Thierry Levade

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.

Ceramide in apoptosis signaling: relationship with oxidative stress

Ceramide is one of the major sphingosine-based lipid second messengers that is generated in response to various extracellular agents. However, while widespread attention has focused on ceramide as a second messenger involved in the induction of apoptosis, important issues with regard to the mechanisms of ceramide formation and mode of action remain to be addressed. Several lines of evidence suggest that ceramide and oxidative stress are intimately related in cell death induction. This review focuses on the putative relationships between oxidative stress and sphingolipid metabolism in the apoptotic process and discusses the potential mechanisms that connect and regulate the two phenomena.

Role of JNK1-dependent Bcl-2 Phosphorylation in Ceramide-induced Macroautophagy

Macroautophagy is a vacuolar lysosomal catabolic pathway that is stimulated during periods of nutrient starvation to preserve cell integrity. Ceramide is a bioactive sphingolipid associated with a large range of cell processes. Here we show that short-chain ceramides (C2-ceramide and C6-ceramide) and stimulation of the de novo ceramide synthesis by tamoxifen induce the dissociation of the complex formed between the autophagy protein Beclin 1 and the anti-apoptotic protein Bcl-2. This dissociation is required for macroautophagy to be induced either in response to ceramide or to starvation. Three potential phosphorylation sites, Thr69, Ser70, and Ser87, located in the non-structural N-terminal loop of Bcl-2, play major roles in the dissociation of Bcl-2 from Beclin 1. We further show that activation of c-Jun N-terminal protein kinase 1 by ceramide is required both to phosphorylate Bcl-2 and to stimulate macroautophagy. These findings reveal a new aspect of sphingolipid signaling in up-regulating a major cell process involved in cell adaptation to stress.

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)

Regulation of Autophagy by Sphingosine Kinase 1 and Its Role in Cell Survival during Nutrient Starvation

The sphingolipid ceramide induces macroautophagy (here called autophagy) and cell death with autophagic features in cancer cells. Here we show that overexpression of sphingosine kinase 1 (SK1), an enzyme responsible for the production of sphingosine 1-phosphate (S1P), in MCF-7 cells stimulates autophagy by increasing the formation of LC3-positive autophagosomes and the rate of proteolysis sensitive to the autophagy inhibitor 3-methyladenine. Autophagy was blocked in the presence of dimethylsphingosine, an inhibitor of SK activity, and in cells expressing a catalytically inactive form of SK1. In SK1wt-overexpressing cells, however, autophagy was not sensitive to fumonisin B1, an inhibitor of ceramide synthase. In contrast to ceramide-induced autophagy, SK1(S1P)-induced autophagy is characterized by (i) the inhibition of mammalian target of rapamycin signaling independently of the Akt/protein kinase B signaling arm and (ii) the lack of robust accumulation of the autophagy protein Beclin 1. In addition, nutrient starvation induced both the stimulation of autophagy and SK activity. Knocking down the expression of the autophagy protein Atg7 or that of SK1 by siRNA abolished starvation-induced autophagy and increased cell death with apoptotic hallmarks. In conclusion, these results show that SK1(S1P)-induced autophagy protects cells from death with apoptotic features during nutrient starvation.

Recommendations on the diagnosis and management of Niemann-Pick disease type C

Niemann-Pick disease type C (NP-C) is a lysosomal storage disease in which impaired intracellular lipid trafficking leads to excess storage of cholesterol and glycosphingolipids in the brain and other tissues. It is characterized clinically by a variety of progressive, disabling neurological symptoms including clumsiness, limb and gait ataxia, dysarthria, dysphagia and cognitive deterioration (dementia). Until recently, there has been no disease-modifying therapy available for NP-C, with treatment limited to supportive measures. In most countries, NP-C is managed through specialist centers, with non-specialist support provided locally. However, effective patient support is hampered by the absence of national or international clinical management guidelines. In this paper, we seek to address this important gap in the current literature. An expert panel was convened in Paris, France in January 2009 to discuss best care practices for NP-C. This commentary reviews current literature on key aspects of the clinical management of NP-C in children, juveniles and adults, and provides recommendations based on consensus between the experts at the meeting.

Sphingomyelin hydrolysis during apoptosis.

Sphingolipid breakdown products are now being recognized as important players in apoptosis. Ceramide, which is considered to serve as second messenger, is mainly generated by hydrolysis of the membrane sphingophospholipid sphingomyelin (SM) through the action of a sphingomyelinase (SMase). However, little is known about the localization and regulation of this phenomenon. Here, we summarize the current knowledge on the function of SM hydrolysis in apoptosis signaling. In particular, the present review focuses on the role of neutral sphingomyelinase (N-SMase) in the generation of the proapoptotic ceramide. This enzyme is regulated by several mechanisms, including the tumor necrosis factor (TNF) receptor-associated protein FAN (for factor associated with N-SMase activation) and oxidative stress. These observations place SMase activation and SM hydrolysis as early events in the apoptosis signaling cascade.

