Peroxidation and externalization of phosphatidylserine associated with release of cytochrome c from mitochondria

Production of reactive oxygen species (ROS) during apoptosis is associated with peroxidation of phospholipids particularly of phosphatidylserine (PS). The mechanism(s) underlying preferential PS oxidation are not well understood. We hypothesized that cytochrome c (cyt c) released from mitochondria into cytosol acts as a catalyst that utilizes ROS generated by disrupted mitochondrial electron transport for PS oxidation. Selectivity of PS oxidation is achieved via specific interactions of positively charged cyt c with negatively charged PS. To test the hypothesis we employed temporary transfection of Jurkat cells with a pro-apoptotic peptide, DP1, a conjugate consisting of a protein transduction domain, PTD-5, and an antimicrobial domain, KLA [(KLAKLAK)2], known to selectively disrupt mitochondria. We report that treatment of Jurkat cells with DP1 yielded rapid and effective release of cyt c from mitochondria and its accumulation in cytosol accompanied by production of H2O2. Remarkably, this resulted in selective peroxidation of PS while more abundant phospholipids such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE) remained nonoxidized. Neither PTD-5 alone nor KLA alone exerted any effect on PS peroxidation. Redox catalytic involvement of cyt c in PS oxidation was further supported by our data demonstrating that: (i) specific interactions of cyt c with PS resulted in the formation of EPR-detectable protein-centered tyrosyl radicals of cyt c upon its interaction with H2O2 in the presence of PS-containing liposomes, and (ii) integration of cyt c into cytochrome c null (Cyt c -/-) cells or HL-60 cells specifically stimulates PS oxidation in the presence of H2O2 or t-BuOOH, respectively. We further demonstrated that DP1 elicited externalization of PS on the surface of Jurkat cells and enhanced their recognition and phagocytosis by J774A.1 macrophages. Our results are compatible with the hypothesis that catalysis of selective PS oxidation during apoptosis by cytosolic cyt c is important for PS-dependent signaling pathways such as PS externalization and recognition by macrophage receptors.     

Lipid antioxidant, etoposide, inhibits phosphatidylserine externalization and macrophage clearance of apoptotic cells by preventing phosphatidylserine oxidation

Apoptosis is associated with the externalization of phosphatidylserine (PS) in the plasma membrane and subsequent recognition of PS by specific macrophage receptors. Selective oxidation of PS precedes its externalization/recognition and is essential for the PS-dependent engulfment of apoptotic cells. Because etoposide is a potent and selective lipid antioxidant that does not block thiol oxidation, we hypothesized that it may affect PS externalization/recognition without affecting other features of the apoptotic program. We demonstrate herein that etoposide induced apoptosis in HL-60 cells without the concomitant peroxidation of PS and other phospholipids. HL-60 cells also failed to externalize PS in response to etoposide treatment. In contrast, oxidant (H2O2)-induced apoptosis was accompanied by PS externalization and oxidation of different phospholipids, including PS. Etoposide potentiated H2O2-induced apoptosis but completely blocked H2O2-induced PS oxidation. Etoposide also inhibited PS externalization as well as phagocytosis of apoptotic cells by J774A.1 macrophages. Integration of exogenous PS or a mixture of PS with oxidized PS in etoposide-treated HL-60 cells reconstituted the recognition of these cells by macrophages. The current data demonstrate that lipid antioxidants, capable of preventing PS peroxidation, can block PS externalization and phagocytosis of apoptotic cells by macrophages and hence dissociate PS-dependent signaling from the final common pathway for apoptosis.     

