key: cord-0007929-39windv4
authors: Nikolova-Karakashian, Mariana N.; Rozenova, Krassimira A.
title: Ceramide in Stress Response
date: 2010-12-24
journal: Sphingolipids as Signaling and Regulatory Molecules
DOI: 10.1007/978-1-4419-6741-1_6
sha: 11cbdd4c1c43fe5443920f7c35dc13496cff6bca
doc_id: 7929
cord_uid: 39windv4

Evidence has consistently indicated that activation of sphingomyelinases and/or ceramide synthases and the resulting accumulation of ceramide mediate cellular responses to stressors such as lipopolysaccharide, interleukin 1ß, tumor necrosis factor a, serum deprivation, irradiation and various antitumor treatments. Recent studies had identified the genes encoding most of the enzymes responsible for the generation of ceramide and ongoing research is aimed at characterizing their individual functions in cellular response to stress. This chapter discusses the seminal and more recent discoveries in regards to the pathways responsible for the accumulation of ceramide during stress and the mechanisms by which ceramide affects cell functions. The former group includes the roles of neutral sphingomyelinase 2, serine palmitoyltransferase, ceramide synthases, as well as the secretory and endosomal/lysosomal forms of acid sphingomyelinase. The latter summarizes the mechanisms by which ceramide activate its direct targets, PKC?, PP2A and cathepsin D. The ability of ceramide to affect membrane organization is discussed in the light of its relevance to cell signaling. Emerging evidence to support the previously assumed notion that ceramide acts in a strictly structure-specific manner are also included. These findings are described in the context of several physiological and pathophysiological conditions, namely septic shock, obesity-induced insulin resistance, aging and apoptosis of tumor cells in response to radiation and chemotherapy.

Cells and organisms have developed various strategies to deal with adverse changes in their environment. Cellular insult by infectious agents, toxins, nutrient deprivation, or genotoxic stress produces a coordinated systemic response (generally referred to as inflammation), which is aimed at neutralization of the insult and initiation of tissue repair. The first line of defense at systemic level is stimulation of the innate immune response, which consists of regulated production of inflammatory mediators, including cytokines like IL-1`, TNF_ and IL-6. The typical cellular response to environmental stressors includes the induction of cellular apoptosis (in response to either genotoxic stress or cytokines like TNF_), growth arrest (during nutrient deprivation), increased eicosanoid production, cell migration and adhesion (in the presence of infectious agent). While acute inflammation is protective for the organisms, excessive and long-standing inflammation is harmful and underlies diseases like septic shock, atherosclerosis, asthma, rheumatoid arthritis and inflammatory bowel disease.

Diverse signaling pathways mediate cellular response to stress. The sphingolipid second messengers ceramide, ceramide-1-phosphate, sphingosine and sphingosine-1-phosphate play important role as mediators in many of these pathways. This chapter is focused on the role of ceramide in cellular stress response and the mechanisms of its generation and action.

Ceramides (Fig. 1) form the hydrophobic backbone of all complex sphingolipids 1 and consist of a long chain sphingoid base and amid linked fatty acid which is either saturated or unsaturated and vary in length from two to 28 carbon atoms. In mammalian cells, the most commonly found ceramides have D-erythro-sphingosine and a saturated fatty acyl chain of 16 carbon atoms and are among the most hydrophobic lipids in the membrane. Free ceramide has a very low critical micellar concentration (cmc 10 <10 M) and cannot exist in aqueous solutions. 2 Nevertheless, ceramides are still considered as amphiphiles because the hydroxyl group at the first carbon and the amide bond are hydrophilic moieties. Dihydroceramide differs from ceramide inasmuch as the latter contains a trans 4, 5 double bond, which is essential for some of the bioactive roles of ceramide.

The structural features in the ceramide molecule that are required for its biological properties are not well understood, however the two hydroxyl groups, amid group protons and the trans-double bond seems to be involved. 3 A network of intramolecular hydrogen bonds involving the OH and NH groups establishes a unique conformation arrangement of ceramide molecule.

The basal ceramide concentrations in the cells are low and may change during cellular dif-f f ferentiation or progression through the cell cycle. Various inducers of cellular stress however lead to an accumulation of ceramide that promotes apoptotic, inflammatory and growth inhibitory signals and also mediates the onset of a specific response. The data in Table 1 illustrate what is the magnitude of changes in ceramide levels under various conditions of stress. These reported differences however, are likely to be an underestimation since they are measured in total cell preparations and not in the specific membrane fractions where ceramide was generated. Furthermore, with the exception of mass spectrometry-based assays, the most commonly used methods of ceramide quantification do not separate the individual ceramide species. 4 This might be important as the impact ceramide has on cell functions seemingly depends on the type of fatty acid attached to the sphingoid base. Recent data indeed support the notion that the different biological effects caused by ceramide may be mediated by distinct molecular species of the lipid, underscoring the necessity to evaluate changes in ceramide content in a structure-specific manner.

A comparison of the magnitudes of ceramide accumulation observed under various treatments reveals that the changes are more robust in response to chemotherapeutics or irradiation. As the outcome of these treatments has been the induction of cell death via apoptosis, some investigators 

Ceramide does not move spontaneously between cellular membranes and is transported either during normal membrane biogenesis, or with the aid of CERT, a protein that transfers ceramides synthesized at the endoplasmic reticulum to the Golgi apparatus. This insolubility of ceramide suggests that once generated, ceramide is likely to remain localized at the place of its synthesis until metabolized to other sphingolipids. Consequently, the increases in ceramide concentration during stress response are compartmentalized in distinct locations within the cells and might affect diverse sets of down stream targets and responses.

The two main metabolic pathways for generation of excess ceramide during stress response are (i) the de novo synthesis in the endoplasmic reticulum and (ii) the turnover of sphingomyelin (SM) either at the plasma membrane or in the endosomal/lysosomal compartment (Fig. 2) . A number of agonists have been shown to activate these pathways leading to transient elevation in ceramide. The magnitude and temporal pattern of ceramide accumulation is further influenced by the activity of ceramidases, SM synthases, ceramide kinase and glucosyl/galactosyl ceramide synthases. These enzymes catalyze the conversion of ceramide to other sphingolipids and some agonists seem to coordinately regulate both, the ceramide production and turnover. 20, 96 

The de novo pathway for synthesis of ceramide consists of 4 reactions: the serine palmitoyltransferase (SPT), which condenses palmitoyl-CoA and serine into 3-ketosphinganine, the 3-ketosphinganine reductase that generates sphinganine, the (dihydro)ceramide synthase, which acylates sphinganine to dihydroceramide and the dihydroceramide desaturase, which converts relatively inactive dihydroceramides to ceramides. Stimulation of the de novo pathway during cellular stress response happens through up-regulation of the activity of SPT and/or (dihydro) ceramide synthase. The enzymes of the de novo synthesis of ceramide are in the endoplasmic reticulum. The newly generated ceramide is actively transported to the Golgi apparatus, where it serves as a rate-limiting substrate in the synthesis of complex sphingolipids, like SM and glycosphingolipids. It is noteworthy that some of the known ceramidases may also act as ceramide synthases in a CoA-dependent and independent manner as they exhibit reverse activity. This alternative route for ceramide synthesis is often referred to as the salvage pathway and its role in stress response is not well understood.

The activation of the de novo pathway apparently leads not only to an elevation in ceramide but also to increases in the concentration of SM and glycosphingolipids. Such parallel accumulation is documented in response to LPS, palmitate treatment and heat shock, among others and implies that a wide range of cell functions might be affected through specific and nonspecific mechanisms. Inhibitors of glucosylceramide synthase and labeling with radioactive precursors have been successfully used to elucidate the specific role of ceramide in each case.

