HindIII liposomes suppress delayed-type hypersensitivity responses in vivo and induce epidermal IL-10 in vitro

The purpose of this study was to evaluate the effects of resident islet macrophage activation on beta cell function. Treatment of freshly isolated rat islets with TNF-alpha and LPS results in a potent inhibition of glucose-stimulated insulin secretion. The inhibitory actions of TNF + LPS are mediated by the intraislet production and release of IL-1 followed by IL-1-induced inducible nitric oxide synthase (iNOS) expression by beta cells. The IL-1R antagonist protein completely prevents TNF + LPS-induced nitrite production, iNOS expression and the inhibitory effects on glucose-stimulated insulin secretion by rat islets. Resident macrophages appear to be the source of IL-1, as a 7-day culture of rat islets at 24 degrees C (conditions known to deplete islets of lymphoid cells) prevents TNF + LPS-induced iNOS expression, nitrite production, and the inhibitory effects on insulin secretion. In addition, macrophage depletion also inhibits TNF + LPS-induced IL-1alpha and IL-1beta mRNA expression in rat islets. Immunocytochemical colocalization of IL-1beta with the macrophage-specific marker ED1 was used to provide direct support for resident macrophages as the islet cellular source of IL-1. IL-1beta appears to mediate the inhibitory actions of TNF + LPS on beta cell function as TNF + LPS-induced expression of IL-1beta is fourfold higher than IL-1alpha, and Ab neutralization of IL-1beta prevents TNF + LPS-induced nitrite production by rat islets. These findings support a mechanism by which the activation of resident islet macrophages and the intraislet release of IL-1 may mediate the initial dysfunction and destruction of beta cells during the development of autoimmune diabetes.     

Mature macrophage cell lines exhibit variable responses to LPS

Bacterial lipopolysaccharide (LPS) is a potent activator of cells of the macrophage/monocyte lineage. Two mature macrophage cell lines, P388D1 and RAW264.7, exhibit very different biological responses to LPS. Although RAW264.7 cells release arachidonic acid from phospholipid in response to LPS stimulation, P388D1 cells do not respond in this manner. However, LPS primes P388D1 cells to release arachidonic acid in response to other stimuli. The goal of this work is to contrast the biochemical events that occur in LPS-treated P388D1 and RAW264.7 macrophages. Enzyme assays indicate that LPS treatment induces the activation of cytosolic PLA2 in RAW264.7, but not in P388D1 cells. Phorbol ester (PMA), a receptor-independent stimulus, also fails to induce arachidonic acid release from P388D1 cells, suggesting that these cells may have a defect in the signal transduction machinery that is common to LPS and PMA. This hypothesis is supported by the observation that the expression of the LPS receptors CD14 and CD11b/CD18 is similar on P388D1 and RAW264.7 cells. Western blot analyses indicate that the erk kinases are activated upon LPS treatment of RAW264.7 but not P388D1 cells. LPS-induced arachidonic acid release is reduced in cells treated with the MEK inhibitor PD98059, suggesting that activated erk kinases mediate the phosphorylation and activation of cPLA2 in this system. Interestingly, the p42 isoform of erk (erk2) appears to be activated in resting P388D1 cells. This observation indicates that the MAP kinase cascade may be constitutively activated in P388D1 cells which may in turn limit their ability to respond to LPS. Together, these data provide evidence that mature macrophages from different sources can exhibit variable responses to LPS and highlight the danger of making generalizations regarding the effects of LPS on macrophages.     

Enhancement of fibroblast collagenase (matrix metalloproteinase-1) gene expression by ceramide is mediated by extracellular signal-regulated and stress-activated protein kinase pathways

