Ceramide and other Sphingolipids in Apoptosis
Membrane sphingolipids, sphingomyelin (SM) and glycosphingolipids (GSLs) had long been regarded as metabolically inactive, and rather stable structural components of the membrane, in contrast to glycerophospholipids which were long known to play an important role in lipid-mediated signal transduction. Only since about 1990, a number of studies reported that extracellular stimuli (e.g. Vitamin D3, TNF alpha, FasL, IL-1beta, gamma irradiation and others) cause the activation of sphingomyelinase (SMase) and the release as a second messenger of stress-related diverse responses such as cell-cycle arrest, apoptosis, and cell-senescence. This pathway is known as the "SM-ceramide pathway", but it is now recognized that not only ceramide, but other bioactive sphingolipids are produced through this degrading pathway, and that they also play a variety of roles in regulation of cellular activities (Igarashi, 1997, J. Biochem., 122: 1080-87).
Ceramide (N-Acyl-Sphingosine) is the precuror of sphingophospholipids which are part of the cell membrane, especially in neuronal cells. Naturally occurring ceramides consit of a long-chain sphingoid base with an amid-linked fatty acid substituent (typically 16-24 carbon atoms long). There is evidence that the generation of ceramide by hydrolysis of sphingophospholipids (e.g. sphingomyelin) is associated with the induction of apoptotic cell death. Ceramide is generated in cells as a result of three distinct enzymes: neutral and acidic sphingomyelinase (N-SMase and A-SMase) and ceramide synthase.
Ligation of TNFR1 by TNF alpha or Fas receptor by FasL promotes activation of N-SMase and A-SMase, leading to increased formation of ceramide. It is not clear if A-SMase or N-SMase is responsible for the generation of apoptosis-inducing ceramide. It was supposed that in TNF- or Fas-meditated apoptosis the apoptotic effect might be mediated by ceramide that was generated by A-SMase activity though it was also reported that Fas induces ceramide formation in the absence of functional A-SMase (Cock et al., 1998, J. Biol. Chem., 273(13): 7560-65). In contrast, ceramide generated by N-SMase might result in the activation of NF-kB and pro-inflammatory events of TNF alpha though there are also contradictory reports against a role of ceramide in TNF induced NF-kB activation (Aggarwal, 1997, Biochem Soc. Trans., vol.25:1166-1171).
Other apoptotic stimuli also activate ceramide generation, e.g. IFN-gamma, daunorubicin, radiation or serum withdrawal. Therefore, a conserved role for ceramide as a primary effector in apoptosis has been proposed. In support of this view, experimental manipulations that increase intracellular ceramide levels (e.g., overexpression of sphingomyelinase or treatment of cells with permeant ceramide analogs like C2-ceramide = N-Acetyl-sphingosine) potently induce apoptosis in mammalian cells.
It has to be mentioned that there are also reports which provide evidence against a (early signaling) role for ceramide in Fas-mediated apoptosis (Hsu et al., 1998, Blood, 91(8):2658-63). Tepper et al. (1997, J. Biol Chem., vol.272, no. 39, p24308-12), for example, measured the kinetics with which Ceramide is generated in Jurkat cells upon Fas-treatment. In contrast to other previous reports (Gulbins et al., 1995, Immunity, 2: 341-351), they found that ceramide levels begin to increase quite late (4-5 h with a maximum - 7 fold increase - after 8 h) after Fas-stimulation, almost in parallel with the onset of apoptosis measured by nuclear fragmentation (they used another methodology than the common but maybe not appropriate E.coli diglyceride kinase assay!). Since the tetrapeptide inhibitor DEVD-CHO does not block Ceramide formation but Caspase-3 processing and apoptosis, it was proposed, that ceramide acts in an intermediate phase of the apoptotic process: not in early events but still upstream of the executioner caspases (Caspases-3, -6, -7).
The mechanism by which ceramide mediates its apoptotic effect (the putative link between ceramide and the activation of the caspase cascade) is not known..
It was reported that ceramide can induce apoptotic events in (membrane- and mitochondria-free) cytoplasmic extracts from untreated cells in a cell free system (Martin et al., 1995,EMBO, vol.14, no.21, 5191-5200) but now it appears that ceramide itself does not trigger apoptosis but relies on the presence of membranes and/or mitochondria (Farschon et al, 1997, The J. Cell Biol., vol. 137, no.5, 1117-1125; Susin et al., 1997, Exp. Cell Res., 236, 397-403).
