A neutral sphingomyelinase in spermatozoal plasma membranes

A highly active neutral sphingomyelinase was observed for the first time in ram spermatozoal plasma membranes. The optimal conditions for the enzyme activity are pH 7.4, 40 mM MgCl2, 40 min of incubation, and 267 nmol sphingomyelin. Ca2+ and cholesterol were found to inhibit sphingomyelinase activity.     

Analysis of membrane topology of neutral sphingomyelinase 2

Neutral sphingomyelinase 2 (nSMase2), which has two hydrophobic segments at its NH(2)-terminus, plays an important role in ceramide-mediated cell regulation. Here, we investigated the membrane topology of nSMase2. When a double-tagged nSMase2 at both the NH(2) and COOH termini, was overexpressed in MCF-7 cells, the signals from both tags were detected in the inner leaflet of the plasma membrane. Furthermore, insertion of a tag into the internal sequence and green fluorescent protein-fused deletion mutants revealed that the entire catalytic region of the protein was located on the cytosolic face of the membranes and each hydrophobic segment is integrated into the membranes, but unlikely to span the entire membrane. These results indicate the presence of the enzyme in the inner leaflet of plasma membrane.     

A cell-autonomous requirement for the cell cycle regulatory protein, Rb, in neuronal migration

Precise cell cycle regulation is critical for nervous system development. To assess the role of the cell cycle regulator, retinoblastoma (Rb) protein, in forebrain development, we studied mice with telencephalon-specific Rb deletions. We examined the role of Rb in neuronal specification and migration of diverse neuronal populations. Although layer specification occurred at the appropriate time in Rb mutants, migration of early-born cortical neurons was perturbed. Consistent with defects in radial migration, neuronal cell death in Rb mutants specifically affected Cajal-Retzius neurons. In the ventral telencephalon, although calbindin- and Lhx6-expressing cortical neurons were generated at embryonic day 12.5, their tangential migration into the neocortex was dramatically and specifically reduced in the mutant marginal zone. Cell transplantation assays revealed that defects in tangential migration arose owing to a cell-autonomous loss of Rb in migrating interneurons and not because of a defective cortical environment. These results revealed a cell-autonomous role for Rb in regulating the tangential migration of cortical interneurons. Taken together, we reveal a novel requirement for the cell cycle protein, Rb, in the regulation of neuronal migration.     

Role for mammalian neutral sphingomyelinase 2 in confluence-induced growth arrest of MCF7 cells

Recently, we reported that neutral sphingomyelinase 2 (nSMase2) functions as a bona fide neutral sphingomyelinase and that overexpression of nSMase2 in MCF7 breast cancer cells caused a decrease in cell growth (Marchesini, N., Luberto, C., and Hannun, Y. A. (2003) J. Biol. Chem. 278, 13775-13783). In this study, the role of endogenous nSMase2 in regulating growth arrest was investigated. The results show that endogenous nSMase2 mRNA was up-regulated approximately 5-fold when MCF7 cells became growth-arrested at confluence, and total neutral SMase activity was increased by 119 +/- 41% with respect to control. Cell cycle analysis showed that up-regulation of endogenous nSMase2 correlated with G(0)/G(1) cell cycle arrest and an increase in total ceramide levels (2.4-fold). Analysis of ceramide species showed that confluence caused selective increases in very long chain ceramide C(24:1) (370 +/- 54%) and C(24:0) (266 +/- 81%) during arrest. The role of endogenous nSMase2 in growth regulation and ceramide metabolism was investigated using short interfering RNA (siRNA)-mediated loss-of-function analysis. Down-regulation of nSMase2 with specific siRNA increased the cell population of cells in S phase of the cell cycle by 59 +/- 14% and selectively reverted the effects of growth arrest on the increase in levels of very long chain ceramides. Mechanistically, confluence arrest also induced hypophosphorylation of the retinoblastoma protein (6-fold) and induction of p21(WAF1) (3-fold). Down-regulation of nSMase2 with siRNA largely prevented the dephosphorylation of the retinoblastoma protein and the induction of p21(WAF1), providing a link between the action of nSMase2 and key regulators of cell cycle progression. Moreover, studies on nSMase2 localization in MCF7 cells showed that nSMase2 distributed throughout the cells in subconfluent, proliferating cultures. In contrast, nSMase2 became nearly exclusively located at the plasma membrane in confluent, contact-inhibited cells. Hence, we demonstrate for the first time that nSMase2 functions as a growth suppressor in MCF7 cells, linking confluence to the G(0)/G(1) cell cycle check point.     

