Sphingolipid Pathway Research Articles

Cholesterol regulation of rab-mediated sphingolipid endocytosis

Despite a tight regulation of its intracellular content, cholesterol is found accumulated in pathological conditions such as sphingolipidosis as well as after cell treatment with drugs like hydrophobic amines. Furthermore, cellular cholesterol increases when cultured cells approach confluence. Under these conditions, the endocytic pathways of plasma membrane sphingolipids are differently affected. In this short review, we will summarize recent results from our laboratory as well as those of other groups, indicating that the intracellular accumulation of cholesterol inhibits the dissociation of rab GTPases from the target membranes, causing the alteration of rab-mediated membrane traffic.

A fluorescent glycolipid-binding peptide probe traces cholesterol dependent microdomain-derived trafficking pathways.

BackgroundThe uptake and intracellular trafficking of sphingolipids, which self-associate into plasma membrane microdomains, is associated with many pathological conditions, including viral and toxin infection, lipid storage disease, and neurodegenerative disease. However, the means available to label the trafficking pathways of sphingolipids in live cells are extremely limited. In order to address this problem, we have developed an exogenous, non-toxic probe consisting of a 25-amino acid sphingolipid binding domain, the SBD, derived from the amyloid peptide Aβ, and conjugated by a neutral linker with an organic fluorophore. The current work presents the characterization of the sphingolipid binding and live cell trafficking of this novel probe, the SBD peptide. SBD was the name given to a motif originally recognized by Fantini et al [1] in a number of glycolipid-associated proteins, and was proposed to interact with sphingolipids in membrane microdomains.Methodology/Principal FindingsIn accordance with Fantini's model, optimal SBD binding to membranes depends on the presence of sphingolipids and cholesterol. In synthetic membrane binding assays, SBD interacts preferentially with raft-like lipid mixtures containing sphingomyelin, cholesterol, and complex gangliosides in a pH-dependent manner, but is less glycolipid-specific than Cholera toxin B (CtxB). Using quantitative time-course colocalization in live cells, we show that the uptake and intracellular trafficking route of SBD is unlike that of either the non-raft marker Transferrin or the raft markers CtxB and Flotillin2-GFP. However, SBD traverses an endolysosomal route that partially intersects with raft-associated pathways, with a major portion being diverted at a late time point to rab11-positive recycling endosomes. Trafficking of SBD to acidified compartments is strongly disrupted by cholesterol perturbations, consistent with the regulation of sphingolipid trafficking by cholesterol.Conclusions/SignificanceThe current work presents the characterization and trafficking behavior of a novel sphingolipid-binding fluorescent probe, the SBD peptide. We show that SBD binding to membranes is dependent on the presence of cholesterol, sphingomyelin, and complex glycolipids. In addition, SBD targeting through the endolysosomal pathway in neurons is highly sensitive to cholesterol perturbations, making it a potentially useful tool for the analysis of sphingolipid trafficking in disease models that involve changes in cholesterol metabolism and storage.

Molecular profiling of LGL leukemia reveals role of sphingolipid signaling in survival of cytotoxic lymphocytes

AbstractT-cell large granular lymphocyte (LGL) leukemia is characterized by clonal expansion of CD3+CD8+ cells. Leukemic LGLs correspond to terminally differentiated effector-memory cytotoxic T lymphocytes (CTLs) that escape Fas-mediated activation-induced cell death (AICD) in vivo. The gene expression signature of peripheral blood mononuclear cells from 30 LGL leukemia patients showed profound dysregulation of expression of apoptotic genes and suggested uncoupling of activation and apoptotic pathways as a mechanism for failure of AICD in leukemic LGLs. Pathway-based microarray analysis indicated that balance of proapoptotic and antiapoptotic sphingolipid-mediated signaling was deregulated in leukemic LGLs. We further investigated sphingolipid pathways and found that acid ceramidase was constitutively overexpressed in leukemic LGLs and that its inhibition induced apoptosis of leukemic LGLs. We also showed that S1P5 is the predominant S1P receptor in leukemic LGLs, whereas S1P1 is down-regulated. FTY720, a functional antagonist of S1P-mediated signaling, induced apoptosis in leukemic LGLs and also sensitized leukemic LGLs to Fas-mediated death. Collectively, these results show a role for sphingolipid-mediated signaling as a mechanism for long-term survival of CTLs. Therapeutic targeting of this pathway, such as use of FTY720, may have efficacy in LGL leukemia.

