Sustained activation of M-Ras induced by nerve growth factor is essential for neuronal differentiation of PC12 cells
Sustained activation of M-Ras induced by nerve growth factor is essential for neuronal differentiation of PC12 cells
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DOI：
10.1111/j.1365-2443.2006.01002.x
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发表时间：
2006-09-01
期刊：
影响因子：
2.1
通讯作者：
Endo, Takeshi
中科院分区：
文献类型：
--
作者：
Sun, Peng;Watanabe, Haruko;Endo, Takeshi
神经生长因子(NGF)诱导PC12细胞分化需要ERK/MAPK通路的持续激活(Raf-MEK-ERK级联)。尽管NGF信号激活经典RAS(H-RAS、K-RAS和N-RAS)可诱导ERK通路的激活，但这种激活是暂时的，不足以促进PC12细胞的分化。相反，已被广泛接受的是NGF信号介导的Rap1激活导致ERK途径的持续激活。然而，目前还没有直接证据表明RAP1参与神经元分化。在这里，我们发现NGF信号诱导了M-RAS的持续激活，随后ERK通路和转录因子CREB的持续激活，导致PC12细胞分化。外源表达的结构性活性突变体M-RAS可诱导PC12细胞突起生长并激活ERK的磷酸化，而激活的Rap1则不能。小干扰RNA对内源性M-RAS的抑制以及M-RAS显性负性突变体的表达干扰了NGF诱导的神经发生。由于MEK抑制剂阻止了M-RAS诱导的轴突生长，ERK通路参与了这一分化途径。此外，M-RAS还可通过ERK途径激活CREB的磷酸化，进而激活CREB的转录。此外，CREB的显性-负性突变体抑制了M-RAS诱导的神经再生。综上所述，NGF诱导PC12细胞分化需要M-RAS-ERK通路介导的CREB活化。M-RAS主要表达于小鼠脑内的海马区和小脑以及脊髓灰质。M-RAS的所有这些特性显然与H-RAS的特性难以区分。然而，NGF刺激导致经典的RAS蛋白短暂激活，而M-RAS持续激活，ERK和CREB持续激活。因此，M-RAS通过诱导ERK通路的持续激活对PC12细胞的神经元分化是必不可少的。
Neuronal differentiation in PC12 cells induced by nerve growth factor (NGF) requires sustained activation of ERK/MAP kinase pathway (Raf-MEK-ERK cascade). Although classical Ras (H-Ras, K-Ras, and N-Ras) activated by NGF signaling induces activation of ERK pathway, the activation is transient and not sufficient for PC12 cell differentiation. Instead, it has been widely accepted that NGF signaling-mediated Rap1 activation causes sustained activation of ERK pathway. There has been no direct evidence, however, that Rap1 participates in neuronal differentiation. Here we show that NGF signaling induces sustained activation of M-Ras and subsequent sustained activation of ERK pathway and the transcription factor CREB leading to PC12 cell differentiation. Exogenously expressed constitutively active mutant of M-Ras caused neurite outgrowth in PC12 cells and activating phosphorylation of ERK, whereas activated Rap1 did not. Knockdown of endogenous M-Ras by small interfering RNAs as well as the expression of a dominant-negative mutant of M-Ras interfered with NGF-induced neuritogenesis. Since MEK inhibitors prevented M-Ras-induced neurite outgrowth, ERK pathway participates in this differentiation pathway. Furthermore, M-Ras brought about ERK pathway-mediated activating phosphorylation of CREB and the CREB-mediated transcription. In addition, a dominant-negative mutant of CREB inhibited M-Ras-induced neuritogenesis. Taken together, NGF-induced PC12 cell differentiation requires M-Ras-ERK pathway-mediated activation of CREB. M-Ras was predominantly expressed in the hippocampus and cerebellum of mouse brain and in the gray matter of the spinal cord. All these properties of M-Ras were apparently indistinguishable from those of H-Ras. However, NGF stimulation caused transient activation of classical Ras proteins but sustained activation of M-Ras as well as sustained activating phosphorylation of ERK and CREB. Therefore, M-Ras is essential for neuronal differentiation in PC12 cells by inducing sustained activation of ERK pathway.