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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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bFGF and IGF-1 promote necroptosis in concert with zVAD.fmk.(A) L929 cells were treated with TNFα or zVAD.fmk under normal serum (10% FBS) or serum free conditions. Cell viability was determined after 24 hr using the CellTiter-Glo Viability assay. The concentrations of all necroptosis-inducing agents are listed in the Materials and Methods section or indicated in the figures. (B) Cells were treated with zVAD.fmk, the indicated growth factors, and Nec-1 under serum free conditions for 24 hrs followed by measurement of cell viability. (C) Cells under serum free conditions were treated with FGF, zVAD.fmk, or both for 24 hrs followed by viability assay. (D) Cell death was induced by zVAD.fmk or TNFα under full serum condition in the presence of 2 µM PD173074 and 20 µM PD166866. In all graphs, average±SD was plotted.
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pone-0056576-g001: bFGF and IGF-1 promote necroptosis in concert with zVAD.fmk.(A) L929 cells were treated with TNFα or zVAD.fmk under normal serum (10% FBS) or serum free conditions. Cell viability was determined after 24 hr using the CellTiter-Glo Viability assay. The concentrations of all necroptosis-inducing agents are listed in the Materials and Methods section or indicated in the figures. (B) Cells were treated with zVAD.fmk, the indicated growth factors, and Nec-1 under serum free conditions for 24 hrs followed by measurement of cell viability. (C) Cells under serum free conditions were treated with FGF, zVAD.fmk, or both for 24 hrs followed by viability assay. (D) Cell death was induced by zVAD.fmk or TNFα under full serum condition in the presence of 2 µM PD173074 and 20 µM PD166866. In all graphs, average±SD was plotted.

Mentions: It has been established that mouse fibrosarcoma L929 cells undergo necroptotic cell death following stimulation with TNFα [10], [17]. In addition, inhibition of caspase-8 activity alone, either through siRNA knockdown or by using the pan-caspase inhibitor, zVAD.fmk, is sufficient to trigger necroptosis in these cells [10], [14]. Interestingly, while necroptosis was initially identified as a back-up form of cell death triggered by pro-apoptotic stimuli in the presence of apoptosis inhibitors [17], recent analysis of physiological cell death during mouse development has suggested that the loss of apoptotic regulators, such as caspase-8 and FADD [18], [19], [20], leads to robust induction of necroptosis and death of E10.5 embryos even though apoptosis is not normally induced in wild type embryos. These data are reminiscent of the observations in L929 cells where the loss of caspase activity in healthy cells is sufficient to trigger necroptosis and prompted us to explore the extrinsic or intrinsic cellular factors that promote necroptosis once caspase-8 activity, which cleaves and inactivates RIP1 kinase and the RIP1 deubiquitinase CYLD [21], [22], is removed in L929 cells. Consistent with a previous report [16], we found that serum starvation of L929 cells prevented necroptosis in response to zVAD.fmk (Fig. 1A). The addition of growth factors, such as bFGF, restored zVAD.fmk induced death under serum free conditions (Fig. 1B). Interestingly, this does not reflect a generic requirement for growth factor signaling, as only some growth factors (bFGF and IGF-1, but not EGF and PDGF) promoted death (Fig. 1B). Furthermore, growth factor-dependent necroptosis required the inhibition of caspase activity, as bFGF alone did not induce cell death (Fig. 1C). In contrast, TNFα triggered necroptosis equally efficiently in the absence of serum (Fig. 1A), suggesting that either growth factors and zVAD.fmk or TNFα are required for necroptotic death in L929 cells.


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)

bFGF and IGF-1 promote necroptosis in concert with zVAD.fmk.(A) L929 cells were treated with TNFα or zVAD.fmk under normal serum (10% FBS) or serum free conditions. Cell viability was determined after 24 hr using the CellTiter-Glo Viability assay. The concentrations of all necroptosis-inducing agents are listed in the Materials and Methods section or indicated in the figures. (B) Cells were treated with zVAD.fmk, the indicated growth factors, and Nec-1 under serum free conditions for 24 hrs followed by measurement of cell viability. (C) Cells under serum free conditions were treated with FGF, zVAD.fmk, or both for 24 hrs followed by viability assay. (D) Cell death was induced by zVAD.fmk or TNFα under full serum condition in the presence of 2 µM PD173074 and 20 µM PD166866. In all graphs, average±SD was plotted.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0056576-g001: bFGF and IGF-1 promote necroptosis in concert with zVAD.fmk.(A) L929 cells were treated with TNFα or zVAD.fmk under normal serum (10% FBS) or serum free conditions. Cell viability was determined after 24 hr using the CellTiter-Glo Viability assay. The concentrations of all necroptosis-inducing agents are listed in the Materials and Methods section or indicated in the figures. (B) Cells were treated with zVAD.fmk, the indicated growth factors, and Nec-1 under serum free conditions for 24 hrs followed by measurement of cell viability. (C) Cells under serum free conditions were treated with FGF, zVAD.fmk, or both for 24 hrs followed by viability assay. (D) Cell death was induced by zVAD.fmk or TNFα under full serum condition in the presence of 2 µM PD173074 and 20 µM PD166866. In all graphs, average±SD was plotted.
Mentions: It has been established that mouse fibrosarcoma L929 cells undergo necroptotic cell death following stimulation with TNFα [10], [17]. In addition, inhibition of caspase-8 activity alone, either through siRNA knockdown or by using the pan-caspase inhibitor, zVAD.fmk, is sufficient to trigger necroptosis in these cells [10], [14]. Interestingly, while necroptosis was initially identified as a back-up form of cell death triggered by pro-apoptotic stimuli in the presence of apoptosis inhibitors [17], recent analysis of physiological cell death during mouse development has suggested that the loss of apoptotic regulators, such as caspase-8 and FADD [18], [19], [20], leads to robust induction of necroptosis and death of E10.5 embryos even though apoptosis is not normally induced in wild type embryos. These data are reminiscent of the observations in L929 cells where the loss of caspase activity in healthy cells is sufficient to trigger necroptosis and prompted us to explore the extrinsic or intrinsic cellular factors that promote necroptosis once caspase-8 activity, which cleaves and inactivates RIP1 kinase and the RIP1 deubiquitinase CYLD [21], [22], is removed in L929 cells. Consistent with a previous report [16], we found that serum starvation of L929 cells prevented necroptosis in response to zVAD.fmk (Fig. 1A). The addition of growth factors, such as bFGF, restored zVAD.fmk induced death under serum free conditions (Fig. 1B). Interestingly, this does not reflect a generic requirement for growth factor signaling, as only some growth factors (bFGF and IGF-1, but not EGF and PDGF) promoted death (Fig. 1B). Furthermore, growth factor-dependent necroptosis required the inhibition of caspase activity, as bFGF alone did not induce cell death (Fig. 1C). In contrast, TNFα triggered necroptosis equally efficiently in the absence of serum (Fig. 1A), suggesting that either growth factors and zVAD.fmk or TNFα are required for necroptotic death in L929 cells.

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