SARS-CoV-2 infection induces the activation of tissue factor-mediated coagulation via activation of acid sphingomyelinase.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with the hypercoagulable state. Tissue factor (TF) is the primary cellular initiator of coagulation. Most of the TF expressed on cell surfaces remains cryptic. Sphingomyelin (SM) is responsible for maintaining TF in the encrypted state, and hydrolysis of SM by acid sphingomyelinase (ASMase) increases TF activity. ASMase was shown to play a role in virus infection biology. In the present study, we investigated the role of ASMase in SARS-CoV-2 infection-induced TF procoagulant activity. Infection of human monocyte-derived macrophages (MDMs) with SARS-CoV-2 spike protein pseudovirus (SARS-CoV-2-SP-PV) markedly increased TF procoagulant activity at the cell surface and released TF+ extracellular vesicles. The pseudovirus infection did not increase either TF protein expression or phosphatidylserine externalization. SARS-CoV-2-SP-PV infection induced the translocation of ASMase to the outer leaflet of the plasma membrane, which led to the hydrolysis of SM in the membrane. Pharmacologic inhibitors or genetic silencing of ASMase attenuated SARS-CoV-2-SP-PV-induced increased TF activity. Inhibition of the SARS-CoV-2 receptor, angiotensin-converting enzyme-2, attenuated SARS-CoV-2-SP-PV-induced increased TF activity. Overall, our data suggest that SARS-CoV-2 infection activates the coagulation by decrypting TF through activation of ASMase. Our data suggest that the US Food and Drug Administration-approved functional inhibitors of ASMase may help treat hypercoagulability in patients with COVID-19.

Secreted sphingomyelins modulate low mammary cancer incidence observed in certain mammals.

Determining mechanisms that naturally protect species from developing cancer is critical in order to prevent and treat cancer. Here, we describe a novel cancer-suppressing mechanism, via the secretion of bioactive factors by mammary cells, that is present in domesticated mammals with a low mammary cancer incidence. Specifically, these bioactive factors induced triple-negative breast cancer cell (TNBC) death in vitro and reduced tumorigenicity in a xenograft TNBC mouse model in vivo. RNA deep sequencing showed significant downregulation of genes associated with breast cancer progression in secretome-cultured TNBC cells. Further in-depth multi-omics analysis identified sphingomyelins as key secreted factors, and their role was confirmed via inhibition of the sphingomyelin signaling pathway. We speculate that secreted sphingomyelins in the mammary gland of mammals with a naturally low incidence of mammary cancer mediate the elimination of cancer cells. This study contributes to the growing list of protective mechanisms identified in cancer-proof species.

Role and Function of Sphingomyelin Biosynthesis in the Development of Cancer.

Sphingomyelin (SM) biosynthesis represents a complex, finely regulated process, mostly occurring in vertebrates. It is intimately linked to lipid transport and it is ultimately carried out by two enzymes, SM synthase 1 and 2, selectively localized in the Golgi and plasma membrane. In the course of the SM biosynthetic reaction, various lipids are metabolized. Because these lipids have both structural and signaling functions, the SM biosynthetic process has the potential to affect diverse important cellular processes (such as cell proliferation, cell survival, and migration). Thus defects in SM biosynthesis might directly or indirectly impact the normal physiology of the cell and eventually of the organism. In this chapter, we will focus on evidence supporting a role for SM biosynthesis in specific cellular functions and how its dysregulation can affect neoplastic transformation.

Effects of cannabinoids and related fatty acids upon the viability of P19 embryonal carcinoma cells.

