Biomedical subjects

Charles E Chalfant

Publications and source records attributed to Charles E Chalfant.

At least 19 recordsLinked to original sources

PubMed2006

Ceramide-activated protein phosphatase involvement in insulin resistance via Akt, serine/arginine-rich protein 40, and ribonucleic acid splicing in L6 skeletal muscle cells.

Elevated TNFalpha levels are associated with insulin resistance, but the molecular mechanisms linking cytokine signaling to impaired insulin function remain elusive. We previously demonstrated a role for Akt in insulin regulation of protein kinase CbetaII alternative splicing through phosphorylation of serine/arginine-rich protein 40, a required mechanism for insulin-stimulated glucose uptake. We hypothesized that TNFalpha attenuated insulin signaling by dephosphorylating Akt and its targets via ceramide-activated protein phosphatase. Western blot analysis of L6 cell lysates demonstrated impaired insulin-stimulated phosphorylation of Akt, serine/arginine-rich protein 40, and glycogen synthase kinase 3beta in response to TNFalpha and the short chain C6 ceramide analog. TNFalpha increased serine/threonine phosphatase activity of protein phosphatase 1 (PP1) in response to C6, but not insulin, suggesting a ceramide-specific effect. Myriocin, an inhibitor of de novo ceramide synthesis, blocked stimulation of the PP1 activity. Ceramide species measurement by liquid chromatography-mass spectrometry showed consistent increases in C24:1 and C16 ceramides. Effects of TNFalpha and C6 on insulin-stimulated phosphorylation of glycogen synthase kinase 3beta were prevented by myriocin and tautomycin, a PP1 inhibitor, further implicating a de novo ceramide-PP1 pathway. Alternative splicing assays demonstrated that TNFalpha abolished insulin-mediated inclusion of the protein kinase CbetaII exon. Collectively, our work demonstrates a role for PP1-like ceramide-activated protein phosphatase in mediating TNFalpha effects blocking insulin phosphorylation cascades involved in glycogen metabolism and alternative splicing.

Alternative Splicing↗

PubMed2006

The immunosuppressant drug FTY720 inhibits cytosolic phospholipase A2 independently of sphingosine-1-phosphate receptors.

FTY720 is a potent immunomodulator drug that inhibits the egress of lymphocytes from secondary lymphoid tissues and thymus. FTY720 is phosphorylated in vivo by sphingosine kinase 2 to FTY720-phosphate, which acts as a potent sphingosine-1-phosphate (S1P) receptor agonist. However, in contrast to S1P, FTY720 has no effect on mast-cell degranulation, yet significantly reduces antigen-induced secretion of PGD2 and cysteinyl-leukotriene. Unexpectedly, this effect of FTY720 was independent of its phosphorylation and S1P receptor functions. The rate-limiting step in the biosynthesis of all eicosanoids is the phospholipase A2 (PLA2)-mediated release of arachidonic acid from glycerol phospholipids. Although FTY720 also reduced arachidonic acid release in response to antigen, it had no effect on translocation of cPLA2 or ERK1/2 activation, suggesting that it does not interfere with FcepsilonRI-mediated events leading to cPLA2 activation. Remarkably, however, FTY720 drastically inhibited recombinant cPLA2alpha activity, whereas FTY720-phosphate, sphingosine, or S1P had no effect. This study has uncovered a unique action of FTY720 as an inhibitor of cPLA2alpha and hence on production of all eicosanoids. Our results have important implications for the potential therapeutic mechanism of action of FTY720 in eicosanoid-driven inflammatory disorders such as asthma and multiple sclerosis.

Animals↗

PubMed2006

SAP155 Binds to ceramide-responsive RNA cis-element 1 and regulates the alternative 5' splice site selection of Bcl-x pre-mRNA.

