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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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Akt contributes to necroptosis induced by zVAD.fmk and TNFα.(A,B) Necroptosis was induced by zVAD.fmk or TNFα (full serum, A) or growth factors/zVAD.fmk (serum free, B) in the presence of inhibitors of Akt (Akt inhibitor VIII), JNK (SP600125), p38 (PD169316), and Erk (UO126). Cell viability was determined after 24 hrs. (C) L929 cells transfected with Akt1, Akt2, and Akt3 siRNAs for 72 hrs were treated with zVAD.fmk or TNFα for 9 hrs. Cell viability and Akt expression levels were determined after 24 hrs. In all graphs, average±SD was plotted.
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pone-0056576-g002: Akt contributes to necroptosis induced by zVAD.fmk and TNFα.(A,B) Necroptosis was induced by zVAD.fmk or TNFα (full serum, A) or growth factors/zVAD.fmk (serum free, B) in the presence of inhibitors of Akt (Akt inhibitor VIII), JNK (SP600125), p38 (PD169316), and Erk (UO126). Cell viability was determined after 24 hrs. (C) L929 cells transfected with Akt1, Akt2, and Akt3 siRNAs for 72 hrs were treated with zVAD.fmk or TNFα for 9 hrs. Cell viability and Akt expression levels were determined after 24 hrs. In all graphs, average±SD was plotted.

Mentions: Given our observation that growth factors are important for zVAD.fmk induced death, we examined the contribution of several pathways, including MAPK pathways and Akt, which are known to be activated following growth factor receptor activation (Fig. 2A). Inhibition of Akt (Akt inhibitor VIII) strongly protected the cells from growth factor-sensitive necroptosis induced by zVAD.fmk [16] as well as cell death triggered by bFGF or IGF-1/zVAD.fmk under serum free conditions (Fig. 2B). Inhibition of Akt also protected the cells from growth-factor insensitive death by caused by TNFα (Fig. 2A). Consistent with previous reports, the JNK inhibitor SP600125 protected the cells from both zVAD.fmk and TNFα induced death (Fig. 2A,B and Fig. S2A) [12], [14]. In contrast, inhibition of two other MAPKs, p38 and ERK, previously reported not to be activated during necroptosis [14], did not protect from either zVAD.fmk or TNFα induced death (Fig. 2A).


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)

Akt contributes to necroptosis induced by zVAD.fmk and TNFα.(A,B) Necroptosis was induced by zVAD.fmk or TNFα (full serum, A) or growth factors/zVAD.fmk (serum free, B) in the presence of inhibitors of Akt (Akt inhibitor VIII), JNK (SP600125), p38 (PD169316), and Erk (UO126). Cell viability was determined after 24 hrs. (C) L929 cells transfected with Akt1, Akt2, and Akt3 siRNAs for 72 hrs were treated with zVAD.fmk or TNFα for 9 hrs. Cell viability and Akt expression levels were determined after 24 hrs. In all graphs, average±SD was plotted.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056576-g002: Akt contributes to necroptosis induced by zVAD.fmk and TNFα.(A,B) Necroptosis was induced by zVAD.fmk or TNFα (full serum, A) or growth factors/zVAD.fmk (serum free, B) in the presence of inhibitors of Akt (Akt inhibitor VIII), JNK (SP600125), p38 (PD169316), and Erk (UO126). Cell viability was determined after 24 hrs. (C) L929 cells transfected with Akt1, Akt2, and Akt3 siRNAs for 72 hrs were treated with zVAD.fmk or TNFα for 9 hrs. Cell viability and Akt expression levels were determined after 24 hrs. In all graphs, average±SD was plotted.
Mentions: Given our observation that growth factors are important for zVAD.fmk induced death, we examined the contribution of several pathways, including MAPK pathways and Akt, which are known to be activated following growth factor receptor activation (Fig. 2A). Inhibition of Akt (Akt inhibitor VIII) strongly protected the cells from growth factor-sensitive necroptosis induced by zVAD.fmk [16] as well as cell death triggered by bFGF or IGF-1/zVAD.fmk under serum free conditions (Fig. 2B). Inhibition of Akt also protected the cells from growth-factor insensitive death by caused by TNFα (Fig. 2A). Consistent with previous reports, the JNK inhibitor SP600125 protected the cells from both zVAD.fmk and TNFα induced death (Fig. 2A,B and Fig. S2A) [12], [14]. In contrast, inhibition of two other MAPKs, p38 and ERK, previously reported not to be activated during necroptosis [14], did not protect from either zVAD.fmk or TNFα induced death (Fig. 2A).

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