Signal transduction and the regulation of apoptosis: roles of ceramide

Knowledge about the molecular regulators of apoptosis is rapidly expanding. Cell death signals emanating from death receptors or internal cell injury detectors launch a number of signaling pathways which converge on several key families of proteins including specialized proteases and endonucleases which play a critical role in the execution of the death order. In this review, we summarize recent discoveries relating to the signaling pathways involved, the death receptors, the caspase family of apoptotic proteases, Bcl-2 family members, the sphingolipid ceramide, and the tumor suppressor p53. In particular, we focus on the role played by ceramide as a coordinator of the stress response and as a candidate biostat in the detection of cell injury.     

Ceramide induces caspase-dependent and -independent apoptosis in A-431 cells

We investigated the ceramide-induced apoptosis and potential mechanism in A-431 cells. Ceramide treatment causes the round up and the death of A-431 cells that is associated with p38 activation and can be observed in 10 h. Short-time ceramide treatment-induced cell death is not associated with the typical apoptotic phenotypes, such as the translocation of phosphatidylserine (PS) from inner layer to outer layer of the plasma membrane, loss of mitochondrial membrane potential, DNA fragmentation, caspase activation, and PARP or PKC-delta degradation. SB202190, a specific inhibitor of p38 mitogen-activated protein (MAP) kinase, but not caspase inhibitor, blocks the cell death induced by short-time ceramide treatment (within 12 h). Whereas neither inhibition of p38 MAP kinase nor inhibition of caspases blocks cell death induced by prolonged ceramide treatment. Moreover, incubation of cells with ceramide for a long time (over 12 h) results in the reduction of proportion of S phase accompanied with typical apoptotic cell death phenotypes that are different from the cell death induced by short-time ceramide treatment. Our data demonstrated that ceramide-induced apoptotic cell death involves both caspase-dependent and caspase-independent signaling pathways. The caspase-independent cell death that occurred in relatively early stage of ceramide treatment is mediated via p38 MAP kinase, which can progress into a stage that is associated with changes of cell cycle events and involves both caspase-dependent and -independent mechanisms.     

Distinct stress and cell destruction pathways are engaged by TNF and ceramide during apoptosis of MCF-7 cells

Ceramide has been proposed to be an important signaling intermediate in tumor necrosis factor (TNF)-induced apoptosis in human MCF-7 breast adenocarcinoma cells. We compared cell death and signal transduction pathways induced by TNF and ceramide in TNF-sensitive, parental MCF-7 cells to those in TNF-resistant, MCF-7 cells (3E9). TNF caused proteolysis of the caspase substrate, polyADP-ribose polymerase (PARP) in parental cells, but not in 3E9 cells. Both apoptosis and PARP cleavage were strongly prevented by co-incubation with caspase inhibitors. In contrast, ceramide-induced cell death was neither affected by TNF resistance nor was it associated with PARP cleavage, and death could not be prevented by co-incubation with caspase inhibitors in either cell line. TNF was able to activate JNK/SAPK approximately 30-fold and approximately 5-fold in parental MCF-7 and 3E9 cells, respectively; in contrast, cell-permeable ceramide only weakly stimulated JNK/SAPK activity in either cell type. Although JNK was activated by TNF, pharmacological blockade of the JNK pathway did not inhibit TNF- or ceramide-mediated cell death. Using mass spectroscopic analysis for ceramide, no increase, rather, a decrease in total ceramide content in TNF-treated parental cells was observed. These results suggest that the cell death signaling and execution pathways utilized by ceramide are distinct from those activated by TNF in MCF-7 cells.     

