Costello B, Meymandi A, Freeman J A
Department of Cell Biology, Vanderbilt Medical School, Nashville, Tennessee 37232.
J Neurosci. 1990 Apr;10(4):1398-406. doi: 10.1523/JNEUROSCI.10-04-01398.1990.
We have studied factors controlling message levels for the neuronal growth- and plasticity-associated protein, GAP-43. Following exposure of PC12 cells to various effectors, cytoplasmic RNA was isolated and analyzed by Northern transfer and autoradiography using a GAP-43 cDNA probe. Induction by NGF is apparent after 3 hr exposure and reaches maximal levels at 24 hr. Beyond 24 hr, levels remain constant in the continued presence of NGF. Induction is insensitive to variations in culture conditions, such as plating density or substrate, which influence NGF-induced neurite outgrowth. Other inducers, in order of decreasing efficacy, are FGF, dBcAMP, TPA, K+, and EGF. Insulin and retinoic acid are ineffective. Dexamethasone partially inhibited basal expression as well as induction by NGF, FGF, dBcAMP, and TPA. The methyltransferase inhibitor 5'-S-(2-methyl-propyl)adenosine completely inhibited induction by NGF, FGF, and dBcAMP. Inhibition of protein synthesis by cycloheximide partially decreased induction by NGF, FGF, and TPA but slightly enhanced dBcAMP induction. Complete down-regulation of protein kinase C by chronic TPA treatment completely eliminated the TPA response but slightly enhanced induction by NGF. These findings and the results of additivity experiments in which cells were stimulated with various combinations of NGF, dBcAMP and TPA suggest that NGF induction of GAP-43 RNA (1) does not involve activation of protein kinase C but (2) may be mediated partially via activation of protein kinase A.
我们研究了控制神经元生长和可塑性相关蛋白GAP-43信息水平的因素。将PC12细胞暴露于各种效应物后，分离细胞质RNA，并使用GAP-43 cDNA探针通过Northern印迹和放射自显影进行分析。暴露于神经生长因子（NGF）3小时后诱导作用明显，24小时达到最高水平。24小时后，在持续存在NGF的情况下水平保持恒定。诱导作用对影响NGF诱导的神经突生长的培养条件变化不敏感，如接种密度或底物。其他诱导剂，按效力递减顺序为成纤维细胞生长因子（FGF）、二丁酰环磷腺苷（dBcAMP）、佛波酯（TPA）、钾离子（K+）和表皮生长因子（EGF）。胰岛素和视黄酸无效。地塞米松部分抑制基础表达以及NGF、FGF、dBcAMP和TPA的诱导作用。甲基转移酶抑制剂5'-S-(2-甲基丙基)腺苷完全抑制NGF、FGF和dBcAMP的诱导作用。环己酰亚胺抑制蛋白质合成部分降低了NGF、FGF和TPA的诱导作用，但略微增强了dBcAMP的诱导作用。通过长期TPA处理完全下调蛋白激酶C可完全消除TPA反应，但略微增强NGF的诱导作用。这些发现以及用NGF、dBcAMP和TPA的各种组合刺激细胞的加和性实验结果表明，NGF诱导GAP-43 RNA：（1）不涉及蛋白激酶C的激活，但（2）可能部分通过蛋白激酶A的激活介导。
