Evidence for JNK-dependent up-regulation of proteoglycan synthesis and for activation of JNK1 following cyclical mechanical stimulation in a human chondrocyte culture model

Objective: To examine the expression of mitogen-activated protein kinases (MAPKs) in human chondrocytes, to investigate whether selective activation of MAPKs is involved in up-regulation of proteoglycan (PG) synthesis following cyclical mechanical stimulation (MS), and to examine whether MS is associated with integrin-dependent or independent activation of MAPKs. Methods: The C-28/I2 and C-20/A4 human chondrocyte cell lines were mechanically stimulated in monolayer cell culture. PG synthesis was assessed by [35S]-sulphate incorporation in the presence and absence of the p38 inhibitor SB203580, and the extracellular-regulated kinase (ERK1/2) inhibitor PD98059. Kinase expression and activation were assessed by Western blotting using phosphorylation status-dependent and independent antibodies, and by kinase assays. The Jun N-terminal kinase (JNK) inhibitor SP600125 and the anti-β1 integrin (CD29) function-blocking antibody were used to assess JNK activation and integrin dependence, respectively. Results: Increased PG synthesis following 3 h of cyclic MS was abolished by pretreatment with 10 μM SB203580, but was not affected by 50 μM PD98059. The kinases p38, ERK1/ERK2 and JNKs were expressed in both stimulated and unstimulated cells. Phosphorylated p38 was detected at various time points following 0.5, 1, 2 and 3 h MS in C-28/I2, but not detected in C-20/A4 cell lines. Phosphorylation of ERK1 and ERK2 was not significantly affected by MS. Phosphorylation of the 54 and 46 kDa JNKs increased following 0.5, 1, 2 and 3 h of MS, and following CO2 deprivation. MS-induced JNK phosphorylation was inhibited by SB203580 at concentrations ≥5 μM and activation of JNK1 following MS was blocked by SP600125 and partially inhibited by anti-CD29. Conclusions: The data suggest JNK, rather than p38 or ERK dependent increases in PG synthesis, and selective, partially integrin-dependent, activation of JNK kinases in human chondrocyte cell lines following cyclical MS. JNK activation is also very sensitive to changes in CO2/pH in this chondrocyte culture model. © 2007 Osteoarthritis Research Society International.