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TAK1 kinase switches cell fate from apoptosis to necrosis following TNF stimulation.

Morioka S, Broglie P, Omori E, Ikeda Y, Takaesu G, Matsumoto K, Ninomiya-Tsuji J - J. Cell Biol. (2014)

Bottom Line: We found that prolonged and hyperactivation of TAK1 induced phosphorylation and activation of RIPK3, leading to necrosis without caspase activation.Conversely, ablation of TAK1 caused caspase-dependent apoptosis, in which Ripk3 deletion did not block cell death either in vivo or in vitro.Our results reveal that TAK1 activation drives RIPK3-dependent necrosis and inhibits apoptosis.

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Affiliation: Department of Biological Sciences, North Carolina State University, Raleigh, NC 27695.

ABSTRACT
TNF activates three distinct intracellular signaling cascades leading to cell survival, caspase-8-mediated apoptosis, or receptor interacting protein kinase 3 (RIPK3)-dependent necrosis, also called necroptosis. Depending on the cellular context, one of these pathways is activated upon TNF challenge. When caspase-8 is activated, it drives the apoptosis cascade and blocks RIPK3-dependent necrosis. Here we report the biological event switching to activate necrosis over apoptosis. TAK1 kinase is normally transiently activated upon TNF stimulation. We found that prolonged and hyperactivation of TAK1 induced phosphorylation and activation of RIPK3, leading to necrosis without caspase activation. In addition, we also demonstrated that activation of RIPK1 and RIPK3 promoted TAK1 activation, suggesting a positive feedforward loop of RIPK1, RIPK3, and TAK1. Conversely, ablation of TAK1 caused caspase-dependent apoptosis, in which Ripk3 deletion did not block cell death either in vivo or in vitro. Our results reveal that TAK1 activation drives RIPK3-dependent necrosis and inhibits apoptosis. TAK1 acts as a switch between apoptosis and necrosis.

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TNF-induced cell death in Tab2-deficient cells is rescued by inhibition of RIPK1. (A) Tab2 WT and Tab2 KO fibroblasts were pretreated with either vehicle (DMSO) or Nec-1 (30 µM) for 1 h, and then treated with 2, 20, or 200 ng/ml of TNF for 24 h. Cells attached on the plates were determined by the crystal violet assay. Values of unstimulated fibroblasts were set at 100%. The x axis is a log scale (three independent experiments; mean ± SD; **, P = 0.0066). (B) Tak1 KO and Tab2 KO fibroblasts were pretreated with vehicle (DMSO) or Nec-1 (30 µM) for 1 h and stimulated with TNF (20 ng/ml for Tak1 fibroblasts or 200 ng/ml for Tab2 fibroblasts) for 0, 3, 6, and 9 h. Caspase-3 was analyzed by immunoblotting. Immunoblots of TAK1, TAB2, and β-actin are shown as controls. Asterisk indicates a nonspecific band. Caspase-8 activity in cellular extracts from samples treated with the same procedure was measured. Data are shown as caspase-8 activity relative to that in unstimulated Tak1 KO samples (three independent experiments; mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001; N.S., not significant; P = 0.012, P = 0.00016, P = 0.0012, P = 0.97, P = 0.99, and P = 0.33 from the left).
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fig2: TNF-induced cell death in Tab2-deficient cells is rescued by inhibition of RIPK1. (A) Tab2 WT and Tab2 KO fibroblasts were pretreated with either vehicle (DMSO) or Nec-1 (30 µM) for 1 h, and then treated with 2, 20, or 200 ng/ml of TNF for 24 h. Cells attached on the plates were determined by the crystal violet assay. Values of unstimulated fibroblasts were set at 100%. The x axis is a log scale (three independent experiments; mean ± SD; **, P = 0.0066). (B) Tak1 KO and Tab2 KO fibroblasts were pretreated with vehicle (DMSO) or Nec-1 (30 µM) for 1 h and stimulated with TNF (20 ng/ml for Tak1 fibroblasts or 200 ng/ml for Tab2 fibroblasts) for 0, 3, 6, and 9 h. Caspase-3 was analyzed by immunoblotting. Immunoblots of TAK1, TAB2, and β-actin are shown as controls. Asterisk indicates a nonspecific band. Caspase-8 activity in cellular extracts from samples treated with the same procedure was measured. Data are shown as caspase-8 activity relative to that in unstimulated Tak1 KO samples (three independent experiments; mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001; N.S., not significant; P = 0.012, P = 0.00016, P = 0.0012, P = 0.97, P = 0.99, and P = 0.33 from the left).

