p38 MAPK mediates the regulation of alpha1(I) procollagen mRNA levels by TNF-alpha and TGF-beta in a cell line of rat hepatic stellate cells(1).

The role of members of the mitogen-activated protein kinase (MAPK) family on tumor necrosis factor alpha (TNF-alpha)-mediated down-regulation of col1a1 gene was studied. TNF-alpha increased extracellular-regulated kinase and Jun-N-terminal kinase phosphorylation, but these effects were not related to its inhibitory effect on alpha1(I) procollagen (col1a1) mRNA levels. Phosphorylation of p38 MAPK was decreased in response to TNF-alpha, and the specific p38 MAPK inhibitor SB203580 mimicked the effect of TNF-alpha on col1a1 mRNA levels. Transforming growth factor beta (TGF-beta) increased p38 MAPK phosphorylation and SB203580 prevented the induction of col1a1 mRNA levels by TGF-beta. These results suggest that p38 MAPK plays an important role in regulating the expression of col1a1 in hepatic stellate cells in response to cytokines.

Signaling pathways activated by daunorubicin.

The anthracycline daunorubicin is widely used in the treatment of acute nonlymphocytic leukemia. The drug has, of course, been the object of intense basic research, as well as preclinical and clinical study. As reviewed in this article, evidence stemming from this research clearly demonstrates that cell response to daunorubicin is highly regulated by multiple signaling events, including a sphingomyelinase-initiated sphingomyelin-ceramide pathway, mitogen-activated kinase and stress-activated protein/c-Jun N-terminal kinase activation, transcription factors such as nuclear factor kappa B, as well as the Fas/Fas-ligand system. These pathways are themselves influenced by a number of lipid products (diacylglycerol, sphingosine-1 phosphate, and glucosyl ceramide), reactive oxygen species, oncogenes (such as the tumor suppressor gene p53), protein kinases (protein kinase C and phosphoinositide-3 kinase), and external stimuli (hematopoietic growth factors and the extracellular matrix). In light of the complexity and diversity of these observations, a comprehensive review has been attempted toward the understanding of their individual implication (and regulation) in daunorubicin-induced signaling. (Blood. 2001;98:913-924)

Cell surface-directed interaction of anthracyclines leads to cytotoxicity and nuclear factor kappaB activation but not apoptosis signaling.

Anthracyclines are, above all, DNA intercalators, which induce genetic damage leading to cell death. However, increasing evidence firmly suggests that the underlying mechanism for anthracycline cytotoxicity is the induction of apoptosis through intracellular-mediated signaling pathways. Whether drug/DNA interaction is necessary for such apoptosis signaling is unknown. We investigated the cellular effects of the anthracyclines daunorubicin (DNR) and doxorubicin (DOX) using the myeloid leukemia cell line U937. By comparing free drug against agarose bead-immobilized drug iDNR and iDOX (which cannot accumulate within the cell), we observed that whereas both free and immobilized anthracyclines were cytotoxic, only the former induced apoptosis; the latter induced necrosis. Indeed, we did not observe ceramide generation, neutral sphingomyelinase activation, poly (ADP-ribose) polymerase cleavage, or other apoptotic events with iDNR or iDOX. However, both free and immobilized drug were similarly capable of triggering nuclear factor kappaB activation. These observations demonstrate that whereas activation of certain cellular signaling pathways can be achieved solely through membrane interaction, apoptosis signaling requires anthracycline internalization. These results also show that the initiation of cell survival pathways (illustrated by nuclear factor kappaB activation) is independent of intracellular drug/target interaction.

Lysosomal sphingomyelinase is not solicited for apoptosis signaling.

