Doxorubicin bypasses the cytoprotective effects of eIF2α phosphorylation and promotes PKR-mediated cell death

Abstract

The eukaryotic cell responds to various forms of environmental stress by adjusting the rates of mRNA translation thus facilitating adaptation to the assaulting stress. One of the major pathways that control protein synthesis involves the phosphorylation of the α-subunit of eukaryotic initiation factor eIF2 at serine 51. Different forms of DNA damage were shown to induce eIF2α phosphorylation by using PERK, GCN2 or PKR. However, the specificity of the eIF2α kinases and the biological role of eIF2α phosphorylation pathway in the DNA damage response (DDR) induced by chemotherapeutics are not known. Herein, we show that PKR is the eIF2α kinase that responds to DDR induced by doxorubicin. We show that activation of PKR integrates two signaling pathways with opposing biological outcomes. More specifically, induction of eIF2α phosphorylation has a cytoprotective role, whereas activation of c-jun N-terminal kinase (JNK) by PKR promotes cell death in response to doxorubicin. We further show that the proapoptotic effects of JNK activation prevail over the cytoprotection mediated by eIF2α phosphorylation. These findings reveal that PKR can be an important inducer of cell death in response to chemotherapies through its ability to act independently of eIF2α phosphorylation.

Figure 1

PKR promotes doxorubicin-induced cell death. (a) PKR^+/+ and PKR^−/− MEFs were left untreated (con) or treated with 1 μM doxorubicin (dox) for the indicated time periods. Cells were subjected to FACS analysis after propidium iodide staining. Cell death is represented by the percentage (%) of cells in sub-G₁. Histograms represent the mean cell death from five independent experiments after subtraction of background cell death (untreated control cells) (n=5). Statistical significance of the difference as calculated by Student's t-test is with ^*P<0.0003, ^**P<0.0009. (b) PKR^+/+ and PKR^−/− MEFs were left untreated (con) or treated with 1 μM doxorubicin (dox) for the indicated time periods. Protein extracts (50 μg) were subjected to western blot analysis for cleaved caspase-3 (a) and actin (b). Histograms represent the mean value of the ratio of cleaved caspase-3 to actin after normalization to that of lane 4 for the indicated lanes of the western blot from five independent experiments (n=5). ^*P<0.005, ^**P<0.006

Figure 2

PERK and GCN2 do not contribute to doxorubicin-induced cell death. PERK^+/+ and PERK^−/− MEFs (a) or GCN2^+/+ and GCN2^−/− MEFs (b) were left untreated (con) or treated with 1 μM doxorubicin (dox) for the indicated time periods and subjected to FACS analysis after propidium iodide staining. Cell death is represented by the percentage (%) of cells in sub-G₁. Histograms represent the mean cell death from three independent experiments after subtraction of background cell death (untreated control cells) (n=3)

Figure 3

PKR induces eIF2α phosphorylation in response to doxorubicin treatment. The indicated types of MEFs were left untreated (con) or treated with 1 μM doxorubicin (dox) for the indicated time periods. Protein extracts (50 μg) were subjected to immunoblot analysis for phosphorylated eIF2α (a, c, e) and total eIF2α (b, d, f). Histograms represent the mean value of the ratio of eIF2α phosphorylation levels to total eIF2α levels for the treated cells normalized to that of their untreated control from six independent experiments for PKR (n=6), five independent experiments for PERK (n=5) and five independent experiments for GCN2 (n=5). Statistical significance of the difference as calculated by Student's t-test is with ^*P<0.05, ^**P<0.02

