The present research investigates the molecular mechanism of neurite outgrowth (protrusion elongation) under pituitary adenylate cyclase-activating polypeptide (PACAP) 38 treatments using a rat adrenal-derived pheochromocytoma cell line—PC12. This study specifically looks into the regulation of PACAP38-induced collapsing response mediator protein 2 (CRMP2) previously identified in a mouse brain ischemia model and which could be recovered by PACAP38 treatment. Previously, DNA microarray analysis revealed that PACAP 38-mediated neuroprotection involved not only CRMP2 but also pathways related to glycogen synthase kinase-3β (GSK-3β) and other signaling components. Thus, to clarify whether CRMP2 acts directly on PACAP38 or through GSK-3β as part of the mechanism of PACAP38-induced neurite outgrowth, we observed neurite outgrowth in the presence of GSK-3β inhibitors and activators. PC12 cells were treated with PACAP38 being added to the cell culture medium at concentrations of 10⁻⁷ M, 10⁻⁸ M, and 10⁻⁹ M. Post PACAP38 treatment, immunostaining was used to confirm protrusion elongation of the PC12 cells, while RT-PCR, two-dimensional gel electrophoresis in conjunction with Western blotting, and inhibition experiments were performed to confirm the expression of the PACAP gene, its receptors, and downstream signaling components. Our data show that neurite protrusion elongation by PACAP38 (10⁻⁷ M) in PC12 cells is mediated through the PAC1-R receptor as demonstrated by its suppression by a specific inhibitor PA-8. Inhibitor experiments suggested that PACAP38-triggered neurite protrusion follows a GSK-3β-regulated pathway, where the AKT and cAMP/ERK pathways are involved and where the inhibition of Rho/Roc could enhance neurite protrusion under PACAP38 stimulation. Although we could not yet confirm the exact role and position of CRMP2 in PACAP38-mediated PC12 cell elongation, it appears that its phosphorylation and dephosphorylation have a correlation with the neurite protrusion elongation through the interplay of CDK5, which needs to be investigated further.

中文翻译：

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PC12 细胞中 PACAP 38 诱导的神经突生长的分子机制

本研究使用大鼠肾上腺源性嗜铬细胞瘤细胞系 PC12 研究垂体腺苷酸环化酶激活多肽 (PACAP) 38 处理下神经突向外生长（突起伸长）的分子机制。这项研究专门研究了以前在小鼠脑缺血模型中发现的 PACAP38 诱导的塌陷反应介质蛋白 2 (CRMP2) 的调节，该蛋白可以通过 PACAP38 治疗恢复。此前，DNA 微阵列分析显示，PACAP 38 介导的神经保护不仅涉及 CRMP2，还涉及与糖原合酶激酶 3 β (GSK-3 β ) 和其他信号成分相关的通路。因此，为了澄清 CRMP2 是直接作用于 PACAP38 还是通过 GSK-3 β作为 PACAP38 诱导的神经突生长机制的一部分，我们在 GSK-3 β抑制剂和激活剂存在下观察到神经突生长。^{PC12细胞用PACAP38处理，PACAP38以10 -7}  M、10 ^-8  M和10 ^-9 M的浓度添加到细胞培养基中 。PACAP38处理后，免疫染色用于确认PC12细胞的突起伸长，而RT -PCR、二维凝胶电泳结合Western印迹和抑制实验以确认PACAP基因、其受体和下游信号成分的表达。我们的数据表明，PACAP38 (10 ^-7 M) 在 PC12 细胞中是通过 PAC1-R 受体介导的，正如其被特异性抑制剂 PA-8 抑制所证明的那样。抑制剂实验表明，PACAP38 触发的神经突突出遵循 GSK-3 β调节的途径，其中涉及 AKT 和 cAMP/ERK 途径，并且 Rho/Roc 的抑制可以增强 PACAP38 刺激下的神经突突出。虽然我们还不能确定 CRMP2 在 PACAP38 介导的 PC12 细胞伸长中的确切作用和位置，但似乎其磷酸化和去磷酸化通过 CDK5 的相互作用与神经突突起伸长有关，这需要进一步研究。