Compounds acting on the cannabinoid (CB) receptors are involved in the control of cell fate, and there is an emerging consensus that CBs have anticancer effects. However, the CB-mediated effects are contradictory since some studies suggest stimulatory effects on cancer cell proliferation, and CBs have been shown to stimulate both proliferation and differentiation of other mitotic cells such as stem and progenitor cells. In this study, the concentration-dependent effects of synthetic and endogenous CBs on the viability of mouse P19 embryonal carcinoma (EC) cells have been examined by using fluorescence assays of cell membrane integrity, cell proliferation, oxidative stress, and detection of apoptosis and necrosis. All compounds examined produced a concentration-dependent decrease in cell viability in the micromolar range, with the potent CB receptor agonist HU 210 and the enantiomer HU 211 (with no CB receptor activity) being the most potent compounds examined with apparent IC50 values of 1 and 0.6 µM, respectively. The endogenous CB anandamide showed similar potency and efficacy as structurally related polyunsaturated fatty acids with no reported activity at the CB receptors. The rapid (within hours) decrease in cell viability induced by the examined CBs suggests cytocidal rather than antiproliferative effects and is dependent on the plating cell population density with the highest toxicity around 100 cells/mm(2). The CB-induced cytotoxicity, which appears to involve CB receptors and the sphingomyelin-ceramide pathway, is a mixture of both apoptosis and necrosis that can be blocked by the antioxidants α-tocopherol and N-acetylcysteine. In conclusion, both synthetic and endogenous CBs produce seemingly unspecific cytotoxic effects in the P19 EC cells.

Mechanisms of Apoptin-induced cell death.

Apoptin, a 13.6-kD protein encoded by chicken anemia virus, is paid more and more attention, since it selectively induces apoptosis in tumor cells while abolishes cytotoxic effect in normal cells. In addition, Apoptin shows different localization in tumor cells and normal cells: it predominantly accumulates in nucleus of tumor cells, whereas in normal cells, it is detected mainly in cytoplasm. There are various mechanisms implicated in the program of Apoptin-mediated cell death. Up to now, the interpretations have been recognized including that the particular domains control nucleocytoplasmic shuttling of Apoptin, phosphorylation on specific residue and varies relevant signaling contribute to Apoptin's activity, and the partners interacted with Apoptin regulate activity or subcellular localization of Apoptin. In this review, we make a comprehensive survey of the existing evidence about mechanisms of Apoptin's action, which might provide scientific basis to make progress in novel targeted tumor therapy.

Regulation of death and growth signals at the plasma membrane by sphingomyelin synthesis: implications for hematological malignancies.

Resistance to death receptor ligands (such as FasL and TRAIL) and anticancer treatments is a hallmark of cancer cells. Ceramide, a biologically active sphingolipid, antagonizes cell growth and promotes apoptosis and non-apoptotic forms of cell death. The intracellular levels of ceramide are highly regulated via complex metabolic pathways. Sphingomyelin synthases (SMS) 1 and 2 convert ceramide to sphingomyelin (SM), a ubiquitous phospholipid in mammals. A growing body of evidence in the literature indicates that SMSs likely modulate hematological cell growth and sensitivity to stress-induced apoptosis. On one hand, complete and sustained inhibition of SMS activity is likely to alter membrane composition and properties through membrane SM depletion, perturbing intracellular signaling pathways and leukemia cell growth and conferring partial resistance to death receptor ligands. On the other hand, different patents & reports point to anti-apoptotic functions for SMSs. In patients with chemoresistant leukemia, a decreased intracellular ceramide level was associated with a higher SMS activity. Thus, SMSs and cofactors may constitute original pharmacological targets to treat leukemia.

Differential regulation of sphingomyelin synthesis and catabolism in oligodendrocytes and neurons.

