Redox regulation and signaling lipids in mitochondrial apoptosis

BBRC Biochemical and Biophysical Research Communications 304 (2003) 471–479 www.elsevier.com/locate/ybbrc

Redox regulation and signaling lipids in mitochondrial apoptosis Jos
e C. Fern
andez-Checa* Liver Unit, Instituto Malalties Digestives, Hospital Clinic i Provincial, Instituto Investigaciones Biomedicas August Pi I Sunyer, Department of Experimental Pathology, Instituto Investigaciones Biom
edicas de Barcelona, Consejo Superior Investigaciones Cient
ıficas, Barcelona 08036, Spain Received 15 January 2003

Abstract Apoptosis can be regulated at multiple levels. A number of proteins with regulatory function in cell death are sensitive to cellular redox environment. The antioxidant glutathione (GSH) and redox-sensitive proteins, thioredoxin and glutathione S-transferase, thus regulate cell death pathways by modulating the redox state of specific thiol residues of target proteins including stress kinases, transcription factors, and caspases. GSH in mitochondria plays an important role in the integrity of mitochondrial proteins and lipids known to play a vital role in the permeabilization of mitochondrial membranes and release of proapoptotic factors. The regulation of mitochondrial GSH (mGSH) is determined by its uptake from the cytosol which is dependent on appropriate membrane dynamics. The deposition of cholesterol in mitochondria induced by alcohol intake impairs this translocation, resulting in severe depletion of mGSH and in sensitization to apoptosis stimuli. Although the interaction of proapoptotic proteins with mitochondria initiates apoptotic pathways, recent data indicate that the mitochondrial trafficking of glycosphingolipids, e.g., ganglioside GD3, induced by apoptotic stimuli is a key event that sets off mitochondrial-dependent apoptotic cascades. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Reactive oxygen species; Glutathione; Ceramide; Gangliosides; Necrosis; Apoptosis

Apoptosis, a genetically controlled form of cell death, is an integral part of life. Discovered in the middle of the 19th century as the morphology of dying cells during the metamorphosis of amphibians, apoptosis is an essential process in development and tissue homeostasis. Since its rediscovery about 30 years ago [1], it has become an intense field of research due to its involvement in the development of pathological states either characterized by decreased incidence of apoptosis (e.g., neoplasia) as well as acute and chronic pathologies with a heightened apoptotic incidence (e.g., stroke and neurodegenerative disorders). Although it was first thought that apoptosis affected mainly the nuclei of cells, this process affects several intracellular organelles including endoplasmic reticulum, lysosomes and most notably mitochondria [2]. Indeed, mitochondria are now considered as strategic centers of cell death regulation where many effectors generated during the initial phase of apoptosis converge and stimulate the release of specialized mitochondrial * Fax: +34-934-51-52-72. E-mail address: [email protected].

proteins that actively trigger apoptosis cascades [2–4]. Several proteins released from mitochondria into the cytosol of cells challenged with apoptosis stimuli have been identified which contribute to caspase-dependent and caspase-independent death pathways. While cytochrome c [5] but not apocytochrome c [6] drives the assembly of a high molecular weight caspase activating complex termed the apoptosome that culminates in the activation of executioner caspase 3, the release of Smac/ Diablo into the cytosol ensures the efficiency of caspase 3 in proteolyzing target proteins through inhibition of inhibitor of apoptosis proteins (IAPs) [7,8]. Furthermore, the mitochondrial protein Omi/HtrA2 promotes cell death in a dual fashion. Besides its IAP activity Omi/ HtrA2 also functions as a serine protease, thus contributing to both caspase-dependent and caspase-independent cell death [9,10]. Moreover, other specialized mitochondria-residing proteins, such as the apoptosis inducing factor (AIF) [11] and endonuclease G [12], are translocated to the nuclei following their release from mitochondria and promote peripheral chromatin condensation and high molecular weight DNA

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fragmentation. The interaction of proapoptotic proteins most notably Bcl-2 family members as well as signaling enzymes, transcription factors or viral-encoded proteins with mitochondrial components stimulates the release of mitochondrial apoptotic proteins [2–4,13] by mechanisms that are still unclear [14,15]. The redox environment can regulate death cascades by modulating the redox state of cysteine residues in specific proteins and this process can play a role in the transduction of reactive oxygen species (ROS) generation. In the present review I will summarize the redox regulation of cell death, particularly in mitochondria, and the role of the emerging interaction of signaling lipids with mitochondria in mitochondrial apoptosis.