De novo synthesis of ceramide is target of a number of fungal inhibitors like fumonisin B1, which inhibits dihydroceramide synthase and the reverse reaction of some ceramidases 97 and myriocin/ISP-1, 98 cycloserine 99 and beta-chloroalanine, 100 all of which inhibit SPT. These widely used inhibitors remain a critical test of establishing whether the de novo pathway is involved in a particular cellular response (reviewed in Merrill 101 and in Perry 96 ).

The activation of the de novo pathway during conditions of stress was initially discovered in yeast, where it regulates growth and the response to heat or osmotic stress (reviewed in Dickson 102 and in Meier et al 103 ) . However, in yeast the signaling mediator is not ceramide but the free long chain bases, phytosphingosine and dihydrosphingosine, most likely reflecting the specifics of sphingolipid synthesis in yeast. Later, the activation of the de novo pathway in C. Elegans in response to ionizing radiation was described and found to be involved in activation of the CED-3 caspase 104 and the resulting apoptosis of germ cells.

In mammalian systems, the de novo pathway seems to play a prominent role in cellular response to heat or chemical stress, 105 septic shock, 106 lipo-apoptosis 107 and insulin resistance, 108 as well as in receptor-dependent and -independent induction of apoptosis by variety of chemotherapeutic agents like etoposides 109 

In the human acute lymphoblastic leukemia cell line Molt-4, heat shock induces more than twofold increase in total ceramide levels with C16 ceramide being the major species affected. This accumulation of ceramide has been linked to the induction of c-jun and apoptosis. 105 Labeling with tritiated palmitate has shown an accumulation of ceramides, but not sphingoid bases, thus confirming that in contrast to yeast, where sphingoid bases mediate heat shock, in mammals, ceramide is apparently involved. Both, myriocin and fumonisin B1 inhibit the increase in total ceramide mass thus confirming that ceramides produced upon heat shock were products of SPT and ceramide synthase activity.

Similar observations delineate a role of the de novo synthesized ceramide in septic shock in vitro as well as in vivo. 106 Administration of LPS or cytokines to rabbits increases hepatic sphingolipid biosynthesis leading to the accumulation of ceramide, SM and glycosphingolipids. Studies in cell lines link the stimulation of the de novo pathway to the activation of the MAP kinases and NFgB and respectively to the innate immune response. In nonhepatic tissues the elevation in ceramide biosynthesis during septic shock is correlated with the elevated glycosphingolipid synthesis that seems to play a role in pathogen recognition.

Activation of ceramide synthesis in the liver has been linked to production and secretion of lipoproteins enriched in ceramides, sphingomyelins and glycosphingolipids. The functions of these lipoproteins with "altered" sphingolipid content was not well understood; it became clear however that the activation of the de novo synthesis during septic shock is paralleled by activation of another ceramide-generating enzyme, the Zn 2 -dependent secretory form of ASMase, termed SSMase. This enzyme hydrolyses SM in the Low Density Lipoproteins (LDL), leading to a robust increase in LDL ceramide content. Several lines of evidence suggest that LDL that are rich in ceramide might mediate injury to the arterial wall during inflammation. LDL extracted from atherosclerotic plaques contain higher ceramide content as compared to LDL isolated from the plasma. 111 Experimentally-induced elevation in LDL ceramide content has been further linked to higher rate of LDL aggregation 112 and oxidation, as well as with an increased potential to induce apoptosis in microvascular endothelial cell. 113

Consumption of diet rich in saturated fats (also known as Western diet) is the main risk factor for the development of insulin resistance, hyperglycemia and atherosclerosis. Palmitic acid is the main component of the Western diet and is linked to excessive accumulation of lipids in lean tissues, mainly muscle and liver, lipotoxicity and insulin resistance. Because SPT has almost exclusive preference for the CoA-thiol ester of palmitate, numerous studies have investigated whether Western diet affects de novo synthesis of ceramide. These studies have shown that excess palmitate (delivered via the consumption of Western diet, i.v. infusion, or directly added to cells in culture) stimulates the flux through the de novo pathway resulting in accumulation of ceramide, SM and glycosphingolipids. 58, 114 Inhibition of SPT by myriocin prevents not only the palmitate flux through the pathway but also inhibits lipotoxicity, improves insulin response and leads to better glucose regulation.

The exact mechanism by which ceramide affects insulin response is not completely understood. Schmitz-Peiffer et al 115 observed that the accumulation of ceramide in myotubes exposed to palmitate was paralleled by inhibition of Akt/PKB. Cotreatment with myriocin, cycloserine, or fumonisin B1 restored insulin-stimulated Akt phosphorylation, even in the presence of excess palmitate, suggesting that palmitate-induced stimulation of de novo synthesis is required for inhibition of insulin responsiveness. Recent data however imply that in vivo, the diet-induced ceramide increases have to be accompanied by increased triacylglycerides synthesis and accumulation in order to affect the overall insulin response. Furthermore, stimulation of the de novo synthesis of sphingolipids and that of triacylglycerides seems to be correlated in liver. 59

Perhaps, the most extensively studied cellular response to ceramide is the induction of programmed cell death or apoptosis. Activation of (dihydro)ceramide synthase in particular is implicated in endothelial cell death induced by TNF_, 116 in daunorubicin, 110 doxorubicin and gemcitabine-induced apoptosis and may account for some aspects of the toxicity of phorbol esters, 117 angiotensin II, 118 cannabinoids 119 and ischemia-reperfusion. In the latter case, investigation of intracellular sites of ceramide accumulation reveals that the elevation of ceramide is in mitochondria and is caused by the activation of a mitochondrial ceramide synthase via posttranslational mechanisms. Furthermore, ceramide accumulation appears to cause mitochondrial respiratory chain damage that could be mimicked by exogenously added natural ceramide to mitochondria. 12 The recent cloning and characterization of 6 members of the ceramide synthase family CerS1-6 (also known as longevity assurance gene 1-6 (LASS1-6)) provided the opportunity to finally begin studying the role of these enzymes in apoptosis in more details. CerS1 but not CerS2-6 for example, was found to be sufficient and required for apoptosis in response to gemcitabine/ doxorubicin treatment. 120 Notably, the gemcitabine/doxorubicin combination treatment was discovered to increase only the levels of C18-ceramide, the preferable substrate for CerS1. These and other studies have provided experimental evidence to the earlier assumptions that not only the type of sphingoid base (i.e., sphingosine vs sphinganine) but also the fatty acid length and degree of saturation influences the biological effectiveness of ceramide.

The role of the de novo pathway in apoptosis had also been investigated in different animal models. Inhibition of the enzymes controlling the de novo pathway prevents alveolar cell apoptosis, oxidative stress and emphysema (the prevalent disease caused by cigarette smoking) in both rats and mice, 121 delays the progression of atherosclerotic plaques in mice 122 and ameliorates some of the pathological consequences of spinal cord injury. 123

The final paradigm to be discussed is the role of the de novo synthesis of ceramide in autophagy, an evolutionary conserved cytoprotective mechanism that sustains cells during periods of nutrient limitation, but under certain conditions may lead to mammalian cell death. Ceramide addition is sufficient to induce autophagy in some cells and a correlation between the rate of autophagy and ceramide synthesis has been observed in same pathological conditions suggesting a mechanistic link between ceramide, autophagy and disease (reviewed in Zheng et al 2 ). Activation of de novo ceramide synthesis mediates autophagy in response to a bioenergetic crisis resulting in the rapid and profound down regulation of nutrient transporter proteins. 124 Ceramide is also shown to mediate the tamoxifen-dependent accumulation of autophagic vacuoles in the human breast cancer MCF-7 cells and to counteract interleukin 13-dependent inhibition of macroautophagy in HT29 cells. 125 The mechanism seems to involve the pro-autophagic protein beclin 1.