Neurons (both primary cultures of 3-day rat hippocampal neurons and embryonic chick neurons) rapidly converted exogenous NBD-sphingomyelin (SM) to NBD-Cer but only slowly converted NBD-Cer to NBD-SM. This was confirmed by demonstrating low in vitro sphingomyelin synthase (SMS) and high sphingomyelinase (SMase) activity in neurons. Similar results were observed in a human neuroblastoma cell line (LA-N-5). In contrast, primary cultures of 3-day-old rat oligodendrocytes only slowly converted NBD-SM to NBD-Cer but rapidly converted NBD-Cer to NBD-SM. This difference was confirmed by high in vitro SMS and low SMase activity in neonatal rat oligodendrocytes. Similar results were observed in a human oligodendroglioma cell line. Mass-Spectrometric analyses confirmed that neurons had a low SM/Cer ratio of (1.5 : 1) whereas oligodendroglia had a high SM/Cer ratio (9 : 1). Differences were also confirmed by [(3)H]palmitate-labeling of ceramide, which was higher in neurons compared with oligodendrocytes. Stable transfection of human oligodendroglioma cells with neutral SMase, which enhanced the conversion of NBD-SM to NBD-Cer and increased cell death, whereas transfection with SMS1 or SMS2 enhanced conversion of NBD-Cer to NBD-SM and was somewhat protective against cell death. Thus, SMS rather than SMases may be more important for sphingolipid homeostasis in oligodendrocytes, whereas the reverse may be true for neurons.

The role of the phospholipid sphingomyelin in heart disease.

Sphingomyelin (SM) is an integral component of mammalian cell membranes and nerves. However, the inability to catabolize SM may lead to its accumulation in various tissues and organs, resulting in pathological disorders such as Niemann Pick disease. Elevated levels of SM have also been identified as an independent risk factor for coronary heart disease. During the past two decades, data have emerged that support an important role for metabolites of SM, such as ceramide and sphingosine-1-phosphate, in the regulation of phenotypic changes such as cell proliferation, cell-cycle arrest, apoptosis and angiogenesis. Further studies of the molecular and pathobiological basis of these phospholipids may facilitate advances in the discovery of drugs with which to mitigate diseases that may result from an elevation in SM and its metabolites.

The role of intranuclear lipids.

The presence of phospholipids as a component of chromatin is now well documented and many enzymes such as sphingomyelinase, sphingomyelin-synthase, reverse sphingomyelin-synthase and phosphatidylcholine-dependent phospholipase C have been described and characterised. Other lipids were demonstrated inside the nucleus especially plasmalogens and cholesterol. The chromatin phospholipids, comprising 10% of that present in the nucleus, show a different metabolism with respect to those present in either microsomes or in nuclear membranes; they increase also during the DNA duplication as shown during both liver regeneration and cell maturation. They appear localised near newly synthesized RNA in decondensed chromatin. Digestion of chromatin with RNase, but not with DNase, causes a loss of phospholipids. The composition of the chromatin phospholipid fraction shows an enrichment in sphingomyelin and phosphatidylserine. In this review the behaviour of single lipids in relation to cell proliferation, cell differentiation and apoptosis is described. Sphingomyelin, the lipid most represented in chromatin with respect to microsomes and nuclear membranes, is localised near to newly synthesized RNA, its presence appearing to protect RNA from RNase digestion. This effect is reversed by sphingomyelinase which digests sphingomyelin and, as a consequence, RNA may be hydrolysed. The amount of sphingomyelin is restored by sphingomyelin-synthase. Sphingomyelin increases during the differentiation process and apoptosis. An increase of sphingomyelinase with consequent decrease in sphingomyelin is observed at the beginning of S-phase of the cell cycle. A possible role in stabilising the DNA double helix is indicated. Phosphatidylserine behaves similarly during differentiation and appears to stimulate both RNA and DNA polymerases. Phosphatidylcholine is implicated in cell proliferation through the activation of intranuclear phosphatidylcholine-dependent phospholipase C and diacylglycerol production. The increase in diacylglycerol stimulates phosphatidylcholine synthesis through the major pathway from cytidyltriphosphate. An inhibition of phosphatidylcholine synthesis is responsible for the initiation of apoptosis. The presence of reverse sphingomyelin-synthase favours the formation of phosphatidylcholine, the donor of phosphorylcholine, from sphingomyelin. Little information has been reported for phospatidylethanolamine, but phosphtidylinositol appears to influence cell differentiation and proliferation. This last effect is due to the action of two enzymes: PI-PLCss1 having a role in the onset of DNA synthesis and PC-PLCgamma1 acting in G2 transit. Phosphoinositides also may have an important role: in membrane-stripped nuclei isolated from mitogen stimulated cells a decrease in PIP and PIP2 followed by an increase in diacylglycerol and a translocation of protein kinase C inside the nucleus is observed. On the other hand, overexpression of the enzyme inositol polysphosphate-1-phosphatase reduced DNA synthesis by 50%. Nevertheless, an enhanced rate of phosphorylation has been demonstrated in cells induced to differentiate. These molecules probably favour RNA transcription, counteracting the inhibition of H1 on RNA polymerase II. Plasmalogens were demonstrated in the nucleus and their increase favours the increased activity of phosphatidylcholine-dependent phospholipase C when DNA synthesis starts. Moreover, two forms of cholesterol has been described in chromatin: one, a less soluble sphingomyelin-linked form and a free fraction. Cholesterol increases during liver regeneration, first as a linked fraction and then, when DNA synthesis starts, as a free fraction. The changes of these components have been summarised in relation to cell function in order to give an overview of their possible roles in the different phases of cell duplication and their influence on cell differentiation and during apoptosis. Finally, the relevance of these molecules as intranuclear signals is discussed and future directions are indicated in clarifying pathological process such as tumour cell transformation and the possibility in finding new therapeutic tools.