Relationships of apoptotic signaling mediated by ceramide and TNF-alpha in U937 cells

It is commonly assumed that ceramide is a second messenger that transduces signaling leading to apoptosis. We tested this hypothesis by investigating the role of ceramide in TNF-alpha-initiated apoptotic signaling using the histiocytic lymphoma cell line U937. We found considerable differences between cell killing by TNF-alpha and by ceramide. U937 cells treated with TNF-alpha are committed early and irreversibly to the apoptotic pathway and start to die 90 min after treatment. U937 cells treated with ceramide start to die 12 h after the initial treatment. The cell death signaling initiated by TNF-alpha is transduced within minutes of exposure to TNF-alpha and it is irreversible. Exogenous ceramide increases the intracellular level of ceramide rapidly, significantly, and well above the physiological levels, within minutes, but cellular commitment to death does not occur until after the first 6 h of incubation. Furthermore, the endogenous ceramide in U937 cells treated with TNF-alpha increases well after the commitment to the apoptotic pathway. The differences between ceramide and TNF-alpha in the kinetics and the commitment to the apoptotic pathway suggest that, (a) ceramide is not a second messenger in the apoptotic signaling of TNF-alpha, (b) ceramide elevations, in TNF-alpha treated cells, are a consequence rather than a cause of apoptosis and (c) exogenously added ceramide and TNF-alpha kill cells via different pathways.     

Sphingolipids and cell death

Sphingolipids (SLs) have been considered for many years as predominant building blocks of biological membranes with key structural functions and little relevance in cellular signaling. However, this view has changed dramatically in recent years with the recognition that certain SLs such as ceramide, sphingosine 1-phosphate and gangliosides, participate actively in signal transduction pathways, regulating many different cell functions such as proliferation, differentiation, adhesion and cell death. In particular, ceramide has attracted considerable attention in cell biology and biophysics due to its key role in the modulation of membrane physical properties, signaling and cell death regulation. This latter function is largely exerted by the ability of ceramide to activate the major pathways governing cell death such as the endoplasmic reticulum and mitochondria. Overall, the evidence so far indicates a key function of SLs in disease pathogenesis and hence their regulation may be of potential therapeutic relevance in different pathologies including liver diseases, neurodegeneration and cancer biology and therapy.     

Glycosphingolipids and mitochondria: role in apoptosis and disease

Glycosphingolipids (GSLs) comprise a class of lipids with important structural and signaling functions. Synthesized from ceramide in the Golgi, they are subsequently distributed to different compartments, most predominantly in the plasma membrane where they integrate signaling platforms. A recently characterized trafficking of ganglioside GD3 (GD3), a GSLs with two sialic-acid residues, to mitochondria has revealed a novel function of this lipid as a death effector. In addition to the interaction of GD3 with mitochondria recruiting these organelles to apoptotic pathways, GD3 disables survival paths dependent on NF-kappaB, thus favoring the balance towards cell death. The present review gathers the evidence documenting this emerging function of GSLs in cell death and their involvement in pathological states.     

Glycosphingolipids and mitochondria: Role in apoptosis and disease

Glycosphingolipids (GSLs) comprise a class of lipids with important structural and signaling functions. Synthesized from ceramide in the Golgi, they are subsequently distributed to different compartments, most predominantly in the plasma membrane where they integrate signaling platforms. A recently characterized trafficking of ganglioside GD3 (GD3), a GSLs with two sialic-acid residues, to mitochondria has revealed a novel function of this lipid as a death effector. In addition to the interaction of GD3 with mitochondria recruiting these organelles to apoptotic pathways, GD3 disables survival paths dependent on NF-B, thus favoring the balance towards cell death. The present review gathers the evidence documenting this emerging function of GSLs in cell death and their involvement in pathological states. Published in 2004..     

