In this study, we demonstrated that OSM activates the JAK/STAT and MAPK pathways in FLS. Furthermore, CP690,550 blocked the OSM-induced STAT phosphorylation, probably by affecting upstream JAKs. These results strongly suggest that the JAK/STAT pathways are essential for OSM-mediated rheumatoid inflammatory responses, and can be blocked by CP690,550. The IL-6 type cytokine OSM is known to be involved in the pathogenesis of RA [
4]. It is secreted by activated T cells, monocytes, and neutrophils, and elevated levels of OSM are detected in the synovial fluid of patients with RA but not OA [
4,
5]. OSM is known to activate the JAK/STAT signaling cascade in other systems [
17], but this has not been properly elucidated in rheumatoid synoviocytes. Our demonstration of OSM-triggered phosphorylation of JAKs/STATs in primary rheumatoid synoviocytes suggests that these cells could be a source of activated STATs in rheumatoid synovial tissues [
18]. CP690,550 is an orally available JAK antagonist that is currently in development for the treatment of RA and other autoimmune conditions [
11‐
13]. Blockade of OSM-induced phosphorylation of JAK1, JAK2 and JAK3 by CP690,550 suggests that this JAK inhibitor also affected JAK1 and JAK2, in addition to JAK3. This notion was further supported by the abolishment of downstream STAT1, STAT3 and STAT5 activation and OSM-induced IL-6 production in rheumatoid synoviocytes. These results suggest the pivotal role of the JAK/STAT pathway in OSM signaling leading to rheumatoid inflammatory responses.
The biological agents targeted against tumor necrosis factor-α (TNF-α) have been shown to successfully suppress rheumatoid inflammation and have been thought to be more efficacious than disease-modifying anti-rheumatic drugs (DMARDs) in controlling joint damage [
19]. The combination of DMARDs with biological agents can increase the response rate; however, about 30% of patients do not respond to initial treatment [
20]. This has prompted research into alternative methods to suppress rheumatoid disease activity. JAK3 is critical for signal transduction from the common γ-chain of the receptors for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 on the plasma membrane to the nuclei of immune cells [
21]. These cytokines bind to cytokine receptors and signal through the JAK3-signal transducer and activator of transcription (STAT) pathways [
22]. Therefore, a better understanding of JAK/STAT activation in the rheumatoid synovium may allow the development of a novel therapeutic strategy. Agents that selectively inhibit JAK3 have the potential to mediate potent immune modulation, affecting lymphocytes, macrophages and NK cells [
23,
24]. CP690,550 was originally believed to be a JAK3 inhibitor. However, it is now clear that in addition to its effect on JAK3, this compound inhibits JAK1 and JAK2 at similar concentrations [
25]. Interestingly, CP690,550 was shown to be effective in preventing joint damage in collagen-induced arthritis (CIA), an animal model of rheumatoid arthritis [
26]. In CIA, IL-6 is a critical cytokine to induce arthritis [
27]. In this study, we have demonstrated that CP690,550 inhibited OSM-induced IL-6 production from rheumatoid synoviocytes by affecting JAK/STAT signaling. The exact mechanism by which CP690,550 prevents CIA remains to be determined, it is possible that CP690,550 inhibits IL-6 induction as well as IL-6-mediated signaling by affecting the JAK/STAT pathway. We have provided evidence that CP690,550 is a potent inhibitor of the JAK/STAT pathway with
in vitro activity in rheumatoid synoviocytes consistent with
in vitro enzyme assay. Although the signaling pathways for IL-6 induction by proinflammatory cytokines and stimuli have been reported in many cell types [
28], no data have been available on the regulation of IL-6 by OSM-signaling pathway. Our data clearly demonstrated that JAK/STAT activation play a pivotal role in OSM-mediated IL-6 up-regulation in RA. IL-6 is considered to be a critical cytokine that drives inflammatory joint destruction in RA [
28]. Furthermore, the targeting of IL-6 has been shown to induce a therapeutic benefit in RA [
29]. CP690,550 could be useful in blocking the JAK/STAT-mediated proinflammatory responses including IL-6-mediated signaling in RA.
The MAPKs play an important role in the induction of pro-inflammatory cytokines in rheumatoid synovitis [
30]. From the experiments using the specific MAPKs inhibitors, we concluded that OSM-mediated IL-6 release is weakly dependent on the ERK1/2 or JNK signaling, but depended mostly on the p38 signaling pathway. Our findings are consistent with previous studies showing the essential role of p38 in IL-6 production from activated rheumatoid synoviocytes [
31]. Dimerization of IL-6 type cytokine receptors does not only lead to activation of the JAK/STAT-signaling pathway, but also to the induction of MAPK [
17]. The relationship between the JAK/STAT pathway and the Ras/MAPK pathway is complex and these pathways cross at multiple levels [
32]. The molecular mechanism linking JAK/STAT engagement to the activation of MAPKs remains to be analyzed in detail. Our findings provide the possibility that p38 MAPK may be partly involved in the JAK/STAT-mediated IL-6 induction.
JAK1 and JAK2 are more widely expressed, whereas JAK3 expression is mostly limited to haematopietic cell lines [
33]. Although JAK3 has been identified as a potential therapeutic target in autoimmune disease, its role in rheumatoid synovitis has not been fully elucidated. In this study, we clearly demonstrated the JAK-family including JAK3 could be activated in FLS in response to an IL-6-type cytokine, OSM. Our data suggest that cytokine-activated JAKs in FLS could be an appropriate therapeutic target in inflammatory arthritis including RA.