Nerve Growth Factor Treatment Prevents the Increase in Superoxide Produced by Epidermal Growth Factor in PC12 Cells

Abstract

Stimulation of pheochromocytoma (PC12) cells with the mitogen epidermal growth factor (EGF) produced a rapid and robust accumulation of intracellular reactive oxygen species (ROS), an accumulation which, in other systems, has been shown to be essential for mitogenesis. Brief pretreatment of the cells with nerve growth factor (NGF) suppressed the EGF-mediated ROS increase. EGF failed to produce elevations in ROS in a PC12 variant stably expressing a dominant-negative p21ras construct (PC12-N17) or in cells pretreated with the MEK inhibitor PD098059. NGF failed to suppress the increase in ROS in the PC12 variant nnr5, which lacks p140 trk receptors. The suppression of the increase in ROS by NGF was restored in nnr5 cells stably expressing p140 trk (nnr5-trk), but NGF failed to prevent the increase in ROS in nnr cells expressing mutant p140 trk receptors that lack binding sites for Shc and phospholipase C␥. Among several inhibitors of superoxide-generating enzymes, only the lipoxygenase inhibitor, nordihydroguaiaretic acid reduced EGF-mediated ROS accumulation. The inhibitory action of NGF on ROS production was mimicked by the nitric oxide donor, sodium nitroprusside, and was blocked by an inhibitor of nitric-oxide synthetase, Lnitroarginine methyl ester. These results suggest a novel mechanism for the rapid interruption of mitogenic signaling by the neurotrophin NGF.The neurotrophins are a family of polypeptides that are involved in the survival and differentiation of several different classes of neurons in both the central and peripheral nervous systems. Although some aspects of the intracellular mechanisms by which they act are now clear (1, 2), the way in which they produce global changes in phenotype, such as differentiation or survival, is not known. One general effect of the neurotrophins on neurons is to inhibit cell division. The mechanisms by which this occurs are a matter of substantial research interest because they may have relevance to the overall control of cell division.It was recently reported that one of the early requirements for the promotion of cell division by mitogenic growth factors involves a rapid intracellular production of reactive oxygen species (ROS), 1 such as hydrogen peroxide and superoxide (3, 4). In related experiments exogenously added ROS stimulated mitogenesis in various cell types and was associated with increased expression of cellular markers of proliferation (5). Neurotrophin-stimulated production of reactive nitrogen species, such as nitric oxide, is reportedly required for neurotrophininduced cytostasis and differentiation (6). Together these reports suggest that the signal transduction mechanisms leading to either mitogenesis or differentiation may be controlled by the relative balance between distinct forms of oxidant and nitroxide levels within cells. The PC12 cell model (7, 8) has been quite informative in providing an understanding of neurotrophin-induced signal transduction. PC12 cells stimulated with the neurotrophin nerve growth factor (NGF) undergo many of the gl...

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“…Equal amounts of protein were loaded on a non-denaturing polyacrylamide gel and processed as described (46).…”

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Protein Kinase B Activation by Reactive Oxygen Species Is Independent of Tyrosine Kinase Receptor Phosphorylation and Requires Src Activity

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