Protection against peroxynitrite-induced fibroblast injury and arthritis development by inhibition of poly(ADP-ribose) synthase (nitric oxideysuperoxideyinf lammationyinducible nitric-oxide synthaseyDNA single-strand break)

Peroxynitrite, a cytotoxic oxidant formed from nitric oxide (NO) and superoxide, induces DNA strand breakage, which activates the nuclear enzyme poly(ADPribose) synthase (PARS; EC 2.4.2.30). The cellular function of PARS was determined in fibroblast lines from PARS knockout animals (PARS2/2) and corresponding wild-type animals (PARS1/1), with the aid of the lipophilic PARS inhibitor 5-iodo-6-amino-1,2-benzopyrone (INH2BP). We investigated the role of PARS in peroxynitrite-induced fibroblast injury in vitro and also in the development of arthritis in vivo. Exposure of embryonic fibroblasts from the PARS1/1 animals to peroxynitrite caused DNA single-stand breakage and PARS activation and caused an acute suppression of mitochondrial respiration. INH2BP protected the PARS1/1 cells against the suppression of mitochondrial respiration in response to peroxynitrite (50–100 mM). Similarly to PARS inhibition with INH2BP, the PARS2/2 cells were protected against peroxynitrite-induced injury. The protection against cellular injury by PARS2/2 phenotype or INH2BP waned when cells were challenged with higher concentrations of the oxidant. Inhibition of PARS by INH2BP or by PARS2/2 phenotype reduced inducible nitric-oxide synthase (iNOS; EC 1.14.13.39) mRNA levels and inhibited production of NO in immunostimulated cells. INH2BP had no peroxynitrite scavenging or hydroxyl radical scavenging effects, and it exerted no additional (nonspecific) effects in the PARS2/2 cells. In collagen-induced arthritis, significant staining for nitrotyrosine, a marker of peroxynitrite formation, was found in the inf lamed joints. Oral treatment with INH2BP (0.5 gykg, daily), starting at the onset of arthritis (day 25), delayed the development of the clinical signs at days 26–35 and improved histological status in the knee and paw. Our data demonstrate that deletion of PARS by genetic manipulation or pharmacological inhibition of PARS protects against oxidant-induced cellular injury in vitro and exhibits anti-inf lammatory effects in vivo. Nitric oxide (NO), superoxide, and their cytotoxic reaction product peroxynitrite (ONOO2) are terminal mediators of cellular injury in various forms of inflammation. In vitro studies employing conventional inhibitors of the nuclear enzyme poly(ADP-ribose) synthase (PARS; EC 2.4.2.30) suggested that the oxidative injury in response to oxy radicals and peroxynitrite is related to DNA single-strand breakage and consequent activation of PARS (1, 2). Massive ADPribosylation of nuclear proteins by PARS then results in cellular energy depletion and injury, reminiscent of necrosis (1–3). However, objections can be raised against the conclusions of these studies, because the commonly used relatively high concentrations of PARS inhibitors (e.g., nicotinamide and benzamide analogs), have additional effects as free radical scavengers, and have short cellular residence time (4–6). More recently a potent pharmacologically active inhibitor of PARS, the lipophilic 6-iodo-5-amino-1,2-benzopyrone (INH2BP), was developed (7, 8). Moreover, a genetically engineered mouse line that lacks PARS is now available: a fibroblast cell line from these animals can be used for in vitro investigations (9). These tools allow a direct testing of the role of PARS. The present work was designed to elucidate (i) whether inhibition of PARS by INH2BP protects against cellular oxidant injury triggered by peroxynitrite, a cytotoxic oxidant produced by the reaction of superoxide and NO (10–14); (ii) whether the PARS2/2 cells are protected against cellular injury compared with the PARS1/1 cells; (iii) whether the PARS2/2 phenotype or pharmacological inhibition of PARS affects NO production in response to immunostimulation; and (iv) whether INH2BP exerts any oxidant scavenging or nonspecific effects. In addition, to elucidate the role of the peroxynitrite-PARS pathway in the pathogenesis of inflammation, we evaluated (v) whether inhibition of PARS with INH2BP affects the course of collagen-induced arthritis. The results of the current study support the role of PARS activation in the peroxynitrite-mediated cellular oxidant injury and inflammation, and they demonstrate that either deletion of PARS or its selective inhibition by INH2BP protects against inflammatory cell injury.