Redox regulation of cell death Cellular redox signaling involves the post-transcriptional modification of proteins that use redox chemistry. A redox reaction involves the transfer of electrons between two centers, resulting in their reduction (gain electrons) and oxidation (lose electrons). The paradigm of a redox reaction in cell signaling centers on the reduction/oxidation state of cysteine residues of proteins, resulting in the breaking or formation of a protein disulfide bridge with the interconversion of cysteine into cystine. Redox changes of target proteins are initiated by the generation of ROS and reactive nitrogen species (RNS), leading to the generation of disulfide bridges between two adjacent cysteine residues in a protein or formation of S-nitrosothiols from the attack of nitric oxide or peroxynitrate to protein cysteines. These thiol modifications which have the possibility of eliciting different biological responses can be regulated by factors that act on or modulate ROS/RNS generation. An important strategy to control the fate of ROS in cells is the function of antioxidant defense systems, of which the GSH redox cycle [16] and the redox sensitive proteins including glutathione S-transferase (GST) and thioredoxin [17] are of primary importance. GSH, the most important non-protein thiol of cells, fulfills many important functions. As an antioxidant, GSH metabolizes ROS and peroxides primarily by serving as a cofactor for GSH-dependent enzymes such as GSH peroxidase. Since the concentration of GSH prevails over its oxidized GSSG form, the oxidation of a limited amount of GSH to GSSG can dramatically change this ratio affecting the redox status of the cell. Thioredoxin, a family of small proteins that contain a conserved redox active center, is known to protect cells against oxidant stress-mediated cell killing. The reversible redox reaction in the active center enables thioredoxin to transfer electrons to protein disulfide substrates. The action of thioredoxin reductase and NADPH reduces the oxidized thioredoxin and completes its cycling.

ROS-mediated response in cell death involves direct alteration of stress kinases and transcription factors. It has been reported that GST mu1 (GST M1) protected primary hepatocytes against TGF b1-induced apoptosis by blocking ASK1, a MAP kinase kinase kinase (MAPKKK) ubiquitously expressed that mediates the activation of downstream targets including JNK (c-Jun N-terminal kinase) and p38 MAP kinase by inflammatory cytokines [18]. In addition, oxidation of thioredoxin by GSSG formation promoted the activation of ASK1 mediating the apoptosis of 293 cells induced by TNF or hydrogen peroxide [19]. The stress kinase, JNK, is maintained inactive in cells through its association with GST Pi forming a GST Pi–JNK complex. Treatment of cells with UV irradiation or hydrogen peroxide causes the formation of GSSG, resulting in the oligomerization of GST Pi and liberation of the active JNK [20]. Thus, as guardians of ASK1 and JNK activities in normal growing cells, GSTs and reduced thioredoxin function as sensors of intracellular changes in redox potential that are elicited by various forms of stresses. Through maintenance of protein sulfhydryls in the appropriate redox state GSH can regulate important death/survival pathways which modulate the fate of cells in response to apoptosis stimuli. A number of studies have shown that intracellular GSH loss such as that induced by stimulated efflux out of the cell or its consumption sensitizes different cell types to a variety of apoptosis stimuli [21,22]. Aplidin, a novel antitumor agent isolated from the Mediterranean Aplidium albicans, induces apoptosis in the highly invasive and proliferative breast cell line MDA-MB-231 with mutated p53 and ras genes [23]. While Aplidin induced sustained activation of epidermal growth factor receptor, Src, JNK, and p38 MAPK, these effects were dependent on low GSH levels. Furthermore, epithelial HeLa cells expressing mutated cystic fibrosis transmembrane conductance regulator (CFTR) displayed resistance to hydrogen peroxide-mediated apoptosis accompanied by higher intracellular GSH stores and lower mitochondrial Bax levels [24]. Although these data suggest a GSH-dependent BAX activation as an early step in hydrogen peroxide-induced apoptosis of HeLa cells, the influence of cytosolic pH changes was not evaluated. Recent studies in mouse hepatocytes in which the GSH levels were depleted by diethylmaleate or acetaminophen indicated a sensitization to TNF-induced apoptosis [25]. In examining the activation of stress kinases and NF-jB-dependent survival genes, it was found that GSH depletion in the cytosol/nuclei resulted in sustained activation of JNK by TNF. Intriguingly, while GSH depletion did not impair the nuclear DNA binding of NF-jB induced by TNF, it did prevent the induction of NF-jB-dependent survival genes such as iNOS. These data show a differential dependence on GSH levels between the NF-jB DNA binding activity

