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

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 p21 ras 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 Cγ. 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, l-nitroarginine 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 differentia-tion 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 global changes in phenotype exhibited by normal neurons, including a cessation of cell division. PC12 cells are unique in that they contain functional receptors for the mitogen epidermal growth factor (EGF), as well as for NGF (9). One way in which the neurotrophins promote cellular differentiation may be to interfere with mitogenic signals. Indeed, previous data from this laboratory suggest that one action of NGF on PC12 cells is the long term down-regulation of mitogen receptors (9,10). The present experiments demonstrate that NGF also has profound and immediate effects on mitogen signaling.
The data show that treatment of PC12 cells with NGF suppresses the increase in ROS produced by the mitogen EGF. Both the increase in ROS produced by EGF and the decrease in ROS produced by NGF appear to be dependent on an intact Ras signal transduction pathway. The generation of ROS produced by EGF is blocked by an inhibitor of lipoxygenase, and the decrease produced by NGF is blocked by an inhibitor of nitricoxide synthetase. Furthermore, a nitric oxide donor mimicked NGF suppression of ROS. Overall, these data elucidate a mechanism for the acute action of the neurotrophins in blocking signal transduction from mitogen receptors.

EXPERIMENTAL PROCEDURES
Cell Culture-PC12 cells were cultured in DMEM containing 7% fetal bovine serum, 7% horse serum, 100 g of streptomycin/ml, and 100 units of penicillin/ml. nnr5 and nnr5 variants were grown in RPMI containing the same additives. One day before each experiment cells were seeded onto collagen-coated 35-mm dishes at a density of 500,000 cells per well in DMEM plus serum, streptomycin, and penicillin as above and allowed to equilibrate overnight.  were loaded with DCHF-DA in Ringer's solution for 5 min, and then the medium was replaced with serum-free DMEM.

Measurement of Intracellular Oxidant Concentration-
The fluorescence of intracellular dichlorofluorescein was quantitated by confocal laser scanning fluorescence microscopy (Leica TCS4D, Leica Lasertechnik Heidelberg, Germany) using excitation and emission wavelengths of 488 and 520 nm, respectively. Images were collected using a 512 ϫ 512 pixel format and archived for later analysis. The intensity of the fluorescence in single cells was quantified using Leica quantitation software. For each treatment, three different fields were recorded. The relative fluorescence intensity was measured in 10 cells per field (total: 30 cells per dish) and expressed on a 0-to 255-step gray scale of intensity of fluorescence emission. Two dishes were evaluated in each experiment.
Enzyme Inhibitors and Growth Factors-EGF and NGF (Collaborative Bioproducts, Boston, MA) were diluted in DMEM and added to cultures at final concentrations of 100 ng/ml each. Sodium nitroprusside and nitro blue tetrazolium (Sigma) were dissolved in sterile H 2 O. L-NAME (Sigma) was dissolved in ethanol. Diphenyliodonium chloride (Calbiochem), allopurinol (Sigma), PD098095 (2Ј-amino-3Ј-methoxyflavone; Calbiochem), and NDGA (Calbiochem) were dissolved in Me 2 SO. All inhibitors were added to cells 30 min before EGF stimulation. NGF was added either 5 or 10 min before EGF.

