Acid sphingomyelinase activation requires caspase-8 but not p53 nor reactive oxygen species during Fas-induced apoptosis in human glioma cells

During apoptosis of human glioma cells induced by anti-Fas antibody, ceramide formation with activation of acid, but not neutral sphingomyelinase (SMase), was observed. A potent inhibitor of acid SMase, SR33557, effectively inhibited ceramide formation and apoptosis. Fas-induced apoptosis and ceramide formation proceeded regardless of p53 status. The agents, which modify intracellular levels of reactive oxygen species (ROS) and reduced glutathione (GSH), failed to modulate Fas-induced acid SMase activation and apoptosis. Moreover, expression of functional p53 protein using a temperature-sensitive human p53val(138) induced ceramide generation by activation of neutral SMase but not acid SMase through ROS formation. Peptide inhibitors for caspases-8 (z-IETD-fmk) and -3 (z-DEVD-fmk) suppressed Fas-induced apoptosis. However, activation of acid SMase was inhibited only by z-IETD-fmk. Thus, ceramide generated by acid SMase may take a part in Fas-induced apoptosis of human glioma cells and acid SMase activation may be dependent on caspase-8 activation, but not on p53 nor ROS.     

p53-Independent ceramide formation in human glioma cells during gamma-radiation-induced apoptosis

Although the p53 tumor-suppressor gene product plays a critical role in apoptotic cell death induced by DNA-damaging chemotherapeutic agents, human glioma cells with functional p53 were more resistant to gamma-radiation than those with mutant p53. U-87 MG cells with wild-type p53 were resistant to gamma-radiation. U87-W E6 cells that lost functional p53, by the expression of type 16 human papillomavirus E6 oncoprotein, became susceptible to radiation-induced apoptosis. The formation of ceramide by acid sphingomyelinase (A-SMase), but not by neutral sphingomyelinase, was associated with p53-independent apoptosis. SR33557 (2-isopropyl-1-(4-[3-N-methyl-N-(3,4-dimethoxybphenethyl)amino]propyloxy)benzene-sulfonyl) indolizine, an inhibitor of A-SMase, suppressed radiation-induced apoptotic cell death. In contrast, radiation-induced A-SMase activation was blocked in glioma cells with endogenous functional p53. The expression of acid ceramidase was induced by gamma-radiation, and was more evident in cells with functional p53. N-oleoylethanolamine, which is known to inhibit ceramidase activity, unexpectedly downregulated acid ceramidase and accelerated radiation-induced apoptosis in U87-W E6 cells. Moreover, cells with functional p53 could be sensitized to gamma-radiation by N-oleoylethanolamine, which suppressed radiation-induced acid ceramidase expression and then enhanced ceramide formation. Sensitization to gamma-radiation was also observed in U87-MG cells depleted of functional p53 by retroviral expression of small interfering RNA. These results indicate that ceramide may function as a mediator of p53-independent apoptosis in human glioma cells in response to gamma-radiation, and suggest that p53-dependent expression of acid ceramidase and blockage of A-SMase activation play pivotal roles in protection from gamma-radiation of cells with endogenous functional p53.     

Roles for C16-ceramide and sphingosine 1-phosphate in regulating hepatocyte apoptosis in response to tumor necrosis factor-alpha

Tumor necrosis factor (TNF)-alpha signals cell death and simultaneously induces the generation of ceramide, which is metabolized to sphingosine and sphingosine 1-phosphate (S1P) by ceramidase (CDase) and sphingosine kinase. Because the dynamic balance between the intracellular levels of ceramide and S1P (the "ceramide/S1P rheostat") may determine cell survival, we investigated these sphingolipid signaling pathways in TNF-alpha-induced apoptosis of primary hepatocytes. Endogenous C16-ceramide was elevated during TNF-alpha-induced apoptosis in both rat and mouse primary hepatocytes. The putative acid sphingomyelinase (ASMase) inhibitor imipramine inhibited TNF-alpha-induced apoptosis and C16-ceramide increase as did the knock out of ASMase. Overexpression of neutral CDase (NCDase) inhibited the TNF-alpha-induced increase of C16-ceramide and apoptosis in rat primary hepatocytes. Moreover, NCDase inhibited liver injury and hepatocyte apoptosis in mice treated with D-galactosamine plus TNF-alpha. This protective effect was abrogated by the sphingosine kinase inhibitor N,N-demethylsphingosine, suggesting that the survival effect of NCDase is due to not only C16-ceramide reduction but also S1P formation. Administration of S1P or overexpression of NCDase activated the pro-survival kinase AKT, and overexpression of dominant negative AKT blocked the survival effect of NCDase. In conclusion, activation of ASMase and generation of C16-ceramide contributed to TNF-alpha-induced hepatocyte apoptosis. NCDase prevented apoptosis both by reducing C16-ceramide and by activation of AKT through S1P formation. Therefore, the cross-talk between sphingolipids and AKT pathway may determine hepatocyte apoptosis by TNF-alpha.     

