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.     

CD95 signaling via ceramide-rich membrane rafts

Clustering seems to be employed by many receptors for transmembrane signaling. Here, we show that acid sphingomyelinase (ASM)-released ceramide is essential for clustering of CD95. In vitro and in vivo, extracellularly orientated ceramide, released upon CD95-triggered translocation of ASM to the plasma membrane outer surface, enabled clustering of CD95 in sphingolipid-rich membrane rafts and apoptosis induction. Whereas ASM deficiency, destruction of rafts, or neutralization of surface ceramide prevented CD95 clustering and apoptosis, natural ceramide only rescued ASM-deficient cells. The data suggest CD95-mediated clustering by ceramide is prerequisite for signaling and death.     

A novel role for sphingolipid intermediates in activation-induced cell death in T cells

Activation-induced cell death (AICD), a process mediated by CD95 and CD95 ligand (CD95L), plays a critical role in regulating homeostasis of the immune system. Although the role of sphingolipids such as ceramides has been suggested to participate in CD95-mediated apoptosis, the exact role of these molecules in this process remains controversial. We employed myriocin, a specific inhibitor of serine palmitoyl-CoA transferase that mediates the first commitment step in sphingolipid synthesis. We found that myriocin could effectively block AICD in T-cell hybridomas and T-cell blasts. However, fumonisin B1, an inhibitor of the final step of ceramide synthesis, or inhibitors of sphingomyelinases did not prevent AICD. Furthermore, ceramide analogues, such as C2 and C6, could not reverse the inhibitory effect of myriocin. Interestingly, sphinganine, an intermediate of ceramide synthesis, completely reversed the inhibitory effect of myriocin, indicating a critical role of sphinganine. Myriocin did not modulate the expression of CD95 or CD95L, instead, it interfered with the early steps of CD95-mediated caspase activation. Therefore, we have uncovered a novel mechanism by which sphingolipid intermediates regulate CD95-mediated apoptosis.     

Identification and characterization of a ligand-independent oligomerization domain in the extracellular region of the CD95 death receptor

The CD95 death receptor plays an important role in several physiological and pathological apoptotic processes involving in particular the immune system. CD95 ligation leads to clustering of the receptor cytoplasmic "death domains" and recruitment of the zymogen form of caspase-8 to the cell surface. Activation of this protease through self-cleavage, followed by activation of downstream effector caspases, culminates in cleavage of a set of cellular proteins resulting in apoptosis with disassembly of the cell. It is very well known that the extracellular region of the CD95 receptor is required for CD95L interaction and that the death domain is necessary for the induction of the apoptotic signaling. Here, we identified and characterized a novel CD95 ligand- and death domain-independent oligomerization domain mapping to the NH(2)-terminal extracellular region of the CD95 receptor. In vitro and in vivo studies indicated that this domain, conserved among all soluble CD95 variants, mediates homo-oligomerization of the CD95 receptor and of the soluble CD95 proteins, as well as hetero-oligomerization of the receptor with the soluble variants. These results offer new insight into the mechanism of apoptosis inhibition mediated by the soluble CD95 proteins and suggest a role of the extracellular oligomerization domain in the regulation of the non-signaling state of the CD95 receptor.     

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.     

Crm-A, bcl-2 and NDGA inhibit CD95L-induced apoptosis of malignant glioma cells at the level of caspase 8 processing

Susceptibility to CD95 (Fas/APO-1)-mediated apoptosis in human glioma cells depends on CD95 expression and unknown factors that regulate signal transduction. Thus, LN-18 cells are highly sensitive to CD95 ligand (CD95L) whereas LN-229 cells require coexposure to inhibitors of RNA or protein synthesis for induction of apoptosis. Here, we report that caspase 8 and 3 activation, poly(ADP-ribose)polymerase cleavage and apoptosis are inhibited by the lipoxygenase inhibitor, nordihydroguaretic acid (NDGA), or ectopic expression of crm-A or bcl-2. CD95L-induced glioma cell apoptosis does not involve ceramide generation. Apoptosis induced by exogenous ceramide resembles CD95-mediated apoptosis in that bcl-2 is protective but differs in that NDGA and crm-A have no effect and in that cycloheximide (CHX) inhibits rather than potentiates ceramide-induced cell death. We conclude that caspase 8 and caspase 3 activation, but not ceramide generation, are required for CD95 ligand-induced apoptosis of glioma cells and that bcl-2, crm-A and NDGA all act upstream of caspases to inhibit apoptosis.     