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and its transactivation with the latter showing a requirement for critical GSH levels. Although the DNA binding of NF-jB can be modulated by redox environment, recent findings indicated a novel pathway for the redox regulation of NF-jB [26]. Using an in vitro model in which the ratio GSH/GSSG was changed from 100 to 0.1, the S-glutathionylation of cysteine 62 of the p50 subunit of NF-jB was shown as demonstrated by mass spectrometry data and molecular modeling. Thus, these data propose an attractive additional means by which oxidative environment (accumulation of GSSG) can promote cell death by interfering with the induction of NF-jB-dependent survival genes, although the significance of this mechanism in living cells remains to be established. Thus, depending on the extent and mechanism of GSH depletion (efflux from cytosol of cells vs. decreased GSH/GSSG ratio) a limited storage of cell GSH can promote cell death through sustained JNK activation and/or suppression of NF-jB-dependent survival pathways. In addition to the redox regulation of these death promoting components, the engagement of the deathinducing signaling complex (DISC) and caspase-8 activated by ligation of death receptors relies on appropriate redox environment for proper function. Through this mechanism, a requirement for adequate GSH levels to ensure active DISC complex and caspase 8 activity after Fas stimulation in CEM and H9 cells [27] has been shown. However, this dependence was not observed in HepG2 or Hepa1–6 cells, indicating that the requirement of GSH for active DISC is cell-type specific. In hepatocytes, for instance, the role of GSH in sensitization to Fas or TNF is controversial [28,29]. The length (prolonged vs acute) and extent of GSH depletion (cytosolic vs mitochondrial) may discriminate the fate of hepatocytes after Fas or TNF challenge. Thus, the existence of additional factors, e.g., the presence of redoxactive thioredoxin in different cell types, may determine the outcome of the delicate balance between physiological antioxidants (GSH) and endogenously produced oxidants (ROS and nitric oxide) in the progression of apoptosis signaling.

Mitochondrial ROS and glutathione in cell death ROS are normally produced as by-products of metabolic pathways, particularly from the physiological mitochondrial respiration. In mitochondria the consumption of oxygen functions as an energy generating device although anaerobic mitochondria can generate ATP without the need of oxygen to do so. The partial reduction of oxygen during the oxidative phosphorylation generates superoxide anion, which is then transformed in other species, including hydrogen peroxide [16]. Previous studies indicated that one of the earliest