Sphingomyelin and cholesterol: from membrane biophysics and rafts to potential medical applications.

The preferential sphingomyelin-cholesterol interaction which results from the structure and the molecular properties of these two lipids seems to be the physicochemical basis for the formation and maintenance of cholesterol/sphingolipid-enriched nano- and micro-domains (referred to as membrane "rafts") in the plane of plasma and other organelle (i.e., Golgi) membranes. This claim is supported by much experimental evidence and also by theoretical considerations. However, although there is a large volume of information about these rafts regarding their lipid and protein composition, their size, and their dynamics, there is still much to be clarified on these issues, as well as on how rafts are formed and maintained. It is well accepted now that the lipid phase of the rafts is the liquid ordered (LO) phase. However, other (non-raft) parts of the membrane may also be in the LO phase. There are indications that the raft LO phase domains are more tightly packed than the non-raft LO phase, possibly due to intermolecular hydrogen bonding involving sphingolipid and cholesterol. This also explains why the former are detergent-resistant membranes (DRM), while the non-raft LO phase domains are detergent-soluble (sensitive) membranes (DSM). Recent findings suggest that protein-protein interactions such as cross-linking can be controlled by protein distribution between raft and non-raft domains, and, as well, these interactions affect raft size distribution. The cholesterol/sphingomyelin-enriched rafts seem to be involved in many biological processes, mediated by various receptors, as exemplified by various lipidated glycosylphosphatidylinositol (GPI)- and acyl chain-anchored proteins that reside in the rafts. The rafts serve as signaling platforms in the cell. Various pathogens (viruses and toxins) utilize the raft domains on the host cell membrane as a port of entry, site of assembly (viruses), and port of exit (viral budding). Existence and maintenance of cholesterol-sphingomyelin rafts are dependent on the level of membrane cholesterol and sphingomyelin. This explains why reduction of cholesterol level--either through reverse cholesterol transport, using cholesterol acceptors such as beta-cyclodextrin, or through cholesterol biosynthesis inhibition using statins--interferes with many processes which involve rafts and can be applied to treating raft-related infections and diseases. Detailed elucidation of raft structure and function will improve understanding of biological membrane composition-structure-function relationships and also may serve as a new avenue for the development of novel treatments for major diseases, including viral infections, neurodegenerative diseases (Alzheimer's), atherosclerosis, and tumors.

Role for matrix metalloproteinase-2 in oxidized low-density lipoprotein-induced activation of the sphingomyelin/ceramide pathway and smooth muscle cell proliferation.