Regulation of Neutral Sphingomyelinase-2 (nSMase2) by Tumor Necrosis Factor-α Involves Protein Kinase C-δ in Lung Epithelial Cells

Neutral sphingomyelinases (N-SMases) are major candidates for stress-induced ceramide production, but there is still limited knowledge of the regulatory mechanisms of the cloned N-SMase enzyme-nSMase2. We have reported that p38 mitogen-activated protein kinase (MAPK) was upstream of nSMase2 in tumor necrosis-alpha (TNF-alpha)-stimulated A549 cells ( J Biol Chem 282: 1384-1396, 2007 ). Here, we report a role for protein kinase C (PKC) in mediating TNF-induced translocation of nSMase2 from the Golgi to the plasma membrane (PM). Pharmacological inhibition of PKCs prevented TNF-stimulated nSMase2 translocation to the PM in A549 cells. Using phorbol 12-myristate 13-acetate (PMA) as a tool to dissect PKC responses, we found that PMA induced nSMase2 translocation to the PM in a time- and dose-dependent manner. Pharmacological inhibitors and specific siRNA implicated the novel PKCs, specifically PKC-delta, in both TNF and PMA-stimulated nSMase2 translocation. However, PMA did not increase in vitro N-SMase activity and PKC-delta did not regulate TNF-induced N-SMase activity. Furthermore, PKC-delta and nSMase2 did not coimmunoprecipitate, suggesting that other signaling proteins may be involved. PMA-stimulated nSMase2 translocation was independent of p38 MAPK, and neither PKC inhibitors nor small interfering RNA had significant effects on TNF-stimulated p38 MAPK activation, indicating that PKC-delta does not act through p38 MAPK in regulating nSMase2. Finally, down-regulation of PKC-delta inhibited induction of vascular cell and intercellular adhesion molecules, previously identified as downstream of nSMase2 in A549 cells. Taken together, these data implicate PKC-delta as a regulator of nSMase2 and, for the first time, identify nSMase2 as a point of cross-talk between the PKC and sphingolipid pathways.

Opposite Effects of Dihydrosphingosine 1-Phosphate and Sphingosine 1-Phosphate on Transforming Growth Factor-β/Smad Signaling Are Mediated through the PTEN/PPM1A-dependent Pathway

Transforming growth factor-beta (TGF-beta) is an important regulator of physiological connective tissue biosynthesis and plays a central role in pathological tissue fibrosis. Previous studies have established that a biologically active lipid mediator, sphingosine 1-phosphate (S1P), mimics some of the profibrotic functions of TGF-beta through cross-activation of Smad signaling. Here we report that another product of sphingosine kinase, dihydrosphingosine 1-phosphate (dhS1P), has an opposite role in the regulation of TGF-beta signaling. In contrast to S1P, dhS1P inhibits TGF-beta-induced Smad2/3 phosphorylation and up-regulation of collagen synthesis. The effects of dhS1P require a lipid phosphatase, PTEN, a key modulator of cell growth and survival. dhS1P stimulates phosphorylation of the C-terminal domain of PTEN and its subsequent translocation into the nucleus. We demonstrate a novel function of nuclear PTEN as a co-factor of the Smad2/3 phosphatase, PPM1A. Complex formation of PTEN with PPM1A does not require the lipid phosphatase activity but depends on phosphorylation of the serine/threonine residues located in the C-terminal domain of PTEN. Upon complex formation with PTEN, PPM1A is protected from degradation induced by the TGF-beta signaling. Consequently, overexpression of PTEN abrogates TGF-beta-induced Smad2/3 phosphorylation. This study establishes a novel role for nuclear PTEN in the stabilization of PPM1A. PTEN-mediated cross-talk between the sphingolipid and TGF-beta signaling pathways may play an important role in physiological and pathological TGF-beta signaling.

