Characterization of arachidonic acid-induced apoptosis

Tumor necrosis factor (TNF) can induce apoptosis in a number of different cell types. This response often depends on the activity of cytosolic phospholipase A₂ (cPLA₂), which catalyzes the release of arachidonic acid from the sn-2 position of membrane phospholipids. In this study, we investigate the ability of arachidonic acid itself to cause cell death. We show that in assays with 10% fetal bovine serum (FBS) arachidonic acid will not kill, nor does act synergistically with TNF. In contrast, by lowering the concentration of FBS to 2% it is possible to use arachidonic acid to induce cell death. Arachidonic acid-induced cell death was judged to be apoptotic based on morphology and the cleavage of poly (ADP) ribose polymerase. Arachidonic acid was able to kill all cell lines tested including two human melanoma-derived cell lines, and susceptibility to arachidonic acid was not influenced by adenovirus gene products that control susceptibility to TNF. Finally, we show that arachidonic acid is unique among 20 carbon fatty acids for its ability to induce apoptosis and that several other unsaturated, but not saturated fatty acids can also induce apoptosis.     

Adenovirus genes that modulate the sensitivity of virus-infected cells to lysis by TNF

TNF is a key inflammatory cytokine with antiviral properties. Human adenoviruses encode several intracellular proteins that mediate the effects of TNF. Expression of the adenovirus immediate early E1A proteins induces viral genes and a host of cellular genes, drives G₀ cells into S-phase, and induces apoptosis and susceptibility to TNF-induced apoptosis. The adenovirus E1B-19K protein inhibits both E1A- and TNF-induced apoptosis. The E3-14.7K protein and the E3-10.4K/14.5K complex of proteins inhibit TNF- but not E1A-induced apoptosis. The E3 14.7K and 10.4K/14.5K proteins inhibit TNF activation of cytosolic phospholipase A₂ (cPLA₂), which may explain how they inhibit TNF cytolysis. Since eicosinoids produced from arachidonic acid (the product of cPLA₂) are potent mediators of inflammation, the E3 proteins may block the inflammatory response to adenovirus infection. These adenovirus proteins should be novel tools to understand adenovirus pathogenesis, TNF signal transduction, and TNF cytolysis.     

Activities and interactions among phospholipases A2 during thapsigargin-induced S49 cell death

The purpose of this study was to determine the roles of calcium-dependent phospholipase A₂ (cPLA₂) and calcium-independent phospholipase A₂ (iPLA₂) in thapsigargin-induced membrane susceptibility to secretory phospholipase A₂ (sPLA₂) and programmed cell death. ³H-arachidonic acid release was observed in the presence of thapsigargin. This release was inhibited partially by an inhibitor of iPLA₂ (BEL) and completely by an inhibitor of both cPLA₂ and iPLA₂ (MAFP) suggesting that these enzymes were active during apoptosis. The process of cell death did not require the activity of either enzyme since neither inhibitor impeded the progression of apoptosis. However, both inhibitors increased the susceptibility of the membrane to sPLA₂ in the presence of thapsigargin. In the case of BEL, this effect appeared to involve direct induction of apoptosis in a sub-population of the cells independent of the action of iPLA₂. In conclusion, the results suggested that cPLA₂ and iPLA₂ are active during thapsigargin-induced apoptosis in S49 cells and that cPLA₂ tempers the tendency of the cells to become susceptible to sPLA₂ during apoptosis.     

Eicosanoid Signaling and Vascular Dysfunction: Methylmercury-Induced Phospholipase D Activation in Vascular Endothelial Cells