A novel mechanism of lysosomal acid sphingomyelinase maturation: requirement for carboxyl-terminal proteolytic processing

Acid sphingomyelinase (aSMase) catalyzes the hydrolysis of sphingomyelin (SM) to form the bioactive lipid ceramide (Cer). Notably, aSMase exists in two forms: a zinc (Zn(2+))-independent lysosomal aSMase (L-SMase) and a Zn(2+)-dependent secreted aSMase (S-SMase) that arise from alternative trafficking of a single protein precursor. Despite extensive investigation into the maturation and trafficking of aSMase, the exact identity of mature L-SMase has remained unclear. Here, we describe a novel mechanism of aSMase maturation involving C-terminal proteolytic processing within, or in close proximity to, endolysosomes. Using two different C-terminal-tagged constructs of aSMase (V5, DsRed), we demonstrate that aSMase is processed from a 75-kDa, Zn(2+)-activated proenzyme to a mature 65 kDa, Zn(2+)-independent L-SMase. L-SMase is recognized by a polyclonal Ab to aSMase, but not by anti-V5 or anti-DsRed antibodies, suggesting that the C-terminal tag is lost during maturation. Furthermore, indirect immunofluorescence staining demonstrated that mature L-SMase colocalized with the lysosomal marker LAMP1, whereas V5-aSMase localized to the Golgi secretory pathway. Moreover, V5-aSMase possessed Zn(2+)-dependent activity suggesting it may represent the common protein precursor of S-SMase and L-SMase. Importantly, the 65-kDa L-SMase, but not V5-aSMase, was sensitive to the lysosomotropic inhibitor desipramine, co-fractionated with lysosomes, and migrated at the same M(r) as partially purified human aSMase. Finally, three aSMase mutants containing C-terminal Niemann-Pick mutations (R600H, R600P, ΔR608) exhibited defective proteolytic maturation. Taken together, these results demonstrate that mature L-SMase arises from C-terminal proteolytic processing of pro-aSMase and suggest that impaired C-terminal proteolysis may lead to severe defects in L-SMase function.     

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.     

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.     

A role for neutral sphingomyelinase activation in the inhibition of LPS action by phospholipid oxidation products

Previous studies from our laboratory and others presented evidence that oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphatidylcholine (OxPAPC) and oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphatidylethanolamine can inhibit lipopolysaccharide (LPS)-mediated induction of interleukin-8 (IL-8) in endothelial cells. Using synthetic derivatives of phosphatidylethanolamine, we now demonstrate that phospholipid oxidation products containing alpha,beta-unsaturated carboxylic acids are the most active inhibitors we examined. 5-Keto-6-octendioic acid ester of 2-phosphatidylcholine (KOdiA-PC) was 500-fold more inhibitory than OxPAPC, being active in the nanomolar range. Our studies in human aortic endothelial cells identify one important mechanism of the inhibitory response as involving the activation of neutral sphingomyelinase. There is evidence that Toll-like receptor-4 and other members of the LPS receptor complex must be colocalized to the caveolar/lipid raft region of the cell, where sphingomyelin is enriched, for effective LPS signaling. Previous work from our laboratory suggested that OxPAPC could disrupt this caveolar fraction. These studies present evidence that OxPAPC activates sphingomyelinase, increasing the levels of 16:0, 22:0, and 24:0 ceramide and that the neutral sphingomyelinase inhibitor GW4869 reduces the inhibitory effect of OxPAPC and KOdiA-PC. We also show that cell-permeant C6 ceramide, like OxPAPC, causes the inhibition of LPS-induced IL-8 synthesis and alters caveolin distribution similar to OxPAPC. Together, these data identify a new pathway by which oxidized phospholipids inhibit LPS action involving the activation of neutral sphingomyelinase, resulting in a change in caveolin distribution. Furthermore, we identify specific oxidized phospholipids responsible for this inhibition.     