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changes during apoptosis was the loss of mitochondrial membrane potential and ROS generation derived from the activity of the mitochondrial electron transport chain [30,31]. The precise site, however, within the mitochondrial electron transport complexes responsible for the ROS formation is not well established. Recent data have pointed to the flavin mononucleotide group of complex I as an important source of ROS through reverse electron transfer [32], deduced from the ability of diphenyleneiodonium to inhibit succinate-supported ROS without affecting the flavin group of complex II. Recently, it has been shown that caspase 3 activated after the release of cytochrome c from mitochondria disrupted electron transport complexes I and II, resulting in loss of DWm and ROS generation [33]. In addition, complex III of respiration is also known to be an important source of ROS generation [34,35]. Respiratory chain complex III has two ubiquinone-reactive sites, Qo, where ubiquinol is oxidized by redox-reactive centers, cytochrome c1 and the Rieske [2Fe–2S] protein, and Qi, where ubiquinone is reduced by the redox center cytochrome b. The Rieske cluster is a mobile structure and this mobility may facilitate rapid electron transfer between cytochrome b and c1 [36]. Furthermore, although AIF has an apoptosis-inducing function [11], its oxidoreductase function has been recently characterized biochemically [37]. Purified AIF exhibits a NADH oxidase activity which can result in the generation of ROS, including superoxide anion and hydrogen peroxide. However, it is unclear whether this emerging function of AIF as a ROS-generating enzyme contributes to mitochondrial-dependent apoptosome activation. Despite the continued formation of ROS as byproducts of the aerobic respiration, mitochondria are endowed with antioxidant defenses to control the mitochondrial formation of ROS. The dismutation of superoxide anion by the Mn-SOD generates hydrogen peroxide which is then metabolized by the matrix GSH redox cycle. A coordinated function of Mn-SOD and the GSH redox cycle is vital to avoid accumulation of hydrogen peroxide, a potent oxidant with a long range of action that may oxidize critical mitochondrial targets [16] (Fig. 1). A vital factor for the efficient detoxification of hydrogen peroxide produced endogenously during mitochondrial respiration is the level of mGSH. By serving as a cofactor for mitochondrial GSH peroxidase GSH ensures optimal elimination of hydrogen peroxide. Thus, the depletion of mGSH to levels below the Km of GSH for GSH peroxidase (3 mM) would compromise this detoxification system allowing the accumulation of hydrogen peroxide and lipid hydroperoxides to toxic levels [38]. Indeed, a variety of studies have indicated the crucial role of GSH in mitochondria in apoptotic cell death. Recent studies showed that GSH depletion by buthionine sulfoximine, an inhibitor of c-glutamylcysteine synthetase, or diethylmaleate, a GSH-depleting

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Fig. 1. Schematic illustration depicting the regulation of mitochondrial apoptosis by ROS and GSH. The overgeneration of ROS within mitochondria by sphingolipid formation (e.g., GD3 generated from ASMase) can target specific proteins (e.g., redox sensitive components of the permeabilization transition, ANT) that regulate mitochondrial permeabilization and subsequent release into the cytosol such as AIF and cytochrome c (Cyt c), responsible for caspase-independent and caspase-dependent cell death. In addition, ROS stimulation can attack vital lipids such as cardiolipin (CL), known to regulate the conformation/binding state of Cyt c and its release into the cytosol. GSH in the matrix derives from its import from the cytosol by a mitochondrial carrier whose activity is modulated by the physico-chemical properties of the mitochondrial inner membrane. Agents that induce endoplasmic reticulum (ER) stress stimulate cholesterol synthesis through induction of hydroxymethylglutaryl coenzyme A reductase (HMGCoA R) via activation of ER-bound transcription factor SREBP. Cholesterol (Ch) deposition in mitochondria with the aid of StAR impairs the carrier activity through altered membrane microviscosity, leading to severe mGSH depletion, ROS generation, and caspase-dependent and caspase-independent cell death.

