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Multiple sclerosis (MS) is a CNS disease characterized by immune-mediated demyelination and progressive axonal loss. MS-related CNS damage and its clinical course have two main phases: active and inactive/progressive. Reliable biomarkers are being sought to allow identification of MS pathomechanisms and prediction of its course. The purpose of this study was to identify sphingolipid (SL) species as candidate biomarkers of inflammatory and neurodegenerative processes underlying MS pathology. We performed sphingolipidomic analysis by HPLC-tandem mass spectrometry to determine the lipid profiles in post mortem specimens from the normal-appearing white matter (NAWM) of the normal CNS (nCNS) from subjects with chronic MS (active and inactive lesions) as well as from patients with other neurological diseases. Distinctive SL modification patterns occurred in specimens from MS patients with chronic inactive plaques with respect to NAWM from the nCNS and active MS (Ac-MS) lesions. Chronic inactive MS (In-MS) lesions were characterized by decreased levels of dihydroceramide (dhCer), ceramide (Cer), and SM subspecies, whereas levels of hexosylceramide and Cer 1-phosphate (C1P) subspecies were significantly increased in comparison to NAWM of the nCNS as well as Ac-MS plaques. In contrast, Ac-MS lesions were characterized by a significant increase of major dhCer subspecies in comparison to NAWM of the nCNS. These results suggest the existence of different SL metabolic pathways in the active versus inactive phase within progressive stages of MS. Moreover, they suggest that C1P could be a new biomarker of the In-MS progressive phase, and its detection may help to develop future prognostic and therapeutic strategies for the disease.

Sphingolipid biology has enjoyed a remarkable rise to fame over the last two decades. Various molecules from this lipid family have been implicated in a variety of cellular functions in health and disease. Ceramides, which constitute the hub of sphingolipid metabolism, are apoptogenic molecules that have many proposed mechanisms of actions. Enigmas revolving around this area of research are slowly being cleared with the advent of better laboratory techniques and data analyses. In this chapter, a general introduction of the topics presented in this book is undertaken highlighting the main ideas of each chapter.

This study aimed to identify circulating levels of neutral sphingomyelinase activity (N-SMase), ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P), C16-C24 sphingomyelin (SM), C16-C24 ceramide (CER) in acne vulgaris (AV) patients and controls. Serum was collected from AV patients and age, gender matched controls. Serum levels of C16-C24 SMs and C16-C24 CERs were determined by multiple reaction monitoring (MRM) method using ultrafast-liquid chromatography coupled with tandem mass spectrometry. Serum activity of N-SMase was assayed by standard kit methods, C1P and S1P levels were determined by enzyme-linked immunosorbent assay (ELISA). A significant increase was observed in serum levels of C16 SM in patients with AV compared to controls. Very-long-chain C24 CER was significantly decreased in AV patients compared to controls. A significant positive correlation was found between serum total cholesterol levels and all measured SMs and CERs in both the control and patient groups. Patients with AV had increased circulating levels of C16 SM, C1P and lower circulating levels of C24 CER compared to healthy controls, which may provide prognostic value for the disease. Future studies are needed to understand the role of altered sphingolipid levels in the pathophysiology of AV.

Cleanup technology and mass spectrometric determination of sphingosine-1-phosphate (S1P) using a phosphate capture molecule are shown. The protocol is rapid, requires neither thin-layer chromatography nor liquid chromatography, and is applicable to both blood and solid tissue samples. The mass spectrometric method is also applicable to ceramide-1-phosphate.