Mercury, especially methylmercury (MeHg), is implicated in the etiology of cardiovascular diseases. Earlier, we have reported that MeHg induces phospholipase D (PLD) activation through oxidative stress and thiol-redox alteration. Hence, we investigated the mechanism of the MeHg-induced PLD activation through the upstream regulation by phospholipase A₂ (PLA₂) and lipid oxygenases such as cyclooxygenase (COX) and lipoxygenase (LOX) in the bovine pulmonary artery endothelial cells (BPAECs). Our results showed that MeHg significantly activated both PLA₂ (release of [³H]arachidonic acid, AA) and PLD (formation of [³²P]phosphatidylbutanol) in BPAECs in dose- (0–10 μM) and time-dependent (0–60 min) fashion. The cPLA₂-specific inhibitor, arachidonyl trifluoromethyl ketone (AACOCF₃), significantly attenuated the MeHg-induced [³H]AA release in ECs. MeHg-induced PLD activation was also inhibited by AACOCF₃ and the COX- and LOX-specific inhibitors. MeHg also induced the formation of COX- and LOX-catalyzed eicosanoids in ECs. MeHg-induced cytotoxicity (based on lactate dehydrogenase release) was protected by PLA₂-, COX-, and LOX-specific inhibitors and 1-butanol, the PLD-generated PA quencher. For the first time, our studies showed that MeHg activated PLD in vascular ECs through the upstream action of cPLA₂ and the COX- and LOX-generated eicosanoids. These results offered insights into the mechanism(s) of the MeHg-mediated vascular endothelial cell lipid signaling as an underlying cause of mercury-induced cardiovascular diseases.     

Turning point in apoptosis/necrosis induced by hydrogen peroxide

The turning point between apoptosis and necrosis induced by hydrogen peroxide (H₂O₂) have been investigated using human T-lymphoma Jurkat cells. Cells treated with 50 μM H₂O₂ exhibited caspase-9 and caspase-3 activation, finally leading to apoptotic cell death. Treatment with 500 μM H₂O₂ did not exhibit caspase activation and changed the mode of death to necrosis. On the other hand, the release of cytochrome c from the mitochondria was observed under both conditions. Treatment with 500 μM H₂O₂, but not with 50 μM H₂O₂, caused a marked decrease in the intracellular ATP level; this is essential for apoptosome formation. H₂O₂-reducing enzymes such as cellular glutathione peroxidase (cGPx) and catalase, which are important for the activation of caspases, were active under the 500 μM H₂O₂ condition. Prevention of intracellular ATP loss, which did not influence cytochrome c release, significantly activated caspases, changing the mode of cell death from necrosis to apoptosis. These results suggest that ATP-dependent apoptosome formation determines whether H₂O₂-induced cell death is due to apoptosis or necrosis.     

Synergistic Effect of Interleukin-1β and Tumor Necrosis Factor α on PGE2Production by Articular Chondrocytes Does Not Involve PLA2Stimulation

This study investigates the ways in which two proinflammatory cytokines, tumor necrosis factor α (TNF) and interleukin-1β (IL1), cause increased production of prostaglandin E₂(PGE₂) in rabbit articular chondrocytes (RAC). Rabbit articular chondrocytes in primary culture were incubated with IL1, TNF, or both. Arachidonic acid (AA) release, PGE₂production, and the activities of cytosolic phospholipase A₂(cPLA₂), secreted phospholipase A₂(sPLA₂), and cyclooxygenase (COX) were measured. The mRNA levels of cPLA₂, sPLA₂, and COX-2 were also measured by Northern blotting, using specific complementary DNA probes. Incubation of IL1-stimulated RAC with TNF further increased PGE₂production. This synergy did not involve PLA₂stimulation, as there were no increases in AA release, cPLA₂and sPLA₂activities, or mRNA. In contrast, TNF increased the effect of IL1 on COX-2 activity and mRNA level. These results show that TNF and IL1 act in synergy in PGE₂production in articular chondrocytes. As sPLA₂and cPLA₂do not seem to be involved, COX-2 appears to be the best target for a specific anti-inflammatory strategy against cartilage degradation.     

TAK1 kinase switches cell fate from apoptosis to necrosis following TNF stimulation

TNF activates three distinct intracellular signaling cascades leading to cell survival, caspase-8–mediated apoptosis, or receptor interacting protein kinase 3 (RIPK3)–dependent necrosis, also called necroptosis. Depending on the cellular context, one of these pathways is activated upon TNF challenge. When caspase-8 is activated, it drives the apoptosis cascade and blocks RIPK3-dependent necrosis. Here we report the biological event switching to activate necrosis over apoptosis. TAK1 kinase is normally transiently activated upon TNF stimulation. We found that prolonged and hyperactivation of TAK1 induced phosphorylation and activation of RIPK3, leading to necrosis without caspase activation. In addition, we also demonstrated that activation of RIPK1 and RIPK3 promoted TAK1 activation, suggesting a positive feedforward loop of RIPK1, RIPK3, and TAK1. Conversely, ablation of TAK1 caused caspase-dependent apoptosis, in which Ripk3 deletion did not block cell death either in vivo or in vitro. Our results reveal that TAK1 activation drives RIPK3-dependent necrosis and inhibits apoptosis. TAK1 acts as a switch between apoptosis and necrosis.     