Figure 4

Phosphorylation of eIF2α protects cells from doxorubicin-induced death. (a) eIF2α^S/S and eIF2α^A/A MEFs were left untreated (con) or treated with 1 μM doxorubicin (dox) for the indicated time periods. Protein extracts (50 μg) were subjected to immunoblotting for phosphorylated eIF2α (a) and total eIF2α (b). Histograms represent the mean value of the ratio of eIF2α phosphorylation levels to eIF2α total levels for the treated cells normalized to that of their untreated control from four independent experiments (n=4) for the eIF2α^S/S MEFs. (b) eIF2α^S/S and eIF2α^A/A MEFs were left untreated (con) or treated with 1 μM doxorubicin (dox) for the indicated time periods and subjected to FACS analysis after propidium iodide staining. Cell death is represented by the percentage (%) of cells in sub-G₁. Histograms represent the mean cell death from five independent experiments after subtraction of background cell death (untreated control cells) (n=5). Statistical significance of the difference as calculated by Student's t-test is with ^*P<0.002, ^**P<0.02. (c) eIF2α^S/S and eIF2α^A/A MEFs were left untreated (con) or treated with 1 μM doxorubicin (dox) for the indicated times. Protein extracts (50 μg) were subjected to western blot analysis for cleaved caspase-3 (a) and actin (b). Histograms represent the mean value of the ratio of cleaved caspase-3 to actin after normalization to the ratio of lane 2 for the indicated lanes from four independent experiments (n=4). Statistical significance of the difference as calculated by Student's t-test is with ^*P<0.006, ^**P<0.04

Figure 5

PKR induces JNK phosphorylation in response to doxorubicin. (a) PKR^+/+ and PKR^−/− MEFs were left untreated (con) or treated with 1 μM doxorubicin (dox) for the indicated times. Protein extracts (50 μg) were subjected to immunoblotting for phosphorylated JNK (a), total JNK1 (b), phosphorylated ERK (c), total ERK (d), phosphorylated p38 (e), total p38 (f). Histograms represent the mean value of the ratio of the phosphorylated protein levels to their total levels for the treated cells normalized to that of their untreated control from four independent experiments for JNK (n=4), four independent experiments for ERK (n=4) and four independent experiments for p38 (n=4). Statistical significance of the difference as calculated by Student's t-test is with ^*P<0.03. (b) PKR^+/+ and PKR^−/− MEFs were left untreated (con) or treated with 1 μM doxorubicin (dox) for the indicated times. Protein extracts (50 μg) were subjected to immunoblotting for phosphorylated JNK (a) and total JNK1 (b). Histograms represent the mean value of the ratio of the phosphorylated protein levels to their total levels for the treated cells normalized to that of their untreated control from six independent experiments (n=6). Statistical significance of the difference as calculated by Student's t-test is with ^*P<0.02, ^**P<0.002. (c) eIF2α^S/S and eIF2α^A/A MEFs were left untreated (con) or treated with 1 μM doxorubicin (dox) for the indicated times. Protein extracts (50 μg) were subjected to immunoblotting for phosphorylated JNK (a) and total JNK1 (b). Histograms represent the mean value of the ratio of the phosphorylated protein levels to their total levels for the treated cells normalized to that of their untreated control from six independent experiments (n=6)

Figure 6

Pharmacological inhibition of JNK blocks PKR-mediated cell death induced by doxorubicin. PKR^+/+ and PKR^−/− MEFs were treated with 10 μM SP600125 (SP600125), 1 μM doxorubicin (dox) or both drugs (dox+SP600125) for the indicated time periods and subjected to FACS analysis using propidium iodide staining. Control cells and doxorubicin-treated cells received the equivalent amount of DMSO in which SP600125 was dissolved. DMSO or SP600125 was added an hour before doxorubicin. Cell death is represented by the percentage (%) of cells in sub-G₁. Histograms represent the mean cell death from three independent experiments (n=3). Group statistical significance of the differences as calculated by ANOVA is with ^*P<0.0001

Figure 7

A model for PKR function in response to doxorubicin. Activation of PKR by doxorubicin leads to the induction of eIF2α phosphorylation, which exerts a cytoprotective effect. Induction of PKR activity also leads to the activation of JNK, which promotes cell death. JNK activation proceeds independently of eIF2α phosphorylation and is sufficient to overcome the cytoprotective effects of eIF2α phosphorylation in immortalized MEFs