Research ArticleImmunology

The ASK family kinases differentially mediate induction of type I interferon and apoptosis during the antiviral response

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Science Signaling  04 Aug 2015:
Vol. 8, Issue 388, pp. ra78
DOI: 10.1126/scisignal.aab1883
  • Fig. 1 The RLR pathway is necessary for effective activation of the p38 and JNK pathways.

    (A to C) HeLa S3 cells were transfected with shRNA expression vectors targeting green fluorescent protein (GFP) (control), MDA5, RIG-I, or IPS-1 and were cultured for 4 days. The cells were then transfected with the indicated concentrations of poly(I:C) and incubated for 6 hours (A), infected (ifx) with increasing titers of EMCV for 3 hours (B), or infected with increasing titers of NDV for 9 hours (C). Cells were then lysed and subjected to Western blotting analysis with antibodies specific for the indicated proteins. Black vertical bars indicate noncontiguous blots. Western blots are representative of at least three independent experiments.

  • Fig. 2 TRAFs mediate the IPS-1–dependent activation of the p38 and JNK pathways.

    (A) Scheme showing wild-type (WT) and the indicated mutant IPS-1 proteins. TM, transmembrane domain. (B) HeLa S3 cells were transiently transfected with empty plasmid (vector) or with plasmids encoding WT IPS-1 or the indicated TRAF-binding mutants of IPS-1 (100 or 500 ng, as indicated) together with the PRD4-Renilla luciferase reporter and pGL3-control as an internal control. The cells were then lysed, and luciferase assays were performed. Data are means ± SD of triplicate wells from a single experiment and are representative of three independent experiments. (C) HeLa S3 cells were transiently transfected with empty plasmid (vector) or with plasmid encoding the dT2/dT3/dT6 IPS-1 mutant and were cultured 24 hours. Cells were then left untreated (0 hour) or were transfected with poly(I:C) (0.25 μg/ml) and then incubated for the indicated times. The amounts of IFNB mRNA were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis and are expressed relative to those of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Data are means ± SD of triplicate samples from a single experiment and are representative of three independent experiments. *P < 0.05. (D) HeLa S3 cells expressing the indicated shRNAs (as described in Fig. 1A) were left untreated or were transfected with the indicated concentrations of poly(I:C) and incubated for 6 hours. Cell lysates were then subjected to Western blotting analysis with antibodies specific for the indicated proteins. Western blots are representative of three independent experiments.

  • Fig. 3 ASK1 is activated in response to viral infection and poly(I:C).

    (A to C) HeLa S3 cells were transfected with the indicated concentrations of poly(I:C) and incubated for 3 hours (A), infected with increasing titers of EMCV for 3 hours (B), or transfected with control vector or plasmid encoding Flag–IPS-1 (C). The cells were then lysed and subjected to Western blotting analysis with antibodies specific for the indicated proteins. H2O2 was used as a positive control to activate ASK1. (D) HeLa S3 cells were transfected with poly(I:C) and incubated for the indicated times before being lysed and subjected to immunoprecipitation (IP) with anti-ASK1 antibody or control immunoglobulin G (IgG), followed by Western blotting analysis with antibodies against ASK1 and IPS-1. As a control, total cell lysates were analyzed by Western blotting with antibodies against the indicated proteins. All Western blots are representative of three independent experiments.

  • Fig. 4 ASK1 plays a role in inducing IFNB expression and activation of the p38 and JNK pathways.

    (A to C) HeLa S3 cells expressing the indicated shRNAs (as described in Fig. 1A) were transfected with the indicated concentrations of poly(I:C) and incubated for 6 hours [A (left), B, and C], infected with increasing titers of EMCV for 3 hours [A (middle) and C], or infected with increasing titers of NDV for 9 hours [A (right) and C] before being subjected to Western blotting analysis with antibodies against the indicated proteins (A and B) and qRT-PCR analysis to determine the abundance of IFNB mRNA relative to that of GAPDH mRNA (C). (A, middle) The asterisk indicates a nonspecific band. (B) Whole-cell extracts were analyzed by native polyacrylamide gel electrophoresis (PAGE) to examine IκBα degradation and IRF3 dimerization. (D to F) ASK1+/+ and ASK1–/– MEFs were transfected with poly(I:C) and incubated for the indicated times or were infected with increasing titers of NDV for 9 hours (D and E), or were infected with the highest titer of NDV used in (E) for 24 hours (F). (D) Cell lysates were subjected to Western blotting analysis with antibodies specific for the indicated proteins. (E and F) The relative amounts of IFNB and GAPDH mRNAs and of NDV matrix protein (M) RNA were determined by qRT-PCR analysis. (G) ASK1+/+ and ASK1–/– MEFs were infected with SeV-GFP [103 plaque-forming units (PFU)] and then were analyzed by immunofluorescence microscopy. Scale bar, 200 μm. Data in (C, left) and (F) are means ± SEM of three independent experiments or are means ± SD of triplicate samples from a single experiment and are representative of three independent experiments [A, B, C (middle and right), D, E, and G]. *P < 0.05, ***P < 0.005. Black vertical lines in (A) and (D) denote noncontiguous Western blots.

