Regulation of JNK by MKK‐7 in fibroblast‐like synoviocytes

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Abstract
Objective: JNK regulates matrix metalloproteinase (MMP) gene expression and joint destruction in rheumatoid arthritis (RA). Previous studies demonstrated that the 2 upstream MAPK kinases (MKK‐4 and MKK‐7) are phosphorylated in RA synovium and form a complex with JNK in fibroblast‐like synoviocytes (FLS). However, the functional hierarchy of MKK‐4 and MKK‐7 in FLS has not been determined. We determined the relative contributions of these MKKs by evaluating the effect of MKK‐4 and MKK‐7 gene knockdown in cultured FLS.Methods: FLS were transfected with MKK‐4 and/or MKK‐7 small interfering RNA, and protein levels were determined by immunoblotting. After stimulation with interleukin‐1β (IL‐1β), tumor necrosis factor α (TNFα), or anisomycin, kinase function was determined by in vitro kinase assay. Activator protein 1 (AP‐1) binding and transcriptional activity were determined by electrophoretic mobility shift assay and AP‐1–luciferase promoter assay, respectively. MMP‐3 expression was determined by enzyme‐linked immunosorbent assay and quantitative polymerase chain reaction.Results: IL‐1β–induced JNK phosphorylation was dependent on MKK‐7 but not on MKK‐4; however, anisomycin‐activated JNK required both kinases. In vitro kinase assay demonstrated that IL‐1β– or TNFα‐induced JNK activity was only MKK‐7 dependent, while anisomycin‐activated JNK was both MKK‐4 and MKK‐7 dependent. IL‐1β–induced AP‐1 binding activity and AP‐1–driven gene expression were strictly MKK‐7 dependent. Finally, MMP‐3 production only required MKK‐7, and there was no effect of MKK‐4 deficiency.Conclusion: These data indicate that only MKK‐7 is required for JNK activation in FLS after cytokine stimulation; however, other forms of cellular stress utilize MKK‐4. Thus, JNK function might be modulated by targeting MKK‐7 to suppress cytokine‐mediated FLS activation while leaving other stress responses intact.

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