agent, resulted in overproduction of endogenous ROS from complex III of respiration and apoptosis of HL-60 cells [39]. Interestingly, this outcome was mediated by ROS-induced mitochondrial membrane permeabilization, despite overexpression of Bcl-2. These findings highlight the relevance of this specific pool of GSH in controlling the endogenous generation of ROS, which otherwise could target vital mitochondrial components including the adenine nucleotide translocator (ANT) resulting in release of cytochrome c and caspase activation [39]. In addition, the overexpression of c-Myc and E2F1 in NIH3T3 and Saos-2 cells potentiated apoptosis by preventing the Mn-SOD-mediated ROS elimination [40]. Indeed, the relevance of endogenous ROS generation in mitochondrial apoptosis was reinforced by recent findings showing the fundamental role of thioredoxin-2, a mitochondrial specific member of the

thioredoxin superfamily, in the regulation of mitochondrial-dependent apoptosis through control of endogenous ROS formation [41]. In addition, mitochondrial targeting of antioxidant enzymes blocks apoptosis in several models [42]. Thus, the mitochondrial ROS generation can accelerate and contribute to apoptosis by targeting specific mitochondrial components, including members of the permeability transition pore with redox sensitive cysteine residues such as ANT [43]. Moreover, superoxide anion caused a selective permeabilization of the mitochondrial outer membrane via a process requiring voltage-dependent anion channel (VDAC) without the concurrence of Bax translocation to mitochondria [44]. Exploiting the selective biotransformation of (R,S)-3hydroxy-4-pentenoate (HP) into a Michael acceptor within mitochondria by the (R)-3-hydroxybutanoate: NADþ oxidoreductase, it has been possible to selectively deplete the mitochondrial GSH (mGSH) pool in intact cells with the sparing of cytosol GSH levels to underscore specifically the role of mGSH in apoptosis induced by TNF. Preincubation of primary culture rat hepatocytes with HP resulted in sensitization to TNF-mediated cell death in the absence of any other sensitization approach such as blocking protein or total RNA synthesis or NF-jB activation [45,46]. mGSH-depleted hepatocytes exposed to TNF exhibited a significant and early generation of peroxides that preceded the loss of mitochondrial membrane potential, release of cytochrome c, and apoptotic and necrotic demise [46]. Similar findings were observed when the depletion of the mGSH was induced by chronic ethanol intake [45,47,48] that was reversed upon repletion of mGSH. On the other hand, overexpression of a mitochondrial GSH transporter, the dicarboxylate carrier, which resulted in substantially higher mGSH levels, protected NKK-52E cells from oxidant-induced apoptosis [49]. Thus, mitochondria are both a source and target of ROS. Through maintenance of superoxide anion-induced hydrogen peroxide generated on the matrix side of the mitochondrial inner membrane [50], the pool of mGSH serves as a critical line of defense that controls the fate of cells in response to apoptosis stimuli. In addition to ensuring appropriate redox state of critical mitochondrial proteins (e.g., components of the mitochondrial permeability transition pore complex), GSH in mitochondria may also be vital in guarding the integrity of lipids. A recent study demonstrated the critical role of cardiolipin in collaboration with proapoptotic Bcl-2 family members in the formation of supramolecular openings in the outer mitochondrial membrane [51]. In addition to its fundamental role as a housekeeping lipid in the organization of individual complexes into functional units of the respiratory chain, cardiolipin has a defined distribution pattern within mitochondria and may work as a functional link in the

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action of BH1-3 multidomain proteins, e.g., Bax, or BH3-only Bid, to elicit the release of cytochrome c [51,52]. Cytochrome c is bound to the inner membrane by cardiolipin. Recent studies in rat liver mitochondria have characterized a two-step process in the release of cytochrome c [53], consisting of the detachment of this protein from its membrane-anchoring lipid, cardiolipin, followed by permeabilization of the outer membrane allowing the release of cytochrome c into the extramitochondrial environment. Since the peroxidation of cardiolipin was shown to contribute to the transition from tight to loose conformation of cytochrome c [53], an additional aspect of the protective role of mGSH might involve protection of cardiolipin from ROS attack. Thus, either through maintenance of vital mitochondrial proteins and/or cardiolipin, mGSH depletion associated with disease states (such as hypoxia-referpusion injury, toxic bile acids-induced damage, and chronic alcohol intake) will favor conditions for cell damage.