Characterization and localization of neutral sphingomyelinase in bovine adrenal medulla

Homogenates of bovine adrenal medullae hydrolyzed exogenous sphingomyelin at 4.3 +/- 1.6 nmol X mg-1 X min-1 and 97% of this sphingomyelinase activity was sedimentable at 110,000 g. The sphingomyelinase had a broad pH optimum centered at pH 7. Enzymatic activity was maximal with 80 microM added Mn2+; Mg2+ supported less than half maximal activity and both Ca2+ and EDTA inhibited activity. No activity was detected in the absence of Triton X-100. Response to detergent was biphasic with dose-dependent stimulation from 0.02% to 0.05% Triton X-100 followed by inhibition with increasing concentrations of detergent. Activity in response to detergent was also modulated by protein concentration. Sphingomyelinase activity was associated with a plasma membrane-microsomal fraction. Phosphatidylcholine was not hydrolyzed under optimal conditions for sphingomyelin hydrolysis and a variety of other conditions. Neutral-active sphingomyelinase activity in adrenal medulla was similar in magnitude to that observed in other non-neural bovine tissues. This study demonstrates the presence of a potent neutral-active sphingomyelinase in a plasma membrane-microsomal fraction of bovine adrenal medulla. This enzyme may be involved in membrane fusion and lysis during catecholamine secretion through its ability to alter membrane composition.     

Transient mechanoactivation of neutral sphingomyelinase in caveolae to generate ceramide

The vascular endothelium acutely autoregulates blood flow in vivo in part through unknown mechanosensing mechanisms. Here, we report the discovery of a new acute mechanotransduction pathway. Hemodynamic stressors from increased vascular flow and pressure in situ rapidly and transiently induce the activity of neutral sphingomyelinase but not that acid sphingomyelinase in a time- and flow rate-dependent manner, followed by the generation of ceramides. This acute mechanoactivation occurs directly at the luminal endothelial cell surface primarily in caveolae enriched in sphingomyelin and neutral sphingomyelinase, but not acid sphingomyelinase. Scyphostatin, which specifically blocks neutral but not acid sphingomyelinase, inhibits mechano-induced neutral sphingomyelinase activity as well as downstream activation of extracellular signal-regulated kinase 1 and 2 (ERK1 and ERK2) by increased flow in situ. We postulate a novel physiological function for neutral sphingomyelinase as a new mechanosensor initiating the ERK cascade and possibly other mechanotransduction pathways.     

The subcellular localization of neutral sphingomyelinase in rat liver.

The subcellular distribution of neutral sphingomyelinase activity has been determined in rat liver. Neutral sphingomyelinase is present in the plasma membrane. This enzyme requires either Mg2+ or Mn2+ for full activity; these cations cannot be replaced by Co2+ or Ca2+. The plasma membrane sphingomyelinase is strongly inhibited by Hg2+. A small amount of neutral spingomyelinase activity appears to be present in microsomes. No neutral sphingomyelinase activity is present in liver mitochondria or bytosol. Lysosomal sphingomyelinase is fully active at pH 4.4--4.8 without added divalent cations. However, between pH 5.0 and 7.5 lysosomal sphingomyelinase activity is stimulated by Mg2+, Mn2+, Co2+, and Ca2+. Below pH 4.8, Mg2+ inhibits the reaction. In contrast to the results obtained with the neutral sphingomyelinase activity of plasma membranes and microsomes, lysosomal sphingomyelinase is unaffected by sulfhydryl inhibitors.     

Protein phosphatase 2A and neutral sphingomyelinase 2 regulate IRAK-1 protein ubiquitination and degradation in response to interleukin-1beta

The IL-1β signaling cascade is initiated by the phosphorylation of IL-1β receptor-associated kinase-1 (IRAK-1), followed by its ubiquitination and degradation. This paper investigates the regulation of IRAK-1 degradation in primary hepatocytes and in HEK cells overexpressing the IL-1β receptor. We provide evidence that protein phosphatase 2A (PP2A) is a negative regulator of the phosphorylation, Lys(48)-linked ubiquitination, and degradation of IRAK-1. PP2A catalytic activity increased within 30 min of stimulation with IL-1β. siRNA against PP2A catalytic subunit (PP2Ac) or treatment with pharmacological inhibitor, okadaic acid, enhanced IRAK-1 Lys(48)-linked ubiquitination and degradation. Direct interaction between PP2Ac and IRAK-1 was observed, suggesting that IRAK-1 might be a PP2A substrate. The mechanisms of PP2A activation by IL-1β involved neutral sphingomyelinase-2 (NSMase-2) and an accumulation of ceramide. Overexpression of NSMase-2 delayed IRAK-1 degradation in a PP2A-dependent manner, whereas NSMase-2 silencing had the opposite effect. The addition of sphingomyelinase, ceramide, or a proteasome inhibitor all led to retention of IRAK-1 at the cell membrane and to increased JNK phosphorylation. This study suggests that NSMase-2- and PP2A-dependent regulation of IRAK-1 degradation is a novel mechanism to fine tune the magnitude of IL-1β response.     

