Protection against peroxynitrite-induced fibroblast injury and arthritis development by inhibition of poly(ADP-ribose) synthase.
Peroxynitrite, a cytotoxic oxidant formed from nitric oxide (NO) and superoxide, induces DNA strand breakage, which activates the nuclear enzyme poly(ADP-ribose) synthase (PARS; EC 2.4.2.30). The cellular function of PARS was determined in fibroblast lines from PARS knockout animals (PARS-/-) and corresponding wild-type animals (PARS+/+), 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 PARS+/+ animals to peroxynitrite caused DNA single-stand breakage and PARS activation and caused an acute suppression of mitochondrial respiration. INH2BP protected the PARS+/+ cells against the suppression of mitochondrial respiration in response to peroxynitrite (50-100 microM). Similarly to PARS inhibition with INH2BP, the PARS-/- cells were protected against peroxynitrite-induced injury. The protection against cellular injury by PARS-/- phenotype or INH2BP waned when cells were challenged with higher concentrations of the oxidant. Inhibition of PARS by INH2BP or by PARS-/- 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 PARS-/- cells. In collagen-induced arthritis, significant staining for nitrotyrosine, a marker of peroxynitrite formation, was found in the inflamed joints. Oral treatment with INH2BP (0.5 g/kg, 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-inflammatory effects in vivo.
Szabó
C
C
Division of Critical Care, Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229, USA. csaba.szabo@chmcc.org
Virág
L
L
Cuzzocrea
S
S
Scott
G S
GS
Hake
P
P
O'Connor
M P
MP
Zingarelli
B
B
Salzman
A
A
Kun
E
E
D000818:Animals; D001168:Arthritis / Q000139:chemically induced / Q000473:pathology / Q000517:prevention & control*; D003374:Coumarins / Q000494:pharmacology* / Q000627:therapeutic use; D004249:DNA Damage / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology* / Q000627:therapeutic use; D005347:Fibroblasts / Q000187:drug effects / Q000473:pathology; D051379:Mice; D009566:Nitrates / Q000633:toxicity*; D016877:Oxidants / Q000633:toxicity*; D000067856:Poly(ADP-ribose) Polymerase Inhibitors
D003374:Coumarins; D004791:Enzyme Inhibitors; D009566:Nitrates; D016877:Oxidants; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; C071383:5-iodo-6-amino-1,2-benzopyrone; C039642:peroxynitric acid