Recent studies have established specific cellular functions for different bioactive sphingolipids in skeletal muscle cells. Ceramide 1-phosphate (C1P) is an important bioactive sphingolipid that has been involved in cell growth and survival. However its possible role in the regulation of muscle cell homeostasis has not been so far investigated. In this study, we show that C1P stimulates myoblast proliferation, as determined by measuring the incorporation of tritiated thymidine into DNA, and progression of the myoblasts through the cell cycle. C1P induced phosphorylation of glycogen synthase kinase-3β and the product of retinoblastoma gene, and enhanced cyclin D1 protein levels. The mitogenic action of C1P also involved activation of phosphatidylinositol 3-kinase/Akt, ERK1/2 and the mammalian target of rapamycin. These effects of C1P were independent of interaction with a putative Gi-coupled C1P receptor as pertussis toxin, which maintains Gi protein in the inactive form, did not affect C1P-stimulated myoblast proliferation. By contrast, C1P was unable to inhibit serum starvation- or staurosporine-induced apoptosis in the myoblasts, and did not affect myogenic differentiation. Collectively, these results add up to the current knowledge on cell types targeted by C1P, which so far has been mainly confined to fibroblasts and macrophages, and extend on the mechanisms by which C1P exerts its mitogenic effects. Moreover, the biological activities of C1P described in this report establish that this phosphosphingolipid may be a relevant cue in the regulation of skeletal muscle regeneration, and that C1P-metabolizing enzymes might be important targets for developing cellular therapies for treatment of skeletal muscle degenerative diseases, or tissue injury., Highlights ► In this study we show that C1P stimulates C2C12 myoblast proliferation. ► C1P does not affect myogenic differentiation or survival of myoblasts. ► The mitogenic action of C1P is mediated by PI3K/Akt, ERK1/2 and mTOR activation. ► C1P effects are pertussis toxin insensitive.

El pdf del artículo es el manuscrito de autor.-- et al., The biogenesis of lipid droplets (LD) induced by serum depends on group IVA phospholipase A2 (cPLA2α). This work dissects the pathway leading to cPLA2α activation and LD biogenesis. Both processes were Ca2+-independent, as they took place after pharmacological blockade of Ca2+ transients elicited by serum or chelation with 1,2-bis(2-aminophenoxy)ethaneN′,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester). The single mutation D43N in cPLA2α, which abrogates its Ca2+ binding capacity and translocation to membranes, did not affect enzyme activation and formation of LD. In contrast, the mutation S505A did not affect membrane relocation of the enzyme in response to Ca2+ but prevented its phosphorylation, activation, and the appearance of LD. Expression of specific activators of different mitogen-activated protein kinases showed that phosphorylation of cPLA2α at Ser-505 is due to JNK. This was confirmed by pharmacological inhibition and expression of a dominant-negative form of the upstream activator MEKK1. LD biogenesis was accompanied by increased synthesis of ceramide 1-phosphate. Overexpression of its synthesizing enzyme ceramide kinase increased phosphorylation of at Ser-505 and formation of LD, and its down-regulation blocked the phosphorylation of cPLA2α and LD biogenesis. These results demonstrate that LD biogenesis induced by serum is regulated by JNK and ceramide kinase. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc., This work was supported, in whole or in part, by National Institutes of Health Grants HL-072925 and CA-117990, Grants SAF 2004-01698, SAF 2007-60055, SAF 2007-67154, and BFU2009-07823 from the Spanish Ministry of Education and Science, and Grant PI05/1723 from the Spanish Ministry of Health.

Lipid phosphate phosphatases (LPPs) regulate cell signaling by modifying the concentrations of lipid phosphates versus their dephosphorylated products. The ecto-activity regulates the availability of extracellular lysophosphatidate (LPA) and sphingosine 1-phosphate (S1P) and thereby signaling by their respective receptors. LPP products (monoacylglycerol or sphingosine) are taken up by cells and rephosphorylated to produce LPA and S1P, respectively, which activate intracellular signaling cascades. The proposed integrin binding domain on the external surface of LPP3 modifies cell/cell interactions. Expression of LPPs on internal membranes controls signaling depending on the access of lipid phosphates to their active sites. Different LPPs perform distinct functions, probably based on integrin binding, their locations, and their abilities to metabolize different lipid phosphates in vivo.

