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Akt Regulates TNFα synthesis downstream of RIP1 kinase activation during necroptosis.

McNamara CR, Ahuja R, Osafo-Addo AD, Barrows D, Kettenbach A, Skidan I, Teng X, Cuny GD, Gerber S, Degterev A - PLoS ONE (2013)

Bottom Line: In L929 cells, this activation requires independent signaling inputs from both growth factors and RIP1.Akt controls necroptosis through downstream targeting of mammalian Target of Rapamycin complex 1 (mTORC1).Overall, our results provide new insights into the mechanism of necroptosis and the role of Akt kinase in both cell death and inflammatory regulation.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Biochemistry, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, Massachussets, United States of America.

ABSTRACT
Necroptosis is a regulated form of necrotic cell death that has been implicated in the pathogenesis of various diseases including intestinal inflammation and systemic inflammatory response syndrome (SIRS). In this work, we investigated the signaling mechanisms controlled by the necroptosis mediator receptor interacting protein-1 (RIP1) kinase. We show that Akt kinase activity is critical for necroptosis in L929 cells and plays a key role in TNFα production. During necroptosis, Akt is activated in a RIP1 dependent fashion through its phosphorylation on Thr308. In L929 cells, this activation requires independent signaling inputs from both growth factors and RIP1. Akt controls necroptosis through downstream targeting of mammalian Target of Rapamycin complex 1 (mTORC1). Akt activity, mediated in part through mTORC1, links RIP1 to JNK activation and autocrine production of TNFα. In other cell types, such as mouse lung fibroblasts and macrophages, Akt exhibited control over necroptosis-associated TNFα production without contributing to cell death. Overall, our results provide new insights into the mechanism of necroptosis and the role of Akt kinase in both cell death and inflammatory regulation.

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Over expression of constitutively active Akt restores necroptosis under serum free conditions.(A,B) L929 cells were stably infected with empty MSCV retrovirus or viruses encoding Myr-Akt or the catalytically inactive Myr-Akt K179M. Necroptosis was induced by the addition of zVAD.fmk under serum free conditions (A) or serum or serum free conditions with Nec-1 (B). Viability assays were performed after 24 hr. (C) Myr-Akt and Myr-Akt K179M cells were treated with zVAD.fmk and/or Nec-1 under serum free conditions for 9 hr, followed by western blot using the indicated antibodies. Endogenous Akt (∼) and Myr-Akt (*) bands are indicated. (D) L929 cells, stably infected with Myr-Akt and Myr-Akt K179KM, were stimulated with zVAD.fmk for 9 hr under serum free conditions. TNFα mRNA levels were determined by qRT-PCR and normalized using mouse 18S RNA. (E-G) L929 cells expressing Myr-Akt and Ala and Asp mutants of Thr308 and Ser473 were treated with zVAD.fmk under serum free conditions, followed by viability assay at 24 hr (E), western blot at 9 hr (F), or evaluation of TNFα mRNA levels by qRT-PCR at 9 hrs (G). In all graphs, average±SD was plotted.
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pone-0056576-g007: Over expression of constitutively active Akt restores necroptosis under serum free conditions.(A,B) L929 cells were stably infected with empty MSCV retrovirus or viruses encoding Myr-Akt or the catalytically inactive Myr-Akt K179M. Necroptosis was induced by the addition of zVAD.fmk under serum free conditions (A) or serum or serum free conditions with Nec-1 (B). Viability assays were performed after 24 hr. (C) Myr-Akt and Myr-Akt K179M cells were treated with zVAD.fmk and/or Nec-1 under serum free conditions for 9 hr, followed by western blot using the indicated antibodies. Endogenous Akt (∼) and Myr-Akt (*) bands are indicated. (D) L929 cells, stably infected with Myr-Akt and Myr-Akt K179KM, were stimulated with zVAD.fmk for 9 hr under serum free conditions. TNFα mRNA levels were determined by qRT-PCR and normalized using mouse 18S RNA. (E-G) L929 cells expressing Myr-Akt and Ala and Asp mutants of Thr308 and Ser473 were treated with zVAD.fmk under serum free conditions, followed by viability assay at 24 hr (E), western blot at 9 hr (F), or evaluation of TNFα mRNA levels by qRT-PCR at 9 hrs (G). In all graphs, average±SD was plotted.