Stress-induced activation of an acidic sphingomyelinase leading to generation of ceramide, an important lipid mediator, has been associated with apoptosis; however, the implication of this hydrolase has been questioned. The present study aimed at re-evaluating the role of this lysosomal enzyme in apoptosis initiated by different apoptotic inducers. The sensitivity of a series of acid sphingomyelinase-deficient cell lines derived from Niemann-Pick disease patients to stress-induced apoptosis was investigated. We have now shown that stress stimuli, such as anthracyclines, ionizing radiation, and Fas ligation trigger similar apoptotic hallmarks in normal and acid sphingomyelinase-deficient cell lines. Retrovirus-mediated gene correction of enzyme deficiency in Niemann-Pick cells does not modify response to apoptosis. Ceramide production is comparable in normal and Niemann-Pick cells, and increased activity of neutral sphingomyelinase is observed. Thus, our findings cast serious doubts that lysosomal sphingomyelinase activation is responsible for stress-induced apoptosis of cultured cells.

Activation of a nuclear sphingomyelinase in radiation-induced apoptosis.

The subcellular origin of ceramide signaling in ionizing radiation-triggered apoptosis was investigated using two previously described subclones of the autonomous erythro-myeloblastic cell line TF-1, radio-resistant and -sensitive TF-1-34 and TF-1-33, respectively. We show in nuclei-free lysates and cytoplasts that both cell lines failed to generate ceramide in response to ionizing radiation. Moreover, whereas cytoplasts did respond to anti-Fas stimulation through phosphatidylserine externalization, no effect was observed with ionizing radiation. Only in highly purified nuclei preparations did we observe ceramide generation, neutral sphingomyelinase activation, and apoptotic features (PARP cleavage, nuclear fragmentation, DNA laddering) in TF-1-33, but not in TF-1-34 cells. These observations suggest that nuclear sphingomyelinase and ceramide formation may contribute to ionizing radiation-triggered apoptosis.

Phosphatidylcholine-specific phospholipase C and phospholipase D are respectively implicated in mitogen-activated protein kinase and nuclear factor kappaB activation in tumour-necrosis-factor-alpha-treated immature acute-myeloid-leukaemia cells.

Tumour necrosis factor-alpha (TNFalpha) has been reported to induce potent growth inhibition of committed myeloid progenitor cells, whereas it is a potential growth stimulator of human CD34(+)CD38(-) multipotent haematopoietic cells. The present study was aimed at evaluating the respective role of two phospholipases, phosphatidylcholine-specific phospholipase C (PC-PLC) and phospholipase D (PLD) in the response of the CD34(+) CD38(-) KG1a cells to TNFalpha. In these cells TNFalpha triggered phosphoinositide 3-kinase (PI3K)-dependent PC hydrolysis within 4-8 min with concomitant production of both diacylglycerol (DAG) and phosphocholine (P-chol). DAG and P-chol production was accompanied by extracellular-signal-related protein kinase-1 ('ERK-1') activation and DNA-synthesis stimulation. PC-PLC stimulation was followed by PI3K-independent PLD activation with concomitant phosphatidic acid (PA) production followed by PA-derived DAG accumulation and sustained nuclear factor kappaB (NF-kappaB) activation. PLD/NF-kappaB signalling activation played no role in the TNFalpha proliferative effect and conferred no consistent protection of KG1a cells towards antileukaemic agents. Altogether these results suggest that, in KG1a cells, TNFalpha can stimulate in parallel PC-PLC and PLD, whose lipid products activate in turn mitogen-activated protein kinase (MAP kinase) and NF-kappaB signalling respectively. Finally, our study suggests that PC-PLC, but not PLD, plays a role in the TNFalpha proliferative effect in immature myeloid cells.

Molecular mechanisms of apoptosis induced by an immunomodulating peptide on human monocytes.