Apoptotic interactions of cytochrome c: redox flirting with anionic phospholipids within and outside of mitochondria

Since the (re)discovery of cytochrome c (cyt c) in the early 1920s and subsequent detailed characterization of its structure and function in mitochondrial electron transport, it took over 70 years to realize that cyt c plays a different, not less universal role in programmed cell death, apoptosis, by interacting with several proteins and forming apoptosomes. Recently, two additional essential functions of cyt c in apoptosis have been discovered that are carried out via its interactions with anionic phospholipids: a mitochondria specific phospholipid, cardiolipin (CL), and plasma membrane phosphatidylserine (PS). Execution of apoptotic program in cells is accompanied by substantial and early mitochondrial production of reactive oxygen species (ROS). Because antioxidant enhancements protect cells against apoptosis, ROS production was viewed not as a meaningless side effect of mitochondrial disintegration but rather playing some - as yet unidentified - role in apoptosis. This conundrum has been resolved by establishing that mitochondria contain a pool of cyt c, which interacts with CL and acts as a CL oxygenase. The oxygenase is activated during apoptosis, utilizes generated ROS and causes selective oxidation of CL. The oxidized CL is required for the release of pro-apoptotic factors from mitochondria into the cytosol. This redox mechanism of cyt c is realized earlier than its other well-recognized functions in the formation of apoptosomes and caspase activation. In the cytosol, released cyt c interacts with another anionic phospholipid, PS, and catalyzes its oxidation in a similar oxygenase reaction. Peroxidized PS facilitates its externalization essential for the recognition and clearance of apoptotic cells by macrophages. Redox catalysis of plasma membrane PS oxidation constitutes an important redox-dependent function of cyt c in apoptosis and phagocytosis. Thus, cyt c acts as an anionic phospholipid specific oxygenase activated and required for the execution of essential stages of apoptosis. This review is focused on newly discovered redox mechanisms of complexes of cyt c with anionic phospholipids and their role in apoptotic pathways in health and disease.     

Externalization of Phosphatidylserine May Not Be an Early Signal of Apoptosis in Neuronal Cells, but Only the Phosphatidylserine-Displaying Apoptotic Cells Are Phagocytosed by Microglia

Abstract: Earlier reports on nonneural cells have shown that the normally inner plasma membrane lipid, phosphatidylserine (PS), flip-flops out during the early stages of apoptosis, whereas DNA laddering and plasma membrane permeabilization occur during the late stages. In this study, the applicability of these parameters to CNS-derived neuronal cells was tested using hippocampal HN2-5, cells that undergo apoptosis under anoxia. Because such insults on unsynchronized cells, e.g., undifferentiated HN2-5 cells, result in both early and late apoptotic cells, we mechanically separated these cells into three fractions containing (a) cells that had completely detached during anoxia, (b) cells that remained weakly attached to the tissue culture dish and, once detached by trituration in serum-containing medium, did not reattach, and (c) cells that reattached in 2–3 h. Fractions a and b contained cells that showed pronounced DNA laddering, whereas cells in fraction c did not show any DNA laddering. Double staining with fluorescein isothiocyanate-annexin V (which binds to PS) and propidium iodide (which stains the DNA in cells with a permeable cell membrane) revealed that all cells in fraction a had a permeable cell membrane (propidium iodide-positive) and PS molecules in the outer leaflet of the plasma membrane (fluorescein isothiocyanate-annexin V-positive). By contrast, fractions b and c contained cells with no externalized PS molecules. Cells in fractions a–c also showed, respectively, 50-, 21-, and 5.5-fold higher caspase-3 (CPP32) activity than that in healthy control cells. All these results show that fraction a contained late apoptotic cells, which also had the highest CPP32 activity; cells in fraction b were at an intermediate stage, when DNA laddering had already occurred; and fraction c contained very early apoptotic cells, in which no DNA laddering had yet occurred. Therefore, in the neuronal HN2-5 cells, externalization of PS occurs only during the final stages of apoptosis when the cells have completely lost their adhesion properties. Further experiments showed that ameboid microglial cells isolated from neonatal mouse brain phagocytosed only the cells in fraction a. These results show that in CNS-derived HN2-5 cells, (a) PS externalization is a late apoptotic event and is concomitant with a complete loss of surface adhesion of the apoptotic cells and (b) PS externalization is crucial for microglial recognition and phagocytosis of the apoptotic HN2-5 cells. Thus, PS externalization could be causally linked to the final detachment of apoptotic neuronal cells, which in turn prepares them for rapid phagocytosis by microglia.     