BACKGROUND: Oxidized LDLs (oxLDLs) and matrix metalloproteinases (MMPs) are present in atherosclerotic lesions. OxLDLs activate various signaling pathways potentially involved in atherogenesis. OxLDLs induce smooth muscle cell (SMC) proliferation mediated by the activation of the sphingomyelin/ceramide pathway and tyrosine kinase receptors. MMPs are also able to induce SMC migration and proliferation in addition to extracellular matrix degradation. The present study was designed to investigate whether MMPs play a role in the mitogenic effect of oxLDLs. METHODS AND RESULTS: OxLDLs induce the release of activated MMP-2 in SMC culture medium. MMP-2 was identified by its 65-kDa gelatinase activity on zymography and by using specific blocking antibodies and MMP-2-/- cells. MMP inhibitors (batimastat and Ro28-2653) and the blocking antibodies anti-MMP-2 and anti-membrane type 1-MMP inhibited the oxLDL-induced sphingomyelin/ceramide pathway activation and subsequent activation of ERK1/2 and DNA synthesis but did not inhibit the oxLDL-induced epidermal growth factor receptor and platelet-derived growth factor receptor activation. Exogenously added activated MMP-2 or membrane type 1-MMP-1 triggered the activation of both sphingomyelin/ceramide and ERK1/2 pathways and DNA synthesis. Conversely, suppression of MMP-2 expression in MMP-2-/- cells or in SMCs treated by small-interference RNA also blocked both sphingomyelin/ceramide signaling and DNA synthesis. CONCLUSIONS: Together, these data demonstrate that MMP-2 plays a pivotal role in oxLDL-induced activation of the sphingomyelin/ceramide signaling pathway and subsequent SMC proliferation. These pathways may constitute a potential therapeutic target for modulating the oxLDL-induced proliferation of SMCs in atherosclerosis or restenosis.

Effects of sphingomyelin on apolipoprotein E- and lipoprotein lipase-mediated cell uptake of lipid particles.

It has been reported that human plasma sphingomyelin (SM) levels are positively and independently related to coronary artery disease. The lipoprotein surface is mainly formed by phosphatidylcholine (PC) and SM together with cholesterol and apolipoproteins. However, the influence of SM on the cell uptake of triglyceride-rich lipoproteins and remnants is poorly understood. To clarify the role of SM in lipoprotein uptake, we prepared lipid emulsions containing triolein, PC and SM as model particles of lipoproteins. Apolipoprotein E (ApoE) binding studies revealed that incorporation of SM into the emulsion surface reduced the binding capacity of apoE without changing the affinity. Surface SM reduced apoE-mediated uptake of emulsions by HepG2 cells because of the decreased amount of binding apoE. Apolipoproteins C-II and C-III inhibited the apoE-mediated uptake of SM containing emulsions more effectively. The stimulatory effect of lipoprotein lipase (LPL) on emulsion uptake was decreased by replacing surface PC with SM. These results suggest that SM-induced changes in the binding properties of apolipoproteins and LPL correlate with decreased hepatic uptake of lipid particles.

Oxidative stress mediates tumor necrosis factor-alpha-induced mitochondrial DNA damage and dysfunction in cardiac myocytes.

