Mitochondrial reactive oxygen species in cell death signaling

During apoptosis, mitochondrial membrane permeability (MMP) increases and the release into the cytosol of pro-apoptotic factors (procaspases, caspase activators and caspase-independent factors such as apoptosis-inducing factor (AIF)) leads to the apoptotic phenotype. Apart from this pivotal role of mitochondria during the execution phase of apoptosis (documented in other reviews of this issue), it appears that reactive oxygen species (ROS) produced by the mitochondria can be involved in cell death. These toxic compounds are normally detoxified by the cells, failing which oxidative stress occurs. However, ROS are not only dangerous molecules for the cell, but they also display a physiological role, as mediators in signal transduction pathways. ROS participate in early and late steps of the regulation of apoptosis, according to different possible molecular mechanisms. In agreement with this role of ROS in apoptosis signaling, inhibition of apoptosis by anti-apoptotic Bcl-2 and Bcl-x(L) is associated with a protection against ROS and/or a shift of the cellular redox potential to a more reduced state. Furthermore, the fact that active forms of cell death in yeast and plants also involve ROS suggests the existence of an ancestral redox-sensitive death signaling pathway that has been independent of caspases and Bcl-2.     

Somatic gonad sheath cells and Eph receptor signaling promote germ-cell death in C. elegans

Programmed cell death eliminates unwanted cells during normal development and physiological homeostasis. While cell interactions can influence apoptosis as they do other types of cell fate, outside of the adaptive immune system little is known about the intercellular cues that actively promote cell death in healthy cells. We used the Caenorhabditis elegans germline as a model to investigate the extrinsic regulators of physiological apoptosis. Using genetic and cell biological methods, we show that somatic gonad sheath cells, which also act as phagocytes of dying germ cells, promote death in the C. elegans germline through VAB-1/Eph receptor signaling. We report that the germline apoptosis function of VAB-1 impacts specific cell death pathways, and may act in parallel to extracellular signal-regulated kinase MAPK signaling. This work defines a non-autonomous, pro-apoptotic signaling for efficient physiological cell death, and highlights the dynamic nature of intercellular communication between dying cells and the phagocytes that remove them.     

Appetizing rancidity of apoptotic cells for macrophages: oxidation,externalization, and recognition of phosphatidylserine

Programmed cell death (apoptosis) functions as a mechanism to eliminate unwanted or irreparably damaged cells ultimately leading to their orderly phagocytosis in the absence of calamitous inflammatory responses. Recent studies have demonstrated that the generation of free radical intermediates and subsequent oxidative stress are implicated as part of the apoptotic execution process. Oxidative stress may simply be an unavoidable yet trivial byproduct of the apoptotic machinery; alternatively, intermediates or products of oxidative stress may act as essential signals for the execution of the apoptotic program. This review is focused on the specific role of oxidative stress in apoptotic signaling, which is realized via phosphatidylserine-dependent pathways leading to recognition of apoptotic cells and their effective clearance. In particular, the mechanisms involved in selective phosphatidylserine oxidation in the plasma membrane during apoptosis and its association with disturbances of phospholipid asymmetry leading to phosphatidylserine externalization and recognition by macrophage receptors are at the center of our discussion. The putative importance of this oxidative phosphatidylserine signaling in lung physiology and disease are also discussed.     

Reactive oxygen species,cellular redox systems,and apoptosis

Reactive oxygen species (ROS) are products of normal metabolism and xenobiotic exposure, and depending on their concentration, ROS can be beneficial or harmful to cells and tissues. At physiological low levels, ROS function as “redox messengers” in intracellular signaling and regulation, whereas excess ROS induce oxidative modification of cellular macromolecules, inhibit protein function, and promote cell death. Additionally, various redox systems, such as the glutathione, thioredoxin, and pyridine nucleotide redox couples, participate in cell signaling and modulation of cell function, including apoptotic cell death. Cell apoptosis is initiated by extracellular and intracellular signals via two main pathways, the death receptor- and the mitochondria-mediated pathways. Various pathologies can result from oxidative stress-induced apoptotic signaling that is consequent to ROS increases and/or antioxidant decreases, disruption of intracellular redox homeostasis, and irreversible oxidative modifications of lipid, protein, or DNA. In this review, we focus on several key aspects of ROS and redox mechanisms in apoptotic signaling and highlight the gaps in knowledge and potential avenues for further investigation. A full understanding of the redox control of apoptotic initiation and execution could underpin the development of therapeutic interventions targeted at oxidative stress-associated disorders.     