Regulation of SPT at a transcriptional level has been seen with a number of agents, including endotoxin and cytokines, 106 UVB irradiation 126 and others. 127 Induction of both form of SPT, SPT1 and SPT2, occurs in balloon-injured rat carotid artery. 128 In contrast, long-term consumption of food rich in palmitate is correlated with increases in SPT1 protein but not mRNA level. 59 Activation of SPT occurs also posttranslationally in response to etoposide 33 and to heat shock in yeast. 34 Mitochondrial injury in cerebral ischemia/reperfusion activates ceramide synthases via posttranslational mechanism which is dependent of JNK. 12 Cers1 mRNA transcription is up-regulated in response to a gemcitabine/doxorubicin combination treatment, 120 while cisplatin is shown to cause a specific translocation of Cers1 from the endoplasmic reticulum to the Golgi apparatus. 129 The de novo pathway is also modulated through a negative feedback mechanism determined by the rate of sphingolipid degradation in the lysosomes since lipoproteins, sphingosine phosphate 130 and ASMase activity 59 seems to inhibit the flux through it. This might be a mechanism to prevent excessive synthesis of ceramide during normal healthy state of the cell.

The SMase family is a group of biochemically and genetically different enzymes all of which hydrolyze SM to ceramide and phosphorylcholine. SMase activities with neutral and acidic pH optima are found in most mammalian cells and an enzyme active in alkaline pH is localized in the intestinal wall. Currently, research is focused on 4 genes encoding different mammalian SMases: smpd1 encodes two forms of acidic SMase, one associated with the endosomal/lysosomal compartment (ASMase) and a second one found in the plasma and the conditioned medium of stimulated cells (SSMase). smpd2 and smpd3 encode the Neutral SMase 1 (nSMase1) and 2 (nSMase2), both of which are Mg 2 g g -dependent but differ in their subcellular localization and role in signaling. Data from several labs have shown that in mammalian cells, nSMase1 is a housekeeping enzyme with no particular function in signaling. The recently cloned zebra fish nSMase1, however seems to mediate heat-induced apoptosis in zebra fish embryonic cells. 131 In mammalian cells, nSMase2 is regulated by cytokines like IL-1`and TNF_ and mediates some of the cytokine effects. [132] [133] [134] The recently cloned smpd4, is suggested to encode a novel form of NSMase, nSMase3 that is found predominantly in skeletal muscle and heart.

The acute phase response of liver is an essential component of the systemic host response to bacterial infection and injury and requires IL-1`, a prototypic inflammatory cytokine. Activation of NSMase by IL-1`resulting in transient elevation in ceramide concentration is observed in number of cells including hepatocytes, mesangial cells, EL-4 cells and it has been related to activation of TAK-1, JNK and NF-gB, all of which have important roles in the IL-1`cascade. [135] [136] [137] In hepatocytes, specific silencing of nSMase2 with siRNA has only a minimal effect on the basal cellular NSMase activity, however it results in a complete inhibition of the IL-1`-stimulated NSMase activity. 138 Therefore, nSMase2 is probably an inducible enzyme that contributes little to the basal turnover of SM, but at the same time it is also the only neutral SMase activated by IL-1`. The role of NSMase2 in the IL-1`signaling cascade is rather complex: seemingly, the activation of nSMase2 modulates the pattern of phosphorylation of JNK by IL-1` and respectively the magnitude of transcriptional induction of the hepatic acute phase proteins like C-Reactive Protein, _1 Acid Glycoprotein and Insulin-like Growth Factor Binding Protein-1. nSMase2 activation in hepatocytes leads to potentiation of JNK phosphorylation due to stabilization of the IL-1`receptor-associated kinase-1. The latter most likely involves a phosphatase, like the ceramide-activated protein phosphatase 2A (PP2A). 139

Activation of nSMase2 by another pro-inflammatory cytokine, TNF_ is well documented in cells of the vasculature and various cancer cells. 132,140,141 TNF_-induced nSMase2 activation is a prerequisite for endothelial nitric oxide synthase activation in HUVEC cells, 134 

Studies into the activation of NSMase during apoptosis have focused mainly on the role of NSMase-mediated ceramide production in the apoptosis induced by 55 kDa receptor for TNF_. 142 A protein factor associated with NSMase activation, FAN, which interacts with the membrane-proximal domain of the p55 receptor and couples stimulation of the receptor to neutral SMase activation, is required for TNF_-induced ceramide generation, caspase processing and apoptosis. 143 Subsequent work in MCF-7 cells found that activation of NSMase in TNF_-stimulated cell death is upstream of mitochondrial changes, cytochrome C release and caspase-9 activation. 144 Oxidative stress-induced cell death may also be attributed to activation of NSMase. Free oxy-y y gen (H 2 O 2 ), but not nitrogen (ONOO < ) radicals specifically activate nSMase2, while silencing nSMase2 prevents H 2 O 2 -induced apoptosis, but had no effect on ONOO < -induced apoptosis. 83 Very recent studies had began to suggest that a novel form of neutral SMase, nSMase3 is a DNA damage and nongenotoxic stress-regulated gene that is deregulated in human malignancies and modu-u u late the sensitivity of cancer cells to adriamycin-induced cell killing. 145 Early studies seem to suggest that at least the overexpressed nSMase3 can be activated by TNF_ within seconds of stimulation.

A number of studies have established a role for NSMase-generated ceramide in regulating the cell cycle and mediating growth arrest, possibly through dephosphorylation of retinoblastoma protein and/or regulation of cyclin dependent kinases. Serum withdrawal causes activation of NSMase and cell cycle arrest at G0/G1 in Molt-4 cells. 146 Interestingly, a study focusing on genes up-regulated during confluence-induced arrest had initially identified nSMase2 as a confluence-arrest gene, CCA1, in rat 3Y1 cells. 147 In line with that, nSMase2 seems to mediate confluence-induced growth arrest of MCF-7 cells. The latter was preceded by confluence-induced translocation of nSMase2 to the plasma membrane. 148

Constitutive up-regulation of nSMase2 and elevation of ceramide concentration during aging has been observed in liver, brain, macrophages and other tissues. 149 This aging-associated elevation in ceramide seems important for the onset of aging process since it has been causatively linked to hyperresponsiveness to IL-1`7 0 and LPS 41 and to deregulation of nitric oxide production in endothelium. 71 A substantial decline in hepatic GSH content, which is characteristic for the aging process is responsible for this constitutive increase in nSMase2 activity. 150 nSMase2 apparently follows a pattern of regulation consistent with "developmental-aging" continuum, since in animal models of delayed aging, like calorie-restricted animals, the aging-associated changes in NSMase activity and function are reversed. 150 In cellular model of senescence, similar induction of NSMase activity and accumulation of ceramide has been observed in senescent cells and linked to the decline in proliferative capacity and onset of senescence. 72 In contrast, a somatic homozygous deletion specifically targeting nSMase2 (Smpd3) is found in a genomic screen for gene copy losses contributing to tumorigenesis in a mouse osteosarcoma model, while loss-of-function mutations in smpd3 gene were identified in 5% of acute myeloid leukemia and 6% of acute lymphoid leukemia cancers. It has been suggested that the mutation could be linked to a defect in plasma membrane translocation of nSMase2 and decreased responsiveness to TNF_. Reconstitution of smpd3 expression in mouse tumor cells lacking the endogenous gene enhances the TNF_-induced reduction of cell viability. 151