Fas, ceramide and serum withdrawal induce apoptosis via a common pathway in a type II Jurkat cell line

Ceramide is a key mediator of apoptosis, yet its role in Fas-mediated apoptosis is controversial. Some reports have indicated that ceramide is either a primary signaling molecule in Fas-induced cell death, or that it functions upstream of Fas by increasing FasL expression. Other studies have suggested that ceramide is not relevant to Fas-induced cell death. We have approached this problem by studying ceramide-induced apoptosis in unique Jurkat cell clones selected for resistance to membrane-bound FasL-induced death. Resistance of the mutant Jurkat cells was specific for FasL killing, since the mutant clones were sensitive to other apoptotic stimuli such as cycloheximide and staurosporine. We tested the effects of serum withdrawal, one of the strongest inducers of ceramide, and of exogenous ceramide on apoptosis of both wild-type and FasL-resistant clones. Wild-type Jurkat cells were remarkably sensitive to serum withdrawal and to exogenous ceramide. In contrast all FasL-resistant mutant clones were resistant to these apoptosis-inducing conditions. In contrast to previous work, we did not detect an increase in FasL in either wild-type or mutant clones. Moreover activation of stress-activated protein kinases (JNK/SAPKs) after serum withdrawal and exogenous ceramide treatment was detected only in the wild-type and not in the resistant clones. Because of the parallel resistance of the mutant clones to Fas and to ceramide-induced apoptosis, our data support the notion that ceramide is a second messenger for the Fas/FasL pathway and that serum withdrawal, through production of ceramide, shares a common step with the Fas-mediated apoptotic pathway. Finally, our data suggest that activation of JNK/SAPKs is a common mediator of the three pathways tested.     

细胞凋亡的分子机制

多细胞生物在发生、发展过程中,为了保持正常的生理机能,一部分的细胞发生自发性细胞死亡,这种细胞死亡是被细胞内一系列相关的分子所调控,并伴随有典型的形态学改变,这种现象被称为细胞凋亡（ａｐｏｐｔｏｓｉｓ）［１］。作为一种积极排除生物体内的过剩细胞和有害细胞的机制在个体形态形成、形态改变等发生过程中,成体的恒常性的维持以及生物体的防御等方面发挥作用。此外在许多病理状态下也存在着细胞凋亡。近年的研...     

Regulation of DNA fragmentation: the role of caspases and phosphorylation

DNA fragmentation is a hallmark of apoptosis that is induced by apoptotic stimuli in various cell types. Apoptotic signal pathways, which eventually cause DNA fragmentation, are largely mediated by the family of cysteinyl aspartate-specific protease caspases. Caspases mediate apoptotic signal transduction by cleavage of apoptosis-implicated proteins and the caspases themselves. In the process of caspase activation, reversible protein phosphorylation plays an important role. The activation of various proteins is regulated by phosphorylation and dephosphorylation, both upstream and downstream of caspase activation. Many kinases/phosphatases are involved in the control of cell survival and death, including the mitogen-activated protein kinase signal transduction pathways. Reversible protein phosphorylation is involved in the widespread regulation of cellular signal transduction and apoptotic processes. Therefore, phosphatase/kinase inhibitors are commonly used as apoptosis inducers/inhibitors. Whether protein phosphorylation induces apoptosis depends on many factors, such as the type of phosphorylated protein, the degree of activation and the influence of other proteins. Phosphorylation signaling pathways are intricately interrelated; it was previously shown that either induction or inhibition of phosphorylation causes cell death. Determination of the relationship between protein and phosphorylation helps to reveal how apoptosis is regulated. Here we discuss DNA fragmentation and protein phosphorylation, focusing on caspase and serine/threonine protein phosphatase activation.     