Mentions: We used L929 cells stably expressing constitutively active wild type Akt1 (Myr-Akt) or the catalytically inactive mutant K179M in order to further understand the contribution of growth factors and RIP1 kinase to Akt activation during necroptosis. Constitutively active Akt1 (Myr-Akt) was generated as previously described [37] by the addition of a myristoylation signal which provides constitutive localization to the plasma membrane and by the deletion of the auto-inhibitory PH domain (Fig. 7A) resulting in an Akt that is active under serum free. It is important to note that the cells expressing Myr-Akt were viable, grew in a manner indistinguishable from the empty vector control cells, and were not triggered to induce necroptosis by serum starvation (Fig. 7B). This indicates that active Akt alone is not sufficient to induce necroptotic cell death. Under serum free conditions Myr-Akt, but not the K179M mutant, fully restored zVAD.fmk-induced necroptosis (Fig. 7A,B). Nec-1 prevented both Myr-Akt dependent cell death and the necroptosis-specific delayed increase in Akt Thr308 phosphorylation (Fig. 7B,C). Myr-Akt also allowed other zVAD.fmk-dependent events, including activation of JNK and c-Jun phosphorylation (Fig. 7C) and upregulation of TNFα mRNA (Fig. 7D) to occur under serum free conditions, confirming an important role for Akt at the apex of necroptotic signaling. These data demonstrated that the presence of active and membrane localized Akt is sufficient to uncouple Akt activation during necroptosis from growth factor signaling. RIP1 kinase was still able to regulate Akt activation during necroptosis, suggesting that growth factors and RIP1 kinase provide two independent inputs required for Akt changes during necroptosis.


Akt Regulates TNFα synthesis downstream of RIP1 kinase activation during necroptosis.

McNamara CR, Ahuja R, Osafo-Addo AD, Barrows D, Kettenbach A, Skidan I, Teng X, Cuny GD, Gerber S, Degterev A - PLoS ONE (2013)

Over expression of constitutively active Akt restores necroptosis under serum free conditions.(A,B) L929 cells were stably infected with empty MSCV retrovirus or viruses encoding Myr-Akt or the catalytically inactive Myr-Akt K179M. Necroptosis was induced by the addition of zVAD.fmk under serum free conditions (A) or serum or serum free conditions with Nec-1 (B). Viability assays were performed after 24 hr. (C) Myr-Akt and Myr-Akt K179M cells were treated with zVAD.fmk and/or Nec-1 under serum free conditions for 9 hr, followed by western blot using the indicated antibodies. Endogenous Akt (∼) and Myr-Akt (*) bands are indicated. (D) L929 cells, stably infected with Myr-Akt and Myr-Akt K179KM, were stimulated with zVAD.fmk for 9 hr under serum free conditions. TNFα mRNA levels were determined by qRT-PCR and normalized using mouse 18S RNA. (E-G) L929 cells expressing Myr-Akt and Ala and Asp mutants of Thr308 and Ser473 were treated with zVAD.fmk under serum free conditions, followed by viability assay at 24 hr (E), western blot at 9 hr (F), or evaluation of TNFα mRNA levels by qRT-PCR at 9 hrs (G). In all graphs, average±SD was plotted.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585731&req=5