Oxidative stress inhibits the phagocytosis of apoptotic cells that have externalized phosphatidylserine

The efficient phagocytosis of apoptotic cells by macrophages reduces the potential for an inflammatory response by ensuring that the dying cells are cleared before their intracellular contents are released. Early apoptotic cells are targeted for phagocytosis through the translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane. In this report, we show that the oxidant H(2)O(2) inhibits phagocytosis of apoptotic cells even though the cells express functional PS on their surface. Thus, B lymphoma cells induced to undergo apoptosis by the chemotherapy drug etoposide are efficiently phagocytosed by macrophages in a process that is mediated by PS (inhibitable by PS liposomes). Exposure of the apoptotic cells to H(2)O(2) inhibits phagocytosis even though the cells still express functional PS on their surface. In addition, Jurkat cells and thymocytes induced to undergo apoptosis by H(2)O(2) alone are poorly phagocytosed. Inhibition of phagocytosis by H(2)O(2) cannot be attributed to oxidative inactivation or redistribution of PS on the cell surface. The results indicate that PS externalization is necessary but is not sufficient to target apoptotic cells for phagocytosis. Another phagocytosis recognition factor must therefore exist to facilitate uptake of apoptotic cells, and this factor is sensitive to modification by H(2)O(2).     

Nitrosative stress inhibits the aminophospholipid translocase resulting in phosphatidylserine externalization and macrophage engulfment: implications for the resolution of inflammation

Macrophage recognition of apoptotic cells depends on externalization of phosphatidylserine (PS), which is normally maintained within the cytosolic leaflet of the plasma membrane by aminophospholipid translocase (APLT). APLT is sensitive to redox modifications of its -SH groups. Because activated macrophages produce reactive oxygen and nitrogen species, we hypothesized that macrophages can directly participate in apoptotic cell clearance by S-nitrosylation/oxidation and inhibition of APLT causing PS externalization. Here we report that exposure of target HL-60 cells to nitrosative stress inhibited APLT, induced PS externalization, and enhanced recognition and elimination of "nitrosatively" modified cells by RAW 264.7 macrophages. Using S-nitroso-L-cysteine-ethyl ester (SNCEE) and S-nitrosoglutathione (GSNO) that cause intracellular and extracellular trans-nitrosylation of proteins, respectively, we found that SNCEE (but not GSNO) caused significant S-nitrosylation/oxidation of thiols in HL-60 cells. SNCEE also strongly inhibited APLT, activated scramblase, and caused PS externalization. However, SNCEE did not induce caspase activation or nuclear condensation/fragmentation suggesting that PS externalization was dissociated from the common apoptotic pathway. Dithiothreitol reversed SNCEE-induced S-nitrosylation, APLT inhibition, and PS externalization. SNCEE but not GSNO stimulated phagocytosis of HL-60 cells. Moreover, phagocytosis of target cells by lipopolysaccharide-stimulated macrophages was significantly suppressed by an NO. scavenger, DAF-2. Thus, macrophage-induced nitrosylation/oxidation plays an important role in cell clearance, and hence in the resolution of inflammation.     

