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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.

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.

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Ripk3 knockdown rescues TNF-induced cell death in Tab2-deficient but not Tak1-deficient fibroblasts. (A) Tab2 WT and Tab2 KO fibroblasts were transfected with Ripk3 siRNA #1 and #2 and stimulated with 50, 100, or 200 ng/ml of TNF at 72 h after transfection. The protein levels of RIPK3 were determined by immunoblotting. Cell viability was determined at 24 h after TNF stimulation 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 < 0.001; P = 0.026 and P = 0.000024 from the bottom). (B) Tak1 WT and Tak1 KO fibroblasts were transfected with Ripk3 siRNA #2, and stimulated with 0.2, 2, or 20 ng/ml of TNF at 72 h after transfection. Cell viability was determined at 24 h after TNF stimulation 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; N.S., not significant; P = 0.99). (C) Tak1 KO fibroblasts were transfected with Ripk3 siRNA #2, and stimulated with 20 ng/ml TNF at 72 h after transfection. Caspase-3 was analyzed by immunoblotting. Immunoblots of RIPK3 and β-actin are shown as controls.
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fig3: Ripk3 knockdown rescues TNF-induced cell death in Tab2-deficient but not Tak1-deficient fibroblasts. (A) Tab2 WT and Tab2 KO fibroblasts were transfected with Ripk3 siRNA #1 and #2 and stimulated with 50, 100, or 200 ng/ml of TNF at 72 h after transfection. The protein levels of RIPK3 were determined by immunoblotting. Cell viability was determined at 24 h after TNF stimulation 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 < 0.001; P = 0.026 and P = 0.000024 from the bottom). (B) Tak1 WT and Tak1 KO fibroblasts were transfected with Ripk3 siRNA #2, and stimulated with 0.2, 2, or 20 ng/ml of TNF at 72 h after transfection. Cell viability was determined at 24 h after TNF stimulation 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; N.S., not significant; P = 0.99). (C) Tak1 KO fibroblasts were transfected with Ripk3 siRNA #2, and stimulated with 20 ng/ml TNF at 72 h after transfection. Caspase-3 was analyzed by immunoblotting. Immunoblots of RIPK3 and β-actin are shown as controls.

Mentions: RIPK3 has been shown to interact with RIPK1 and induce necrotic cell death (Vandenabeele et al., 2010; Yuan and Kroemer, 2010; Green et al., 2011; Oberst and Green, 2011; Vandenabeele and Melino, 2012). Thus, we examined whether RIPK3 is required for TNF-induced cell death in Tab2-deficient fibroblasts. Ripk3 knockdown effectively blocked TNF-induced cell death in Tab2-deficient fibroblasts (Fig. 3 A). To confirm this result, we generated Tab2 and Ripk3 double-deficient mice using a ubiquitously expressing inducible Cre transgene system, Rosa26-CreERT (Badea et al., 2003). Dermal fibroblasts were isolated from Rosa26-CreERT Tab2flox/flox Ripk3−/− and control mice, and Tab2 deletion was induced in vitro by a CreERT activator, 4-hydroxytamoxifen. Tab2 and Ripk3 double-deficient fibroblasts were found to be resistant to TNF-induced cell death (Fig. S2 B). Thus, TNF-induced cell death in Tab2-deficient fibroblasts is RIPK3 dependent. In contrast, ablation of Ripk3 did not block TNF-induced cell death in Tak1-deficient fibroblasts (Fig. 3 B), and caspase activation in Tak1-deficient fibroblasts was not reduced by Ripk3 knockdown (Fig. 3 C). These results suggest that TNF induces RIPK1–RIPK3-dependent cell death in Tab2-deficient fibroblasts, whereas Tak1 deficiency engages RIPK3-independent cell death.


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)

Ripk3 knockdown rescues TNF-induced cell death in Tab2-deficient but not Tak1-deficient fibroblasts. (A) Tab2 WT and Tab2 KO fibroblasts were transfected with Ripk3 siRNA #1 and #2 and stimulated with 50, 100, or 200 ng/ml of TNF at 72 h after transfection. The protein levels of RIPK3 were determined by immunoblotting. Cell viability was determined at 24 h after TNF stimulation 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 < 0.001; P = 0.026 and P = 0.000024 from the bottom). (B) Tak1 WT and Tak1 KO fibroblasts were transfected with Ripk3 siRNA #2, and stimulated with 0.2, 2, or 20 ng/ml of TNF at 72 h after transfection. Cell viability was determined at 24 h after TNF stimulation 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; N.S., not significant; P = 0.99). (C) Tak1 KO fibroblasts were transfected with Ripk3 siRNA #2, and stimulated with 20 ng/ml TNF at 72 h after transfection. Caspase-3 was analyzed by immunoblotting. Immunoblots of RIPK3 and β-actin are shown as controls.
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fig3: Ripk3 knockdown rescues TNF-induced cell death in Tab2-deficient but not Tak1-deficient fibroblasts. (A) Tab2 WT and Tab2 KO fibroblasts were transfected with Ripk3 siRNA #1 and #2 and stimulated with 50, 100, or 200 ng/ml of TNF at 72 h after transfection. The protein levels of RIPK3 were determined by immunoblotting. Cell viability was determined at 24 h after TNF stimulation 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 < 0.001; P = 0.026 and P = 0.000024 from the bottom). (B) Tak1 WT and Tak1 KO fibroblasts were transfected with Ripk3 siRNA #2, and stimulated with 0.2, 2, or 20 ng/ml of TNF at 72 h after transfection. Cell viability was determined at 24 h after TNF stimulation 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; N.S., not significant; P = 0.99). (C) Tak1 KO fibroblasts were transfected with Ripk3 siRNA #2, and stimulated with 20 ng/ml TNF at 72 h after transfection. Caspase-3 was analyzed by immunoblotting. Immunoblots of RIPK3 and β-actin are shown as controls.
Mentions: RIPK3 has been shown to interact with RIPK1 and induce necrotic cell death (Vandenabeele et al., 2010; Yuan and Kroemer, 2010; Green et al., 2011; Oberst and Green, 2011; Vandenabeele and Melino, 2012). Thus, we examined whether RIPK3 is required for TNF-induced cell death in Tab2-deficient fibroblasts. Ripk3 knockdown effectively blocked TNF-induced cell death in Tab2-deficient fibroblasts (Fig. 3 A). To confirm this result, we generated Tab2 and Ripk3 double-deficient mice using a ubiquitously expressing inducible Cre transgene system, Rosa26-CreERT (Badea et al., 2003). Dermal fibroblasts were isolated from Rosa26-CreERT Tab2flox/flox Ripk3−/− and control mice, and Tab2 deletion was induced in vitro by a CreERT activator, 4-hydroxytamoxifen. Tab2 and Ripk3 double-deficient fibroblasts were found to be resistant to TNF-induced cell death (Fig. S2 B). Thus, TNF-induced cell death in Tab2-deficient fibroblasts is RIPK3 dependent. In contrast, ablation of Ripk3 did not block TNF-induced cell death in Tak1-deficient fibroblasts (Fig. 3 B), and caspase activation in Tak1-deficient fibroblasts was not reduced by Ripk3 knockdown (Fig. 3 C). These results suggest that TNF induces RIPK1–RIPK3-dependent cell death in Tab2-deficient fibroblasts, whereas Tak1 deficiency engages RIPK3-independent cell death.

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