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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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mTORC1 contributes to the regulation of necroptosis.(A) L929 cells were treated with zVAD.fmk or TNFα for 9 hr and harvested for western blot. (B) Cell under serum free condition were treated with bFGF or bFGF/zVAD.fmk for the indicated amounts of time, followed by western blotting using the indicated antibodies. (C) Necroptosis was induced by zVAD.fmk or TNFα in L929 cell in the presence of inhibitors of Akt(Akt inh. VIII) and mTOR (rapamycin, Torin-1 and PI-103). (D) L929 cells with mTOR siRNA knockdown were harvested for western blot or treated with zVAD.fmk or TNFα for 24 hrs. Cell viability was determined 24 hr after activation of necroptosis. In all graphs, average±SD was plotted.
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pone-0056576-g005: mTORC1 contributes to the regulation of necroptosis.(A) L929 cells were treated with zVAD.fmk or TNFα for 9 hr and harvested for western blot. (B) Cell under serum free condition were treated with bFGF or bFGF/zVAD.fmk for the indicated amounts of time, followed by western blotting using the indicated antibodies. (C) Necroptosis was induced by zVAD.fmk or TNFα in L929 cell in the presence of inhibitors of Akt(Akt inh. VIII) and mTOR (rapamycin, Torin-1 and PI-103). (D) L929 cells with mTOR siRNA knockdown were harvested for western blot or treated with zVAD.fmk or TNFα for 24 hrs. Cell viability was determined 24 hr after activation of necroptosis. In all graphs, average±SD was plotted.

Mentions: We next determined whether the necroptosis-associated increase in Thr308 phosphorylation results in an increase in Akt kinase activity. Under necroptotic conditions, we observed an increase in the phosphorylation of multiple known Akt substrates (Forkhead box class O (FoxO) proteins, GSK-3 kinases and mouse double minute 2 (MDM2)) as well as downstream molecules (p70 ribosomal protein S6 Kinase (p70S6K), S6) (Fig. 5A). In some cases (FoxO1, FoxO4, MDM2), a robust increase was observed. In other cases (FoxO3a, GSK-3α/β, p70S6K and its substrate S6), the changes were less pronounced (Fig. 5A). The timing of the phosphorylation changes paralleled the increase in Akt phosphorylation (Fig. 5B, S5A, B). In the case of pFoxO1 we occasionally observed a shift in migration rather than an increase in band intensity (e.g. comparing Fig. 5A and B), suggesting that phosphorylation events in addition to Thr24 take place during necroptosis. Notably, in all cases the necroptosis-associated increases in Akt substrates were abrogated by Nec-1 (Fig. 5A, Fig. S5A, B). Overall, these data suggested that a significant part of the “canonical” Akt signaling network is activated at the onset of necroptotic cell death in a RIP1 dependent fashion.


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)

mTORC1 contributes to the regulation of necroptosis.(A) L929 cells were treated with zVAD.fmk or TNFα for 9 hr and harvested for western blot. (B) Cell under serum free condition were treated with bFGF or bFGF/zVAD.fmk for the indicated amounts of time, followed by western blotting using the indicated antibodies. (C) Necroptosis was induced by zVAD.fmk or TNFα in L929 cell in the presence of inhibitors of Akt(Akt inh. VIII) and mTOR (rapamycin, Torin-1 and PI-103). (D) L929 cells with mTOR siRNA knockdown were harvested for western blot or treated with zVAD.fmk or TNFα for 24 hrs. Cell viability was determined 24 hr after activation of necroptosis. In all graphs, average±SD was plotted.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056576-g005: mTORC1 contributes to the regulation of necroptosis.(A) L929 cells were treated with zVAD.fmk or TNFα for 9 hr and harvested for western blot. (B) Cell under serum free condition were treated with bFGF or bFGF/zVAD.fmk for the indicated amounts of time, followed by western blotting using the indicated antibodies. (C) Necroptosis was induced by zVAD.fmk or TNFα in L929 cell in the presence of inhibitors of Akt(Akt inh. VIII) and mTOR (rapamycin, Torin-1 and PI-103). (D) L929 cells with mTOR siRNA knockdown were harvested for western blot or treated with zVAD.fmk or TNFα for 24 hrs. Cell viability was determined 24 hr after activation of necroptosis. In all graphs, average±SD was plotted.
Mentions: We next determined whether the necroptosis-associated increase in Thr308 phosphorylation results in an increase in Akt kinase activity. Under necroptotic conditions, we observed an increase in the phosphorylation of multiple known Akt substrates (Forkhead box class O (FoxO) proteins, GSK-3 kinases and mouse double minute 2 (MDM2)) as well as downstream molecules (p70 ribosomal protein S6 Kinase (p70S6K), S6) (Fig. 5A). In some cases (FoxO1, FoxO4, MDM2), a robust increase was observed. In other cases (FoxO3a, GSK-3α/β, p70S6K and its substrate S6), the changes were less pronounced (Fig. 5A). The timing of the phosphorylation changes paralleled the increase in Akt phosphorylation (Fig. 5B, S5A, B). In the case of pFoxO1 we occasionally observed a shift in migration rather than an increase in band intensity (e.g. comparing Fig. 5A and B), suggesting that phosphorylation events in addition to Thr24 take place during necroptosis. Notably, in all cases the necroptosis-associated increases in Akt substrates were abrogated by Nec-1 (Fig. 5A, Fig. S5A, B). Overall, these data suggested that a significant part of the “canonical” Akt signaling network is activated at the onset of necroptotic cell death in a RIP1 dependent fashion.

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