Two splice variants are derived from the BCL-x gene, proapoptotic Bcl-x(s) and antiapoptotic Bcl-x(L), via alternative 5' splice site selection. In previous studies, our laboratory identified an RNA cis-element within exon 2 of Bcl-x pre-mRNA that is a ceramide responsive termed CRCE 1. In this study, mass spectrometric analysis identified the splicing factor SAP155, as an RNA trans-acting factor binding to the purine-rich CRCE 1. The interaction of SAP155 with CRCE 1 was confirmed by the addition of an anti-SAP155 antibody (Ab) to EMSA decreasing the mobility of a protein:CRCE 1 complex (SuperShift). Furthermore, the down-regulation of SAP155 in A549 cells by RNA interference (RNAi) technology resulted in the loss of a 155 kDa protein complexed with CRCE 1. Moreover, this down-regulation of SAP155 induced an increase in the Bcl-x(s) with a concomitant decrease in the Bcl-x(L) splice variants and immunoreactive protein levels, thereby decreasing the Bcl-x(L)/Bcl-x(s) ratio. Specific down-regulation of SAP155 also inhibited the ability of exogenous ceramide treatment to further induce the activation of the Bcl-x(s) 5' splice site. Additionally, the specific down-regulation of SAP155 sensitized cells to undergo apoptosis in response to daunorubicin in a manner similar to ceramide. Therefore, we have identified SAP155 as an RNA trans-acting factor that binds to CRCE 1, functions to regulate the alternative 5' splice site selection of Bcl-x pre-mRNA, and is required for ceramide to induce the activation of the Bcl-x(s) 5' splice site. Furthermore, we have demonstrated that activation of the Bcl-x(s) 5' splice site can increase the effectiveness of chemotherapeutic drug treatment, thus establishing a role for the alternative splicing mechanism of Bcl-x in chemotherapeutic sensitivity.

Antineoplastic Agents↗

PubMed2006

SRp30a (ASF/SF2) regulates the alternative splicing of caspase-9 pre-mRNA and is required for ceramide-responsiveness.

Two splice variants are derived from the caspase-9 gene, proapoptotic caspase-9a and antiapoptotic caspase-9b, by either the inclusion or exclusion of an exon 3, 4, 5, and 6 cassette. Previous studies from our laboratory have shown that the alternative splicing of caspase-9 and the phosphorylation status of SR proteins, a conserved family of splicing factors, are regulated by chemotherapy and ceramide via the action of protein phosphatase-1. In this study, a link between ceramide, SR proteins, and the alternative splicing of caspase-9 was established. The downregulation of SRp30a in A549 cells by RNA interference technology resulted in an increase in the caspase-9b splice variant, with a concomitant decrease in the caspase-9a splice variant, thereby significantly decreasing the caspase-9a/9b ratio from 1.67 +/- 0.11 to 0.56 +/- 0.08 (P < 0.005). The specific downregulation of SRp30a also inhibited the ability of exogenous ceramide treatment to induce the inclusion of the exon 3, 4, 5, and 6 cassette. Therefore, we have identified SRp30a as an RNA trans-acting factor that functions as a major regulator of caspase-9 pre-mRNA processing and is required for ceramide to mediate the alternative splicing of caspase-9.

Alternative Splicing↗

PubMed2006

Fine tuning therapeutic targeting of the sphingolipid biosynthetic pathway to treat atherosclerosis.

The accumulation of sphingolipids, including sphingomyelin and glycosphingolipids, in atherosclerotic lesions is well known. Plasma sphingomyelin concentration is correlated with atherosclerosis development and is an independent predictor of coronary artery disease. Similarly, plasma glycosphingolipid levels are increased in conditions associated with atherosclerosis risk. Recent studies have focused on understanding the mechanisms by which specific intermediates and end-products of the sphingolipid biosynthetic pathway, such as sphingomyelin, glycosphingolipids, ceramide and sphingosine-1-phosphate may modulate vascular biology and atherosclerosis. Here we focus on recent work indicating that pharmacological modulation of the sphingolipid biosynthetic pathway could offer a novel treatment for atherosclerosis or, at the very least, provide mechanistic insights concerning the etiology of this disease which is the major cause of death in developed countries.

Animals↗

PubMed2005

Ceramide regulates SR protein phosphorylation during adenoviral infection.

In this study, we show that adenoviral infection induced accumulation of the sphingolipid ceramide in a dose- and time-dependent manner. This accumulation preceded cell lysis, occurred in the absence of biochemical evidence of apoptosis, and was derived from de novo synthesis of ceramide. An adenovirus mutant that lacks the adenovirus death protein (ADP) produced ceramide accumulation in the absence of cell lysis. This suggested that ceramide accumulation was either driven by adenovirus or was a cellular stress response but was unlikely a result of cell death. The use of inhibitors of ceramide synthesis resulted in a significant delay in cell lysis, suggesting that ceramide was necessary for the lytic phase of the infection. Serine/arginine-rich (SR) proteins were dephosphorylated during the late phase of the viral cycle, and inhibitors of ceramide synthesis reversed this. These findings suggest that adenovirus utilizes the ceramide pathway to regulate SR proteins during infection.