(i) Translocation: Studies have shown that nSMase2 can be translocated to the plasma membrane when cell confluence is reached. 132 This translocation of nSMase2 is also required for the confluence-induced cell cycle arrest to ensue. 152 In oligodendroma-derived cells, a regulated translocation of NSMase2 to the caveolae, which are the signaling domains of the plasma membrane 153 has been observed, while studies with highly dif-f f ferentiated nonproliferating primary hepatocytes have shown that the overexpressed nSMase2 is localized constitutively at the plasma membrane. 133 These data suggest that translocation of nSMase2 to the plasma membrane might be important mechanisms for regulation of its activity in situ by bringing the enzyme into contact with its substrate. Pharmacological inhibitors and specific siRNA has implicated the novel PKC, specifically PKCb, in TNF and PMA-stimulated nSMase2 translocation to the plasma membrane. 52 (ii) NSMase as a redox-sensitive enzyme. The major scavenger of reactive oxygen species, GSH, has been found to be a reversible inhibitor of cellular NSMase activity. 33, 154 This is noteworthy, because depletion of cellular GSH content is observed in conditions of increased oxidative stress. The modulation of NSMase activity by GSH was first established in the context of regulation of TNF_ signaling and apoptosis. 33, 154 Later, the ability of GSH to affect the sensitivity of T47D/H3 breast cancer cells to doxorubicin was attributed to the inhibitory effect GSH has on NSMase activity. 155 A correlation between oxidative stress and NSMase activity was also found in long-lived rats on vitamin Q10 enriched diet 156 and in astrocytes treated with vitamin E. 157 Finally, recent research has shown that specific downregulation of nSMase2 with siRNA blocks H 2 O 2 -induced apoptosis of human aortic endothelial cells, identifying nSMase2 as a redox-sensitive protein. 158 Detailed analyses of sensitivity of nSMase2 to GSH in hepatocytes suggest that GSH depletion exerts its effect on NSMase activity following a sigmoid dose dependent curve. A rapid and significant activation of NSMase is observed only when hepatic GSH concentration drops below 30% of its basal level, implying that there is a threshold required for NSMase activation during oxidative stress. 150 Interestingly, nSMase1 is also sensitive to changes in the GSH/GSSG ratio. 159 This suggests that redox sensitivity might be a common property of neutral SMases.

Patients with severe sepsis exhibit an enhanced SMase activity in plasma. The increase is correlated with the severity of illness and the fatal outcome. 160 Studies in humans and mice administered with LPS confirm these observations. 39 Deletion of ASMase protects against LPS-induced elevation in the plasma SMase activity 161 and attenuates endothelial apoptosis and animal death 43 implicating a secretory isoform of ASMase (SSMase) in apoptosis and organ failure in sepsis. The physiological and biochemical properties of this SSMase are not well understood. It has been postulated that SSMase degrades SM in the secreting cell outer membrane leaflet, which contains almost ¾ of the cellular SM. This SM pool is seemingly inaccessible for other cellular SMases, since the active center of nSMase2, the sole plasma membrane-associated SMase, is facing the cytosolic leaflet of the membrane. These proposed autocrine functions of SSMase however had not been rigorously tested. In turn, substantial evidence had implicated SSMase in modifying the SM/ ceramide content of circulating LDL as discussed earlier in the chapter.

Irradiation of tumor cells with ionizing radiation 52,163-165 transiently activates ASMase with maximal activity detected between 1 to 10 min post irradiation. The activation of ASMase has been linked to the ability of radiation to induce apoptosis since ASMase deficiency causes apoptosis resistance in various tissues in ex vivo or in vivo experiments. [163] [164] [165] Genetic restoration of the activity also restores the radiation effects indicating that ASMase mediates apoptosis via ceramide, at least in some cells including B cells, endothelial cells or mesothelium, lung epithelial cells, MCF-7 breast cancer cells, etc. However, some other cell types, for example, thymocytes, remained sensitive to radiation in ASMase-deficient mice suggesting that radiation effects are mediated by different mechanisms depending on the cell type.

ASMase is also involved in death receptor-mediated apoptosis. Stimulation via the CD95 receptor leads to ASMase activation and ceramide accumulation that precedes the induction of cell death. [166] [167] [168] Furthermore, studies with fibroblasts from Niemann-Pick disease type A patients, who exhibit deficiency of ASMase and hepatocytes from ASMase null mice show that ASMase activation is required and sufficient for CD95 induced apoptosis. 168, 169 

Cellular ASMase activity seems to play an important role in susceptibility of mammalian organisms to microbial infections. ASMase-deficient mice were found to be more sensitive to infections with the Gram-negative bacteria L. monocytogenes, due to a defect in lyso-phagosomal fusion. In wild-type macrophages, the phagosome rapidly fuse with the lysosomes to form a phago-lysosome and to kill and digest bacteria, while in macrophages deficient in ASMase the process is slower and leads to inefficient transfer of lysosomal antibacterial hydrolyses into phagosomes. 170 Apparently, ASMase is required for the proper fusion of late phagosomes with lysosomes. 171 ASMase-deficient mice were also more susceptible to infection with Sindbis virus, an enveloped virus with a single-stranded RNA that is involved in fatal alpha virus encephalomyelitis, due to more rapid replication and spread of the virus in the nervous system. 172

One mechanism for activation of ASMase involves its phosphorylation by PKCb at Ser 508 which mediates UV light-induced ASMase activation and cell death in MCF-7 breast cancer cells. 52 Phosphorylation of ASMase seems to be correlated with its translocation to the plasma membrane (see below) in irradiated cells.

A handful of studies suggest that the activity of the two SMases and the de novo pathway might be regulated in a coordinated fashion by the same agonist, resulting in the transient generation of distinct "waves" of ceramide increases that might serve to diversify the biological effects of ceramide. These studies also emphasize the significance of specific ceramide species in defined stages of cellular stress response.

For example, a transient increase of ceramide is observed within minutes after exposure to ionizing irradiation which is a consequence of DNA damage-independent acid SMase 173 or neutral SMase activation. 47 Several hours after irradiation, a second wave of ceramide accumulation is observed depending on the DNA damage-dependent activation of ceramide synthase. It seems that the late ceramide accumulation is also dependent on the first one and is rate limiting for the apoptotic process induced by irradiation. 173 Kroesen et al 174 on the other hand, showed that cross linking of the B-cell receptor generates C16-ceramide upstream of the mitochondria in a caspase independent manner and that inhibition of C16 ceramide generation rescues from cell death. 56 C24 ceramide however was generated downstream of mitochondrial dysfunction in a caspase dependent manner. All of the increases are seemingly due to activation of de novo synthesis and apparently different members of the ceramide synthase family might be differentially regulated.

A classical example of coordinated regulation of neutral and acidic SMase is the early work of Kronke and colleagues. 175 In these studies, stimulation with TNF_ lead to activation of both enzymes through apparently separate mechanisms since different domains of the cytosolic tail of the TNF_ receptor were responsible. Furthermore, while the activation of ASMase was linked to NFgB activation, NSMase seemed to regulate the activation of proline-directed serine/ threonine protein kinase(s).

PKC c, atypical PKC isoform, was identified as a molecule that ceramide directly binds and activate. Notably, ceramide-induced activation of PKCc is linked to inhibition of Akt-1. Akt-1 is a key regulatory molecule for various metabolic cellular functions, cell proliferation and cell death, which has been known for a long time to be inhibited by ceramide. Ceramide-activated PKCc seems to phosphorylate Ser 34 of the Akt-1 pleckstrin homology domain, thus preventing the interaction of Akt-1 with PIP 3 and respectively with the plasma membrane. [176] [177] [178] In addition to stimulating the kinase activity of PKCc, ceramide binding seems to affect the ability of PKCc to interact with other proteins. In vascular smooth muscle, ceramide stabilizes the interaction of Akt-1 and PKCc within caveolin-enriched lipid microdomains to inactivate Akt and specifically reduces the association of PKCc with 14-3-3, a scaffold protein localized to less structured regions within membranes. 179 In differentiating embryonic stem cells, ceramide binding to PKCc similarly c leads to activation of its kinase activity but also increases PKCc binding to its inhibitor protein, prostate apoptosis response-4 (PAR-4), thus compromising the antiapoptotic activity of PKCc and inducing apoptosis. 180

Long chain D-erythro-C18-ceramide has been shown to activate PP2A (more specifically the AB'C trimer), PP2AC and PP1-aC and -_C in vitro suggesting a direct and stereospecific effect of ceramide on the phosphatase activity. This ability of ceramide to activate PP2A was later found to mediate the ceramide effects on various substrates relevant to the induction of apoptosis, growth arrest or inflammation, including c-Jun, Bcl-2, Akt/PKB, Rb, PKC_, ERK1/2, IRAK-1, SR proteins and many others.