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) and angiotensin II (Ang II) are implicated in the development and further progression of heart failure, which might be, at least in part, mediated by the production of reactive oxygen species (ROS). However, the cause and consequences of this agonist-mediated ROS production in cardiac myocytes have not been well defined. Recently, we demonstrated that increased ROS production was associated with mitochondrial DNA (mtDNA) damage and dysfunction in failing hearts. We thus investigated whether the direct exposure of cardiac myocytes to TNF-alpha and Ang II in vitro could induce mtDNA damage via production of ROS. METHODS AND RESULTS: TNF-alpha increased ROS production within cultured neonatal rat ventricular myocytes after 1 hour, as assessed by 2',7'-dichlorofluorescin diacetate fluorescence microscopy. TNF-alpha also decreased mtDNA copy number by Southern blot analysis in association with complex III activity, which was prevented in the presence of the antioxidant alpha-tocopherol. A direct exposure of myocytes to H2O2 caused a similar decrease in mtDNA copy number. In contrast, Ang II did not affect mtDNA copy number, despite the similar increase in ROS production. TNF-alpha-mediated ROS production and a decrease in mtDNA copy number were inhibited by the sphingomyelinase inhibitor D609. Furthermore, N-acetylsphingosine (C2-ceramide), a synthetic cell-permeable ceramide analogue, increased myocyte ROS production, suggesting that TNF-alpha-mediated ROS production and subsequent mtDNA damage were mediated by the sphingomyelin-ceramide signaling pathway. CONCLUSIONS: The intimate link between TNF-alpha, ROS, and mtDNA damage might play an important role in myocardial remodeling and failure.

Oxidized LDL-induced smooth muscle cell proliferation involves the EGF receptor/PI-3 kinase/Akt and the sphingolipid signaling pathways.

OBJECTIVE: Oxidized low-density lipoprotein (oxLDL)-induced smooth muscle cell (SMC) proliferation requires the coactivation of various signaling pathways, namely sphingomyelin/ceramide/sphingosine-1-phosphate, epithelial growth factor receptor (EGFR), and phosphoinositide 3-kinase (PI-3K) pathways. This study aimed to clarify the respective role and the hypothetical cross-talk between sphingomyelin/ceramide/sphingosine-1-phosphate, EGFR, and PI-3K/Akt pathways in the balance between mitogenic and cytotoxic effects elicited by oxLDL. METHODS AND RESULTS: Coimmunoprecipitation experiments and the use of inhibitors and dominant-negative mutant showed that oxLDL-induced PI-3K activation is dependent on EGFR. PI-3K activation is independent of the sphingomyelin/ceramide/sphingosine-1-phosphate pathway, because PI-3K inhibition by LY294002 or dominant-negative Deltap85 mutant does not abrogate sphingomyelin hydrolysis, and, conversely, the use of permeant C2-ceramide and of N,N-dimethyl-sphingosine, a sphingosine kinase inhibitor, does not alter PI-3K activity. Activation of Akt/PKB by oxLDL requires PI-3K, as shown by the inhibition by LY204002 and in Deltap85 SMC. The inhibition of Akt/PKB by PI-3K inhibitor LY204002 or by overexpression of kinase-dead Akt shifted the mitogenic effect of oxLDL toward apoptosis, thus suggesting that the PI-3K/Akt pathway acts as a survival pathway. CONCLUSIONS: SMC proliferation elicited by moderate concentrations of oxLDL involves the sphingomyelin/ceramide/sphingosine-1-phosphate pathway, which leads to extracellular regulated kinase 1/2 activation and DNA synthesis, and the EGFR/PI-3K/Akt pathway, which prevents the apoptotic effect of oxLDL.

Extracellular membrane vesicles from tumor cells promote angiogenesis via sphingomyelin.

Actively growing tumor cells shed membrane vesicles into the extracellular milieu both in vivo and in vitro. Extracellular membrane vesicles from tumor cells contain most surface antigens and proteases present on these cells. They facilitate the escape of tumors from immune surveillance and promote tumor cell invasion. Here, we demonstrate that tumor membrane vesicles stimulate an additional important activity for tumor growth and metastasis by promoting endothelial cell migration, invasion, and tube formation, and inducing in vivo neovascularization. Our data show that tumor vesicles are one of the multiple effectors involved in tumor-induced angiogenesis. Heat-treated vesicles and lipid extracts from the vesicles also induce endothelial cell migration and in vivo angiogenesis. We identify sphingomyelin as the active component for vesicle-induced endothelial cell migration, tube formation, and neovascularization. Together with previously reported results, our data demonstrate that shed tumor vesicles play multiple roles in tumor growth and metastasis by promoting angiogenesis, tumor invasion, and immune escape.