Mentions: RIPK1 catalytic activity is generally implicated in necrotic cell death in TNF signaling pathways (Vandenabeele et al., 2010; Yuan and Kroemer, 2010). Necrotic morphology seen in TNF-treated Tab2-deficient fibroblasts prompted us to investigate the involvement of RIPK1. Treatment of Tab2-deficient fibroblasts with a specific inhibitor of RIPK1 kinase, necrostatin-1 (Nec-1), completely blocked TNF-induced cell death, indicating that Tab2 deficiency causes RIPK1-dependent cell death in response to TNF (Fig. 2 A). Although Tak1-deficient cells showed apoptotic morphology in response to TNF, Nec-1 also blocked TNF-induced cell death in Tak1-deficient fibroblasts (Fig. S2 A), consistent with previous studies (Arslan and Scheidereit, 2011; Vanlangenakker et al., 2011; Lamothe et al., 2013). This rescue effect may be caused by an inhibitory effect of Nec-1 on apoptosis, as RIPK1 catalytic activity was previously shown to be also required for induction of apoptosis (Vanlangenakker et al., 2012). Consistently, Nec-1 inhibited TNF-dependent caspase-3 and caspase-8 activation in Tak1-deficient cells, whereas Tab2 deficiency did not induce TNF-induced caspase activation (Fig. 2 B). Tab2-deficient fibroblasts undergo RIPK1-dependent cell death without engaging caspase activation, whereas Tak1-deficient fibroblasts die with RIPK1-dependent caspase activation.


TAK1 kinase switches cell fate from apoptosis to necrosis following TNF stimulation.

Morioka S, Broglie P, Omori E, Ikeda Y, Takaesu G, Matsumoto K, Ninomiya-Tsuji J - J. Cell Biol. (2014)