Ceramide specifically binds and activates the endosomal acidic aspartate protease cathepsin D. Direct interaction of ceramide with cathepsin D results in autocatalytic proteolysis of the 52 kDa procathepsin D to the enzymatically active 48/32 kDa isoforms that can subsequently cleave and activate the apoptotic regulator Bid. 182 Studies in ASMase deficient cells strongly suggest that ASMase activity is responsible for the generation of ceramide that can activate the protease. 183 This is in contrast to the ability of ceramide to activate PKCc and PP2A, which seemingly requires activation of the de novo pathway or NSMase.

The members of the Bcl-2 family of proteins are essential modulators of apoptotic cell death following genotoxic and nongenotoxic stress. Strong evidence links ceramide to the regulation of two members of that family, the antiapoptotic Bcl-2 and the pro-apoptotic Bax. The mechanisms involved are far from understood and apparently quite complex. TNF_-and etoposide-induced activation of NSMase leads to apoptosis via a pathway where ceramide is upstream of the antiapoptotic member of the family, Bcl-2 and lead to its inhibition (5) . 185 These effects seem to involve PP2A. Bcl-2, whose phosphorylation at Ser 70 is required for its anti-apoptotic function, becomes dephosphorylated in response to ceramide in a PP2A-dependent manner and consequently is degraded in the proteasomes. 186 In turn, gemcitabine-induced ceramide generation is found to enhance the expression of proapoptotic Bcl-x. 187 Ceramide accumulation is also linked to the activation of the execution caspases, but it is downstream of the initiator caspases. 188, 189 C16-ceramide, which is generated by ASMase in response to irradiation is shown to induce a conformation change of the pro-apoptotic member Bax leading to its activation and cytochrome C release. 190 It was suggested that only the production of ceramide in the mitochondria can induce the oligomerization of Bax that drives cell death. 191 As Bcl-2, Bax is also regulated by ceramide via PP2A, since its dephosphorylation is associated with conformational change and release of cytochrome C from the mitochondria. Ceramide generation by ASMase has been suggested to mediate cell death by caspase-dependent and independent mechanisms depending on the death stimulus. 192

Dephosphorylation of the Retinoblastoma protein, activation of the cyclin dependent kinase inhibitor p21 and inhibition of the cyclin dependent kinase 2 193,194 are essential steps in the pathway 4 leading to cell cycle arrest in response to ceramide accumulation. Ceramide induced-dephosphory-y y lation of Retinoblastoma protein is mediated through PP2A, for which Retinoblastoma protein is a direct substrate. The hypophosphorylated Retinoblastoma protein binds and sequesters E2F, an essential factor for progression through the cell cycle. The senescent-associated growth may be attributed to a defect in the phospholipase D/protein kinase C (PLD/PKC) pathway and ceramide can inhibit both PLD and PKC. 195, 196 

Studies by Hannun and Brenner were the first to show that TNF_ activates the stress-activated protein kinases JNKs, resulting in the stimulation of AP-1-transcription factor and induces the translocation of NFgB to the nucleus, resulting in the stimulation of NFgB-dependent gene transcription, through ceramide. 197 Ceramide induced JNK activation has also been reported in response to FAS activation, 198 irradiation 199 and many cytokines. The activation of JNK by ceramide involves Rac-1 200 in the case of radiation-induced apoptosis, PKCc in response to IL-1 c`2 01 and TAK-1 in response to TNF-_, IL-1`, or anti-Fas antibody. 202 In hepatocytes, nSMase2-generated ceramide has been shown to modulate the effects of IL-1`on JNK phosphorylation and activity in PP2A and IRAK-1-depndent manner. 133 Ceramide-mediated JNK activation inhibits differentiation of skeletal muscle progenitor cells to myoblasts 203 and induces the expression of various acute phase proteins like Insulin-like Growth Factor Binding Protein-1 in liver. 204 JNK activation mediates apoptosis in neurons in response to ceramide and Amyloid `, the protein involved in the ethiology of Alzheimer's disease. In that system, an inhibition of NSMase was found to attenuate Amyloid `-induced JNK phosphorylation and AP-1 DNA binding activity suggesting the involvement of the plasma membrane-associated SMase activity. 205

Some of the biological responses to ceramide generation are probably due to the effects ceramide has on the structure of membrane rafts and caveolae, on the membrane curvature and the membrane permeability to aqueous solutes. The two hydroxyl groups, the amid group protons and the trans-double bond in ceramide molecule, together with the lack of large hydrophilic head group determine the ability of ceramide to affect the properties and organization of biological membrane. The long-chain base and long saturated N-acyl chains accompanied by a very small hydrophilic head promote the partitioning of ceramide into ordered membrane domains. Together with SM, which has a strong affinity for interacting with membrane cholesterol, ceramide participate in the formation of the caveolae, the signaling platforms on the cell surface. While SM is by far much more abundant than ceramide in the lipid rafts, the generation of ceramide within rafts dramatically alters the biophysical properties of these membrane domains. Ceramide molecules have the tendency to spontaneously self associate to small ceramide-enriched membrane microdomains. 206 Gulbins and colleagues had proposed that these ceramide microdomains spontaneously fuse to form large ceramide-enriched macrodomains or platforms. The formation of such platforms is proposed to underline the process of agonist induced receptor clustering and consequently, the reorganization of intracellular signaling molecules to transmit a signal into the cell. Among the receptors affected by ceramide-mediated aggregation/clustering are CD95, CD28, TNF_, CD40, FcgRII. 207 Intriguingly, the acidic form of SMase seems to be involved in the formation of these signaling platforms, since it has been shown to translocate to the plasma membrane under certain conditions. In further support to that extend, overexpression of the SM synthase, SMS-1, which enriches plasma membrane with SM also enhances the translocation of Fas into lipid rafts leading to subsequent Fas clustering, DISC formation, activation of caspases and apoptosis, suggesting that SM levels in the lipid rafts might also be a critical factor possible by acting as a source for ceramide. 208 Another intriguing property of ceramide is its ability to spontaneously form channels into planer bilayers, liposomes and biological membranes indicating that ceramide might regulate membrane leakiness. Experiments with unilamellar vesicles show that ceramide forms large pores that can allow the efflux of proteins as large as 60 kDa. Studies by Colombini and colleagues had shown that at physiologically relevant levels, ceramides form stable channels in mitochondrial outer membranes capable of passing the largest proteins known to exit mitochondria during apoptosis including cytochrome C. 209 Ongoing efforts on characterizing the ceramide metabolism in this organelle will shed more light into the physiological function of ceramide in the mitochondria.

Finally, studies in cells isolated from ASMase-deficient mice or Niemann-Pick patients had shown defects in the phagolysosomal fusion and rab-4-mediated endocytosis 210 indicating that ASMase regulates select vesicular fusion processes by modifying the steric conformation of cellular membranes. 211 These conclusions are supported by series of studies on the infectivity of Listeria monocytogenes and s Mycobacterium avium discussed earlier.