Inflammatory cytokines tumor necrosis factor-alpha and interleukin-1 trigger the ceramide signaling pathway, initiated by neutral sphingomyelinase-elicited hydrolysis of cell membrane phospholipid sphingomyelin to ceramide, a new lipid second messenger. Here, we show that triggering the ceramide pathway by sphingomyelinase or C2- and C6-ceramide enhances collagenase-1 (matrix metalloproteinase-1; MMP-1) gene expression by fibroblasts. C2-ceramide activates three distinct mitogen-activated protein kinases (MAPKs) in dermal fibroblasts, i.e. extracellular signal-regulated kinase 1/2 (ERK1/2), stress-activated protein kinase/Jun N-terminal-kinase (SAPK/JNK), and p38. Stimulation of MMP-1 promoter activity by C2-ceramide is dependent on the presence of a functional AP-1 cis-element and is entirely inhibited by overexpression of MAPK inhibitor, dual specificity phosphatase CL100 (MAPK phosphatase-1). Activation of MMP-1 promoter by C2-ceramide is also effectively inhibited by kinase-deficient forms of ERK1/2 kinase (MEK1/2) activator Raf-1, ERK1 and ERK2, SAPK/JNK activator SEK1, or SAPKbeta. In addition, ceramide-dependent induction of MMP-1 expression is potently prevented by PD 98059, a selective inhibitor of MEK1 activation, and by specific p38 inhibitor SB 203580. These results show that triggering the ceramide signaling pathway activates MMP-1 gene expression via three distinct MAPK pathways, i.e. ERK1/2, SAPK/JNK, and p38, and suggest that targeted modulation of the ceramide signaling pathway may offer a novel therapeutic approach for inhibiting collagenolytic activity, e.g. in inflammatory disorders.     

Induction of intracellular ceramide by interleukin-1 beta in oligodendrocytes

The sphingomyelin pathway has been implicated in mediating the effect of several extracellular agents leading to important biochemical and cellular changes. The aim of this investigation is to study interleukin-1 beta (IL-1 beta) signaling in oligodendrocytes. For this purpose, the CG4 oligodendrocyte cells were differentiated and incubated with IL-1 beta. This treatment induced a time- and dose-dependent increase of the endocellular ceramide. To mimic the effect of the elevation of endogenous ceramide, the CG4 cells were treated with the ceramide analogue C2-ceramide. Cell survival, measured with the MTT assay, showed that, by increasing the concentration of ceramide, up to 40% of CG4 cells were dying within 6 h, similar data were obtained with the primary differentiated oligodendrocytes. Condensation of chromatin, nuclear fragmentation, and formation of apoptotic bodies indicated that apoptosis was the cause of death. Surprisingly, long-term exposure (72 h) to increasing concentrations of IL-1 beta, which increases intracellular ceramide, did not induce oligodendroglial cell death. These results show that an increase of intracellular ceramide is not sufficient to induce apoptosis in oligodendrocytes and that IL-1 beta signaling through the ceramide pathway in these cells can mediate functions other than programmed cell death.     

The mitogen-activated protein kinase pathway inhibits ceramide-induced terminal differentiation of a human monoblastic leukemia cell line, U937

This communication describes an extracellular signal-regulated kinase kinase (MEK)-dependent signal transduction pathway that prevents the terminal differentiation of a hemopoietic cell line. Both PMA and the cell-permeable ceramide, C2-ceramide, caused differentiation of U937 cells, but with distinct cell morphology and CD11b/CD14 surface expression. While PMA activated extracellular signal-regulated kinase (ERK), a downstream kinase of Raf-MEK signaling, C2-ceramide activated c-Jun NH2-terminal kinase (JNK), an anchor kinase of stress-induced signaling. Furthermore, only C2-ceramide stimulated an induction of cell cycle arrest that was associated with stable expression of p21CIP1 and retinoblastoma nuclear phosphoprotein dephosphorylation. Expression of p21CIP1 and JNK activation were also observed in sphingosine-treated cells, whereas sphingosine did not induce detectable differentiation. Concomitant stimulation with C2-ceramide and PMA resulted in the PMA phenotype, and cell cycle arrest was absent. ERK activation was enhanced by C2-ceramide plus PMA stimulation, whereas the activation of JNK was aborted. Strikingly, the inhibition of MEK with PD98059 altered the phenotype of C2-ceramide- and PMA-stimulated U937 cells to that of cells treated with C2-ceramide alone. Thus, ERK and JNK pathways deliver distinct signals, and the ERK pathway is dominant to the JNK cascade. Furthermore, differentiation and cell cycle arrest caused by C2-ceramide rely on independent signaling pathways, and JNK is an unlikely signaling element for this differentiation. Importantly, during C2-ceramide and PMA costimulation, the JNK pathway is not simply blocked by ERK activation; rather, cross-talk between these MAP kinase pathways acts to simultaneously augment ERK activity and down-regulate JNK activity.     

