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Dexamethasone Promotes Toxicity in U937 Cells Exposed to Otherwise Non-toxic Concentrations of Peroxynitrite: Pivotal Role for Lipocortin 1 Mediated Inhibition of Cytosolic Phospholipase A2
No full textPretreatment with dexamethasone (Dex) was not toxic for U937 cells but caused a rapid lethal response upon subsequent exposure to otherwise nontoxic concentrations of peroxynitrite. This effect was not associated with enhanced formation of hydrogen peroxide taking place after peroxynitrite and was shown previously to play a pivotal role in the ensuing lethal response. Further analyses revealed that although Dex did not affect cytosolic phospholipase A(2) (cPLA(2)) expression, it markedly reduced the extent of arachidonic acid (AA) release mediated by peroxynitrite-dependent stimulation of cPLA(2). This event, as well as the enhanced toxicity, was abolished by mifepristone, a glucocorticoid receptor antagonist. The outcome of various approaches, using phospholipase A(2) inhibitors, cPLA(2) antisense oligonucleotide-transfected cells, and supplementation with exogenous AA, led to the demonstration that inhibition of cPLA(2) activity is causally linked to the increased susceptibility to peroxynitrite caused by Dex. Finally, the effects of Dex were shown to be mediated by enhanced expression of lipocortin 1 (LC1), a cPLA(2) inhibitory protein. These results indicate that Dex promotes toxicity in U937 cells exposed to otherwise nontoxic concentrations of peroxynitrite and that this event is causally linked to enhanced expression of LC1 leading to inhibition of cPLA(2). Thus, the increased lethal response arises because of LC1-dependent impairment of the AA-induced cytoprotective mechanism triggered by peroxynitrite
Peroxynitrite promotes mitochondrial permeability transition-dependent rapid U937 cell necrosis: survivors proliferate with kinetics superimposable on those of untreated cells.
No full textNON-TOXIC CONCENTRATIONS OF PEROXYNITRITE COMMIT U937 CELLS TO MITOCHONDRIAL PERMEABILITY TRANSITION-DEPENDENT NECROSIS THAT IS HOWEVER PREVENTED BY ENDOGENOUS ARACHIDONIC ACID
No full textThe present study was aimed at examining the mechanism whereby an otherwise non-toxic concentration of peroxynitrite promotes a rapid necrotic response in U937 cells in which cytosolic phospholipase A(2) is pharmacologically inhibited or genetically depleted. We found that loss of viable cells is appreciable 15 min after addition of peroxynitrite, does not further increase at 30 min and is mediated by mitochondrial permeability transition (MPT). Both MPT and toxicity were prevented by exogenous arachidonic acid (AA). Various experimental approaches produced results consistent with the notion that the AA-dependent protective mechanism takes place 10-15 min after addition of peroxynitrite. The observation that the extent of DNA strand scission induced by peroxynitrite did not vary under conditions of different AA availability suggests that this event is either upstream to mitochondrial dysfunction or irrelevant for cytotoxicity. Collectively, these data indicate that a non-toxic concentration of peroxynitrite commits U937 cells to MTP-dependent necrosis that is however prevented by endogenous AA. Thus, mitochondria are a likely target of the cytoprotective signalling triggered by AA
Delayed formation of hydrogen peroxide mediates the lethal response evoked by peroxynitrite in U937 cells
Full text linkThe toxicity paradigm used in the present study involves exposure of U937 cells to a concentration of authentic peroxynitrite, leading to a rapid necrotic response mediated by mitochondrial permeability transition. We found that addition of catalase after treatment with peroxynitrite specifically prevents the loss of mitochondrial membrane potential and the ensuing lethal response. The protective effects of catalase were mimicked by the cocktail glutathione peroxidase/reduced glutathione. A defensive role of intracellular catalase was implied by experiments showing that catalase-depleted cells are hypersensitive to peroxynitrite and that cells with an increased catalase content, selected for their resistance to H2O2, are cross-resistant to peroxynitrite. Further experiments demonstrated that H2O2 formation takes place after peroxynitrite exposure. Various approaches using inhibitors of the mitochondrial respiratory chain as well as respiration-deficient cells revealed that the oxidant is produced upon dismutation of superoxides generated at the level of complex III. Interestingly, respiration-deficient cells were found to be resistant to peroxynitrite toxicity, and all those treatments increasing formation of H2O2produced a parallel increase in toxicity. In conclusion, the results presented in this study indicate that peroxynitrite-induced impairment of electron transport from cytochrome b to cytochromec1 leads to delayed formation of hydrogen peroxide, which plays a pivotal role in the ensuing necrotic response
The arachidonate-dependent survival signaling preventing toxicity in monocytes/macrophages exposed to peroxynitrite.
