c-Jun氨基末端激酶和细胞外调节蛋白激酶信号通路对苯并(a)芘诱导人胚肺成纤维细胞c-Jun活化的调控

目的探讨丝裂原活化蛋白激酶（MAPKs）信号通路在调控苯并（a）芘[B（a）P]诱导人胚肺成纤维细胞（HELF）c—Jun活化中的作用。方法2．0μmol/LB（a）P处理HELF0、3、6、12、24h后或加入不同浓度B（a）P（0．0、0．5、1．0、2．0μmol/L）处理12h后，通过免疫印迹法检测B（a）P对细胞c-Jun活性的影响；利用p38、c—Jun氨基末端激酶（JNK）以及细胞外调节蛋白激酶（ERK）的显性失活突变体分别阻断p38、JNK和ERK活性后，观察3种MAPKs信号分子与B（a）P诱导c-Jun活化之间的关系。结果在所观察的B（a）P作用时间和浓度范围内，c—Jun蛋白表达量无明显变化；B（a）P可诱导细胞内磷酸化c—Jun（Ser63／Ser73）水平增高，并随着作用时间的延长，细胞内c—Jun的磷酸化水平也逐渐增强，至12h达到峰值（1．61±0．12，1．82±0．18），c-Jun磷酸化Ser63、Ser73与actin灰度比值分别是对照组的20．1倍、15．2倍，作用24h时细胞内c-Jun磷酸化水平呈现下降趋势，而且随着B（a）P浓度的增加，细胞内c-Jun的磷酸化水平也逐渐升高，呈现剂量-反应关系；使用显性失活突变体分别阻断JNK和ERK活性均可明显抑制B（a）P诱导细胞c—Jun磷酸化水平增加，但是阻断p38活性对B（a）P诱导细胞c—Jun磷酸化水平升高无明显影响。结论JNK和ERK信号通路调控B（a）P诱导的HELF细胞c—Jun活化，B（a）P促进c-Jun磷酸化的过程与p38信号通路无关。

c-Jun NH2-terminal kinase and extracellular signal-regulated protein kinase signaling pathways in regulation of benzo(a) pyrene-induced c-Jun activation in human embryo lung fibroblasts

OBJECTIVE: To investigate the role of mitogen activated protein kinases (MAPKs) signaling pathways in the regulation of benzo(a)pyrene (B(a)P)-induced c-Jun activation in human embryo lung fibroblasts (HELFs). METHODS: HELFs were cultured with 2.0 micromol/L B(a)P for various time (0, 3, 6, 12, 24 h) or with various concentration of B(a)P (0.0, 0.5, 1.0, 2.0 micromol/L) for 12 h. Western blot was performed to examine the effect of B(a)P on c-Jun activation. The dominant negative mutants of p38, c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated protein kinase (ERK) were applied to establish stable transfectant, and to detect the relationship of MAPK signal molecules and c-Jun activation in B (a) P-treated cells. RESULTS: B(a)P treatment resulted in a marked activation of c-Jun in time-dependent manner with a peak at 12 h (the densitometric ratios of phosphorylated c-Jun Ser63, Ser73 to actin were 20.1, 15.2 times for control respectively) and in dose-dependent manner. However, there was no evident change on total c-Jun expression in B(a)P-treated HELFs. Moreover, B(a)P-induced activation of c-Jun was inhibited by stable expression of dominant negative mutants of JNK or ERK, but not by dominant negative mutant of p38. CONCLUSION: JNK and ERK signaling pathways, but not p38 pathway regulate B(a)P-induced c-Jun activation in HELFs.