Evidence for JNK-dependent up-regulation of proteoglycan synthesis and for activation of JNK1 following cyclical mechanical stimulation in a human chondrocyte culture model.
Evidence for JNK-dependent up-regulation of proteoglycan synthesis and for activation of JNK1 following cyclical mechanical stimulation in a human chondrocyte culture model.
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DOI：
10.1016/j.joca.2007.02.001
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发表时间：
2007-08
影响因子：
7
通讯作者：
Y. Zhou;S. J. Millward-Sadler;H. Lin;H. Robinson;M. Goldring;D. Salter;G. Nuki
中科院分区：
文献类型：
--
作者：
Y. Zhou;S. J. Millward-Sadler;H. Lin;H. Robinson;M. Goldring;D. Salter;G. Nuki
目的检测人软骨细胞中丝裂原活化蛋白激酶（MAPKs）的表达，探讨周期性机械刺激（MS）后，MAPKs的选择性激活是否参与了蛋白多糖（PG）合成的上调，以及MS是否与整合素依赖或独立的MAPKs激活有关。方法采用机械刺激法培养C-28/I2和C-20/A4人软骨细胞系。在p38抑制剂SB203580和细胞外调节激酶（ERK1/2）抑制剂PD98059存在和不存在的情况下，通过[35S]-硫酸盐掺入来评估PG的合成。激酶的表达和激活通过磷酸化状态依赖和独立抗体的Western blotting和激酶测定来评估。使用Jun n-末端激酶（JNK）抑制剂SP600125和抗β1整合素（CD29）功能阻断抗体分别评估JNK激活和整合素依赖性。结果10μM SB203580预处理可消除3h后PG合成的增加，而50μM PD98059预处理对PG合成无影响。激酶p38、ERK1/ERK2和JNKs在受刺激和未受刺激的细胞中均有表达。p38磷酸化在C-28/I2细胞株中分别于0.5、1、2和3h出现，但在C-20/A4细胞株中未出现。MS对ERK1和ERK2的磷酸化没有显著影响，MS作用0.5、1、2和3h以及co2剥夺后，54和46kDa JNKs的磷酸化增加。MS诱导的JNK磷酸化被浓度≥5μM的SB203580抑制，MS后JNK1的活化被SP600125阻断，部分被抗cd29抑制。结论数据表明，在周期性ms后，人软骨细胞系中JNK激酶的选择性、部分整合素依赖性激活对CO2/pH的变化也非常敏感，而不是p38或ERK依赖性的PG合成增加。
OBJECTIVETo 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.METHODSThe 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-β1integrin (CD29) function-blocking antibody were used to assess JNK activation and integrin dependence, respectively.RESULTSIncreased PG synthesis following 3h of cyclic MS was abolished by pretreatment with 10μM SB203580, but was not affected by 50μM 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 3h 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 46kDa JNKs increased following 0.5, 1, 2 and 3h of MS, and following CO2deprivation. MS-induced JNK phosphorylation was inhibited by SB203580 at concentrations ≥5μM and activation of JNK1 following MS was blocked by SP600125 and partially inhibited by anti-CD29.CONCLUSIONSThe 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.