Sphingomyelinases in cell regulation

Sphingomyelin hydrolysis and ceramide generation have emerged as key events in cellular regulation. Sphingomyelinases (SMases) catalyse the breakdown of sphingomyelin to form ceramide and phosphorylcholine. Ceramide formed through activation of SMases may function as a second messenger in mediating cell growth, differentiation, stress responses, and programmed cell death (apoptosis). So far, five types of SMases have been described and they include the acidic, the acidic zinc-dependent, the neutral magnesium-dependent, the neutral magnesium-independent, and the alkaline SMase. These SMases differ in tissue distribution, cofactor dependence, mechanism for regulation, and involvement in diverse cellular processes. At least two of these sphingomyelinases may regulate the intracellular levels of ceramide and subsequent ceramide-mediated responses. This review will focus on the identification, regulation and roles of SMases in cell function.     

Ceramide and reactive oxygen species generated by H2O2 induce caspase-3-independent degradation of Akt/protein kinase B

This study was designed to elucidate the mechanisms leading to down-regulation of the Akt/protein kinase B (PKB) survival pathway during H2O2-induced cell death. H2O2 produced early activation of Akt/PKB and also DNA damage that was followed by stabilization of p53 levels, formation of reactive oxygen species (ROS), and generation of ceramide through activation of a glutathione-sensitive neutral sphingomyelinase. These events correlated with long term dephosphorylation and subsequent degradation of Akt. A membrane-targeted active Akt version attenuated apoptosis but not necrosis induced by H2O2 and was more resistant to dephosphorylation and proteolysis induced by apoptotic concentrations of H2O2. Proteolysis of Akt was prevented by exogenous addition of glutathione, indicating a role of ROS and ceramide in Akt degradation. However, Akt was degraded similarly in cells transfected with wild type and dominant negative p53 mutant, indicating that degradation of Akt under oxidative injury may be p53-independent. Specific inhibitors of caspase groups I and III prevented proteolysis of Akt/PKB and poly(ADP-ribose) polymerase in cells submitted to apoptotic but not necrotic H2O2 concentrations. Surprisingly, in caspase-3-deficient MCF-7 cells Akt was more sensitive to H2O2-induced degradation than the caspase-3 substrate poly(ADP-ribose) polymerase. Moreover, the Akt/PKB double mutant Akt(D108A,D119A), which is not cleaved by caspase-3, and a triple mutant (D453A,D455A,D456A), which lacks the consensus sequence for caspase-3 cleavage, were also degraded in H2O2-treated cells. Our results suggest that strong oxidants generate intracellular ROS and ceramide which in term lead to down-regulation of Akt by dephosphorylation and caspase-3-independent proteolysis.     

PG490 (triptolide) cooperates with tumor necrosis factor-alpha to induce apoptosis in tumor cells

Progress in the treatment of solid tumors has been slow and sporadic. The efficacy of conventional chemotherapy in solid tumors is limited because tumors frequently have mutations in the p53 gene. Also, chemotherapy only kills rapidly dividing cells. Members of the tumor necrosis factor (TNF) family, however, induce apoptosis regardless of the p53 phenotype. Unfortunately, the cytotoxicity of TNF-alpha is limited by its activation of NF-kappaB and activation of NF-kappaB is proinflammatory. We have identified a compound called PG490, that is composed of purified triptolide, which induces apoptosis in tumor cells and sensitizes tumor cells to TNF-alpha-induced apoptosis. PG490 potently inhibited TNF-alpha-induced activation of NF-kappaB. PG490 also blocked TNF-alpha-mediated induction of c-IAP2 (hiap-1) and c-IAP1 (hiap-2), members of the inhibitor of apoptosis (IAP) family. Interestingly, PG490 did not block DNA binding of NF-kappaB, but it blocked transactivation of NF-kappaB. Our identification of a compound that blocks TNF-alpha-induced activation of NF-kappaB may enhance the cytotoxicity of TNF-alpha on tumors in vivo and limit its proinflammatory effects.     

