Prevention of PC12 cell death by N-acetylcysteine requires activation of the Ras pathway

Abstract

We have shown that N-acetylcysteine (NAC) promotes survival of sympathetic neurons and pheochromocytoma (PC12) cells in the absence of trophic factors. This action of NAC was not related to its antioxidant properties or ability to increase intracellular glutathione levels but was instead dependent on ongoing transcription and seemed attributable to the action of NAC as a reducing agent. Here, we investigate the mechanism by which NAC promotes neuronal survival. We show that NAC activates the Ras-extracellular signal-regulated kinase (ERK) pathway in PC12 cells. Ras activation by NAC seems necessary for survival in that it is unable to sustain serum-deprived PC12 MM17-26 cells constitutively expressing a dominant-negative form of Ras. Promotion of PC12 cell survival by NAC is totally blocked by PD98059, an inhibitor of the ERK-activating MAP kinase/ERK kinase, suggesting a required role for ERK activation in the NAC mechanism. In contrast, LY294002 and wortmannin, inhibitors of phosphatidylinositol 3-kinase (PI3K) that partially block NGF-promoted PC12 cell survival, have no effect on prevention of death by NAC. We hypothesized previously that the ability of NAC to promote survival correlates with its antiproliferative properties. However, although NAC does not protect PC12 MM17-26 cells from loss of trophic support, it does inhibit their capacity to synthesize DNA. Thus, the antiproliferative effect of NAC does not require Ras activation, and inhibition of DNA synthesis is insufficient to mediate NAC-promoted survival. These findings highlight the role of Ras-ERK activation in the mechanism by which NAC prevents neuronal death after loss of trophic support.

Fig. 1.

NAC increases the expression of immediate early genes. PC12 cells in serum-containing medium were treated with 100 ng/ml NGF or 60 mm NAC. At the indicated times, the RNA was collected, blotted, probed with radiolabeled TIS1, c-fos, or c-jun, stripped, and reprobed with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Similar findings were achieved in another independent experiment.

Fig. 2.

Time course of NAC-induced ERK-1 activity. PC12 cells grown in medium with serum were treated with 100 ng/ml NGF for 5 min or 60 mm NAC for the indicated times, after which the cells were collected and submitted to anti-ERK-1 immunoprecipitation (see Materials and Methods) and assessment of ERK-1 activity using myelin basic protein (MBP) as substrate. ERK-1 activity is expressed as the amount of ³²P incorporated into MBP per 30 min.

Fig. 3.

NAC activates Ras. PC12 cells loaded with [³²P]orthophosphate were treated with no additives (control), with 100 ng/ml NGF for 5 min, or with 60 mm NAC for the indicated times. Ras activity is measured as the ratio of [³²P]GTP/total nucleotides associated with Ras after immunoprecipitation (see Materials and Methods). The results are expressed relative to the activity present in untreated cells; n = number of independent experiments; data are expressed as the mean ± SEM.

Fig. 4.

NAC does not protect MM17-26 cells from apoptosis caused by withdrawal of trophic support. Replicate cultures of PC12 cells transfected with control vector (M7) or dominant-negative Ras (MM17-26) were cultured in serum-free medium treated with no additives (control) or 40 or 60 mm NAC for 2 d. Cells were counted, and results are expressed relative to initial plating number (n = 3).

Fig. 5.

NAC induces ERK-1 activity in PC12 but not in MM17-26 cells. Cells grown in medium supplemented with serum were treated with no additives (control), 100 ng/ml NGF, or 60 mm NAC for 30 min, after which ERK-1 was immunoprecipitated and assessed for activity as described in Materials and Methods. Activity is expressed as the amount of ³²P incorporated into myelin basic protein in 30 min. This experiment is representative of three others.

Fig. 6.

Effect of MEK and PI3 kinase inhibitors on NAC-mediated PC12 cell survival (A) and of MEK inhibition on NAC-induced ERK-1 activity (B).A, For the survival experiments, cells in serum-free medium were pretreated with no inhibitors, with 100 μmPD98059, and/or with 10 μm LY294002 for 1 hr before addition of no additives (CONTROL), 100 ng/ml NGF, 100 μm 8-(4-chlorophenylthio)-cAMP (CPT-cAMP), or 60 mm NAC. Surviving cells were counted after 24 hr, and results are expressed relative to NGF-treated cells in the absence of inhibitors (n = 3). B, For the assessment of ERK-1 activity, cells in complete medium were pretreated with no inhibitor or with PD98059 for 1 hr before addition of no additives (CONTROL), 100 ng/ml NGF or 60 mm NAC for 30 min, after which ERK-1 was immunoprecipitated and assessed for kinase activity (see Materials and Methods). Activity is expressed as the amount of ³²P incorporated into myelin basic protein in 30 min.

Fig. 7.

The PI3 kinase inhibitor wortmannin does not affect NAC-mediated survival. PC12 cells in serum-free medium were pretreated with 150 nm wortmannin for 30 min and then 60 mm NAC for 9 hr. Wortmannin was added every 2 hr to compensate for breakdown. Results are expressed relative to survival obtained in NGF-treated cells in the absence of wortmannin (n = 3).

Fig. 8.

NAC inhibits thymidine incorporation in both M7 and MM17-26 cells. The cells were cultured in serum-free medium supplemented with 100 μm insulin and were treated with increasing concentrations of NAC overnight. Results are expressed relative to cells that were not treated with NAC (n= 3).

Fig. 9.

MEK and PI3 kinase inhibitors do not reverse NAC-induced cell cycle arrest. PC12 cells in serum-containing medium were preincubated with 100 μm PD98059 or 10 μm LY294002 for 1 hr and then were incubated with or without 60 mm NAC overnight. Measurements of thymidine incorporation were performed as described in Materials and Methods. Results are expressed relative to the untreated control (n = 3).