Ceramide-enriched membrane domains

Cellular activation involves the re-organization of receptor molecules and the intracellular signalosom in the cell membrane. Recent studies indicate that specialized domains of the cell membrane, termed rafts, are central for the spatial organization of receptors and signaling molecules. Rafts are converted into larger membrane platforms by activity of the acid sphingomyelinase, which hydrolyses raft-sphingomyelin to ceramide. Ceramide molecules spontaneously associate to form ceramide-enriched microdomains, which fuse to large ceramide-enriched membrane platforms. The acid sphingomyelinase is activated by multiple stimuli including CD95, CD40, DR5/TRAIL, CD20, FcgammaRII, CD5, LFA-1, CD28, TNF, the Interleukin-1 receptor, the PAF-receptor, CD14, infection with P. aeruginosa, S. aureus, N. gonorrhoeae, Sindbis-Virus, Rhinovirus, treatment with gamma-irradiation, UV-light, doxorubicin, cisplatin, disruption of integrin-signaling and under some conditions of developmental death. Ceramide-enriched membrane platforms serve the clustering of receptors, the recruitment of intracellular signaling molecules and the exclusion of inhibitory signaling factors and, thus, facilitate signal transduction initiated by the specific stimulus.     

Conjugation of doxorubicin to cell penetrating peptides sensitizes human breast MDA-MB 231 cancer cells to endogenous TRAIL-induced apoptosis

Previous work from our laboratory has shown that coupling doxorubicin (Dox) to cell penetrating peptides (Dox–CPPs) is a good strategy to overcome Dox resistance in MDA-MB 231 breast cancer cells. We also reported that, in contrast to unconjugated Dox-induced cell death, the increase in apoptotic response does not involve the mitochondrial apoptotic pathway. In this study, we demonstrate that both Dox and Dox–CPPs can increase the density of the TRAIL receptors DR4 and DR5 at the plasma membrane and moderately sensitize MDA-MB 231 cells to exogeneously added recombinant TRAIL, as has already been shown for other chemotherapeutic drugs. Moreover, we show that Dox–CPPs, used alone, induce the clustering of TRAIL receptors into ceramide-enriched membrane lipid rafts, a property not shared by unconjugated Dox and that this process is due to the generation of ceramide during Dox–CPPs treatment. In addition, MDA-MB 231 cells were found to express TRAIL and we show that the increased apoptotic rate induced by Dox–CPPs is due to the sensitization of MDA-MB 231 cells to endogenous TRAIL. The capacity of Dox–CPPs to sensitize cancer cells to physiologic amounts of TRAIL suggests that, in addition to their efficiency in combination chemotherapy, these compounds might increase the response of tumor cells to cytotoxic lymphocyte-mediated killing via TRAIL.     

Rhinoviruses infect human epithelial cells via ceramide-enriched membrane platforms

The cell membrane contains very small distinct membrane domains enriched of sphingomyelin and cholesterol that are named rafts. We have shown that the formation of ceramide via activation of the acid sphingomyelinase transforms rafts into ceramide-enriched membrane platforms. These platforms are required for infection of mammalian cells with Pseudomonas aeruginosa, Staphylococcus aureus, or Neisseriae gonorrhoeae. In the present study we determined whether the acid sphingomyelinase, ceramide, and ceramide-enriched membrane platforms are also involved in the infection of human cells with pathogenic rhinoviruses. We demonstrate that infection of human epithelial cells with several rhinovirus strains triggers a rapid activation of the acid sphingomyelinase correlating with microtubules- and microfilament-mediated translocation of the enzyme from an intracellular compartment onto the extracellular leaflet of the cell membrane. The activity of the acid sphingomyelinase results in the formation of ceramide in the cell membrane and, finally, large ceramide-enriched membrane platforms. Rhinoviruses colocalize with ceramide-enriched membrane platforms during the infection. The significance of ceramide-enriched membrane platforms for rhinoviral uptake is demonstrated by the finding that genetic deficiency or pharmacological inhibition of the acid sphingomyelinase prevented infection of human epithelial cells by rhinoviruses. The data identify the acid sphingomyelinase and ceramide as key molecules for the infection of human cells with rhinoviruses.     