Lipopolysaccharide-related stimuli induce expression of the secretory leukocyte protease inhibitor, a macrophage-derived lipopolysaccharide inhibitor

Mouse secretory leukocyte protease inhibitor (SLPI) was recently characterized as a lipopolysaccharide (LPS)-induced product of macrophages that antagonizes their LPS-induced activation of NF-kappaB and production of NO and tumor necrosis factor (TNF) (F. Y. Jin, C. Nathan, D. Radzioch, and A. Ding, Cell 88:417-426, 1997). To better understand the role of SLPI in innate immune and inflammatory responses, we examined the kinetics of SLPI expression in response to LPS, LPS-induced cytokines, and LPS-mimetic compounds. SLPI mRNA was detectable in macrophages by Northern blot analysis within 30 min of exposure to LPS but levels peaked only at 24 to 36 h and remained elevated at 72 h. Despite the slowly mounting and prolonged response, early expression of SLPI mRNA was cycloheximide resistant. Two LPS-induced proteins-interleukin-10 (IL-10) and IL-6-also induced SLPI, while TNF and IL-1beta did not. The slow attainment of maximal induction of SLPI by LPS in vitro was mimicked by infection with Pseudomonas aeruginosa in vivo, where SLPI expression in the lung peaked at 3 days. Two LPS-mimetic molecules-taxol from yew bark and lipoteichoic acid (LTA) from gram-positive bacterial cell walls-also induced SLPI. Transfection of macrophages with SLPI inhibited their LTA-induced NO production. An anti-inflammatory role for macrophage-derived SLPI seems likely based on SLPI's slowly mounting production in response to constituents of gram-negative and gram-positive bacteria, its induction both as a direct response to LPS and as a response to anti-inflammatory cytokines induced by LPS, and its ability to suppress the production of proinflammatory products by macrophages stimulated with constituents of both gram-positive and gram-negative bacteria.     

Macrophage inflammatory protein-1 alpha production in lipopolysaccharide-stimulated human adherent blood mononuclear cells is inhibited by the nitric oxide synthase inhibitor N(G)-monomethyl-L-arginine

The release of chemokines such as macrophage-inflammatory protein-1 alpha (MIP-1 alpha) from activated macrophages is a crucial step in cell recruitment necessary for establishing local inflammatory responses. To ascertain the importance of the L-arginine/nitric oxide (NO) pathway in LPS-induced MIP-1 alpha release, we stimulated human adherent PBMC with LPS in the presence of the NO synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA). L-NMMA decreased LPS-induced MIP-1 alpha protein release (45.5% inhibition) and steady state levels of mRNA (48% inhibition) in adherent PBMC. The concentration of L-NMMA for inhibition of MIP-1 alpha release was dependent on the concentration of L-arginine in the cell culture medium, emphasizing the L-arginine-related action of the drug. Most of the MIP-1 alpha release was attributed to the activity of IL-1 and TNF, since coincubation of LPS-stimulated PBMC with IL-1R antagonist and TNF-binding protein abrogated LPS-induced MIP-1 alpha release (by 76.8%). Analysis of cytokine secretion revealed that, in addition to MIP-1 alpha, L-NMMA inhibited the release of mature IL-1 beta (by 70%) and TNF-alpha (by 53%). In contrast, release of macrophage chemoattractant protein-1 was unaffected; IL-10 was augmented (123.4%) by L-NMMA. In the presence of exogenous NO provided by NO donors, LPS-induced MIP-1 alpha release was enhanced. We concluded that endogenous NO acts as a mediator of inflammation. Since IL-10 is a potent anti-inflammatory cytokine, these data also suggest that L-NMMA acts as an anti-inflammatory agent by specifically altering the balance of pro- and anti-inflammatory cytokines released from LPS-stimulated human PBMC.     