TNF-induced cell death in Tab2-deficient cells is rescued by inhibition of RIPK1. (A) Tab2 WT and Tab2 KO fibroblasts were pretreated with either vehicle (DMSO) or Nec-1 (30 µM) for 1 h, and then treated with 2, 20, or 200 ng/ml of TNF for 24 h. Cells attached on the plates were determined by the crystal violet assay. Values of unstimulated fibroblasts were set at 100%. The x axis is a log scale (three independent experiments; mean ± SD; **, P = 0.0066). (B) Tak1 KO and Tab2 KO fibroblasts were pretreated with vehicle (DMSO) or Nec-1 (30 µM) for 1 h and stimulated with TNF (20 ng/ml for Tak1 fibroblasts or 200 ng/ml for Tab2 fibroblasts) for 0, 3, 6, and 9 h. Caspase-3 was analyzed by immunoblotting. Immunoblots of TAK1, TAB2, and β-actin are shown as controls. Asterisk indicates a nonspecific band. Caspase-8 activity in cellular extracts from samples treated with the same procedure was measured. Data are shown as caspase-8 activity relative to that in unstimulated Tak1 KO samples (three independent experiments; mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001; N.S., not significant; P = 0.012, P = 0.00016, P = 0.0012, P = 0.97, P = 0.99, and P = 0.33 from the left).
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fig2: TNF-induced cell death in Tab2-deficient cells is rescued by inhibition of RIPK1. (A) Tab2 WT and Tab2 KO fibroblasts were pretreated with either vehicle (DMSO) or Nec-1 (30 µM) for 1 h, and then treated with 2, 20, or 200 ng/ml of TNF for 24 h. Cells attached on the plates were determined by the crystal violet assay. Values of unstimulated fibroblasts were set at 100%. The x axis is a log scale (three independent experiments; mean ± SD; **, P = 0.0066). (B) Tak1 KO and Tab2 KO fibroblasts were pretreated with vehicle (DMSO) or Nec-1 (30 µM) for 1 h and stimulated with TNF (20 ng/ml for Tak1 fibroblasts or 200 ng/ml for Tab2 fibroblasts) for 0, 3, 6, and 9 h. Caspase-3 was analyzed by immunoblotting. Immunoblots of TAK1, TAB2, and β-actin are shown as controls. Asterisk indicates a nonspecific band. Caspase-8 activity in cellular extracts from samples treated with the same procedure was measured. Data are shown as caspase-8 activity relative to that in unstimulated Tak1 KO samples (three independent experiments; mean ± SD; *, P < 0.05; **, P < 0.01; ***, P < 0.001; N.S., not significant; P = 0.012, P = 0.00016, P = 0.0012, P = 0.97, P = 0.99, and P = 0.33 from the left).
Mentions: RIPK1 catalytic activity is generally implicated in necrotic cell death in TNF signaling pathways (Vandenabeele et al., 2010; Yuan and Kroemer, 2010). Necrotic morphology seen in TNF-treated Tab2-deficient fibroblasts prompted us to investigate the involvement of RIPK1. Treatment of Tab2-deficient fibroblasts with a specific inhibitor of RIPK1 kinase, necrostatin-1 (Nec-1), completely blocked TNF-induced cell death, indicating that Tab2 deficiency causes RIPK1-dependent cell death in response to TNF (Fig. 2 A). Although Tak1-deficient cells showed apoptotic morphology in response to TNF, Nec-1 also blocked TNF-induced cell death in Tak1-deficient fibroblasts (Fig. S2 A), consistent with previous studies (Arslan and Scheidereit, 2011; Vanlangenakker et al., 2011; Lamothe et al., 2013). This rescue effect may be caused by an inhibitory effect of Nec-1 on apoptosis, as RIPK1 catalytic activity was previously shown to be also required for induction of apoptosis (Vanlangenakker et al., 2012). Consistently, Nec-1 inhibited TNF-dependent caspase-3 and caspase-8 activation in Tak1-deficient cells, whereas Tab2 deficiency did not induce TNF-induced caspase activation (Fig. 2 B). Tab2-deficient fibroblasts undergo RIPK1-dependent cell death without engaging caspase activation, whereas Tak1-deficient fibroblasts die with RIPK1-dependent caspase activation.

Bottom Line: We found that prolonged and hyperactivation of TAK1 induced phosphorylation and activation of RIPK3, leading to necrosis without caspase activation.Conversely, ablation of TAK1 caused caspase-dependent apoptosis, in which Ripk3 deletion did not block cell death either in vivo or in vitro.Our results reveal that TAK1 activation drives RIPK3-dependent necrosis and inhibits apoptosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biological Sciences, North Carolina State University, Raleigh, NC 27695.

ABSTRACT
TNF activates three distinct intracellular signaling cascades leading to cell survival, caspase-8-mediated apoptosis, or receptor interacting protein kinase 3 (RIPK3)-dependent necrosis, also called necroptosis. Depending on the cellular context, one of these pathways is activated upon TNF challenge. When caspase-8 is activated, it drives the apoptosis cascade and blocks RIPK3-dependent necrosis. Here we report the biological event switching to activate necrosis over apoptosis. TAK1 kinase is normally transiently activated upon TNF stimulation. We found that prolonged and hyperactivation of TAK1 induced phosphorylation and activation of RIPK3, leading to necrosis without caspase activation. In addition, we also demonstrated that activation of RIPK1 and RIPK3 promoted TAK1 activation, suggesting a positive feedforward loop of RIPK1, RIPK3, and TAK1. Conversely, ablation of TAK1 caused caspase-dependent apoptosis, in which Ripk3 deletion did not block cell death either in vivo or in vitro. Our results reveal that TAK1 activation drives RIPK3-dependent necrosis and inhibits apoptosis. TAK1 acts as a switch between apoptosis and necrosis.

Show MeSH
Related in: MedlinePlus