Acid sphingomyelinase amplifies redox signaling in Pseudomonas aeruginosa-induced macrophage apoptosis

Recent studies indicate that distinct membrane microdomains, also named lipid rafts, and ceramide play an important role in infectious biology. Ceramide forms larger ceramide-enriched membrane platforms that are required for diverse signal transduction. In this study, we demonstrate that ceramide-enriched membrane platforms are critically involved in redox signaling that regulates alveolar macrophage apoptosis upon infection with Pseudomonas aeruginosa. In freshly isolated alveolar macrophages, P. aeruginosa infection results in rapid activation of acid sphingomyelinase (Asm), release of ceramide, and formation of ceramide-enriched membrane platforms, which are required for P. aeruginosa-induced activation of NADPH oxidase and production of reactive oxygen species (ROS). Inhibition of NADPH oxidase or removal of intracellular ROS reduced P. aeruginosa-induced activation of the Asm and formation of ceramide-enriched membrane platforms, suggesting that NADPH oxidase-derived ROS regulate Asm-initiated redox signaling in a positive feedback manner. Furthermore, stimulation of JNK and induction of apoptosis upon P. aeruginosa infections are dependent on NADPH oxidase-derived ROS. These findings indicate that ceramide-enriched membrane platforms are essential for amplification of Asm-mediated redox signaling, which mediates JNK activation and thereby apoptosis of alveolar macrophages upon P. aeruginosa infection.     

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.     

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.     

Infections with human rhinovirus induce the formation of distinct functional membrane domains.

The plasma membrane contains distinct domains that are characterized by a high concentration of sphingolipids and cholesterol. These membrane microdomains also referred to as rafts, seem to be intimately involved in transmembranous signaling and often initiate interactions of pathogens and the host cell membranes. Here, we investigated the further reorganization of membrane rafts in cultured epithelial cells and ex vivo isolated nasal cells after infection with rhinoviruses. We demonstrate the formation of ceramide-enriched membrane platforms and large glycosphingolipid-enriched membrane domains and the co-localization of fluorochrome-labeled rhinoviruses with these membrane domains during attachment and uptake of human rhinovirus. Destruction of glycosphingolipid-enriched membrane domains blocked infection of human cells with rhinovirus. Furthermore, our studies indicate that the activation of the acid sphingomyelinase (ASM) is intrigued in the formation of ceramide- or GM1- enriched membrane platforms. Inhibition of the ASM reduces the number of ceramide-enriched platforms and glycosphingolipid-enriched membrane domains. These data reveal a critical role of the ASM for the formation of membrane platforms and infection of human cells with rhinoviruses.     

Host defense against Pseudomonas aeruginosa requires ceramide-rich membrane rafts

Pseudomonas aeruginosa infection is a serious complication in patients with cystic fibrosis and in immunocompromised individuals. Here we show that P. aeruginosa infection triggers activation of the acid sphingomyelinase and the release of ceramide in sphingolipid-rich rafts. Ceramide reorganizes these rafts into larger signaling platforms that are required to internalize P. aeruginosa, induce apoptosis and regulate the cytokine response in infected cells. Failure to generate ceramide-enriched membrane platforms in infected cells results in an unabated inflammatory response, massive release of interleukin (IL)-1 and septic death of mice. Our findings show that ceramide-enriched membrane platforms are central to the host defense against this potentially lethal pathogen.     

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 gamma-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.     

Biological aspects of ceramide-enriched membrane domains

Ceramide has been shown to be critically involved in many aspects of cellular responses to receptor-dependent and -independent stimuli. For instance, ceramide was demonstrated to be a central component of the signaling cascades mediating apoptosis after death receptor stimulation, treatment with chemotherapy or exposure to gamma-irradiation or UV-A light. Further studies indicated the importance of ceramide for the infection of mammalian cells with bacterial, viral and parasitic pathogens. Ceramide is released by the activity of acid, neutral or alkaline sphingomyelinases or de novo synthesized. A concept unifying the diverse biological functions of ceramide indicates that ceramide forms distinct membrane domains, named ceramide-enriched membrane domains or platforms. These domains serve the clustering of receptor molecules, the re-organization of signaling proteins, the exclusion of inhibitory signals and, thus, initiate and greatly amplify a primary signal. In addition, ceramide directly interacts with and stimulates intracellular enzymes that may act together with signals initiated in ceramide-enriched membrane domains to transmit signals into a cell.     

