Activation of the ERK 1/2 and STAT3 signaling pathways is required for 661W cell survival following oxidant injury

Abstract

Aim: To evaluate the influence of hydrogen peroxide (H(2)O(2)) on mouse photoreceptor-derived 661W cell survival and to determine the effect of PD98059, an inhibitor for MEK1 (the direct upstream activator of ERK1/2), and S3I201, a STAT3- specific inhibitor on 661W cell survival after H(2)O(2) exposure.

Methods: The mouse photoreceptor-derived 661W cells were cultured. 661W cells were treated for 12 hours with different concentrations (0, 0.25, 0.50, 0.75, 1mmol/L) of H(2)O(2) and cell viability was determined by 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide ) (MTT) assay. 661W cells were treated with different concentrations H(2)O(2) (0, 5, 10, 50, 500, 1000 µmol/L) for 15 minutes or 1mmol/L H(2)O(2) for different time points (0,5,10,15,30 minutes), and p-Tyr705-STAT3, STAT3, Phospho-p44/42 MAPK (Thr202/Tyr204), ERK1/2 were surveyed by immunoblot analysis. After treatment with 50µmol/L PD98059, or S3I201 for 1 hour, the inhibition efficiency of cell signal pathways was analyzed by immunoblot analysis and the effects of inhibitors on cell viability were determined by MTT.

Results: After treating with different concentrations of H(2)O(2) for 12 hours, the cell viability of 661W cells decreased in concentration-dependent manner (P<0.05). Moreover, H(2)O(2) induced phosphorylation of ERK1/2 and STAT3 in 661W cells (P<0.05). After pretreatment with 50µmol/L PD98059 or S3I201 for 1 hour, H(2)O(2)-induced phosphorylation of ERK1/2 or STAT3 was suppressed separately (P<0.05). Using PD98059 or S3I201 to inhibit ERK1/2 or STAT3 signal pathway, the cell viability of 661W cells decreased significantly (P<0.05).

Conclusion: We demonstrated that the exposure of 661W cells to H(2)O(2) increased the activation of ERK1/2 and STAT3 signal pathways. Activation of these pathways is required for 661W cell survival following oxidant injury.

Keywords: 661W cells; ERK1/2; STAT3; oxidant injury; survival.

Figure 1. Effect of H₂O₂ on cell viability of 661W cells

Freshly grown 661W cells were exposed to different indicated concentrations of H₂O₂ for 12 hours. Cell viability was assessed by MTT assay. Values are means ±SD of 3 independent experiments conducted in triplicates and expressed as the percentage of control. *P<0.05 compared with control.

Figure 2. H₂O₂–induced activation of ERK1/2 and STAT3. 661W cells were exposed to different indicated concentrations of H₂O₂ for 15 minutes

Cell lysates were prepared and subject to immunoblot analysis with antibodies for Phospho-p44/42 MAPK (Thr202/Tyr204), ERK1/2, p-Tyr705-STAT3, STAT3. Representative immunoblots from 3 experiments are shown (A). Quantiative analysis of the expression of p-ERK1/2 and p-Tyr705-STAT3 was conducted. Values are means ±SD of 3 independent experiments conducted in triplicates and expressed as the percentage of ERK1/2 and STAT3, respectively (B, C). *P<0.05 compared with control.

Figure 3. H₂O₂–induced activation of ERK1/2 and STAT3 signal pathways

661W cells were exposed to 1mM H₂O₂ for indicated time. Cell lysates were prepared and subject to immunoblot analysis with antibodies for Phospho-p44/42 MAPK (Thr202/Tyr204), ERK1/2, p-Tyr705-STAT3, STAT3 (A). Quantiative analysis of the expression of p-ERK1/2 and p-Thy705-STAT3 was conducted. Values are means ±SD of 3 independent experiments conducted in triplicates and expressed as the percentage of ERK1/2 and STAT3, respectively (B, C). *P<0.05 compared with control.

Figure 4. PD98059 and S3I201 reduced H₂O₂ –induced ERK1/2 and STAT3 activation, respectively

After pretreating with PD98059 and S3I201 for 1 hour and then exposing them to 1mM H₂O₂ for 15 minutes, 661W cell lysates were prepared and subjected to immunoblot analysis with antibodies for Phospho-p44/42 MAPK (Thr202/Tyr204), ERK1/2, p-Tyr705-STAT3, STAT3 (A). Quantiative analysis of the expression of p-ERK1/2 and p-Thy705-STAT3 was conducted. Values are means ±SD of 3 independent experiments conducted in triplicates and expressed as the percentage of ERK1/2 and STAT3, respectively (B, C). *P<0.05 compared with control. # P<0.05 compared with the group treated with H₂O₂ only.

Figure 5. Inhibition of ERK1/2 or STAT3 decreases the viability of 661W cells

661W cells were incubated with PD98059 (50µmol/L) or S3I201 (50µmol/L) for 1 hour and then exposed to 1mmol/L H₂O₂ for 12 hours. Cell viability was determined by MTT assay. Values are means ±SD of 3 independent experiments conducted in triplicates and expressed as the percentage of control. *P<0.05 compared with control.