Coordination of the dynamics of yeast sphingolipid metabolism during the diauxic shift

BackgroundThe diauxic shift in yeast requires cells to coordinate a complicated response that involves numerous genes and metabolic processes. It is unknown whether responses of this type are mediated in vivo through changes in a few "key" genes and enzymes, which are mathematically characterized by high sensitivities, or whether they are based on many small changes in genes and enzymes that are not particularly sensitive. In contrast to global assessments of changes in gene or protein interaction networks, we study here control aspects of the diauxic shift by performing a detailed analysis of one specific pathway–sphingolipid metabolism–which is known to have signaling functions and is associated with a wide variety of stress responses.ResultsThe approach uses two components: publicly available sets of expression data of sphingolipid genes and a recently developed Generalized Mass Action (GMA) mathematical model of the sphingolipid pathway. In one line of exploration, we analyze the sensitivity of the model with respect to enzyme activities, and thus gene expression. Complementary to this approach, we convert the gene expression data into changes in enzyme activities and then predict metabolic consequences by means of the mathematical model. It was found that most of the sensitivities in the model are low in magnitude, but that some stand out as relatively high. This information was then deployed to test whether the cell uses a few of the very sensitive pathway steps to mount a response or whether the control is distributed throughout the pathway. Pilot experiments confirm qualitatively and in part quantitatively the predictions of a group of metabolite simulations.ConclusionThe results indicate that yeast coordinates sphingolipid mediated changes during the diauxic shift through an array of small changes in many genes and enzymes, rather than relying on a strategy involving a few select genes with high sensitivity. This study also highlights a novel approach in coupling data mining with mathematical modeling in order to evaluate specific metabolic pathways.

MAO-A-induced mitogenic signaling is mediated by reactive oxygen species, MMP-2, and the sphingolipid pathway

The degradation of biogenic amines by monoamine oxidase A (MAO-A) generates reactive oxygen species (ROS) which participate in serotonin and tyramine signaling. This study aimed to investigate the role of ROS in the mitogenic signaling activated during tyramine and serotonin oxidation by MAO-A in smooth muscle cells (SMC). Incubation of SMC with serotonin or tyramine induced intracellular ROS generation, and a signaling cascade involving metalloproteases and the neutral sphingomyelinase-2 (nSMase2, the initial step of the sphingolipid pathway), ERK1/2 phosphorylation, and DNA synthesis. Silencing MAO-A by siRNA, pharmacological MAO-A inhibitors (pargyline and Ro41-1049), and the antioxidant/ROS scavenger butylated hydroxytoluene (BHT) inhibited the signaling cascade, suggesting that ROS generated during tyramine oxidation by MAO-A are required. The MMP inhibitor Batimastat, MMP2-specific siRNA, and MMP2 deletion (MMP2 −/− fibroblasts) blocked nSMase activation and SMC proliferation, suggesting a role for MMP2 in this signaling pathway. Silencing nSMase2 by siRNA did not inhibit ROS generation and MMP2 activation, but blocked SMC proliferation induced by tyramine, suggesting that nSMase2 is downstream MMP2. These findings demonstrate that H 2O 2-generated during tyramine oxidation by MAO-A triggers a stress-induced mitogenic signaling via the MMP2/sphingolipid pathway, which could participate in excessive remodeling and alteration of the vascular wall.

Endogenous sphingolipid metabolites related to the growth inSphingomonas chungbukensis

Sphingolipids are present in animals, plants, fungi, yeasts and some bacteria. In mammalian cells sphingolipids act as lipid mediators for cell growth, differentiation, apoptosis and angiogenesis. In contrast, in bacteria the biological significance of sphingolipids has not been fully elucidated and sphingolipid metabolism has not been investigated. The aim of this study was to compare the pattern of sphingolipid metabolites in HIT-T15 beta cells originating from hamster pancreas to that in the bacterial strain Sphingomonas chungbukensis DJ77, under various culture conditions. It was found that the concentration of cellular sphinganine (Sa) in S. chungbukensis was higher than that of sphingosine (So), while the level of cellular So in HIT-T15 cells was higher than that of Sa. Aeration and shaking during culture increased bacterial growth in S. chungbukensis, and the contents of So and Sa were also elevated. These results indicate that a de novo sphingolipid pathway appeared to be active in bacteria and that bacterial growth may be closely related to Sa levels.

