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To elucidate the role of endogenous inducible nitric oxide (NO) on the regulation of NF-kappaB activity in human chondrocytes, we evaluated (i) the pattern of expression of the neuronal (nNOS) and inducible (iNOS) NO synthase isoforms and the basal NF-kappaB activity in normal and osteoarthritic (OA) human chondrocytes, (ii) the role of cytokines and growth factors in modulating the protein levels of the two NOS isoforms, and (iii) the effect of inhibiting endogenous inducible NO production on the ability of interleukin-1beta (IL-1) to induce NF-kappaB activation. nNOS was more frequently expressed in normal than in OA chondrocytes, whereas the opposite was found for iNOS. IL-1 induced the degradation of both enzymes, but iNOS disappeared more rapidly. Although IkappaB-alpha was present in all the normal samples and in the majority of the OA samples, NF-kappaB-DNA binding activity in OA chondrocytes was increased approximately twofold relatively to normal cells. Addition of a NOS inhibitor, after induction of iNOS expression, induced IkappaB-alpha degradation and potenciated the effect of IL-1, indicating that endogenous inducible NO inhibits NF-kappaB activation. Taken together, these findings favor an inhibitory role of high NO levels on the regulation of NF-kappaB activation in chondrocytes, indicating that NF-kappaB activity is regulated, at least in part, by the balanced production of NO resulting from a dynamic process that, at any given moment, determines the availability of the constitutive and inducible NOS isoforms. Moreover, the down-regulatory role of NO on NF-kappaB activation warrants caution as to the possible utilization of NO inhibitors in the therapy of OA.

Nitric oxide (NO), produced by the inducible isoform of the NO synthase (iNOS), plays an important role in the pathophysiology of arthritic diseases. This work aimed at elucidating the role of the mitogen-activated protein kinases (MAPK), p38MAPK and p42/44MAPK, and of protein tyrosine kinases (PTK) on interleukin-1beta (IL-1)-induced iNOS expression in bovine articular chondrocytes. The specific inhibitor of the p38MAPK, SB 203580, effectively inhibited IL-1-induced iNOS mRNA and protein synthesis, as well as NO production, while the specific inhibitor of the p42/44MAPK, PD 98059, had no effect. These responses to IL-1 were also inhibited by treatment of the cells with the tyrosine kinase inhibitors, genistein and tyrphostin B42, which also prevented IL-1-induced NF-kappaB activation. The p38MAPK inhibitor, SB 203580, had no effect on IL-1-induced NF-kappaB activation. Finally, the p42/44MAPK inhibitor, PD 98059, prevented IL-1-induced AP-1 activation in a concentration that did not inhibit iNOS expression. In conclusion, this study shows that (1) PTK are part of the signaling pathway that leads to IL-1-induced NF-kappaB activation and iNOS expression; (2) the p38MAPK cascade is required for IL-1-induced iNOS expression; (3) the p42/44MAPK and AP-1 are not involved in IL-1-induced iNOS expression; and (4) NF-kappaB and the p38MAPK lie on two distinct pathways that seem to be independently required for IL-1-induced iNOS expression. Hence, inhibition of any of these two signaling cascades is sufficient to prevent iNOS expression and the subsequent production of NO in articular chondrocytes.