Ceramide-enriched Membrane Domains Research Articles

Ceramide-Enriched Membrane Domains Contribute to Targeted and Nontargeted Effects of Radiation through Modulation of PI3K/AKT Signaling in HNSCC Cells.

We investigated the potential involvement of ceramide-enriched membrane domains in radiation-induced targeted and nontargeted effects using head and neck squamous cell carcinoma with opposite radiosensitivities. In radiosensitive SCC61 cells, the proportion of targeted effects was 34% and nontargeted effects killed 32% of cells. In contrast, only targeted effects (30%) are involved in the overall death of radioresistant SQ20B cells. We then demonstrated in SCC61 cells that nontargeted cell response was driven by the formation of the radiation-induced ceramide-enriched domain. By contrast, the existence of these platforms in SQ20B cells confers a permissive region for phosphatidylinositol-3-kinase (PI3K)/AKT activation. The disruption of lipid raft results in strong inhibition of PI3K/AKT signaling, leading to radiosensitization and apparition of nontargeted effects. These results suggest that ceramide-enriched platforms play a significant role in targeted and nontargeted effects during radiotherapy and that drugs modulating cholesterol levels may be a good alternative for improving radiotherapy effectiveness.

Ceramide-Enriched Membrane Domains Contribute to Targeted and Nontargeted Effects of Radiation Through Modulation of PI3K/AKT Signaling in HNSCC Cells

Background: It is now established that radiation can affect membranes of mammalian cells in several ways. Depending on the degree of conditioning and mobility of phospholipidic molecules within the plasma membrane, cells exhibit different degrees of sensitivity to radiation. Methods: Here, we investigated the potential involvement of ceramide-enriched membrane domains in radiation-induced targeted and nontargeted effects. Two cell lines of head and neck squamous cell carcinoma (HNSCC) with opposite radiosensitivity were used. Findings: In radiosensitive SCC61 cells, the proportion of targeted effects was 34% and nontargeted effects killed about 32% of cells. In contrast, only targeted effects (30%) are involved in the overall death of radioresistant SQ20B cells. Using methyl-β-cyclodextrin (MBCD), a lipid raft disruptor, we found in SCC61 cells that the formation of a radiation-induced ceramide-enriched domain was suppressed which resulted in a significant reduction of nontargeted effects. By contrast, the existence of ceramide-enriched platforms in radioresistant SQ20B cells, blocks the response to bystander signaling and also confers a protective effect against radiation. We also showed that lipid raft disruption was followed with strong inhibition of PI3K/AKT signaling in SQ20B cells. Combining wortmannin, a PI3K inhibitor with radiation resulted in an increase of cell death and induction of nontargeted response when compared with radiation alone in these cells. Interpretation: These results show in HNSCC cells that ceramide-enriched platforms play a significant role in targeted and nontargeted effect during radiotherapy. They also suggest the potential utility of combining drugs modulating cholesterol levels such as statins with radiation to improve the HNSCC treatment. Funding: This study was supported by LabEx Primes (ANR-11 LABX-0063). Declaration of Interests: No potential conflicts of interest are disclosed.

Acid Sphingomyelinase-Ceramide System in Bacterial Infections.

Acid sphingomyelinase hydrolyzes sphingomyelin to ceramide and phosphorylcholine. Ceramide molecules spontaneously interact with each other and generate ceramide-enriched membrane domains. These ceramide-enriched domains further fuse, forming large ceramideenriched platforms that participate in the organization of receptors and in the amplification of signaling molecules. Recent studies have suggested several bacteria and bacterial toxins that stimulate the activation and the translocation of acid sphingomyelinase, which leads to the release of ceramide. The acid sphingomyelinase/ceramide system also regulates the internalization of bacteria into the host cell, the subsequent cytokine release, inflammatory response, and initiation of host cell apoptosis. In addition, ceramide has been implicated in the fusion of phagosomes and lysosomes upon bacterial infection. Thus, this system modulates the reorganization of cell membrane receptors and intracellular signaling molecules during bacteria-host interactions. The acid sphingomyelinase and ceramide system may thus serve as a novel therapeutic target for treating infections.

Role of caspases in CD95-induced biphasic activation of acid sphingomyelinase.