Full text linkCelts belonging to the monocyte/macrophage lineage are in general highly resistant to peroxynitrite. Resistance is not dependent on the scavenging of peroxynitrite itself, or of other secondary reactive species, but is rather associated with the prompt activation of a survival signaling leading to the prevention of toxicity in cells otherwise committed to mitochondrial permeability transition (MPT)-dependent necrosis. The signaling pathway is triggered by cytosolic phospholipase A(2)-released arachidonic acid, leading to the sequential activation of 5-lipoxygenase (5-LO) and protein kinase C alpha, an event associated with the cytosolic accumulation of Bad. Hence, inhibition of 5-1-0 (or that of any of the aforementioned enzymes involved in the signaling cascade) was associated with the mitochondrial accumulation of Bad and Bax and with a rapid MPT-dependent toxicity. These results contribute to the definition of the mechanism(s) whereby monocytes/macrophages survive to peroxynitrite in inflamed tissues and provide insights for the development of novel anti-inflammatory therapies based on the suppression of inflammatory cell survival
ERK1/2-dependent regulation of U937 cell survival after exposure to peroxynitrite.
No full textA short-term growth of U937 cells in serum-free medium causes a prompt, mitochondrial permeability transition (MPT)-dependent necrotic response after exposure to an otherwise non-toxic concentration of peroxynitrite. This event is mediated by inhibition of extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation, essential for the cytosolic phospholipase A(2)-dependent arachidonic acid (AA) release evoked by peroxynitrite. Reduced availability of the lipid messenger would therefore limit the efficiency of the AA-dependent survival signalling and cause an MPT-based necrosis. Since peroxynitrite further reduces the extent of ERK1/2 phosphorylation, regardless of whether cells had been grown in serum-free or -containing medium, it appears that basal ERK1/2 phosphorylation is a critical determinant for the survival response of U937 cells to a non-toxic, but nevertheless MPT-committing, concentration of peroxynitrite
Hydrogen peroxide generated at the level of mitochondria in response to peroxynitrite promotes U937 cell death via inhibition of the cytoprotective signalling mediated by cytosolic phospholipase A2
Full text linkWe have studied the relationships existing between delayed formation of H2O2 and activation of cytosolic phospholipase A2 (cPLA2), events respectively promoting toxicity or survival in U937 cells exposed to peroxynitrite. The outcome of an array of different approaches using phospholipase A2 inhibitors, or cPLA2 antisense oligonucleotides, as well as specific respiratory chain inhibitors and respiration-deficient cells led to the demonstration that H2O2 does not mediate toxicity by producing direct molecular damage. Rather, the effects of H2O2 were found to be upstream to the arachidonic acid (AA)-mediated cytoprotective signalling and in fact causally linked to inhibition of cPLA2. Thus, it appears that U937 cells exposed to nontoxic concentrations of peroxynitrite are nevertheless committed to death, which however is normally prevented by the activation of parallel pathways resulting in cPLA2-dependent release of AA. A rapid necrotic response, however, takes place when high concentrations of peroxynitrite promote formation of H2O2 at levels impairing the cPLA2 cytoprotective signalling
Prostaglandin E2 signals monocyte/macrophage survival to peroxynitrite via protein kinase A converging in bad phosphorylation with the protein kinase C alpha-dependent pathway driven by 5-hydroxyeicosatetraenoic acid.
