SMase-resistant pool of SM. Importantly, pretreatment of MCF-7 cells with GSH, N-acetylcysteine, an antioxidant, or GW69A, a specific N-SMase inhibitor, prevented diamide-induced degradation of SM to ceramide, suggesting that intracellular levels of GSH regulate the extent to which SM is degraded to ceramide and that this probably involves a GW69A-sensitive N-SMase. Unexpectedly, expression of Bcl-xL prevented tumor necrosis factor--induced SM hydrolysis and ceramide accumulation but not the decrease in intracellular GSH. Furthermore, Bcl-xL inhibited diamide-induced SM hydrolysis and ceramide accumulation but not the decrease in intracellular GSH. These results suggest that the site of action of Bcl-xL is downstream of GSH depletion and upstream of ceramide accumulation, and that GSH probably does not exert direct physiologic effects on N-SMase.     

Effect of Procysteine on aging-associated changes in hepatic GSH and SMase: evidence for transcriptional regulation of smpd3

In hepatocytes, aging-associated decline in GSH has been linked to activation of neutral SMase (nSMase), accumulation of bioactive ceramide, and inflammation. In this study, we seek to test whether dietary supplementation with the cysteine precursor, L-2-oxothiazolidine-4-carboxylic acid (OTC), would correct the aging-associated differences in hepatic GSH, nSMase, and ceramide. Young and aged mice were placed on a diet that either lacked sulfur-containing amino acids (SAAs) or had 0.5% OTC for 4 weeks. Mice fed standard chow were used as an additional control. SAA-deficient mice exhibited significant aging-associated differences in hepatic GSH, GSH/GSSG, ceramide, and nSMase. C24:1 ceramide, the major ceramide species in liver, was affected the most by aging, followed by the less abundant C16:0 ceramide. OTC supplementation eliminated the aging-associated differences in hepatic GSH and GSH/GSSG ratio. Surprisingly, however, instead of decreasing, the nSMase activity and ceramide increased in the OTC-fed mice irrespective of their age. These effects were due to elevated nSMase-2 mRNA and protein and appeared to be direct. Similar increases were seen in HepG2 cells following treatment with OTC. The OTC-fed aged mice also exhibited hepatic steatosis and triacylglyceride accumulation. These results suggest that OTC is a potent stimulant of nSMase-2 expression and that there may be unanticipated complications of OTC supplementation.