RhoA/Rho Kinase Mediates Neuronal Death Through Regulating cPLA<Subscript>2</Subscript> Activation

Activation of RhoA/Rho kinase leads to growth cone collapse and neurite retraction. Although RhoA/Rho kinase inhibition has been shown to improve axon regeneration, remyelination and functional recovery, its role in neuronal cell death remains unclear. To determine whether RhoA/Rho kinase played a role in neuronal death after injury, we investigated the relationship between RhoA/Rho kinase and cytosolic phospholipase A₂ (cPLA₂), a lipase that mediates inflammation and cell death, using an in vitro neuronal death model and an in vivo contusive spinal cord injury model performed at the 10th thoracic (T10) vertebral level. We found that co-administration of TNF-α and glutamate induced spinal neuron death, and activation of RhoA, Rho kinase and cPLA₂. Inhibition of RhoA, Rho kinase and cPLA₂ significantly reduced TNF-α/glutamate-induced cell death by 33, 52 and 43 %, respectively (p < 0.001). Inhibition of RhoA and Rho kinase also significantly downregulated cPLA₂ activation by 66 and 60 %, respectively (p < 0.01). Furthermore, inhibition of RhoA and Rho kinase reduced the release of arachidonic acid, a downstream substrate of cPLA₂. The immunofluorescence staining showed that ROCK₁ or ROCK₂, two isoforms of Rho kinase, was co-localized with cPLA₂ in neuronal cytoplasm. Interestingly, co-immunoprecipitation (Co-IP) assay showed that ROCK₁ or ROCK₂ bonded directly with cPLA₂ and phospho-cPLA₂. When the Rho kinase inhibitor Y27632 was applied in mice with T10 contusion injury, it significantly decreased cPLA₂ activation and expression and reduced injury-induced apoptosis at and close to the lesion site. Taken together, our results reveal a novel mechanism of RhoA/Rho kinase-mediated neuronal death through regulating cPLA₂ activation.     

Quinacrine protects neuronal cells against heat-induced injury

The effects of quinacrine (QA) on heat-induced neuronal injury have been explored, with the intention of understanding the mechanisms of QA protection. Primary cultivated striatum neurons from newborn rats were treated with QA 1 h before heat treatment at 43 °C which lasted for another 1 h, and necrosis and apoptosis were detected by Annexin-V-FITC and propidium iodide (PI) double staining. Neuronal apoptosis was determined using terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) techniques. Cell membrane fluidity, activity of cytosolic phospholipase A₂ (cPLA₂) and the level of arachidonic acid (AA) were also examined. Membrane surface ultrastructure of striatum neurons was investigated by atomic force microscopy (AFM). Results showed that heat treatment induced great striatum neurons death, with many dying neurons undergoing necrosis rather than apoptosis. QA alone had little effect on the survival of striatum neurons, while QA pretreatment before heat treatment decreased necrosis. Heat treatment also resulted in decreased membrane fluidity and increased cPLA₂ activity as well as arachidonic acid level; these effects were reversed by QA pretreatment. QA pretreatment also significantly prevented damage to the membrane surface ultrastructure of heat-treated neurons. These results suggest that QA protects striatum neurons against heat-induced neuronal necrosis, and also demonstrate that inhibition of cPLA₂ activity and stabilization of membranes may contribute to protective effect of quinacrine.     

