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 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.
Cassano
Silvana
S
Dipartimento di Biologia e Patologia Molecolare e Cellulare, Istituto di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche.
Agnese
Savina
S
D'Amato
Valentina
V
Papale
Massimo
M
Garbi
Corrado
C
Castagnola
Patrizio
P
Ruocco
Maria Rosaria
MR
Castellano
Immacolata
I
De Vendittis
Emmanuele
E
Santillo
Mariarosaria
M
Amente
Stefano
S
Porcellini
Antonio
A
Avvedimento
Enrico Vittorio
EV
D000818:Animals; D002454:Cell Differentiation; D003599:Cytoskeleton / Q000378:metabolism; D011905:Genes, ras / Q000235:genetics*; D006801:Humans; D020935:MAP Kinase Signaling System; D008928:Mitochondria / Q000201:enzymology; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D009154:Mutation; D020932:Nerve Growth Factor / Q000378:metabolism*; D016716:PC12 Cells; D051381:Rats; D017382:Reactive Oxygen Species; D013482:Superoxide Dismutase / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism*; D000099156:Superoxide Dismutase 2
D048052:Mitogen-Activated Protein Kinase 3; D020932:Nerve Growth Factor; D017382:Reactive Oxygen Species; D013482:Superoxide Dismutase; D018631:ras Proteins; D000099156:Superoxide Dismutase 2