Perrone-Bizzozero N I, Cansino V V, Kohn D T
Department of Biochemistry, University of New Mexico School of Medicine, Albuquerque 87131-5221.
J Cell Biol. 1993 Mar;120(5):1263-70. doi: 10.1083/jcb.120.5.1263.
We have previously shown that nerve growth factor (NGF) selectively stabilizes the GAP-43 mRNA in PC12 cells. To study the cellular mechanisms for this post-transcriptional control and to determine the contribution of mRNA stability to GAP-43 gene expression, we examined the effects of several agents that affect PC12 cell differentiation on the level of induction and rate of degradation of the GAP-43 mRNA. The NGF-mediated increase in GAP-43 mRNA levels and neurite outgrowth was mimicked by the phorbol ester TPA, but not by dibutyryl cAMP or the calcium ionophore A12783. Downregulation of protein kinase C (PKC) by high doses of phorbol esters or selective PKC inhibitors prevented the induction of this mRNA by NGF, suggesting that NGF and TPA act through a common PKC-dependent pathway. In mRNA decay studies, phorbol esters caused a selective 6-fold increase in the half-life of the GAP-43 mRNA, which accounts for most of the induction of this mRNA by TPA. The phorbol ester-induced stabilization of GAP-43 mRNA was blocked by the protein kinase inhibitor polymyxin B and was partially inhibited by dexamethasone, an agent that blocks GAP-43 expression and neuronal differentiation in PC12 cells. In contrast, the rates of degradation and the levels of the GAP-43 mRNA in control and TPA-treated cells were not affected by cycloheximide treatment. Thus, changes in GAP-43 mRNA turnover do not appear to require continuous protein synthesis. In conclusion, these data suggest that PKC activity regulates the levels of the GAP-43 mRNA in PC12 cells through a novel, translation-independent mRNA stabilization mechanism.
我们之前已经表明，神经生长因子（NGF）可选择性地使PC12细胞中的GAP-43 mRNA稳定。为了研究这种转录后调控的细胞机制，并确定mRNA稳定性对GAP-43基因表达的贡献，我们检测了几种影响PC12细胞分化的试剂对GAP-43 mRNA的诱导水平和降解速率的影响。佛波酯TPA模拟了NGF介导的GAP-43 mRNA水平升高和神经突生长，但二丁酰cAMP或钙离子载体A23187则不能。高剂量佛波酯或选择性PKC抑制剂对蛋白激酶C（PKC）的下调可阻止NGF对该mRNA的诱导，这表明NGF和TPA通过共同的PKC依赖性途径发挥作用。在mRNA降解研究中，佛波酯使GAP-43 mRNA的半衰期选择性地增加了6倍，这是TPA诱导该mRNA的主要原因。佛波酯诱导的GAP-43 mRNA稳定被蛋白激酶抑制剂多粘菌素B阻断，并被地塞米松部分抑制，地塞米松可阻断PC12细胞中GAP-43的表达和神经元分化。相反，对照细胞和TPA处理细胞中GAP-43 mRNA的降解速率和水平不受环己酰亚胺处理的影响。因此，GAP-43 mRNA周转的变化似乎不需要持续的蛋白质合成。总之，这些数据表明PKC活性通过一种新的、不依赖翻译的mRNA稳定机制调节PC12细胞中GAP-43 mRNA的水平。
