Erich Gulbins

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.

Ceramide enables fas to cap and kill.

Recent studies suggest that trimerization of Fas is insufficient for apoptosis induction and indicate that super-aggregation of trimerized Fas might be prerequisite. For many cell surface receptors, cross-linking by multivalent ligands or antibodies induces their lateral segregation within the plasma membrane and co-localization into “caps” on one pole of the cell. In this study, we show that capping of Fas is essential for optimal function and that capping is ceramide-dependent. In Jurkat T lymphocytes and in primary cultures of hepatocytes, ceramide elevation was detected as early as 15–30 s and peaked at 1 min after CH-11 and Jo2 anti-Fas antibody treatment, respectively. Capping was detected 30 s after Fas ligation, peaked at 2 min, and was maintained at a lower level for as long as 30 min in both cell types. Ceramide generation appeared essential for capping. Acid sphingomyelinase− /− hepatocytes were defective in Jo2-induced ceramide generation, capping, and apoptosis, and nanomolar concentrations of C16-ceramide restored these events. To further explore the role of ceramide in capping of Fas, we employed FLAG-tagged soluble Fas ligand (sFasL), which binds trimerized Fas but is unable to induce capping or apoptosis in Jurkat cells. Cross-linking of sFasL with M2 anti-FLAG antibody induced both events. Pretreatment of cells with natural C16-ceramide bypassed the necessity for forced antibody cross-linking and enabled sFasL to cap and kill. The presence of intact sphingolipid-enriched membrane domains may be essential for Fas capping since their disruption with cholesterol-depleting agents abrogated capping and prevented apoptosis. These data suggest that capping is a ceramide-dependent event required for optimal Fas signaling in some cells.

The LPS receptor (CD14) links innate immunity with Alzheimer's disease

To rapidly respond to invading microorganisms, humans call on their innate immune system. This occurs by microbe‐detecting receptors, such as CD14, that activate immune cells to eliminate the pathogens. Here, we link the lipopolysaccharide receptor CD14 with Alzheimers disease, a severe neurodegenerative disease resulting in dementia. We demonstrate that this key innate immunity receptor interacts with fibrils of Alzheimer amyloid peptide. Neutralization with antibodies against CD14 and genetic deficiency for this receptor significantly reduced amyloid peptide induced microglial activation and microglial toxicity. The observation of strongly enhanced microglial expression of the LPS receptor in brains of animal models of Alzheimers disease indicates a clinical relevance of these findings. These data suggest that CD14 may significantly contribute to the overall neuroinflammatory response to amyloid peptide, highlighting the possibility that the enormous progress currently being made in the field of innate immunity could be extended to research on Alzheimers disease.

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 bacteria induced apoptosis

Interaction of mammalian cells with pathogenic bacteria results in a whole variety of responses in the infected cells including internalization or phagocytosis of the bacterium, release of cytokines, secretion of defensins or production of oxygen radicals. However, recent studies pointed out that many bacteria are able to trigger apoptosis in the host cell. The induction of apoptosis upon infection results from a complex interaction of bacterial proteins with cellular proteins finally mediating apoptosis. Thus, bacteria are able to activate several pro-apoptotic proteins, e.g. caspases, to inactivate anti-apoptotic proteins, e.g. NFκB or MAP-kinases, or to upregulate endogenous receptor/ligand systems, that induce apoptosis, on the surface of the infected cell. Host cell apoptosis very often serves the bacteria to attack the host and to gain access to the tissue. However, in some infections, apoptosis of mammalian cells significantly contributes to the host defense against the bacteria further indicating the role of apoptosis in host-pathogen interactions.

Natural Ceramide Reverses Fas Resistance of Acid Sphingomyelinase −/− Hepatocytes

The role of the second messenger ceramide in Fas-mediated death requires clarification. To address this issue, we generated hepatocytes from paired acid sphingomyelinase(ASMase; asmase) +/+ andasmase − /− mice.asmase − /− hepatocytes, derived from 8-week-old mice, manifested normal sphingomyelin content and normal morphological, biochemical, and biologic features. Nonetheless, ASMase-deficient hepatocytes did not display rapid ceramide elevation or apoptosis in response to Jo2 anti-Fas antibody.asmase − /− hepatocytes were not inherently resistant to apoptosis because staurosporine, which did not induce early ceramide elevation, stimulated a normal apoptotic response. The addition of low nanomolar quantities of natural C16-ceramide, which by itself did not induce apoptosis, completely restored the apoptotic response to anti-Fas inasmase − /− hepatocytes. Other sphingolipids did not replace natural ceramide and restore Fas sensitivity. Overcoming resistance to Fas inasmase − /− hepatocytes by natural ceramide is evidence that it is the lack of ceramide and not ASMase which determines the apoptotic phenotype. The ability of natural ceramide to rescue the phenotype without reversing the genotype provides evidence that ceramide is obligate for Fas induction of apoptosis in hepatocytes.

Mechanisms of Staphylococcus aureus induced apoptosis of human endothelial cells

Staphylococcus aureus plays an important role in sepsis, pneumonia and wound infections. Here, we demonstrate that infection with several S. aureus strains results in apoptosis of human endothelial cells. S. aureus induced an activation of cellular caspases, the acid sphingomyelinase, a release of cytochrome c and a stimulation of Jun NH2-terminal kinase (JNK). The significance of these findings is indicated by a prevention of S. aureus triggered apoptosis of human cells deficient for ASM or upon genetic or pharmacological inhibition of JNK or caspases, respectively.

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.

Actinomycin D-induced apoptosis involves the potassium channel Kv1.3.

Several cytostatic agents are known to induce apoptosis in T-leukemic cells. Although a variety of studies show the central role of apoptosis in cytostatic drug-induced cell death, many molecular details require definition. Here, we demonstrate that cells genetically deficient for the potassium channel Kv1.3 are resistant to apoptosis initiated by the cytostatic drug actinomycin D. Retransfection of Kv1.3 restores sensitivity of the cells to actinomycin D. Cells lacking Kv1.3 fail to respond to actinomycin D with DNA fragmentation, release of cytochrome c, and loss of mitochondrial membrane potential (Delta Psi(m)), while cells functionally expressing Kv1.3 rapidly undergo those changes indicative for apoptosis. The data indicate a central role of the ion channel Kv1.3 in actinomycin D-triggered apoptosis.

Acid sphingomyelinase-derived ceramide signaling in apoptosis

A variety of studies demonstrate a central role for ASM and ceramide in several forms of apoptosis. Ceramide seems to regulate the activity of certain proteins and, thus, may function, in some circumstances, as a second messenger. In addition, the concept of raft modification by ceramide provides a comprehensive model for cellular effects of ceramide, and perhaps a biophysical explanation for the diverse functions of this lipid.