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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 deletion rescues cell death in the Tab2-deficient skin in vivo. (A) Rosa26-CreERTTab2flox/+ Ripk3−/−, Rosa26-CreERT Tab2flox/flox, and Rosa26-CreERT Tab2flox/flox Ripk3−/− mice were treated with tamoxifen at 50 mg/kg for three consecutive days to generate Tab2 Het Ripk3 KO, Tab2 KO, and Tab2 Ripk3 DKO, respectively. Tab2flox/flox mice were treated with the same dose of tamoxifen and studied in parallel as Tab2 WT. 2 wks after the injection, protein extracts from spleen were isolated to confirm the deletion of Tab2 gene by immunoblotting. RIPK3 and β-actin are shown as controls. (B) 1 µg TNF was intradermally injected in Tab2 WT (n = 3), Tab2 KO (n = 3), and Tab2 Ripk3 DKO mice (n = 3). After 6 h, the skin was isolated and TUNEL staining was conducted on the sections. The arrows indicate TUNEL-positive dermal fibroblasts. epi, epidermis; der, dermis. Bars, 40 µm. The percentages of TUNEL-positive cells in the dermal fibroblasts are shown (mean ± SD; **, P < 0.01; P = 0.0018, P = 0.0035, and P = 0.0027 from the left). (C and D) Skin wound surgery was conducted on Tab2 WT (n = 6), Tab2 KO (n = 5), Tab2 Het Ripk3 KO (n = 4), and Tab2 Ripk3 DKO (n = 5) mice. Wound area was expressed as a percentage of initial wound size. Statistical significance between Tab2 WT and Tab2 KO or between Tab2 KO and Tab2 Ripk3 DKO is indicated by # or *, respectively (mean ± SD; ## and **, P < 0.01; # and *, P < 0.05; #, P = 0.034; #, P = 0.011; #, P = 0.014; #, P = 0.045; #, P = 0.038; #, P = 0.027; **, P = 0.0042; **, P = 0.0022; **, P = 0.01; *, P = 0.030; and *, P = 0.028 from the left). 4 d after skin surgery, the skin was isolated from the mice and TUNEL staining was conducted on the sections (D). Arrows indicate TUNEL-positive cells. epi, epidermis; der, dermis; scab, a scab formed over the wound. Bars, 40 µm.
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fig4: Ripk3 deletion rescues cell death in the Tab2-deficient skin in vivo. (A) Rosa26-CreERTTab2flox/+ Ripk3−/−, Rosa26-CreERT Tab2flox/flox, and Rosa26-CreERT Tab2flox/flox Ripk3−/− mice were treated with tamoxifen at 50 mg/kg for three consecutive days to generate Tab2 Het Ripk3 KO, Tab2 KO, and Tab2 Ripk3 DKO, respectively. Tab2flox/flox mice were treated with the same dose of tamoxifen and studied in parallel as Tab2 WT. 2 wks after the injection, protein extracts from spleen were isolated to confirm the deletion of Tab2 gene by immunoblotting. RIPK3 and β-actin are shown as controls. (B) 1 µg TNF was intradermally injected in Tab2 WT (n = 3), Tab2 KO (n = 3), and Tab2 Ripk3 DKO mice (n = 3). After 6 h, the skin was isolated and TUNEL staining was conducted on the sections. The arrows indicate TUNEL-positive dermal fibroblasts. epi, epidermis; der, dermis. Bars, 40 µm. The percentages of TUNEL-positive cells in the dermal fibroblasts are shown (mean ± SD; **, P < 0.01; P = 0.0018, P = 0.0035, and P = 0.0027 from the left). (C and D) Skin wound surgery was conducted on Tab2 WT (n = 6), Tab2 KO (n = 5), Tab2 Het Ripk3 KO (n = 4), and Tab2 Ripk3 DKO (n = 5) mice. Wound area was expressed as a percentage of initial wound size. Statistical significance between Tab2 WT and Tab2 KO or between Tab2 KO and Tab2 Ripk3 DKO is indicated by # or *, respectively (mean ± SD; ## and **, P < 0.01; # and *, P < 0.05; #, P = 0.034; #, P = 0.011; #, P = 0.014; #, P = 0.045; #, P = 0.038; #, P = 0.027; **, P = 0.0042; **, P = 0.0022; **, P = 0.01; *, P = 0.030; and *, P = 0.028 from the left). 4 d after skin surgery, the skin was isolated from the mice and TUNEL staining was conducted on the sections (D). Arrows indicate TUNEL-positive cells. epi, epidermis; der, dermis; scab, a scab formed over the wound. Bars, 40 µm.