Dual role of ceramide in the control of apoptosis following IL-2 withdrawal

Ceramide is largely known as a lipid second messenger with pleiotropic effects. Increases in ceramide levels have been related to the onset of apoptosis, terminal differentiation, or growth suppression. In this study, addition of exogenous C2-ceramide to CTLL-2 cells is found to block IL-2-induced cell cycle entry, as well as the apoptosis triggered by IL-2 deprivation. The protective effect of C2-ceramide is achieved only in the early stages following cytokine deprivation and is related to the inhibition of bcl-xL degradation and the induction of a G0 arrest of cells. The same treatment over a longer time when, as we demonstrate, ceramide is produced physiologically, enhances cell death by apoptosis. The dual effect of ceramide both in protecting from or inducing apoptosis is discussed further.     

CD14-dependent internalization of bacterial lipopolysaccharide (LPS) is strongly influenced by LPS aggregation but not by cellular responses to LPS

We analyzed the impact of ligand aggregation and LPS-induced signaling on CD14-dependent LPS internalization kinetics in human monocytic THP-1 cells and murine macrophages. Using two independent methods, we found that the initial rate and extent of LPS internalization increased with LPS aggregate size. In the presence of LPS binding protein (LBP), large LPS aggregates were internalized extremely rapidly (70% of the cell-associated LPS was internalized in 1 min). Smaller LPS aggregates were internalized more slowly than the larger aggregates, and LPS monomers, complexed with soluble CD14 in the absence of LBP, were internalized very slowly after binding to membrane CD14 (5% of the cell-associated LPS was internalized in 1 min). In contrast, the initial aggregation state had little or no effect on the stimulatory potency of the LPS. Previous studies suggest that LPS-induced signal responses may influence the intracellular traffic and processing of LPS. We found that elicited peritoneal macrophages from LPS-responsive (C3H/HeN) and LPS-hyporesponsive (C3H/HeJ) mice internalized LPS with similar kinetics. In addition, pre-exposure of THP-1 cells to LPS had no effect on their ability to internalize subsequently added LPS, and pre-exposure of the cells to the LPS-specific inhibitor, LA-14-PP, inhibited stimulation of the cells without inhibiting LPS internalization. In these cells, LPS is thus internalized by a constitutive cellular mechanism(s) with kinetics that depend importantly upon the physical state in which the LPS is presented to the cell.     

Interleukin-13 is a more potent inhibitor of the expression of inducible nitric oxide synthase in smooth muscle cells than in macrophages: a comparison with interleukin-4 and interleukin-10

This study compares the effects of interleukin (IL)-13, a cytokine with anti-inflammatory properties, with those of IL-4 or IL-10 on the expression of inducible nitric oxide synthase (iNOS) protein and activity in 1) a murine macrophage cell line (J774.2) activated with lipopolysaccharide (LPS) and 2) rat aortic smooth muscle cells (RASM) activated with LPS plus interferon-gamma. Pretreatment of macrophages with IL-4 or IL-13 caused a similar, concentration-dependent inhibition of the formation of nitrite and the expression of iNOS protein elicited by LPS. In contrast, IL-13 was a much more potent inhibitor of the formation of nitrite and the expression of iNOS protein in activated RASM than IL-4. IL-10 caused only a small, but significant, inhibition of the nitrite formation induced by LPS in macrophages and RASM. Pretreatment of J774.2 macrophages, but not of RASM, with the phosphatidylinositol-3-kinase inhibitor, wortmannin (10-100 nM), attenuated the inhibition by either IL-13 or IL-4 of the LPS-induced increase in nitrite in a dose-related fashion. Thus, IL-13 is more potent than IL-4 in preventing the expression of iNOS protein and activity in activated RASM, whereas IL-13 and IL-4 are equipotent in inhibiting the expression of iNOS protein and activity in J774.2 macrophages.     

Acid sphingomyelinase-derived ceramide is not required for inflammatory cytokine signalling in murine macrophages

