Bruno Ségui

Involvement of FAN in TNF-induced apoptosis

TNF-alpha is a pleiotropic cytokine activating several signaling pathways initiated at distinct intracellular domains of the TNF receptors. Although the C-terminal region is believed to be responsible for apoptosis induction, the functions of more membrane-proximal domains, including the domain that couples to neutral sphingomyelinase activation, are not yet fully elucidated. The roles of this region and of the associated adapter protein FAN (factor associated with neutral SMase activation) in the cytotoxic response to TNF have been investigated. We have now shown that stable expression in human fibroblasts of a dominant negative form of FAN abrogates TNF-induced ceramide generation from sphingomyelin hydrolysis and reduces caspase processing, thus markedly inhibiting TNF-triggered apoptosis. However, the cytotoxic responses to daunorubicin and exogenous ceramide remain unaltered, as do the TNF-induced p42/p44 MAPK activation and CD54 expression. Fibroblasts from FAN-knockout mice also proved to be resistant to TNF toxicity. These findings highlight the previously unrecognized role of the adapter protein FAN in signaling cell death induction by TNF.

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)

Ceramide in apoptosis: a revisited role.

The sphingolipid ceramide has recently emerged as a new transducer or modulator of apoptotic cell death. This function, however, has recently been challenged. Here, in the light of recent observations, the role of ceramide in apoptosis signaling is discussed.

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.

IL-6 Deficiency Attenuates Murine Diet-Induced Non-Alcoholic Steatohepatitis

Background The role of inflammation in the pathogenesis of non-alcoholic steatohepatitis (NASH), a common cause of liver disease, is still poorly understood. This study aimed at assessing the involvement of a major inflammatory cytokine, IL-6, in NASH. Materials and Methods Steatohepatitis was induced by feeding wild-type or IL-6−/− mice for 5 weeks with a methionine and choline-deficient (MCD) diet. Results Whereas MCD diet-induced weight loss and decreases in serum glucose, cholesterol and triglyceride levels were similar in both genotypes, serum alanine aminotransferase was less elevated in IL-6−/− mice than in wild-type animals. Despite having a comparable liver steatosis score, IL-6-deficient mice exhibited less lobular inflammation than their wild-type littermates. Liver gene expression of TGF-β and MCP-1 was also strongly attenuated in mutant mice; a more modest reduction was observed for PPAR-γ and F4/80 transcripts as well as proteins. Chromatographic analysis of liver lipids demonstrated that MCD diet induced in normal and mutant mice a similar decrease in the ratio of phosphatidylcholine to phosphatidylethanolamine. However, the diet-induced increase in the levels of sphingomyelin and ceramide was less important in IL-6−/− mice. Conclusion Altogether, these results indicate that IL-6 deficiency does not block the development of NASH; yet, IL-6 plays a critical role in the accompanying liver inflammation.

Caspase-mediated inhibition of sphingomyelin synthesis is involved in FasL-triggered cell death

Ceramide can be converted into sphingomyelin by sphingomyelin synthases (SMS) 1 and 2. In this study, we show that in human leukemia Jurkat cells, which express mainly SMS1, Fas ligand (FasL) treatment inhibited SMS activity in a dose- and time-dependent manner before nuclear fragmentation. The SMS inhibition elicited by FasL (1) was abrogated by benzyloxycarbonyl valyl-alanyl-aspartyl-(O-methyl)-fluoromethylketone (zVAD-fmk), a broad-spectrum caspase inhibitor; (2) did not occur in caspase-8-deficient cells and (3) was not affected in caspase-9-deficient cells. Western blot experiments showed SMS1 cleavage in a caspase-dependent manner upon FasL treatment. In a cell-free system, caspase-2, -7, -8 and -9, but not caspase-3 and -10, cleaved SMS1. In HeLa cells, SMS1 was Golgi localized and relocated throughout the cytoplasm in cells exhibiting an early apoptotic phenotype on FasL treatment. zVAD-fmk prevented FasL-induced SMS1 relocation. Thus, FasL-mediated SMS1 inhibition and relocation depend on caspase activation and likely represent proximal events in Fas signaling. FasL-induced ceramide production and cell death were enhanced in cells stably expressing an siRNA against SMS1. Conversely, in cells stably overexpressing SMS1, FasL neither increased ceramide generation nor efficiently induced cell death. Altogether, our data show that SMS1 is a novel caspase target that is functionally involved in the regulation of FasL-induced apoptosis.