RESULTS AND DISCUSSION
Treatment of PC12 cells with EGF produced a rapid and robust increase in ROS in the cells (Fig. 1, A and B). Treatment with NGF had no comparable effect (Fig. 1C), however pretreatment of cells for 10 min with NGF completely abolished the increase in ROS seen upon subsequent treatment with EGF (Fig. 1E).
The EGF-induced increase in ROS appears to require signaling through the Ras pathway. PD098059, a MEK inhibitor, blocked the EGF-induced increase in ROS (Fig. 2a, A-C) Ras dominant-negative cells (PC12-N17) treated with EGF did not show any increase in ROS (Fig. 2a, D and E), although, clearly, these cells have EGF receptors (11). The NGF-induced decrease in mitogen-generated ROS also appears to depend on the Ras pathway. PC12 nnr5 cells (12), which lack p140 trk receptors for NGF, respond normally to EGF (Fig. 2b, A and B) but are not affected by pretreatment with NGF (Fig. 2b, C). When p140 trk receptors are heterologously expressed in nnr5 cells, the ability of NGF to prevent the EGF-induced rise in ROS is restored (Fig. 2b, D and E). However PC12 nnr5 cells transfected with a mutant p140 trk , which lacks binding sites for PLC␥ and for Shc (13), and thus do not show NGF-induced activation of the Ras pathway, did not show a suppressive effect of NGF on EGFinduced ROS formation (Fig. 2b, F and G). The dual reliance of EGF-induced ROS generation and NGF-induced ROS suppression on the Ras pathway is probably predictable; mitogeninduced ROS generation in fibroblasts is suppressed by the expression of dominant-negative isoforms of Ras (14), and the suppression of endogenous ROS by NGF in GT1-1 trk cells is dependent on the Ras pathway (15).
It is likely that the species of ROS generated by EGF stimulation is superoxide (O 2 Ϫ ). Among a number of drugs that are known to inhibit superoxide-producing enzymes, including NADPH oxidase, cyclooxygenase, and lipoxygenase, only pretreatment of cells with the lipoxygenase inhibitor NDGA sup- pressed EGF-induced ROS production (Fig. 3a, A-D). In addition, nitro blue tetrazolium (NBT), a specific chemical scavenger of superoxide, suppressed EGF-induced ROS accumulation (Fig.  3a, E and F).
It is also likely that the quenching by NGF pretreatment of the superoxide generated is a result of the production of nitric oxide. Only an inhibitor of nitric-oxide synthetase, L-NAME, prevented the action of NGF (Fig. 3b, A-F). In addition, the nitric oxide donor sodium nitroprusside (SNP) mimicked the suppressive action of NGF on ROS generation by EGF (Fig. 3b,  G and H). It has been shown that nitric oxide will react with superoxide in a highly favorable reaction to produce peroxynitrate anion (OONO Ϫ ) (16), which appears to react readily with reduced glutathione (17). It is also known that nitric oxide donor compounds inhibit lipoxygenase activity (18).
The data show that EGF treatment of PC12 cells produces a marked increase in ROS, that is likely superoxide, and that NGF produces little or none, despite the data indicating that NGF is initially mitogenic for PC12 cells (19). Pretreatment with NGF abolishes that increase in a manner dependent upon p140 trk . Further, both these actions rely on the integrity of the Ras signal transduction pathway. Finally, the inhibitory action of NGF treatment on ROS accumulation appears to involve the generation of nitric oxide and either the reaction of that nitric oxide with superoxide or the inhibition, by nitric oxide, of lipoxygenase. The acute nature of the response to NGF predicts either a very quick increase in inducible nitric-oxide synthetase or a rapid NGF-induced activation of the constitutive nitricoxide synthetase, an effect not hitherto reported.
It is known that treatment of PC12 cells with NGF leads to a rapid down-regulation of the EGF receptor (20,21). However, it is unlikely that this is the reason for the prevention of the ROS increase by NGF because the down-regulation is only partial and a comparable action of NGF is seen when 3T3 cells transfected with p140 trk are treated with PDGF (data not shown), and there is no reason to believe that NGF has any effect on the levels of PDGF receptors in these cells.
There is some evidence for the involvement of Ras in ROS generation. In A431 cells, overexpression of Ras was associated with a 40-fold induction in the promoter activity of human 12-lipoxygenase (22). Arachidonic acid metabolites produced by lipoxygenase have been reported to be Ras-dependent mitogens for certain cells (23) and may also influence Ras-dependent proliferation by modulating its associations with GTPase-activating proteins (24,25). There is only a limited amount of information regarding the involvement of Ras in the regulation of nitric oxide production; lipopolysaccharide induction of nitric-oxide synthetase in astrocytes can be prevented with specific Ras inhibitors (26).
The generation of ROS is an essential element in signaling from mitogen receptors (3,4). If increases in ROS are prevented the cells neither proliferate nor express normal markers of PDGF-induced mitogenic signaling. Treatment of appropriate cells with neurotrophins inhibits their cell division. The present data indicate that this effect of the neurotrophins is, at least in part, due to their ability to generate nitric oxide which, in turn, quenches the ROS generated by mitogen treatment and thus interrupts the signal transduction pathway that leads to cell division.