Oxidative signaling pathway for externalization of plasma membrane phosphatidylserine during apoptosis

Active maintenance of membrane phospholipid asymmetry is universal in normal cell membranes and its disruption with subsequent externalization of phosphatidylserine is a hallmark of apoptosis. Externalized phosphatidylserine appears to serve as an important signal for targeting recognition and elimination of apoptotic cells by macrophages, however, the molecular mechanisms responsible for phosphatidylserine translocation during apoptosis remain unresolved. Studies have focused on the function of aminophospholipid translocase and phospholipid scramblase as mediators of this process. Here we present evidence that unique oxidative events, represented by selective oxidation of phosphatidylserine, occur during apoptosis that could promote phosphatidylserine externalization. We speculate that selective phosphatidylserine oxidation could affect phosphatidylserine recognition by aminophospholipid translocase and/or directly result in enzyme inhibition. The potential interactions between the anionic phospholipid phosphatidylserine and the redox-active cationic protein effector of apoptosis, cytochrome c, are presented as a potential mechanism to account for selective oxidation of phosphatidylserine during apoptosis. Thus, cytochrome c-mediated phosphatidylserine oxidation may represent an important component of the apoptotic pathway.     

Telomerase activity and hepatic functions of rat embryonic liver progenitor cell in nanoscaffold-coated model bioreactor

Phosphatidylserine (PS) externalization is a key feature of apoptotic cell death and plays an important role in clearance of apoptotic cells by phagocytes. PS externalization during apoptosis is generally an irreversible event mediated by caspase activation and is accompanied by other apoptotic events. We report here that an apoptosis inducer α-tocopheryl succinate (TOS) can induce PS externalization that is independent of apoptosis and reversible in the absence of fetal bovine serum (FBS) in histiocytic lymphoma U937 cells. In the presence of FBS, TOS induced PS externalization via a caspase-dependent mechanism accompanied by mitochondrial depolarization, cell shrinkage, increase of caspase-3 activity, and chromatin condensation. In contrast, in the absence of FBS, TOS induced the rapid PS externalization which was not accompanied by other apoptotic events. The PS externalization was reversible by removing TOS and was not involved in Ca²⁺-dependent scramblase activation and thiol oxidation of aminophospholipid translocase. A similar PS externalization was also induced by cholesteryl hemisuccinate (CS), the other succinate ester. These results suggested that the mechanism of TOS- and CS-induced PS externalization in the absence of FBS was different from it occurring during typical apoptosis.     

Increased mitochondrial cytochrome c levels and mitochondrial hyperpolarization precede camptothecin-induced apoptosis in Jurkat cells

Mitochondria play a central role in apoptosis through release of cytochrome c and activation of caspases. In the present study, we showed that, in Jurkat human T cells, camptothecin-induced apoptosis is preceded by (i) an increase in cytochrome c and subunit IV of cytochrome c oxidase (COX IV) levels in mitochondria; and (ii) an elevation of the mitochondrial membrane potential (Delta(Psi)m). These events are followed by cytochrome c release into the cytosol, cytochrome c and COX IV depletion from mitochondria, externalization of phosphatidylserine (PS), disruption of Delta(Psi)m, caspase activation, poly(ADP-ribose)polymerase cleavage and DNA fragmentation. The pan-caspase inhibitor z-VAD.fmk blocked camptothecin-induced PS externalization, disruption of Delta(Psi)m and DNA fragmentation, suggesting that these events are mediated by caspase activation. In contrast, z-VAD did not prevent cytochrome c release, despite preventing cytochrome c and COX IV depletion from mitochondria. Together, these data suggest that mitochondrial cytochrome c and COX IV enrichment are early events preceding the onset of apoptosis and that cytochrome c release is upstream of caspase activation and loss of Delta(Psi)m. Furthermore, prevention by z-VAD of cytochrome c and COX IV depletion in mitochondria suggests the possibility that a caspase-like activity in mitochondria is involved in the proteolytic depletion of respiratory chain proteins. Activation of this activity may play an important role in drug-induced apoptosis.     