Role of tumor necrosis factor alpha and sphingomyelin cycle activation in the induction of apoptosis by ischemia/reperfusion of the liver.

The signal transduction pathways triggering apoptotic mechanisms after ischemia/reperfusion may involve TNF-alpha secretion, ceramide generation, and initiation of lipid peroxidation. In the present study involvement of the TNF-alpha, sphingomyelin cycle, and lipid peroxidation in the initiation of apoptosis induced in liver cells by ischemia and reperfusion was investigated. Wistar rats were subjected to total liver ischemia (for 15, 30 min, and 1 h) followed by subsequent reperfusion. Ischemia caused sharp decrease of neutral sphingomyelinase activity. Activity of acidic sphingomyelinase initially decreased (during 15-30 min ischemia) but then increased (after 1 h of ischemic injury). Reperfusion of the ischemic lobe of the liver caused increase in neutral sphingomyelinase activity and decrease in acidic sphingomyelinase activity. A small amount of TNF-alpha detected by immunoblotting analysis was accumulated in the ischemic area of liver rapidly and the content of this cytokine dramatically increased after the reperfusion. TNF-alpha is known to induce free radical production. We found that the accumulation of TNF and increase of sphingomyelinase activity during the development of ischemic/reperfusion injury coincided with increase in content of lipid peroxidation products (conjugated dienes) and DNA degradation detected by gel electrophoresis. Recently it was shown that superoxide radicals are used as signaling molecules within the sphingomyelin pathway. This suggests the existence of cross-talk between the oxidation system and the sphingomyelin cycle in cells, which may have important implications for the initial phase and subsequent development of post-ischemic injury.

Sphingomyelin and ceramide as regulators of development and lifespan.

Sphingomyelin (SM) is a prominent phospholipid component of cell membranes that has evolved diverse functions in cells beyond its role in membrane structural organization. Cleavage of SM by acid or neutral sphingomyelinase results in the liberation of ceramide, an intracellular messenger that regulates the activities of an array of kinases, phosphatases and transcription factors. Signals that activate sphingomyelinases range from growth factors and cytokines, to neurotransmitters, hormones and reactive oxygen species. Studies of experimental cell culture and animal models, and of patients with inherited defects in sphingomyelin metabolism suggest important roles for SM-ceramide signaling in the regulation of cell proliferation, differentiation and survival. At low concentrations SM and ceramide can stimulate cell proliferation and survival, whereas higher levels can induce cell dysfunction or death. Analyses of development and aging suggest a major role for SM metabolism in regulating development rate and lifespan. Several factors that alter the metabolism of sphingolipids, including oxidative and metabolic stress, also increase risk and progression of age-related diseases. In addition, recent findings have linked alterations in SM metabolism to the pathogenesis of several age-related diseases including cancers and neurodegenerative disorders. The emerging data suggest the possibility that dietary and pharmacological manipulations of SM metabolism might prove effective in extending lifespan and treating various age-related diseases.

Sphingomyelin modulates capacitation of human sperm in vitro.

Ejaculated mammalian sperm must mature (capacitate) before they can undergo acrosomal exocytosis and fertilize an egg. Loss of sperm sterols is an early step in capacitation. Because sphingomyelin slows cholesterol efflux from other cells, the role of sphingomyelin in capacitation was tested. Human sperm were exposed to sphingomyelinase and then incubated for as long as 24 h. The ability of sperm to acrosome-react in response to progesterone was tested to measure capacitation. Sphingomyelinase-treated sperm became responsive to progesterone approximately 10 h earlier than control sperm. Sphingomyelinase also increased spontaneous acrosomal exocytosis. The effects of sphingomyelinase were accompanied by accelerated losses of the inhibitory sterols, cholesterol and desmosterol. To test whether sphingomyelinase-generated ceramide might promote capacitation, sperm were incubated for 8 h with the cell-permeable ceramide N:-hexanoylsphingosine (25 microM) or with solvent. Ceramide increased the incidence of progesterone-responsive sperm and, at later times, spontaneously reacted sperm. N:-Hexanoylsphinganine, an inactive control ceramide, had no effect. These results suggest that sphingomyelin in the sperm influences the rate of capacitation by slowing the loss of sterols, and that exogenous sphingomyelinase accelerates capacitation by speeding the loss of sterols and by generating ceramide.