Redirection of sphingolipid metabolism toward de novo synthesis of ethanolamine in Leishmania

In most eukaryotes, sphingolipids (SLs) are critical membrane components and signaling molecules. However, mutants of the trypanosomatid protozoan Leishmania lacking serine palmitoyltransferase (spt2-) and SLs grow well, although they are defective in stationary phase differentiation and virulence. Similar phenotypes were observed in sphingolipid (SL) mutant lacking the degradatory enzyme sphingosine 1-phosphate lyase (spl-). This epistatic interaction suggested that a metabolite downstream of SLs was responsible. Here we show that unlike other organisms, the Leishmania SL pathway has evolved to be the major route for ethanolamine (EtN) synthesis, as EtN supplementation completely reversed the viability and differentiation defects of both mutants. Thus Leishmania has undergone two major metabolic shifts: first in de-emphasizing the metabolic roles of SLs themselves in growth, signaling, and maintenance of membrane microdomains, which may arise from the unique combination of abundant parasite lipids; Second, freed of typical SL functional constraints and a lack of alternative routes to produce EtN, Leishmania redirected SL metabolism toward bulk EtN synthesis. Our results thus reveal a striking example of remodeling of the SL metabolic pathway in Leishmania.

SVF1 Regulates Cell Survival by Affecting Sphingolipid Metabolism in Saccharomyces cerevisiae

Sphingolipid signaling plays an important role in the regulation of central cellular processes, including cell growth, survival, and differentiation. Many of the essential pathways responsible for sphingolipid biogenesis, and key cellular responses to changes in sphingolipid balance, are conserved between mammalian and yeast cells. Here we demonstrate a novel function for the survival factor Svf1p in the yeast sphingolipid pathway and provide evidence that Svf1p regulates the generation of a specific subset of phytosphingosine. Genetic analyses suggest that Svf1p acts in concert with Lcb4p and Lcb3p to generate a localized pool of phytosphingosine distinct from phytosphingosine generated by Sur2p. This subset is implicated in cellular responses to stress, as loss of SVF1 is associated with defects in the diauxic shift and the oxidative stress response. A genetic interaction between SVF1 and SUR2 demonstrates that both factors are required for optimal growth and survival, and phenotypic similarities between svf1delta sur2delta and ypk1delta suggest that pathways controlled by Svf1p and Sur2p converge on a signaling cascade regulated by Ypk1p. Loss of YPK1 together with disruption of either SVF1 or SUR2 is lethal. Together, these data suggest that compartmentalized generation of distinct intracellular subsets of sphingoid bases may be critical for activation of signaling pathways that control cell growth and survival.

Necessary Role for the Lag1p Motif in (Dihydro)ceramide Synthase Activity

Lag1 (longevity assurance gene 1) homologues, a family of transmembrane proteins found in all eukaryotes, have been shown to be necessary for (dihydro)ceramide synthesis. All Lag1 homologues contain a highly conserved stretch of 52 amino acids known as the Lag1p motif. However, the functional significance of the conserved Lag1p motif for (dihydro)ceramide synthesis is currently unknown. In this work, we have investigated the function of the motif by introducing eight point mutations in the Lag1p motif of the mouse LASS1 (longevity assurance homologue 1 of yeast Lag1). The (dihydro)ceramide synthase activity of the mutants was tested using microsomes in HeLa cells and in vitro. Six of the mutations resulted in loss of activity in cells and in vitro. In addition, our results showed that C18:0 fatty acid CoA (but not cis-C18:1 fatty acid CoAs) are substrates for LASS1 and that LASS1 in HeLa cells is sensitive to fumonisin B1, an in vitro inhibitor of (dihydro)ceramide synthase. Moreover, we mutated the Lag1p motif of another Lag homologue, human LASS5. The amino acid substitutions in the human LASS5 were the same as in mouse LASS1, and had the same effect on the in vitro activity of LASS5, suggesting the Lag1p motif appears to be essential for the enzyme activity of all Lag1 homologues.

How do sphingolipids and lipid rafts relate to pathology?