Adenoviridae↗

PubMed2005

Sphingosine 1-phosphate and ceramide 1-phosphate: expanding roles in cell signaling.

The phosphorylated sphingolipid metabolites sphingosine 1-phosphate (S1P) and ceramide 1-phosphate (C1P) have emerged as potent bioactive agents. Recent studies have begun to define new biological functions for these lipids. Generated by sphingosine kinases and ceramide kinase, they control numerous aspects of cell physiology, including cell survival and mammalian inflammatory responses. Interestingly, S1P is involved in cyclooxygenase-2 induction and C1P is required for the activation and translocation of cPLA2. This suggests that these two sphingolipid metabolites may act in concert to regulate production of eicosanoids, important inflammatory mediators. Whereas S1P functions mainly via G-protein-coupled receptors, C1P appears to bind directly to targets such as cPLA2 and protein phosphatase 1/2A. S1P probably also has intracellular targets, and in plants it appears to directly regulate the G protein alpha subunit GPA1.

Cell Movement↗

PubMed2005

Substrate specificity of human ceramide kinase.

Previous studies in our laboratory have established ceramide kinase (CERK) as a critical mediator of eicosanoid synthesis. To date, CERK has not been well characterized in vitro. In this study, we investigated the substrate specificity of CERK using baculovirus-expressed human CERK (6 x His) and a newly designed assay based on mixed micelles of Triton X-100. The results indicate that the ability of CERK to recognize ceramide as a substrate is stereospecific. A minimum of a 12 carbon acyl chain was required for normal CERK activity, and the 4-5 trans double bond was important for substrate recognition. A significant discrimination by CERK was not observed between ceramides with long saturated and long unsaturated fatty acyl chains. Methylation of the primary hydroxyl group resulted in a loss of activity, confirming that CERK produces ceramide-1-phosphate versus ceramide-3-phosphate. In addition, methylation of the secondary hydroxyl group drastically decreased the phosphorylation by CERK. These results also indicated that the free hydrogen of the secondary amide group is critical for substrate recognition. Lastly, the sphingoid chain was also required for substrate recognition by CERK. Together, these results indicate a very high specificity for substrate recognition by CERK, explaining the use of ceramide and not sphingosine or diacylglycerol as substrates.

Amides↗

PubMed2005

The coordination of prostaglandin E2 production by sphingosine-1-phosphate and ceramide-1-phosphate.

The ability of pro-inflammatory cytokines such as interleukin-1beta (IL-1beta) to induce the major inflammatory mediator prostaglandin (PG) E(2) depends on the activation of two rate-limiting enzymes, phospholipase A(2) (PLA(2)) and cyclooxygenase 2 (COX-2). PLA(2) acts to generate arachidonic acid, which serves as the precursor substrate for COX-2 in the metabolic pathway leading to PGE(2) production. However, less is known about the mechanisms that coordinate the regulation of these two enzymes. We have provided prior evidence that sphingosine kinase 1 and its bioactive lipid product sphingosine-1-phosphate (S1P) mediate the effects of cytokines on COX-2 induction, whereas ceramide kinase and its distinct product, ceramide-1-phosphate (C1P), are required for the activation and translocation of cPLA(2) (FASEB J 17:1411-1421. 2003; J Biol Chem 278:38206-38213, 2003; J Biol Chem 279:11320-11326, 2004). Herein, we show that these two pathways are independent but coordinated, resulting in synergistic induction of PGE(2). Moreover, the combination of both S1P and C1P recapitulates the temporal and spatial activation of cPLA(2) and with COX-2 seen IL-1beta. Taken together, the results provide, for the first time, a mechanism that assures the coordinate expression and activation in time and space of COX-2 and cPLA(2), assuring maximal production of PGE(2).

Cell Line, Tumor↗

PubMed2005

Ceramide 1-phosphate acts as a positive allosteric activator of group IVA cytosolic phospholipase A2 alpha and enhances the interaction of the enzyme with phosphatidylcholine.

