Neuronal nitric oxide synthase, as a downstream signaling molecule of c-jun, regulates the survival of differentiated PC12 cells
Neuronal nitric oxide synthase, as a downstream signaling molecule of c-jun, regulates the survival of differentiated PC12 cells
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DOI：
10.3892/mmr.2014.2415
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发表时间：
2014-10-01
影响因子：
3.4
通讯作者：
Zhuo, Lihua
中科院分区：
文献类型：
--
作者：
Cheng, Xiao;Luo, Haoxuan;Zhuo, Lihua
c-jun和神经元型一氧化氮合酶（nNOS）的高表达通常发生在产生各种中枢神经元疾病动物模型后的神经元中。然而，它们在神经元疾病中的作用机制仍有待阐明。我们的前期研究表明，在运动神经元损伤中，c-jun的表达总是先于nNOS的表达，c-jun siRNA抑制c-jun表达可降低分化的PC 12细胞中nNOS的表达。本研究旨在研究c-jun和nNOS在神经元中是否存在上下游调节或串扰。用体外培养的分化的PC 12细胞，用免疫荧光法检测nNOS和c-jun在细胞中的表达。用nNOS抑制剂7-硝基吲唑（7-NI）下调分化后的PC 12细胞中nNOS的表达。用3-（4，5-二甲基噻唑-2-基）-2，5-二苯基四氮唑（DTT）法筛选7-NI对分化的PC 12细胞活力和存活的最佳浓度，并用NOS活性检测试剂盒检测7-NI对分化的PC 12细胞中组成型一氧化氮合酶（cNOS）活性的影响。Western blot检测7-NI对nNOS和c-jun基因表达的影响。免疫荧光结果显示，PC 12细胞中c-jun和nNOS蛋白持续表达。用200和400 μ mol/l 7-NI处理分化的PC 12细胞，其细胞活力明显受到抑制，用200 μ mol/l 7-NI处理，nNOS蛋白的表达水平明显受到抑制。而7-NI对c-jun蛋白表达水平和cNOS总活性无明显影响。本实验室前期研究表明，nNOS基因是JNK/c-jun信号通路的下游信号分子，nNOS下游的表达受c-jun的调控。因此，这些结果表明，它们之间的关联涉及上调和下调，而不是串扰。
The high expression of c-jun and neuronal nitric oxide synthase (nNOS) generally occurs in neurons following the generation of various animal models of central neuronal diseases. However, the mechanism between them in neuronal disease remains to be elucidated. Our previous studies demonstrated that the expression of c-jun always occurs prior to expression of nNOS in motoneuron injuries and suppression of c-jun expression by c-jun siRNA decreased nNOS expression in differentiated PC12 cells. The present study aimed to examine whether there was an association of up and downstream regulation or crosstalk between c-jun and nNOS in neurons. Using a culture of differentiated PC12 cells in vitro, the expression of nNOS and c-jun in cells was investigated by immunofluorescence. The nNOS inhibitor 7-nitroindazole (7-NI) was used in differentiated PC12 cells to downregulate the expression of nNOS. The optimal concentration of 7-NI on the viability and survival of cultured differentiated PC12 cells was selected using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay and the effects of 7-NI on the activity of constitutive nitric oxide synthase (cNOS) in differentiated PC12 cells were determined using a NOS Activity Detection kit. The effects of 7-NI on the gene expression of nNOS and c-jun were detected by western blot analysis. The results from the immunofluorescence demonstrated that the c-jun and nNOS protein were constantly expressed in PC12 cells. The cell viability of differentiated PC12 cells were significantly inhibited by treatment with 200 and 400 mu mol/l 7-NI, and the expression levels of the nNOS protein were significantly inhibited by treatment with 200 mu mol/l 7-NI. However, 7-NI had no significant effect on the protein expression level of c-jun and the total activities of cNOS. Based on our previous studies, which revealed that the nNOS gene was a downstream signaling molecule of the JNK/c-jun signaling pathway in cultured neurons, the expression of nNOS downstream was able to be regulated by c-jun which was the upstream molecule. Therefore, these results indicated that the association between them involved up and downregulation instead of crosstalk.