Nitric oxide induced cell death in human osteoarthritic synoviocytes is mediated by tyrosine kinase activation and hydrogen peroxide and/or superoxide formation.
OBJECTIVE
To investigate the regulation of osteoarthritis (OA) synovial fibroblast nitric oxide (NO) induced cell death.


METHODS
Cultured synovial fibroblasts from human OA synovium were incubated with NO donor sodium nitroprusside (SNP) in the absence or presence of specific inhibitors of different protein kinases, cyclooxygenase-2 (COX-2), caspase-3 and caspase-9, inducible NO synthase, and in the absence or presence of prostaglandin E2 (PGE2). Experiments were also performed using scavengers of NO (carboxy-PXTO), peroxynitrite (uric acid), and superoxide (taxifolin). The level of cell death was measured by MTT and DNA fragmentation.


RESULTS
Human OA synovial fibroblasts incubated with SNP decreased cell viability and increased DNA fragmentation in a dose dependent manner. This was associated with increased levels of both COX-2 and PGE2 production. Selective inhibition of COX-2 by NS-398 significantly inhibited SNP induced cell death, even in the presence of exogenously added PGE2. Experiments revealed that SNP treated cells expressed increased levels of active caspase-3 and caspase-9, while Bcl-2 was downregulated. Incubation of these treated cells with inhibitors of caspase-3 (Z-DEVD-FMK) or caspase-9 (Z-LEHD-FMK) protected viability of SNP treated OA synovial fibroblasts, indicating that NO mediated cell death was mainly related to apoptosis. This was also confirmed by measuring the DNA fragmentation (TUNEL method) and the level of active caspase-3 (immunocytochemistry) in these cells. Data also showed that SNP induces the activation of kinases MEK 1/2, p38, and tyrosine kinases. Specific inhibition of tyrosine kinases completely abrogated the SNP induced cell death. In turn, this cell death protection was associated with a marked inhibition of caspase-3 and caspase-9 activities, as well as COX-2/PGE2 production. Moreover, data showed that the NO donor SNP induced cell death was not solely related to the production of NO or peroxynitrite, but to the generation of reactive oxygen species (ROS) such as hydrogen peroxide and/or superoxide.


CONCLUSIONS
Our results provided strong evidence of the role of tyrosine kinase and mitogen activated protein kinase activation, by upregulation of COX-2 expression, in NO induced OA synovial fibroblast death. The generation of ROS such as hydrogen peroxide and superoxide appeared to be a major factor in the death of these cells.
Jovanovic
Dragan V
DV
Osteoarthritis Research Unit, Hôpital Notre-Dame, Centre Hospitalier de l'Université de Montréal (CHUM), Montréal, Québec, Canada.
Mineau
François
F
Notoya
Kohei
K
Reboul
Pascal
P
Martel-Pelletier
Johanne
J
Pelletier
Jean-Pierre
JP
D002470:Cell Survival / Q000187:drug effects; D051546:Cyclooxygenase 2; D053938:DNA Fragmentation / Q000187:drug effects; D015232:Dinoprostone / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004790:Enzyme Induction / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D006801:Humans; D020287:In Situ Nick-End Labeling; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D008297:Male; D008565:Membrane Proteins; D008875:Middle Aged; D009569:Nitric Oxide / Q000502:physiology*; D009599:Nitroprusside / Q000494:pharmacology; D020370:Osteoarthritis, Knee / Q000201:enzymology* / Q000473:pathology; D010545:Peroxides / Q000378:metabolism*; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D013583:Synovial Membrane / Q000187:drug effects / Q000201:enzymology* / Q000473:pathology
D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008565:Membrane Proteins; D010545:Peroxides; D009599:Nitroprusside; D009569:Nitric Oxide; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D011505:Protein-Tyrosine Kinases; D015232:Dinoprostone