Phospholipase D protects ECV304 cells against TNFalpha-induced apoptosis

Tumor necrosis factor alpha (TNFalpha), a pleiotropic cytokine, activates both apoptotic and pro-survival signals depending on the cell model. Using ECV304 cells, which can be made TNFalpha-sensitive by cycloheximide (CHX) co-treatment, we evaluated the potential roles of ceramide and phospholipase D (PLD) in TNFalpha-induced apoptosis. TNFalpha/CHX induced a robust increase in ceramide levels after 16 h of treatment when cell death was maximal. PLD activity was increased at early time point (1h) whereas both PLD activity and PLD1 protein were strongly decreased after 24h. TNFalpha/CHX-induced cell death was significantly lowered by exogenous bacterial PLD and phoshatidic acid, and in cells overexpressing PLD1. Conversely, cells depleted in PLD proteins by small interference RNA (siRNA) treatment exhibited higher susceptibility to apoptosis. These results show that PLD exerts a protective role against TNFalpha-induced cell death.     

Nicotinamide "protects" resting lymphocytes exposed to hydrogen peroxide from necrosis but not from apoptosis

The aim of this work was to investigate the relationship between mechanisms of DNA repair and apoptosis induced by oxidative stress (H₂O₂) in human lymphocytes. Using the comet assay, fluorescent microscopy, and DNA electrophoresis, we studied the DNA damage induced by hydrogen peroxide (H₂O₂) treatment, the time and the amount of repair of strand breaks, the type of cell death, and the influence of inhibitors of repair (nicotinamide). When lymphocytes were treated with H₂O₂, we observed an increased in necrosis compared to apoptosis. However, when nicotinamide (which inhibits DNA repair) was added, the mode of death reversed to increased apoptosis. These results indicate that nicotinamide "protects" resting lymphocytes exposed to H₂O₂ from necrosis but not from apoptosis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Lack of Alpha-Synuclein Modulates Microglial Phenotype In Vitro

Alpha (α)-synuclein neuronal effects are continually being defined although its role in regulating glial phenotypes remains unclear. An ability to regulate microglial activation was investigated using primary cultures from wild type and α-synuclein deficient mice (Snca ^−/−). Snca ^−/− microglia demonstrated increased secretion of the cytokine tumor necrosis factor-alpha (TNF-α), impaired phagocytic ability, elevated prostaglandin levels, and increased protein levels of key enzymes in lipid-mediated signaling events, cytosolic phospholipase (cPLA₂), cyclooxygenase-2 (Cox-2) and phospholipase D2 (PLD2) when compared to wild type cells. Increased cytokine secretion and cPLA₂ and Cox-2 levels in Snca ^−/− microglia were partially attenuated by inhibiting PLD-dependent signaling with n-butanol treatment.     

Tumor necrosis factor α‐mediated activation of c‐Jun NH2‐terminal kinase as a mechanism for fumonisin B1 induced apoptosis in murine primary hepatocytes

Fumonisin B₁ is a mycotoxin produced by Fusarium verticillioides, frequently associated with corn. It produces species‐specific and organ‐specific toxicity, including equine leukoencephalomalacia, porcine pulmonary edema, and hepatic or renal damage in most animal species. Fumonisin B₁ perturbs sphingolipid metabolism by inhibiting ceramide synthase. Our previous studies indicated that fumonisin B₁ caused localized activation of cytokines in liver produced by macrophages and other cell types that modulate fumonisin B₁ induced hepatic apoptosis in mice. The role of tumor necrosis factor α (TNFα) in fumonisin B₁ mediated hepatocyte apoptosis has been established; not much is known about the downstream events leading to apoptosis. In the current study, fumonisin B₁ induced apoptosis in primary culture of liver cells. In consistence with previous reports, fumonisin B₁ caused accumulation of sphingoid bases and led to increase in TNFα expression. Phosphorylated and total c‐Jun NH₂‐terminal kinase (JNK) activities were increased after 24 h fumonisin B₁ treatment. JNK inhibitor (SP600125) and anti‐TNFα reduced the apoptosis induced by fumonisin B₁. The role of JNK signaling in fumonisin B₁ induced apoptosis is downstream of TNFα production, as fumonisin B₁‐mediated activation of JNK was reduced by the presence of anti‐TNFα in the medium, whereas the presence of JNK inhibitor did not change the fumonisin B₁ induced TNFα expression. Results of this study imply that generation of fumonisin B₁ induced TNFα results in modulation of mitogen activated protein kinases, particularly of JNK, and provides a possible mechanism for apoptosis in murine hepatocytes. © 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 19:359‐367, 2005; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20102     

