酪氨酸激酶在TNF-α诱导类风湿关节炎成纤维样滑膜细胞MAPKs活化中的作用

摘要

Objective To study on MAPKs (mitogen-activated protein kinases)activation in rheumatoid arthritis fibroblast-like synoviocytes (RA FLS) under the stimulation of TNF-α, and to elucidate the role of protein tyrosine kinase (PTK) in activation of MAPKs. Methods RA FLS were primarily cultured. Western blots were applied to examine transient change of protein tyrosine phosphorylation status and MAPKs activation in RA FLS stimulated with various doses and periods of TNF-α. Genistein, the specific PTK inhibitor was used to evaluate the inhibitory role in activation of MAPKs by TNF-α. Results TNF-α transiently increased protein tyrosine phosphorylation, and activated MAPKs cascades (mainly ERK2, JNK2, P38) in RA FLS. The peak activation of ERK2 and JNK2 were obtained at 10IU/ml, but that of P38 was at 100IU/ml. The maximal activation of ERK2, JNK2 and P38 was observed at 5, 15 and 15 minutes respectively after stimulation with TNF-α. The activation of ERK2 was strongly inhibited by genistein, but the inhibitory role on that of JNK and P38 was relatively weak. Conclusion During signal transduction of TNF-α in RA FLS, tyrosine phosphorylation were increased transiently, MAPKs cascades were activated in a few minutes, PTK plays a significant role in the activation of ERK, but has only weak effect on that of JNK and P38.%目的 研究在类风湿关节炎成纤维样滑膜细胞（RAFLS）信号转导中，酪氨酸激酶在TNF-α刺激下丝裂原活化蛋白激酶(mitogen-activatedproteinkinases，MAPKs)活化中的作用。方法 原代培养类风湿关节炎成纤维样滑膜细胞。应用Westernblot检测TNF-α短时间内引起RAFLS蛋白质酪氨酸磷酸化状态改变，及其对MAPKs家族成员活化的浓度效应和时相特点；并应用genistein，酪氨酸激酶（PTK）抑制剂观察对MAPKs活化的抑制情况。结果 TNF-α可以瞬时引起RAFLS蛋白质酪氨酸磷酸化程度增加；并在短时间内激活MAPKs通路（ERK2、JNK2、P38）。不同浓度梯度TNF-α作用显示：10IU/ml时对ERK2、JNK2即可达到峰值活化，100IU/ml时P38达到最大活化。时间上，ERK2、JNK2、P38的活化分别在TNF-α作用后5min、15min、15min最明显；genistein对TNF-α诱导的ERK2活化抑制作用显著，而对于JNK2、P38的抑制则较弱。结论 TNF-α在RAFLS信号转导中，可以瞬时导致蛋白质酪氨酸磷酸化程度增加，并同时激活MAPKs3条通路，但是对MAPKs3个亚家族成员的活化具有异质性；PTK在TNF-α导致ERK活化中发挥作用，对JNK、P38活化无明显影响。