Mitogen-activated protein kinase/extracellular signal-regulated kinase signaling in activated T cells abrogates TRAIL-induced apoptosis upstream of the mitochondrial amplification loop and caspase-8

Fas ligand and TNF-related apoptosis-inducing ligand (TRAIL) induce apoptosis in many different cell types. Jurkat T cells die rapidly by apoptosis after treatment with either ligand. We have previously shown that mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) can act as a negative regulator of apoptosis mediated by the Fas receptor. In this study we examined whether MAPK/ERK can also act as a negative regulator of apoptosis induced by TRAIL. Activated Jurkat T cells were efficiently protected from TRAIL-induced apoptosis. The protection was shown to be MAPK/ERK dependent and independent of protein synthesis. MAPK/ERK suppressed TRAIL-induced apoptosis upstream of the mitochondrial amplification loop because mitochondrial depolarization and release of cytochrome c were inhibited. Furthermore, caspase-8-mediated relocalization and activation of Bid, a proapoptotic member of the Bcl family, was also inhibited by the MAPK/ERK signaling. The protection occurred at the level of the apoptotic initiator caspase-8, as the cleavage of caspase-8 was inhibited but the assembly of the death-inducing signaling complex was unaffected. Both TRAIL and Fas ligand have been suggested to regulate the clonal size and persistence of different T cell populations. Our previous results indicate that MAPK/ERK protects recently activated T cells from Fas receptor-mediated apoptosis during the initial phase of an immune response before the activation-induced cell death takes place. The results of this study show clearly that MAPK/ERK also participates in the inhibition of TRAIL-induced apoptosis after T cell activation.     

Caspases activation in hyperthermia-induced stimulation of TRAIL apoptosis

In leukemia cells, hyperthermia enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. The phenomenon is caspase-dependent and results in membrane changes leading to an increased recognition of TRAIL death receptors by TRAIL. Because either caspase-2 or an apical proteolytic event has been recently proposed to act as an initiator of the cell death mechanism induced by heat shock, we have investigated the hierarchy of caspase activation in cells exposed to the combined heat shock plus TRAIL treatment. We report here that caspases-2, -3, and -8 were the first caspases to be activated. As expected, caspase-8 is required and indispensable during the initiation of this death signaling. Caspase-2 may also participate in the phenomenon but, in contrast to caspase-8, its presence appears dispensable because its depletion by small interfering RNA is devoid of effects. Our observations also suggest a role of caspase-3 and of a particular cleaved form of this caspase during the early signals of heat shock plus TRAIL-induced apoptosis.     

Redox signaling: Potential arbitrator of autophagy and apoptosis in therapeutic response

Redox signaling plays important roles in the regulation of cell death and survival in response to cancer therapy. Autophagy and apoptosis are discrete cellular processes mediated by distinct groups of regulatory and executioner molecules, and both are thought to be cellular responses to various stress conditions including oxidative stress, therefore controlling cell fate. Basic levels of reactive oxygen species (ROS) may function as signals to promote cell proliferation and survival, whereas increase of ROS can induce autophagy and apoptosis by damaging cellular components. Growing evidence in recent years argues for ROS that below detrimental levels acting as intracellular signal transducers that regulate autophagy and apoptosis. ROS-regulated autophagy and apoptosis can cross-talk with each other. However, how redox signaling determines different cell fates by regulating autophagy and apoptosis remains unclear. In this review, we will focus on understanding the delicate molecular mechanism by which autophagy and apoptosis are finely orchestrated by redox signaling and discuss how this understanding can be used to develop strategies for the treatment of cancer.     

A cross talk between cellular signalling and cellular redox state during heat-induced apoptosis in a rat histiocytoma

Increasing evidence provides support for oxidative stress to be closely linked to apoptosis. Reactive oxygen species (ROS) are thought to be involved in many forms of programmed cell death. Though heat shock is a universal phenomenon, BC-8, a macrophage-like cell line failed to mount a typical heat shock response. In the absence of heat shock proteins and functional p53, BC-8 cells undergo apoptosis through CD95 signaling. In the present study, we have investigated the role of ROS in the regulation of apoptosis in these cells. We show that cells transfected with hsp70 and functional p53 are resistant to heat-induced apoptosis through inhibition of CD95 expression and ROS induction. Furthermore, apoptosis in BC-8 cells resulted in two bursts of ROS generation, one correlated with heat stress and intracellular depletion of GSH and the other with Bax overexpression and cytochrome c release. Antioxidants could not protect these cells from heat-induced apoptosis and the death pathway seems to be dependent on initial signaling cascade subsequently altering the intracellular redox. Hence, our data suggest that ROS generation in BC-8 cells upon heat shock is facultative but not obligatory for apoptosis.     