Somatostatin receptor subtype 2 sensitizes human pancreatic cancer cells to death ligand-induced apoptosis

Somatostatin receptor subtype 2 (sst2) gene expression is lost in 90% of human pancreatic adenocarcinomas. We previously demonstrated that stable sst2 transfection of human pancreatic BxPC-3 cells, which do not endogenously express sst2, inhibits cell proliferation, tumorigenicity, and metastasis. These sst2 effects occur as a consequence of an autocrine sst2-dependent loop, whereby sst2 induces expression of its own ligand, somatostatin. Here we investigated whether sst2 induces apoptosis in sst2-transfected BxPC-3 cells. Expression of sst2 induced a 4.4- ± 0.05-fold stimulation of apoptosis in BxPC-3 through the activation of tyrosine phosphatase SHP-1. sst2 also sensitized these cells to apoptosis induced by tumor necrosis factor α (TNFα), enhancing it 4.1- ± 1.5-fold. Apoptosis in BxPC-3 cells mediated by TNF-related apoptosis-inducing ligand (TRAIL) and CD95L was likewise increased 2.3- ± 0.5-fold and 7.4- ± 2.5-fold, respectively. sst2-dependent activation and cell sensitization to death ligand-induced apoptosis involved activation of the executioner caspases, key factors in both death ligand- or mitochondria-mediated apoptosis. sst2 affected both pathways: first, by up-regulating expression of TRAIL and TNFα receptors, DR4 and TNFRI, respectively, and sensitizing the cells to death ligand-induced initiator capase-8 activation, and, second, by down-regulating expression of the antiapoptotic mitochondrial Bcl-2 protein. These results are of interest for the clinical management of chemoresistant pancreatic adenocarcinoma by using a combined gene therapy based on the cotransfer of genes for both the sst2 and a nontoxic death ligand.

Antiproliferative properties of sphingosine 1-phosphate in human hepatic myofibroblasts. A cyclooxygenase-2 mediated pathway.

Proliferation of hepatic myofibroblasts (hMF) is central for the development of fibrosis during liver injury, and factors that may limit their growth are potential antifibrotic agents. Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid with growth-regulating properties, either via Edg receptors or through intracellular actions. In this study, we examined the effects of S1P on the proliferation of human hMF. Human hMF expressed mRNAs for the S1P receptors Edg1, Edg3, and Edg5. These receptors were functional at nanomolar concentrations and coupled to pertussis toxin-sensitive and -insensitive G proteins, as demonstrated in guanosine 5′-3-O-(thio)triphosphate binding assays. S1P potently inhibited hMF growth (IC50 = 1 μm), in a pertussis toxin-insensitive manner. Analysis of the mechanisms involved in growth inhibition revealed that S1P rapidly increased prostaglandin E2 production and in turn cAMP, two growth inhibitory messengers for hMF; C2-ceramide and sphingosine, which inhibited hMF proliferation, did not affect cAMP levels. Production of cAMP by S1P was abolished by NS-398, a selective inhibitor of COX-2. Also, S1P potently induced COX-2 protein expression. Blocking COX-2 by NS-398 blunted the antiproliferative effect of S1P. We conclude that S1P inhibits proliferation of hMF, probably via an intracellular mechanism, through early COX-2-dependent release of prostaglandin E2 and cAMP, and delayed COX-2 induction. Our results shed light on a novel role for S1P as a growth inhibitory mediator and point out its potential involvement in the negative regulation of liver fibrogenesis.

Oxidized LDL-Induced Smooth Muscle Cell Proliferation Involves the EGF Receptor/PI-3 Kinase/Akt and the Sphingolipid Signaling Pathways

Objective—Oxidized low-density lipoprotein (oxLDL)-induced smooth muscle cell (SMC) proliferation requires the coactivation of various signaling pathways, namely sphingomyelin/ceramide/sphingosine-1-phosphate, epithelial growth factor receptor (EGFR), and phosphoinositide 3-kinase (PI-3K) pathways. This study aimed to clarify the respective role and the hypothetical cross-talk between sphingomyelin/ceramide/sphingosine-1-phosphate, EGFR, and PI-3K/Akt pathways in the balance between mitogenic and cytotoxic effects elicited by oxLDL. Methods and Results—Coimmunoprecipitation experiments and the use of inhibitors and dominant-negative mutant showed that oxLDL-induced PI-3K activation is dependent on EGFR. PI-3K activation is independent of the sphingomyelin/ceramide/sphingosine-1-phosphate pathway, because PI-3K inhibition by LY294002 or dominant-negative &Dgr;p85 mutant does not abrogate sphingomyelin hydrolysis, and, conversely, the use of permeant C2-ceramide and of N,N-dimethyl-sphingosine, a sphingosine kinase inhibitor, does not alter PI-3K activity. Activation of Akt/PKB by oxLDL requires PI-3K, as shown by the inhibition by LY204002 and in &Dgr;p85 SMC. The inhibition of Akt/PKB by PI-3K inhibitor LY204002 or by overexpression of kinase-dead Akt shifted the mitogenic effect of oxLDL toward apoptosis, thus suggesting that the PI-3K/Akt pathway acts as a survival pathway. Conclusions—SMC proliferation elicited by moderate concentrations of oxLDL involves the sphingomyelin/ceramide/sphingosine-1-phosphate pathway, which leads to extracellular regulated kinase 1/2 activation and DNA synthesis, and the EGFR/PI-3K/Akt pathway, which prevents the apoptotic effect of oxLDL.