The
mitogen-activated protein kinase (MAPK)
c-Jun N-terminal kinase (JNK) is a critical regulator of
collagenase-1 production in
rheumatoid arthritis (RA). The MAPKs are regulated by upstream
kinases, including
MAPK kinases (
MAPKKs) and
MAPK kinase kinases (MAP3Ks). The present study was designed to evaluate the expression and regulation of the JNK pathway by MAP3K in
arthritis. RT-PCR studies of MAP3K gene expression in RA and
osteoarthritis synovial tissue demonstrated
mitogen-activated protein kinase/ERK
kinase kinase (
MEKK) 1, MEKK2, apoptosis-signal regulating kinase-1,
TGF-beta activated kinase 1 (TAK1) gene expression while only trace amounts of MEKK3, MEKK4, and MLK3
mRNA were detected. Western blot analysis demonstrated immunoreactive MEKK2, TAK1, and trace amounts of MEKK3 but not MEKK1 or apoptosis-signal regulating kinase-1. Analysis of MAP3K
mRNA in cultured fibroblast-like synoviocytes (FLS) showed that all of the MAP3Ks examined were expressed. Western blot analysis of FLS demonstrated that MEKK1, MEKK2, and TAK1 were readily detectable and were subsequently the focus of functional studies. In vitro
kinase assays using MEKK2 immunoprecipitates demonstrated that
IL-1 increased MEKK2-mediated phosphorylation of the key
MAPKKs that activate JNK (
MAPK kinase (MKK)4 and MKK7). Furthermore, MEKK2 immunoprecipitates activated c-Jun in an
IL-1 dependent manner and this activity was inhibited by the selective JNK inhibitor
SP600125. Of interest, MEKK1 immunoprecipitates from IL-1-stimulated FLS appeared to activate c-Jun through the JNK pathway and TAK1 activation of c-Jun was dependent on JNK, ERK, and p38. These data indicate that MEKK2 is a potent activator of the JNK pathway in FLS and that signal complexes including MEKK2, MKK4, MKK7, and/or JNK are potential therapeutic targets in RA.