Amyloid-beta peptide enhances tumor necrosis factor-alpha-induced iNOS through neutral sphingomyelinase/ceramide pathway in oligodendrocytes

Although accumulating evidence demonstrates that white matter degeneration contributes to pathology in Alzheimer's disease (AD), the underlying mechanisms are unknown. In order to study the roles of the amyloid-beta peptide in inducing oxidative stress damage in white matter of AD, we investigated the effects of amyloid-beta peptide 25-35 (Abeta) on proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha)-induced inducible nitric oxide synthase (iNOS) in cultured oligodendrocytes (OLGs). Although Abeta 25-35 by itself had little effect on iNOS mRNA, protein, and nitrite production, it enhanced TNF-alpha-induced iNOS expression and nitrite generation in OLGs. Abeta, TNF-alpha, or the combination of both, increased neutral sphingomyelinase (nSMase) activity, but not acidic sphingomyelinase (aSMase) activity, leading to ceramide accumulation. Cell permeable C2-ceramide enhanced TNF-alpha-induced iNOS expression and nitrite generation. Moreover, the specific nSMase inhibitor, 3-O-methyl-sphingomyelin (3-OMS), inhibited iNOS expression and nitrite production induced by TNF-alpha or by the combination of TNF-alpha and Abeta. Overexpression of a truncated mutant of nSMase with a dominant negative function inhibited iNOS mRNA production. 3-OMS also inhibited nuclear factor kappaB (NF-kappaB) binding activity induced by TNF-alpha or by the combination of TNF-alpha and Abeta. These results suggest that neutral sphingomyelinase/ceramide pathway is required but may not be sufficient for iNOS expression induced by TNF-alpha and the combination of TNF-alpha and Abeta.     

Tumor necrosis factor alpha-induced apoptosis requires p73 and c-ABL activation downstream of RB degradation

The retinoblastoma protein (RB) suppresses cell proliferation and apoptosis. We have previously shown that RB degradation is required for tumor necrosis factor alpha (TNF-alpha) to induce apoptosis. We show here the identification of two apoptotic effectors, i.e., c-ABL tyrosine kinase and p73, which are activated by TNF-alpha following RB degradation. In cells expressing a degradation-resistant RB protein (RB-MI), TNF-alpha does not activate c-ABL. RB-MI also inhibits TNF-alpha-mediated activation of p73. Genetic deletion and pharmacological inhibition of c-ABL or p73 diminish the apoptotic response to TNF-alpha in human cell lines and mouse fibroblasts. Thymocytes isolated from Rb(MI/MI), Abl(-/-), or p73(-/-) mice are resistant to TNF-alpha-induced apoptosis compared to their wild-type counterparts. This is in contrast to p53(-/-) thymocytes, which exhibit a wild-type level of apoptosis in response to TNF-alpha. Thus, c-ABL and p73 contribute to apoptosis induced by TNF-alpha, in addition to their role in promoting DNA damage-associated cell death.     

Overexpression of catalase in the mitochondrial or cytosolic compartment increases sensitivity of HepG2 cells to tumor necrosis factor-alpha-induced apoptosis

The sensitivity of HepG2 cells overexpressing catalase in either the cytosolic or mitochondrial compartment to tumor necrosis factor-alpha (TNF-alpha) and cycloheximide was studied. Cells overexpressing catalase in the cytosol (C33 cells) and especially in mitochondria (mC5 cells) were more sensitive to TNF-alpha-induced apoptosis than were control cells (Hp cells). The activities of caspase-3 and -8 were increased by TNF-alpha, with the highest activities found in mC5 cells. Sodium azide, an inhibitor of catalase, reduced the increased sensitivity of mC5 and C33 cells to TNF-alpha to the level of toxicity found with control Hp cells. Azide also decreased the elevated caspase-3 activity of mC5 cells. A pan-caspase inhibitor prevented the TNF-alpha-induced apoptosis and toxicity produced by catalase overexpression. Addition of H(2)O(2) prevented TNF-alpha-induced apoptosis and caspase activation, an effect prevented by simultaneous addition of catalase. TNF-alpha plus cycloheximide increased ATP levels, with higher levels in C33 and mC5 cells compared with Hp cells. TNF-alpha did not produce apoptosis in mC5 cells maintained in a low energy state. TNF-alpha signaling was not altered by the overexpression of catalase, as activation of nuclear factor kappaB and AP-1 by TNF-alpha was similar in the three cell lines. These results suggest that catalase, overexpressed in the cytosolic or especially the mitochondrial compartment, potentiates TNF-alpha-induced apoptosis and activation of caspases by removal of H(2)O(2).     