Cell surface ceramide generation precedes and controls FcgammaRII clustering and phosphorylation in rafts

Despite the role of sphingolipid/cholesterol rafts as signaling platforms for Fcgamma receptor II (FcgammaRII), the mechanism governing translocation of an activated receptor toward the rafts is unknown. We show that at the onset of FcgammaRII cross-linking acid sphingomyelinase is rapidly activated. This enzyme is extruded from intracellular compartments to the cell surface, and concomitantly, exofacially oriented ceramide is produced. Both non-raft and, to a lesser extent, raft sphingomyelin pools were hydrolyzed at the onset of FcgammaRII cross-linking. The time course of ceramide production preceded the recruitment of FcgammaRII to rafts and the receptor phosphorylation. Exogenous C(16)-ceramide facilitated clustering of FcgammaRII and its association with rafts. In contrast, inhibition of acid sphingomyelinase diminished both the ceramide generation and clustering of cross-linked FcgammaRII. Under these conditions, tyrosine phosphorylation of FcgammaRII and receptor-accompanying proteins was also reduced. All the inhibitory effects were bypassed by treatment of cells with exogenous ceramide. These data provide evidence that the generation of cell surface ceramide is a prerequisite for fusion of cross-linked FcgammaRII and rafts, which triggers the receptor tyrosine phosphorylation and signaling.     

Ordering of ceramide formation and caspase-9 activation in CD95L-induced Jurkat leukemia T cell apoptosis

Ceramide, a biologically active sphingolipid in cell death signaling, accumulates upon CD95L treatment, concomitantly to apoptosis induction in Jurkat leukemia T cells. Herein, we show that ceramide did not increase in caspase-8 and -10-doubly deficient Jurkat cells in response to CD95L, indicating that apical caspases are essential for CD95L-triggered ceramide formation. Jurkat cells are typically defined as type 2 cells, which require the activation of the mitochondrial pathway for efficient apoptosis induction in response to CD95L. Caspase-9-deficient Jurkat cells significantly resisted CD95L-induced apoptosis, despite ceramide accumulation. Knock-down of sphingomyelin synthase 1, which metabolizes ceramide to sphingomyelin, enhanced (i) CD95L-triggered ceramide production, (ii) cytochrome c release from the mitochondria and (iii) caspase-9 activation. Exogenous ceramide-induced caspase-3 activation and apoptosis were impaired in caspase-9-deficient Jurkat cells. Conversely, caspase-9 re-expression in caspase-9-deficient Jurkat cells restored caspase-3 activation and apoptosis upon exogenous ceramide treatment. Collectively, our data provide genetic evidence that CD95L-triggered endogenous ceramide increase in Jurkat leukemia T cells (i) is not a mere consequence of cell death and occurs mainly in a caspase-9-independent manner, (ii) is likely involved in the pro-apoptotic mitochondrial pathway leading to caspase-9 activation.     

MHC class II proteins contain a potential binding site for the verotoxin receptor glycolipid CD77.

Globotriaosyl ceramide or CD77 functions as a cell surface receptor for toxins of the Shiga toxin/verotoxin family and as a marker for germinal center stage B-cells. The B-cell protein CD19 and the interferon-alpha receptor possess verotoxin-like amino acid sequences in their extracellular domains, and CD77 has been shown to function in CD19-mediated adhesion and interferon-induced growth inhibition. The Burkitt's lymphoma cell line, Daudi, is similar to germinal center B-cells in their expression of CD77, CD19 and MHC class II molecules. Using the multiple sequence alignment program, ClustalW, we have identified a verotoxin-like amino acid sequence on the beta-chain of human and murine MHC class II molecules. Binding of CD77 at this site could modulate the peptide-binding properties of these MHC class II molecules. Using Western blot analysis of whole cell extracts, we found that CD77-positive Daudi cells have higher levels of HLA-D proteins than VT500 cells, a Daudi-derived CD77-deficient mutant cell line. In contrast, MHC class II-mediated adhesion and surface expression are similar in the two cell lines. Therefore, CD77 could play a functional or regulatory role in MHC class II-mediated functions specifically relating to antigen presentation by B-cells to T helper cells.     