Identification of novel anionic phospholipid binding domains in neutral sphingomyelinase 2 with selective binding preference

Sphingolipids such as ceramide are recognized as vital regulators of many biological processes. Neutral sphingomyelinase 2 (nSMase2) is one of the key enzymes regulating ceramide production. It was previously shown that the enzymatic activity of nSMase2 was dependent on anionic phospholipids (APLs). In this study, the structural requirements for APL-selective binding of nSMase2 were determined and characterized. Using lipid-protein overlay assays, nSMase2 interacted specifically and directly with several APLs, including phosphatidylserine and phosphatidic acid. Lipid-protein binding studies of deletion mutants identified two discrete APL binding domains in the N terminus of nSMase2. Further, mutagenesis experiments pinpointed the core sequences and major cationic amino acids in the domains that are necessary for the cooperative activation of nSMase2 by APLs. The first domain included the first amino-terminal hydrophobic segment and Arg-33, which were essential for nSMase2 to interact with APLs. The second binding domain was comprised of the second hydrophobic segment and Arg-92 and Arg-93. Moreover, mutation of one or both domains decreased APL binding and APL-dependent catalytic activity of nSMase2. Further, mutation of both domains in nSMase2 reduced its plasma membrane localization. Finally, these binding domains are also important for the capability of nSMase2 to rescue the defects of yeast lacking the nSMase homologue, ISC1. In conclusion, these data have identified the APL binding domains of nSMase2 for the first time. The analysis of interactions between nSMase2 and APLs will contribute to our understanding of signaling pathways mediated by sphingolipid metabolites.     

Triploid Atlantic salmon Salmo salar have a higher dietary phosphorus requirement for bone mineralization during early development

The effect of a dietary phosphorus regime in freshwater on vertebra bone mineralization was assessed in diploid and triploid Atlantic salmon, Salmo salar. Fish were fed either a low phosphorus (LP) diet containing 10.5 g kg⁻¹ total phosphorus or a normal phosphorus (NP) diet containing 17.4 g kg⁻¹ total phosphorus from ∼3 to ∼65 g (day 126) in body weight. Two further groups were fed the NP diet from ∼3 g in body weight, but were then switched to the LP diet after 38 (∼10 g in body weight) or 77 (∼30 g in body weight) days. Growth, vertebral ash content (% ash) and radiologically detectable vertebra pathologies were assessed. Triploids were initially smaller than diploids, and again on day 77, but there was no ploidy effect on days 38 or 126. Vertebral ash content increased with increasing body size and those fish fed the NP diet had higher vertebral ash content than those groups fed the LP diet during the intervening time period, but this diet effect became less apparent as fish grew, with all groups having relatively equal vertebral ash content at termination. In general, triploids had lower vertebral ash content than diploids on day 38 and this was most evident in the group fed the LP diet. On day 77, those triploids fed the LP diet during the intervening time period had lower vertebral ash content than diploids. At termination on day 126, the triploids had the same vertebral ash content as diploids, irrespective of diet. There was a ploidy × diet interaction on vertebral deformities, with triploids having higher prevalences of fish with ≥1 deformed vertebra in all dietary groups except continuous NP. In conclusion, between days 0 and 77 (3–30 g body size), triploids required more dietary phosphorus than diploids in order to maintain similar vertebral ash content. A possible link between phosphorus feeding history and phosphorus demand is also discussed.     

