Presentation: Differential Activation of c-Jun N-terminal Kinase (JNK) In Synoviocytes by MKK4 and MKK7 (2007)

148 Differential Activation of c-Jun N-terminal Kinase (JNK) In Synoviocytes by MKK4 and MKK7

Purpose: JNK is a key regulator of matrix metalloproteinase (MMP) production in activated fibroblast-like synoviocytes (FLS). JNK is, in turn, activated by two upstream kinases known as MKK4 and MKK7. Recent studies show that only MKK7 is required for TNF-mediated JNK activation and MMP expression. However, the relative contributions of MKK4 and MKK7 have not been determined in FLS after stimulation by Toll-like receptor (TLR) ligands. To evaluate this question, we studied the effect of MKK4 or MKK7 deficiency on FLS activation by LPS, peptidoglycan (PGN), and poly (I-C), which are ligands for TLR4, 2, and 3, respectively.
Methods: MKK4 or MKK7 deficiency was induced in FLS by transfection with gene specific siRNA or scrambled siRNA. Cells were stimulated with LPS (1 ug/ml), PGN (50 ug/ml), or poly (I-C) (20 ug/ml) for 15 min to 24 hr. Phospho-JNK (P-JNK) and P-c-Jun were determined by Western blot analysis. Gene expression was determined by quantitative real time PCR.
Results: LPS, PGN, and poly (I-C) increased phosphorylation of JNK within 15 min. MKK4 deficiency had no effect on LPS or PGN induced P-JNK and P-c-Jun induction or MMP3 expression. In contrast, MKK7 knockdown suppressed LPS and PGN induced P-JNK (72-76% inhibition, p<0.05), P-c-Jun (38-69% inhibition, p<0.05), and MMP3 gene expression (57-60% inhibition, p<0.05). Surprisingly, poly (I-C) utilizes both MKK4 and MKK7: MKK4 deficiency decreased P-JNK (29% inhibition, p<0.05), P-c-Jun (43% inhibition, p<0.05) and MKK7 deficiency decreased P-JNK and P-c-Jun by 93 and 85% (p<0.005, respectively). Anti-viral genes (IP-10, IFN-ß) were also significantly lower in MKK4 knockdown FLS (28-34% inhibition, p<0.05) and MKK7 knockdown FLS (30-45% inhibition, p<0.05) after poly (I-C) stimulation compared with scrambled siRNA controls. Therefore, poly (I-C) can utilize both MKK4 and MKK7 while LPS and PGN only require MKK7. To investigate the mechanism of differential MKK utilization, we evaluated the time course of MKK4 and MKK7 activation after TLR stimulation. PGN and LPS, like TNF, induced rapid and transient MKK4 phosphorylation while intense P-MKK7 persisted for over an hour. However, this pattern was reversed after poly (I-C) stimulation, with persistent MKK4 activation and transient MKK7 activation.
Conclusion: JNK utilizes distinct signaling pathways depending on the specific TLR ligand. TLR2 and TLR4 ligands only require MKK7 while poly (I-C) and TLR3 signaling utilizes both MKK4 and MKK7. Distinct kinetics of MKK activation might account for the unique MKK profile of TLR3 compared with cytokines and other TLRs. Targeting an upstream kinase like MKK7 rather than JNK can potentially modulate cytokine and TLR2/4 responses while leaving anti-viral responses mediated by TLR3 intact.

 T. Yoshizawa, None; D. Hammaker, None; D.L. Boyle, None; G.S. Firestein, None.