GSK-3beta inhibition by lithium confers resistance to chemotherapy-induced apoptosis through the repression of CD95 (Fas/APO-1) expression

Lithium exerts neuroprotective actions that involve the inhibition of glycogen synthase kinase-3beta (GSK-3beta). Otherwise, recent studies suggest that sustained GSK-3beta inhibition is a hallmark of tumorigenesis. In this context, the present study was undertaken to examine whether lithium modulated cancer cell sensitivity to apoptosis induced by chemotherapy agents. We observed that, in different human cancer cell lines, lithium significantly reduced etoposide- and camptothecin-induced apoptosis. In HepG2 cells, lithium repressed drug induction of CD95 expression and clustering at the cell surface as well as caspase-8 activation. Lithium acted through deregulation of GSK-3beta signaling since (1) it provoked a rapid and sustained phosphorylation of GSK-3beta on the inhibitory serine 9 residue; (2) the GSK-3beta inhibitor SB-415286 mimicked lithium effects by repressing drug-induced apoptosis and CD95 membrane expression; and (3) lithium promoted the disruption of nuclear GSK-3beta/p53 complexes. Moreover, the overexpression of an inactivated GSK-3beta mutant counteracted the stimulatory effects of etoposide and camptothecin on a luciferase reporter plasmid driven by a p53-responsive sequence from the CD95 gene. In conclusion, we provide the first evidence that lithium confers resistance to apoptosis in cancer cells through GSK-3beta inhibition and subsequent repression of CD95 gene expression. Our study also highlights the concerted action of GSK-3beta and p53 on CD95 gene expression.     

Filamin-A regulates actin-dependent clustering of HIV receptors

Human immunodeficiency virus (HIV)-1 infection requires envelope (Env) glycoprotein gp120-induced clustering of CD4 and coreceptors (CCR5 or CXCR4) on the cell surface; this enables Env gp41 activation and formation of a complex that mediates fusion between Env-containing and target-cell membranes. Kinetic studies show that viral receptors are actively transported to the Env-receptor interface in a process that depends on plasma membrane composition and the actin cytoskeleton. The mechanisms by which HIV-1 induces F-actin rearrangement in the target cell remain largely unknown. Here, we show that CD4 and the coreceptors interact with the actin-binding protein filamin-A, whose binding to HIV-1 receptors regulates their clustering on the cell surface. We found that gp120 binding to cell receptors induces transient cofilin-phosphorylation inactivation through a RhoA-ROCK-dependent mechanism. Blockade of filamin-A interaction with CD4 and/or coreceptors inhibits gp120-induced RhoA activation and cofilin inactivation. Our results thus identify filamin-A as an adaptor protein that links HIV-1 receptors to the actin cytoskeleton remodelling machinery, which may facilitate virus infection.     

Role of sphingomyelinase and ceramide in modulating rafts: do biophysical properties determine biologic outcome?

Recent biophysical data suggest that the properties of ceramide observed in model membranes may apply to biological systems. In particular, the ability of ceramide to form microdomains, which coalesce into larger platforms or macrodomains, appears to be important for some cellular signaling processes. Several laboratories have now demonstrated similar reorganization of plasma membrane sphingolipid rafts, via ceramide generation, into macrodomains. This event appeared necessary for signaling upon activation of a specific set of cell surface receptors. In this article, we review the properties and functions of rafts, and the role of sphingomyelinase and ceramide in the biogenesis and re-modeling of these rafts. As clustering of some cell surface receptors in these domains may be critical for signal transduction, we propose a new model for transmembrane signal transmission.     

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 sphingomyelinase present in acidic compartments has been reported to mediate, via the production of ceramide, 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 apoptosis. A series of observations strongly suggests that ceramide involved in stress-induced apoptosis is not endolysosomal: 1) Although short-chain ceramides induced apoptosis, loading cells with natural ceramide through receptor-mediated endocytosis did not result in cell death. 2) Neither TNF-alpha 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-alpha 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 ceramide. Also, induction of apoptosis using anti-CD95 (Fas) or anti-CD40 antibodies, TNF-alpha, 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) Retroviral-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-alpha and CD40 ligand.     

