Evidence for JNK-dependent up-regulation of proteoglycan synthesis and for activation of JNK(1) following cyclical mechanical stimulation in a human chondrocyte culture model

Y. Zhou, S. J. Millward-Sadler, H. Lin, H. Robinson, M. Goldring, D. M. Salter, G. Nuki

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Abstract / Description of output

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-28/12 and C-20/A4 human chondrocyte cell lines were mechanically stimulated in monolayer cell culture. PG synthesis was assessed by [S-35]-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-beta(1) 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 mu M SB203580, but was not affected by 50 mu M 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-28/12, 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 mu M 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. (c) 2007 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Original languageEnglish
Pages (from-to)884-893
Number of pages10
JournalOsteoarthritis and Cartilage
Volume15
Issue number8
DOIs
Publication statusPublished - Aug 2007

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