Regulation of apoptosis by protein S-nitrosylation

Summary. S-nitrosylation/denitrosylation of critical cysteine residues on proteins serves as a redox switch that regulates the function of a wide array of proteins. A key signaling pathway that is regulated by S-nitrosylation is apoptotic cell death. Here we will review the proteins in apoptotic pathways that are known to be S-nitrosylated by endogenous NO production. The targets and functional consequences of S-nitrosylation during apoptosis are multifaceted, allowing cells to fine tune their response to apoptotic signals.     

Newcastle disease virus exerts oncolysis by both intrinsic and extrinsic caspase-dependent pathways of cell death

Newcastle disease virus (NDV), an avian paramyxovirus, is tumor selective and intrinsically oncolytic. Here, we present evidence that genetically modified, recombinant NDV strains are cytotoxic to human tumor cell lines of ecto-, endo-, and mesodermal origin. We show that cytotoxicity against tumor cells is due to multiple caspase-dependent pathways of apoptosis independent of interferon signaling competence. The signaling pathways of NDV-induced, cancer cell-selective apoptosis are not well understood. We demonstrate that NDV triggers apoptosis by activating the mitochondrial/intrinsic pathway and that it acts independently of the death receptor/extrinsic pathway. Caspase-8-methylated SH-SY5Y neuroblastoma cells are as sensitive to NDV as other caspase-8-competent cells. This demonstrates that NDV is likely to act primarily through the mitochondrial death pathway. NDV infection results in the loss of mitochondrial membrane potential and the subsequent release of the mitochondrial protein cytochrome c, but the second mitochondrion-derived activator of caspase (Smac/DIABLO) is not released. In addition, we describe early activation of caspase-9 and caspase-3. In contrast, cleavage of caspase-8, which is predominantly activated by the death receptor pathway, is a TNF-related, apoptosis-inducing ligand (TRAIL)-induced late event in NDV-mediated apoptosis of tumor cells. Our data, therefore, indicate that the death signal(s) generated by NDV in tumor cells ultimately converges at the mitochondria and that it acts independently of the death receptor pathway. Our cytotoxicity studies demonstrate that recombinant NDV could be developed as a cancer virotherapy agent, either alone or in combination with therapeutic transgenes. We have also shown that trackable oncolytic NDV could be developed without any reduction in oncolytic efficacy.     

Paracrine-mediated apoptosis in reproductive tract development

In mammalian development, the signaling pathways that couple extracellular death signals with the apoptotic machinery are still poorly understood. We chose to examine Müllerian duct regression in the developing reproductive tract as a possible model of apoptosis during morphogenesis. The TGFbeta-like hormone, Müllerian inhibiting substance (MIS), initiates regression of the Müllerian duct or female reproductive tract anlagen; this event is essential for proper male sexual differentiation and occurs between embryonic days (E) 14 and 17 in the rat. Here, we show that apoptosis occurs during Müllerian duct regression in male embryos beginning at E15. Female Müllerian ducts exposed to MIS also exhibited prominent apoptosis within 13 h, which was blocked by a caspase inhibitor. In both males and females the MIS type-II receptor is expressed exclusively in the mesenchymal cell layer surrounding the duct, whereas apoptotic cells localize to the epithelium. In addition, tissue recombination experiments provide evidence that MIS does not act directly on the epithelium to induce apoptosis. Based on these data, we suggest that MIS triggers cell death by altering mesenchymal-epithelial interactions.     

Hydrogen peroxide and redox modulation sensitize primary mouse hepatocytes to TNF-induced apoptosis