Doxorubicin enhances TRAIL-induced cell death via ceramide-enriched membrane platforms

Previous studies indicated that signalling via CD95 and DR5 is greatly enhanced by the formation of ceramide-enriched membrane platforms. Here, we employed this concept to convert doses of subtherapeutic TRAIL that were unable to release ceramide and kill leukemic B-cells or ex vivo T lymphocytes, into a very effective apoptotic stimulus. Ceramide production was induced by application of sub-toxic doses of doxorubicin that resulted in an activation of the acid sphingomyelinase (ASM), release of ceramide and formation of ceramide-enriched membrane platforms. The latter served DR5 to cluster after application of very low doses of TRAIL in combination with doxorubicin. Genetic deficiency of the ASM abrogated doxorubicin-induced ceramide release, as well as clustering of DR5 and apoptosis induced by the combined treatment of doxorubicin and TRAIL. These data show that local release of ceramide potentiates very low, otherwise inactive doses of TRAIL that may represent a novel therapeutic concept to treat tumors.     

Ultraviolet Light Induces Apoptosis via Direct Activation of CD95 (Fas/APO-1) Independently of Its Ligand CD95L

Induction of apoptosis in keratinocytes by UV light is a critical event in photocarcinogenesis. Although p53 is of importance in this process, evidence exists that other pathways play a role as well. Therefore, we studied whether the apoptosis-related surface molecule CD95 (Fas/APO-1) is involved. The human keratinocyte cell line HaCaT expresses CD95 and undergoes apoptosis after treatment with UV light or with the ligand of CD95 (CD95L). Incubation with a neutralizing CD95 antibody completely prevented CD95L-induced apoptosis but not UV-induced apoptosis, initially suggesting that the CD95 pathway may not be involved. However, the protease CPP32, a downstream molecule of the CD95 pathway, was activated in UV-exposed HaCaT cells, and UV-induced apoptosis was blocked by the ICE protease inhibitor zVAD, implying that at least similar downstream events are involved in CD95- and UV-induced apoptosis. Activation of CD95 results in recruitment of the Fas-associated protein with death domain (FADD) that activates ICE proteases. Immunoprecipitation of UV-exposed HaCaT cells revealed that UV light also induces recruitment of FADD to CD95. Since neutralizing anti-CD95 antibodies failed to prevent UV-induced apoptosis, this suggested that UV light directly activates CD95 independently of the ligand CD95L. Confocal laser scanning microscopy showed that UV light induced clustering of CD95 in the same fashion as CD95L. Prevention of UV-induced CD95 clustering by irradiating cells at 10°C was associated with a significantly reduced death rate. Together, these data indicate that UV light directly stimulates CD95 and thereby activates the CD95 pathway to induce apoptosis independently of the natural ligand CD95L. These findings further support the concept that UV light can affect targets at the plasma membrane, thereby even inducing apoptosis.     

Palmitate promotes monocyte atherogenicity via de novo ceramide synthesis

Elevated plasma free fatty acids (FAs) are associated with increased risk of cardiovascular disease. This study investigates the effects of the saturated FA palmitate and unsaturated FA oleate on monocyte phenotype and function. Incubation of human U937 and THP-1 monocytes with palmitate for 24h increased cell surface expression of integrin CD11b and scavenger receptor CD36 in a concentration-dependent manner with some decrease in mitochondrial reducing capacity at high concentration (300μM). Monocytes incubated with palmitate, but not oleate, showed increased uptake of oxidized LDL and increased adhesion to rat aortic endothelium, particularly at bifurcations. The palmitate-induced increase in CD11b and CD36 expression was associated with increased cellular C16 ceramide and sphingomyelin, loss of reduced glutathione, and increased reactive oxygen species (ROS). Increased monocyte surface CD11b and CD36 was inhibited by fumonisin B1, an inhibitor of de novo ceramide synthesis, but not by the superoxide dismutase mimetic MnTBap. In contrast, MnTBap prevented the mitochondrial ROS increase and metabolic inhibition due to 300μM palmitate. This study demonstrates that in viable monocytes, palmitate but not oleate increases expression of surface CD11b and CD36. Palmitate increases monocyte adhesion to the aortic wall and promotes uptake of oxidized LDL and this involves de novo ceramide synthesis.     