Rac1 mediates intestinal epithelial cell apoptosis via JNK

Apoptosis plays a key role in the maintenance of a constant cell number and a low incidence of cancer in the mucosa of the intestine. Although the small GTPase Rac1 has been established as an important regulator of migration of intestinal epithelial cells, whether Rac1 is also involved in apoptosis is unclear. The present study tested the hypothesis that Rac1 mediates TNF-alpha-induced apoptosis in IEC-6 cells. Rac1 is activated during TNF-alpha-induced apoptosis as judged by the level of GTP-Rac1, the level of microsomal membrane-associated Rac1, and lamellipodia formation. Although expression of constitutively active Rac1 does not increase apoptosis in the basal condition, inhibition of Rac1 either by NSC-23766 (Rac1 inhibitor) or expression of dominant negative Rac1 protects cells from TNF-alpha-induced apoptosis by inhibiting caspase-3, -8, and -9 activities. Inhibition of Rac1 before the administration of apoptotic stimuli significantly prevents TNF-alpha-induced activation of JNK1/2, the key proapoptotic regulator in IEC-6 cells. Inhibition of Rac1 does not modulate TNF-alpha-induced ERK1/2 and Akt activation. Inhibition of ERK1/2 and Akt activity by U-0126 and LY-294002, respectively, increased TNF-alpha-induced apoptosis. However, inhibition of Rac1 significantly decreased apoptosis in the presence of ERK1/2 and Akt inhibitors, similar to the effect observed with NSC-23766 alone in response to TNF-alpha. Thus, Rac1 inhibition protects cells independently of ERK1/2 and Akt activation during TNF-alpha-induced apoptosis. Although p38 MAPK is activated in response to TNF-alpha, inhibition of p38 MAPK did not decrease apoptosis. Rac1 inhibition did not alter p38 MAPK activity. Thus, these results indicate that Rac1 mediates apoptosis via JNK and plays a key role in proapoptotic pathways in intestinal epithelial cells.     

Tumor necrosis factor alpha-induced activation of downstream NF-kappaB site of the promoter mediates epithelial ICAM-1 expression and monocyte adhesion. Involvement of PKCalpha, tyrosine kinase, and IKK2, but not MAPKs, pathway

TNF-alpha induced an increase in intercellular adhesion molecule-1 (ICAM-1) expression in human A549 epithelial cells and immunofluorescence staining confirmed this result. The enhanced ICAM-1 expression was shown to increase the adhesion of U937 cells to A549 cells. Tyrosine kinase inhibitors (genistein or tyrphostin 23) or phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor (D 609) attenuated TNF-alpha-induced ICAM-1 expression. TNF-alpha produced an increase in protein kinase C (PKC) activity and this effect was inhibited by D 609. PKC inhibitors (staurosporine, Ro 31-8220, calphostin C, or Go 6976) also inhibited TNF-alpha-induced response. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a PKC activator, stimulated ICAM-1 expression, this effect was inhibited by genistein or tyrphostin 23. Treatment of cells with TNF-alpha resulted in stimulation of p44/42 MAPK, p38, and JNK. However, TNF-alpha-induced ICAM-1 expression was not affected by either MEK inhibitor, PD 98059, or p38 inhibitor, SB 203580. A cell-permeable ceramide analog, C(2) ceramide, also stimulated the activation of these three MAPKs, but had no effect on ICAM-1 expression. NF-kappaB DNA-protein binding and ICAM-1 promoter activity were enhanced by TNF-alpha and these effects were inhibited by D 609, calphostin C, or tyrphostin 23, but not by PD 98059 or SB 203580. TPA also stimulated NF-kappaB DNA-protein binding and ICAM-1 promoter activity, these effects being inhibited by genistein or tyrphostin 23. TNF-alpha- or TPA-induced ICAM-1 promoter activity was inhibited by dominant negative PKCalpha or IKK2, but not IKK1 mutant. IKK activity was stimulated by both TNF-alpha and TPA, and these effects were inhibited by Ro 31-8220 or tyrphostin 23. These data suggest that, in A549 cells, TNF-alpha activates PC-PLC to induce activation of PKCalpha and protein tyrosine kinase, resulting in the stimulation of IKK2, and NF-kappaB in the ICAM-1 promoter, then initiation of ICAM-1 expression and neutrophil adhesion. However, activation of p44/42 MAPK, p38, and JNK is not involved in this event.     

