Activation of stress-activated protein kinase in osteoarthritic cartilage: evidence for nitric oxide dependence.
OBJECTIVE
We have demonstrated in bovine chondrocytes that nitric oxide (NO) mediates IL1 dependent apoptosis under conditions of oxidant stress. This process is accompanied by activation of c-Jun NH2-terminal kinase (JNK; also called stress-activated protein kinase). In these studies we examined activation of JNK in explant cultures of human osteoarthritic cartilage obtained at joint replacement surgery and we characterized the role of peroxynitrite to act as an upstream trigger.


METHODS
A novel technique to isolate chondrocyte proteins (<10% of total cartilage protein) from cartilage specimens was developed. It was used to analyse 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 incubated 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 synthase, resulted in an activation of JNK; this effect was reversed by N-monomethylarginine (NMA). TNFalpha treated chondrocytes at 48 h produce superoxide anion (EPR method). Exposure of cells to peroxynitrite led to an accumulation of intracellular oxidants, in association with JNK activation and cell death by apoptosis.


CONCLUSIONS
We suggest that JNK activation is among the IL-1 elicited responses that injure articular chondrocytes and this activation of JNK is dependent on intracellular oxidant formation (including NO peroxynitrite). In addition, the extraction technique here described is a novel method that permits the quantitation and study of proteins such as JNK involved in the signaling pathways of chondrocytes within osteoarthritic cartilage.
Clancy
R
R
Department of Rheumatology, Hospital for Joint Diseases and Division of Medicine, NYU School of Medicine, New York, New York, 10003, USA. rmc4@is2.nyu.edu
Rediske
J
J
Koehne
C
C
Stoyanovsky
D
D
Amin
A
A
Attur
M
M
Iyama
K
K
Abramson
S B
SB
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D015153:Blotting, Western; D002357:Cartilage Diseases / Q000201:enzymology*; D002358:Cartilage, Articular; D002417:Cattle; D019902:Chondrocytes / Q000201:enzymology; D005260:Female; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D008297:Male; D020929:Mitogen-Activated Protein Kinase Kinases / Q000502:physiology*; D009569:Nitric Oxide / Q000502:physiology*; D019001:Nitric Oxide Synthase / Q000502:physiology*; D052247:Nitric Oxide Synthase Type II; D020370:Osteoarthritis, Knee / Q000201:enzymology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D007375:Interleukin-1; D014409:Tumor Necrosis Factor-alpha; D009569:Nitric Oxide; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D048031:JNK Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4; D020929:Mitogen-Activated Protein Kinase Kinases