Laboratory of Neurobiology, Department of Life Science and Biotechnology, Faculty of Chemistry, Materials and Bioengineering, Kansai University, 3-3-35, Yamate-cho, Suita, Osaka, 564-8680, Japan.
Sci Rep. 2020 Apr 14;10(1):6325. doi: 10.1038/s41598-020-62968-y.
Forskolin promotes neuronal differentiation of PC12 cells via the PKA-CREB-dependent signaling pathway. Activation of PKA by forskolin phosphorylates CREB, which then binds to CRE sites in numerous gene promoters. However, it is unclear which gene contains the CRE sites responsible for forskolin-induced neuronal differentiation. In this study, we investigated how an immediate early gene, nur77, which has CRE sites in the promoter region, contributes to the early stage of differentiation of forskolin-treated PC12 cells. After treatment with forskolin, expression of Nur77 was upregulated within 1 hr. In addition, knockdown of nur77 inhibited neurite outgrowth induced by forskolin. We also revealed that the specific four CRE sites near the transcriptional start site (TSS) of nur77 were strongly associated with phosphorylated CREB within 1 hr after treatment with forskolin. To analyze the roles of these four sites, reporter assays using the nur77 promoter region were performed. The results showed that nur77 expression was mediated through three of the CRE sites, -242, -222, and -78, and that -78, the nearest of the three to the TSS of nur77, was particularly important. An analysis of neuronal markers controlled by Nur77 after A-CREB-Nur77-Synapsin1 signaling pathway plays a pivotal role in differentiation of forskolin-induced PC12 cells.
毛喉素通过 PKA-CREB 依赖的信号通路促进 PC12 细胞的神经元分化。毛喉素通过激活 PKA 使 CREB 磷酸化，然后 CREB 与众多基因启动子中的 CRE 位点结合。然而，尚不清楚哪个基因包含负责毛喉素诱导神经元分化的 CRE 位点。在这项研究中，我们研究了即刻早期基因 nur77 如何参与毛喉素处理的 PC12 细胞分化的早期阶段，该基因在启动子区域具有 CRE 位点。用毛喉素处理后，Nur77 的表达在 1 小时内上调。此外，nur77 的敲低抑制了毛喉素诱导的突起生长。我们还揭示了在处理毛喉素后 1 小时内，nur77 转录起始位点（TSS）附近的四个特定 CRE 位点与磷酸化的 CREB 强烈相关。为了分析这四个位点的作用，使用 nur77 启动子区域进行了报告基因实验。结果表明，nur77 的表达是通过三个 CRE 位点 -242、-222 和 -78 介导的，而最靠近 nur77 TSS 的 -78 尤为重要。在 A-CREB-Nur77-Synapsin1 信号通路后对 Nur77 控制的神经元标记物进行分析表明，它在毛喉素诱导的 PC12 细胞分化中起着关键作用。