Crm-A, bcl-2 and NDGA inhibit CD95L-induced apoptosis of malignant glioma cells at the level of caspase 8 processing

Susceptibility to CD95 (Fas/APO-1)-mediated apoptosis in human glioma cells depends on CD95 expression and unknown factors that regulate signal transduction. Thus, LN-18 cells are highly sensitive to CD95 ligand (CD95L) whereas LN-229 cells require coexposure to inhibitors of RNA or protein synthesis for induction of apoptosis. Here, we report that caspase 8 and 3 activation, poly(ADP-ribose)polymerase cleavage and apoptosis are inhibited by the lipoxygenase inhibitor, nordihydroguaretic acid (NDGA), or ectopic expression of crm-A or bcl-2. CD95L-induced glioma cell apoptosis does not involve ceramide generation. Apoptosis induced by exogenous ceramide resembles CD95-mediated apoptosis in that bcl-2 is protective but differs in that NDGA and crm-A have no effect and in that cycloheximide (CHX) inhibits rather than potentiates ceramide-induced cell death. We conclude that caspase 8 and caspase 3 activation, but not ceramide generation, are required for CD95 ligand-induced apoptosis of glioma cells and that bcl-2, crm-A and NDGA all act upstream of caspases to inhibit apoptosis.     

Interleukin-10 and interleukin-13 inhibit proinflammatory cytokine-induced ceramide production through the activation of phosphatidylinositol 3-kinase

Ceramide produced by hydrolysis of plasma membrane sphingomyelin (SM) in different cells including brain cells in response to proinflammatory cytokines [tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta)] plays an important role in coordinating cellular responses to stress, growth suppression, and apoptosis. The present study underlines the importance of IL-10 and IL-13, cytokines with potent antiinflammatory properties, in inhibiting the proinflammatory cytokine (TNF-alpha and IL-1beta)-mediated degradation of SM to ceramide in rat primary astrocytes. Treatment of rat primary astrocytes with TNF-alpha or IL-1beta led to rapid degradation of SM to ceramide, whereas IL-10 and IL-13 by themselves were unable to induce the degradation of SM to ceramide. Interestingly, both IL-10 and IL-13 prevented proinflammatory cytokine-induced degradation of SM to ceramide. Both IL-10 and IL-13 caused rapid activation of phosphatidylinositol (PI) 3-kinase, and inhibition of that kinase activity by wortmannin and LY294002 potently blocked the inhibitory effect of IL-10 and IL-13 on proinflammatory cytokine-mediated induction of ceramide production. This study suggests that the inhibition of proinflammatory cytokine-mediated degradation of SM to ceramide by IL-10 and IL-13 is mediated through the activation of PI 3-kinase. As ceramide induces apoptosis and IL-10 and IL-13 inhibit the induction of ceramide production, we examined the effect of IL-10 and IL-13 on proinflammatory cytokine-mediated apoptosis. Inhibition of TNF-alpha-induced apoptosis by IL-10 and IL-13 suggests that the antiapoptotic nature of IL-10 and IL-13 is probably due to the inhibition of ceramide production.     

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

Neutral sphingomyelinases (SMases) are involved in the induction of ceramide-mediated proapoptotic signaling under heat stress conditions. Although ceramide is an important mediator of apoptosis, the neutral SMase that is activated under heat stress has not been identified. In this study, we cloned an Mg(2+)-dependent neutral SMase from a zebrafish embryonic cell cDNA library using an Escherichia coli expression-cloning vector. Screening of the clones using an SMase activity assay with C(6)-7-nitro-2-1,3-benzoxadiazol-4-yl-sphingomyelin as the substrate resulted in the isolation of one neutral SMase cDNA clone. This cDNA encoded a polypeptide of 420 amino acids (putative molecular weight: 46,900) containing two predicted transmembrane domains in its C-terminal region. The cloned neutral SMase 1 acted as a mediator of stress-induced apoptosis. Bacterially expressed recombinant neutral SMase 1 hydrolyzed [choline-methyl-(14)C]sphingomyelin optimally at pH 7.5 in the presence of an Mg(2+) ion. In zebrafish embryonic cells, the endogenous SMase enzyme was localized in the microsomal fraction. In FLAG-tagged SMase-overexpressing cells, neutral SMase 1 colocalized with a Golgi marker in a cytochemical analysis. Inactivation of the enzyme by an antisense phosphorothioate oligonucleotide repressed the induction of ceramide generation, caspase-3 activation, and apoptotic cell death by heat stress. Thus, neutral SMase 1 participates in an inducible ceramide-mediating, proapoptotic signaling pathway that operates in heat-induced apoptosis in zebrafish embryonic cells.     

