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

Silvana Cassano,Savina Agnese,Valentina D'Amato,Massimo Papale,Corrado Garbi,Patrizio Castagnola,Maria Rosaria Ruocco,Immacolata Castellano,Emmanuele De Vendittis,Mariarosaria Santillo,Stefano Amente,Antonio Porcellini,Enrico Vittorio Avvedimento

doi:10.1074/jbc.m109.098525

Abstract

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.

Highlights

Reactive oxygen species (ROS)2 are continuously generated by metabolic reactions in all cellular compartments
We measured the mitochondrial ROS levels, the mitochondrial membrane potential, and the activity of MnSOD, a key enzyme buffering mitochondrial superoxide, in PC12 cells exposed to nerve growth factor (NGF)
Lower mitochondrial ROS levels indicate that dissipation of the mitochondrial electrochemical gradient is reduced and the respiration is more efficient in the presence of NGF

Summary

EXPERIMENTAL PROCEDURES

Materials and Antibodies—Mn(III) tetrakis(1-methyl-4pyridyl)porphyrin pentachloride (MnTMpyP), a cell-permeable superoxide dismutase mimetic, was obtained from Calbiochem. 68 h after the transfection the cells were serum-deprived for 4 h and induced with EGF or NGF for 15 min and 3 h, respectively. Fluorescence Microscopy and Flow Cytometry—Mitochondria were stained with Mitotracker Red CM-H2XRos at nM final concentration (Molecular Probes) for min at 37 °C. The superoxide indicator MitoSOX Red (Molecular Probes, Invitrogen) was used at a final concentration of 5 ␮M, incubated for 10 min at 37 °C, and washed 2 times with PBS. The left upper panel shows the immunoblot with Ha- and Ki-Ras-specific antibodies of extracts derived from cells transfected with various concentrations of Ha- or Ki-Ras expression vectors. Lysis—Lysates were prepared by dissolving the cell pellet in cold Manganese Superoxide Dismutase Activity—Cells from two radioimmune precipitation assay buffer

RESULTS

DISCUSSION

Vittorio Avvedimento