  • Fig. 5 ASK1 is required for apoptosis induced by poly(I:C).

    (A) HeLa S3 cells expressing the indicated shRNAs were left untreated (control) or were transfected with poly(I:C) (2.5 μg/ml) and then incubated for 6 hours. (Left) Cells were stained with Hoechst 33342 and analyzed by microscopy. White arrowheads indicate apoptotic cells with pyknotic nuclei. (Right) The percentages of cells with pyknotic nuclei were determined. Data are means ± SD of values obtained from three fields of 150 to 200 cells in each of three independent experiments (~600 cells in total). Scale bar, 300 μm. (B) HeLa S3 cells were treated as described in (A), and apoptotic cells were stained with annexin V. Data are presented as the fold change in annexin V–positive cells relative to untreated control cells. Data are means ± SEM of four independent experiments. (C to E) HeLa S3 cells expressing the indicated shRNAs were transfected with the indicated concentrations of poly(I:C) and incubated for 6 hours (C), infected with NDV for 9 hours (D), or infected with EMCV for 12 hours (E). Cells were then lysed and subjected to Western blotting analysis with antibodies specific for the indicated proteins. Black vertical lines in (E) denote noncontiguous Western blots. Data in (A) and (C) to (E) are representative of at least three independent experiments. *P < 0.05, **P < 0.01.

  • Fig. 6 ASK1 mediates antiviral responses in vivo.

    (A to C) ASK1+/+ and ASK1–/– mice were intranasally inoculated with phosphate-buffered saline (PBS) (Control) or 3.2 × 102 PFU (50 μl) of influenza virus CA04. (A) The amounts of IFNB mRNA relative to that of GAPDH mRNA in the lungs of the indicated mice were determined by qRT-PCR analysis 1 day after infection. Data are means ± SEM of four independent experiments (from 9 to 11 mice per group). (B) (Left) Influenza virus–infected cells in the lungs of the indicated mice were analyzed 3 days after infection by immunohistochemical staining for cleaved caspase-3 (red) and the viral M1 protein (green). The numbers of cells in the bronchioles that were positive for both active caspase-3 and M1 (white arrowheads) were counted. (Right) The percentages of infected cells with active caspase-3 were determined. Data in the bar graph are means ± SD of values obtained from three to eight fields of 150 to 200 cells per mouse in each of four independent experiments (~900 cells in total) (Control, n = 5 mice; Influenza, n = 7 mice). Scale bars, 10 μm. Note that although the anti-M1 antibody showed a nonspecific signal in uninfected (control) lungs, as indicated by asterisks, bronchiolar epithelial cells were labeled by the anti-M1 antibody only after influenza infection. (C) The viral titers in lungs 3 days after infection were determined by plaque assays in Madin-Darby canine kidney (MDCK) cells. Data are means ± SEM of four independent experiments (n = 15 to 16 mice per group). (D) Age- and sex-matched ASK1+/+ mice (n = 17 mice) and ASK1−/− mice (n = 12 mice) were inoculated intraperitoneally with 1 × 105 PFU of EMCV and then were monitored daily for 9 days for survival. Data were analyzed by the log-rank test. (E) Age- and sex-matched ASK1+/+ mice (n = 16 mice) and ASK1−/− mice (n = 11 mice) were infected intraperitoneally with 1 × 102 PFU of EMCV. Two days after infection, the mice were sacrificed, and viral titers in the hearts were determined by standard plaque assay in MEFs from IFN-α receptor 1 (IFNAR1)−/− mice. Data are pooled from four independent experiments. *P < 0.05, **P < 0.01, ***P < 0.005.

  • Fig. 7 ASK2 selectively activates an apoptotic response and inhibits viral replication.

    (A to D) HeLa S3 cells expressing the indicated shRNAs were transfected with poly(I:C) and incubated for 6 hours. The amounts of ASK2 (A) and IFNB (B) mRNAs relative to that of GAPDH mRNA were determined by qRT-PCR analysis. (C and D) The extent of apoptosis was determined as described in Fig. 5 (A and B). Scale bar, 300 μm. (E) HeLa S3 cells ex????pressing the indicated shRNAs were left untreated or were transfected with the indicated concentrations of poly(I:C) and incubated for 6 hours. Cells were then lysed and subjected to West????ern blotting analysis with antibodies specific for the indi?????cated proteins. Black vertical lines denote noncontiguous Western blots. Data are representative of three independent experiments. (F and G) 293-ASK2/1KN cells were left untreated or were pretreated with the indicated concentrations of tetracycline (Tet) (to induce expression of ASK2) and then were transfected with poly(I:C) (7.5 μg/ml) (left) or infected with NDV for the indicated times (right). (F) The amounts of IFNB mRNA relative to that of GAPDH mRNA were determined by qRT-PCR analysis. (G) Cells were then lysed and subjected to Western blotting analysis with antibodies specific for the indicated proteins. Black vertical lines denote noncontig????uous Western blots. Data are means ± SEM of four indepen????dent experiments (D) or are means ± SD of triplicate sam????ples from a single experiment and are representative of three independent experiments (A to C, F, and G). (H to J) ASK2+/+ and ASK2–/– mice were intranasally inoculated with PBS or 3.2 × 102 PFU (50 μl) of influenza virus CA04. (H) The amounts of IFNB mRNA relative to that of GAPDH mRNA in the lungs of the indicated mice were determined by qRT-PCR analysis 1 day after infection. Data are means ± SEM of four independent experiments (from 11 to 14 mice per group). (I) (Left) Influenza virus–infected cells in the lung were analyzed 3 days after infection by immunohistochemical staining for cleaved caspase-3 (red) and the viral M1 protein (green). The numbers of cells in the bronchioles that were positive for both active caspase-3 and M1 (white arrowheads) were counted. (Right) The percentages of infected cells with active caspase-3 were determined. Data in the bar graphs are means ± SD of values obtained from three to eight fields of 150 to 200 cells per mouse in each of three independent experiments (~900 cells in total) (Control, n = 3 mice; Influenza, n = 5 to 6 mice). Scale bars, 10 μm. (J) The viral titers in the lungs of the indicated mice 3 days after infection were determined by plaque assays in MDCK cells. Data are means ± SEM of three independent experiments. (n = 10 to 11 mice per group). *P < 0.05, **P < 0.01, ***P < 0.005.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/388/ra78/DC1