Phosphatidylserine exposure during apoptosis is a cell-type-specific event and does not correlate with plasma membrane phospholipid scramblase expression

Phosphatidylserine (PS) exposure on the surface of cells has been considered a characteristic feature of apoptosis. However, we demonstrate herein that externalization of PS occurs in a cell-type-specific, albeit caspase-dependent, manner. Moreover, we could find no correlation in six different cell lines between the level of expression of the phospholipid (PL) scramblase and the capacity of these cells to externalize PS during apoptosis. Overexpression of PL scramblase in Raji cells, which exhibit low constitutive expression of this enzyme, by retroviral transduction of PL scramblase or treatment of the cells with interferon-alpha, failed to confer the capacity to expose PS in response to apoptotic stimuli. However, the lack of PS exposure in some cell types was not due to their inability to translocate PS molecules to the cell surface, since incubation with thiol reactive agents, such as N-ethylmaleimide, disulfiram and diamide, yielded rapid and pronounced PS exposure in all cell lines. These data suggest that plasma membrane PS exposure is not an obligatory component of the apoptotic phenotype, and that PL scramblase is not the sole determinant of PS externalization in apoptotic cells when this occurs.     

Oxidation of phosphatidylserine: a mechanism for plasma membrane phospholipid scrambling during apoptosis?

Selective oxidation of phosphatidylserine (PS) during apoptosis precedes its externalization in plasma membrane and is essential for the engulfment of apoptotic cells. To experimentally test whether PS oxidation stimulates its externalization via its effects on aminophospholipid translocase (APT) or by enhanced PS scrambling, action of oxidized PS (PSox) was studied using leukemia HL-60 cells and lymphoma Raji cells. Both PS and PSox were equally well recognized by APT. PSox did not inhibit APT. Rate of transmembrane PS diffusion was fourfold higher in cells with integrated PSox than with PS. Thus, PSox acts as a "non-enzymatic scramblase" likely contributing to PS externalization.     

Macrophage recognition of externalized phosphatidylserine and phagocytosis of apoptotic Jurkat cells--existence of a threshold

Phosphatidylserine (PS) is predominantly confined to the inner leaflet of plasma membrane in cells, but it is externalized on the cell surface during apoptosis. This externalized PS is required for effective phagocytosis of apoptotic cells by macrophages. Because PS trans-bilayer asymmetry is not absolute in different types of nonapoptotic cells, we hypothesized that the amounts of externalized PS may be critical for macrophage discrimination between apoptotic and nonapoptotic cells. We developed a sensitive electron paramagnetic resonance method to quantify the amounts of externalized PS based on specific binding of paramagnetic annexin V-microbead conjugates with PS on cell surfaces. Using this technique, we found that nonapoptotic Jurkat cells externalize 0.9 pmol of endogenous PS/10(6) Jurkat cells. For cells with different amounts of integrated exogenous PS on their surface, no phagocytic response was observed at PS levels <5 pmol/10(6) Jurkat cells; at higher PS concentrations, phagocytosis increased in a concentration-dependent manner. Apoptosis in Jurkat cells caused externalization of approximately 240 pmol PS/10(6) Jurkat cells; these amounts of externalized PS are manyfold higher than the threshold amounts of PS required for phagocytosis. Thus, macrophages have a sensitivity threshold for PS externalized on the cell surface that provides for reliable recognition and distinction between normal cells with low contents of externalized PS and apoptotic cells with remarkably elevated PS levels.     

Oxidative lipidomics of apoptosis: redox catalytic interactions of cytochrome c with cardiolipin and phosphatidylserine