CD95 triggers Orai1-mediated localized Ca2+ entry, regulates recruitment of protein kinase C (PKC) β2, and prevents death-inducing signaling complex formation

The death receptor CD95 plays a pivotal role in immune surveillance and immune tolerance. Binding of CD95L to CD95 leads to recruitment of the adaptor protein Fas-associated death domain protein (FADD), which in turn aggregates caspase-8 and caspase-10. Efficient formation of the CD95/FADD/caspase complex, known as the death-inducing signaling complex (DISC), culminates in the induction of apoptosis. We show that cells exposed to CD95L undergo a reorganization of the plasma membrane in which the Ca2+ release-activated Ca2+ channel Orai1 and the endoplasmic reticulum-resident activator stromal interaction molecule 1 colocalize with CD95 into a micrometer-sized cluster in which the channel elicits a polarized entry of calcium. Orai1 knockdown and expression of a dominant negative construct (Orai1E106A) reveal that on CD95 engagement, the Orai1-driven localized Ca2+ influx is fundamental to recruiting the Ca2+-dependent protein kinase C (PKC) β2 to the DISC. PKCβ2 in turn transiently holds the complex in an inactive status, preventing caspase activation and transmission of the apoptotic signal. This study identifies a biological role of Ca2+ and the Orai1 channel that drives a transient negative feedback loop, introducing a lag phase in the early steps of the CD95 signal. We suggest that these localized events provide a time of decision to prevent accidental cell death.

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.

Downregulation of ceramide synthase-6 during epithelial-to-mesenchymal transition reduces plasma membrane fluidity and cancer cell motility.

Epithelial-to-mesenchymal transition (EMT) promotes cell motility, which is important for the metastasis of malignant cells, and blocks CD95-mediated apoptotic signaling triggered by immune cells and chemotherapeutic regimens. CD95L, the cognate ligand of CD95, can be cleaved by metalloproteases and released as a soluble molecule (cl-CD95L). Unlike transmembrane CD95L, cl-CD95L does not induce apoptosis but triggers cell motility. Electron paramagnetic resonance was used to show that EMT and cl-CD95L treatment both led to augmentation of plasma membrane fluidity that was instrumental in inducing cell migration. Compaction of the plasma membrane is modulated, among other factors, by the ratio of certain lipids such as sphingolipids in the membrane. An integrative analysis of gene expression in NCI tumor cell lines revealed that expression of ceramide synthase-6 (CerS6) decreased during EMT. Furthermore, pharmacological and genetic approaches established that modulation of CerS6 expression/activity in cancer cells altered the level of C16-ceramide, which in turn influenced plasma membrane fluidity and cell motility. Therefore, this study identifies CerS6 as a novel EMT-regulated gene that has a pivotal role in the regulation of cell migration.

Expression of membrane-bound and soluble FasL in Fas- and FADD-dependent T lymphocyte apoptosis induced by mildly oxidized LDL

Apoptosis plays an essential role in atherosclerosis. Oxidized low‐density lipoproteins (oxLDL) and activated T lymphocytes are present in atherosclerotic lesions, and we have previously reported that oxLDL induce apoptosis of activated T lymphocytes. We now show that this is preceded by an increase of Fas and FasL expression. Fas and FasL overexpression was dependent on reactive oxygen species (ROS) production as well as ERK and JNK activation. In addition, oxLDL triggered an early production of soluble FasL by T lymphocytes. Blocking anti‐ Fas antibody or Fas‐Fc protein, but also antioxidant molecules and inhibitors of ERK and JNK, decreased oxLDL‐mediated apoptosis. Moreover, PHA‐activated murine lymphocytes lacking a functional Fas receptor were partially resistant to oxLDL. Finally, Jurkat T cells deficient for FADD, an adaptor protein required for Fas signaling, resisted oxLDL‐induced apoptosis. OxLDL triggered caspase 8 and 3 activation as well as ceramide production in PHA‐activated lymphocytes and in Jurkat cells. Caspase activation was completely impaired in FADD‐deficient cells, but ceramide production was not affected. Altogether, our results highlight the putative role of both membrane‐bound and soluble FasL in oxLDL‐induced Fas and FADD‐dependent apoptosis of T lymphocytes and suggest an involvement of ROS, ERK, and JNK in this process.