It was realized long ago that sphingolipid structure is highly diverse and encodes distinct chemical information that can be conveyed during cell signaling. 212 Research spanning through the last two decades established experimentally the role of this novel family of bioactive lipids in signaling. Ceramide emerged as the key metabolite in several sphingolipid-based signaling networks and was recognized to mediate conserved pathways of cellular stress response. With the genetic cloning and characterization of the majority of ceramide metabolizing enzymes, it also became clear that ceramide metabolism is finely tuned leading to spatial, temporal and species-specific accumulation during stress. A strict substrate specificity and distinct cellular localization of the enzymes that catalyze ceramide synthesis and degradation has brought diversity in the signaling molecules and cellular responses regulated by ceramide. The distinct ability of ceramide to alter the biophysical properties of cellular membrane adds additional layer of complexity in our understanding of the roles of ceramide in cellular stress response.

Biophysics of sphingolipids I. Membrane properties of sphingosine, ceramides and other simple sphingolipids

Ceramides and other bioactive sphingolipid backbones in health and disease: Lipidomic analysis, metabolism and roles in membrane structure, dynamics, signaling and autophagy

Sphingolipids and the formation of sterol-enriched ordered membrane domains

Quantitative analysis of sphingolipids for lipidomics using triple quadrupole and quadrupole linear ion trap mass spectrometers

Bcl-2 interrupts the ceramide-mediated pathway of cell death

Ceramide generated by sphingomyelin hydrolysis and the salvage pathway is involved in hypoxia/reoxygenation-induced Bax redistribution to mitochondria in NT-2 cells

Rapid activation of neutral sphingomyelinase by hypoxia-reoxygenation of cardiac myocytes

Ceramide synthase is essential for endonuclease-mediated death of renal tubular epithelial cells induced by hypoxia-reoxygenation

Ceramide formation leads to caspase-3 activation during hypoxic PC12 cell death. Inhibitory effects of Bcl-2 on ceramide formation and caspase-3 activation

Reactive oxygen species production by mitochondria in endothelial cells exposed to reoxygenation after hypoxia and glucose depletion is mediated by ceramide

Regulatory role of sphingomyelin metabolites in hypoxia-induced vascular smooth muscle cell proliferation

JNK3 signaling pathway activates ceramide synthase leading to mitochondrial dysfunction

Ceramide is involved in triggering of cardiomyocyte apoptosis induced by ischemia and reperfusion

Altered ceramide and sphingosine expression during the induction phase of ischemic acute renal failure

Critical role of acidic sphingomyelinase in murine hepatic ischemia-reperfusion injury

Cytokine-mediated induction of ceramide production is redox-sensitive. Implications to proinflammatory cytokine-mediated apoptosis in demyelinating diseases

Induction of intracellular ceramide by interleukin-1 beta in oligodendrocytes

Involvement of ceramide in inhibitory effect of IL-1 beta on L-type Ca2 current in adult rat ventricular myocytes

Negative regulation of interleukin-1beta-activated neutral sphingomyelinase by protein kinase C in rat mesangial cells

Bimodal regulation of ceramidase by interleukin-1beta. Implications for the regulation of cytochrome p450 2C11

Regulation of cytochrome P450 2C11 (CYP2C11) gene expression by interleukin-1, sphingomyelin hydrolysis and ceramides in rat hepatocytes

Regulatory role of ceramide in interleukin (IL)-1 beta-induced E-selectin expression in human umbilical vein endothelial cells. Ceramide enhances IL-1 beta action, but is not sufficient for E-selectin expression

Ceramide inhibits antigen uptake and presentation by dendritic cells

Interleukin 1 beta (IL-1 beta) action in porcine thyroid cells involves the ceramide signalling pathway

Hypoxic preconditioning protects cultured neurons against hypoxic stress via TNF-alpha and ceramide

Molecular mechanisms of TNF-alpha-induced ceramide formation in human glioma cells: P53-mediated oxidant stress-dependent and -independent pathways

Ceramide-induced enhancement of secretory phospholipase A2 expression via generation of reactive oxygen species in tumor necrosis factor-alpha-stimulated mesangial cells

Defective TNF-alpha-mediated hepatocellular apoptosis and liver damage in acidic sphingomyelinase knockout mice

TNF-alpha increases ceramide without inducing apoptosis in alveolar type II epithelial cells

Alveolar sphingolipids generated in response to TNF-alpha modifies surfactant biophysical activity

Apoptotic sphingolipid signaling by ceramides in lung endothelial cells

Tumor necrosis factor alpha signaling pathway and apoptosis in pancreatic beta cells

Glutathione regulation of neutral sphingomyelinase in tumor necrosis factor-alpha-induced cell death

Inhibition of tumor necrosis factor-induced cell death in MCF7 by a novel inhibitor of neutral sphingomyelinase

Overexpression of acid ceramidase protects from tumor necrosis factor-induced cell death

Role of an acidic compartment in tumor-necrosis-factor-alpha-induced production of ceramide, activation of caspase-3 and apoptosis

Role of ceramide kinase in peroxisome proliferator-activated receptor beta-induced cell survival of mouse keratinocytes

Lipopolysaccharide-induced ischemic tolerance is associated with increased levels of ceramide in brain and in plasma

Elevation of ceramide in serum lipoproteins during acute phase response in humans and mice: role of serine-palmitoyl transferase

Phosphatidylcholine-specific phospholipase C (PC-PLC) is required for LPS-mediated macrophage activation through CD14

Ceramide mediates age-associated increase in macrophage cyclooxygenase-2 expression

Ceramide regulates lipopolysaccharide-induced phosphatidylinositol 3-kinase and Akt activity in human alveolar macrophages

Lipopolysaccharide induces disseminated endothelial apoptosis requiring ceramide generation

The group VIA calcium-independent phospholipase A2 participates in ER stress-induced INS-1 insulinoma cell apoptosis by promoting ceramide generation via hydrolysis of sphingomyelins by neutral sphingomyelinase

Radiation-induced apoptosis of oligodendrocytes and its association with increased ceramide and down-regulated protein kinase B/Akt activity

De novo ceramide synthesis participates in the ultraviolet B irradiation-induced apoptosis in undifferentiated cultured human keratinocytes

Ionizing radiation acts on cellular membranes to generate ceramide and initiate apoptosis

Elevated sphingomyelinase activity and ceramide concentration in serum of patients undergoing high dose spatially fractionated radiation treatment: implications for endothelial apoptosis

Non-enzymatic triggering of the ceramide signalling cascade by solar UVA radiation

Mass spectrometric identification of increased C16 ceramide levels during apoptosis

Cell line dependent involvement of ceramide in ultraviolet light-induced apoptosis

A novel role for protein kinase Cdelta-mediated phosphorylation of acid sphingomyelinase in UV light-induced mitochondrial injury

Mitochondrial ceramide increases in UV-irradiated HeLa cells and is mainly derived from hydrolysis of sphingomyelin

UV-C light induces raft-associated acid sphingomyelinase and JNK activation and translocation independently on a nuclear signal

Lipotoxic heart disease in obese rats: implications for human obesity

Ceramide content is increased in skeletal muscle from obese insulin-resistant humans

1,2-Diacylglycerol and ceramide levels in insulin-resistant tissues of the rat in vivo

Inhibition of ceramide synthesis ameliorates glucocorticoid-, saturated-fat-and obesity-induced insulin resistance

Acid sphingomyelinase deficiency prevents diet-induced hepatic triacylglycerol accumulation and hyperglycemia in mice

Plasma Ceramides Are Elevated in Obese Subjects with Type 2 Diabetes and Correlate with the Severity of Insulin Resistance

Protection from high fat diet-induced increase in ceramide in mice lacking plasminogen activator inhibitor 1

Altered adipose and plasma sphingolipid metabolism in obesity: a potential mechanism for cardiovascular and metabolic risk

Adipose tissue inflammation and increased ceramide content characterize subjects with high liver fat content independent of obesity

