Dipartimento di Biologia e Patologia Molecolare e Cellulare, Istituto di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche.
J Biol Chem. 2010 Jul 30;285(31):24141-53. doi: 10.1074/jbc.M109.098525. Epub 2010 May 21.
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.
神经生长因子（NGF）诱导 PC12 细胞，即一种嗜铬细胞瘤衍生细胞系的终末分化。NGF 与细胞膜上的特定受体结合，触发 ERK1/2 级联反应，从而刺激神经基因的转录。我们报告称，NGF 通过减少线粒体产生的活性氧物质并稳定电化学梯度，显著影响线粒体代谢。这是通过刺激线粒体锰过氧化物酶（MnSOD）的转录和转录后水平来实现的，其通过 Ki-Ras 和 ERK1/2 来实现。MnSOD 的激活对于完成神经元分化至关重要，因为 1）MnSOD 的表达诱导神经元特异性启动子的转录和突起生长，2）通过小干扰 RNA 沉默内源性 MnSOD 会显著降低 NGF 诱导的转录，以及 3）在羧基末端多赖氨酸延伸处的 Ki-Ras 突变体，无法刺激 MnSOD，无法诱导完全分化。MnSOD 的过表达可恢复表达该突变体的细胞中的分化。ERK1/2 也是 MnSOD 的下游，因为 SOD 模拟药物以与 NGF 相同的动力学刺激 ERK1/2，并且 MnSOD 的沉默会降低 NGF 诱导的晚期 ERK1/2。NGF 通过 ERK1/2 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 刺激含有膜结合的 Ki-Ras、微管和线粒体的复合物的形成。我们提出，活性 NGF 受体诱导线粒体与质膜的结合。Ki-Ras 局部激活 ERK1/2 可刺激线粒体 SOD，从而减少活性氧物质并产生 H2O2。低水平和空间限制的 H2O2 诱导并维持长期的 ERK1/2 活性，并最终导致 PC12 细胞的分化。