Acid sphingomyelinase (A-SMase) plays an important role in the initiation of CD95 signaling by forming ceramide-enriched membrane domains that enable clustering and activation of the death receptors. In TNF-R1 and TRAIL-R1/R2 signaling, A-SMase also contributes to the lysosomal apoptosis pathway triggered by receptor internalization. Here, we investigated the molecular mechanism of CD95-mediated A-SMase activation, demonstrating that A-SMase is located in internalized CD95-receptosomes and is activated by the CD95/CD95L complex in a biphasic manner.Since several caspases have been described to be involved in the activation of A-SMase, we evaluated expression levels of caspase-8, caspase-7 and caspase-3 in CD95-receptosomes. The occurrence of cleaved caspase-8 correlated with the first peak of A-SMase activity and translocation of the A-SMase to the cell surface which could be blocked by the caspase-8 inhibitor IETD.Inhibition of CD95-internalization selectively reduced the second phase of A-SMase activity, suggesting a fusion between internalized CD95-receptosomes and an intracellular vesicular pool of A-SMase. Further analysis demonstrated that caspase-7 activity correlates with the second phase of the A-SMase activity, whereas active caspase-3 is present at early and late internalization time points. Blocking caspases-7/ -3 by DEVD reduced the second phase of A-SMase activation in CD95-receptosomes suggesting the potential role of caspase-7 or -3 for late A-SMase activation.In summary, we describe a biphasic A-SMase activation in CD95-receptosomes indicating (I.) a caspase-8 dependent translocation of A-SMase to plasma membrane and (II.) a caspase-7 and/or -3 dependent fusion of internalized CD95-receptosomes with intracellular A-SMase-containing vesicles.

Lipid Domain Morphologies in Phosphatidylcholine−Ceramide Monolayers

In cells, one of the main roles of ceramide-enriched membrane domains is to recruit or exclude intracellular signaling molecules and receptors, thereby facilitating signal transduction cascades. Accordingly, in model membranes, even low contents of ceramide segregate into lateral domains. The impact of the N-acyl chain on this segregation and on the morphology of the domains remains to be explored. Using Langmuir monolayers, we have systematically studied binary mixtures of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and ceramide (2:1, molar ratio) and varied the N-acyl chain length of ceramide from 2 to 24 carbon atoms (Cer2 to Cer24). Fluid Cer2, Cer6, and Cer8/DMPC mixtures were miscible at all surface pressures. Longer ceramides, however, formed surface pressure-dependent immiscible mixtures with DMPC. The domain morphology under fluorescence microscopy after including a trace amount of fluorescent NBD-phosphatidylcholine into DMPC/Cer mixtures was found to be very sensitive to the N-acyl chain length. Shorter ceramides (Cer10-Cer14) formed flower-like (seaweed) domains, whereas longer ceramides (N-acyl chain length>14 carbon atoms) formed round and regular domains. We attribute the formation of the flower patterns to diffusive morphological instabilities during domain growth.

Ceramide-Enriched Membrane Domains in Red Blood Cells and the Mechanism of Sphingomyelinase-Induced Hot-Cold Hemolysis

Hot-cold hemolysis is the phenomenon whereby red blood cells, preincubated at 37 degrees C in the presence of certain agents, undergo rapid hemolysis when transferred to 4 degrees C. The mechanism of this phenomenon is not understood. PlcHR 2, a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa, induces hot-cold hemolysis. We found that the sphingomyelinase, but not the phospholipase C activity, is essential for hot-cold hemolysis because the phenomenon occurs not only in human erythrocytes that contain both phosphatidylcholine (PC) and sphingomyelin (SM) but also in goat erythrocytes, which lack PC. Fluorescence microscopy observations confirm the formation of ceramide-enriched domains as a result of PlcHR 2 activity. After cooling down to 4 degrees C, the erythrocyte ghost membranes arising from hemolysis contain large, ceramide-rich domains. We suggest that formation of these rigid domains in the originally flexible cell makes it fragile, thus highly susceptible to hemolysis. We also interpret the slow hemolysis observed at 37 degrees C as a phenomenon of gradual release of aqueous contents, induced by the sphingomyelinase activity. These hypotheses are supported by the fact that ceramidase, which is known to facilitate slow hemolysis at 37 degrees C, actually hinders hot-cold hemolysis. Differential scanning calorimetry of erythrocyte membranes treated with PlcHR 2 demonstrates the presence of ceramide-rich domains that are rigid at 4 degrees C but fluid at 37 degrees C. Ceramidase treatment causes the disapperance of the calorimetric signal assigned to ceramide-rich domains. Finally, in liposomes composed of SM, PC, and cholesterol, which exhibit slow release of aqueous contents at 37 degrees C, addition of 10 mol % ceramide and transfer to 4 degrees C cause a large increase in the rate of solute efflux.

