Chang Jay H, Mellon Eric, Schanen N Carolyn, Twiss Jeffery L
Cellular and Molecular Pathology Graduate Program, Department of Pathology, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA.
J Biol Chem. 2003 Oct 31;278(44):42877-85. doi: 10.1074/jbc.M308155200. Epub 2003 Aug 8.
Neurotrophins are required for the differentiation and survival of several different neuronal subpopulations in the developing nervous system. The PC12 cell line responds to nerve growth factor (NGF) by withdrawing from the cell cycle and acquiring a sympathetic neuron-like phenotype. Previous studies have shown that the activation kinetics of the NGF receptor, TrkA, and downstream protein kinases appear rapid and seemingly transient after NGF treatment of naive PC12 cells. However, maintenance of the neuronal phenotype and survival of differentiated PC12 cells under serum-free conditions require constant NGF exposure. In this study we have addressed the mechanisms that NGF uses to maintain neuronal PC12 cells. We show that TrkA remains phosphorylated at a basal level throughout differentiation of the PC12 cells. The phospho-TrkA levels in the differentiated PC12 cells were diminished by both complete NGF withdrawal and pharmacological inhibition of Trk kinase activity. Intracellular sequestration of the majority of TrkA molecules (both phosphorylated and non-phosphorylated TrkA) and persistent dephosphorylation of the small pool of cell surface TrkA renders the persistent phospho-TrkA signal in the differentiated PC12 cells resistant to partial NGF withdrawal as well as exposure to additional NGF. NGF regulated both extracellular-regulated kinases 1/2 and Akt activity in the differentiated PC12 cells via sustained TrkA activity. Moreover, analysis of transcription using activating protein 1-, serum response element-, and cyclic AMP response element-Luc reporter constructs showed that NGF regulated these promoters through TrkA activity in differentiated PC12 cells. Interestingly, the initial response of the cyclic AMP response element promoter to NGF was delayed, becoming Trk-dependent well beyond the peaks in TrkA and downstream protein kinase signal transduction.
神经营养因子是发育中的神经系统中几种不同神经元亚群分化和存活所必需的。PC12细胞系对神经生长因子（NGF）作出反应，退出细胞周期并获得交感神经元样表型。先前的研究表明，在未处理的PC12细胞用NGF处理后，NGF受体TrkA和下游蛋白激酶的激活动力学似乎很快且看似短暂。然而，在无血清条件下维持神经元表型和分化的PC12细胞的存活需要持续暴露于NGF。在本研究中，我们探讨了NGF用于维持神经元样PC12细胞的机制。我们发现，在PC12细胞的整个分化过程中，TrkA一直保持在基础水平的磷酸化状态。完全撤除NGF和对Trk激酶活性进行药理学抑制均可降低分化的PC12细胞中的磷酸化TrkA水平。细胞内大部分TrkA分子（磷酸化和未磷酸化的TrkA）的隔离以及一小部分细胞表面TrkA的持续去磷酸化，使得分化的PC12细胞中持续的磷酸化TrkA信号对部分撤除NGF以及暴露于额外的NGF具有抗性。NGF通过持续的TrkA活性调节分化的PC12细胞中的细胞外调节激酶1/2和Akt活性。此外，使用激活蛋白1、血清反应元件和环磷酸腺苷反应元件荧光素酶报告构建体进行的转录分析表明，NGF在分化的PC12细胞中通过TrkA活性调节这些启动子。有趣的是，环磷酸腺苷反应元件启动子对NGF的初始反应延迟，在TrkA和下游蛋白激酶信号转导达到峰值很久之后才变得依赖于Trk。
