ERK和JNK/AP-1通路参与石英诱导的细胞周期改变

ERK,JNK/AP-1 pathway was involved in silica-induced cell cycle changes

目的探讨在石英刺激的人胚肺成纤维细胞（human embryo lung fibroblasts，HELF）中转录因子活化蛋白-1（activator protein-1，AP-1）的活性改变，以及丝裂原活化蛋白激酶（mitogen activated protein kinase，MAPK）／AP-1通路在石英诱导的细胞周期改变中的作用。方法用200μg／ml石英处理HELF细胞；用免疫荧光法检测细胞外调节蛋白激酶（extracellular signal-regulated protein kinase，ERK）和c-Jun氨基末端激酶（c-Jun N-terminal kinase，JNK）蛋白磷酸化水平及细胞分布；运用AP-1荧光素酶报告基因技术检测AP-1荧光素酶活力；用MAPK显性失活突变体（dominant negative mutant,DN）（DNERK2、DN-JNK1和DN-p38）证明通路的上下游关系；用流式细胞术检测细胞周期变化。结果用200μg／ml石英分别处理转染AP-1报告基因的细胞（HELF-AP-1）6、12、24h，结果显示，AP-1活性随着时间变化而发生变化，6h活性增强，12h活性达峰值，24h活性略有降低；用200μg／ml石英分别处理细胞1和2h，结果显示，ERK和JNK在石英刺激1h后，磷酸化水平升高，主要集中于胞浆，2h后磷酸化水平进一步升高，并主要集中于胞核；200μg／ml石英处理细胞24h，G1期细胞所占比例从（63．80±9．57）％下降到（32.23±7.22）％，S期细胞所占比例从（35．17±10．33）％升高到（66．00±8．07）％；AP-1化学抑制剂姜黄素（20μmol／L）可明显减弱石英引起的G1期细胞比例减少和S期细胞比例增加；DN-ERK和DN-JNK的过表达均可明显降低石英诱导的AP-1活性增强，DN-p38的过表达对石英诱导的AP-1活性增强无明显影响。结论200μg／ml石英可诱导AP-1活性增强，并通过ERK、JNK／AP-1通路诱导细胞周期改变。

Objective To investigate the alteration of activator protein-1 （AP- 1 ） luciferase activity in human embryo lung fibroblasts（HELF） after exposed to silica, and the role of mitogen activated protein kinese （MAPK）/AP-lpathway on silica-induced cell cycle changes. Methods After HELF cells were treated with 200 μg/ml silica, immunofluorescence assays were employed to detect the translocation and the phosphorylation level of extracellular singal-regulated protein kinase （ERK） and c-Jun N-terminal kinase （JNK）,flow cyo tometry was used to detect the distributions of cell cycle,the dominant negative mutant of ERK,JNK and p38 were applied to detect the upstream or downstream relationship of signaling pathways. Results After HELFAP-1 cells were exposed to 200 μg/ml silica 6,12,24 h respectively,silica exposure lead to AP-1 activation in a time-dependent manner, inducing significant AP-1 activation at 6 h, reaching a maximum activation at 12 h, and having a little decrease at 24 h. After silica exposure 1 h, phosphorylation level of ERK and JNK increased mainly in cytoplasm,however,after exposure 2 h,they tranlocated to nucleus. The proportion of cells in G, phases was decreased from （63.80±9.57）% to （32.23±7.22）% ,and the proportion of cells in S phases was increased from（ 35.17±10.33 ）% to （66.00±8.07）% after exposed to silica 24 h. Curcumin, a chemical inhibitor of AP-1 ,impaired the decrease of cells in G, phases. Furthermore we found expression of dominant-negative mutant of ERK and JNK impaired silica-induced AP-1 activation,whereas,dominant-negative mutant of p38 did not show the effect. Conclusion These result suggested that 200 μg/ml silica exposure can induce AP-1 activation, induce cell cycle changes through ERK, JNK/AP-1-dependent pathway.