Previous findings from our laboratory have demonstrated that cPLA(2)alpha is directly activated by the emerging bioactive sphingolipid, ceramide 1-phosphate (C-1-P) (1). In this study, a Triton X-100/phosphatidylcholine (PC) mixed micelle assay was utilized to determine the kinetics and specificity of this lipid-enzyme interaction. Using this assay, the addition of C-1-P induced a dramatic increase in the activity of cPLA(2)alpha (>15-fold) with a K(a) of 2.4 mol % C-1-P/Triton X-100 micelle. This activation was highly specific as the addition of other lipids had insignificant effects on cPLA(2)alpha activity. Studies using surface-dilution kinetics revealed that C-1-P had no effect on the Michaelis-Menten constant, K(m)(B), but decreased the dissociation constant (K (A)(s)) value by 87%. Thus, C-1-P not only increases the membrane affinity of cPLA(2)alpha but also may act as an allosteric activator of the enzyme. Surface plasmon resonance analysis of the C-1-P/cPLA(2)alpha interaction verified a decrease in the dissociation constant, demonstrating that cPLA(2)alpha bound PC vesicles containing C-1-P with increased affinity (5-fold) compared with PC vesicles alone. The effect on the dissociation rate of cPLA(2)alpha was also found to be lipid-specific with the exception of phosphatidylinositol 4,5-bisphosphate, which caused a modest increase in vesicle affinity (2-fold). Lastly, the binding site for C-1-P was determined to be within the C2-domain of cPLA(2)alpha, unlike phosphatidylinositol 4,5-bisphosphate. These data demonstrate a novel interaction site for C-1-P and suggest that C-1-P may function to recruit cPLA(2)alpha to intracellular membranes as well as allosterically activate the membrane-associated enzyme.

Allosteric Regulation↗

PubMed2005

Ceramide-1-phosphate: the "missing" link in eicosanoid biosynthesis and inflammation.

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.

Animals↗

PubMed2004

Insulin regulates protein kinase CbetaII alternative splicing in multiple target tissues: development of a hormonally responsive heterologous minigene.

Cells respond to external signals like insulin to alter metabolic pathways in response to varying physiological environments. Insulin stimulates the protein kinase C beta (PKCbeta) isozymes and preferentially switches the expression to PKCbetaII isozyme, which is shown to have a crucial role in glucose uptake, cellular proliferation, and differentiation. We have developed an insulin-responsive PKCbetaII heterologous minigene to identify cis-elements in vivo in eukaryotes by cloning the PKCbetaII exon and its flanking intronic sequences into the splicing vector pSPL3. The transfected minigene mimicked the endogenous insulin response of PKCbetaII alternative splicing in five distinct cell types, i.e. L6 skeletal muscle, 3T3-L1 pre-adipocytes, HepG2 human hepatoma cells, A10 vascular smooth muscle cells, and murine embryonic fibroblasts within 30 min of insulin stimulation. Sequential deletions of the flanking introns in the minigene demonstrated that insulin regulated elements within the 5'-intron flanking the PKCbetaII exon. Mutational studies indicated the SRp40 binding site promotes splice site selection. In these cases, splicing appears to be regulated by a phosphatidylinositol 3-kinase signaling pathway because LY294002 and wortmannin, its specific inhibitors, blocked exon inclusion. Cotransfection with constitutively active Akt2 kinase mimicked insulin action. Signal-dependent regulation of splicing by insulin is unique from tissue-specific and developmentally regulated mechanisms previously reported and serves as a prototype for studies of alternative splicing involving protein phosphorylation.

Adipocytes↗

PubMed2004

Identification of two RNA cis-elements that function to regulate the 5' splice site selection of Bcl-x pre-mRNA in response to ceramide.

