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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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Late Thr308 phosphorylation of Akt contributes to necroptosis.(A) L929 cells were treated with zVAD.fmk and bFGF or PDGF, with or without Nec-1, for the indicated periods of time. (B,C) L929 cells were stimulated by zVAD.fmk or TNFα (B) or bFGF/zVAD.fmk under serum free conditions (C). Akt inh. VIII was added 15 min before necroptotic stimulation (Pre) or at indicated times after stimulation. Viability was measured 24 hr after activation of necroptosis. (D) L929 cells were stimulated with bFGF/zVAD under serum free conditions. PD173074 was added 15 min before or 1 hr after FGF/zVAD. Samples for western blot were collected at 15 min and 9 hr time points. (E) Cells were pretreated with PD173074 or it was added 1 hr after bFGF/zVAD.fmk, followed by viability assessment at 24 hr. In all graphs, average±SD was plotted.
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pone-0056576-g004: Late Thr308 phosphorylation of Akt contributes to necroptosis.(A) L929 cells were treated with zVAD.fmk and bFGF or PDGF, with or without Nec-1, for the indicated periods of time. (B,C) L929 cells were stimulated by zVAD.fmk or TNFα (B) or bFGF/zVAD.fmk under serum free conditions (C). Akt inh. VIII was added 15 min before necroptotic stimulation (Pre) or at indicated times after stimulation. Viability was measured 24 hr after activation of necroptosis. (D) L929 cells were stimulated with bFGF/zVAD under serum free conditions. PD173074 was added 15 min before or 1 hr after FGF/zVAD. Samples for western blot were collected at 15 min and 9 hr time points. (E) Cells were pretreated with PD173074 or it was added 1 hr after bFGF/zVAD.fmk, followed by viability assessment at 24 hr. In all graphs, average±SD was plotted.

Mentions: We next investigated if the delayed RIP1 kinase-dependent increase in Akt Thr308 phosphorylation functionally contributes to the execution of necroptotic cell death. Firstly, PDGF/zVAD.fmk, which cannot induce necroptosis (Fig. 2A), triggered only the initial, rapid Akt and JNK phosphorylation changes and not the delayed activation (Fig. 4A), indicating that late, rather than early Akt phosphorylation correlates with necroptosis. Secondly, we saw that the ability of the Akt inhibitor to protect cells from necroptosis rapidly declined after 6 hrs of stimulation with zVAD.fmk, TNFα or bFGF/zVAD.fmk and no protection was observed when the inhibitor was added at 9 hrs (Fig. 4B,C). This time frame coincides with the timing of the secondary Akt Thr308 phosphorylation. Finally, we terminated the bFGF signal one hour after addition of bFGF by the addition of PD173074. This allowed us to retain early Akt activation, but to suppress the secondary increase (Fig. 4D). Both pre-addition and delayed addition of PD173074 fully prevented necroptosis (Fig. 4E). Overall, these data, while correlative, indicate that early Akt activation is insufficient to promote necroptosis and are strongly supportive of an important role for the delayed activation of Akt in the induction of necroptotic cell death.


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)

Late Thr308 phosphorylation of Akt contributes to necroptosis.(A) L929 cells were treated with zVAD.fmk and bFGF or PDGF, with or without Nec-1, for the indicated periods of time. (B,C) L929 cells were stimulated by zVAD.fmk or TNFα (B) or bFGF/zVAD.fmk under serum free conditions (C). Akt inh. VIII was added 15 min before necroptotic stimulation (Pre) or at indicated times after stimulation. Viability was measured 24 hr after activation of necroptosis. (D) L929 cells were stimulated with bFGF/zVAD under serum free conditions. PD173074 was added 15 min before or 1 hr after FGF/zVAD. Samples for western blot were collected at 15 min and 9 hr time points. (E) Cells were pretreated with PD173074 or it was added 1 hr after bFGF/zVAD.fmk, followed by viability assessment at 24 hr. In all graphs, average±SD was plotted.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585731&req=5

pone-0056576-g004: Late Thr308 phosphorylation of Akt contributes to necroptosis.(A) L929 cells were treated with zVAD.fmk and bFGF or PDGF, with or without Nec-1, for the indicated periods of time. (B,C) L929 cells were stimulated by zVAD.fmk or TNFα (B) or bFGF/zVAD.fmk under serum free conditions (C). Akt inh. VIII was added 15 min before necroptotic stimulation (Pre) or at indicated times after stimulation. Viability was measured 24 hr after activation of necroptosis. (D) L929 cells were stimulated with bFGF/zVAD under serum free conditions. PD173074 was added 15 min before or 1 hr after FGF/zVAD. Samples for western blot were collected at 15 min and 9 hr time points. (E) Cells were pretreated with PD173074 or it was added 1 hr after bFGF/zVAD.fmk, followed by viability assessment at 24 hr. In all graphs, average±SD was plotted.
Mentions: We next investigated if the delayed RIP1 kinase-dependent increase in Akt Thr308 phosphorylation functionally contributes to the execution of necroptotic cell death. Firstly, PDGF/zVAD.fmk, which cannot induce necroptosis (Fig. 2A), triggered only the initial, rapid Akt and JNK phosphorylation changes and not the delayed activation (Fig. 4A), indicating that late, rather than early Akt phosphorylation correlates with necroptosis. Secondly, we saw that the ability of the Akt inhibitor to protect cells from necroptosis rapidly declined after 6 hrs of stimulation with zVAD.fmk, TNFα or bFGF/zVAD.fmk and no protection was observed when the inhibitor was added at 9 hrs (Fig. 4B,C). This time frame coincides with the timing of the secondary Akt Thr308 phosphorylation. Finally, we terminated the bFGF signal one hour after addition of bFGF by the addition of PD173074. This allowed us to retain early Akt activation, but to suppress the secondary increase (Fig. 4D). Both pre-addition and delayed addition of PD173074 fully prevented necroptosis (Fig. 4E). Overall, these data, while correlative, indicate that early Akt activation is insufficient to promote necroptosis and are strongly supportive of an important role for the delayed activation of Akt in the induction of necroptotic cell death.

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