No full textMonocytes/macrophages committed to death by peroxynitrite nevertheless survive with a signaling response promoting Bad
phosphorylation, as well as its cytosolic localization, via upstream activation of cytosolic phospholipase A2, 5-lipoxygenase, and protein kinase C. We now report evidence for an alternative mechanism converging in Bad phosphorylation when the expression/activity of the above enzymes are suppressed. Under these conditions, also associated with peroxynitrite-dependent severe inhibition
of Akt, an additional Bad kinase, Bad dephosphorylation promoted its accumulation in the mitochondria and a prompt
lethal response. PGE2 prevented toxicity via EP2 receptor-mediated protein kinase A-dependent Bad phosphorylation. This notion was established in U937 cells by the following criteria: 1) there was a strong correlation between survival and cAMP accumulation, both in the absence and presence of phosphodiesterase inhibitors; 2) direct activation of adenylyl cyclase afforded cytoprotection; and 3) PGE2 promoted loss of mitochondrial Bad and cytoprotection, mimicked by EP2 receptor agonists, and prevented by EP2 receptor antagonists or protein kinase A inhibitors. Finally, selected experiments performed in human monocytes/macrophages
and in rat peritoneal macrophages indicated that the above cytoprotective pathway is a general response of cells belonging to the monocyte/macrophage lineage to both exogenous and endogenous peroxynitrite. The notion that two different pathways mediated by downstream products of arachidonic acid metabolism converge in Bad phosphorylation emphasizes the relevance of this strategy for the regulation of macrophage survival to peroxynitrite at the inflammatory sites
Reduced mitochondrial formation of H(2)O(2) is responsible for resistance of dimethyl sufoxide differentiated U937 cells to peroxynitrite
Full text linkPrevious studies performed in our laboratory indicated that non-toxic concentrations of peroxynitrite nevertheless commit U937 cells to a rapid necrosis that is however prevented by a survival signaling driven by cytosolic phospholipase AZ-released arachidonic acid. Toxicity was mediated by concentrations of peroxynitrite resulting in H2O2-dependent inhibition of arachidonic acid release. The present study shows that U937 cells differentiated to monocytes by prolonged exposure to dimethyl sulfoxide are resistant to peroxynitrite because able to respond with enhanced release of arachidonic acid. An additional important observation was that these cells require more arachidonate than the undifferentiated cells to support the survival signaling. The enhanced arachidonic acid release was not associated with changes in cytosolic phospholipase AZ expression but was rather dependent on the increased responsiveness of the enzyme to calcium-dependent stimulation as well as on reduced mitochondrial formation of H2O2. The latter event was found to be critical, since differentiated and undifferentiated cells were equally sensitive to peroxynitrite when the accumulation of H2O2 was enhanced via depletion of catalase, or addition of a complex III inhibitor. Thus, the strategy selected by the differentiation process to allow monocytes to cope with peroxynitrite appears to involve some specific mechanism preventing the mitochondrial formation of H2O2
The arachidonate-dependent cytoprotective signaling evoked by peroxynitrite is a general response of the monocyte/macrophage lineage
No full textU937, THP-1, and J774 cells or human monocytes and macrophages display similar levels of sensitivity to peroxynitrite and exposure to an otherwise non-toxic concentration of the oxidant in the presence of a phospholipase A, inhibitor was invariably associated with the onset of mitochondrial permeability transition (MPT)-dependent toxicity. These events were prevented by exogenous arachidonic acid (AA). In general, the protective concentrations of AA were greater in those cell types releasing more AA. Thus, non-toxic concentrations of peroxynitrite commit cells belonging to the monocyte/macrophage lineage to MPT-dependent toxicity that is however prevented by endogenous AA
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