The primary life-supporting function of cytochrome c (cyt c) is control of cellular energetic metabolism as a mobile shuttle in the electron transport chain of mitochondria. Recently, cyt c's equally important life-terminating function as a trigger and regulator of apoptosis was identified. This dreadful role is realized through the relocalization of mitochondrial cyt c to the cytoplasm where it interacts with Apaf-1 in forming apoptosomes and mediating caspase-9 activation. Although the presence of heme moiety of cyt c is essential for the latter function, cyt c's redox catalytic features are not required. Lately, two other essential functions of cyt c in apoptosis, that may rely heavily on its redox activity have been suggested. Both functions are directed toward oxidation of two negatively charged phospholipids, cardiolipin (CL) in the mitochondria and phosphatidylserine (PS) in the plasma membrane. In both cases, oxidized phospholipids seem to be essential for the transduction of two distinctive apoptotic signals: one is participation of oxidized CL in the formation of the mitochondrial permeability transition pore that facilitates release of cyt c into the cytosol and the other is the contribution of oxidized PS to the externalization and recognition of PS (and possibly oxidized PS) on the cell surface by specialized receptors of phagocytes. In this review, we present a new concept that cyt c actuates both of these oxidative roles through a uniform mechanism: its specific interactions with each of these phospholipids result in the conversion and activation of cyt c, transforming it from an innocuous electron transporter into a calamitous peroxidase capable of oxidizing the activating phospholipids. We also show that this new concept is compatible with a leading role for reactive oxygen species in the execution of the apoptotic program, with cyt c as the main executioner.     

Oxidatively modified phosphatidylserines on the surface of apoptotic cells are essential phagocytic ‘eat-me' signals: cleavage and inhibition of phagocytosis by Lp-PLA2

Diversified anionic phospholipids, phosphatidylserines (PS), externalized to the surface of apoptotic cells are universal phagocytic signals. However, the role of major PS metabolites, such as peroxidized species of PS (PSox) and lyso-PS, in the clearance of apoptotic cells has not been rigorously evaluated. Here, we demonstrate that H₂O₂ was equally effective in inducing apoptosis and externalization of PS in naive HL60 cells and in cells enriched with oxidizable polyunsaturated species of PS (supplemented with linoleic acid (LA)). Despite this, the uptake of LA-supplemented cells by RAW264.7 and THP-1 macrophages was more than an order of magnitude more effective than that of naive cells. A similar stimulation of phagocytosis was observed with LA-enriched HL60 cells and Jurkat cells triggered to apoptosis with staurosporine. This was due to the presence of PSox on the surface of apoptotic LA-supplemented cells (but not of naive cells). This enhanced phagocytosis was dependent on activation of the intrinsic apoptotic pathway, as no stimulation of phagocytosis occurred in LA-enriched cells challenged with Fas antibody. Incubation of apoptotic cells with lipoprotein-associated phospholipase A₂ (Lp-PLA₂), a secreted enzyme with high specificity towards PSox, hydrolyzed peroxidized PS species in LA-supplemented cells resulting in the suppression of phagocytosis to the levels observed for naive cells. This suppression of phagocytosis by Lp-PLA₂ was blocked by a selective inhibitor of Lp-PLA₂, SB-435495. Screening of possible receptor candidates revealed the ability of several PS receptors and bridging proteins to recognize both PS and PSox, albeit with diverse selectivity. We conclude that PSox is an effective phagocytic ‘eat-me'' signal that participates in the engulfment of cells undergoing intrinsic apoptosis.     

Fas-induced B cell apoptosis requires an increase in free cytosolic magnesium as an early event

Ligation of the Fas molecule expressed on the surface of a cell initiates multiple signaling pathways that result in the apoptotic death of that cell. We have examined Mg2+ mobilization as well as Ca2+ mobilization in B cells undergoing Fas-initiated apoptosis. Our results indicate that cytosolic levels of free (non-complexed) Mg2+ ([Mg2+]i) and Ca2+ ([Ca2+]i) increase in cells undergoing apoptosis. Furthermore, the percentages of cells mobilizing Mg2+, fragmenting DNA, or externalizing phosphatidylserine (PS) increase in parallel as the concentration of anti-Fas monoclonal antibody is raised. Kinetic analysis suggests that Mg2+ mobilization is an early event in apoptosis, clearly preceding DNA fragmentation and probably occurring prior to externalization of PS as well. The source of Mg2+ that produces the increases in [Mg2+]i is intracellular and most likely is the mitochondria. Extended pretreatment of B cells with carbonyl cyanide m-chlorophenylhydrazone, an inhibitor of mitochondrial oxidative phosphorylation, produces proportional decreases in the percentage of cells mobilizing Mg2+, fragmenting DNA, and externalizing PS in response to anti-Fas monoclonal antibody treatment. These observations are consistent with the hypothesis that elevated [Mg2+]i is required for apoptosis. Furthermore, we propose that the increases in [Mg2+]i function not only as cofactors for Mg2+-dependent endonucleases, but also to facilitate the release of cytochrome c from the mitochondria, which drives many of the post-mitochondrial, caspase-mediated events in apoptotic cells.     

