Jean-Pierre Jaffrézou

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.

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 µM) 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 Vmax. Pretreatment of cardiac myocytes with L‐carnitine (200 µg/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 Vmax. 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 Vmax 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.—Andrieu‐Abadie, N., Jaffrézou, J.‐P., Hatem, S., Laurent, G., Levade, T., Mercadier, J.‐J. L‐carnitine prevents doxorubicin‐induced apoptosis of cardiac myocytes: role of inhibition of ceramide generation. FASEB J. 13, 1501–1510 (1999)

UV-C light induces raft-associated acid sphingomyelinase and jnk activation and translocation independently on a nuclear signal

The initiation of UV light-induced signaling in mammalian cells is largely considered to be subsequent to DNA damage. Several studies have also described ceramide (CER), a lipid second messenger, as a major contributor in mediating UV light-induced c-Jun N-terminal kinase (JNK) activation and cell death. It is demonstrated here that UV-C light irradiation of U937 cells results in the activation and translocation of a Zn2+-independent acid sphingomyelinase, leading to CER accumulation in raft microdomains. These CER-enriched rafts aggregate and play a functional role in JNK activation. The observation that UV-C light also induced CER generation and the externalization of acid sphingomyelinase and JNK in human platelets conclusively rules out the involvement of a nuclear signal generated by DNA damage in the initiation of a UV light response, which is generated at the plasma membrane.

p38 MAPK mediates the regulation of α1(I) procollagen mRNA levels by TNF-α and TGF-β in a cell line of rat hepatic stellate cells1

The role of members of the mitogen‐activated protein kinase (MAPK) family on tumor necrosis factor α (TNF‐α)‐mediated down‐regulation of col1a1 gene was studied. TNF‐α increased extracellular‐regulated kinase and Jun‐N‐terminal kinase phosphorylation, but these effects were not related to its inhibitory effect on α1(I) procollagen (col1a1) mRNA levels. Phosphorylation of p38 MAPK was decreased in response to TNF‐α, and the specific p38 MAPK inhibitor SB203580 mimicked the effect of TNF‐α on col1a1 mRNA levels. Transforming growth factor β (TGF‐β) increased p38 MAPK phosphorylation and SB203580 prevented the induction of col1a1 mRNA levels by TGF‐β. These results suggest that p38 MAPK plays an important role in regulating the expression of col1a1 in hepatic stellate cells in response to cytokines.

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

OBJECTIVESnAnthracyclines 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.nnnMETHODSnAdult 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.nnnRESULTSnMyocyte 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).nnnCONCLUSIONnOur 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.

Serine protease inhibitors block neutral sphingomyelinase activation, ceramide generation, and apoptosis triggered by daunorubicin.

To address the role of a plausible protease cascade in daunorubicin‐triggered apoptosis, we evaluated the effect of cell‐permeant protease inhibitors on its signal transduction pathway. Treatment of U937 and HL‐60 cells with 0.5‐1 μM of the chemotherapeutic drug daunorubicin induced a greater than 30% activation of neutral sphingomyelinase activity within 4‐10 min with concomitant sphingomyelin hydrolysis and ceramide generation. DNA fragmentation and the classical morphological features of apoptosis were observed within 4–6 h. Pretreatment of cells with the serine protease inhibitors N‐tosyl‐L‐phenylalanyl chloromethyl ketone (20 μM) or dichloroisocoumarin (20 μM) for 30 min inhibited daunorubicin‐induced neutral sphingomyelinase activation, sphingomyelin hydrolysis, ceramide generation, and apoptosis. Other cell‐permeant protease inhibitors such as pepstatin, leupeptin, and antipain had no such effect. The apoptotic response could be restored by the addition of 25 μM cell‐permeant C6‐ceramide. Daunorubicin‐induced NF‐κB activation was inhibited by dichloroisocoumarin but not by N‐tosyl‐L‐phenylalanyl chloromethyl ketone, suggesting that this transcription factor can be activated independently of ceramide and is not directly implicated in the apoptotic pathway. These results suggest that inhibitors of serine proteases can act upstream of ceramide in drug‐triggered apoptosis and that neutral sphingomyelinase activation is either directly or indirectly serine protease dependent.—Mansat, V., Bettaïeb, A., Levade, T., Laurent, G., Jaffrézou, J.‐P. Serine protease inhibitors block neutral sphingomyelinase activation, ceramide generation, and apoptosis triggered by daunorubicin. FASEB J. 11, 695–702 (1997)

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 sphingomyeli‐nase present in acidic compartments has been reported to mediate, via the production of cer‐amide, 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 apopto‐sis. A series of observations strongly suggests that ceramide involved in stress‐induced apoptosis is not endolysosomal: 1) Although short‐chain cer‐amides induced apoptosis, loading cells with natural ceramide through receptor‐mediated endocy‐tosis did not result in cell death. 2) Neither TNF‐α 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‐α 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 cer‐amide. Also, induction of apoptosis using anti‐CD95 (Fas) or anti‐CD40 antibodies, TNF‐α, 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) Retro‐viral‐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‐α and CD40 ligand.—Ségui, B., Bezombes, C., Uro‐Coste, E., Medin, J. A., Andrieu‐Abadie, N., Augé, N., Brouchet, A., Laurent, G., Salvayre, R., Jaffrézou, J.‐P., Levade, T. Stress‐induced apoptosis is not mediated by endolysosomal ceramide. FASEB J. 14, 36–47(2000)

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.—Jaffrezou, J.‐P., Bruno, A. P., Moisand, A., Levade, T., Laurent, G. Activation of a nuclear sphingomyelinase in radiation induced apoptosis. FASEB J. 15, 123–133 (2001)

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.

Ceramide in Regulation of Apoptosis

Studying cell death signaling pathways may provide a few answers in the field of anticancer pharmacology and may be useful for tailoring new therapies. Indeed, while extensive studies of the biochemical and molecular pharmacology of chemotherapeutic drugs have revealed intimate drug-target interactions, how and why such interactions should bring about cell death has been hitherto unclear. In fact, in many cases cellular damage actually caused by active doses of these agents (intercalators, topoisomerase inhibitors, antimitotics, etc.) is not sufficient to explain the observed toxicity (Chabner and Myers, 1989; Dive and Hickman, 1991). There is mounting evidence that anticancer agents kill by activating a metabolic pathway, rather than by crippling cellular metabolism (Fisher, 1994). The identification of apoptosis as a common response of neoplastic cells suggests that the cytotoxic action of chemotherapeutic drugs requires the active participation of the target cell (Dive and Hickman, 1991; Hickman, 1992; Fisher, 1994; Martin and Green, 1994). Although it remains to be determined if this participation occurs before or after effector induced cell damage; this observation has forced a reconsideration of the mechanism(s) whereby tumor cells respond to cytotoxic agents. Until recently, anticancer research has been, for the most part, more empirical that rational. In our endeavour to combat neoplastic diseases, researchers have developed a battery of treatments, most based on a simple