Sphingomyelin hydrolysis is induced in myeloid cell-lines by tumour necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1beta), and interferon gamma (IFN-gamma). Ceramide, a product of sphingomyelin hydrolysis, recapitulates many of the cellular responses elicited by these cytokines, and this has lead to the hypothesis that ceramide is a second messenger of cytokine signalling. Sphingomyelin hydrolysis is catalysed by an acid spingomyelinase (ASMase) and one or more neutral sphingomyelinases (NSMase); both ASMase and NSMase are activated during cytokine signalling. In the present study, the contribution of ASMase to TNF-alpha, IL-1beta, and IFN-gamma signalling in murine macrophages was addressed. Cytokine-induced responses were compared in macrophages derived from the bone marrow of AMSase null and wild-type mice. Specifically, TNF-alpha-and IFN-gamma-induced nitric oxide production and TNF-alpha- and IL-1beta-induced expression of the alpha-chemokine, KC, were intact in ASMase null macrophages. Furthermore, TNF-alpha induction of p42/p44 ERK and p38-MAPK phosphorylation, c-jun kinase activation, and IkappaBalpha degradation were normal. Also normal in ASMase null macrophages was TNF-alpha-, IL-1beta- and IFN-gamma-induced expression of a panel of early response genes. It is concluded that ASMase is non-essential for the inflammatory signals activated in murine macrophages by TNF-alpha, IL-1beta and IFN-gamma.     

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

The sphingomyelin derivative ceramide is a signaling molecule implicated in numerous physiological events. Recently published reports indicate that ceramide levels are elevated in insulin-responsive tissues of diabetic animals and that agents which trigger ceramide production inhibit insulin signaling. In the present series of studies, the short-chain ceramide analog C2-ceramide inhibited insulin-stimulated glucose transport by approximately 50% in 3T3-L1 adipocytes, with similar reductions in hormone-stimulated translocation of the insulin-responsive glucose transporter (GLUT4) and insulin-responsive aminopeptidase. C2-ceramide also inhibited phosphorylation and activation of Akt, a molecule proposed to mediate multiple insulin-stimulated metabolic events. C2-ceramide, at concentrations which antagonized activation of both glucose uptake and Akt, had no effect on the tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1) or the amounts of p85 protein and phosphatidylinositol kinase activity that immunoprecipitated with anti-IRS-1 or antiphosphotyrosine antibodies. Moreover, C2-ceramide also inhibited stimulation of Akt by platelet-derived growth factor, an event that is IRS-1 independent. C2-ceramide did not inhibit insulin-stimulated phosphorylation of mitogen-activated protein kinase or pp70 S6-kinase, and it actually stimulated phosphorylation of the latter in the absence of insulin. Various pharmacological agents, including the immunosuppressant rapamycin, the protein synthesis inhibitor cycloheximide, and several protein kinase C inhibitors, were without effect on ceramide's inhibition of Akt. These studies demonstrate ceramide's capacity to inhibit activation of Akt and imply that this is a mechanism of antagonism of insulin-dependent physiological events, such as the peripheral activation of glucose transport and the suppression of apoptosis.     

Evidence for the involvement of interleukin 10 in the differential deactivation of murine peritoneal macrophages by prostaglandin E2

Among other effects, prostaglandins (PG) of the E series are known to inhibit several acute and chronic inflammatory conditions in vivo and proinflammatory cytokine production by activated macrophages in culture. The research presented here demonstrates that the inhibitory effect of PGE2 on tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) production by lipopolysaccharide (LPS)-stimulated murine peritoneal macrophages involves IL-10. In a dose-dependent manner, PGE2 inhibits LPS-induced release of TNF-alpha and IL-6, but not of lactate or nitric oxide. The decrease in the level of these cytokines is inversely proportional to the increase in immunoreactive IL-10. This differential inhibitory effect of PGE2 is mimicked by agents that elevate intracellular levels of cAMP, but not cGMP. Neutralizing anti IL-10 antibody but not neutralizing antibodies against other macrophage secretory products (IL-6, leukemia inhibitory factor, and transforming growth factor beta [TGF-beta]), significantly reverse the potent inhibitory effect of PGE2. In vivo, the administration of PGE2 before LPS challenge significantly reduces circulating TNF-alpha and IL-6 levels. Anti-IL-10 antibody substantially enhanced the LPS-induced TNF-alpha and IL-6 levels in mice that received either LPS alone or LPS plus PGE2. These results suggest that the anti-inflammatory effect of PGE2 on mononuclear phagocytes is mediated in part by an autocrine feedback mechanism involving IL-10.     