Sphingolipids and their metabolites have emerged as a new class of lipid mediator of various cell functions. It has been over a decade since the sphingolipid, ceramide-1-phosphate, was described. Until recently, only sparse reports on possible biological functions for this lipid have been published. In the last few years, a large number of reports have surfaced demonstrating distinct biological mechanisms regulated by ceramide-1-phosphate. The following is a review of the current literature on this overlooked lipid, with emphasis on its newly discovered role in signal transduction.

Sphingolipids are major constituents of biological membranes of eukaryotic cells. Many studies have shown that sphingomyelin (SM) is a major phospholipid in cell bilayers and is mainly localized to the plasma membrane of cells, where it serves both as a building block for cell architecture and as a precursor of bioactive sphingolipids. In particular, upregulation of (C-type) sphingomyelinases will produce ceramide, which regulates many physiological functions including apoptosis, senescence, or cell differentiation. Interestingly, the venom of some arthropodes including spiders of the genus Loxosceles, or the toxins of some bacteria such as Corynebacterium tuberculosis, or Vibrio damsela possess high levels of D-type sphingomyelinase (SMase D). This enzyme catalyzes the hydrolysis of SM to yield ceramide 1-phosphate (C1P), which promotes cell growth and survival and is a potent pro-inflammatory agent in different cell types. In particular, C1P stimulates cytosolic phospholipase A2 leading to arachidonic acid release and the subsequent formation of eicosanoids, actions that are all associated to the promotion of inflammation. In addition, C1P potently stimulates macrophage migration, which has also been associated to inflammatory responses. Interestingly, this action required the interaction of C1P with a specific plasma membrane receptor, whereas accumulation of intracellular C1P failed to stimulate chemotaxis. The C1P receptor is coupled to Gi proteins and activates of the PI3K/Akt and MEK/ERK1-2 pathways upon ligation with C1P. The proposed review will address novel aspects on the control of inflammatory responses by C1P and will highlight the molecular mechanisms whereby C1P exerts these actions. Work in AGM lab is supported by Departamento de Educacion, Universidades e Investigacion del Gobierno Vasco (Gazteiz-Vitoria, Basque Country), and Ministerio de Economia y Competitividad (Madrid, Spain).

The Arabidopsis accelerated-cell-death11 (acd11) mutant provides a genetic model for studying localized cell suicide to halt the spread of pathogen infection in plants. Recently, we showed that ACD...

Ceramide kinase (CERK) converts ceramide (Cer) to ceramide-1-phosphate (C1P), which has recently emerged as a new bioactive molecule capable of regulating diverse cellular functions. The N-terminus of the CERK protein encompasses a sequence motif known as a pleckstrin homology (PH) domain. Although the PH domain was previously demonstrated to be an important domain for the subcellular localization of CERK, the precise properties of this domain remained unclear. In this study, we reveal that the PH domain of CERK exhibits high affinity for phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2)[0], among other lipids. Furthermore, in COS7 cells, GFPfused CERK translocated rapidly from the cytoplasm to the plasma membrane in response to hyper-osmotic stress, which is known to increase the intracellular PI(4,5)P2 levels, whereas a PH-domain deletion mutant did not. Additionally, in [32P]orthophosphate-labeled COS7 cells, the translocation of CERK to the plasma membrane induced a 2.8 fold increase in C1P levels. The study presented here provides insight into the crucial role of the CERK[0]-PH domain in plasma membrane targeting, through its binding to PI(4,5)P2, and subsequent induction of C1P production in the vicinity of the membrane

