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 [(35)S]-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 microM SB203580, but was not affected by 50 microM 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 CO(2) deprivation. MS-induced JNK phosphorylation was inhibited by SB203580 at concentrations > or =5 microM 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 CO(2)/pH in this chondrocyte culture model.
Zhou
Y
Y
University of Edinburgh, Osteoarticular Research Group, Queen's Medical Research Institute, Edinburgh, Scotland, UK.
Millward-Sadler
S J
SJ
Lin
H
H
Robinson
H
H
Goldring
M
M
Salter
D M
DM
Nuki
G
G
D002245:Carbon Dioxide / Q000494:pharmacology; D018929:Cell Culture Techniques; D002461:Cell Line, Transformed; D019902:Chondrocytes / Q000166:cytology / Q000201:enzymology*; D015194:Diffusion Chambers, Culture; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology*; D048055:Mitogen-Activated Protein Kinase 8 / Q000378:metabolism*; D010766:Phosphorylation; D011509:Proteoglycans / Q000096:biosynthesis*; D011725:Pyridines / Q000494:pharmacology; D013314:Stress, Mechanical; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology
D004791:Enzyme Inhibitors; D007093:Imidazoles; D011509:Proteoglycans; D011725:Pyridines; D002245:Carbon Dioxide; D048055:Mitogen-Activated Protein Kinase 8; C093642:SB 203580