Dept. of Physiology, School of Medicine, National Yang Ming University, Taipei 11221, Taiwan, Republic of China.
Am J Physiol Cell Physiol. 2010 Aug;299(2):C516-27. doi: 10.1152/ajpcell.00534.2009. Epub 2010 May 12.
The signaling mechanisms underlying cell differentiation have been extensively studied with the use of rat PC12 cells as a model system. Nerve growth factor (NGF) is a trophic factor inducing PC12 cell differentiation through the activation of the p35/cyclin-dependent kinase 5 (Cdk5) complex. It has been reported that adenylyl cyclase activation and cAMP production may be involved in NGF-dependent actions. Our previous results indicate that cAMP activates the p35/Cdk5 complex in reproductive cells. Therefore, the role of cAMP in NGF-triggered p35/Cdk5 activation and PC12 differentiation was interesting to explore. Our results indicate that roscovitine, a molecular inhibitor of Cdk5, blocks cAMP-triggered PC12 differentiation, which was evaluated by neurite initiation, a decrease in proliferation, and cell cycle G(1) arrest. The following data show that cAMP treatment increased Cdk5 activity through p35 upregulation. cAMP downstream components, protein kinase A (PKA) and phosphorylated cAMP response element binding protein (CREB), are involved in this regulation. The immunocytochemical results indicate that PKA inhibition disrupted cAMP-triggered p35/Cdk5 localization in PC12 cells. In addition, adenylyl cyclase inhibition was found to diminish NGF-induced intracellular cAMP production, CREB phosphorylation, and p35 expression. The cAMP antagonist and the PKA inhibitors reduced NGF-induced p35 expression. Finally, NGF-triggered PC12 differentiation was partially decreased by adenylyl cyclase or PKA inhibitors. In conclusion, these results demonstrate that cAMP may play a role in NGF-p35/Cdk5-dependent PC12 differentiation.
细胞分化的信号机制已经在使用大鼠 PC12 细胞作为模型系统进行了广泛的研究。神经生长因子（NGF）是一种营养因子，通过激活 p35/细胞周期蛋白依赖性激酶 5（Cdk5）复合物诱导 PC12 细胞分化。据报道，腺苷酸环化酶的激活和 cAMP 的产生可能参与 NGF 依赖性作用。我们之前的结果表明 cAMP 在生殖细胞中激活 p35/Cdk5 复合物。因此，cAMP 在 NGF 触发的 p35/Cdk5 激活和 PC12 分化中的作用值得探讨。我们的结果表明，罗氏考维丁（一种 Cdk5 的分子抑制剂）阻断 cAMP 触发的 PC12 分化，这可以通过神经突起始、增殖减少和细胞周期 G1 期阻滞来评估。以下数据表明，cAMP 通过上调 p35 增加 Cdk5 活性。cAMP 的下游成分，蛋白激酶 A（PKA）和磷酸化 cAMP 反应元件结合蛋白（CREB），参与这种调节。免疫细胞化学结果表明，PKA 抑制破坏了 cAMP 触发的 PC12 细胞中 p35/Cdk5 的定位。此外，发现腺苷酸环化酶抑制减少了 NGF 诱导的细胞内 cAMP 产生、CREB 磷酸化和 p35 表达。cAMP 拮抗剂和 PKA 抑制剂降低了 NGF 诱导的 p35 表达。最后，腺苷酸环化酶或 PKA 抑制剂部分减少了 NGF 触发的 PC12 分化。总之，这些结果表明 cAMP 可能在 NGF-p35/Cdk5 依赖的 PC12 分化中发挥作用。
