Chondrocyte death in experimental osteoarthritis is mediated by MEK 1/2 and p38 pathways: role of cyclooxygenase-2 and inducible nitric oxide synthase.
OBJECTIVE
To explore the mechanisms responsible for in situ induction of chondrocyte death in experimental dog osteoarthritic (OA) cartilage. The roles of 2 mitogen activated protein kinases (MAPK), MEK 1/2 and p38, nuclear factor-kappaB (NF-kappaB), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and the caspase cascade were investigated.


METHODS
OA knee cartilage was obtained from dogs that had received sectioning of the anterior cruciate ligament and were sacrificed 12 weeks after surgery. Cartilage explants were cultured in different inhibitors: Z-DEVD-FMK (caspase 3 inhibitor), Z-LEHD-FMK (caspase 9 inhibitor), PD 98059 (MEK 1/2 inhibitor). SB 202190 (p38 inhibitor), SN-50 (NF-kappaB inhibitor), NS-398 (COX-2 inhibitor), N-iminoethyl-l-lysine (L-NIL) (iNOS inhibitor). Cartilage specimens were stained for TUNEL reaction and immunostained using specific antibodies for caspase 3, COX-2, iNOS, and nitrotyrosine. Morphometric analyses were performed.


RESULTS
The significant level of chondrocyte death in OA cartilage was markedly decreased by caspase 3 and caspase 9 inhibitors. The two MAPK inhibitors, but not the NF-kappaB inhibitor, decreased chondrocyte death concomitant with the levels of caspase 3 and iNOS. COX-2 level was reduced by all 3 inhibitors. Specific inhibition of either COX-2 or iNOS reduced the level of chondrocyte death and caspase 3. There was evidence of crosstalk between these 2 latter systems; specific inhibition of COX-2 reduced the iNOS level, and selective inhibition of iNOS reduced COX-2 expression. COX-2 and iNOS seem to function in a positive autoregulatory manner that triggers transcription of their own biosynthetic machinery, since the specific inhibition of each system downregulates its expression.


CONCLUSIONS
This study shows that in the early lesions of experimental OA cartilage in situ, activation of the caspase cascade is responsible for induction of chondrocyte death. Marked inhibition of cell death by caspase inhibitors indicates a significant participation of apoptosis in the phenomenon. This phenomenon is linked to the activation of at least 2 major kinase pathways, MEK 1/2 and p38. These pathways are responsible for upregulating the expression of iNOS and COX-2, each of which seems essential for the induction of apoptosis. Data are provided about possible regulation and interregulation of the COX-2 and iNOS systems by prostaglandin E2 and NO.
Pelletier
J P
JP
Osteoarthritis Research Unit, Hôpital Notre-Dame, Centre hospitalier de l'Université de Montréal, Québec, Canada.
Fernandes
J C
JC
Jovanovic
D V
DV
Reboul
P
P
Martel-Pelletier
J
J
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D002358:Cartilage, Articular / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D051546:Cyclooxygenase 2; D004285:Dogs; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020287:In Situ Nick-End Labeling; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D007719:Knee Joint; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D010003:Osteoarthritis / Q000378:metabolism* / Q000473:pathology; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases
D002352:Carrier Proteins; D061945:Caspase Inhibitors; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D016328:NF-kappa B; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D011505:Protein-Tyrosine Kinases; D017346:Protein Serine-Threonine Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D020929:Mitogen-Activated Protein Kinase Kinases; D020169:Caspases