A short immunomodulating peptide (Pa) containing a defined structural motif present in a number of extracellular matrix proteins and autoantigens was found to stimulate human monocytes. Pa-induced apoptosis of isolated monocytes, as indicated by internucleosomal DNA cleavage, increased annexin V binding capacity and cleavage of caspase substrates, such as poly(ADP)ribosylpolymerase. In addition, Bcl-2 protein levels were downregulated during Pa-induced cell death. Nuclear extracts of monocytes incubated with Pa showed higher neutral, Ca(2+)-dependent DNase activity than those obtained from nontreated monocytes. Caspase inhibitors prevented Pa-induced apoptosis, Bcl-2 depletion, and DNase activation. Treatment of monocytes with Pa activated c-Jun N-terminal kinases and p38 kinase, in an acidic sphingomyelinase- and caspase-dependent fashion. Pa-induced apoptosis was blocked by selective inhibitors of p38 kinase (SB203580) and acidic sphingomyelinase (SR33557). These results indicate that JNK and p38 kinase stimulation as well as monocyte apoptosis induced by Pa could depend, at least in part, on early activation of acidic sphingomyelinase.

Stress-induced apoptosis is not mediated by endolysosomal ceramide.

A major lipid-signaling pathway in mammalian cells implicates the generation of ceramide from the ubiquitous sphingolipid sphingomyelin (SM). Hydrolysis of SM by a sphingomyelinase present in acidic compartments has been reported to mediate, via the production of ceramide, the apoptotic cell death triggered by stress-inducing agents. In the present study, we investigated whether the ceramide formed within or accumulated in lysosomes indeed triggers apoptosis. A series of observations strongly suggests that ceramide involved in stress-induced apoptosis is not endolysosomal: 1) Although short-chain ceramides induced apoptosis, loading cells with natural ceramide through receptor-mediated endocytosis did not result in cell death. 2) Neither TNF-alpha nor anti-CD95 induced the degradation to ceramide of a natural SM that had been first introduced selectively into acidic compartments. 3) Stimulation of SV40-transformed fibroblasts by TNF-alpha or CD40 ligand resulted in apoptosis equally well in cells derived from control individuals and from patients affected with Farber disease, having a genetic defect of acid ceramidase activity leading to lysosomal accumulation of ceramide. Also, induction of apoptosis using anti-CD95 (Fas) or anti-CD40 antibodies, TNF-alpha, daunorubicin, and ionizing radiation was similar in control and Farber disease lymphoid cells. In all cases, apoptosis was preceded by a comparable increase of intracellular ceramide levels. 4) Retroviral-mediated gene transfer and overexpression of acid ceramidase in Farber fibroblasts, which led to complete metabolic correction of the ceramide catabolic defect, did not affect the cell response to TNF-alpha and CD40 ligand.

The acid sphingomyelinase inhibitor SR33557 counteracts TNF-alpha-mediated potentiation of IL-1beta-induced NF-kappaB activation in the insulin-producing cell line Rinm5F.

Cytokines induce nitric oxide (NO) production and cell death in insulin-producing cells in vitro but the signaling pathways mediating the cytokine effects are not well characterized. The aim of this study was to determine whether sphingomyelinase (SMase) participates in cytokine signaling leading to NF-kappaB activation, iNOS induction and cell death in insulin-producing cells. Acute exposure to IL-1beta or TNF-alpha did not affect SMase activities in rat insulinoma (RINm5F) cells. TNF-alpha activated NF-kappaB in gel shift experiments without inducing iNOS--as assessed by nitrite formation--whereas IL-1beta stimulated both NF-kappaB activation and iNOS induction. Natural ceramide did not activate NF-kappaB or iNOS. However, both ceramide and TNF-alpha potentiated IL-1beta- induced activation of NF-kappaB and iNOS. Moreover, the potentiating effects of TNF-alpha were counteracted by the acid SMase inhibitor SR33557. The combination of IL-1beta and IFN-gamma induced apoptosis in RINm5F cells, which was paralleled by a modest increase in acid SMase, whereas ceramide mainly induced necrosis. It is concluded that cytokine-induced beta-cell signaling is associated with the induction of iNOS but not with enhanced SMase activities. However, TNF-alpha-mediated potentiation of the IL-1beta effect may involve an increased sensitivity to basal acid SMase activity. An increased acid SMase activity may participate in the execution of cytokine-induced beta-cell apoptosis.

