Chiara Luberto

Ceramide in the eukaryotic stress response.

Several extracellular agents and stress stimuli, such as tumour necrosis factor alpha, chemotherapeutic agents and heat, cause ceramide accumulation. They do this by regulating enzymes involved in its metabolism. Ceramide modulates a number of biochemical and cellular responses to stress, including apoptosis, cell-cycle arrest and cell senescence.

Inhibition of tumor necrosis factor-induced cell death in MCF7 by a novel inhibitor of neutral sphingomyelinase.

A high throughput screen for neutral, magnesium-dependent sphingomyelinase (SMase) was performed. One inhibitor discovered in the screen, GW4869, functioned as a noncompetitive inhibitor of the enzyme in vitro with an IC50 of 1 μm. It did not inhibit acid SMase at up to at least 150 μm. The compound was then evaluated for its ability to inhibit tumor necrosis factor (TNF)-induced activation of neutral SMase (N-SMase) in MCF7 cells. GW4869 (10 μm) partially inhibited TNF-induced sphingomyelin (SM) hydrolysis, and 20 μm of the compound was protected completely from the loss of SM. The addition of 10–20 μmGW4869 completely inhibited the initial accumulation of ceramide, whereas this effect was partially lost at later time points (24 h). These data therefore support the inhibitory action of GW4869 on N-SMase not only in vitro but also in a cellular model. The addition of GW4869 at both 10 and 20 μm did not modify cellular glutathione levels in response to TNF, suggesting that the action of GW4869 occurred downstream of the drop in glutathione, which was shown previously to occur upstream of the activation of N-SMase. Further, whereas TNF treatment also caused a 75% increase ofde novo synthesized ceramide after 20 h of incubation, GW4869, at either 10 or 20 μm, had no effect on this pathway of ceramide generation. In addition, GW4869 did not significantly impair TNF-induced NF-κB translocation to nuclei. Therefore, GW4869 does not interfere with other key TNF-mediated signaling effects. GW4869 was able, in a dose-dependent manner, to significantly protect from cell death as measured by nuclear condensation, caspase activation, PARP degradation, and trypan blue uptake. These protective effects were accompanied by significant inhibition of cytochrome c release from mitochondria and caspase 9 activation, therefore localizing N-SMase activation upstream of mitochondrial dysfunction. In conclusion, our results indicate that N-SMase activation is a necessary step for the full development of the cytotoxic program induced by TNF.

Induction of Apoptosis through B-cell Receptor Cross-linking Occurs via de Novo Generated C16-Ceramide and Involves Mitochondria

B-cells, triggered via their surface B-cell receptor (BcR), start an apoptotic program known as activation-induced cell death (AICD), and it is widely believed that this phenomenon plays a role in the restriction and focusing of the immune response. Although both ceramide and caspases have been proposed to be involved in AICD, the contribution of either and the exact molecular events through which AICD commences are still unknown. Here we show that in Ramos B-cells, BcR-triggered cell death is associated with an early rise of C16 ceramide that derives from activation of the de novopathway, as demonstrated using a specific inhibitor of ceramide synthase, fumonisin B1 (FB1), and using pulse labeling with the metabolic sphingolipid precursor, palmitate. There was no evidence for activation of sphingomyelinases or hydrolysis of sphingomyelin. Importantly, FB1 inhibited several specific apoptotic hallmarks such as poly(A)DP-ribose polymerase cleavage and DNA fragmentation. Electron microscopy revealed morphological evidence of mitochondrial damage, suggesting the involvement of mitochondria in BcR-triggered apoptosis, and this was inhibited by FB1. Moreover, a loss of mitochondrial membrane potential was observed in Ramos cells after BcR cross-linking, which was inhibited by the addition of FB1. Interestingly, benzyloxycarbonyl-Val-Ala-dl-Asp, a broad spectrum caspase inhibitor did not inhibit BcR-induced mitochondrial membrane permeability transition but did block DNA fragmentation. These results suggest a crucial role for de novo generated C16 ceramide in the execution of AICD, and they further suggest an ordered and more specific sequence of biochemical events in which de novo generated C16 ceramide is involved in mitochondrial damage resulting in a downstream activation of caspases and apoptosis.

Glucosylceramide synthase is an essential regulator of pathogenicity of Cryptococcus neoformans

The pathogenic fungus Cryptococcus neoformans infects humans upon inhalation and causes the most common fungal meningoencephalitis in immunocompromised subjects worldwide. In the host, C. neoformans is found both intracellularly and extracellularly, but how these two components contribute to the development of the disease is largely unknown. Here we show that the glycosphingolipid glucosylceramide (GlcCer), which is present in C. neoformans, was essential for fungal growth in host extracellular environments, such as in alveolar spaces and in the bloodstream, which are characterized by a neutral/alkaline pH, but not in the host intracellular environment, such as in the phagolysosome of macrophages, which is characteristically acidic. Indeed, a C. neoformans mutant strain lacking GlcCer did not grow in vitro at a neutral/alkaline pH, yet it had no growth defect at an acidic pH. The mechanism by which GlcCer regulates alkali tolerance was by allowing the transition of C. neoformans through the cell cycle. This study establishes C. neoformans GlcCer as a key virulence factor of cryptococcal pathogenicity, with important implications for future development of new antifungal strategies.

