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 c hondrocyte death in experimental dog osteoarthritic (OA) cartilage. The rol es 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 section ing of the anterior cruciate ligament and were sacrificed 12 weeks after su rgery. 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). Cartila ge specimens were stained for TUNEL reaction and immunostained using specif ic antibodies for caspase 3, COX-2, iNOS, and nitrotyrosine. Morphometric a nalyses were per-formed. Results. The significant level of chondrocyte death in OA cartilage was mar kedly decreased by caspase 3 and caspase 9 inhibitors. The two MAPK inhibit ors, but not the NF-kappaB inhibitor, decreased chondrocyte death concomita nt 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 o f 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 WOS reduced COX-2 expression. COX-2 and WOS s eem to function in a positive autoregulatory manner that triggers transcrip tion of their own biosynthetic machinery, since the specific inhibition of each system downregulates its expression. Conclusion. This study shows that in the early lesions of experimental OA c artilage in situ, activation of the caspase cascade is responsible for indu ction of chondrocyte death. Marked inhibition of cell death by caspase inhi bitors indicates a significant participation of apoptosis in the phenomenon . This phenomenon is linked to the activation of at least 2 major kinase pa thways, MEK 1/2 and p38. These pathways are responsible for upregulating th e expression of iNOS and COX-2, each of which seems essential for the induc tion of apoptosis. Data are provided about possible regulation and inter-re gulation of the COX-2 and iNOS systems by prostaglandin E-2 and NO.