Dijkmans T F, van Hooijdonk L W A, Schouten T G, Kamphorst J T, Fitzsimons C P, Vreugdenhil E
Division of Medical Pharmacology, Leiden/Amsterdam Center for Drug Research, Leiden University Medical Center, PO Box 9502, 2300 RA, Leiden, The Netherlands.
Brain Res. 2009 Jan 16;1249:19-33. doi: 10.1016/j.brainres.2008.10.050. Epub 2008 Oct 31.
Stimulation of the PC12 pheochromocytoma cell line with the prototypical neurotrophin Nerve Growth Factor (NGF) induces a cellular response of neuronal differentiation and is therefore a widely used model to gain molecular insight into this process. Classically, the transcriptional response to extracellular stimuli such as NGF is divided in genes that require no protein synthesis prior to their induction (immediate-early genes) and genes that do (delayed-response genes). Because an increasing number of studies have reported important roles for immediate-early genes (IEGs) in neuronal differentiation, the goal of the present study was to identify previously unrecognized NGF-responsive IEGs. Stimulation with NGF for 15, 30, 60 and 120 min resulted in a typical transient induction of many known NGF-responsive IEGs. To identify candidate new genes, we analyzed 27000 measured expression profiles and selected 10 genes for further study. Five genes, including Cbp/p300-interacting transactivator 2 (Cited2), Kruppel-like factor 4 (Klf4), v-Maf musculoaponeurotic fibrosarcoma oncogene family, protein F (Maff), Kruppel-like factor 10 (Klf10 or Tieg) and Activating transcription factor 3 (Atf3) were selected and positively validated by qPCR. NGF-induced activation of all five genes seems to be mediated by MAPK and PI3K-mediated pathways. Additionally, we tested translation-independent induction and showed that NGF induced upregulation of these genes in both the subclonal Neuroscreen-1 PC12 and parental PC12 cell line. These 5 transcription factors have not been previously reported as NGF-responsive IEGs, however have previously been reported as important regulators of cell differentiation and proliferation in different systems. These observations may therefore provide important new information on the molecular mechanisms underlying NGF-induced differentiation.
用典型的神经营养因子神经生长因子（NGF）刺激PC12嗜铬细胞瘤细胞系可诱导神经元分化的细胞反应，因此它是一个被广泛用于深入了解这一过程分子机制的模型。传统上，对诸如NGF等细胞外刺激的转录反应分为诱导前无需蛋白质合成的基因（即早基因）和需要蛋白质合成的基因（延迟反应基因）。由于越来越多的研究报道即早基因（IEGs）在神经元分化中发挥重要作用，本研究的目的是鉴定此前未被识别的NGF反应性即早基因。用NGF刺激15、30、60和120分钟导致许多已知的NGF反应性即早基因出现典型的瞬时诱导。为了鉴定候选新基因，我们分析了27000个测量的表达谱，并选择了10个基因进行进一步研究。包括Cbp/p300相互作用反式激活因子2（Cited2）、 Kruppel样因子4（Klf4）、v-Maf肌腱膜纤维肉瘤癌基因家族蛋白F（Maff）、Kruppel样因子10（Klf10或Tieg）和激活转录因子3（Atf3）在内的5个基因被选中，并通过qPCR进行了阳性验证。NGF诱导的所有这5个基因的激活似乎是由MAPK和PI3K介导的途径介导的。此外，我们测试了非翻译依赖性诱导，并表明NGF在亚克隆的Neuroscreen-1 PC12和亲本PC12细胞系中均诱导了这些基因的上调。这5个转录因子此前未被报道为NGF反应性即早基因，但此前在不同系统中被报道为细胞分化和增殖的重要调节因子。因此，这些观察结果可能为NGF诱导分化的分子机制提供重要的新信息。
