TNF-Induced Apoptosis Implicating Sphingolipids

Various lipid molecules serve as second messengers for transducing signals from the cell surface to the cell interior and trigger specific cellular responses. Recently, several sphingolipids have emerged as cellular constituents that are able to promote, mediate or counterbalance apoptosis. Sphingolipids are a family of membrane lipids whose structure is made up of a long-chain sphingoid base backbone (such as sphingosine), an amide-linked fatty acid of varying chain and one of various polar head groups (hydroxyl for ceramide, phosphorylcholine for sphingomyelin, and carbohydrate residues for glycosphingolipids). Addition of a phosphocholine substituent or sugar to ceramide gives rise to the major sphingolipid SM (sphingomyelin) or to glycosphingolipids. Ceramide is also produced by breakdown of all sphingolipids by glycosidases and sphingomyelinases (Smases; for SM degradation). These hydrolytic steps occur in the acidic organelles as well as in several other subcellular compartments. Ceramide is deacylated by ceramidase to release sphingosine, which is then phosphorylated by SphK (Sphingosine Kinase) (Ref.1).

TNF-Alpha (Tumor Necrosis Factor-Alpha) promotes SM turnover and/or ceramide generation in various cell types. This production involves TNFR1 (TNF Receptor-1) and occurs with differing kinetics. Different subcellular pools of SM serve as reservoirs for agonist-induced ceramide production, including acidic compartments, the inner leaflet of the plasma membrane or caveolae. After activation of TNFR1 by binding of TNF-Alpha, the adaptor proteins TRADD (TNFR-Associated Death Domain), FADD (Fas-Associated Death Domain) and FLICE (FADD-Like IL-1Beta Converting Enzyme)-like protease stimulate A-Smase (Acid Smase), whereas FAN (Factor Associated with Neutral Smase activation) is required for N-Smase (Neutral Smase) activation; by contrast, the adaptors TRAF2 (TNF Receptor-Associated Factors) and RIP (Receptor-Interacting Protein) do not affect A-Smase (Ref.2). One primary target of ceramide is CAPK (Ceramide-Activated Protein Kinase), a membrane-associated proline-directed serine/threonine kinase. CAPK phosphorylate and activate Raf1 kinase. Activation of Raf1 triggers the MEK (MAPK/ERK Kinase)-ERK (Extracellular signal-Regulated Kinase) cascade, which may lead to Arachidonic acid production by PLA2 (Phospholipase-A2). Environmental stresses (UV and ionizing irradiation, heat shock, and oxidative stress) act directly on membranes and activate A-Smase, generating ceramide, which induces apoptosis by activating pro-death pathways through activation of caspases or JNK/SAPK (c-Jun N-terminal kinase/Stress-Activated Protein Kinase), and by promoting dephosphorylation of the pro-survival protein BCl2 (B-Cell CLL/Lymphoma-2), BAX (BCL2 Associated-X Protein) and the pro-apoptotic protein BAD (BCL2 Associated Death Promoter). The SAPK/JNK signaling system involves sequential activation of kinases MEKK1 (MAPK/ERK kinase kinase-1), SEK1, and SAPK/JNK, which eventually leads to phosphorylation of c-Jun. The TNFR1 initiates apoptosis via formation of a death domain-adaptor protein complex that links downstream to A-Smase and to the SAPK/JNK and the ICE/Ced-3 proteases to signal apoptosis. A membrane proximal region of the cytoplasmic domain links the adaptor protein FAN to N-Smase, ceramide generation, and stimulation of CAPK, Raf1, and the ERK cascade. In addition, TRADD may link the TNF receptor to NF-kappaB activation via RIP and/or TRAF2 (Ref.3). Another mechanism that account for ceramide production after TNF-Alpha activation is stimulation of de novo synthesis. This de novo ceramide synthesis account for the late, sustained generation of ceramide that also participate in TNF-Alpha-induced cell death. Furthermore, ligation of other members of the TNFR family, including CD40, CD95, the p75NGF (Nerve Growth Factor) and TRAIL (TNF-Related Apoptosis-Inducing Ligand) receptors, induces apoptosis and engages sphingolipid second messengers (Ref.1).

The bioactive sphingolipid metabolite S1P, formed by activation of SphK in response to diverse stimuli, is an important lipid mediator that has novel dual actions both inside and outside of cells. Activation of GPCRs (G Protein-Coupled Receptors) by S-1P or DH-S-1P (Dihydro-S-1P) regulates diverse processes, including cell migration, angiogenesis, vascular maturation, heart development, and neurite retraction. S-1P also function as a second messenger important for regulation of intracellular calcium homeostasis, stimulates fibroblast proliferation, DNA synthesis, cell growth, adhesion molecule expression, and inhibits cellular apoptosis and cell migration (Ref.4). In many cases, the intracellular level of S-1P and ceramide, associated with cell death and cell growth arrest, coordinately determine cell fate. Depending upon the cell type (monoblastic leukemia cells, endothelial cells, fibroblasts, pheochromocytoma cells, and oligodendrocytes), ceramides also act as modulator of immune cell differentiation, mitochondrial respiration releasing CytoC (Cytochrome C), inflammation, cell cycle progression, apoptosis, and the stress response. Changes in S-1P and ceramide have been implicated in a number of pathological conditions in which apoptosis plays an important role. Various stressful stimuli, including radiation, as well as proinflammatory cytokines, anticancer drugs, and growth factor withdrawal, activate sphingomyelinase (Ref.5).