Identification of ISC1 (YER019w) as Inositol Phosphosphingolipid Phospholipase C inSaccharomyces cerevisiae

Sphingolipids have emerged as novel bioactive mediators in eukaryotic cells including yeast. It has been proposed that sphingomyelin (SM) hydrolysis and the concomitant generation of ceramide are involved in various stress responses in mammalian cells. The yeast Saccharomyces cerevisiae has inositol phosphosphingolipids (IPS) instead of SM and glycolipids, and synthesis of IPS is indispensable to its growth. Although the genes responsible for the synthesis of IPS have been identified, the gene(s) for the degradation of IPS has not been reported. Here we show thatISC1 (YER019w), which has homology to bacterial neutral sphingomyelinase (SMase), encodes IPS phospholipase C (IPS-PLC). First, we observed that overexpression ofISC1 greatly increased neutral SMase activity, and this activity was dependent on the presence of phosphatidylserine. Cells deleted in ISC1 demonstrated negligible neutral SMase activity. Because yeast cells have IPS instead of SM, we investigated whether IPS are the physiologic substrates of this enzyme. Lysates ofISC1-overexpressing cells demonstrated very high PLC activities on IPS. Deletion of ISC1 eliminated endogenous IPS-PLC activities. Labeling yeast cells with [3H]dihydrosphingosine showed that IPS were increased in the deletion mutant cells. This study identifies the first enzyme involved in catabolism of complex sphingolipids in S. cerevisiae.

A mitochondrial pool of sphingomyelin is involved in TNFα-induced Bax translocation to mitochondria

We recently showed that targeting bSMase (bacterial sphingomyelinase) specifically to mitochondria caused accumulation of ceramide in mitochondria, and induced cytochrome c release and cell death [Birbes, El Bawab, Hannun and Obeid (2001) FASEB J., 15, 2669-2679]. In the present study, we investigated the role of this mitochondrial pool of ceramide in response to a receptor-mediated event, namely TNFalpha (tumour necrosis factor alpha), and the involvement of this mitochondrial pool of ceramide in Bax translocation to mitochondria, an event that precedes cytochrome c release. Treatment of MCF7 cells with TNFalpha caused an increase in ceramide levels in the mitochondrial fraction which accompanied Bax translocation to mitochondria. Targeting bSMase to mitochondria specifically resulted in Bax translocation to mitochondria, suggesting that the mitochondrial ceramide pool is involved in Bax translocation. Moreover, in a reconstituted cell-free system, treatment of isolated mitochondria with bSMase enhanced Bax association with mitochondrial membranes. Collectively, these results suggest that the generation of ceramide in mitochondria in response to TNFalpha is sufficient to induce Bax translocation to mitochondria and subsequent cytochrome c release and cell death.

Role of Sphingolipids in Microbial Pathogenesis

The sphingolipids are a class of lipids that serve as integral components of eukaryotic cell membranes (21, 49). Although previously considered to be simply structural molecules, sphingolipids have more recently been shown to act as signaling molecules in many cellular functions and to play crucial roles in the regulation of pathobiological processes, such as cancer, cardiovascular and neurodegenerative disorders, and inflammation or infectious diseases. In mammalian cells, ceramide, sphingosine, sphingosine-1-phosphate, and glucosylceramide are the most studied sphingolipids, and they regulate important processes, including the stress response, cell proliferation, apoptosis, angiogenesis, genetic diseases, and resistance to chemotherapy (7, 51, 80, 103, 105, 120). In other eukaryotes, such as fungi, sphingolipids have been implicated in the heat stress response (59, 110), endocytosis (97, 151), signal transduction (104), apoptosis (11), and fungal pathogenesis (11, 83). Interestingly, a mathematical model interconnecting the sphingolipid intracellular network has recently been proposed in the nonpathogenic fungus Saccharomyces cerevisiae (2); however, whether this interconnection is also present in pathogenic fungi or in mammalian cells awaits further investigations. In the area of microbial pathogenesis, sphingolipids play a role in the regulation of the delicate balance between the microbe and the host. Microorganisms that do not produce sphingolipids, including most bacteria and viruses, are able to utilize host sphingolipids to promote their virulence. Thus, in the context of bacterium- and virus-host interaction, the host is typically the source of sphingolipids, whereas in the context of the protozoan- and fungus-host interaction, both host and pathogen sphingolipids are involved. The determination of which sphingolipid(s) (host, microbe, or both) modulate the host-parasite interaction is particularly significant not only because it may provide important insights into the development of new therapeutic strategies but also because the outcome of this sphingolipid interaction may either lead to commensalism or to host damage/disease (9), thus expanding the function of a specific sphingolipid outside of the organism from which it originated. For instance, a mammalian sphingolipid acquired by a microbe may be used to exert a novel or different function through conversion by microbial enzyme into new sphingolipids or by hiding microbial cells from the host immune response and allowing colonization or dormancy without damage to the host. On the other hand, a microbial sphingolipid acquired by the host may interfere with host intracellular signaling and thereby alter the removal and destruction of the microbial cell, or it may elicit an autoimmune response through molecular mimicry. The goal of this review is to introduce and discuss microbial sphingolipids and their corresponding metabolizing enzymes as regulators of pathogenesis and to propose new hypotheses and perspectives toward a better understanding of the host-microbe interaction mediated by sphingolipids.