CD40 signals apoptosis through FAN-regulated activation of the sphingomyelin-ceramide pathway.

The possibility that the sphingomyelin (SM)-ceramide pathway is activated by CD40, a transmembrane glycoprotein belonging to the tumor necrosis factor receptor superfamily and that plays a critical role in the regulation of immune responses has been investigated. We demonstrate that incubation of Epstein-Barr virus-transformed lymphoid cells with an anti-CD40 antibody acting as an agonist results in the stimulation of a neutral sphingomyelinase, hydrolysis of cellular SM, and concomitant ceramide generation. In addition, SM degradation was observed in acid sphingomyelinase-deficient cells, as well as after ligation by soluble CD40 ligand. The anti-CD40 antibody, as well as the soluble CD40 ligand induced a decrease in thymidine incorporation and morphological features of apoptosis, which were mimicked by cell-permeant or bacterial sphingomyelinase-produced ceramides. Stable expression of a dominant-negative form of the FAN protein (factor associated with neutral sphingomyelinase activation), which has been reported to mediate tumor necrosis factor-induced activation of neutral sphingomyelinase, significantly inhibited CD40 ligand-induced sphingomyelinase stimulation and apoptosis of transformed human fibroblasts. Transformed fibroblasts from FAN knockout mice were also protected from CD40-mediated cell death. Finally, anti-CD40 antibodies were able to co-immunoprecipitate FAN in control fibroblasts but not in cells expressing the dominant-negative form of FAN, indicating interaction between CD40 and FAN. Altogether, these results strongly suggest that CD40 ligation can activate via FAN a neutral sphingomyelinase-mediated ceramide pathway that is involved in the cell growth inhibitory effects of CD40.

Doxorubicin induces slow ceramide accumulation and late apoptosis in cultured adult rat ventricular myocytes.

OBJECTIVES: Anthracyclines cause apoptotic death in many cell types through activation of the ceramide pathway. We tested the hypothesis that doxorubicin induces cardiac myocyte apoptosis through ceramide generation. METHODS: Adult rat ventricular myocytes were grown in the presence of 10% fetal calf serum, and exposed to 0.5 microM doxorubicin (Dox) for 1 h on the day of cell isolation (day 0). We used the membrane-permeant ceramide analog C2-ceramide (C2-cer) to mimic the effects of endogenous ceramide and PDMP to induce endogenous ceramide accumulation. Apoptosis was assessed upon morphological criteria and DNA fragmentation by the TUNEL method and agarose gel electrophoresis. Ceramide concentration was assessed using the DAG kinase assay. RESULTS: Myocyte exposure to Dox was associated with cellular and nuclear alterations typical of apoptosis on day 7 but not on day 3. At day 7, the percentage of TUNEL-positive myocytes was markedly increased in Dox-treated cultures compared to control (Cl) cultures (82 +/- 3 vs. 12 +/- 1%, n = 7; p < 0.001); internucleosomal DNA fragmentation was confirmed by the observation of DNA ladders. These alterations were associated with an increase in the intracellular ceramide concentration (1715 +/- 243 vs. 785 +/- 99 pmol/mg prot, n = 5; p < 0.01), a phenomenon also detected on day 3 (731 +/- 59 vs. 259 +/- 37 pmol/mg prot, n = 5; p < 0.001). Incubation of myocytes at day 0 with 50 microM C2-cer induced rapid cell shrinkage and DNA fragmentation (45 +/- 3 vs. 10 +/- 1% TUNEL-positive myocytes on day 1 in C2-cer-treated and Cl cultures, respectively; n = 6, p < 0.001). Myocyte exposure to 10 microM PDMP for 7 days (n = 5), caused ceramide accumulation (1.7-fold increase vs. Cl, p < 0.01), and a marked increase in the percentage of TUNEL-positive myocytes (62 +/- 6 vs. 11 +/- 3% in Cl cultures, p < 0.001). Ventricles of rats injected intraperitoneally with a cumulative dose of 14 mg/kg Dox over a period of 2 weeks also showed an increased ceramide concentration 2 weeks later (11.01 +/- 0.64 vs. 5.24 +/- 0.88 pmol/mg prot, n = 6; p < 0.001). CONCLUSION: Our study confirms the existence of a functional ceramide pathway related to apoptosis in cardiac myocytes, and points to its possible involvement in doxorubicin-induced cardiomyopathy.

