FIGURE 5.

MnSOD influences NGF-induced ERK1/2 activity. A, a peroxide scavenger abolishes long term ERK1/2 stimulation by NGF. The time-course of ERK1/2 induction by NGF is shown. PC12 cells were treated with NGF (100 ng/ml) for the times indicated in the absence or presence of the peroxide scavenger (ebselen 20 μm for 30 min). 50 μg of proteins were immunoblotted with anti-p-ERK1/2. The values shown in the histogram represent the means ± S.E. derived from of at least three experiments performed in triplicate. The inset shows a representative experiment. *, p < 0.01 relative to untreated cells. B, a peroxide scavenger inhibits neurite outgrowth induced by NGF. The same cells indicated in A were plated in the presence of NGF for 5 days in the presence or absence of 20 μm ebselen. Viability of the cells, measured by fluorescence-activated cell sorter, was not affected under these conditions. Neurite outgrowth was measured as described under “Experimental Procedures” except that neurites whose length was 0.5 and 1 times the cell body were also measured. 200 cells were counted for each plate, and the percentage of cells with neurites was calculated as described (6). Control, 6 ± 2%: +NGF, 75 ± 10%; +NGF-Ebselen 25 ± 10%; NGF versus control, p < 0.001; NGF versus ebselen + NGF, p < 0.02. The experiment was performed in triplicate. The viability of cells treated with 20 μm ebselen for 5 days was not significantly different from control cells, assayed by cytofluorimetry. C, long term ERK1/2 stimulation by SOD mimetic drugs is shown. Time-course of ERK1/2 induction by MnTMPyP (100 μm), a SOD mimetic drug is shown. PC12 cells were treated with MnTPyP for the times indicated in the absence or presence of ebselen (20 μm for 30 min). ERK1/2 activation was assayed by immunoblot with specific antibodies anti-p-ERK1/2. Values represent the means ± S.E. of at least three experiments performed in triplicate (lower panel). A representative experiment is shown in the upper panel. *, p < 0.01 relative to untreated cells. D, MnSOD stimulates ERK1/2. Several cell lines (HEK293, PC12, HeLa) were transfected with control vector, wild type (WT), and mutant (S82A) MnSOD expression vectors. 24 h later the cells were serum-deprived for 18 h and incubated in the presence of 20 μm ebselen for 3 h. Total extracts were prepared as described under “Experimental Procedures,” and 50 μg of proteins were immunoblotted with anti-p-ERK1/2 and anti-MnSOD antibodies. The values shown in the histogram are the means ± S.E. of at least three experiments, performed in triplicate. A representative experiment performed in HEK293 cells is shown in the upper panel. *, p < 0.01 relative to untreated control cells; **, p < 0.01 relative to untreated cells expressing wild type MnSOD.