Cellular apoptosis is associated with increased caveolin-1 expression in macrophages

Macrophage apoptosis is an important factor in determining the efficiency of the immune response, atherosclerotic lesion stability, and clearance of aged cells by phagocytosis. The involvement of caveolin-1 in the regulation of apoptosis has been previously suggested in fibroblasts and epithelial cells. Here we show that treatment of thioglycollate-elicited mouse peritoneal macrophages with various unrelated apoptotic agents, including simvastatin, camptothecin, or glucose deprivation, is associated with a specific and large increase in caveolin-1 expression. In contrast, caveolin-2 levels remain unaffected. Induction of apoptosis was measured by changes in cell morphology, annexin V-labeling, and DNA fragmentation. We demonstrate that caveolin-1 in macrophages is present in lipid rafts and colocalizes with phosphatidylserine (PS) at the cell surface of apoptotic macrophages. Our data suggest that caveolin-1 increase is an early event, closely accompanied by PS externalization and independent of caspase activation and nuclear DNA fragmentation. The increase in caveolin-1 levels does not require new protein synthesis, as cycloheximide does not prevent the apoptosis-mediated increase in caveolin-1 levels. We propose that increased levels of caveolin-1 characterize the apoptotic phenotype of macrophages. Caveolin-1 may be involved in the efficient externalization of PS at the surface of the apoptotic cells.     

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.     

Cytochrome c is transformed from anti- to pro-oxidant when interacting with truncated oncoprotein prothymosin alpha

Many apoptotic signals are known to induce release to cytosol of cytochrome c, a small mitochondrial protein with positively charged amino acid residues dominating over negatively charged ones. On the other hand, in this group, it was shown that prothymosin alpha (PT), a small nuclear protein where 53 of 109 amino acid residues are negatively charged, is truncated to form a protein of 99 amino acid residues which accumulates in cytosol during apoptosis [FEBS Lett. 467 (2000) 150]. It was suggested that positively charged cytochrome c and negatively charged truncated prothymosin alpha (tPT), when meeting in cytosol, can interact with each other. In this paper, such an interaction is shown. (1) Formation of cytochrome cz.ccirf;tPT complex is demonstrated by a blot-overlay assay. (2) Analytical centrifugation of solution containing cytochrome c and tPT reveals formation of complexes of molecular masses higher than those of these proteins. The masses increase when the cytochrome c/tPT ratio increases. High concentration of KCl prevents the complex formation. (3) In the complexes formed, cytochrome c becomes autoxidizable; its reduction by superoxide or ascorbate as well as its operation as electron carrier between the outer and inner mitochondrial membranes appear to be inhibited. (4) tPT inhibits cytochrome c oxidation by H(2)O(2), catalyzed by peroxidase. Thus, tPT abolishes all antioxidant functions of cytochrome c which, in the presence of tPT, becomes in fact a pro-oxidant. A possible role of tPT in the development of reactive oxygen species- and cytochrome c-mediated apoptosis is discussed.     

