Stimulation of cyclooxygenase-2-activity by nitric oxide-derived species in rat chondrocyte: lack of contribution to loss of cartilage anabolism1

Footnote 1:Abbreviations: ACT D, actinomycine D; CHX, cycloheximide; COX, cyclooxygenase; CuDips, Cu(II) (3,5-diisopropylsalicylate)2; DMSO, dimethyl sulfoxide; D-NMMA, Nω-monomethyl-D-arginine; FCS, heat-inactivated fetal calf serum; H2O2, hydrogen peroxide; IL-1β, interleukin-1β; L-Arg, L-arginine; L-NMMA, Nω-monomethyl-L-arginine; NO, Nitric oxide; NO2−, nitrites; NOS, nitric oxide synthase; NSAID, non steroidal anti-inflammatory drug; PGE2, prostaglandin E2; RT-PCR, reverse transcription-polymerase chain reaction; SIN-1, 3-morpholinosydnonimine; SMT, S-methylisothiourea; SOD, superoxide dismutase.; International audience; Cross-talk between inducible nitric oxide synthase (NOS II) and cyclooxygenase-2 (COX-2) was investigated in rat chondrocytes. In monolayers, interleukin-1beta (IL-1beta) induced COX-2 and NOS II expression in a dose- and time-dependent manner, to produce high prostaglandin E(2) (PGE(2)) and nitrite (NO(2)(-)) levels in an apparently coordinated fashion. COX-2 mRNA was induced earlier (30 min. versus 4 hr) and less markedly (4-fold versus 12-fold at 24 hr) than NOS II, and was poorly affected by the translational inhibitor cycloheximide (CHX). IL-1beta did not stabilize COX-2 mRNA in contrast to CHX. Indomethacin and NS-398 lacked any effect on NO(2)(-) levels whereas L-NMMA and SMT reduced PGE(2) levels at concentration inhibiting NO(2)(-) production from 50 to 90%, even when added at a time allowing a complete expression of both enzymes (8 hr). Basal COX activity was unaffected by NO donors. The SOD mimetic, CuDips inhibited COX-2 activity by more than 75% whereas catalase did not. Inhibition of COX-2 by CuDips was not sensitive to catalase, consistent with a superoxide-mediated effect. In tridimensional culture, IL-1beta inhibited radiolabelled sodium sulphate incorporation while stimulating COX-2 and NOS II activities. Cartilage injury was corrected by L-NMMA or CuDips but not by NSAIDs, consistent with a peroxynitrite-mediated effect. These results show that in chondrocytes: (i) COX2 and NOS II genes are induced sequentially and distinctly by IL-1beta; (ii) COX-1 and COX-2 activity are affected differently by NO-derived species; (iii) peroxynitrite accounts likely for stimulation of COX-2 activity and inhibition of proteoglycan synthesis induced by IL-1beta.