Antigen-induced degranulation of mast cells plays a pivotal role in allergic and inflammatory responses. Recently, ceramide kinase (CERK) and its phosphorylated product ceramide 1-phosphate (C1P) have emerged as important players in mast cell degranulation. Here, we describe the synthesis of a novel F-12509A olefin isomer, K1, as an effective CERK inhibitor. In vitro kinase assays demonstrated that K1 effectively inhibits CERK without inhibiting sphingosine kinase and diacylglycerol kinase. Treating RBL-2H3 cells with K1 reduced cellular C1P levels to 40% yet had no effect on cell growth. Furthermore, treatment with K1 significantly suppressed both calcium ionophore- and IgE/antigen-induced degranulation, indicating that K1 interferes with signals that happen downstream of Ca(2+) mobilization. Finally, we show that K1 affects neither IgE/antigen-induced global tyrosine phosphorylation nor subsequent Ca(2+) elevation, suggesting a specificity for CERK-mediated signals. Our novel CERK inhibitor provides a useful tool for studying the biological functions of CERK and C1P. Moreover, to our knowledge, this is the first report demonstrating that inhibition of CERK suppresses IgE/antigen-induced mast cell degranulation. This finding suggests that CERK inhibitors might be a potential therapeutic tool in the treatment of allergic diseases.

The importance of sphingolipids, not only as components of plasma membranes but also as key players in different physiological and pathophysiological cellular events, is now emerging. This review gathers together what the authors feel are the most relevant data, present in the literature, regarding the roles and the effects of sphingolipids, such as ceramide, ceramide-1-phosphate (C1P), sphingosine (SP) and sphingosine-1-phosphate (S1P), on the development, activation and regulation of the immune system.

The interrelation between the bioeffector functions of sphingolipids and their chemical structure is reviewed. The effects of modifications of sphingoid functional groups on the bioregulatory properties of sphingolipids in cell proliferation, differentiation, and apoptosis are discussed.

Previous work suggested that glucosylceramide (GlcCer) plays a role in the regulation of cell differentiation of HT29 human colon tumor cells [I]. In the present study, we investigated the role of GlcCer in the cellular release of carcinoembryonic antigen (CEA), a marker for cell differentiation. This was done by modulating the intracellular level of the glycolipid, according to two different approaches. The cells were treated with D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), which resulted in a specific lowering of the cellular GlcCer pool. Alternatively, by exogenous addition of a short-chain analog of the lipid, hexanoyl(C-6)-GlcCer, the cellular pool was enhanced. The results demonstrate that PDMP causes an increase in the release of CEA, while exogenous C-6-GlcCer suppresses its release. Furthermore, the enhanced release of CEA in the presence of PDMP, could be completely reversed upon exogenous addition of C-6-GlcCer. Control experiments reveal that a potential interference of the well-known modulator of cell physiology, ceramide (Cer), can be excluded. Long-term depletion of GlcCer resulted in a change in a morphological feature of differentiation of the cells, i.e. an increase in apical membrane surface with microvilli brush borders, accompanied by an enhanced expression of the cytoskeletal protein villin. These results, together with the observations on modulation of the differentiation marker CEA by GlcCer, provide support for the conclusion that GlcCer interferes with the differentiation of HT29 cells.

Model studies for the syntheses of phosphonate analogs of sphingomyelin and ceramide 1-phosphate are described. The pentacovalent oxaphospholene 3b (derived from methyl vinyl ketone and triethyl phosphite) readily condensed with dialkyl azodicarboxylates (R = Et, t-Bu, CH(2)CCl(3)) to form beta-hydrazido gamma-ketophosphonates 5 and 8 in excellent yields. Cleavage of the N-N bond in 5a (R = Et) or 5b (R = t-Bu) via standard methods was unsuccessful. Upon reduction with NaBH(4), 8 produced the oxazolidinone 9 (93%) as a diastereomeric mixture of 3:1. Treatment of 9 with Zn/HOAc/acetone at rt readily cleaved the N-N bond to form 11 (78-83%). Confirmation of stereochemical assignments in 11 (3:1, trans:cis) was accomplished via NOE experiments.

