Forskolin上调MN9D细胞Nurr1的表达及Nurr1过表达对酪氨酸羟化酶的影响

目的寻找能激活MN9D细胞内源性孤儿核受体（Nurr1）表达的信号分子，研究Nurr1表达增高对酪氨酸羟化酶（TH）表达的影响，探讨激活Nurr1表达的信号机制。方法用蛋白激酶A激动剂Forskolin或蛋白激酶C激动剂PMA作用于MN9D细胞，用免疫荧光细胞化学及Westernblot方法检测MN9D细胞内源性Nurr1表达的变化，以及Nurr1表达增加对TH表达的影响。利用PKA信号转导通路的特异性抑制剂H89探讨激活Nurr1表达的信号机制。结果1．从加入Forskolin1h起，直至6h，MN9D细胞Nurr1表达比未加Forskolin组明显增加（P〈0．05）；Forskolin主要增加MN9D细胞核内Nurr1含量，而细胞浆内Nurr1含量变化不明显；Forskolin作用后MN9D细胞TH表达无明显变化。2．用蛋白激酶A的特异性抑制剂H89预处理后，Forskolin仍具有增加Nurrl表达的作用。结论Forskolin可增加MN9D细胞内源性Nurr1表达及核转位，单纯Nurr1表达及核转位增加不影响MN9D细胞TH的表达，TH表达的激活可能还需要其他特殊的细胞（或神经元）环境或共激活因子的共同作用。Forskolin增加MN9D细胞内源性Nurr1表达及核转位的作用不是主要通过激活PKA信号转导通路完成的。

UP-REGULATION OF NURR1 EXPRESSION INDUCED BY FORSKOLIN IN MN9D CELLS AND THE EFFECT OF NURR1 ON TYROSINE HYDROXYLASE EXPRESSION

Objectives To investigate the molecular signaling mechanism activating Nurrl expression in a dopamine-synthesizing cell line(MN9D)with immature characteristics and to study whether tyrosine hydroxylase(TH) expression is up-regulated in response to Nurrl activation. Methods The MN9D cells were treated with protein kinase A activator Forskolin or protein kinase C activator PMA.The changes of Nurrl and TH expression were analyzed by immuneocytochemistry and Western blot.The molecular signaling mechanism that leads to the activation of Nurrl expression was studied by using specific inhibitors H89 for protein kinase A signaling pathways. Results 1.Treatment with Forskolin from 1 h to 6 h up-regulate Nurrl protein level significantly in MN9D cells(P<0.05)and the newly synthesized Nurrl protein is targeted in the nucleus.The expression of TH protein in MN9D cells didn't change significanty following Nurrl up-regulation induced by Forskolin.2.Pretreated with PKA inhibitor H89 couldn't block the up-regulation of Nurrl protein expression induced by Forskolin. Conclusions Forskolin up-regulates Nurrl protein expression and promotes its nuclear translocation in MN9D cells.Up-regulation of Nurrl expression and nuclear translocation alone might be insufficient for activating TH gene expression in MN9D cells,but rather requires specific cellular(or neuronal) environments or cofactors.Activation of Nurrl protein expression and nuclear translocation by Forskolin in MN9D cells is not mediated primarily through the protein kinase A signaling pathway.