    Fig. S1. The RLR pathway is necessary for the activation of IRF3 after infection with EMCV.

    Fig. S2. TRAF2 and TRAF6 mediate the activation of ATF-2 and c-Jun in response to poly(I:C).

    Fig. S3. Quantification of Western blots.

    Fig. S4. Analysis of the effect of TRAF3 knockdown on the activation of the p38 and JNK pathways.

    Fig. S5. ASK1 interacts with IPS-1.

    Fig. S6. TLR3 is necessary for the effective activation of the p38 and JNK pathways after infection with EMCV.

    Fig. S7. ASK1 is necessary for effective activation of caspase-3 and induction of IFNB expression in response to influenza virus.

    Fig. S8. Survival and weight loss of ASK1+/+ and ASK1–/– mice after infection with influenza virus.

    Fig. S9. ASK2 selectively activates an apoptotic response to infection with EMCV.

    Fig. S10. ASK2 selectively activates an apoptotic response to infection with influenza virus.

    Fig. S11. Overexpression of ASK2 selectively promotes the activation of caspase-3 in response to infection by EMCV.

    Fig. S12. Stable association between ASK1 and ASK2 before and after transfection of cells with poly(I:C).

    Fig. S13. Schematic overview of the antiviral strategies mediated by ASK family members.

  • Supplementary Materials for:

    The ASK family kinases differentially mediate induction of type I interferon and apoptosis during the antiviral response

    Tomohiko Okazaki,* Maiko Higuchi, Kohsuke Takeda, Kiyoko Iwatsuki-Horimoto, Maki Kiso, Makoto Miyagishi, Hideyuki Yanai, Atsushi Kato, Mitsutoshi Yoneyama, Takashi Fujita, Tadatsugu Taniguchi, Yoshihiro Kawaoka, Hidenori Ichijo, Yukiko Gotoh

    *Corresponding author. E-mail: tokazaki{at}mol.f.u-tokyo.ac.jp

    This PDF file includes:

    • Fig. S1. The RLR pathway is necessary for the activation of IRF3 after infection with EMCV.
    • Fig. S2. TRAF2 and TRAF6 mediate the activation of ATF-2 and c-Jun in response to poly(I:C).
    • Fig. S3. Quantification of Western blots.
    • Fig. S4. Analysis of the effect of TRAF3 knockdown on the activation of the p38 and JNK pathways.
    • Fig. S5. ASK1 interacts with IPS-1.
    • Fig. S6. TLR3 is necessary for the effective activation of the p38 and JNK pathways after infection with EMCV.
    • Fig. S7. ASK1 is necessary for effective activation of caspase-3 and induction of IFNB expression in response to influenza virus.
    • Fig. S8. Survival and weight loss of ASK1+/+ and ASK1−/− mice after infection with influenza virus.
    • Fig. S9. ASK2 selectively activates an apoptotic response to infection with EMCV.
    • Fig. S10. ASK2 selectively activates an apoptotic response to infection with influenza virus.
    • Fig. S11. Overexpression of ASK2 selectively promotes the activation of caspase-3 in response to infection by EMCV.
    • Fig. S12. Stable association between ASK1 and ASK2 before and after transfection of cells with poly(I:C).
    • Fig. S13. Schematic overview of the antiviral strategies mediated by ASK family members.

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    Citation: T. Okazaki, M. Higuchi, K. Takeda, K. Iwatsuki-Horimoto, M. Kiso, M. Miyagishi, H. Yanai, A. Kato, M. Yoneyama, T. Fujita, T. Taniguchi, Y. Kawaoka, H. Ichijo, Y. Gotoh, The ASK family kinases differentially mediate induction of type I interferon and apoptosis during the antiviral response. Sci. Signal. 8, ra78 (2015).

    © 2015 American Association for the Advancement of Science

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