Two splice variants derived from the BCL-x gene, proapoptotic Bcl-x(s) and anti-apoptotic Bcl-x(L), are produced via alternative 5' splice site selection within exon 2 of Bcl-x pre-mRNA. In previous studies, our laboratory demonstrated that ceramide regulated this 5' splice site selection, inducing the production of Bcl-x(s) mRNA with a concomitant decrease in Bcl-x(L) correlating with sensitization to chemotherapy (Chalfant, C. E., Rathman, K., Pinkerman, R. L., Wood, R. E., Obeid, L. M., Ogretmen, B., and Hannun, Y. A. (2002) J. Biol. Chem. 277, 12587-12595). We have now identified several possible RNA cis-elements within exon 2 of Bcl-x pre-mRNA by sequence analysis. To study the possible roles of these RNA cis-elements in regulating the alternative 5' splice site selection of Bcl-x pre-mRNA, we developed a BCL-x minigene construct which conferred the same ratio of Bcl-x(L)/Bcl-x(s) mRNA as the endogenous Bcl-x and was responsive to ceramide treatment. Mutagenesis of either a purine-rich splicing enhancer or a pyrimidine tract element within exon 2 induced a change in the ratio of Bcl-x(L)/Bcl-x(s) mRNA from 7 to 1 and 0.23, thereby diminishing the selection of the Bcl-x(L) 5' splice site with a concomitant increase in Bcl-x(s) 5' splice site selection. Furthermore, mutagenesis of these cis-elements abolished the ability of ceramide to affect the 5' splice site selection. In vitro binding assays coupled with competitor studies demonstrated specific binding of RNA trans-activating proteins to these regions. SDS-PAGE analysis of cross-linked RNA trans-activating factors with these RNA cis-elements revealed the binding of 215-, 120-, and 30-kDa proteins to the purine-rich element and 120- and 76-kDa proteins to the pyrimidine tract element. In addition, exogenous treatment of A549 cells with ceramide increased the formation of protein complexes with these RNA cis-elements. Therefore, we have identified two ceramide-responsive RNA cis-elements within exon 2 of Bcl-x pre-mRNA, and this is the first report of an RNA cis-element responsive to a bioactive lipid.

Alternative Splicing↗

PubMed2003

Ceramide 1-phosphate is a direct activator of cytosolic phospholipase A2.

Recently, we demonstrated that ceramide kinase, and its product, ceramide 1-phosphate (Cer-1-P), were mediators of arachidonic acid released in cells in response to interleukin-1beta and calcium ionophore (Pettus, B. J., Bielawska, A., Spiegel, S., Roddy, P., Hannun, Y. A., and Chalfant, C. E. (2003) J. Biol. Chem. 278, 38206-38213). In this study, we demonstrate that down-regulation of cytosolic phospholipase A(2) (cPLA(2)) using RNA interference technology abolished the ability of Cer-1-P to induce arachidonic acid release in A549 cells, demonstrating that cPLA(2) is the key phospholipase A(2) downstream of Cer-1-P. Treatment of A549 cells with Cer-1-P (2.5 microm) induced the translocation of full-length cPLA(2) from the cytosol to the Golgi apparatus/perinuclear regions, which are known sites of translocation in response to agonists. Cer-1-P also induced the translocation of the CaLB/C2 domain of cPLA(2) in the same manner, suggesting that this domain is responsive to Cer-1-P either directly or indirectly. In vitro studies were then conducted to distinguish these two possibilities. In vitro binding studies disclosed that Cer-1-P interacts directly with full-length cPLA(2) and with the CaLB domain in a calcium- and lipid-specific manner with a K(Ca) of 1.54 microm. Furthermore, Cer-1-P induced a calcium-dependent increase in cPLA(2) enzymatic activity as well as lowering the EC(50) of calcium for the enzyme from 191 to 31 nm. This study identifies Cer-1-P as an anionic lipid that translocates and directly activates cPLA(2), demonstrating a role for this bioactive lipid in the mediation of inflammatory responses.

Base Sequence↗

PubMed2003

The structural requirements for ceramide activation of serine-threonine protein phosphatases.

The protein phosphatases1 (PP1) and 2A (PP2A) serve as ceramide-activated protein phosphatases (CAPP). In this study, the structural requirements for interaction between ceramide and CAPP were determined. D-erythro-C(6) ceramide activated the catalytic subunit of PP2A (PP2Ac) approximately 3-fold in a stereospecific manner. In contrast, saturation of the 4-5 double bond, producing D-erythro-dihydro C(6) ceramide, inhibited PP2Ac (IC(50) = 8.5 microM). Furthermore, phyto C(6) ceramide, D-erythro-dehydro C(6) ceramide, and D-erythro-cis-C(6) ceramide had no effect on PP2Ac activity. Modification of the sphingoid chain also abolished the ability of ceramide to activate PP2Ac. Further studies demonstrated the requirement for the amide group, the primary hydroxyl group, and the secondary hydroxyl group of the sphingoid backbone for activation of PP2Ac through the synthesis and evaluation of D-erythro-urea C(6) ceramide, L-erythro-urea C(6) ceramide, D-erythro-N-methyl C(6) ceramide, D-erythro-L-O-methyl C(6) ceramide, D-erythro-3-O-methyl C(6) ceramide, and (2S) 3-keto C(6) ceramide. None of these compounds induced significant activation of PP2Ac. Liposome binding studies were also conducted using analogs of D-erythro-C C(6) ceramide, and the results showed that the ability of ceramide analogs to influence CAPP (activation or inhibition) was associated with the ability of the analogs to bind to CAPP. This study demonstrates strict structural requirements for interaction of ceramide with CAPP, and disclose ceramide as a very specific regulator of CAPP. The studies also begin to define features that transform ceramide analogs into inhibitors of CAPP.