Mentions: To investigate the role of TAB2 in vivo, we used the inducible Tab2 deletion (Rosa26-CreERT Tab2flox/flox) mice and generated Tab2-deficient mice by treatment of a CreERT activator, tamoxifen. We confirmed that TAB2 protein was diminished by the gene deletion (Fig. 4 A). Because our fibroblasts were isolated from the dermal layer of the skin, we intradermally injected TNF to examine whether our results in culture cells can be reproduced in the in vivo skin (Fig. 4 B). We found that Tab2-deficient mice exhibited extensive cell death in the dermal fibroblasts in vivo, which was rescued by Ripk3 deletion (Fig. 4 B). This demonstrates that TNF induces RIPK3-dependent cell death in Tab2-deficient mice in vivo. To further examine the relationship between TAB2 and RIPK3 in vivo, we performed a wound-healing assay. The wound-healing process is characterized by regeneration of the cellular and extracellular components of the skin. This process involves inflammation, proliferation, and remodeling (Werner and Grose, 2003). The proliferative phase of healing includes the re-epithelialization of the superficial surface layer and the reconstitution of the underlying dermis by dermal fibroblasts. Upon cutaneous injury, TNF is known to be secreted by immune cells (Werner and Grose, 2003). Thus, we hypothesized that Tab2 deficiency in dermal fibroblasts would induce cell death upon skin injury, which would delay the wound-healing process. Indeed, Tab2-deficient mice showed slower wound healing compared with wild-type mice, and the delay in Tab2-deficient mice was rescued by Ripk3 deletion (Fig. 4 C). TUNEL-positive dermal fibroblasts were increased in the injured Tab2-deficient skin, which was also reduced by Ripk3 deletion (Fig. 4 D). Thus, TAB2 participates in skin wound healing in vivo by preventing RIPK3-dependent 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 deletion rescues cell death in the Tab2-deficient skin in vivo. (A) Rosa26-CreERTTab2flox/+ Ripk3−/−, Rosa26-CreERT Tab2flox/flox, and Rosa26-CreERT Tab2flox/flox Ripk3−/− mice were treated with tamoxifen at 50 mg/kg for three consecutive days to generate Tab2 Het Ripk3 KO, Tab2 KO, and Tab2 Ripk3 DKO, respectively. Tab2flox/flox mice were treated with the same dose of tamoxifen and studied in parallel as Tab2 WT. 2 wks after the injection, protein extracts from spleen were isolated to confirm the deletion of Tab2 gene by immunoblotting. RIPK3 and β-actin are shown as controls. (B) 1 µg TNF was intradermally injected in Tab2 WT (n = 3), Tab2 KO (n = 3), and Tab2 Ripk3 DKO mice (n = 3). After 6 h, the skin was isolated and TUNEL staining was conducted on the sections. The arrows indicate TUNEL-positive dermal fibroblasts. epi, epidermis; der, dermis. Bars, 40 µm. The percentages of TUNEL-positive cells in the dermal fibroblasts are shown (mean ± SD; **, P < 0.01; P = 0.0018, P = 0.0035, and P = 0.0027 from the left). (C and D) Skin wound surgery was conducted on Tab2 WT (n = 6), Tab2 KO (n = 5), Tab2 Het Ripk3 KO (n = 4), and Tab2 Ripk3 DKO (n = 5) mice. Wound area was expressed as a percentage of initial wound size. Statistical significance between Tab2 WT and Tab2 KO or between Tab2 KO and Tab2 Ripk3 DKO is indicated by # or *, respectively (mean ± SD; ## and **, P < 0.01; # and *, P < 0.05; #, P = 0.034; #, P = 0.011; #, P = 0.014; #, P = 0.045; #, P = 0.038; #, P = 0.027; **, P = 0.0042; **, P = 0.0022; **, P = 0.01; *, P = 0.030; and *, P = 0.028 from the left). 4 d after skin surgery, the skin was isolated from the mice and TUNEL staining was conducted on the sections (D). Arrows indicate TUNEL-positive cells. epi, epidermis; der, dermis; scab, a scab formed over the wound. Bars, 40 µm.
