Ilaria Tommasini

Essential Role of the Mitochondrial Respiratory Chain in Peroxynitrite‐Induced Strand Scission of Genomic DNA

A large body of experimental evidence suggests that DNA damage and cytotoxicity mediated by peroxynitrite are linked by a causal relationship and important events in various pathological conditions. In the present study, we investigated the mechanism whereby peroxynitrite causes DNA single strand breakage in intact cells and found that the respiratory chain plays a pivotal role in this response. In particular, peroxynitrite mediates inhibition of complex III and, under these conditions, electrons are directly transferred from ubisemiquinone to molecular oxygen. Hydrogen peroxide produced by the dismutation of superoxides is the species mediating the peroxynitrite‐dependent DNA cleavage.

Peroxynitrite promotes mitochondrial permeability transition-dependent rapid U937 cell necrosis: survivors proliferate with kinetics superimposable on those of untreated cells.

A short term exposure to peroxynitrite promotes a time- and concentration-dependent lethal response in U937 cells. The mode of cell death was necrosis and rapid (within minutes) cell lysis was found to occur via a mechanism involving mitochondrial permeability transition. Apoptosis was not detected in cells exposed to low levels of peroxynitrite, or in cells which survived a treatment with toxic amounts of peroxynitrite, neither after the 60 min exposure nor following increasing time intervals of growth in fresh culture medium. Rather, cells treated with peroxynitrite concentrations which were not immediately lethal, as well as the survivors of treatments with toxic levels of peroxynitrite, proliferated with kinetics superimposable on those observed in untreated cells.

The Arachidonate-Dependent Survival Signaling Preventing Toxicity in Monocytes/Macrophages Exposed to Peroxynitrite

Cells 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 A2-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-LO (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.

5-Hydroxyeicosatetraenoic acid is a key intermediate of the arachidonate-dependent protective signaling in monocytes/macrophages exposed to peroxynitrite

Endogenous generation of arachidonic acid via selective activation of cytosolic phospholipase A2 has been implicated in the mechanism of monocytes/macrophage survival in the presence of peroxynitrite. In particular, the lipid messenger was shown to prevent the otherwise rapid onset of a mitochondrial permeability‐transition (MPT)‐dependent necrosis by causing the mitochondrial translocation of protein kinase Cα (PKCα) and the ensuing cytosolic accumulation of the Bcl‐2‐antagonist of cell death (Bad), an event promoting the anti‐MPT function of Bcl‐2 (or Bcl‐XL). Here, we show that the effects on PKCα are not mediated directly by arachidonate but rather, by downstream products of the enzyme 5‐lipoxygenase (5‐LO). Peroxynitrite elicited the nuclear membrane translocation of 5‐LO and enhanced its enzymatic activity via a mechanism sensitive to low concentrations of inhibitors of 5‐LO or the 5‐LO‐activating protein, as well as to genetic depletion of the latter enzyme. Inhibition of 5‐LO activity was invariably associated with the cytosolic localization of PKCα, the mitochondrial accumulation of Bad, and a rapid MPT‐dependent necrosis. All these events were prevented by nanomolar concentrations of the 5‐LO product 5‐hydroxyeicosatetraenoic acid.

Prostaglandin E2 Signals Monocyte/Macrophage Survival to Peroxynitrite via Protein Kinase A Converging in Bad Phosphorylation with the Protein Kinase Cα-Dependent Pathway Driven by 5-Hydroxyeicosatetraenoic Acid

Monocytes/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 Cα. 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.