Ceramide-Enriched Membrane Domains in Red Blood Cells and the Mechanism of Sphingomyelinase-Induced Hot−Cold Hemolysis

Hot-cold hemolysis is the phenomenon whereby red blood cells, preincubated at 37 degrees C in the presence of certain agents, undergo rapid hemolysis when transferred to 4 degrees C. The mechanism of this phenomenon is not understood. PlcHR 2, a phospholipase C/sphingomyelinase from Pseudomonas aeruginosa, that is the prototype of a new phosphatase superfamily, induces hot-cold hemolysis. We found that the sphingomyelinase, but not the phospholipase C activity, is essential for hot-cold hemolysis because the phenomenon occurs not only in human erythrocytes that contain both phosphatidylcholine (PC) and sphingomyelin (SM) but also in goat erythrocytes, which lack PC. However, in horse erythrocytes, with a large proportion of PC and almost no SM, hot-cold hemolysis induced by PlcHR 2 is not observed. Fluorescence microscopy observations confirm the formation of ceramide-enriched domains as a result of PlcHR 2 activity. After cooling down to 4 degrees C, the erythrocyte ghost membranes arising from hemolysis contain large, ceramide-rich domains. We suggest that formation of these rigid domains in the originally flexible cell makes it fragile, thus highly susceptible to hemolysis. We also interpret the slow hemolysis observed at 37 degrees C as a phenomenon of gradual release of aqueous contents, induced by the sphingomyelinase activity, as described by Ruiz-Arguello et al. [(1996) J. Biol. Chem. 271, 26616]. These hypotheses are supported by the fact that ceramidase, which is known to facilitate slow hemolysis at 37 degrees C, actually hinders hot-cold hemolysis. Differential scanning calorimetry of erytrocyte membranes treated with PlcHR 2 demonstrates the presence of ceramide-rich domains that are rigid at 4 degrees C but fluid at 37 degrees C. Ceramidase treatment causes the disapperance of the calorimetric signal assigned to ceramide-rich domains. Finally, in liposomes composed of SM, PC, and cholesterol, which exhibit slow release of aqueous contents at 37 degrees C, addition of 10 mol % ceramide and transfer to 4 degrees C cause a large increase in the rate of solute efflux.

Ceramide in bacterial infections and cystic fibrosis

Ceramide is formed by the activity of sphingomyelinases, by degradation of complex sphingolipids, reverse ceramidase activity or de novo synthesized. The formation of ceramide within biological membranes results in the formation of large ceramide-enriched membrane domains. These domains serve the spatial and temporal organization of receptors and signaling molecules. The acid sphingomyelinase-ceramide system plays an important role in the infection of mammalian host cells with bacterial pathogens such as Neisseria gonorrhoeae, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Salmonella typhimurium and Pseudomonas aeruginosa. Ceramide and ceramide-enriched membrane platforms are also involved in the induction of apoptosis in infected cells, such as in epithelial and endothelial cells after infection with Pseudomonas aeruginosa and Staphylococcus aureus, respectively. Finally, ceramide-enriched membrane platforms are critical regulators of the release of pro-inflammatory cytokines upon infection. The diverse functions of ceramide in bacterial infections suggest that ceramide and ceramide-enriched membrane domains are key players in host responses to many pathogens and thus are potential novel targets to treat infections.