Acute tumor necrosis factor alpha signaling via NADPH oxidase in microvascular endothelial cells: role of p47phox phosphorylation and binding to TRAF4

Tumor necrosis factor alpha (TNF-alpha) receptor-associated factors (TRAFs) play important roles in TNF-alpha signaling by interacting with downstream signaling molecules, e.g., mitogen-activated protein kinases (MAPKs). However, TNF-alpha also signals through reactive oxygen species (ROS)-dependent pathways. The interrelationship between these pathways is unclear; however, a recent study suggested that TRAF4 could bind to the NADPH oxidase subunit p47phox. Here, we investigated the potential interaction between p47phox phosphorylation and TRAF4 binding and their relative roles in acute TNF-alpha signaling. Exposure of human microvascular endothelial cells (HMEC-1) to TNF-alpha (100 U/ml; 1 to 60 min) induced rapid (within 5 min) p47phox phosphorylation. This was paralleled by a 2.7- +/- 0.5-fold increase in p47phox-TRAF4 association, membrane translocation of p47phox-TRAF4, a 2.3- +/- 0.4-fold increase in p47phox-p22phox complex formation, and a 3.2- +/- 0.2-fold increase in NADPH-dependent O2- production (all P < 0.05). TRAF4-p47phox binding was accompanied by a progressive increase in extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38(MAPK) activation, which was inhibited by an O2- scavenger, tiron. TRAF4 predominantly bound the phosphorylated form of p47phox, in a protein kinase C-dependent process. Knockdown of TRAF4 expression using siRNA had no effect on p47phox phosphorylation or binding to p22phox but inhibited TNF-alpha-induced ERK1/2 activation. In coronary microvascular EC from p47phox-/- mice, TNF-alpha-induced NADPH oxidase activation, ERK1/2 activation, and cell surface intercellular adhesion molecule 1 (ICAM-1) expression were all inhibited. Thus, both p47phox phosphorylation and TRAF4 are required for acute TNF-alpha signaling. The increased binding between p47phox and TRAF4 that occurs after p47phox phosphorylation could serve to spatially confine ROS generation from NADPH oxidase and subsequent MAPK activation and cell surface ICAM-1 expression in EC.     

Daunorubicin attenuates tumor necrosis factor-alpha-induced biosynthesis of plasminogen activator inhibitor-1 in human umbilical vein endothelial cells

The anthracycline antibiotic daunorubicin is reported to induce apoptosis in cells by triggering ceramide generation through de novo synthesis or sphingomyelin hydrolysis. Treatment of human umbilical vein endothelial cells (HUVEC) with daunorubicin markedly decreased the mRNA expression and protein release of plasminogen activator inhibitor-1 (PAI-1). This cellular event was accompanied by a significant increase in the total ceramide content in HUVEC. On the other hand, tumor necrosis factor (TNF)-alpha treatment of HUVEC led to an increase in both PAI-1 mRNA expression and protein release, and an enhancement of total ceramide content was also observed. The stimulating effect of TNF-alpha on PAI-1 synthesis was attenuated by the pretreatment of HUVEC with daunorubicin. Interestingly, the daunorubicin-induced increase in ceramide content was blocked by addition of the potent ceramide synthase inhibitor fumonisin B(1), while the TNF-alpha-induced ceramide increase was not affected by this drug. Fumonisin B(1) treatment restored the daunorubicin-induced decrease in PAI-1 release to approximately 70% of the control, but did not affect the TNF-alpha-induced increase in PAI-1 release. Thus, these data imply the possibility that the subcellular topology of ceramide production determines its lipid mediator function in the regulation of PAI-1 synthesis in HUVEC, because both TNF-alpha and daunorubicin could increase the ceramide levels.