Ceramide-1-phosphate sugars were synthesized by direct glycosyl phosphite/phosphate and O-glycosyl trichloroacetimidate/phosphate exchange reactions, respectively. Thus, ceramide-1-O-phosphoric acid 5 gave with sialyl phosphite 1 as sialyl donor directly beta-linked sialyl phosphate 6; deprotection afforded the corresponding glycophospholipid ceramide-1-phosphate N-acetylneuraminate 7. Similarly, from O-glucosyl- and O-galactosyltrichloroacetimidate 10 and 13 with phosphoric acid derivative 5 glycosyl ceramide-1-phosphate sugars 12 and 15, respectively, were obtained.

Ceramide kinase (CERK) is an enzyme that phosphorylates ceramide to produce ceramide 1-phosphate. Recently, evidence has emerged that CERK has a role in inflammatory signaling of immune cells. Since obesity is accompanied by chronic, low-grade inflammation, we examined whether CERK might be involved using CERK-null mice. We determined that CERK deficiency suppresses diet-induced increases in body weight, and improves glucose intolerance. Furthermore, we demonstrated that CERK deficiency attenuates MCP-1/CCR2 signaling in macrophages infiltrating the adipose tissue, resulting in the suppression of inflammation in adipocytes, which might otherwise lead to obesity and diabetes.

201 p. : gráf. Ceramide 1-phosphate is a bioactive sphingolipid that regulates important cell functions, including cell proliferation, apoptosis, migration or inflammation. Cell homeostasis is essential for the normal development of any organism and alteration of any of these pro¬cesses can lead to metabolic dysfunction or cause illnesses such as autoimmune diseases, chronic inflammation, neural degeneration, cardiovascular disorders, or cancer. The molecular mechanisms involved in the regulation of these cell responses by C1P have only begun to be understood. The aim of this Doctoral Thesis was to study the cellular signaling processes involved in C1P-stimulated cell proliferation and migration in immune cells, mainly macrophages. Concerning the regulation of cell proliferation, this Thesis has contributed to the elucidation of three major mechanisms by which C1P exerts its mitogenic actions: 1) Stimulation of vascular endothelial growth factor (VEGF) secretion, an action that involves activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. 2) Induction of translocation and activation of protein kinase C alpha (PKCα). 3) Activation of NADPH oxidase and the subsequent formation of reactive oxygen species (ROS). With regards to cell migration, we have demonstrated that C1P stimulates the release of macrophage chemoattractant protein-1 (MCP-1), which is essential for the induction of macrophage migration. In addition, we have shown that C1P-stimulated cell migration requires the interaction of C1P with a putative C1P receptor, and the subsequent activation of the PI3K/Akt, MAPK/Erk1-2, p38 and JNK signalling pathways. Finally, this Thesis also shows that C1P increases the expression of CD69 in spleenic mononuclear cells, and that it protects these cells from entering apoptosis. A major pathway involved in this action is the mitogen-activated protein kinase kinase (MEK)/ERK1-2/ NF-кB pathway.

In the last two decades there has been considerable progress in our understanding of the role of sphingolipids in controlling signal transduction processes, particularly in the mechanisms leading to regulation of cell growth and death. Ceramide is a well-characterized sphingolipid metabolite and second messenger that can be produced by cancer cells in response to a variety of stimuli, including therapeutic drugs, leading to cell cycle arrest and apoptosis. Although this is a promising aspect when thinking of treating cancer, it should be borne in mind that ceramide production may not always be a growth inhibitory or pro-apoptotic signal. In fact, ceramide can be readily converted to sphingosine 1-phosphate (S1P) by the concerted actions of ceramidases and sphingosine kinases, or to ceramide 1-phosphate (C1P) by the action of ceramide kinase. In general, S1P and C1P have opposing effects to ceramide, acting as pro-survival or mitogenic signals in most cell types. This review will address our current understanding of the many roles of ceramide, S1P and C1P in the regulation of cell growth and survival with special emphasis to the emerging role of these molecules and their metabolizing enzymes in controlling tumor progression and metastasis.