Ceramide: Physiological and pathophysiological aspects

Ceramide generated in the cell membrane has been shown to be central for the induction of apoptosis by death receptors and many stress stimuli such as γ-irradiation, UV-light or infection with pathogens. Ceramide reorganizes cell membranes and forms large ceramide-enriched membrane domains that serve the spatial and temporal organization of the cellular signalosome upon activation. Thus, ceramide-enriched membrane domains mediate clustering of CD95 and DR5 to facilitate apoptosis, and they are also critically involved in apoptosis after irradiation, UV-light and infection with Pseudomonas aeruginosa. Since ceramide-enriched membrane domains amplify signals, their function is not restricted to the induction of apoptosis and it was shown that ceramide-enriched membrane domains are also involved in internalization of pathogens and the control of cytokine release from infected epithelial cells. Recent studies support the notion that changes of the ceramide metabolism are also critically involved in human diseases, for instance neurological disorders, cancer, infectious diseases and Wilson’s disease.

Ceramide and sphingomyelin species of fibroblasts and neurons in culture

The ceramide (Cer) and sphingomyelin (SM) species of cultured differentiated rat cerebellar granule cells and human fibroblasts were characterized by electrospray ionization-mass spectrometry. We identified 35 different species of Cer and 18 species of SM in human fibroblasts, and 35 different species of Cer and 9 species of SM were characterized in rat neurons. The main Cer species of rat cerebellar granule cells contained d18:1 sphingosine linked with palmitic, stearic, or nervonic fatty acid, and the two main SM species were d18:1,16:0 and d18:1,18:0. Both sphingolipids were enriched in detergent-resistant membranes (DRMs; or lipid rafts), and significant differences were found in the sphingolipid patterns of DRMs and of detergent-soluble fractions (DSF) from these cells. In human fibroblasts, the main Cer species were d18:1,16:0, d18:2,16:0, d18:1,24:0, d18:2,24:0, d18:1,24:1, and d18:2,24:1; the most represented species of SM were d18:1,16:0, d18:1,24:0, and d18:1,24:1. In these cells, SM was highly enriched in DRMs and Cer was mainly associated with DSF, and the species found in DRMs were markedly different from those found in DSF.

TRAIL activates acid sphingomyelinase via a redox mechanism and releases ceramide to trigger apoptosis

We have previously shown that activation of the acid sphingomyelinase (ASM), the release of ceramide and the formation of ceramide-enriched membrane domains are central for the induction of apoptosis by CD95. Here, we demonstrate that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and CD95 activate the ASM via a redox mechanism resulting in release of ceramide and formation of ceramide-enriched membrane platforms. Ceramide-enriched membrane platforms serve to cluster DR5 upon stimulation. Antioxidants prevent TRAIL-mediated stimulation of ASM, the release of ceramide, the formation of ceramide-enriched membrane platforms and the induction of apoptosis by TRAIL. Further, ASM-deficient splenocytes fail to cluster DR5 in ceramide-enriched membrane domains upon TRAIL stimulation and resist TRAIL-induced apoptosis, events that were restored by addition of natural C(16)-ceramide. A dose-response analysis indicates that ceramide-enriched membrane platforms greatly sensitized tumor cells to TRAIL-induced apoptosis. Our data indicate that ceramide-enriched membrane platforms are required for the signaling of TRAIL-DR5 complexes under physiological conditions.

Clustering of CD40 Ligand Is Required to Form a Functional Contact with CD40

Receptor clustering is a key event in the initiation of signaling by many types of receptor molecules. Here, we provide evidence for the novel concept that clustering of a ligand is a prerequisite for clustering of the cognate receptor. We show that clustering of the CD40 receptor depends on reciprocal clustering of the CD40 ligand (gp39, CD154). Clustering of the CD40 ligand is mediated by an association of the ligand with p53, a translocation of acid sphingomyelinase (ASM) to the cell membrane, an activation of the ASM, and a formation of ceramide. Ceramide appears to modify preexisting sphingolipid-rich membrane microdomains to fuse and form ceramide-enriched signaling platforms that serve to cluster CD40 ligand. Genetic deficiency of p53 or ASM or disruption of ceramide-enriched membrane domains prevents clustering of CD40 ligand. The functional significance of CD40 ligand clustering is indicated by the finding that clustering of CD40 on B lymphocytes upon co-incubation with CD40 ligand-expressing T cells depends on clustering of the CD40 ligand and is abrogated by inhibition of CD40 ligand clustering.