Ceramide mediates radiation-induced death of endothelium.

The sphingomyelin (SM) pathway is an ubiquitous, evolutionarily conserved signaling system, analogous to conventional systems such as the cAMP and phosphoinositide pathways. Ceramide is generated from SM by the action of a neutral or acid SMase, or by de novo synthesis coordinated through the enzyme ceramide synthase. Once generated, ceramide may serve as a second messenger in signaling responses to physiologic or environmental stimuli, or may be converted to a variety of structural or effector molecules. In the radiation response, ceramide serves as a second messenger in initiating apoptosis, while some of its metabolites block apoptosis. In certain cells, such as endothelial, lymphoid and haematopoietic cells, ceramide mediates apoptosis while in others ceramide may serve only as a co-signal for or play no role in the death response. Regulated ceramide metabolism may determine the balance between pro- and anti-apoptotic signals, and hence, the intensity of the apoptotic response, thus constituting a mechanism of radiation sensitivity or resistance. This paradigm may offer new opportunities for modulation of the radiation effects in the treatment of cancer. Chemical modifiers of ceramide metabolism may be useful to enhance the therapeutic effects or reduce the toxicity of radiation treatment.

P-glycoprotein plays a drug-efflux-independent role in augmenting cell survival in acute myeloblastic leukemia and is associated with modulation of a sphingomyelin-ceramide apoptotic pathway.

P-glycoprotein (pgp), which is the product of the MDR1 (multidrug resistance-1) gene, has an established role as a mediator of cytotoxic drug resistance in acute myeloid leukemia (AML). To study the role of pgp in mediating apoptosis resistance in AML cells deprived of serum and growth factors, apoptosis was quantified by flow cytometry using uptake of the dye 7-amino-actinomycin D (7-AAD) alongside low forward scatter. In pgp+ve primary AML samples, there was a significant increase in apoptosis in the presence of the pgp-specific antibody UIC2 (mean increase: 58%; range: 11%-95%; P <. 05). Likewise, apoptosis in growth factor-deprived TF1 cells cultured for 30 hours increased 2.5-fold in the presence of 25 microg/mL UIC2. The pgp reversal agent PSC-833 (1 micromol/L) augmented in vitro apoptosis by a median of 52% in pgp+ve patient samples and to a comparable degree in 6 pgp-ve samples. To determine whether the sphingomyelin-ceramide (SM-ceramide) pathway of apoptosis occurs in AML blasts in response to cytotoxic drugs, cells were incubated with daunorubicin at the patient-specific IC(30) (the concentration of daunorubicin that caused apoptotic cell death in 30% of cells) in the presence of the ceramide synthase inhibitor fumonisin B1, which inhibited apoptosis by 18%-81% (median: 40%). Exogenous SM failed to augment apoptosis induced by growth factor withdrawal in pgp+ve TF1 cells and was significantly more effective at augmenting apoptosis in pgp-ve patient blasts (median increase in cell death: 33%; range: 19%-88%) than in pgp+ve samples (median: 7%; range: 0%-27%; P =.028). Cellular accumulation of exogenous SM was associated with apoptosis and also occurred in nonapoptotic patient cells treated with PSC-833. However, this effect was not seen following treatment with the UIC2 antibody. These results indicate that pgp is able to exert a protective effect on AML cell viability and that this is associated with a reduced effect of exogenous SM on apoptosis. The pgp reversal agent PSC-833 acts, at least in part, by a pgp independent mechanism to alter SM distribution and to augment apoptosis induced in AML cells by serum and growth factor withdrawal. (Blood. 2000;95:2897-2904)