Docosahexaenoic acid enrichment can reduce L929 cell necrosis induced by tumor necrosis factor

We previously reported that docosahexaenoic acid (DHA) attenuated tumor necrosis factor (TNF)-induced apoptosis in human monocytic U937 cells (J. Nutr. 130: 1095-1101, 2000). In the present study, we examined the effects of DHA and other polyunsaturated fatty acids (PUFA) on TNF-induced necrosis, another mode of cell death, using L929 murine fibrosarcoma cells. After preincubation with PUFA conjugated with BSA for 24 h, cells were treated with TNF or TNF+actinomycin D (Act D). Preincubation of cells with DHA enriched this polyunsaturated acid in the phospholipids and attenuated cell death induced by either TNF or TNF+Act D. When cells were treated with TNF alone, DNA laddering was not detected, and cells were coincidently stained with both annexin V-FITC and propidium iodide, indicating that the death mode was necrotic. TNF+Act D predominantly induced necrosis, although concurrent apoptotic cell death was also observed in this case. Preincubation with oleic acid, linoleic acid or 20:3(n-3) did not affect TNF-induced necrosis. Conversely, supplementation with n-3 docosapentaenoic acid (DPAn-3) or eicosapentaenoic acid (EPA) reduced necrotic cell death, but to a lesser extent in comparison with DHA. Unlike the case of U937 cell apoptosis, arachidonic acid (AA) significantly attenuated L929 cell necrosis, and 20:3(n-6) or 22:4(n-6) showed similar or less activity, respectively. Statistical evaluation indicated that the order of effective PUFA activity was DHA>DPAn-3> or =EPA>AA approximately 20:3(n-6)> or =22:4(n-6). One step desaturation, C2 elongation or C2 cleavage within the n-6 or n-3 fatty acid group was probably very active in L929 cells, because AA, synthesized from 20:3(n-6) or 22:4(n-6), and C22 fatty acids, synthesized from AA or EPA, were preferentially retained in cellular phospholipids. These observations suggested that attenuation of TNF-induced necrosis by the supplementation of various C20 or C22 polyunsaturated fatty acids is mainly attributable to the enrichment of three kinds of polyunsaturated fatty acids, i.e., DHA, DPAn-3 or AA, in phospholipids. Among these fatty acids, DHA was the most effective in the reduction of L929 necrosis as observed in the case of U937 apoptosis. This suggests that DHA-enriched membranes can protect cell against TNF irrespective of death modes and that membranous DHA may abrogate the death signaling common to necrosis and apoptosis.     

1,25-dihydroxyvitamin D3 pretreatment limits prostaglandin biosynthesis by cytokine-stimulated adult human osteoblast-like cells

The steroid derivative 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) is a regulator of bone biology, and there is evidence that 1,25(OH)₂D₃ modulates arachidonic acid metabolism in osteoblastic cell model systems and in bone organ cultures. In the present studies, 1,25(OH)₂D₃ decreased prostaglandin (PG) biosynthesis by normal adult human osteoblast-like (hOB) cell cultures by about 30%. The decrease was observed under basal incubation conditions, or in specimens stimulated by transforming growth factor-β₁ (TGF-β) or by tumor necrosis factor-α (TNF). The inhibition of the TGF-β-stimulated PG production appeared to reflect a diminished efficiency of arachidonic acid conversion into PGs by the cells, while the efficiency of substrate utilization for PG biosynthesis was unaffected by 1,25(OH)₂D₃ pretreatment in the unstimulated samples, or in samples stimulated with TNF or with TNF plus TGF-β. Free arachidonic acid levels were decreased following 1,25(OH)₂D₃ pretreatment in the TNF stimulated samples. hOB cell phospholipase A₂ activity was measured in subcellular fractions, and this activity was decreased by 20–25% in the 1,25(OH)₂D₃ pretreated samples. The addition of the selective inhibitor AACOCF₃ to the phospholipase A₂ assays provided evidence that it was the cytoplasmic isoform of the enzyme that was affected by the 1,25(OH)₂D₃ pretreatment of the hOB cells. Thus, 1,25(OH)₂D₃ regulation of hOB cell biology includes significant effects on arachidonic acid metabolism. In turn, this could influence the effects of other hormones and cytokines whose actions include the stimulated production of bioactive arachidonic acid metabolites. J. Cell. Biochem. 68:237–246, 1998. © 1998 Wiley-Liss, Inc.     

