Early phosphatidylinositol 3-kinase/Akt pathway activation limits poliovirus-induced JNK-mediated cell death

Abstract

Poliovirus (PV)-induced apoptosis seems to play a major role in tissue injury in the central nervous system (CNS). We have previously shown that this process involves PV-induced Bax-dependent mitochondrial dysfunction mediated by early JNK activation in IMR5 neuroblastoma cells. We showed here that PV simultaneously activates the phosphatidylinositol 3-kinase (PI3K)/Akt survival signaling pathway in these cells, limiting the extent of JNK activation and thereby cell death. JNK inhibition is associated with PI3K-dependent negative regulation of the apoptosis signal-regulating kinase 1, which acts upstream from JNK in PV-infected IMR5 cells. In poliomyelitis, this survival pathway may limit the spread of PV-induced damage in the CNS.

FIG. 1.

PV induces early Akt phosphorylation in a PI3K-dependent manner in IMR5 neuroblastoma cells. (A) Kinetics of Akt activation in PV-infected neuronal cells. (Top panel) Akt activation was analyzed in whole-cell lysates at the indicated times p.i. by Western blotting with a specific anti-phospho (Ser473)-Akt antibody (Cell Signaling). Whole-cell lysates from mock-infected cells were analyzed at 30 min (first lane) and 240 min (last lane), respectively, after mock infection. The blots were then stripped and reprobed with an antibody recognizing all forms of Akt (Cell Signaling) to confirm equal protein loading. (Bottom panels) Western blot analyses of Akt activation 30 min p.i. (Bottom left panel) Cells were infected with viral stock (PV) or viral stock depleted of PV (PV ^depleted) with anti-PV antibody. (Bottom right panel) Cells were infected with viral stock (PV) or CsCl-purified PV (PV ^purified). (B) Inhibition of Akt phosphorylation during PV infection in IMR5 cells treated with the PI3K inhibitor wortmannin (100 nM and 500 nM; Calbiochem). (Top panel) Cells were incubated or not incubated with the PI3K inhibitor for 2 h before PV infection, and the concentration of the inhibitor was maintained during the adsorption period and throughout PV infection. The levels of phospho (Ser473)-Akt in whole-cell lysates were determined by Western blotting 30 min p.i. The blots were then stripped and reprobed with an antibody recognizing all forms of Akt to confirm equal protein loading. (Bottom panel) Mock- and PV-infected IMR5 cells (30 min p.i.), treated or not treated with wortmannin (100 nM), were stained for immunofluorescence with a specific antibody against phospho (Ser473)-Akt and a secondary, fluorescein isothiocyanate-conjugated antibody (green; middle panels). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue; left panels). The merged image is an overlay of the DAPI image with the anti-phospho (Ser473)-Akt image (right panels).

FIG. 2.

UV-inactivated PV induces early Akt activation in IMR5 cells. Akt activation was analyzed by Western blotting whole-cell lysates from cells infected with infectious or UV-inactivated PV (30 min p.i.) with a specific anti-phospho (Ser473)-Akt antibody. The blots were then stripped and reprobed with an antibody recognizing all forms of Akt to confirm equal protein loading.

FIG. 3.

Inhibition of the PI3K/Akt signaling pathway enhances PV-induced apoptosis in IMR5 cells. (A) Enhancement of Bax activation in PV-infected cells treated with wortmannin. (Top panel) Cells were mock infected or infected with PV (8 h p.i.) in the presence or absence of wortmannin (100 nM). The cells were lysed in immunoprecipitation buffer. Conformationally active Bax protein was immunoprecipitated (IP) with anti-Bax 6A7 antibody (Santa Cruz), and the precipitates were immunoblotted with anti-Bax antibody. The asterisk indicates immunoglobulin light chains. (Bottom panel) Whole-cell lysates not incubated with 6A7 antibody were similarly tested for total Bax by immunoblotting with a specific antibody (Upstate) to check that the amounts of Bax protein in the samples before immunoprecipitation were equivalent. Actin was used as a control for protein loading. (B) There was a greater level of cytochrome c (Cyt c) release in PV-infected cells treated with wortmannin. Cytochrome c release was analyzed in the cytosolic fractions of mock-infected and PV-infected IMR5 cells (8 h p.i.) treated or not treated with wortmannin (100 nM) by Western blotting with a specific antibody (BD Pharmingen). Actin was used as a protein-loading control. Protein levels were determined by densitometry and plotted as ratios relative to the actin levels. (C) Enhancement of apoptosis in PV-infected cells treated with wortmannin. Mock-infected and PV-infected IMR5 cells treated (black) or not treated (light gray) with wortmannin (100 nM) were analyzed at the indicated times p.i. by flow cytometry after AO (Molecular Probes) staining, and the increase (n-fold) in apoptosis was calculated as the ratio of the percentage of PV-infected IMR5 cells that were apoptotic to the percentage of mock-infected cells that were apoptotic. The data shown are the means from three independent experiments. Error bars represent the standard errors of the means. *, P < 0.05 by Student's t test comparing untreated IMR5 cells to treated IMR5 cells. (D) Higher levels of apoptosis were observed after the knockdown of Akt expression in PV-infected cells. (Left panel) IMR5 cells were transfected with Akt siRNA (Cell Signaling) or nontargeted control siRNA (Cell Signaling) or left untreated. Akt protein was then assayed by immunoblotting with extracts from nontargeted control siRNA-transfected, Akt siRNA-transfected, or untreated cells. Actin was used as a protein loading control. (Right panel) Cells were mock infected or infected (8 h p.i.) with PV 72 h after transfection and then analyzed by flow cytometry after AO staining. The increase (n-fold) in apoptosis was calculated as the ratio of the percentage of PV-infected IMR5 cells that were apoptotic to the percentage of mock-infected cells that were apoptotic. The data shown are the means from three independent experiments. Error bars represent the standard errors of the means. *, P < 0.05 by a Student's t test comparing untreated IMR5 cells to treated IMR5 cells.