Regulation of mitochondrial glutathione by cholesterol mGSH being an important line of defense against apoptosis, factors that regulate this pool of GSH may be of relevance in the control of cell death. Despite its universal presence in cells, GSH is not homogeneously distributed within cellular organelles. Most of the GSH is found in cytosol, comprising 80–85% of total cellular content, while, 10–15% of the cellular pool is found in mitochondria where it reaches a concentration similar to that of cytosol [54]. The mGSH pool originates from a specific transport system located in the inner mitochondrial membrane that translocates GSH from the cytosol into the mitochondrial matrix [45,48,49,55]. Although the molecular identification of this carrier and the driving forces for its operation are unclear, its functional activity depends on appropriate fluidity range of inner mitochondrial membrane. Alcohol feeding is known to profoundly deplete the mGSH levels [45,48,54–58]. Studies in intact mitochondria and mitoplasts from alcohol-fed rat liver indicated that the normalization of mitochondrial inner membrane fluidity in vitro by the fluidizing agent A2 C, or in vivo by SAM or tauroursodeoxycholic acid, restores the kinetics of the mGSH carrier replenishing mGSH levels. Membrane dynamics are controlled by lipid composition, particularly cholesterol/phospholipid ratio, and our findings with mitochondria and mitoplasts showed that chronic alcohol enhanced the levels of total cholesterol [55]. Indeed, cholesterol enrichment of mitochondria from normal rat liver impaired selectively the uptake of GSH into mitochondria that was restored upon fluidization with A2 C [59]. Recently, we have demonstrated that

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acetaldehyde reproduces the disturbing effects of alcohol on the impairment of mitochondrial GSH transport. Notably, acetaldehyde stimulated the accumulation of cholesterol in mitochondria, resulting in decreased membrane fluidity. Consequently, acetaldehyde impaired the mitochondrial transport of GSH decreasing the Km of both the low and high affinity components [59]. Due to the lowering of mGSH levels, acetaldehyde sensitized HepG2 cells to TNF-mediated cell death, prevented by cyclosporin A and GSH ethyl ester. The increase of mitochondrial cholesterol by acetaldehyde was mediated by endoplasmic reticulum (ER) stress, a process characterized by the accumulation of unfolded or misfolded proteins in the ER which signals the induction of responsive genes. In this case, acetaldehyde increased the levels of GADD153, an ER stress-specific chaperon, and the ER associated transcription factor SREBP, which regulates the cholesterol synthesis by activation of the rate-limiting enzyme hydroxyglutaryl CoA reductase (HMGCoAR). Interestingly, the stimulating effect of acetaldehyde on cholesterol was inhibited by lovastatin, preventing the increase in mitochondrial membrane microviscosity, impairment of mGSH transport, and sensitization to TNF [59]. Thus these observations suggest a novel role of ER stress in the sensitization of hepatocytes to TNF via mGSH depletion (Fig. 1) and add to previous data, indicating a functional link between ER stress and mitochondrial control of apoptosis [60]. The role of steroidogenic acute regulatory protein (StaR) in the mitochondrial cholesterol transport has been characterized in steroidogenic cells [61], which has been recently identified in hepatocytes [62]. The peripheral benzodiazepine receptor (PBR) has been suggested to function as a cholesterolbinding protein and in the transport of cholesterol into the mitochondrial inner membrane of steroidogenic cells [63]. Thus, it is possible that the partnership between StaR and PBR may actually mediate the acetaldehydeinduced cholesterol deposition in mitochondria, resulting in selective mGSH depletion and sensitization of hepatocytes to apoptotic stimuli.