Consistent with this, it was reported that - upon treatment of living cells - ceramide is able to induce mitochondrial Permeability Transition (PT) and release of AIF, however not directly but mediated by yet unknown factors. The ceramide induced PT and AIF release of mitochondria is blocked by Bcl-2. In contrast, ceramide does not induce PT and associated AIF release in isolated mitochondria, and ceramide also does not induce apoptotic events when added to cytosol of untreated cells: it was stated that in order to reveal the apoptogenic effect of ceramide, it is necessary to treat intact cells (rather than its isolated components) with ceramide (Susin et al, 1997, J. Exp. Med., vol. 186, No. 1, 25-37). In contrast, in the Xenopus cell free system ceramide is reported to induce nuclear apoptosis in the presence of part of the heavy membrane fraction (Farschon et al, 1997, The J. Cell Biol., vol. 137, no.5, 1117-1125)!
At present, the corresponding proximal target(s) for ceramide in the initiation of lethal signaling is unknown, although several downstream effector elements essential for the lethal actions of ceramide have been identified. Ceramide-mediated induction of cell death seems to require activation of the SAPK (stress activated protein kinase) cascade: the SAPK cascade is closely associated with multiple ceramide-dependent proapoptotic stimuli (Verhejl et al., 1996, Nature, 380: 75-79), and interruption of the SAPK cascade abolishes the apoptotic responses to ceramide or ceramide-dependent lethal stimuli. One proximal target for ceramide is the serine/threonine kinase CAPK (ceramide activated protein kinase), which possibly (hypothesis!) recruits MAPK/ERK kinase kinase (MEKK1). MEKK1 activates SAPK-kinase (SEK1) which engages the Jun N-terminal kinases (JNK1/2). The mechanism by which c-Jun mediates apoptosis is unknown, but in rat neuronal cells, for example, apoptosis (by NGF deprivation) was inhibited by a c-Jun dominant negative mutant and also by actinomycin and cycloheximide (Johnson, 1993, Rev. Neurosci, 16: 31-46). It was suggested that c-Jun might regulate genes as yet unknown, that are involved in cell proliferation, and that strong growth-promoting signals in non-cycling neurons might trigger apoptosis (Ham et al.,1995, Cell, 14: 927-939). In contrast, inhibition of protein synthesis in U937 cells does not inhibit apoptosis: perhaps activated c-Jun may sequester and regulate unknown proteins required in the apoptotic response (Verhejl et al., 1996, Nature, 380: 75-79).
Thus, ceramide activated CAPK can induce the SAPK cascade which contributes to cell death. But under circumstances that lead to inflammatory responses CAPK also can activate Raf-1 which is involved in the activation of the mitogen-activated protein kinase (MAPK) cascade. The MAPK cascade is a cytoprotective signaling pathway!
Another proximal target for ceramide is the heterotrimeric cytosolic serine/threonine (class 2A) phosphoprotein phosphatase CAPP (ceramide-activated protein phosphatase). CAPP might be implicated in apoptosis because class-2A phosphatase inhibitors (e.g. okadaic acid) antagonize ceramide-related apoptosis. CAPP mediates dephosphorylation and consequent inactivation of cPKC. Active cPKC promotes activation of Raf-1 which is involved in cytoprotective signaling through the MAPK cascade. Thus, the apoptotic effect of ceramide might be regulated by the CAPK mediated activation of the cytotoxic SAPK cascade and - at the same time - by the CAPP mediated inhibition of the cytoprotective MAPK cascade.
In some systems ceramide might mediate its apoptotic effect in combination with its metabolites, the sphingoid bases sphingosine and dihydrosphingosine (= sphinganine). In some systems (e.g. human neutrophils) only exogenous sphingosine but not ceramide induced apoptosis (Igarashi, 1997, J. Biochem., 122: 1080-87). Sphingosine and sphinganine inhibit the "conventional" and "novel" isoforms of PKC (cPKC/nPKC) and by this the MAPK cascade: by blocking this cytoprotective pathway, they might support the apoptotic effect of ceramide. In cooperation with N,N-dimethylsphingosine, sphingosine might also be involved in other events effecting apoptosis (Igarashi, 1997, J. Biochem., 122: 1080-87). Additionally, both ceramide and sphingosine promote dephosphorylation of the retinoblastoma gene product (RB) and by this Go/G1 cell cycle arrest (Ariga et al, 1998, J Lipid Res., 39: 1-16). Sphingosine-1-phosphate (the initial intermediate product in the catabolism of sphingosine) was reported to prevent ceramide-mediated apoptosis (Cuvillier et al., 1998, J. Biol. Chem., 273(5): 2910-16).
In summary, the roles of the different sphingolipids (ceramide, sphingosine, sphingosine-1-phosphate ...) in apoptotic pathways and the underlying mechanisms are obviously distinct: these sphingolipids act cooperatively in some cells, but in other cells they act oppositely or independently.
Igarashi, 1997, J. Biochem., 122: 1080-87;
Ariga et al, 1998, J Lipid Res., 39: 1-16;
Hannun, 1996, Science, 174: 1855-1859)