Reactive Oxygen Species, Ki-Ras, and Mitochondrial Superoxide Dismutase Cooperate in Nerve Growth Factor-induced Differentiation of PC12 Cells
Reactive Oxygen Species, Ki-Ras, and Mitochondrial Superoxide Dismutase Cooperate in Nerve Growth Factor-induced Differentiation of PC12 Cells
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DOI：
10.1074/jbc.m109.098525
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发表时间：
2010-07-30
影响因子：
4.8
通讯作者：
Avvedimento, Enrico Vittorio
中科院分区：
文献类型：
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作者：
Cassano, Silvana;Agnese, Savina;Avvedimento, Enrico Vittorio
神经生长因子（NGF）诱导嗜铬细胞瘤衍生细胞系PC 12的终末分化。NGF结合膜上的特异性受体并触发ERK 1/2级联，其刺激神经基因的转录。我们报告说，神经生长因子显着影响线粒体代谢，通过减少神经元产生的活性氧和稳定的电化学梯度。这是通过Ki-Ras和ERK 1/2在转录和转录后刺激线粒体锰超氧化物歧化酶（MnSOD）来实现的。MnSOD的激活对于完成神经元分化是必不可少的，因为1）MnSOD的表达诱导神经元特异性启动子的转录和神经突生长，2）通过小干扰RNA沉默内源性MnSOD显著降低由NGF诱导的转录，以及3）在COOH末端的聚赖氨酸段中的Ki-Ras突变体不能刺激MnSOD，不能诱导完全分化。MnSOD的过表达恢复表达这种突变体的细胞的分化。ERK 1/2也是MnSOD的下游，因为SOD模拟药物以与NGF相同的动力学刺激ERK 1/2，并且MnSOD的沉默降低了NGF诱导的晚期ERK 1/2。NGF对ERK 1/2的长期激活需要SOD激活、低水平的过氧化氢和微管细胞骨架的完整性。共聚焦免疫荧光显示，NGF刺激含有膜结合Ki-Ras，微管和线粒体的复合物的形成。我们建议，活性神经生长因子受体诱导协会的线粒体与质膜。Ki-Ras局部激活ERK 1/2可刺激线粒体SOD，从而减少活性氧并产生H（2）O（2）。低水平和空间受限的H2 O2诱导并维持PC 12细胞的长期ERK 12活性并最终分化。
Nerve growth factor (NGF) induces terminal differentiation in PC12, a pheochromocytoma-derived cell line. NGF binds a specific receptor on the membrane and triggers the ERK1/2 cascade, which stimulates the transcription of neural genes. We report that NGF significantly affects mitochondrial metabolism by reducing mitochondrial-produced reactive oxygen species and stabilizing the electrochemical gradient. This is accomplished by stimulation of mitochondrial manganese superoxide dismutase (MnSOD) both transcriptionally and post-transcriptionally via Ki-Ras and ERK1/2. Activation of MnSOD is essential for completion of neuronal differentiation because 1) expression of MnSOD induces the transcription of a neuronal specific promoter and neurite outgrowth, 2) silencing of endogenous MnSOD by small interfering RNA significantly reduces transcription induced by NGF, and 3) a Ki-Ras mutant in the polylysine stretch at the COOH terminus, unable to stimulate MnSOD, fails to induce complete differentiation. Overexpression of MnSOD restores differentiation in cells expressing this mutant. ERK1/2 is also downstream of MnSOD, as a SOD mimetic drug stimulates ERK1/2 with the same kinetics of NGF and silencing of MnSOD reduces NGF-induced late ERK1/2. Long term activation of ERK1/2 by NGF requires SOD activation, low levels of hydrogen peroxide, and the integrity of the microtubular cytoskeleton. Confocal immunofluorescence shows that NGF stimulates the formation of a complex containing membrane-bound Ki-Ras, microtubules, and mitochondria. We propose that active NGF receptor induces association of mitochondria with plasma membrane. Local activation of ERK1/2 by Ki-Ras stimulates mitochondrial SOD, which reduces reactive oxygen species and produces H(2)O(2). Low and spatially restricted levels of H(2)O(2) induce and maintain long term ERK1/2 activity and ultimately differentiation of PC12 cells.