FIG. 4.

Effect of PI3K/Akt signaling inhibition on PV growth and externalization. IMR5 cells were infected with PV in the presence or absence of wortmannin (100 nM). Total virus yield (extracellular and intracellular) was determined by TCID₅₀ assay at the indicated times after three cycles of freezing and thawing to release intracellular viruses. The extracellular virus titers were determined from the supernatant of PV-infected cells at the indicated times after the removal of detached cells by centrifugation. Each point represents the mean virus titers for two independent experiments. Standard errors of the mean are indicated. *, P < 0.05 by a Student's t test comparing untreated and treated IMR5 cells.

FIG. 5.

The PI3K/Akt signaling pathway limits JNK activation by promoting ASK1 phosphorylation in PV-infected IMR5 cells. (A) JNK activation levels are higher in PV-infected cells treated with wortmannin. Cells were mock infected or infected with PV (30 min p.i.) in the presence or absence of wortmannin (100 nM). JNK activation was analyzed in whole-cell lysates by Western blotting with a specific anti-phospho (Thr183/Tyr185)-JNK (p46 [JNK1] and p54 [JNK2/3]) antibody, as previously described (6). The blots were then stripped and reprobed with an antibody recognizing all forms of JNK to confirm equal protein loading. (B) Inhibition of JNK activation after the knockdown of ASK1 expression in PV-infected IMR5 cells. (Left panel) IMR5 cells were transfected with ASK1 siRNA (37) or nontargeted control siRNA (Cell Signaling) or left untreated. ASK1 protein was then assayed by immunoblotting with extracts from nontargeted control siRNA-transfected, ASK1 siRNA-transfected, or untreated cells. Actin was used as a protein loading control. (Right panel) Untreated, nontargeted control, and ASK1 siRNA-transfected IMR5 cells were mock infected or infected with PV. JNK activation was analyzed (30 min p.i.) in whole-cell lysates by Western blotting with a specific anti-phospho (Thr183/Tyr185)-JNK antibody. The blots were then stripped and reprobed with an antibody recognizing all forms of JNK to confirm equal protein loading. Phosphorylated JNK protein levels were determined by densitometry and plotted as ratios relative to the levels of total JNK. Phosphorylated JNK levels following PV infection in untreated cells were taken as 100%. The data are the means from three independent experiments. Error bars represent the standard errors of the means. *, P < 0.05 by a Student's t test comparing nontargeted control siRNA-transfected IMR5 cells to ASK1-transfected IMR5 cells. (C) Phosphorylation of ASK1 in PV-infected neuronal cells. ASK1 phosphorylation was analyzed in whole-cell lysates at the indicated times p.i. by Western blotting with a specific anti-phospho (Ser83)-ASK1 antibody (Cell Signaling). The blots were then stripped and reprobed with an antibody recognizing all forms of ASK1 (Cell Signaling) to confirm equal protein loading. (D) Inhibition of PV-induced ASK1 phosphorylation by the PI3K/Akt pathway inhibitor wortmannin. Cells were mock infected or infected with PV in the presence or absence of wortmannin (100 nM). ASK1 phosphorylation was analyzed (30 min p.i.) in whole-cell lysates by Western blotting with a specific anti-phospho (Ser83)-ASK1 antibody. The blots were then stripped and reprobed with an antibody recognizing all forms of ASK1 to confirm equal protein loading. Phosphorylated ASK1 protein levels were determined by densitometry and plotted as ratios relative to the levels of total ASK1.