Exploring the link between ceramide and ionizing radiation

The aim of radiotherapy is to eradicate cancer cells with ionizing radiation; tumor cell death following irradiation can be induced by several signaling pathways, most of which are triggered as a consequence of DNA damage, the primary and major relevant cell response to radiation. Several lines of evidence demonstrated that ceramide, a crucial sensor and/or effector of different signalling pathways promoting cell cycle arrest, death and differentiation, is directly involved in the molecular mechanisms underlying cellular response to irradiation. Most of the studies strongly support a direct relationship between ceramide accumulation and radiation-induced cell death, mainly apoptosis; for this reason, defining the contribution of the multiple metabolic pathways leading to ceramide formation and the causes of its dysregulated metabolism represent the main goal in order to elucidate the ceramide-mediated signaling in radiotherapy. In this review, we summarize the current knowledge concerning the different routes leading to ceramide accumulation in radiation-induced cell response with particular regard to the role of the enzymes involved in both ceramide neogenesis and catabolism. Emphasis is placed on sphingolipid breakdown as mechanism of ceramide generation activated following cell irradiation; the functional relevance of this pathway, and the role of glycosphingolipid glycohydrolases as direct targets of ionizing radiation are also discussed. These new findings add a further attractive point of investigation to better define the complex interplay between sphingolipid metabolism and radiation therapy.     

Regulation of caspase activity in apoptosis

Intracellular cysteine aspartate-specific proteases (caspases) play both signaling and effector roles in realizing the program of cell death. Caspases function as proteolytic cascades unique for each cell type and signal triggering apoptosis. All parts of the proteolytic cascades are duplicated and controlled by feedback signals. Amplification cycles between pairs of caspases (the third and the sixth, the ninth and the third, the twelfth and the sixth, and others) help multiply the initial apoptotic signal. The presence of physiological inhibitors of apoptosis that directly interact with caspases creates a multilevel regulatory network of apoptosis in cell. The caspase proteolytic cascades are also regulated by sphingolipid secondary messengers, among them ceramide, sphingosine, and their phosphates. Moreover, an association of the caspase signaling with ubiquitin-dependent proteolysis is shown in cells. In particular, the use of extracellular activators and inhibitors of caspases allows irreversible activation of apoptosis in tumor cells or the prevention of neuron death in neurodegenerative diseases.     

Role of ceramide in mediating apoptosis of irradiated LNCaP prostate cancer cells

The sphingomyelin metabolites ceramide and sphingosine are mediators of cell death induced by gamma-irradiation. We studied the production of ceramide and the effects of exogenous ceramide on apoptosis in LNCaP prostate cancer cells that are highly resistant to gamma-irradiation-induced cell death. LNCaP cells can be sensitized to gamma-irradiation by tumor necrosis factor alpha (TNF-alpha) and, to a lesser degree, by the agonistic FAS antibody CH-11. TNF-alpha activated intrinsic and extrinsic apoptosis pathways and increased ceramide and sphingosine levels in irradiated LNCaP cells. CH-11 activated only the extrinsic apoptosis pathways and had a negligible effect on ceramide and sphingosine levels in irradiated LNCaP cells. Exogenous ceramide and bacterial sphingomyelinase sensitized LNCaP cells to radiation-induced apoptosis and had a synergistic effect on cell death after irradiation with TNF-alpha, but not with CH-11. Cell death effects after exposure to ceramide and irradiation were blocked by the serine protease inhibitor TLCK (Na-p-tosyl-L-lysine-chloromethylketone), but not by the caspase inhibitor z-VAD (2-val-Ala-Asp(oMe)-CH(2)F). During LNCaP cell apoptosis induced by exogenous ceramide, we observed activation of caspase-9, but not caspases-8, -3, or -7. The effect of ceramide occurred largely via the intrinsic mitochondrial apoptosis pathway and enhanced TNF-alpha, but not CH-11 effects on irradiated cells. The data show that ceramide enhanced activation of the intrinsic apoptotic pathway and enhanced cell death induced by TNF-alpha with or without gamma-irradiation. TNF-alpha and gamma-irradiation elevated levels of endogenous ceramide and activated the intrinsic cell death pathway.     