European Journal of Lipid Science and TechnologyVolume 108, Issue 10 p. 799-801 EditorialFree Access How do sphingolipids and lipid rafts relate to pathology? Gerrit van Meer, Gerrit van Meer Bijvoet Center and Institute of Biomembranes, Utrecht University, Utrecht, The NetherlandsSearch for more papers by this author Gerrit van Meer, Gerrit van Meer Bijvoet Center and Institute of Biomembranes, Utrecht University, Utrecht, The NetherlandsSearch for more papers by this author First published: 09 October 2006 https://doi.org/10.1002/ejlt.200600184Citations: 1AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. References 1 S. Hakomori: Glycosphingolipids in cellular interaction, differentiation, and oncogenesis. Annu Rev Biochem. 1981, 50, 733– 764. 2 K. Powell: Lipid raft idea is floated. J Cell Biol. 2006, 172, 166– 167. 3 A. H. Futerman, Y. A. Hannun: The complex life of simple sphingolipids. EMBO Rep. 2004, 5, 777– 782. 4 C. S. Ejsing, T. Moehring, U. Bahr, E. Duchoslav, M. Karas, K. Simons, A. Shevchenko: Collision-induced dissociation pathways of yeast sphingolipids and their molecular profiling in total lipid extracts: a study by quadrupole TOF and linear ion trap-orbitrap mass spectrometry. J Mass Spectrom. 2006, 41, 372– 389. 5 B. Brügger, B. Glass, P. Haberkant, I. Leibrecht, F. T. Wieland, H. G. Krausslich: The HIV lipidome: a raft with an unusual composition. Proc Natl Acad Sci U S A. 2006, 103, 2641– 2646. 6 K. I. Willig, S. O. Rizzoli, V. Westphal, R. Jahn, S. W. Hell: STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis. Nature. 2006, 440, 935– 939. 7 F. Spener, S. D. Kohlwein, G. Schmitz: Lipid droplets and lamellar bodies – from innocent bystanders to prime targets of lipid research for combating human diseases. Eur J Lipid Sci Technol. 2006, 108, 541– 543. 8 F. Spener, R. Zechner, J. Borlak: Is lipotoxicity an oxymoron? Eur J Lipid Sci Technol. 2006, 108, 625– 627. 9 W. J. Griffiths: Why steroidomics in brain? Eur J Lipid Sci Technol. 2006, 108, 707– 708. Citing Literature Volume108, Issue10No. 10 October 2006Pages 799-801 ReferencesRelatedInformation

Hydrogen peroxide induces S1P1 receptors and sensitizes vascular endothelial cells to sphingosine 1-phosphate, a platelet-derived lipid mediator

Sphingosine 1-phosphate (S1P) is a platelet-derived angiogenic lipid growth factor, modulating G-protein-coupled S1P(1) receptors (S1P(1)-R) to activate endothelial nitric oxide synthase (eNOS), as well as MAPK pathways in endothelial cells. We explored whether and how hydrogen peroxide (H(2)O(2)), a representative reactive oxygen species, alters S1P(1)-R expression and influences S1P signaling in cultured bovine aortic endothelial cells (BAECs). When BAECs are treated with pathophysiologically relevant concentrations of H(2)O(2) (150 microM for 30 min), S1P(1)-R protein expression levels are acutely augmented by approximately 30-fold in a dose-dependent fashion. When BAECs have been pretreated with H(2)O(2), subsequent S1P stimulation (100 nM) leads to a higher degree of eNOS enzyme activation (assessed as intracellular cGMP content, 1.7 +/- 0.2-fold vs. no H(2)O(2) pretreatment groups, P < 0.05), associated with a higher magnitude of phosphorylation responses of eNOS and MAPK ERK1/2. PP2, an inhibitor of Src-family tyrosine kinase, abolished the effects of H(2)O(2) on both S1P(1)-R protein upregulation and enhanced BAEC responses to S1P. H(2)O(2) does not augment S1P(1) mRNA expression, whereas VEGF under identical cultures leads to increases in S1P(1) mRNA signals. Whereas H(2)O(2) attenuates proliferation of BAECs, addition of S1P restores growth responses of these cells. These results demonstrate that extracellularly administered H(2)O(2) increases S1P(1)-R expression and promotes endothelial responses for subsequent S1P treatment. These results may identify potentially important points of cross-talk between reactive oxygen species and sphingolipid pathways in vascular responses.