Tumor necrosis factor alpha (TNF) plays an important role in mediating hepatocyte injury in various liver pathologies. TNF treatment alone does not cause the death of primary cultured hepatocytes, suggesting other factors are necessary to mediate TNF-induced injury. In this work the question of whether reactive oxygen species can sensitize primary cultured hepatocytes to TNF-induced apoptosis and necrosis was investigated. Sublethal levels of H(2)O(2), either as bolus doses or steady-state levels generated by glucose oxidase, were found to sensitize cultured hepatocytes to TNF-induced apoptosis. High levels of H(2)O(2) also triggered necrosis in hepatocytes regardless of whether TNF was present. Similarly, antimycin, a complex III inhibitor that increases reactive oxygen species generation from mitochondria, sensitized hepatocytes to TNF-induced apoptosis at low doses but caused necrosis at high doses. Redox changes seem to be important in sensitizing primary hepatocytes, because diamide, a thiol-oxidizing agent, and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), an inhibitor of GSSG reductase, also increased TNF-induced apoptosis in cultured primary hepatocytes at sublethal doses. High doses of diamide and BCNU predominantly triggered necrotic cell death. Agents that sensitized hepatocytes to TNF-induced apoptosis -- H(2)O(2), antimycin, diamide, BCNU -- all caused a dramatic fall in the GSH/GSSG ratio. These redox alterations were found to inhibit TNF-induced IkappaB-alpha phosphorylation and NF-kappaB translocation to the nucleus, thus presumably inhibiting expression of genes necessary to inhibit the cytotoxic effects of TNF. Taken together, these results suggest that oxidation of the intracellular environment of hepatocytes by reactive oxygen species or redox-modulating agents interferes with NF-kappaB signaling pathways to sensitize hepatocytes to TNF-induced apoptosis. The TNF-induced apoptosis seems to occur only in a certain redox range -- in which redox changes can inhibit NF-kappaB activity but not completely inhibit caspase activity. The implication for liver disease is that concomitant TNF exposure and reactive oxygen species, either extrinsically generated (e.g., nonparenchymal or inflammatory cells) or intrinsically generated in hepatocytes (e.g., mitochondria), may act in concert to promote apoptosis and liver injury.     

A dominant role of Toll-like receptor 4 in the signaling of apoptosis in bacteria-faced macrophages

Conserved bacterial components potently activate host immune cells through transmembrane Toll-like receptors (TLRs), which trigger a protective immune response but also may signal apoptosis. In this study, we investigated the roles of TLR2 and TLR4 as inducers of apoptosis in Yersinia enterocolitica-infected macrophages. Yersiniae suppress activation of the antiapoptotic NF-kappaB signaling pathway in host cells by inhibiting inhibitory kappaB kinase-beta. This leads to macrophage apoptosis under infection conditions. Experiments with mouse macrophages deficient for TLR2, TLR4, or both receptors showed that, although yersiniae could activate signaling through both TLR2 and TLR4, loss of TLR4 solely diminished Yersinia-induced apoptosis. This suggests implication of TLR4, but not of TLR2, as a proapoptotic signal transducer in Yersinia-conferred cell death. In the same manner, agonist-specific activation of TLR4 efficiently mediated macrophage apoptosis in the presence of the proteasome inhibitor MG-132, an effect that was less pronounced for activation through TLR2. Furthermore, the extended stimulation of overexpressed TLR4 elicited cellular death in epithelial cells. A dominant-negative mutant of Fas-associated death domain protein could suppress TLR4-mediated cell death, which indicates that TLR4 may signal apoptosis through a Fas-associated death domain protein-dependent pathway. Together, these data show that TLR4 could act as a potent inducer of apoptosis in macrophages that encounter a bacterial pathogen.     

Vertebrate cell death in energy-limited conditions and how to avoid it: what we might learn from mammalian hibernators and other stress-tolerant vertebrates

Dormancy in vertebrates may expose cells to acidosis, hypoxia/anoxia, oxidative damage, and extremes in temperature. All of these insults are known to be pro-apoptotic in typical vertebrate cells, especially mammals. Since dormancy is presumably the result of a need for energy conservation, the inherent energetic demand of replenishing cells that underwent apoptosis seems at odds with this strategy. This review will discuss processes to mitigate apoptosis and how these processes might be regulated in stress-tolerant vertebrates such as mammalian hibernators. As data directly addressing such issues are scarce and often conflicting, an apparently complex regulation of apoptosis seems to be at work. For example, apoptosis is mitigated during dormancy, key signaling events including the activation of caspase-3 may still occur. However, both passive, temperature-induced depression of apoptotic signaling as well as active suppression of apoptosis appear to work in synergy in these systems. In many instances cell death is prevented by simply avoiding the cellular triggers (e.g. leakage of proteins from the mitochondria or increases in intracellular calcium) that initiate apoptotic signaling. In this review we discuss what is known about programmed cell death in these under-studied models and highlight features of their physiology that likely support survival in the face of conditions that would induce cell death in typical vertebrate cells.     

