Vitamin E inhibits anti-Fas-induced phosphatidylserine oxidation but does not affect its externalization during apoptosis in Jurkat T cells and their phagocytosis by J774A.1 macrophages.

Apoptosis and phagocytosis of apoptotic cells provide for effective and harmless clearance of unwanted or damaged cells in the body. Preferential oxidation of one particular class of phospholipids, phosphatidylserine (PS), is a typical trait of both oxidant- and nonoxidant-induced apoptosis. PS oxidation is likely to play an important role in phagocytosis either by affecting PS externalization acting as an "eat me" signal or by more effective recognition of apoptotic cells by macrophage receptors. This implies that antioxidants effective in inhibiting PS oxidation may affect PS externalization and/or effective removal of apoptotic cells. Therefore, it is essential to determine whether vitamin E, the major lipid-soluble antioxidant of membranes, inhibits PS oxidation, and hence blocks apoptosis/phagocytosis. To test this, we studied the effects of vitamin E on PS oxidation and signaling using a model of anti-Fas-triggered apoptosis in Jurkat T cells. We found that incubation of cells with vitamin E (0.25-50 micro M) resulted in its integration into cells to reach physiologically relevant concentrations. Using labeling of cell phospholipids with oxidation-sensitive and fluorescent cis-parinaric acid (PnA), we found that anti-Fas exposure caused significant and selective oxidation of PnA-PS in Jurkat T cells (22 +/- 2.1% of its content in nonexposed cells). Vitamin E protected PnA-PS against oxidation in a concentration-dependent way such that at 25 micro M and 50 micro M, a complete inhibition of anti-Fas-induced PS oxidation was achieved. At all concentrations used, vitamin E had no effect on either biomarkers of anti-Fas-induced apoptosis (PS externalization, nuclear fragmentation) or phagocytosis of anti-Fas-induced apoptotic cells by J774A.1 macrophages. We conclude that vitamin E does not significantly interfere with extrinsic (death receptor-triggered) pathways of apoptosis and does not affect phagocytosis of anti-Fas-triggered apoptotic cells.

Cytochrome c release is required for phosphatidylserine peroxidation during Fas-triggered apoptosis in lung epithelial A549 cells.

Oxidation of phosphatidylserine (PtdSer) has been shown to play a pivotal role in signaling during cell apoptosis and subsequent recognition of apoptotic cells by phagocytes. However, the redox catalytic mechanisms involved in selective PtdSer oxidation during apoptosis remain poorly understood. Here we employed anti-Fas antibody CH-11-treated A549 cells as a physiologically relevant model to investigate the involvement of PtdSer oxidation and its potential mechanism during apoptosis. We demonstrated that ligation of CH-11 with its cognate receptor initiated execution of apoptotic program in interferon gamma-pretreated A549 cells as evidenced by activation of caspase and DNA fragmentation. A significant increase of cytochrome c (cyt c) content in the cytosol as early as 2 h after CH-11 exposure was detected indicating that Fas-induced apoptosis in A549 cells proceeds via extrinsic type II pathway and includes mitochondrial signaling. PtdSer was selectively oxidized 3 h after anti-Fas triggering while two more abundant phospholipids--phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn)--and the major intracellular antioxidant, glutathione, remained nonoxidized. A pan-caspase inhibitor, z-VAD, fully blocked cyt c release and oxidation of PtdSer in Fas-treated A549 cells. On the other hand, z-DQMD, a caspase-3 inhibitor, completely inhibited caspase-3 activity but did not fully block caspase-8 activation and release of cyt c. Importantly, z-DQMD failed to protect PtdSer from oxidation. In addition, in a model system, we demonstrated that peroxidase activity of cyt c was greatly enhanced in the presence of dioleoylphosphatidylserine containing liposomes by monitoring oxidation of 2',7'-dichlorodihydrofluorescein to 2',7'-dichlorofluorescein. We further showed that peroxidase activity of cyt c catalyzed oxidation of 1-palmitoyl-2-arachidonoyl-3-glycero-phosphoserine using a newly developed HPLC assay. MS analysis of 1-palmitoyl-2-arachidonoyl-3-glycero-phosphoserine revealed that in addition to its mono- and dihydroperoxides, several different PtdSer oxidation products can be formed. Overall, we concluded that cyt c acts as a catalyst of PtdSer oxidation during Fas-triggered A549 cell apoptosis.

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.

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.

Toward mechanism-based antioxidant interventions: lessons from natural antioxidants.

It is generally accepted that one of the major and important contributions to skin aging, skin disorders, and skin diseases results from reactive oxygen species. More than other tissues, the skin is exposed to numerous environmental chemical and physical agents, such as ultraviolet light, causing oxidative stress. Accelerated cutaneous UV-induced aging, photo aging, is only one of the harmful effects of continual oxygen radical production in the skin. Interestingly, our ELISA assays of 8-oxo-2'-deoxyguanosine in skin of young and old Balb/c mice showed that cumene hydroperoxide-induced accumulation of the biomarker of oxidative DNA damage in skin of 32-week-old mice occurred independently of their vitamin E status, while no accumulation of oxo8-dG was detectable in the skin of young animals. This suggests that vitamin E is not the major protector of skin against cumene hydroperoxide-induced oxidative stress. Production and accumulation of apoptotic cells is one of the characteristic features of skin damage by oxidative stress that, in the absence of effective scavenging by macrophages, dramatically enhances oxidative damage and inflammatory response. In our model experiments, we demonstrated that Cu-OOH induces significant oxidative stress in phospholipids of normal human epidermal keratinocytes (NHEK) whose characteristic feature is an early and profound oxidation of phosphatidylserine (PS), likely related to PS externalization. Since externalized PS is a signal for recognition of apoptotic cells by macrophage scavenger receptors, PS oxidation may be translatable into elimination of thus damaged NHEKs. Experiments are now underway to determine whether inhibition of PS oxidation by antioxidants may interfere with important signaling functions of oxidative stress in eliminating apoptotic cells.