Implication of radical oxygen species in ceramide generation, c-Jun N-terminal kinase activation and apoptosis induced by daunorubicin.

Anthracyclines such as daunorubicin (DNR) generate radical oxygen species (ROS), which account, at least in part, for their cytotoxic effect. We observed that early ceramide generation (within 6-10 min) through neutral sphingomyelinase stimulation was inhibitable by the antioxidants N-acetylcysteine and pyrrolidine dithiocarbamate, which led to a decrease in apoptosis (>95% decrease in DNA fragmentation after 6 h). Furthermore, we observed that DNR triggers the c-Jun N-terminal kinase (JNK) and the transcription factor activated protein-1 through an antioxidant-inhibitable mechanism. Treatment of U937 cells with cell-permeant ceramides induced both an increase in ROS generation and JNK activation, and apoptosis, all of which were antioxidant-sensitive. In conclusion, DNR-triggered apoptosis implicates a ceramide-mediated, ROS-dependent JNK and activated protein-1 activation.

L-carnitine prevents doxorubicin-induced apoptosis of cardiac myocytes: role of inhibition of ceramide generation.

Besides the well-documented effect of the chemotherapeutic drug doxorubicin on free radical generation, the exact signaling mechanisms by which it causes cardiac damage remain largely unknown and are of fundamental importance in understanding anthracycline cardiotoxicity. In this study, we describe that a 1 h treatment of isolated adult rat cardiac myocytes with doxorubicin (0.5 microM) induced DNA fragmentation associated with the classical morphological features of apoptosis observed after 7 days of culture. The doxorubicin toxicity was preceded by an increase in intracellular ceramide levels with a concurrent decrease in sphingomyelin. Anthracycline-induced ceramide accumulation resulted from the activation of a sphingomyelinase assayed under acidic conditions, an effect related to an increase in V(max). Pretreatment of cardiac myocytes with L-carnitine (200 microgram/ml), a compound known for its protective effect on cardiac metabolic injuries, was found to dose-dependently inhibit the doxorubicin-induced sphingomyelin hydrolysis and ceramide generation as well as subsequent cell death. However, L-carnitine did not protect cardiac myocytes from apoptosis induced by exogenous cell-permeant ceramide. L-carnitine pretreatment did not affect the sphingomyelinase basal activity but abolished the doxorubicin-induced increase in V(max). Moreover, in vitro studies conducted on cell extracts or with purified acid sphingomyelinase demonstrated that L-carnitine exerted a dose-dependent, sphingomyelinase inhibitory effect (through V(max) reduction). Taken together, these findings show that by inhibiting a (perhaps novel) drug-activated acid sphingomyelinase and ceramide generation, L-carnitine can prevent doxorubicin-induced apoptosis of cardiac myocytes.

Signalling sphingomyelinases: which, where, how and why?

A major lipid signalling pathway in mammalian cells implicates the activation of sphingomyelinase (SMase), which upon cell stimulation hydrolyses the ubiquitous sphingophospholipid sphingomyelin to ceramide. This review summarizes our current knowledge on the nature and regulation of signalling SMase(s). Because of the controversy on the identity of this(these) phospholipase(s), the roles of various SMases in cell signalling are discussed. Special attention is also given to the subcellular site of action of signalling SMases and to the cellular factors that positively or negatively control their activity. These regulating agents include lipids (arachidonic acid, diacylglycerol and ceramide), kinases, proteases, glutathione and other proteins.