Sphingolipids and cell death Sphingolipids constitute a class of lipids that may function as second messengers in different cellular processes such as cell differentiation, growth, and cell death. In particular ceramide has been shown to play a role in the stress response, whose levels increase before the onset of cell death [64,65]. While Bcl2 family members may induce ROS secondarily to the release of cytochrome c and caspase activation [33], ceramide has been shown to disrupt electron flow at complex III, resulting in enhanced ROS generation, which facilitates cytochrome c release and caspase activation [66–69]. Cellular

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ceramide levels can increase by several means. In addition to the de novo synthesis through activation of serine-palmitoyl transferase, the rate-limiting enzyme in ceramide synthesis, or ceramide synthetase, ceramide can arise from hydrolysis of sphingomyelin-engaging sphingomyelinases (SMases) [70]. This pathway may be of significance in promoting specific macrodomain formation in the plasma membrane, allowing oligomerization of certain cell surface proteins such as ligated receptors (TNF family) [71]. Several SMases have been characterized of which two are of relevance in signaling. The membrane-bound neutral SMase (NSMase) with an optimum pH of approximately 7.5 and an acidic SMase (ASMase) with an optimum pH of about 4.8 further subclassified into an endosomal/lysosomal ASMase and a secretory Zn2þ -dependent SMase. Apoptotic stimuli, such as death ligands (e.g., Fas and TNF), chemotherapeutic agents or ionizing radiation, activate these SMases, which account for the ability of the inducing stimuli to generate ceramide with various kinetics and possibly at different intracellular locations. Although the precise intracellular site of ceramide generation by individual SMases remains to be clearly established, the domains within the intracytoplasmic region of the death ligand receptor responsible for the activation of NSMase and ASMase are distinct [70]. Using mutants of the cytoplasmic domain of TNFR55, specific receptor domains link differentially to NSMase and ASMase. The activation of NSMase is signaled by a cytoplasmic portion of TNFR55 containing a small motif of nine amino acid residues that binds to factor associated with NSMase activation (FAN). In contrast, the domain of TNFR55 activating ASMase corresponds to the death domain signaling the cytotoxic effects of TNF [70]. Although sphingomyelin is thought to be located almost exclusively in the outer leaflet of the plasma membrane, recent evidence indicated that this lipid is also present in mitochondria and the in situ generation of ceramide within this organelle by enforced mitochondrial targeting of bacterial sphingomyelinase induced apoptosis in MCF7 cells [72]. Although the role of individual SMase in apoptosis pathways is not well established and their engagement in apoptosis seems to depend on several conditions, such as the kind of apoptotic stimuli used the cell type studied, cells lacking ASMase but with unimpaired NSMase were found to resist stress-mediated cell death [73]. In line with this, we have recently characterized the role of ASMase in TNF-mediated hepatocellular apoptosis [46]. Using two distinct approaches, the inactivation of endogenous ASMase by imipramine, a trycyclic antideppresant that induces the proteolysis of active ASMase form, and hepatocytes from ASMase knockout mice, ASMase was shown to contribute to TNF-mediated hepatocellular apoptosis [46]. Moreover, ASMase= mice were resis-