Bax-dependent apoptosis induced by ceramide in HL-60 cells

Ceramide is an important lipid messenger involved in mediating a variety of cell functions including apoptosis. In this study, we show that antisense bax inhibits cytochrome c release, poly(ADP-ribose)polymerase cleavage and cell death induced by ceramide in HL-60 cells. In addition, ceramide induces translocation of Bax to mitochondria. The addition of the broad spectrum caspase inhibitor zVAD-fmk prevented ceramide-induced apoptotic cell death but did not inhibit translocation of Bax and mitochondrial cytochrome c release. Furthermore, ceramide inhibits the expression of the antiapoptotic protein Bcl-xL with an increase in the ratio of Bax to Bcl-xL. These data provide direct evidence that Bax plays an important role in regulating ceramide-induced apoptosis.     

Effects of ceramide, the Fas signal intermediate, on apoptosis and phospholipase D activity in mouse ovarian granulosa cells in vitro

Although recent studies have demonstrated that ovarian follicular atresia occurs by apoptosis of granulosa cells, the intracellular signaling pathways involved in apoptotic cell death are still poorly characterized. We examined the role of ceramide as a candidate intracellular mediator of Fas-mediated signaling in cultured granulosa cells. Expression of Fas antigen was demonstrated by Western blot of granulosa cell lysates and immunostaining of cultured granulosa cells. Exposure of granulosa cells to anti-Fas monoclonal antibody (anti-Fas mAb) resulted in significant sphingomyelin hydrolysis, which was accompanied by a progressive increase in endogenous levels of ceramide. The addition of exogenous C6-ceramide induced drastic morphological change, including nuclear fragmentation and typical apoptotic DNA degradation. Furthermore, both anti-Fas mAb and C6-ceramide decreased phospholipase D (PLD) activity and diacylglycerol (DAG) concentrations in a time- or a dose-dependent manner. In addition, treatment with phorbol 12-myristate 13-acetate completely attenuated the ceramide-induced inhibition of PLD activity and partially suppressed ceramide-induced apoptosis. These results indicate that the Fas/ceramide signaling pathway might play a role in granulosa cell apoptosis and suggest that the PLD/DAG pathway might be cross-linked to the Fas/ceramide pathway in apoptotic processes of granulosa cells.     

Beyond apoptosis: nonapoptotic cell death in physiology and disease.

Apoptosis is a morphologically defined form of programmed cell death (PCD) that is mediated by the activation of members of the caspase family. Analysis of death-receptor signaling in lymphocytes has revealed that caspase-dependent signaling pathways are also linked to cell death by nonapoptotic mechanisms, indicating that apoptosis is not the only form of PCD. Under physiological and pathological conditions, cells demonstrate a high degree of flexibility in cell-death responses, as is reflected in the existence of a variety of mechanisms, including necrosis-like PCD, autophagy (or type II PCD), and accidental necrosis. In this review, we discuss recent data suggesting that canonical apoptotic pathways, including death-receptor signaling, control caspase-dependent and -independent cell-death pathways.     

Cell death pathways in juvenile Batten disease

Apoptosis, Golgi fragmentation and elevated ceramide levels occur in Juvenile Neuronal Ceroid Lipofuscinosis (JNCL) neurons, lymphoblasts and fibroblasts. Our purpose was to examine whether apoptosis is the mechanism of cell death in JNCL. This was tested by analyzing caspase-dependent/independent pathways and autophagy, and caspase effects on ceramide and Golgi fragmentation. zVAD prevented caspase activation, but not all cell death. Inhibiting caspase-8 suppressed caspases more than inhibition of any other caspase. Inhibiting caspase-8/6 was synergistic. zVAD suppressed autophagy. 3-methyladenine suppressed caspase activation less than zVAD did. Blocking autophagy/caspase-8/or-6 was synergistic. Blocking autophagy/caspase-3/or-9 was not. Inhibiting caspase-9/3 suppressed autophagy. Golgi fragmentation was suppressed by zVAD, and blocked by CLN3. CLN3, not zVAD, prevented ceramide elevation. In conclusion: caspase-dependent/independent apoptosis and autophagy occur caspase-dependent pathways initiate autophagy Golgi fragmentation results from apoptosis ceramide elevation is independent of caspases, and CLN3 blocks all cell death, prevents Golgi fragmentation and elevation of ceramide in JNCL.     