Inhibition of neutral Mg2+-dependent sphingomyelinase by ubiquinol-mediated plasma membrane electron transport

Summary.  Sphingomyelin is an abundant constituent of the plasma membranes of mammalian cells. Ceramide, its primary catabolic intermediate, has emerged as an important lipid signaling molecule. Previous work carried out by our group has documented that plasma membrane Mg²⁺-dependent neutral sphingomyelinase can be effectively inhibited by exogenous ubiquinol. In this work, we have tested whether or not plasma-membrane-associated electron transport can also achieve this inhibition through endogenous ubiquinol. Our results have shown that Mg²⁺-dependent neutral sphingomyelinase in isolated plasma membranes was inhibited by NAD(P)H under conditions where ubiquinone is reduced to ubiquinol. This inhibition was potentiated in the presence of an extra amount of NAD(P)H:(quinone acceptor) oxidoreductase 1 (EC 1.6.99.2). Depletion of plasma membranes from lipophilic antioxidants by solvent extraction abolished the inhibition by reduced pyridine nucleotides without affecting the sensitivity of the neutral sphingomyelinase to exogenous ubiquinol. Reconstitution of plasma membranes with ubiquinone restored the ability of NAD(P)H to inhibit the enzyme. Our results support that the reduction of endogenous ubiquinone to ubiquinol by NAD(P)H-driven electron transport may regulate the activity of the plasma membrane neutral sphingomyelinase. Received May 20, 2002; accepted September 20, 2002; published online May 21, 2003 RID="**" ID="**" Present address: Department of Biomedical Engineering, School of Medicine, University of Baltimore, Maryland, U.S.A. RID="*" ID="*" Correspondence and reprints: Departamento de Biología Celular, Fisiología e Inmunología, Facultad de Ciencias, Edificio C-6, Campus Rabanales, Universidad de Córdoba, 14014 Córdoba, Spain.     

A novel magnesium-independent neutral sphingomyelinase associated with rat central nervous system meylin.

Purified myelin fractions prepared from young adult rat brain contain a novel sphingomyelinase which has a pH optimum of 7.0 and does not require divalent cations. This sphingomyelinase is different from the two previously known sphingomyelinases in the brain--the acidic sphingomyelinase and the magnesium-dependent neutral sphingomyelinase. When the distributions of the sphingomyelinases among the purified myelin, the total subcellular fractions heavier than myelin (greater than 0.85 M sucrose), and the microsomes were examined, the magnesium-independent sphingomyelinase was detected only in myelin, while the magnesium-dependent sphingomyelinase was present in the other two fractions but not in myelin. Therefore, this new sphingomyelinase appears to be specifically localized in the myelin sheath.     

Phospholipid dependence of the neutral sphingomyelinase in rat liver plasma membranes

Investigations have been carried out on the influence of the phospholipid composition of rat liver plasma membranes and of their physico-chemical properties on the activity of membrane-bound neutral sphingomyelinase. The membrane phospholipid composition was modified by the incorporation of different phospholipids into the membrane bilayer by means of lipid transfer proteins, n-butanol delipidation or exogenous sphingomyelinase (Staphylococcus aureus) treatment. The results indicate that the activity of neutral sphingomyelinase in liver plasma membranes depends upon phosphatidyl choline presence in the membrane bilayer and not upon membrane fluidity.     

Identification of multiple forms of membrane-associated neutral sphingomyelinase in bovine brain

Many different stimuli such as bioactive agents and environmental stresses are known to cause the activation of sphingomyelinase (SMase), which hydrolyzes sphingomyelin to generate ceramide as a second messenger playing a key role in differentiation and apoptosis in various cell types. Here we identified multiple forms of the membrane-associated neutral SMase (N-mSMase) activity in bovine brain. They could be classified into two groups according to extracting agents: group T-mSMase, extracted with 0.2% Triton X-100, and group S-mSMase, extracted with 0.5 M (NH(4))(2)SO(4). Group T-mSMase: alpha, beta, gamma, and delta, which were extensively purified from 40,000-g pellets of bovine brain homogenates by 3,150-, 5,275-, 1,665-, and 2,556-fold over the membrane extracts, respectively, by sequential use of several column chromatographies. On the other hand, S-mSMase was eluted as two active peaks of S-mSMase epsilon and zeta in a phenyl-5PW hydrophobic HPLC column and further purified by 1,119- and 976-fold over 40,000-g pellets of the homogenates, respectively. These highly purified N-mSMase enzyme preparations migrated as several bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed many different features in biochemical properties such as pH dependence, Mg(2+) requirements, and effects of detergents. Taken together, our data strongly suggest that mammalian brain N-mSMase may exist as multiple forms different in both its chromatographic profiles and biochemical properties.