Recent anticancer agents targeting sphingolipid pathways

Sphingolipids are a diverse family of molecules that affect multiple cell signalling pathways to control cell proliferation, survival, vascularisation, migration and interactions with other cells, including those of the immune system. Nearly all categories of sphingolipids are relevant to cancer based on their biological functions; however, there is considerable experimental evidence for the anticancer efficacy of the sphingoid base and ceramide backbones of sphingolipids in both cell culture and in vivo systems, plus an increase in the patent literature dealing with the use of these compounds, analogues or modulators of their endogenous production, to control cancer and other proliferative disorders. This review describes the structural features of these compounds, their potential mechanisms of action and some aspects of their formulation, uptake and transport, metabolism and activities as a new category of anticancer agents.

Sphingolipids as Targets for Microbial Infections

Sphingolipids had long been regarded as merely structural components of eukaryotic cellular membranes. Research has discovered sphingolipids to have crucial roles in cellular processes as bioactive molecules. Lately, there has also been an increased interest in sphingolipids and sphingolipid-metabolizing enzymes as mediators of microbial pathogenicity and as potential targets for the development of new therapeutics. This minireview will provide a comprehensive analysis of sphingolipid pathways in mammalian and microbial cells, highlighting their uniqueness and discussing their potential as therapeutic targets for microbial infections.

Collision‐induced dissociation pathways of yeast sphingolipids and their molecular profiling in total lipid extracts: a study by quadrupole TOF and linear ion trap–orbitrap mass spectrometry

The yeast Saccharomyces cerevisiae synthesizes three classes of sphingolipids: inositolphosphoceramides (IPCs), mannosyl-inositolphosphoceramides (MIPCs), and mannosyl-diinositolphosphoceramides (M(IP)2C). Tandem mass spectrometry of their molecular anions on a hybrid quadrupole time-of-flight (QqTOF) instrument produced fragments of inositol-containing head groups, which were specific for each lipid class. MS(n) analysis performed on a hybrid linear ion trap-orbitrap (LTQ Orbitrap) mass spectrometer with better than 3 ppm mass accuracy identified fragment ions specific for the amide-linked fatty acid and the long chain base moieties in individual molecular species. By selecting m/z of class-specific fragment ions for multiple precursor ion scanning, we profiled yeast sphingolipids in total lipid extracts on a QqTOF mass spectrometer. Thus, a combination of QqTOF and LTQ Orbitrap mass spectrometry lends itself to rapid, comprehensive and structure-specific profiling of the molecular composition of sphingolipids and glycerophospholipids in important model organisms, such as fungi and plants.

Essential requirement for sphingosine kinase activity in eNOS-dependent NO release and vasorelaxation.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that acts both as an extracellular ligand for endothelial differentiation gene receptor family and as an intracellular second messenger. Cellular levels of S1P are low and tightly regulated by sphingosine kinase (SPK). Recent studies have suggested that eNOS pathway may function as a downstream target for the biological effects of receptor-mediated S1P. Here we have studied the possible interplay between intracellular SIP generation and the eNOS activation pathway. S1P causes an endothelium-dependent vasorelaxation in rat aorta that is PTX sensitive, inhibited by L-NAME that involves eNOS phosphorylation, and mainly dependent on hsp90. When rat aorta rings were incubated with the SPK inhibitor DL-threo-dihydrosphingosine (DTD), there was a concentration-dependent reduction of Ach-induced vasorelaxation, implying a consistent contribution of sphingolipid pathway through intracellular sphingosine release and phosphorylation. Co-immunoprecipitation experiments consistently showed increased association of hsp90 with eNOS after exposure of cells to S1P as well to BK or calcium ionophore A-23187. Interestingly, as opposite to A-23187, BK and S1P effect were significantly inhibited by pretreatment with the SPK inhibitor DTD. In conclusion, our data demonstrate that an interplay exists among eNOS, hsp90, and intracellularly generated S1P where eNOS coupling to hsp90 is a major determinant for NO release as confirmed by our functional and molecular studies.

