Coleman E S, Wooten M W
Division of Zoology, Auburn University, AL 36849.
J Mol Neurosci. 1994 Spring;5(1):39-57. doi: 10.1007/BF02736693.
To elucidate the role of protein kinase C (PKC) in nerve growth factor (NGF)-induced differentiation, PMA downregulation of pheochromocytoma (PC12) cells was undertaken. Prolonged treatment (2 d) of PC12 cells with PMA (1 microM) resulted in depleting the cells of alpha, beta, delta, and epsilon-PKC isoforms, but had no effect on the expression of the atypical PKC isoform zeta. PC12 cells, which expressed only PKC zeta, were evaluated for their responses to NGF. Removal of the PMA-sensitive PKC isoforms enhanced the ability of NGF to promote neurite extension. Both the percentage cells with neurites and length of neurites were increased in the PMA-treated cells, whereas no effect was observed on the number of neurites per cell or branching of individual neurites. In addition, PMA downregulation resulted in an increase in the incorporation of 3H-thymidine without any significant effect on the expression of c-fos. Addition of NGF to PC12 cells depleted of the PMA-sensitive PKC isoforms resulted in the activation of PKC zeta (Wooten et al., 1994). To test whether the transient activation of PKC zeta is a necessary component of the neuritogenetic pathway, antisense oligonucleotide strategy was utilized to remove this particular PKC isoform. The addition of a 20-bp antisense oligonucleotide directed against the 5' coding sequence of PKC zeta attenuated NGF-induced neurite outgrowth in PC12 cells lacking PMA-sensitive PKC isoforms. Sense oligonucleotide directed at the same site was without effect on NGF responses. These data indicate that PKC zeta comprises a portion of the NGF pathway and underscores the importance of this isoform in neuronal differentiation. Moreover, these findings demonstrate that the PMA-insensitive pathway, which was previously characterized as PKC-independent, and the neurite induction pathway are synonymous and mediated by PKC zeta.
为阐明蛋白激酶C（PKC）在神经生长因子（NGF）诱导分化中的作用，进行了佛波酯（PMA）对嗜铬细胞瘤（PC12）细胞的下调实验。用PMA（1微摩尔）对PC12细胞进行长时间处理（2天），导致细胞中的α、β、δ和ε-PKC亚型耗竭，但对非典型PKC亚型ζ的表达没有影响。仅表达PKCζ的PC12细胞被评估其对NGF的反应。去除对PMA敏感的PKC亚型增强了NGF促进神经突延伸的能力。在经PMA处理的细胞中，有神经突的细胞百分比和神经突长度均增加，而对每个细胞的神经突数量或单个神经突的分支没有影响。此外，PMA下调导致3H-胸腺嘧啶掺入增加，而对c-fos的表达没有任何显著影响。向缺乏对PMA敏感的PKC亚型的PC12细胞中添加NGF导致PKCζ激活（伍滕等人，1994年）。为了测试PKCζ的瞬时激活是否是神经突生成途径的必要组成部分，采用反义寡核苷酸策略去除这种特定的PKC亚型。添加针对PKCζ 5'编码序列的20个碱基对反义寡核苷酸减弱了缺乏对PMA敏感的PKC亚型的PC12细胞中NGF诱导的神经突生长。针对同一位点的正义寡核苷酸对NGF反应没有影响。这些数据表明PKCζ是NGF途径的一部分，并强调了该亚型在神经元分化中的重要性。此外，这些发现表明，先前被表征为不依赖PKC的PMA不敏感途径与神经突诱导途径是同义的，并由PKCζ介导。