Ceramide-enriched membrane domains—Structure and function

Membrane lipids seem to be organized and not randomly distributed in the cell membrane. In particular, sphingolipids seem to interact with cholesterol in the outer leaflet of the cell membrane resulting in the formation of distinct membrane domains, i.e. rafts. The generation of ceramide within rafts alters their biophysical properties and results in the formation of large ceramide-enriched membrane platforms. These platforms serve to cluster receptor molecules and to organize intracellular signalling molecules to facilitate signal transduction via a receptor upon stimulation. Thus, ceramide-enriched membrane domains amplify not only receptor-, but also stress-mediated signalling events. Although many receptors cluster, the molecular mechanisms mediating this important and general event in signal transduction need to be identified.     

Ceramide-enriched membrane domains--structure and function

Membrane lipids seem to be organized and not randomly distributed in the cell membrane. In particular, sphingolipids seem to interact with cholesterol in the outer leaflet of the cell membrane resulting in the formation of distinct membrane domains, i.e. rafts. The generation of ceramide within rafts alters their biophysical properties and results in the formation of large ceramide-enriched membrane platforms. These platforms serve to cluster receptor molecules and to organize intracellular signalling molecules to facilitate signal transduction via a receptor upon stimulation. Thus, ceramide-enriched membrane domains amplify not only receptor-, but also stress-mediated signalling events. Although many receptors cluster, the molecular mechanisms mediating this important and general event in signal transduction need to be identified.     

Differential Activation of Acid Sphingomyelinase and Ceramide Release Determines Invasiveness of Neisseria meningitidis into Brain Endothelial Cells

The interaction with brain endothelial cells is central to the pathogenicity of Neisseria meningitidis infections. Here, we show that N. meningitidis causes transient activation of acid sphingomyelinase (ASM) followed by ceramide release in brain endothelial cells. In response to N. meningitidis infection, ASM and ceramide are displayed at the outer leaflet of the cell membrane and condense into large membrane platforms which also concentrate the ErbB2 receptor. The outer membrane protein Opc and phosphatidylcholine-specific phospholipase C that is activated upon binding of the pathogen to heparan sulfate proteoglycans, are required for N. meningitidis-mediated ASM activation. Pharmacologic or genetic ablation of ASM abrogated meningococcal internalization without affecting bacterial adherence. In accordance, the restricted invasiveness of a defined set of pathogenic isolates of the ST-11/ST-8 clonal complex into brain endothelial cells directly correlated with their restricted ability to induce ASM and ceramide release. In conclusion, ASM activation and ceramide release are essential for internalization of Opc-expressing meningococci into brain endothelial cells, and this segregates with invasiveness of N. meningitidis strains.     

Induction of Membrane Ceramides: A Novel Strategy to Interfere with T Lymphocyte Cytoskeletal Reorganisation in Viral Immunosuppression

Silencing of T cell activation and function is a highly efficient strategy of immunosuppression induced by pathogens. By promoting formation of membrane microdomains essential for clustering of receptors and signalling platforms in the plasma membrane, ceramides accumulating as a result of membrane sphingomyelin breakdown are not only essential for assembly of signalling complexes and pathogen entry, but also act as signalling modulators, e. g. by regulating relay of phosphatidyl-inositol-3-kinase (PI3K) signalling. Their role in T lymphocyte functions has not been addressed as yet. We now show that measles virus (MV), which interacts with the surface of T cells and thereby efficiently interferes with stimulated dynamic reorganisation of their actin cytoskeleton, causes ceramide accumulation in human T cells in a neutral (NSM) and acid (ASM) sphingomyelinase–dependent manner. Ceramides induced by MV, but also bacterial sphingomyelinase, efficiently interfered with formation of membrane protrusions and T cell spreading and front/rear polarisation in response to β1 integrin ligation or αCD3/CD28 activation, and this was rescued upon pharmacological or genetic ablation of ASM/NSM activity. Moreover, membrane ceramide accumulation downmodulated chemokine-induced T cell motility on fibronectin. Altogether, these findings highlight an as yet unrecognised concept of pathogens able to cause membrane ceramide accumulation to target essential processes in T cell activation and function by preventing stimulated actin cytoskeletal dynamics.     