Sphingosine-1-Phosphate Receptor Expression and Signaling Correlate with Uterine Prostaglandin-Endoperoxide Synthase 2 Expression and Angiogenesis During Early Pregnancy1

Signaling mechanisms coordinating uterine angiogenesis and tissue remodeling during decidualization are not completely understood. Prostanoid signaling is thought to play a functionally important role in each of these events. In the present study, we demonstrate that the subfamily of G-protein-coupled receptors that binds and becomes activated by the terminal signaling lipid in the sphingolipid pathway, sphingosine-1-phosphate (S1P), were expressed during uterine decidualization. Three of the five known S1P receptors, termed endothelial differentiation genes (Edg; Edg1, Edg3, and Edg5) were upregulated in the uterine deciduum from Day of Pregnancy (DOP) 4.5 to 7.5, while Edg6 and Edg8 expression remained unchanged. Consistent with angiogenesis in general during decidualization, we believe EDG1 and EDG5 to be regulated by the embryo because no microvascular expression for these receptors was observed in oil-induced deciduomas. Observed expression of EDG1 and EDG5 showed a similar expression pattern to that previously reported for prostaglandin-endoperoxide synthase 2 (PTGS2), transitioning from the sublumenal stromal compartment in the antimesometrial pole (DOP 5) to the microvasculature of the mesometrial pole (DOP 7). Furthermore, these two receptors colocalized with PTGS2 at three additional sites at the maternal:fetal interface throughout pregnancy. Treatment of cultured predecidualized stromal cells with S1P resulted in upregulation of Ptgs2 mRNA and PTGS2 protein, but not the downstream enzyme prostacyclin synthase. These combined results suggest the existence of a link between the sphingolipid and prostanoid signaling pathways in uterine physiology, and that, based on their expression pattern, S1P receptors function to coordinate uterine mesometrial angiogenesis during the implantation phase of early gestation.

Cytotoxicity of an Anti-cancer Lysophospholipid through Selective Modification of Lipid Raft Composition

Edelfosine is a prototypical member of the alkylphosphocholine class of antitumor drugs. Saccharomyces cerevisiae was used to screen for genes that modulate edelfosine cytotoxicity and identified sterol and sphingolipid pathways as relevant regulators. Edelfosine addition to yeast resulted in the selective partitioning of the essential plasma membrane protein Pma1p out of lipid rafts. Microscopic analysis revealed that Pma1p moved from the plasma membrane to intracellular punctate regions and finally localized to the vacuole. Consistent with altered sterol and sphingolipid synthesis resulting in increased edelfosine sensitivity, mislocalization of Pma1p was preceded by the movement of sterols out of the plasma membrane. Cells with enfeebled endocytosis and vacuolar protease activities prevented edelfosine-mediated (i) mobilization of sterols, (ii) loss of Pma1p from lipid rafts, and (iii) cell death. The activities of proteins and signaling processes are meaningfully altered by changes in lipid raft biophysical properties. This study points to a novel mode of action for an anti-cancer drug through modification of plasma membrane lipid composition resulting in the displacement of an essential protein from lipid rafts.

Using genomic and lipidomic strategies to investigate sphingolipid function in the yeast heat-stress response

In addition to their crucial role in membrane structure in Saccharomyces cerevisiae, sphingolipids serve vital roles in various aspects of yeast biology including endocytosis, intracellular protein transport and stress responses. Although previous studies have unequivocally demonstrated the sphingolipid requirements for these processes, few studies have contributed mechanistic information. We have used a systems approach including microarray, lipidomics and metabolic modelling to better understand (i) biochemical relationships between various branches of sphingolipid metabolism and pathways and contributing pathways such as fatty acid metabolism and phospholipid synthesis, (ii) the changes in cellular sphingolipid composition under various conditions and (iii) the effects of these changes on the transcriptional profiles and subsequently, cell phenotypes. Thus far, these approaches have indicated roles for sphingolipids in major transcriptional changes in response to heat stress, carbon source utilization, sporulation, cell wall integrity and other basic cellular functions. Although the yeast genome is fully sequenced, nearly 50% of all transcribed open reading frames remain uncharacterized with regard to cellular function; therefore, a major advantage of this approach is the ability to identify both biochemical and biological roles for enzymes and their products within broad cellular contexts.