Modulation of lipopolysaccharide-induced macrophage gene expression by Rhodobacter sphaeroides lipid A and SDZ 880.431

Rhodobacter sphaeroides lipid A (RsDPLA) and SDZ 880.431 (3-aza-lipid X-4-phosphate) are prototypic lipopolysaccharide (LPS) antagonists. Herein, we examined the ability of these structures to regulate murine macrophage tumor necrosis factor (TNF) secretion and LPS-inducible gene expression (tumor necrosis factor alpha [TNF-alpha], interleukin-1 beta [IL-1 beta], IP-10, type 2 TNF receptor [TNFR-2], D3, and D8 genes). We report that RsDPLA alone (> 1 microgram/ml) induced low levels of TNF-alpha secretion and a selective pattern of gene expression in peritoneal exudate macrophages; SDZ 880.431 alone was completely inactive. When LPS was present at a low concentration (1 ng/ml), RsDPLA and SDZ 880.431 blocked TNF secretion and gene induction in a concentration-dependent fashion. In general, gene induction was measurably reduced by 10 to 30 ng of RsDPLA per ml or 300 ng of SDZ 880.431 per ml, but inhibition could be uniformly overridden by increasing the concentration of LPS. Although induction of all six genes by LPS was suppressed by either inhibitor, effective inhibitor concentrations depended on the gene of interest. Induction of TNFR-2 by LPS was relatively resistant to inhibition by RsDPLA, and induction of TNFR-2 and D3 was relatively resistant to inhibition by SDZ 880.431. When LPS was present at > or = 100 ng/ml, correspondingly high concentrations (> or = 20 micrograms/ml) of either inhibitor influenced gene expression in a bidirectional manner. Under these conditions, LPS-induced expression of IP-10, D3, and D8 was suppressed regardless of the LPS concentration used (concentrations tested up to 50 micrograms/ml), while expression of TNF-alpha mRNA was enhanced about fourfold. In toto, RsDPLA and SDZ 880.431, when present at low concentrations, act in a manner consistent with competitive inhibition of LPS, while at higher concentrations, these structures inhibit certain LPS responses noncompetitively and synergize with LPS for other responses.     

Inhibition of Helicobacter pylori urease activity by ebrotidine

Lipopolysaccharide (LPS), known as one of the potent activators of macrophages, has inhibitory effects on the proliferation of normal macrophages and macrophage-like cell lines. We report here that LPS dose- and time-dependently suppressed the tritiated thymidine ([3H]TdR) incorporation into the acid-insoluble fraction with a significant inverse correlation to the tumour necrosis factor-alpha (TNF) production in the J774.1 macrophage cell line. Among the three tested enzymes involved in DNA synthesis, only thymidine kinase (TK) activity decreased progressively in parallel with the decline in [3H]TdR incorporation, reaching 97% inhibition within 12 hr of LPS treatment, while changes in the activities of other two enzymes, DNA polymerase alpha and thymidylate synthase (TS), were less significant. On the other hand, LPS inhibited the cell proliferation only incompletely, as judged by 62% inhibition of cell growth at 36 hr. Even in the experiments done in a TdR-free medium, cell growth was inhibited by LPS to the same extent, suggesting that TK was not directly involved in the proliferation of J774 cells. LPS also inhibited the conversion of TdR to thymidine monophosphate (TMP) in murine peritoneal exudate macrophages (PEM). Thus LPS-induced suppression of TdR salvage related to TNF production is common in both normal and neoplastic macrophages, and therefore may be of potential importance in the process of macrophage activation.     

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

Ceramide has been proposed as a second messenger molecule implicated in a variety of biological processes. It has recently been reported that ceramide activates stress-activated protein kinase (SAPK, also known as c-Jun NH2-terminal kinase JNK), a subfamily member of mitogen-activated protein kinase superfamily molecules and that the ceramide/SAPK/JNK signaling pathway is required for stress-induced apoptosis. However, the molecular mechanism by which ceramide induces SAPK/JNK activation is unknown. Here we show that TAK1, a member of the mitogen-activated protein kinase kinase kinase family, is activated by treatment of cells with agents and stresses that induce an increase in ceramide. Ceramide itself stimulated the kinase activity of TAK1. Expression of a constitutively active form of TAK1 resulted in activation of SAPK/JNK and SEK1/MKK4, a direct activator of SAPK/JNK. Furthermore, expression of a kinase-negative form of TAK1 interfered with the activation of SAPK/JNK induced by ceramide. These results indicate that TAK1 may function as a mediator of ceramide signaling to SAPK/JNK activation.