Daunorubicin- and mitoxantrone-triggered phosphatidylcholine hydrolysis: implication in drug-induced ceramide generation and apoptosis.

Several studies have suggested that diacylglycerol can affect the induction of apoptosis induced by toxicants and ceramide. The present study demonstrates that clinically relevant concentrations of the chemotherapeutic drugs daunorubicin and mitoxantrone (0.2-1 microM) transiently stimulated concurrently with sphingomyelin-derived ceramide generation and diacylglycerol and phosphorylcholine production within 4 to 10 min via phospholipase C hydrolysis of phosphatidylcholine. Pretreatment of cells with the xanthogenate compound D609, a potent inhibitor of phosphatidylcholine-phospholipase C, led to significant inhibition of drug triggered diacylglycerol and phosphorylcholine production and to a sustained increase in ceramide levels for a period up to 2 h. Moreover, D609 pretreatment induced both cell death and ceramide generation at daunorubicin and mitoxantrone concentrations previously shown to be ineffective (i.e., 0.1 microM). These results underline the importance of diacylglycerol in the regulation of programmed cell death and strongly argue for a balance between apoptotic (ceramide) and survival (diacylglycerol) signal transducers.

Sphingomyelin-degrading pathways in human cells role in cell signalling.

The ubiquitous sphingophospholipid sphingomyelin (SM) can be hydrolysed in human cells to ceramide by different sphingomyelinases (SMases). These enzymes exert a dual role, enabling not only the turnover of membrane SM and the degradation of exogenous (lipoprotein) SM, but also the signal-induced generation of the lipid second messenger ceramide. This review focuses on the function(s) of the different SMases in living cells. While both lysosomal and non-lysosomal pathways that ensure SM hydrolysis in intact cells can be distinguished, the precise contribution of each of these SM-cleaving enzymes to the production of ceramide as a signalling molecule remains to be clarified.

[Role of lipid second messengers involved in the response of leukemic cells to anticancer drugs]. / Rôle des seconds messagers lipidiques impliqués dans la réponse des cellules leucémiques aux médicaments anticancéreux.

Simple clinical observation suggests that while anti-leukemia agents are efficient at eradicating blasts cells in terminal division, as illustrated, in the case of acute myeloid leukemia, by the high complete remission rate (70%); these agents are relatively inept at eliminating leukemic myeloid progenitors as suggested by the high level of recurrence. This interpretation underlines the apparently natural chemoresistance of cells which compose the myeloid leukemia progenitor compartment. Over the past few years, several studies have shown that similar cellular damage can lead to divers effects such as rapid apoptotic death, differed mitotic death, or a transitory cytostatic effect. Cell response to damage is regulated by a complex and highly regulated network of intracellular signals including cell death signals mediated by ceramide and cell survival signals mediated (at least in part) by diacylglycerol and phosphoinositide-3 phosphates. Cellular fate relies on the balance between these two signaling pathways. This hypothesis opens several prospects on pharmacological manipulation aimed at either favoring cell death or at conferring resistance to anti-cancer agents.

Positive feedback control of neutral sphingomyelinase activity by ceramide.

While ceramide has emerged as a potent signal transducer, inconsistencies in the kinetics of ceramide generation, or its absence, in response to stimuli have led to confusion and skepticism as to its potential role in apoptosis or proliferation. Here we show that in U937 and HL60 myeloid leukemia cells and in normal skin fibroblasts, cell-permeant ceramides can trigger neutral sphingomyelinase activation, sphingomyelin hydrolysis, and endogenous ceramide generation regardless of Bcl2 overexpression. These observations identify neutral sphingomyelinase as a novel target for ceramide and show that this positive feedback mechanism is responsible for signal propagation, as exemplified by mitogen-activated protein kinase activation in daunorubicin-treated cells. This study provides insight into a fundamental process of cell biology. Indeed, such a sustained ceramide-mediated signal throughout the apoptotic process would ensure self-destruction, perhaps by overriding evolutionary conserved primal cell survival mechanisms.