Phospholipid signalling and lipid-derived second messengers in plants

Phospholipid signalling is mediated by phospholipid breakdown products generated by phospholipases. The enzymes from animals and plants generating known or potential lipid-derived second messengers are compared. Plants possess a phospholipase C and a phospholipase A₂ both of which are agonist-activated. These agonists (auxin, elicitors, perhaps others) bind to the external surface of the plasma membrane. The target enzyme for potential plant lipid-derived second messengers is lipid-activated protein kinase but the possibility that other enzymes may be also lipid-modulated should not be precluded.Abbreviations DAG diacylglycerol - CDPK calmodulin-like domain protein kinase - PLA2 phospholipase A₂ - PLC phospholipase C - PLD phospholipase D - PKC protein kinase C - PS phosphatidylserine     

Distinctive roles of receptor‐interacting protein kinases 1 and 3 in caspase‐independent cell death of L929

Upon tumour necrosis factor alpha (TNFα) stimulation, cells respond actively by way of cell survival, apoptosis or programmed necrosis. The receptor‐interacting proteins 1 (RIP1) and 3 (RIP3) are responsible for TNFα‐mediated programmed necrosis. To delineate the differential contributions of RIP3 and RIP1 to programmed necrosis, L929 cells were stimulated with TNFα, carbobenzoxy‐valyl‐alanyl‐aspartyl‐[O‐methyl]‐fluoromethylketone (zVAD) or zVAD along with TNFα following RNA interference against RIP1 and RIP3, respectively. RIP1 silencing did not protect cells from TNFα‐mediated cell death, while RIP3 down‐regulation made them refractory to TNFα. The heat shock protein 90 inhibitor geldanamycin (GA) down‐regulated both RIP1 and RIP3 expression, which rendered cells resistant to zVAD/TNFα‐mediated cell death but not to TNFα‐mediated cell death alone. Therefore, the protective effect of GA on zVAD/TNFα‐stimulated necrosis might be attributed to RIP3, not RIP1, down‐regulation. Pretreatment of L929 cells with rapamycin mitigated zVAD‐mediated cell death, while the autophagy inhibitor chloroquine did not affect necrotic cell death. Meanwhile, necrotic cell death by zVAD and TNFα was caused by reactive oxygen species generation and effectively diminished by lipid‐soluble butylated hydroxyanisole. Taken together, the results indicate that RIP1 and RIP3 can independently mediate death signals being transduced by two different death stimuli, zVAD and TNFα. Copyright © 2013 John Wiley & Sons, Ltd.     

Regulation of tumor necrosis factor cytotoxicity by calcineurin

Cyclosporin (CsA) inhibits mitochondrial death signaling and opposes tumor necrosis factor (TNF)-induced apoptosis in vitro. However, CsA is also a potent inhibitor of calcineurin, a phosphatase that may participate in cell death. Therefore, we tested the hypothesis that calcineurin regulates TNF cytotoxicity in rat hepatoma cells (FTO2B). TNF-treated FTO2B cells appeared apoptotic by DNA fragmentation, nuclear condensation, annexin V binding, and caspase activation. We studied two calcineurin inhibitors, CsA and FK506, and found that each potently inhibited TNF cytotoxicity. Western blot demonstrated calcineurin in FTO2B homogenates. In a model of mitochondrial permeability transition (MPT), we found that CsA prevented MPT and cytochrome c release, while FK506 inhibited neither. In summary, we present evidence that calcineurin participates in an apoptotic death pathway activated by TNF. CsA may oppose programmed cell death by inhibiting calcineurin activity and/or inhibiting mitochondrial signaling.