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

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Presentation transcript:

Evidence for JNK-dependent up-regulation of proteoglycan synthesis and for activation of JNK1 following cyclical mechanical stimulation in a human chondrocyte culture model Y. Zhou, M.D., S.J. Millward-Sadler, Ph.D., H. Lin, M.Phil., H. Robinson, M.D., M. Goldring, Ph.D., D.M. Salter, M.D., G. Nuki, M.B., F.R.C.P. Osteoarthritis and Cartilage Volume 15, Issue 8, Pages 884-893 (August 2007) DOI: 10.1016/j.joca.2007.02.001 Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 1 Apparatus employed for MS. (a) Application of intermittent strain from above the cell culture dishes. (b) Application of intermittent strain from below the cell culture dishes. Osteoarthritis and Cartilage 2007 15, 884-893DOI: (10.1016/j.joca.2007.02.001) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 2 Effects of MS and MAPK inhibitors on PG synthesis in C-20/A4 cells. (a) Effect of SB203580 on PG synthesis following MS. Mean (±s.e.m.) [35S]-sulphate incorporation with or without 30min pretreatment with 10μM SB203580, followed by 3h of MS (n=6). (b) Effect of PD98059 on PG synthesis following MS. Mean (±s.e.m.) [35S]-sulphate incorporation with or without 30min pretreatment with 50μM PD98059 followed by 3h of MS (n=12). CP: cyclical pressure control. MS: cyclical mechanical strain. Osteoarthritis and Cartilage 2007 15, 884-893DOI: (10.1016/j.joca.2007.02.001) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 MAPK expression and activation in C-28/I2 and C-20/A4 cells following MS. Western blots using phosphorylation status-dependent and independent antibodies against p38, ERK1/2 (44 and 42kDa) and JNK kinases (54 and 46kDa) following 0.5, 1, 2, and 3h of cyclical MS. (a) Expression and phosphorylation of p38 following MS (n=3). Western blots show phosphorylation of p38 in C-28/I2 cells, but not in C-20/A4 cells following MS. (b) Expression and phosphorylation of ERK1/2 following MS. Western blots show that there is no significant change in phosphorylation following MS (n=3). (c) Expression and phosphorylation of JNK following MS. Western blots show phosphorylation of 54 and 46kDa JNK following 0.5, 1, 2 and 3h of cyclic MS (n=3). Osteoarthritis and Cartilage 2007 15, 884-893DOI: (10.1016/j.joca.2007.02.001) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 The effects of SB203580 on phosphorylation of JNK in C-28/I2 and C-20/A4 cells. Western blots using phosphorylation dependent antibody show inhibition of JNKs by SB203580 at concentrations ≥5μM. Osteoarthritis and Cartilage 2007 15, 884-893DOI: (10.1016/j.joca.2007.02.001) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 The effects of MS, inhibition of JNK and an anti-β1 integrin antibody (anti-CD29) on JNK activation using a kinase assay. (a) JNK1 activation following MS. Activation of JNK1 following 1h cyclical MS in the C-28/I2 and C-20/A4cell lines (n=3). (b) Effects of the JNK inhibitor SP600125 on JNK1 activity following MS. Pre-incubation with SP600125 blocks the activation of JNK1 in C-28/I2 following 1h cyclical MS at concentrations ≥10μM (n=3). (c) Effects of anti-CD29 on JNK activation following MS. Mouse anti-human CD 29 partially blocked the activation of JNK1 following 1h cyclical MS in C-28I2 and C-20/A4 cells (n=3). Osteoarthritis and Cartilage 2007 15, 884-893DOI: (10.1016/j.joca.2007.02.001) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 The effects of CO2 and MS on JNK phosphorylation in C-28/I2 cells. Western blots showing phosphorylation of 54 and 46kDa JNK protein kinases when cells left in the pressure chamber without CO2 for 3h or following 0.5, 1, 2, and 3h cyclical MS. Ct=Control in CO2 incubator. CL=Control in stimulation chamber without CO2 (n=3). Osteoarthritis and Cartilage 2007 15, 884-893DOI: (10.1016/j.joca.2007.02.001) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions

Fig. 7 The influence of the control environment on JNK phosphorylation in C-28/I2 cells. Western blots showing phosphorylation of JNK protein kinases (54 and 46kDa) in controls. Ct=Control in CO2 incubator. Cb=Control in sealed dishes in pressure incubator. CL=Control in pressure incubator without CO2. Cs=Control in pressure chamber without MS (n=3). Osteoarthritis and Cartilage 2007 15, 884-893DOI: (10.1016/j.joca.2007.02.001) Copyright © 2007 Osteoarthritis Research Society International Terms and Conditions