Over‐expression of HSP70 attenuates caspase‐dependent and caspase‐independent pathways and inhibits neuronal apoptosis

HSP70 is a member of the family of heat‐shock proteins that are known to be up‐regulated in neurons following injury and/or stress. HSP70 over‐expression has been linked to neuroprotection in multiple models, including neurodegenerative disorders. In contrast, less is known about the neuroprotective effects of HSP70 in neuronal apoptosis and with regard to modulation of programmed cell death (PCD) mechanisms in neurons. We examined the effects of HSP70 over‐expression by transfection with HSP70‐expression plasmids in primary cortical neurons and the SH‐SY5Y neuronal cell line using four independent models of apoptosis: etoposide, staurosporine, C2‐ceramide, and β‐Amyloid. In these apoptotic models, neurons transfected with the HSP70 construct showed significantly reduced induction of nuclear apoptotic markers and/or cell death. Furthermore, we demonstrated that HSP70 binds and potentially inactivates Apoptotic protease‐activating factor 1, as well as apoptosis‐inducing factor, key molecules involved in development of caspase‐dependent and caspase‐independent PCD, respectively. Markers of caspase‐dependent PCD, including active caspase‐3, caspase‐9, and cleaved PARP were attenuated in neurons over‐expressing HSP70. These data indicate that HSP70 protects against neuronal apoptosis and suggest that these effects reflect, at least in part, to inhibition of both caspase‐dependent and caspase‐independent PCD pathways.     

Ceramide and other sphingolipids in cellular responses

Formerly considered to serve only as structural components, sphingolipids are emerging as an important group of signaling molecules involved in many cellular events, including cell growth, senescence, meiotic maturation, and cell death. They are also implicated in functions such as inflammation and the responses to heat shock and genotoxic stress. Defects in the metabolism of sphingolipids are related to various genetic disorders, and sphingolipids have the potential to serve as therapeutic agents for human diseases such as colon cancer and viral or bacterial infections. The best-studied member of this family, ceramide, which also serves as the structural back-bone for other sphingolipids, is an important mediator in multiple cellular signaling pathways. The metabolism and functions of sphingolipids are discussed in this review, with a focus on ceramide regulation in various cellular responses.     

Role of ceramide in Ca2+-sensing receptor-induced apoptosis

Increased extracellular Ca(2+) ([Ca(2+)](o)) can damage tissues, but the molecular mechanisms by which this occurs are poorly defined. Using HEK 293 cell lines that stably overexpress the Ca(2+)-sensing receptor (CaR), a G protein-coupled receptor, we demonstrate that activation of the CaR leads to apoptosis, which was determined by nuclear condensation, DNA fragmentation, caspase-3 activation, and increased cytosolic cytochrome c. This CaR-induced apoptotic pathway is initiated by CaR-induced accumulation of ceramide which plays an important role in inducing cell death signals by distinct G protein-independent signaling pathways. Pretreatment of wild-type CaR-expressing cells with pertussis toxin inhibited CaR-induced [(3)H]ceramide formation, c-Jun phosphorylation, and caspase-3 activation. The ceramide accumulation, c-Jun phosphorylation, and caspase-3 activation by the CaR can be abolished by sphingomyelinase and ceramide synthase inhibitors in different time frames. Cells that express a nonfunctional mutant CaR that were exposed to the same levels of [Ca(2+)](o) showed no evidence of activation of the apoptotic pathway. In conclusion, we report the involvement of the CaR in stimulating programmed cell death via a pathway involving GTP binding protein alpha subunit (Galpha(i))-dependent ceramide accumulation, activation of stress-activated protein kinase/c-Jun N-terminal kinase, c-Jun phosphorylation, caspase-3 activation, and DNA cleavage.