Proinflammatory cytokines and MMPs promote synovial inflammation and facilitate cartilage and bone destruction in RA . The MAPKs (ERK, JNK and p38) contribute by phosphorylating key transcription factors, such as activator protein-1 (AP-1), that are required for gene transcription. JNK, in particular, plays a pivotal role in cytokine-mediated AP-1 induction and MMP gene expression in FLS [4, 19]. Three isoforms of JNK have been characterized, namely JNK1, 2 and 3. JNK1 and 2 are ubiquitous while JNK3 is primarily restricted to neurologic tissue . JNK2 deficiency has only modest effects in pre-clinical models of arthritis, but JNK1 deficiency attenuates synovitis and joint destruction in murine antigen-induced arthritis and passive K/BxN serum transfer arthritis [6, 7, 21]. JNK1 also contributes to osteoclast differentiation, since JNK1-deficient osteoclast progenitors do not mature into bone-resorbing osteoclasts . These data suggest that JNK participates in the synovial inflammation and joint destruction of RA and could potentially be targeted in diseases like RA.
While JNKs are attractive targets, they regulate in many normal cell functions, especially in matrix remodeling and host defense [23, 24]. Thus, blocking all JNK activity, or even all JNK1 activity, could affect host defense or matrix homeostasis. As an alternative strategy, targeting an individual upstream kinase like MKK4 or MKK7 could permit some normal JNK functions while interfering with a subset that is pathogenic in synovitis. MKK4 and MKK7, two JNK upstream kinases, exhibit some different properties although they can synergistically activate JNKs . TNF and IL-1 mainly activate MKK7 in murine embryonic fibroblasts, while ultraviolet radiation, anisomycin, heat and osmotic shock activate both MKK4 and MKK7 [8, 9, 25]. These data suggest that MKK4 and MKK7 contribute separately to the activation of JNKs in response to environmental stress or inflammatory cytokines.
We previously showed that MKK7, but not MKK4, is required for IL-1-induced JNK phosphorylation and AP-1-driven MMP expression . Nevertheless, MKK4 is a component of the JNK signal complex and is also readily phosphorylated in FLS . Mice lacking Gadd45β, which serves as an endogenous inhibitor of MKK7, have enhanced JNK activity and disease severity in the passive K/BxN model . These data suggest that selective MKK7 blockade could suppress arthritis and potentially decrease adverse effects by permitting non-pathogenic MKK4-mediated JNK activation. However, there is no direct evidence that MKK7 inhibition would be beneficial in synovitis. Our initial plans to focus on Gadd45β were complicated by the recent observation that Gadd45β deficiency unexpectedly exacerbates disease severity in collagen-induced arthritis .
We, therefore, focused on genetic approaches that circumvent the embryonic lethality of MKK7 deficiency. Several small interfering RNA (siRNA) methods were tested because others have reported success , but we were unable to consistently knockdown endogenous MKK7 expression (data not shown). Chemically modified ASOs were then tested for applications in animal models of RA because of their nuclease-resistant capacity, potency and long half-life [28–30]. Free ASOs are considerably smaller than siRNA-delivery agent complexes and enter many cells types via pinocytosis and phagocytosis, whereas larger siRNA complexes primarily enter macrophages and neutrophils by phagocytosis [31, 32]. Thus, we used single strand, 2'-O-methoxyethylribose modified chimeric ASOs to investigate the effect of MKK7 deficiency in mice. Selectivity was confirmed with MKK7 ASOs, which decreased MKK7 mRNA and protein expression but not MKK3, MKK4 or MKK6.
The ASO studies showed that selective MKK7 deficiency significantly reduced arthritis severity and joint destruction compared with control ASO-injected group even though MKK7 was only partially depleted. Downstream events were consistent with previous in vitro studies by demonstrating reduced phosphorylation of JNK and c-Jun in the inflamed joints of MKK7 ASO-treated mice. Decreased joint damage in mice treated with MKK7 ASOs is consistent with previous observations that MKK7 is a pivotal signaling molecule that regulates JNK and MMP expression in FLS .
Taken together, these results imply that MKK7 plays a pivotal role in inflammatory arthritis and that MKK7 ASO acts through the inhibition of JNK in passive K/BxN arthritis. Because JNK2 does not contribute to this model, the effect is most likely due to decreased JNK1 activation with resultant decreased mast cell activation . That observation is supported by the fact that JNK activation is abolished in mkk7-/- mast cell lines, suggesting that MKK7 is essential for JNK activation in mast cells .