Amides↗

PubMed2003

Ceramide kinase mediates cytokine- and calcium ionophore-induced arachidonic acid release.

Despite the importance of prostaglandins, little is known about the regulation of prostanoid synthesis proximal to the activation of cytosolic phospholipase A2, the initial rate-limiting step. In this study, ceramide-1-phosphate (C-1-P) was shown to be a specific and potent inducer of arachidonic acid (AA) and prostanoid synthesis in cells. This study also demonstrates that two well established activators of AA release and prostanoid synthesis, the cytokine, interleukin-1beta (IL-1beta), and the calcium ionophore, A23187, induce an increase in C-1-P levels within the relevant time-frame of AA release. Furthermore, the enzyme responsible for the production of C-1-P in mammalian cells, ceramide kinase, was activated in response to IL-1beta and A23187. RNA interference targeted to ceramide kinase specifically down-regulated ceramide kinase mRNA and activity with a concomitant decrease of AA release in response to IL-1beta and A23187. Down-regulation of ceramide kinase had no effect on AA release induced by exogenous C-1-P. Collectively, these results indicate that ceramide kinase, via the formation of C-1-P, is an upstream modulator of phospholipase A2 activation. This study identifies previously unknown roles for ceramide kinase and its product, C-1-P, in AA release and production of eicosanoids and provides clues for potential new targets to block inflammatory responses.

Animals↗

PubMed2003

The sphingosine kinase 1/sphingosine-1-phosphate pathway mediates COX-2 induction and PGE2 production in response to TNF-alpha.

In this study we addressed the role of sphingolipid metabolism in the inflammatory response. In a L929 fibroblast model, tumor necrosis factor-alpha (TNF) induced prostaglandin E2 (PGE2) production by 4 h and cyclooxygenase-2 (COX-2) induction as early as 2 h. This TNF-induced PGE2 production was inhibited by NS398, a COX-2 selective inhibitor. GC-MS analysis revealed that only COX-2-generated prostanoids were produced in response to TNF, thus providing further evidence of COX-2 selectivity. As sphingolipids have been implicated in mediating several actions of TNF, their role in COX-2 induction and PGE2 production was evaluated. Sphingosine-1-phosphate (S1P) induced both COX-2 and PGE2 in a dose-responsive manner with an apparent ED50 of 100-300 nM. The related sphingolipid sphingosine also induced PGE2, though with much less efficacy. TNF induced a 3.5-fold increase in sphingosine-1-phosphate levels at 10 min that rapidly returned to baseline by 40 min. Small interfering RNAs (siRNAs) directed against mouse SK1 decreased (typically by 80%) SK1 protein and inhibited TNF-induced SK activity. Treatment of cells with RNAi to SK1 but not SK2 almost completely abolished the ability of TNF to induce COX-2 or generate PGE2. By contrast, cells treated with RNAi to S1P lyase or S1P phosphatase enhanced COX-2 induction leading to enhanced generation of PGE2. Treatment with SK1 RNAi also abolished the effects of exogenous sphingosine and ceramide on PGE2, revealing that the action of sphingosine and ceramide are due to intracellular metabolism into S1P. Collectively, these results provide novel evidence that SK1 and S1P are necessary for TNF to induce COX-2 and PGE2 production. Based on these findings, this study indicates that SK1 and S1P could be implicated in pathological inflammatory disorders and cancer.

Animals↗

PubMed2002

Ceramide in apoptosis: an overview and current perspectives.

Recent years have witnessed significant advances in the understanding of the role of ceramide in apoptosis. This review summarizes these recent findings and discusses insights from studies of ceramide metabolism, topology, and effector actions. The recent identification of several genes for enzymes of ceramide metabolism, the development of mass spectrometric methods for ceramide analysis, and the increasing molecular and pharmacological tools to probe ceramide metabolism and function promise an accelerated phase in defining the molecular and biochemical details of the role of ceramide in apoptosis.

Amidohydrolases↗