Fatty acid-induced beta cell apoptosis: a link between obesity and diabetes

Diabetes alters sphingolipid metabolism in the retina: a potential mechanism of cell death in diabetic retinopathy

An aging pathway controls the TrkA to p75NTR receptor switch and amyloid beta-peptide generation

Peroxisome proliferator-activated receptor alpha down-regulation is associated with enhanced ceramide levels in age-associated cardiac hypertrophy

Aging up-regulates expression of inflammatory mediators in mouse adipose tissue

Activation of sphingolipid turnover and chronic generation of ceramide and sphingosine in liver during aging

Aging in rat causes hepatic hyperresposiveness to interleukin-1beta which is mediated by neutral sphingomyelinase-2

Age-related changes in endothelial nitric oxide synthase phosphorylation and nitric oxide dependent vasodilation: evidence for a novel mechanism involving sphingomyelinase and ceramide-activated phosphatase 2A

Role of ceramide in cellular senescence

Astroglial expression of ceramide in Alzheimer's disease brains: a role during neuronal apoptosis

Involvement of oxidative stress-induced abnormalities in ceramide and cholesterol metabolism in brain aging and Alzheimer's disease

Substantial sulfatide deficiency and ceramide elevation in very early Alzheimer's disease: potential role in disease pathogenesis

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

Oxidative stress kills human primary oligodendrocytes via neutral sphingomyelinase: implications for multiple sclerosis

Oxidative stress induced by ascorbate causes neuronal damage in an in vitro system

Involvement of oxidative stress in ascorbate-induced proapoptotic death of PC12 cells

H2O2 acts on cellular membranes to generate ceramide signaling and initiate apoptosis in tracheobronchial epithelial cells

Oxidized low density lipoproteins induce a pathologic response by retinal pigmented epithelial cells

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

Reactive nitrogen and oxygen species activate different sphingomyelinases to induce apoptosis in airway epithelial cells

Ceramide is a cardiotoxin in lipotoxic cardiomyopathy

Overexpression of vascular endothelial growth factor-B in mouse heart alters cardiac lipid metabolism and induces myocardial hypertrophy

Association of ceramides in human plasma with risk factors of atherosclerosis

Increasing intracellular ceramide: an approach that enhances the cytotoxic response in prostate cancer cells

Resveratrol induces growth inhibition and apoptosis in metastatic breast cancer cells via de novo ceramide signaling

Taxol-induced ceramide generation and apoptosis in human breast cancer cells

Synergistic cytotoxicity between tamoxifen and the plant toxin persin in human breast cancer cells is dependent on Bim expression and mediated by modulation of ceramide metabolism

The multidrug resistance modulator SDZ PSC 833 is a potent activator of cellular ceramide formation

Enhanced de novo ceramide generation through activation of serine palmitoyltransferase by the P-glycoprotein antagonist SDZ PSC 833 in breast cancer cells

Enhanced ceramide generation and induction of apoptosis in human leukemia cells exposed to DT(388)-granulocyte-macrophage colony-stimulating factor (GM-CSF), a truncated diphtheria toxin fused to human GM-CSF

N-(4-hydroxyphenyl)retinamide elevates ceramide in neuroblastoma cell lines by coordinate activation of serine palmitoyltransferase and ceramide synthase

Vitamin E succinate induces ceramide-mediated apoptosis in head and neck squamous cell carcinoma in vitro and in vivo

Sphingolipid metabolites differentially regulate extracellular signal-regulated kinase and stress-activated protein kinase cascades

Fumonisin B1 inhibits sphingosine (sphinganine) N-acyltransferase and de novo sphingolipid biosynthesis in cultured neurons in situ

Inhibition of Carnitine Palmitoyltransferase I Augments Sphingolipid Synthesis and Palmitate-induced Apoptosis

De novo-synthesized ceramide signals apoptosis in astrocytes via extracellular signal-regulated kinase

Activation of the de novo Biosynthesis of Sphingolipids Mediates Angiotensin II Type 2 †Receptor-induced Apoptosis

Novo Sphingolipid Biosynthesis: A Necessary, but Dangerous, Pathway

Thematic Review Series: Sphingolipids. New insights into sphingolipid metabolism and function in budding yeast

Sphingoid base is required for translation initiation during heat stress in Saccharomyces cerevisiae

Ceramide Biogenesis Is Required for Radiation-Induced Apoptosis in the Germ Line of C. elegans

The emerging role for sphingolipids in the eukaryotic heat shock response

Endotoxin and Cytokines Increase Hepatic Sphingolipid Biosynthesis and Produce Lipoproteins Enriched in Ceramides and Sphingomyelin

Lipoapoptosis in beta-cells of obese prediabetic fa/fa rats. Role of serine palmitoyltransferase overexpression

Key role for ceramides in mediating insulin resistance in human muscle cells

Serine Palmitoyltransferase Regulates de Novo Ceramide Generation during Etoposide-induced Apoptosis

Ceramide synthase mediates daunorubicin-induced apoptosis: An alternative mechanism for generating death signals

Rabbit aorta and human atherosclerotic lesions hydrolyze the sphingomyelin of retained low-density lipoprotein. Proposed role for arterial-wall sphingomyelinase in subendothelial retention and aggregation of atherogenic lipoproteins

Effect of sphingomyelinase-mediated generation of ceramide on aggregation of low-density lipoprotein

Uptake and metabolism of low density lipoproteins with elevated ceramide content by human microvascular endothelial cells: implications for the regulation of apoptosis

Insulin Resistance and Metabolic Disease: New Insights from in Vivo Manipulation of Sphingolipid Metabolism

Ceramide Generation Is Sufficient to Account for the Inhibition of the Insulin-stimulated PKB Pathway in C2C12 Skeletal Muscle Cells Pretreated with Palmitate

Involvement of de Novo Ceramide Biosynthesis in Tumor Necrosis Factor-alpha/ Cycloheximide-induced Cerebral Endothelial Cell Death

12-O-tetradecanoylphorbol-13-acetate-induced apoptosis in LNCaP cells is mediated through ceramide synthase

Activation of the de novo biosynthesis of sphingolipids mediates angiotensin II type 2 receptor-induced apoptosis

Ceramide: a new second messenger of cannabinoid action

Role of human longevity assurance gene 1 and C18-ceramide in chemotherapy-induced cell death in human head and neck squamous cell carcinomas

Ceramide upregulation causes pulmonary cell apoptosis and emphysema-like disease in mice

Serine palmitoyltransferase inhibitor myriocin induces the regression of atherosclerotic plaques in hyperlipidemic ApoE-deficient mice

Inhibition of Ceramide Biosynthesis Ameliorates Pathological Consequences of Spinal Cord Injury

Ceramide starves cells to death by downregulating nutrient transporter proteins

Ceramide-mediated Macroautophagy Involves Inhibition of Protein Kinase B and Up-regulation of Beclin 1

UVB irradiation up-regulates serine palmitoyltransferase in cultured human keratinocytes

Regulation of de novo sphingolipid biosynthesis and the toxic consequences of its disruption

Increased expression of serine palmitoyltransferase (SPT) in balloon-injured rat carotid artery

Dihydro)ceramide synthase 1 regulated sensitivity to cisplatin is associated with the activation of p38 mitogen-activated protein kinase and is abrogated by sphingosine kinase 1

Characterization of ceramide synthase 2: tissue distribution, substrate specificity and inhibition by sphingosine 1-phosphate

Identification of Mg2 -dependent neutral sphingomyelinase 1 as a mediator of heat stress-induced ceramide generation and apoptosis

Role for Mammalian Neutral Sphingomyelinase 2 in Confluence-induced Growth Arrest of MCF7 Cells

Expression of neutral sphingomyelinase-2 (NSMase-2) in primary rat hepatocytes modulates IL-beta-induced JNK activation

