Mapping out the mitogen activated protein kinase pathway

The mitogen activated protein (MAP) kinases represent another attractive target for rheumatoid arthritis (RA) because they can regulate cell proliferation, apoptosis, cytokine expression, and metalloproteinase production. They represent a complex, interrelated signal transduction mechanism that integrates extracellular stresses and induces an appropriate cellular response. The three major MAP kinase families, c-Jun-N-terminal kinase (JNK), extracellular regulating kinase (ERK) and p38 kinase, differ in their substrate specificity and subsequent responses to stress depending on the cell type and the environmental influences. The MAP kinases regulate various genes via both transcriptional and post-transcriptional mechanisms. The upstream MAP kinase kinases (MAPKK) serve as regulators of MAP kinase activity by phosphorylating specific threonine and tyrosine residues. MAPKKs are, in turn, regulated, by MAPKK kinases (MAPKKK or MAP3K). The p38 MAP kinase is of particular interest and several inhibitors have progressed into clinical trials. There are at least four isoforms of p38 (α, β, γ and δ), although the α form is probably the most important in macrophages for cytokine regulation. Many of the cytokine regulatory effects appear to be mediated through the downstream p38 substrate MAPKAP-2. Some evidence suggests that tumor necrosis factor production, in particular, is regulated by this kinase. Preclinical models demonstrate that p38 inhibitors are effective in a number of animal models of arthritis, including murine collagen-induced arthritis. Of note, p38 inhibitors also suppress joint destruction in these models, perhaps due to a combination of indirect effects on cytokine expression and a direct effect on metalloproteinase production. In vitro studies previously identified the MAPKKs MKK3 and MKK6 as the primary regulators of p38 phosphorylation and activation. To investigate a potential role for MKK3 and MKK6 in RA, we evaluated their expression and regulation in RA synovium and cultured fibroblast-like synoviocytes (FLS). Immunohistochemistry demonstrated that MKK3 and MKK6 are expressed in RA and osteoarthritis (OA) synovium. Digital image analysis showed no significant differences between OA and RA with regard to expression or distribution. However, phosphorylated MKK3/MKK6 expression was significantly higher in RA synovium and was localized to the sublining mononuclear cells and the intimal lining. Western blot analysis of synovial tissue lysates confirm the increased expression of phosphorylated MKK3/MKK6 in RA. Western blot analysis demonstrated constitutive expression of MKK3 and MKK6 in RA and OA FLS. Phospho-MKK3 levels were low in medium-treated FLS, but were rapidly increased by IL-1 and tumor necrosis factor alpha, although phospho-MKK6 levels only modestly increased. p38 co-immunoprecipitated with MKK3 and MKK6 from cytokine-stimulated FLS, and the complex phosphorylated ATF2 in an in vitro kinase assay. Studies using dominant negative constructs in cultured synoviocytes suggest that both are required for full activation of p38. These data are the first documentation of MKK3 and MKK6 activation in human inflammatory disease. By forming a complex with p38 in synovial tissue and FLS, these kinases can potentially be targeted to regulate the production of proinflammatory cytokine production in inflamed synovium.