Physiological and pathophysiological aspects of ceramide

Activation of cells by receptor- and nonreceptor-mediated stimuli not only requires a change in the activity of signaling proteins but also requires a reorganization of the topology of the signalosom in the cell. The cell membrane contains distinct domains, rafts that serve the spatial organization of signaling molecules in the cell. Many receptors or stress stimuli transform rafts by the generation of ceramide. These stimuli activate the acid sphingomyelinase and induce a translocation of this enzyme onto the extracellular leaflet of the cell membrane. Surface acid sphingomyelinase generates ceramide that serves to fuse small rafts and to form large ceramide-enriched membrane platforms. These platforms cluster receptor molecules, recruit intracellular signaling molecules to aggregated receptors, and seem to exclude inhibitory signaling factors. Thus ceramide-enriched membrane platforms do not seem to be part of a specific signaling pathway but may facilitate and amplify the specific signaling elicited by the cognate stimulus. This general function may enable these membrane domains to be critically involved in the induction of apoptosis by death receptors and stress stimuli, bacterial and viral infections of mammalian cells, and the regulation of cardiovascular functions.     

Hyperthermia-enhanced TRAIL- and mapatumumab-induced apoptotic death is mediated through mitochondria in human colon cancer cells

Colorectal cancer is the third leading cause of cancer-related mortality in the world; death usually results from uncontrolled metastatic disease. Previously, we developed a novel strategy of TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) in combination with hyperthermia to treat hepatic colorectal metastases. However, previous studies suggest a potential hepatocyte cytotoxicity with TRAIL. Unlike TRAIL, anti-human TRAIL receptor antibody induces apoptosis without hepatocyte toxicity. In this study, we evaluated the anti-tumor efficacy of humanized anti-death receptor 4 (DR4) antibody mapatumumab (Mapa) by comparing it with TRAIL in combination with hyperthermia. TRAIL, which binds to both DR4 and death receptor 5 (DR5), was approximately tenfold more effective than Mapa in inducing apoptosis. However, hyperthermia enhances apoptosis induced by either agent. We observed that the synergistic effect was mediated through elevation of reactive oxygen species, c-Jun N-terminal kinase activation, Bax oligomerization, and translocalization to the mitochondria, loss of mitochondrial membrane potential, release of cytochrome c to cytosol, activation of caspases, and increase in poly(ADP-ribose) polymerase cleavage. We believe that the successful outcome of this study will support the application of Mapa in combination with hyperthermia to colorectal hepatic metastases.     

Ceramide acyl chain length markedly influences miscibility with palmitoyl sphingomyelin in bilayer membranes

Ceramides are precursors of major sphingolipids and can be important cellular effectors. The biological effects of ceramides have been suggested to stem from their biophysical effects on membrane structure affecting the lateral and transbilayer organization of other membrane components. In this study we investigated the effect of acyl chain composition in ceramides (C4-C24:1) on their miscibility with N-palmitoyl-sphingomyelin (PSM) using differential scanning calorimetry. We found that short-chain (C4 and C8) ceramides induced phase separation and lowered the T _m and enthalpy of the PSM endotherm. We conclude that short-chain ceramides were more miscible in the fluid-phase than in the gel-phase PSM bilayers. Long-chain ceramides induced apparent heterogeneity in the bilayers. The main PSM endotherm decreased in cooperativity and enthalpy with increasing ceramide concentration. New ceramide-enriched components could be seen in the thermograms at all ceramide concentrations above X _Cer = 0.05. These broad components had higher T _m values than pure PSM. C24:1 ceramide exhibited complex behavior in the PSM bilayers. The miscibility of C24:1 ceramide with PSM at low (X _Cer = 0.05–0.10) concentrations was exceptionally good according to the cooperativity of the transition. At higher concentrations, multiple components were detected, which might have arisen from interdigitated gel-phases formed by this very asymmetric ceramide. The results of this study indicate that short-chain and long-chain ceramides have very different effects on the sphingomyelin bilayers. There also seems to be a correlation between their miscibility in binary systems and the effect of ceramides of different hydrophobic length on sphingomyelin-rich domains in multicomponent membranes.     

Ceramide-rich membrane rafts mediate CD40 clustering.

