Received for publication October 26, 2005.
Revised January 24, 2006.
Accepted for publication January 24, 2006.

The novel Syk inhibitor R406 blocks JNK-mediated gene expression in synoviocytes

Abstract

Spleen tyrosine kinase (Syk) is a key regulator of cell signaling induced by cytokines or Fc receptors engagement. However the role of Syk in rheumatoid arthritis (RA) is not known yet. We investigated the pathways activated by Syk in tumor necrosis factor- (TNF)-stimulated RA fibroblast-like synoviocytes (FLS) using the novel Syk inhibitor, R406. On immunohistochemistry, Syk was detected in RA synovial tissue (ST), primarily in the synovial intimal lining. Western blot analysis demonstrated significantly greater amounts of phospho-Syk expression in RA ST compared with osteoarthritis ST. The kinase was expressed and functionally activated by TNF in FLS and was blocked by R406. Western blot analysis demonstrated that Syk inhibition by R406 markedly suppressed TNF-induced c-Jun N-terminal kinase (JNK) phosphorylation in FLS, with a modest decrease in extracellular signal-regulated kinase (ERK) phosphorylation. Surprisingly, p38 activation was not affected by R406. The Syk inhibitor also decreased TNF-induced mitogen-activated protein kinase kinase (MKK) 4 phosphorylation but not MKK3 and MKK6 phosphorylation, which is consistent with its selective sparing of p38. The connection between Syk and JNK was confirmed by demonstrating decreased phospho-c-Jun protein expression and complete inhibition of JNK function in R406- treated cells. R406 also suppressed downstream actions of JNK as determined by activator protein 1 (AP-1) binding as well as matrix metalloproteinase (MMP)-3 gene expression. These data demonstrate that Syk activation plays an essential role in TNF-induced cytokine and MMP production in RA FLS, especially by suppressing activation of the JNK pathway.

Key words: Inflammation, arthritis, cytokines, fibroblast, signal transduction, synovium