Ceramide (N-acylsphingosine) forms the backbone of all complex sphingolipids. It is composed of the long-chain sphingoid base, sphingosine, in N-linkage to a variety of acyl groups (varying in length from C14 to C26) (Fig. 8.1). In addition to serving as a precursor to complex sphingolipids, ceramide is a potent signaling molecule capable of regulating vital cellular functions.

It has been over a decade since the sphingolipid ceramide‐1‐phosphate (C1P) was described. Until recently, only sparse reports on possible biological functions for this lipid have been published. A large number of reports have now surfaced demonstrating distinct biological mechanisms regulated by C1P produced from ceramide kinase (CERK). In the following methods chapter, the methodologies for examining CERK function in vitro and in cells are outlined in detail. The methodologies for examining C1P levels and the use of exogenous C1P on cells to observe lipid specific effects on a particular biology are also detailed.

It has been over a decade since ceramide kinase (CERK) and its product, ceramide-1-phosphate (C1P), were first reported. Since itscloning, in 2002, CERK has been the subject of an explosion of publications concerning various signal transduction pathways. The roles of this previously overlooked enzyme, as well as those of its product C1P, continue to expand, and their regulatory functions in the production of eicosanoid inflammatory mediators are proving essential to fundamental signal transduction pathways. In particular, C1P is required for the activation and translocation of cPLA(2)alpha, the initial rate-limiting step of eicosanoid synthesis. The potential for inhibitors of CERK to offer a new generation of anti-inflammatory and anti-cancer therapeutics is especially deserving of further study.

In 2004 the WHO estimated that 64 million people worldwide suffered from chronic obstructive pulmonary disease (COPD), with 3 million dying from related complications in 2005. Although many patients go undiagnosed, the number of people with the disease continues to increase, and by 2030 it is thought it will be the third most common cause of death in the world. With no cure for COPD, treatment of symptoms increased the European health bill by USD 10 million last year and the market is thought to increase to approximately USD 37.7 million by 2030. These staggering numbers were presented by Frank Thielman (Novartis Pharma) as he opened SMi's Seventh Annual COPD Conference in London, U.K. The meeting gathered experts and scientific pioneers in the respiratory area to discuss developments in known and new therapies, devices and diagnosis, including biomarker models to gain greater disease understanding. This report covers highlights from the 2-day meeting.

Ceramide and sphingosine and their phosphorylated counterparts are recognized as “bioactive sphingolipids” and modulate membrane integrity, the activity of enzymes, or act as ligands of G protein-coupled receptors. The subcellular distribution of the bioactive sphingolipids is central to their function as the same lipid can mediate diametrically opposite effects depending on its location. To ensure that these lipids are present in the right amount and in the appropriate organelles, cells employ selective lipid transport and compartmentalize sphingolipid-metabolizing enzymes to characteristic subcellular sites. Our knowledge of key mechanisms involved in sphingolipid signaling and trafficking has increased substantially in the past decades–- thanks to advances in biochemical and cell biological methods. In this review, we focus on the bioactive sphingolipids and discuss how the combination of studies in cells and in model membranes have contributed to our understanding of how they behave and function in living organisms.

Non-isosteric phosphonate analogs of sphingomyelin and ceramide 1-phosphate are being synthesized from the condensation product of a pentacovalent oxaphospholene and azodicarboxylates. Model studies are initially described.

Macrophages play vital roles in inflammatory responses, and their number at sites of inflammation is strictly regulated by cell death and division. Here, we demonstrate that production of nitric oxide (NO) is a major mechanism whereby ceramide-1-phosphate (C1P) blocks apoptosis in macrophages. However, NO failed to stimulate macrophage proliferation. The prosurvival effect of C1P was blocked by inhibitors of inducible NO synthase. The antiapoptotic effect of C1P was also blocked by phosphatidylinositol 3-kinase or nuclear factor-kappa B inhibitors. Moreover, NO reversed the inhibitory effect of C1P on acid sphingomyelinase, but the prosurvival effect of C1P was independent of this action.