Many receptor systems use receptor clustering for transmembrane signaling. In this study, we show that acid sphingomyelinase (ASM) is essential for the clustering of CD40. Stimulation of lymphocytes via CD40 ligation results in ASM translocation from intracellular stores, most likely vesicles, into distinct membrane domains on the extracellular surface of the plasma membrane. Surface ASM initiates a release of extracellularly oriented ceramide, which in turn mediates CD40 clustering in sphingolipid-rich membrane domains. ASM, ceramide, and CD40 colocalize in the cap-like structure of stimulated cells. Deficiency of ASM, destruction of sphingolipid-rich rafts, or neutralization of surface ceramide prevents CD40 clustering and CD40-initiated cell signaling. These findings indicate that the ASM-mediated release of ceramide and/or metabolites of ceramide regulate clustering of CD40, which seems to be a prerequisite for cellular activation via CD40.     

Molecular mechanisms of resveratrol (3,4,5-trihydroxy-trans-stilbene) and its interaction with TNF-related apoptosis inducing ligand (TRAIL) in androgen-insensitive prostate cancer cells

Although resveratrol, an active ingredient derived from grapes and red wine, possesses chemopreventive properties against several cancers, the molecular mechanisms by which it inhibits cell growth and induces apoptosis have not been clearly understood. Here, we examined the molecular mechanisms of resveratrol and its interactive effects with TRAIL on apoptosis in prostate cancer PC-3 and DU-145 cells. Resveratrol inhibited cell viability and colony formation, and induced apoptosis in prostate cancer cells. Resveratrol downregulated the expression of Bcl-2, Bcl-X_L and survivin and upregulated the expression of Bax, Bak, PUMA, Noxa, and Bim, and death receptors (TRAIL-R1/DR4 and TRAIL-R2/DR5). Treatment of prostate cancer cells with resveratrol resulted in generation of reactive oxygen species (ROS), translocation of Bax to mitochondria and subsequent drop in mitochondrial membrane potential, release of mitochondrial proteins (cytochrome c, Smac/DIABLO, and AIF) to cytosol, activation of effector caspase-3 and caspase-9, and induction of apoptosis. Resveratrol-induced ROS production, caspase-3 activity and apoptosis were inhibited by N-acetylcysteine. Bax was a major proapoptotic gene mediating the effects of resveratrol as Bax siRNA inhibited resveratrol-induced apoptosis. Resveratrol enhanced the apoptosis-inducing potential of TRAIL, and these effects were inhibited by either dominant negative FADD or caspase-8 siRNA. The combination of resveratrol and TRAIL enhanced the mitochondrial dysfunctions during apoptosis. These properties of resveratrol strongly suggest that it could be used either alone or in combination with TRAIL for the prevention and/or treatment of prostate cancer.     

Deficient tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor transport to the cell surface in human colon cancer cells selected for resistance to TRAIL-induced apoptosis

Many tumor cell types are sensitive to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Incubation of TRAIL-sensitive cells with TRAIL invariably leads to resistant survivors even when high doses of TRAIL are used. Because the emergence of resistance to apoptosis is a major concern in successful treatment of cancer, and TRAIL survivors may contribute to therapeutic failure, we investigated potential resistance mechanisms. We selected TRAIL-resistant SW480 human colon adenocarcinoma cells by repeatedly treating them with high and/or low doses of TRAIL. The resulting TRAIL-resistant clones were not cross-resistant to Fas or paclitaxel. Expression of modulators of apoptosis was not changed in the resistant cells, including TRAIL receptors, cFLIP, Bax, Bid, or IAP proteins. Surprisingly, we found that DISC formation was deficient in multiple selected TRAIL-resistant clones. DR4 was not recruited to the DISC upon TRAIL treatment, and caspase-8 was not activated at the DISC. Although total cellular DR4 mRNA and protein were virtually identical in TRAIL-sensitive parental and TRAIL-resistant clones, DR4 protein expression on the cell surface was essentially undetectable in the TRAIL-resistant clones. Moreover, exogenous DR4 and KILLER/DR5 were not properly transported to the cell surface in the TRAIL-resistant cells. Interestingly, TRAIL-resistant cells were resensitized to TRAIL by tunicamycin pretreatment, which increased cell surface expression of DR4 and KILLER/DR5. Our data suggest that tumor cells may become resistant to TRAIL through regulation of the death receptor cell surface transport and that resistance to TRAIL may be overcome by the glycosylation inhibitor/endoplasmic reticulum stress-inducing agent tunicamycin.