Lipid raft connection between extrinsic and intrinsic apoptotic pathways

Apoptosis in mammalian cells is modulated by extrinsic and intrinsic signaling pathways through the formation of death receptor-mediated death-inducing signaling complex (DISC) and mitochondrial-derived apoptosome, respectively. We found by ultrastructural approaches that the antitumor drug edelfosine induced aggregates of lipid rafts containing Fas/CD95 receptor and Fas-associated death domain-containing protein in leukemic cells. Death receptors together with DISC and apoptosome constituents were recruited in rafts during edelfosine treatment in multiple myeloma cells. This apoptotic response involved caspases-8/-9/-10 that were translocated to rafts. Lipid raft disruption by cholesterol depletion inhibited loss of mitochondrial transmembrane potential, caspase activation and apoptosis, whereas cholesterol replenishment restored these responses. Our data indicate that rafts act as scaffolds where extrinsic and intrinsic apoptotic signaling pathways concentrate, forming clusters of apoptotic signaling molecule-enriched rafts (CASMER), which function as novel supramolecular entities in the triggering of apoptosis, and play an important role in edelfosine-induced apoptosis in blood cancer cells.     

Phosphatidylserine peroxidation/externalization during staurosporine-induced apoptosis in HL-60 cells

Although oxidative stress is commonly associated with apoptosis, its specific role in the execution of the apoptotic program has yet to be described. We hypothesized that catalytic redox interactions between negatively charged phosphatidylserine (PS) and positively charged cytochrome c released into the cytosol, along with the production of reactive oxygen species (ROS), results in pronounced oxidation and externalization of PS, and subsequent recognition of apoptotic cells by macrophages. By using staurosporine, a protein kinase inhibitor that does not act as a prooxidant, we were able to induce apoptosis in HL-60 cells without triggering the confounding effects of non-specific oxidation reactions. Through this approach, we demonstrated for the first time that PS underwent a statistically significant and pronounced oxidation at an early stage (2 h) of non-oxidant-induced apoptosis while the most abundant phospholipid, phosphatidylcholine, did not. Glutathione (GSH), the most abundant cytosolic thiol, also remained unoxidized at this time point. Furthermore, PS oxidation and the appearance of cytochrome c in the cytosol were concurrent; PS externalization was followed by phagocytosis of apoptotic cells. These findings are compatible with our proposed roles for oxidative PS-dependent signaling during apoptosis and phagocytosis.     

Low doses of reactive oxygen species protect endothelial cells from apoptosis by increasing thioredoxin-1 expression

The redox regulator thioredoxin-1 (Trx-1) is required for the redox potential of the cell and exerts important functions in cell growth and apoptosis. Severe oxidative stress has been implicated in the oxidation of proteins and cell death. However, the role of low doses of reactive oxygen species (ROS) is poorly understood. Here, we show that 10 and 50 microM H2O2 and short-term exposure to shear stress significantly increased Trx-1 mRNA and protein levels in endothelial cells. Since it is known that Trx-1 exerts anti-apoptotic functions, we next investigated whether low doses of ROS can inhibit basal and serum-depletion induced endothelial cell apoptosis. Indeed, treatment of endothelial cells with 10 and 50 microM H2O2 significantly reduced apoptosis induction. Reduction of Trx-1 expression using an antisense oligonucleotide approach resulted in the induction of apoptosis and abolished the inhibitory effect of low doses of H2O2. Taken together, our results demonstrate that low doses of ROS act as signaling molecules and exert anti-apoptotic functions in endothelial cells via upregulation of the redox-regulator Trx-1.     

Cytochrome c-mediated apoptosis in cells lacking mitochondrial DNA. Signaling pathway involving release and caspase 3 activation is conserved.

Mitochondria serve as a pivotal component of the apoptotic cell death machinery. However, cells that lack mitochondrial DNA (rho(0) cells) retain apparently normal apoptotic signaling. In the present study, we examined mitochondrial mechanisms of apoptosis in rho(0) osteosarcoma cells treated with staurosporine. Immunohistochemistry revealed that rho(0) cells maintained a normal cytochrome c distribution in mitochondria even though these cells were deficient in respiration. Upon staurosporine treatment, cytochrome c was released concomitantly with activation of caspase 3 and loss of mitochondrial membrane potential (Deltapsi(m)). After mitochondrial loss of cytochrome c, rho(0) cells underwent little change in glutathione (GSH) redox potential whereas a dramatic oxidation in GSH/glutathione disulfide (GSSG) pool occurred in parental rho(+) cells. These results show that mitochondrial signaling of apoptosis via cytochrome c release was preserved in cells lacking mtDNA. However, intracellular oxidation that normally accompanies apoptosis was lost, indicating that the mitochondrial respiratory chain provides the major source of redox signaling in apoptosis.