NADPH oxidase-dependent oxidation and externalization of phosphatidylserine during apoptosis in Me2SO-differentiated HL-60 cells. Role in phagocytic clearance

Resolution of inflammation requires clearance of activated neutrophils by phagocytes in a manner that protects adjacent tissues from injury. Mechanisms governing apoptosis and clearance of activated neutrophils from inflamed areas are still poorly understood. We used dimethylsulfoxide-differentiated HL-60 cells showing inducible oxidase activity to study NADPH oxidase-induced apoptosis pathways typical of neutrophils. Activation of the NADPH oxidase by phorbol myristate acetate caused oxidative stress as shown by production of superoxide and hydrogen peroxide, depletion of intracellular glutathione, and peroxidation of all three major classes of membrane phospholipids, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. In addition, phorbol myristate acetate stimulation of the NADPH oxidase caused apoptosis, as evidenced by apoptosis-specific phosphatidylserine externalization, increased caspase-3 activity, chromatin condensation, and nuclear fragmentation. Furthermore, phorbol myristate acetate stimulation of the NADPH oxidase caused recognition and ingestion of dimethylsulfoxide-differentiated HL-60 cells by J774A.1 macrophages. To reveal the apoptosis-related component of oxidative stress in the phorbol myristate acetate-induced response, we pretreated cells with a pancaspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (z-VAD-fmk), and found that it caused partial inhibition of hydrogen peroxide formation as well as selective protection of only phosphatidylserine, whereas more abundant phospholipids, phosphatidylcholine and phosphatidylethanolamine, were oxidized to the same extent in the absence or presence of z-VAD-fmk. In contrast, inhibitors of NADPH oxidase activity, diphenylene iodonium and staurosporine, as well as antioxidant enzymes, superoxide dismutase/catalase, completely protected all phospholipids against peroxidation, inhibited expression of apoptotic biomarkers and externalization of phosphatidylserine, and reduced phagocytosis of differentiated HL-60 cells by J774A.1 macrophages. Similarly, zymosan-induced activation of the NADPH oxidase resulted in the production of superoxide and oxidation of different classes of phospholipids of which only phosphatidylserine was protected by z-VAD-fmk. Accordingly, zymosan caused apoptosis in differentiated HL-60 cells, as evidenced by caspase-3 activation and phosphatidylserine externalization. Finally, zymosan triggered caspase-3 activation and extensive SOD/catalase-inhibitable phosphatidylserine exposure in human neutrophils. Overall, our results indicate that NADPH oxidase-induced oxidative stress in neutrophil-like cells triggers apoptosis and subsequent recognition and removal of these cells through pathways dependent on oxidation and externalization of phosphatidylserine.     

The "pro-apoptotic genies" get out of mitochondria: oxidative lipidomics and redox activity of cytochrome c/cardiolipin complexes

One of the prominent consequences of the symbiogenic origin of eukaryotic cells is the unique presence of one particular class of phospholipids, cardiolipin (CL), in mitochondria. As the product originated from the evolution of symbiotic bacteria, CL is predominantly confined to the inner mitochondrial membrane in normally functioning cells. Recent findings identified CL and its oxidation products as important participants and signaling molecules in the apoptotic cell death program. Early in apoptosis, massive membrane translocations of CL take place resulting in its appearance in the outer mitochondrial membrane. Consequently, significant amounts of CL become available for the interactions with cyt c, one of the major proteins of the intermembrane space. Binding of CL with cytochrome c (cyt c) yields the cyt c/CL complex that acts as a potent CL-specific peroxidase and generates CL hydroperoxides. In this review, we discuss the catalytic mechanisms of CL oxidation by the peroxidase activity of cyt c as well as the role of oxidized CL (CLox) in the release of pro-apoptotic factors from mitochondria into the cytosol. Potential implications of cyt c/CL peroxidase intracellular complexes in disease conditions (cancer, neurodegeneration) are also considered. The discovery of the new role of cyt c/CL complexes in early mitochondrial apoptosis offers interesting opportunities for new targets in drug discovery programs. Finally, exit of cyt c from damaged and/or dying (apoptotic) cells into extracellular compartments and its accumulation in biofluids is discussed in lieu of the formation of its peroxidase complexes with negatively charged lipids and their significance in the development of systemic oxidative stress in circulation.