Reactive Oxygen Species, Ki-Ras, and Mitochondrial Superoxide Dismutase Cooperate in Nerve Growth Factor-induced Differentiation of PC12 Cells

Nerve Growth Factor (NGF) induces terminal differentiation in PC12, a pheochromocytoma-derived cell line. NGF stimulates a specific receptor on the membrane and ERK1/2 cascade, which induces the transcription of neural genes. We report that NGF significantly affects mitochondrial metabolism by reducing mitochondrial-produced ROS 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 siRNA significantly reduces transcription induced by NGF; 3) a Ki-Ras mutant in the poly-lysine stretch at the COOH terminus, unable to stimulate MnSOD, fails to induce complete differentiation. Differentiation of cells expressing this mutant, is restored over-expressing MnSOD. ERK1/2 is also downstream of MnSOD, since a SOD mimetic drug stimulates ERK1/2 with the same kinetics of NGF and silencing of MnSOD reduces NGF stimulated ERK1/2. Long-term activation of ERK1/2 by NGF requires SOD activation, low levels of hydrogen peroxide and the integrity of microtubular cytoskeleton. Confocal immunofluorescence shows that NGF stimulates the formation of a complex containing membrane-bound Ki-Ras, microtubules and mitochondria. We propose that activation of NGF receptor induces association of mitochondria with plasma membrane. Local activation of ERK1/2 by Ki-Ras stimulates mitochondrial SOD, which reduces ROS 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.