Endothelial Nitric Oxide Synthase Activation by Tumor Necrosis Factor {alpha} Through Neutral Sphingomyelinase 2, Sphingosine Kinase 1 and Sphingosine 1 Phosphate Receptors: A Novel Pathway Relevant to the Pathophysiology of Endothelium

TAK1 mediates the ceramide signaling to stress-activated protein kinase/c-Jun N-terminal kinase

Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis

Ceramide activates the stress-activated protein kinases

Aging in rat causes hepatic hyperresponsiveness to interleukin 1 b which is mediated by neutral sphingomyelinase-2

Long chain ceramides activate protein phosphatase-1 and protein phosphatase-2A. Activation is stereospecific and regulated by phosphatidic acid

Role for furin in tumor necrosis factor alpha-induced activation of the matrix metalloproteinase/sphingolipid mitogenic pathway

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

Ceramide in Apoptosis: A Revisited Role

Involvement of FAN in TNF-induced apoptosis

Inhibition of Tumor Necrosis Factor-induced Cell Death in MCF7 by a Novel Inhibitor of Neutral Sphingomyelinase

Neutral sphingomyelinase-3 is a DNA damage and nongenotoxic stress-regulated gene that is deregulated in human malignancies

Role for ceramide in cell cycle arrest

cca1 Is Required for Formation of Growth-arrested Confluent Monolayer of Rat 3Y1 Cells

Biochemical Properties of Mammalian Neutral Sphingomyelinase2 and Its Role in Sphingolipid Metabolism

Role of neutral sphingomyelinases in aging and inflammation

Regulation of neutral sphingomyelinase-2 by GSH: a new insight to the role of oxidative stress in aging-associated inflammation

Mutations in the neutral sphingomyelinase gene SMPD3 implicate the ceramide pathway in human leukemias

cca1 Is Required for Formation of Growth-arrested Confluent Monolayer of Rat 3Y1 Cells

Differential regulation of ceramide in lipid-rich microdomains (rafts): Antagonistic role of palmitoyl:protein thioesterase and neutral sphingomyelinase 2

Inhibition of the neutral magnesium-dependent sphingomyelinase by glutathione

Andrieu-Abadie. Glutathione peroxidase-1 overexpression prevents ceramide production and partially inhibits apoptosis in doxorubicin-treated human breast carcinoma cells

Enhanced anti-oxidant protection of liver membranes in long-lived rats fed on a coenzyme Q10-supplemented diet

Inflammatory mediator and beta-amyloid (25-35)-induced ceramide generation and iNOS expression are inhibited by vitamin E Free Radic

Reactive nitrogen and oxygen species activate different sphingomyelinases to induce apoptosis in airway epithelial cells

Redox regulation of neutral sphingomyelinase-1 activity in HEK293 cells through a GSH-dependent mechanism

Role of increased sphingomyelinase activity in apoptosis and organ failure of patients with severe sepsis

Acute systemic inflammation up-regulates secretory sphingomyelinase in vivo: a possible link between inflammatory cytokines and atherogenesis

Raft ceramide in molecular medicine

Acid sphingomyelinase-deficient human lymphoblasts and mice are defective in radiation-induced apoptosis

Sphingolipid regulation of female gonadal cell apoptosis

Radiation-induced apoptosis of endothelial cells in the murine central nervous system: protection by fibroblast growth factor and sphingomyelinase deficiency

Apoptotic signaling through CD95 (Fas/Apo-1) activates an acidic sphingomyelinase

FAS-induced apoptosis is mediated via a ceramide-initiated RAS signaling pathway

CD95 signaling via ceramide-rich membrane rafts

Ceramide enables fas to cap and kill

Fusogenicity of membranes: the impact of acid sphingomyelinase on innate immune responses

Acid sphingomyelinase is required for efficient phago-lysosomal fusion

Acid sphingomyelinase deficiency increases susceptibility to fatal alphavirus encephalomyelitis

Role of the ceramide-signaling pathways in ionizing radiation-induced apoptosis

BcR-induced apoptosis involves differential regulation of C16 and C24-ceramide formation and sphingolipid-dependent activation of the proteasome

Functional dichotomy of neutral and acidic sphingomyelinases in tumor necrosis factor signaling

Ceramide-induced Inhibition of Akt Is Mediated through Protein Kinase Czeta . Implications for Growth Arrest

Regulation of insulin-stimulated glucose transporter GLUT4 translocation and Akt kinase activity by ceramide

Ceramide disables 3-phosphoinositide binding to the pleckstrin homology domain of protein kinase B (PKB)/Akt by a PKCzeta-dependent mechanism

Ceramide recruits and activates protein kinase C zeta (PKC zeta) within structured membrane microdomains

Direct binding to ceramide activates protein kinase Czeta before the formation of a pro-apoptotic complex with PAR-4 in differentiating stem cells

Ceramide in apoptosis: an overview and current perspectives

Cathepsin D targeted by acid sphingomyelinase-derived ceramide

Cathepsin D links TNF-induced acid sphingomyelinase to Bid-mediated caspase-9 and -3 activation

A house divided: ceramide, sphingosine and sphingosine-1-phosphate in programmed cell death

Ordering of ceramide formation, caspase activation and Bax/Bcl-2 expression during etoposide-induced apoptosis in C6 glioma cells

Bcl-2 phosphorylation required for anti-apoptosis function

De novo ceramide regulates the alternative splicing of caspase 9 and Bcl-x in A549 lung adenocarcinoma cells. Dependence on protein phosphatase-1

Molecular mechanisms of TNF-[alpha]-induced ceramide formation in human glioma cells:P53-mediated oxidant stress-dependent and -independent pathways

Ordering of ceramide formation, caspase activation and mitochondrial changes during CD95-and DNA damage-induced apoptosis

Acid sphingomyelinase is indispensable for UV light-induced Bax conformational change at the mitochondrial membrane

A mitochondrial pool of sphingomyelin is involved in TNFalpha-induced Bax translocation to mitochondria

Caspase-dependent and -independent Activation of Acid Sphingomyelinase Signaling

Retinoblastoma gene product as a downstream target for a ceramide-dependent pathway of growth arrest

Regulation of Cyclin-Dependent Kinase 2 Activity by Ceramide

Ceramide Inhibits Phospholipase D in a Cell-free System

Ceramide Inactivates Cellular Protein Kinase Calpha

Ceramide activates the stress-activated protein kinases

Fas ligation induces apoptosis and Jun kinase activation independently of CD45 and Lck in human T-cells

Requirement for ceramide-initiated SAPK/JNK signalling in stress-induced apoptosis

Fas-or Ceramide-induced Apoptosis Is Mediated by a Rac1-regulated Activation of Jun N-terminal Kinase/p38 Kinases and GADD153

Ceramide Directly Activates Protein Kinase C zeta to Regulate a Stress-activated Protein Kinase Signaling Complex

TAK1 Mediates the Ceramide Signaling to Stress-activated Protein Kinase/c-Jun N-terminal Kinase

C-jun N-terminal kinase mediates tumor necrosis factor-alpha suppression of differentiation in myoblasts

Regulation of insulin-like growth factor binding protein-1 expression during aging

Amyloid beta peptide increases DP5 expression via activation of neutral sphingomyelinase and JNK in oligodendrocytes

Compartmentalization of ceramide signaling: physical foundations and biological effects

Physiological and pathophysiological aspects of ceramide

Role of membrane sphingomyelin and ceramide in platform formation for Fas-mediated apoptosis

Ceramide Channels Increase the Permeability of the Mitochondrial Outer Membrane to Small Proteins

Elevated endosomal cholesterol levels in Niemann-Pick cells inhibit rab4 and perturb membrane recycling

Fusogenicity of membranes: The impact of acid sphingomyelinase on innate immune responses

An update of the enzymology and regulation of sphingomyelin metabolism