Cholera toxin B-subunit prevents activation and proliferation of human CD4+ T cells by activation of a neutral sphingomyelinase in lipid rafts

The inhibition of human CD4+ T lymphocyte activation and proliferation by cholera toxin B-subunit (CTB) is a well-established phenomenon; nevertheless, the exact mechanism remained unclear. In the present study, we propose an explanation for the rCTB-induced inhibition of CD4+ T lymphocytes. rCTB specifically binds to GM1, a raft marker, and strongly modifies the lipid composition of rafts. First, rCTB inhibits sphingomyelin synthesis; second, it enhances phosphatidylcholine synthesis; and third, it activates a raft-resident neutral sphingomyelinase resembling to neutral sphingomyelinase type 1, thus generating a transient ceramide production. We demonstrated that these ceramides inhibit protein kinase Calpha phosphorylation and its translocation into the modified lipid rafts. Furthermore, we show that rCTB-induced ceramide production activate NF-kappaB. Combined all together: raft modification in terms of lipids, ceramide production, protein kinase Calpha inhibition, and NF-kappaB activation lead to CD4+ T cell inhibition.     

The transmembranous domain of CD40 determines CD40 partitioning into lipid rafts

Stimulation of CD40 has been previously shown to result in a release of ceramide in small sphingolipid-enriched rafts in the cell membrane [Grassmé et al., J. Immunol. 168 (2002) 298-307]. Those rafts fused to larger signaling platforms that served to cluster CD40. Here, we defined molecular mechanisms of CD40 clustering in membrane platforms. To this end, we replaced the transmembranous domain of CD40 with that of CD45, a molecule known to be excluded from lipid rafts. Murine T cells were stably transfected with wild-type CD40 or chimeric CD40/CD45. Flow cytometry confirmed normal binding properties of the mutant to CD40 ligand. Stimulation with CD40 ligand resulted in clustering of wild-type CD40, while the chimeric CD40/45 receptor failed to cluster. This correlated with a deficiency of the chimeric receptor to activate JNK, p38 MAP kinase and SAPK, known signaling molecules of the intracellular pathway initiated by CD40. Forced crosslinking of the CD40/45 chimeric receptor restored, at least partially, these signaling events. The results suggest that the transmembranous domain of CD40 is central for the recruitment to and clustering of CD40 in membrane platforms.     

GSK-3β inhibition by lithium confers resistance to chemotherapy-induced apoptosis through the repression of CD95 (Fas/APO-1) expression

Lithium exerts neuroprotective actions that involve the inhibition of glycogen synthase kinase-3β (GSK-3β). Otherwise, recent studies suggest that sustained GSK-3β inhibition is a hallmark of tumorigenesis. In this context, the present study was undertaken to examine whether lithium modulated cancer cell sensitivity to apoptosis induced by chemotherapy agents. We observed that, in different human cancer cell lines, lithium significantly reduced etoposide- and camptothecin-induced apoptosis. In HepG2 cells, lithium repressed drug induction of CD95 expression and clustering at the cell surface as well as caspase-8 activation. Lithium acted through deregulation of GSK-3β signaling since (1) it provoked a rapid and sustained phosphorylation of GSK-3β on the inhibitory serine 9 residue; (2) the GSK-3β inhibitor SB-415286 mimicked lithium effects by repressing drug-induced apoptosis and CD95 membrane expression; and (3) lithium promoted the disruption of nuclear GSK-3β/p53 complexes. Moreover, the overexpression of an inactivated GSK-3β mutant counteracted the stimulatory effects of etoposide and camptothecin on a luciferase reporter plasmid driven by a p53-responsive sequence from the CD95 gene. In conclusion, we provide the first evidence that lithium confers resistance to apoptosis in cancer cells through GSK-3β inhibition and subsequent repression of CD95 gene expression. Our study also highlights the concerted action of GSK-3β and p53 on CD95 gene expression.