c-Jun N-terminal kinase (JNK) contributes to
metalloproteinase (
MMP) gene expression and joint destruction in inflammatory
arthritis. It is phosphorylated by at least two upstream
kinases, the
mitogen-activated protein kinase kinases (
MEK) MKK4 and MKK7, which are, in turn, phosphorylated by
MEK kinases (
MEKKs). However, the
MEKKs that are most relevant to JNK activation in synoviocytes have not been determined. These studies were designed to assess the hierarchy of upstream
MEKKs, MEKK1, MEKK2, MEKK3, and
transforming growth factor-beta activated
kinase (TAK)1, in
rheumatoid arthritis (RA). Using either
small interfering RNA (
siRNA) knockdown or knockout fibroblast-like synoviocytes (FLSs), MEKK1, MEKK2, or MEKK3 deficiency (either alone or in combination) had no effect on IL-1beta-stimulated phospho-JNK (P-JNK) induction or
MMP expression. However, TAK1 deficiency significantly decreased P-JNK, P-MKK4 and P-MKK7 induction compared with scrambled control. TAK1 knockdown did not affect p38 activation.
Kinase assays showed that TAK1
siRNA significantly suppressed
JNK kinase function. In addition, MKK4 and MKK7
kinase activity were significantly decreased in TAK1 deficient FLSs. Electrophoretic mobility shift assays demonstrated a significant decrease in IL-1beta induced
AP-1 activation due to TAK1 knockdown. Quantitative PCR showed that TAK1 deficiency significantly decreased IL-1beta-induced MMP3 gene expression and
IL-6 protein expression. These results show that TAK1 is a critical pathway for IL-1beta-induced activation of JNK and JNK-regulated gene expression in FLSs. In contrast to other cell lineages, MEKK1, MEKK2, and MEKK3 did not contribute to JNK phosphorylation in FLSs. The data identify TAK1 as a pivotal upstream
kinase and potential therapeutic target to modulate synoviocyte activation in RA.