Activation of stress-activated protein kinase in osteoarthritic cartilage:evidence for nitric oxide dependence

Objective: We have demonstrated in bovine chondrocytes that nitric oxide (N O) mediates IL1 dependent apoptosis under conditions of oxidant stress. Thi s process is accompanied by activation of c-Jun NH2-terminal kinase (JNK; a lso called stress-activated protein kinase). In these studies we examined a ctivation of JNK in explant cultures of human osteoarthritic cartilage obta ined at joint replacement surgery and we characterized the role of peroxyni trite to act as an upstream trigger. Design: A novel technique to isolate chondrocyte proteins (<10% of total ca rtilage protein) from cartilage specimens was developed. It was used to ana lyse JNK activation by a western blot technique. To examine the hypothesis that chondrocyte JNK activation is a result of increased peroxynitrite, in vitro experiments were performed in which cultured chondrocytes were incuba ted with this oxidant. Results: Activated JNK was detected in the cytoplasm of osteoarthritis (OA) affected chondrocytes but not in that of controls. In vitro, chondrocytes produce NO and superoxide anion. IL-1 (48 h), which induces nitric oxide sy nthase, resulted in an activation of JNK; this effect was reversed by N-mon omethylarginine (NMA). TNF<alpha> treated chondrocytes at 48 h produce supe roxide anion (EPR method). Exposure of cells to peroxynitrite led to an acc umulation of intracellular oxidants, in association with JNK activation and cell death by apoptosis. Conclusion: We suggest that JNK activation is among the IL-1 elicited respo nses that injure articular chondrocytes and this activation of JNK is depen dent on intracellular oxidant formation (including NO peroxynitrite). In ad dition, the extraction technique here described is a novel method that perm its the quantitation and study of proteins such as JNK involved in the sign aling pathways of chondrocytes within osteoarthritic cartilage. (C) 2001 Os teoArthritis Research Society International.