tant to endogenous or exogenous TNF-induced liver damage in vivo. Interestingly, the defective TNF-induced hepatocellular apoptosis and liver damage in ASMase deficient mice were accompanied by unaltered Bax translocation into mitochondria, indicating that the signaling events upstream of mitochondria are preserved in ASMase-deficient hepatocytes. These results therefore indicate that the translocation of BH1-3 multidomain Bcl-2 family members, such as Bax, appears to be independent of ASMase, yet downstream steps of mitochondria, including cytochrome c release and caspase activation, seem to require ASMase. Perhaps through altered ceramide generation and/or sphingomyelin metabolism, ASMase may contribute to the maintenance of a mitochondrial lipid environment adequate for the proper mitochondrial docking of Bax to facilitate the apoptosome. In addition to its involvement in apoptotic signaling, ceramide also provides the carbon source for glycosphingolipid (GSL) synthesis in the Golgi network coupled to the exocytotic vesicle flow to the plasma membrane, one of their predominant destinations in cells [74]. Gangliosides are a subfamily of GSLs that are distinguished by the presence of several sialic acid residues. GSLs and gangliosides have been implicated in fundamental cell processes such as growth, differentiation, adhesion, and cell signaling [75]. Ganglioside GD3 (GD3) has emerged as a cell death effector activating the mitochondrial-dependent apoptosome through sequential mitochondrial ROS stimulation, cytochrome c release, and caspase activation [76–78] and this cell death function seems to be modulated by the acetylation state of GD3 [79]. As ceramide, cell GD3 levels increase in response to apoptosis stimuli [77,78,80,81] while the down-regulation of GD3 synthase, the enzyme responsible for GD3 synthesis from its precursor ganglioside GM3, prevents Fas-, TNF- or b-amyloid-induced cell death [81,82]. While most of the evidence for the apoptotic role of GD3 has been derived from in vitro studies with isolated mitochondria, recent studies in CEM cells and cultured hepatocytes indicated the trafficking and physical interaction of GD3 with mitochondria in response to apoptotic stimuli [77,83]. Most of GD3 was present at the plasma membrane in resting hepatocytes, however, in response to TNF exogenous ASMase or ionizing radiation, GD3 underwent a dramatic redistribution that involved first its disappearance from the plasma membrane followed by its trafficking to mitochondria [83]. The colocalization of GD3 with mitochondria was preceded by its location in early to late endosomes via coordinated secretory/endocytic vesicular trafficking, and the disruption of this pathway prevented the interaction of GD3 with mitochondria sparing sensitized hepatocytes to TNF exposure. These findings suggest that endosomal vesicles trafficking through actin

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cytoskeleton may be part of the TNF/Fas multicomponent signaling complex delivering death signals, e.g., GD3, to mitochondria. In addition to the active role of GD3 in promoting apoptosis, GD3 interferes with the nuclear translocation of active members of NF-jB thus suppressing the activation of NF-jB-dependent gene induction including antiapoptotic genes [84]. Using GSL derivatives, it was shown that while the N-fatty acyl sphingosine moiety common to both ceramide and GD3 is necessary for its ROS stimulating effect, the presence of sugar residues in the backbone of ceramide is required in blocking the nuclear translocation of NF-jB [84]. Thus, GD3 has a dual role in apoptosis as a mitochondria-interacting, ROS stimulator, and NF-jB-inactivating agent. The exploitation of this duality has been recently shown in HepG2 cells, a tumor cell line highly resistant to current cancer therapy [85]. The preincubation of HepG2 cells with GD3 blocked the translocation of NF-jB to the nuclei sensitizing cells to radiotherapy due to overaccumulation of ROS/RNS generated from mitochondria.

Concluding remarks The inherent ability of mitochondria as physiological ROS generators regulates mitochondrial-dependent death pathways. Although ROS stimulation may not be required per se for apoptosis, a variety of stimuli (e.g., TNF) that generate sphingolipid synthesis, particularly, ganglioside GD3, facilitate the apoptosome activation via ROS formation and subsequent impact on redox sensitive mitochondrial components including proteins (e.g., ANT) and/or lipids (e.g., cardiolipin); consequently the scavenging of ROS can delay or prevent cell death. In this scenario, caspase inhibitors may have limited therapeutic value. While they can block caspasedependent cell death they may be ineffective in protecting cells from dying by necrosis; in contrast, antioxidant therapy may be a more effective measure to guarantee protection against necrosis and apoptosis. By regulating the efficient removal of hydrogen peroxide and other organic peroxides, GSH in mitochondria may guard the integrity of guarding mitochondrial components, thus playing a determinant role in the fate of cells in response to apoptosis.

Acknowledgments The work presented was supported in part by the Research Center for Liver and Pancreatic Diseases (P50 AA11999) and Grant 1R21 AA014135-01 funded by the US National Institute on Alcohol Abuse and Alcoholism; Plan Nacional de I + D Grants SAF 99-0138 and SAF2001-2118. I want to thank members of my laboratory for valuable comments.

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