Mitogen-activated protein kinase and nuclear factor ?B together regulate interleukin-17-induced nitric oxide production in human osteoarthritic chondrocytes: Possible role of transactivating factor mitogen-activated protein kinase-activated protein kinase (MAPKAPK)

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Abstract
Objective To explore the signaling pathways by which the proinflammatory cytokine interleukin‐17 (IL‐17) may contribute to cartilage catabolism in osteoarthritis (OA) by inducing inducible nitric oxide synthase (iNOS) expression in chondrocytes. Methods We examined the IL‐17–induced NO production in human OA chondrocytes, in combination with the proinflammatory cytokines IL‐1β, tumor necrosis factor α (TNFα), and leukemia inhibitory factor (LIF); the antiinflammatory cytokines IL‐4, IL‐10, and IL‐13; and IL‐1 receptor antagonist (IL‐1Ra). Further, we explored the major intracellular signaling pathways through which IL‐17 induced iNOS expression and NO production. Results Treatment with IL‐17 induced a dose‐dependent increase in the level of NO. When IL‐17 was combined with the above factors, it resulted in a synergistic effect with TNFα, an additive effect with LIF, and no further effect than when used alone with IL‐1β. IL‐4, IL‐10, IL‐13, and IL‐1Ra had no true effect on IL‐17–induced NO production. The cAMP mimetics, 3‐isobutyl‐1‐methyl xanthine plus forskolin, completely blocked IL‐17–induced NO production. KT‐5720, genistein, and Calphostin C, inhibitors of protein kinase A (PKA), tyrosine kinase, and protein kinase C, respectively, reduced the IL‐17–induced NO production by 72%, 56%, and 42%, respectively. Within minutes, IL‐17 induced the phosphorylation of mitogen‐activated protein kinase kinase‐1/2 (MEK‐1/2), ‐3/6 (MKK‐3/6), p44/42, p38, and inhibitor of nuclear factor κB (IκB)‐α, as well as the activation of mitogen‐activated protein kinase–activated protein kinase‐1 and ‐2 (MAPKAPK‐1 and ‐2). Interestingly, IL‐17 induced phosphorylation of the stress‐activated protein kinase/Jun N‐terminal kinase (SAPK/JNK) (p54/46) only when PKA was inhibited. Specific protein kinase inhibitors for MEK‐1/2 (PD98059), p38 (SB202190), and nuclear factor κB (NF‐κB) (pyrrolidine dithiocarbamate) each markedly decreased the IL‐17–increased iNOS level and NO production. Inhibiting MAPK, including MEK‐1/2 and p38, had no effect on the IL‐17–induced activation of IκB‐α, but reversed the IL‐17 activation of MAPKAPK‐1 and ‐2, respectively. Conclusion These findings show that the stimulation of NO production by IL‐17 is mediated mainly by a complex activation of kinases, especially PKA, NF‐κB, and MAPK. NF‐κB appears to require MAPK activation, with downstream activation of MAPKAPK probably acting as a transactivating factor, to induce iNOS expression.

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