摘要
目的利用具有未成熟多巴胺神经前体细胞特性的杂交瘤细胞系MN9D,探讨Forskolin激活Nurr1表达的分子信号机制。Nurr1是未知配体的核转录因子,对中脑多巴胺神经元的发育至关重要。方法①用Forskolin作用MN9D细胞1～6h,提取细胞蛋白,Westernblot方法检测MN9D细胞内源性Nurr1表达的变化,并利用磷酸化的ERK1/2(pERK1/2)抗体检测Forskolin作用后MN9D细胞内ERK信号转导通路是否被激活。②应用ERK信号转导通路的特异性抑制剂U0126预孵育MN9D细胞,再用Forskolin作用,检测MN9D细胞Nurr1表达的变化。结果①从Forskolin作用1h起,直至6h,MN9D细胞核内Nurr1表达均比未加Forskolin作用组明显增加。同时,MN9D细胞内pERK1/2蛋白含量迅速增加,在1h起即达到差异有显著性(P<0.05),并持续保持在较高水平,直至Forskolin作用6h。而Forskolin作用前后MN9D细胞内ERK1/2总蛋白的表达量基本保持不变。②用MEK1/2的特异性抑制剂U0126预处理,可有效抑制MN9D细胞内ERK信号转导通路的激活,并明显阻断Forskolin引起的MN9D细胞内源性Nurr1表达增加。结论ERK信号通路在Forskolin引起的MN9D细胞内源性Nurr1表达及核转位增加中发挥重要作用。
Aim The aim of this study is to investigate the molecular signaling mechanism of activating Nurrl expression by Forskolin in a dopamine-synthesizing cell line （MN9D） with immature characteristics. Nurrl is a transcription factor essential to the development of midbrain dopaminergic neurons. Methods The MN9D cells were treated with Forskolin. The changes of Nurrl expression were analyzed with Western blot. The molecular signaling mechanism and pathways that lead to the activation of Nurrl expression were studied through using some specific antibodies and pharmacological inhibitors of extracellular signal regulated kinase （ERK） signaling pathways. Results Treatment with Forskolin from 1 h to 6h up-regulated Nurrl protein level significantly in MN9D cells（P 〈0.05）. Using phospho-ERK1/2 antibody, we detected the activation of ERK1/2 in MN9D cells following Forskolin treatment. Moreover, treatment with the MEK1/2 inhibitor, U0126,inhibited the ERK1/2 activation in MN9D cells and blocked the Forskolin induced up-regulation of Nurrl protein expression and nuclear translocation.Conclusion Forskolin induced up-regulation of Nurrl protein expression and nuclear translocation in MN9D cells is mediated primarily through the ERK1/2 pathway.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2005年第8期922-926,共5页
Chinese Pharmacological Bulletin
基金
国家重点基础研究发展规划(973规划)资助课题(NoG1999054008)
国家自然科学基金(No30270433)
首都医学发展科研基金资助项目
北京市优秀人才培养专项经费
北京市"科技新星"计划资助项目