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fig4: Ripk3 deletion rescues cell death in the Tab2-deficient skin in vivo. (A) Rosa26-CreERTTab2flox/+ Ripk3−/−, Rosa26-CreERT Tab2flox/flox, and Rosa26-CreERT Tab2flox/flox Ripk3−/− mice were treated with tamoxifen at 50 mg/kg for three consecutive days to generate Tab2 Het Ripk3 KO, Tab2 KO, and Tab2 Ripk3 DKO, respectively. Tab2flox/flox mice were treated with the same dose of tamoxifen and studied in parallel as Tab2 WT. 2 wks after the injection, protein extracts from spleen were isolated to confirm the deletion of Tab2 gene by immunoblotting. RIPK3 and β-actin are shown as controls. (B) 1 µg TNF was intradermally injected in Tab2 WT (n = 3), Tab2 KO (n = 3), and Tab2 Ripk3 DKO mice (n = 3). After 6 h, the skin was isolated and TUNEL staining was conducted on the sections. The arrows indicate TUNEL-positive dermal fibroblasts. epi, epidermis; der, dermis. Bars, 40 µm. The percentages of TUNEL-positive cells in the dermal fibroblasts are shown (mean ± SD; **, P < 0.01; P = 0.0018, P = 0.0035, and P = 0.0027 from the left). (C and D) Skin wound surgery was conducted on Tab2 WT (n = 6), Tab2 KO (n = 5), Tab2 Het Ripk3 KO (n = 4), and Tab2 Ripk3 DKO (n = 5) mice. Wound area was expressed as a percentage of initial wound size. Statistical significance between Tab2 WT and Tab2 KO or between Tab2 KO and Tab2 Ripk3 DKO is indicated by # or *, respectively (mean ± SD; ## and **, P < 0.01; # and *, P < 0.05; #, P = 0.034; #, P = 0.011; #, P = 0.014; #, P = 0.045; #, P = 0.038; #, P = 0.027; **, P = 0.0042; **, P = 0.0022; **, P = 0.01; *, P = 0.030; and *, P = 0.028 from the left). 4 d after skin surgery, the skin was isolated from the mice and TUNEL staining was conducted on the sections (D). Arrows indicate TUNEL-positive cells. epi, epidermis; der, dermis; scab, a scab formed over the wound. Bars, 40 µm.
Mentions: To investigate the role of TAB2 in vivo, we used the inducible Tab2 deletion (Rosa26-CreERT Tab2flox/flox) mice and generated Tab2-deficient mice by treatment of a CreERT activator, tamoxifen. We confirmed that TAB2 protein was diminished by the gene deletion (Fig. 4 A). Because our fibroblasts were isolated from the dermal layer of the skin, we intradermally injected TNF to examine whether our results in culture cells can be reproduced in the in vivo skin (Fig. 4 B). We found that Tab2-deficient mice exhibited extensive cell death in the dermal fibroblasts in vivo, which was rescued by Ripk3 deletion (Fig. 4 B). This demonstrates that TNF induces RIPK3-dependent cell death in Tab2-deficient mice in vivo. To further examine the relationship between TAB2 and RIPK3 in vivo, we performed a wound-healing assay. The wound-healing process is characterized by regeneration of the cellular and extracellular components of the skin. This process involves inflammation, proliferation, and remodeling (Werner and Grose, 2003). The proliferative phase of healing includes the re-epithelialization of the superficial surface layer and the reconstitution of the underlying dermis by dermal fibroblasts. Upon cutaneous injury, TNF is known to be secreted by immune cells (Werner and Grose, 2003). Thus, we hypothesized that Tab2 deficiency in dermal fibroblasts would induce cell death upon skin injury, which would delay the wound-healing process. Indeed, Tab2-deficient mice showed slower wound healing compared with wild-type mice, and the delay in Tab2-deficient mice was rescued by Ripk3 deletion (Fig. 4 C). TUNEL-positive dermal fibroblasts were increased in the injured Tab2-deficient skin, which was also reduced by Ripk3 deletion (Fig. 4 D). Thus, TAB2 participates in skin wound healing in vivo by preventing RIPK3-dependent 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