title: A novel spleen tyrosine kinase inhibitor blocks c-Jun N-terminal kinase-mediated gene expression in synoviocytes.
abstract: Spleen tyrosine kinase (Syk) is a key regulator of cell signaling induced by cytokines or Fc receptor 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-alpha (TNFalpha)-stimulated fibroblast-like synoviocytes (FLS) using the novel Syk inhibitor N4-(2,2-dimethyl-3-oxo-4H-pyrid[1,4]oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine (R406). Using 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 TNFalpha in FLS and was blocked by R406. Western blot analysis demonstrated that Syk inhibition by R406 markedly suppressed TNFalpha-induced c-Jun N-terminal kinase (JNK) phosphorylation in FLS, with a modest decrease in extracellular signal-regulated kinase phosphorylation. Surprisingly, p38 activation was not affected by R406. The Syk inhibitor also decreased TNFalpha-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 binding, as well as matrix metalloproteinase 3 gene expression. These data demonstrate that Syk activation plays an essential role in TNFalpha-induced cytokine and matrix metalloproteinase production in RA FLS, especially by suppressing activation of the JNK pathway.
authors.affiliation: Division of Rheumatology, Allergy and Immunology, University of California, San Diego, School of Medicine, La Jolla, CA 92093, USA.
authors.forename: Hoon-Suk
authors.initials: HS
authors.lastname: Cha
authors.forename: David L
authors.initials: DL
authors.lastname: Boyle
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Inoue
authors.forename: Reineke
authors.initials: R
authors.lastname: Schoot
authors.forename: Paul P
authors.initials: PP
authors.lastname: Tak
authors.forename: Polly
authors.initials: P
authors.lastname: Pine
authors.forename: Gary S
authors.initials: GS
authors.lastname: Firestein
mesh_terms: D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D019278:Matrix Metalloproteinase 3 / Q000235:genetics; D010078:Oxazines / Q000494:pharmacology*; D010766:Phosphorylation; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors* / Q000235:genetics; D011725:Pyridines / Q000494:pharmacology*; D000072377:Syk Kinase; D013583:Synovial Membrane / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; C510788:N4-(2,2-dimethyl-3-oxo-4H-pyrid(1,4)oxazin-6-yl)-5-fluoro-N2-(3,4,5-trimethoxyphenyl)-2,4-pyrimidinediamine; D010078:Oxazines; D011725:Pyridines; D018808:Transcription Factor AP-1; D014409:Tumor Necrosis Factor-alpha; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; D048031:JNK Mitogen-Activated Protein Kinases; D019278:Matrix Metalloproteinase 3