Role of ceramide in mediating the inhibition of telomerase activity in A549 human lung adenocarcinoma cells

This study was designed to analyze whether ceramide, a bioeffector of growth suppression, plays a role in the regulation of telomerase activity in A549 cells. Telomerase activity was inhibited significantly by exogenous C6-ceramide, but not by the biologically inactive analog dihydro-C6-ceramide, in a time- and dose-dependent manner, with 85% inhibition produced by 20 μm C6-ceramide at 24 h. Moreover, analysis of phosphatidylserine translocation from the inner to the outer plasma membrane by flow cytometry and of poly(ADP-ribose) polymerase degradation by Western blotting showed that ceramide treatment (20 μm for 24 h) had no apoptotic effects. Trypan blue exclusion, [3H]thymidine incorporation, and cell cycle analyses, coupled with clonogenic cell survival assay on soft agar, showed that ceramide treatment with a 20 μmconcentration at 24 h resulted in the cell cycle arrest of the majority of the cell population at G0/G1 with no detectable cell death. These results suggest that the inhibition of telomerase by ceramide is not a consequence of cell death but is correlated with growth arrest. Next, to determine the role of endogenous ceramide in telomerase modulation, A549 cells were transiently transfected with an expression vector containing the full-length bacterial sphingomyelinase cDNA (b-SMase). The overexpression of b-SMase, but not exogenously applied purified b-SMase enzyme, resulted in significantly decreased telomerase activity compared with controls, showing that the increased endogenous ceramide is sufficient for telomerase inhibition. Moreover, treatment of A549 cells with daunorubicin at 1 μm for 6 h resulted in the inhibition of telomerase, which correlated with the elevation of endogenous ceramide levels and growth arrest. Finally, stable overexpression of human glucosylceramide synthase, which attenuates ceramide levels by converting ceramide to glucosylceramide, prevented the inhibitory effects of C6-ceramide and daunorubicin on telomerase. Therefore, these results provide novel data showing for the first time that ceramide is a candidate upstream regulator of telomerase.

Identification of App1 as a regulator of phagocytosis and virulence of Cryptococcus neoformans

Cryptococcus neoformans is a fungal pathogen that, after inhalation, can disseminate to the brain. Host alveolar macrophages (AMs) represent the first defense against the fungus. Once phagocytosed by AMs, fungal cells are killed by a concerted mechanism, involving the host-cellular response. If the cellular response is impaired, phagocytosis of the fungus may be detrimental for the host, since C. neoformans can grow within macrophages. Here, we identified a novel cryptococcal gene encoding antiphagocytic protein 1 (App1). App1 is a cryptococcal cytoplasmic protein that is secreted extracellularly and found in the serum of infected patients. App1 does not affect melanin production, capsule formation, or growth of C. neoformans. Treatment with recombinant App1 inhibited phagocytosis of fungal cells through a complement-mediated mechanism, and Deltaapp1 mutant is readily phagocytosed by AMs. Interestingly, the Deltaapp1 mutant strain showed a decreased virulence in mice deficient for complement C5 (A/Jcr), but it was hypervirulent in mice deficient for T and NK cells (Tgepsilon26). This study identifies App1 as a novel regulator of virulence for C. neoformans, and it highlights that internalization of fungal cells by AMs increases the dissemination of C. neoformans when the host cellular response is impaired.

Positively Charged Ceramide Is a Potent Inducer of Mitochondrial Permeabilization

Ceramide-induced cell death is thought to be mediated by change in mitochondrial function, although the precise mechanism is unclear. Proposed models suggest that ceramide induces cell death through interaction with latent binding sites on the outer or inner mitochondrial membranes, followed by an increase in membrane permeability, as an intermediate step in ceramide signal propagation. To investigate these models, we developed a new generation of positively charged ceramides that readily accumulate in isolated and in situ mitochondria. Accumulated, positively charged ceramides increased inner membrane permeability and triggered release of mitochondrial cytochrome c. Furthermore, the positively charged ceramide-induced permeability increase was suppressed by cyclosporin A (60%) and 1,3-dicyclohexylcarbodiimide (90%). These observations suggest that the inner membrane permeability increase is due to activation of specific ion transporters, not the generalized loss of lipid bilayer barrier functions. The difference in sensitivity of ceramide-induced ion fluxes to inhibitors of mitochondrial transporters suggests activation of at least two transport systems: the permeability transition pore and the electrogenic H+ channel. Our results indicate the presence of specific ceramide targets in the mitochondrial matrix, the occupation of which triggers permeability alterations of the inner and outer mitochondrial membranes. These findings also suggest a novel therapeutic role for positively charged ceramides.