pone-0056576-g007: Over expression of constitutively active Akt restores necroptosis under serum free conditions.(A,B) L929 cells were stably infected with empty MSCV retrovirus or viruses encoding Myr-Akt or the catalytically inactive Myr-Akt K179M. Necroptosis was induced by the addition of zVAD.fmk under serum free conditions (A) or serum or serum free conditions with Nec-1 (B). Viability assays were performed after 24 hr. (C) Myr-Akt and Myr-Akt K179M cells were treated with zVAD.fmk and/or Nec-1 under serum free conditions for 9 hr, followed by western blot using the indicated antibodies. Endogenous Akt (∼) and Myr-Akt (*) bands are indicated. (D) L929 cells, stably infected with Myr-Akt and Myr-Akt K179KM, were stimulated with zVAD.fmk for 9 hr under serum free conditions. TNFα mRNA levels were determined by qRT-PCR and normalized using mouse 18S RNA. (E-G) L929 cells expressing Myr-Akt and Ala and Asp mutants of Thr308 and Ser473 were treated with zVAD.fmk under serum free conditions, followed by viability assay at 24 hr (E), western blot at 9 hr (F), or evaluation of TNFα mRNA levels by qRT-PCR at 9 hrs (G). In all graphs, average±SD was plotted.
Mentions: We used L929 cells stably expressing constitutively active wild type Akt1 (Myr-Akt) or the catalytically inactive mutant K179M in order to further understand the contribution of growth factors and RIP1 kinase to Akt activation during necroptosis. Constitutively active Akt1 (Myr-Akt) was generated as previously described [37] by the addition of a myristoylation signal which provides constitutive localization to the plasma membrane and by the deletion of the auto-inhibitory PH domain (Fig. 7A) resulting in an Akt that is active under serum free. It is important to note that the cells expressing Myr-Akt were viable, grew in a manner indistinguishable from the empty vector control cells, and were not triggered to induce necroptosis by serum starvation (Fig. 7B). This indicates that active Akt alone is not sufficient to induce necroptotic cell death. Under serum free conditions Myr-Akt, but not the K179M mutant, fully restored zVAD.fmk-induced necroptosis (Fig. 7A,B). Nec-1 prevented both Myr-Akt dependent cell death and the necroptosis-specific delayed increase in Akt Thr308 phosphorylation (Fig. 7B,C). Myr-Akt also allowed other zVAD.fmk-dependent events, including activation of JNK and c-Jun phosphorylation (Fig. 7C) and upregulation of TNFα mRNA (Fig. 7D) to occur under serum free conditions, confirming an important role for Akt at the apex of necroptotic signaling. These data demonstrated that the presence of active and membrane localized Akt is sufficient to uncouple Akt activation during necroptosis from growth factor signaling. RIP1 kinase was still able to regulate Akt activation during necroptosis, suggesting that growth factors and RIP1 kinase provide two independent inputs required for Akt changes during necroptosis.

Bottom Line: In L929 cells, this activation requires independent signaling inputs from both growth factors and RIP1.Akt controls necroptosis through downstream targeting of mammalian Target of Rapamycin complex 1 (mTORC1).Overall, our results provide new insights into the mechanism of necroptosis and the role of Akt kinase in both cell death and inflammatory regulation.

View Article: PubMed Central - PubMed

Affiliation: Graduate Program in Biochemistry, Sackler School of Graduate Biomedical Sciences, Tufts University, Boston, Massachussets, United States of America.

ABSTRACT
Necroptosis is a regulated form of necrotic cell death that has been implicated in the pathogenesis of various diseases including intestinal inflammation and systemic inflammatory response syndrome (SIRS). In this work, we investigated the signaling mechanisms controlled by the necroptosis mediator receptor interacting protein-1 (RIP1) kinase. We show that Akt kinase activity is critical for necroptosis in L929 cells and plays a key role in TNFα production. During necroptosis, Akt is activated in a RIP1 dependent fashion through its phosphorylation on Thr308. In L929 cells, this activation requires independent signaling inputs from both growth factors and RIP1. Akt controls necroptosis through downstream targeting of mammalian Target of Rapamycin complex 1 (mTORC1). Akt activity, mediated in part through mTORC1, links RIP1 to JNK activation and autocrine production of TNFα. In other cell types, such as mouse lung fibroblasts and macrophages, Akt exhibited control over necroptosis-associated TNFα production without contributing to cell death. Overall, our results provide new insights into the mechanism of necroptosis and the role of Akt kinase in both cell death and inflammatory regulation.

Show MeSH
Related in: MedlinePlus