PMC:3585731 / 19836-25597
Annnotations
testone
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Akt Signaling Pathway Contributes to the Regulation of Necroptosis\nWe 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.\n10.1371/journal.pone.0056576.g005 Figure 5 mTORC1 contributes to the regulation of necroptosis.\n(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. Akt kinase is considered to be a pro-survival protein that inhibits apoptosis through the control of multiple effectors including mTORC1, GSK-3 and others [28]. An important question is whether these same molecules reverse their pro-survival roles during necroptosis. We found that inhibition of mTORC1 by rapamycin, an inhibitor of the mTOR co-factor Raptor [29], protected cells from necroptosis (Fig. 5C). Similarly, the direct mTOR kinase inhibitor Torin1 [30] and the dual PI3K/mTOR inhibitor PI-103 [31] also efficiently inhibited necroptosis (Fig. 5C). Knockdown of mTOR using siRNA further validated the small-molecule inhibitor data indicating a role for mTOR in necroptosis by protecting cells from both zVAD.fmk and TNFα induced death (Fig. 5D).\nmTORC1 regulates translation through activation of p70S6 kinase and, subsequently, ribosomal protein S6 [32]. Notably, a genome-wide siRNA screen [10] suggested an important role for protein translation in necroptosis. Consistently, we found that the small molecule inhibitor of p70S6K PF-4708671 [33] attenuated necroptosis at the concentrations required to block S6 phosphorylation (Fig. S5C, D). Partial siRNA knockdown of S6 protein attenuated necroptosis as well (Fig. S5E), suggesting that translational control by p70S6K/S6 may play a role in necroptosis. Overall, while the full repertoire of Akt targets during necroptosis remains to be fully explored, our data provide evidence that the activity of an anti-apoptotic branch of Akt signaling can promote necroptosis.\nRIP1 kinase, Akt, mTORC1 and JNK control the upregulation of TNFα accompanying necroptosis. Hitomi et al. [10] have recently reported that the induction of necroptosis by zVAD.fmk in L929 cells is associated with increased synthesis of TNFα, which potentiates cell death. Therefore, we examined whether Akt and its effectors contribute to TNFα synthesis. Consistent with a RIP1-dependent increase in TNFα protein (Fig. S6A, B), we found that TNFα mRNA levels increased during necroptosis in L929 cells in a RIP1 (Fig. S6C. Under serum free conditions, bFGF alone triggered some induction of TNFα mRNA, while its combination with zVAD.fmk (but not zVAD.fmk alone) caused a pronounced further increase (Fig. 6A). Conversely, PDGF caused a modest upregulation of TNFα mRNA, which was not further increased in the presence of zVAD.fmk (Fig. 6A), demonstrating that activation of necroptosis is specifically accompanied by a marked increase in autocrine TNFα synthesis.\n10.1371/journal.pone.0056576.g006 Figure 6 Akt and mTORC1 control autocrine TNFα synthesis and JNK activation during necroptosis.\n(A) Cells were treated under serum free conditions with bFGF or PDGF with or without zVAD.fmk for 9 hr, followed by qRT-PCR analysis of mTNFα. Data was normalized to mouse 18S RNA. (B) Necroptosis was induced by zVAD.fmk or TNFα in cells treated with Nec-1, rapamycin (rapa), or Akt inh. VIII inh. followed by qRT-PCR analysis of TNFα mRNA levels. (C-F) L929 cells with siRNA knockdown of Akt isoforms (C,E) or mTOR (D,F) were stimulated with zVAD.fmk or TNFα for 9 hr, followed by qRT-PCR analysis of mTNFα (C,D) or western blot (E,F). In all graphs, average±SD was plotted. Further analysis suggested that both Akt and mTORC1 contribute to the upregulation of TNFα mRNA during necroptosis as both small-molecule inhibition and siRNA knockdown of Akt and mTOR reduced TNFα mRNA levels in necroptotic cells (Fig. 6B,C,D). Notably, RIP1 and Akt inhibitors had no effect on the levels of TNFα mRNA in control cells or in the cells stimulated with bFGF alone (Fig. 6A,B, Fig. S6C), suggesting that these kinases specifically mediate necroptosis-dependent increase in TNFα synthesis."}
2_test
{"project":"2_test","denotations":[{"id":"23469174-18955974-90505435","span":{"begin":2209,"end":2211},"obj":"18955974"},{"id":"23469174-12150925-90505436","span":{"begin":2413,"end":2415},"obj":"12150925"},{"id":"23469174-20860370-90505437","span":{"begin":2514,"end":2516},"obj":"20860370"},{"id":"23469174-16697955-90505438","span":{"begin":2559,"end":2561},"obj":"16697955"},{"id":"23469174-12374276-90505439","span":{"begin":2915,"end":2917},"obj":"12374276"},{"id":"23469174-19109899-90505440","span":{"begin":2957,"end":2959},"obj":"19109899"},{"id":"23469174-20704563-90505441","span":{"begin":3108,"end":3110},"obj":"20704563"},{"id":"23469174-19109899-90505442","span":{"begin":3693,"end":3695},"obj":"19109899"}],"text":"The Akt Signaling Pathway Contributes to the Regulation of Necroptosis\nWe 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.\n10.1371/journal.pone.0056576.g005 Figure 5 mTORC1 contributes to the regulation of necroptosis.\n(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. Akt kinase is considered to be a pro-survival protein that inhibits apoptosis through the control of multiple effectors including mTORC1, GSK-3 and others [28]. An important question is whether these same molecules reverse their pro-survival roles during necroptosis. We found that inhibition of mTORC1 by rapamycin, an inhibitor of the mTOR co-factor Raptor [29], protected cells from necroptosis (Fig. 5C). Similarly, the direct mTOR kinase inhibitor Torin1 [30] and the dual PI3K/mTOR inhibitor PI-103 [31] also efficiently inhibited necroptosis (Fig. 5C). Knockdown of mTOR using siRNA further validated the small-molecule inhibitor data indicating a role for mTOR in necroptosis by protecting cells from both zVAD.fmk and TNFα induced death (Fig. 5D).\nmTORC1 regulates translation through activation of p70S6 kinase and, subsequently, ribosomal protein S6 [32]. Notably, a genome-wide siRNA screen [10] suggested an important role for protein translation in necroptosis. Consistently, we found that the small molecule inhibitor of p70S6K PF-4708671 [33] attenuated necroptosis at the concentrations required to block S6 phosphorylation (Fig. S5C, D). Partial siRNA knockdown of S6 protein attenuated necroptosis as well (Fig. S5E), suggesting that translational control by p70S6K/S6 may play a role in necroptosis. Overall, while the full repertoire of Akt targets during necroptosis remains to be fully explored, our data provide evidence that the activity of an anti-apoptotic branch of Akt signaling can promote necroptosis.\nRIP1 kinase, Akt, mTORC1 and JNK control the upregulation of TNFα accompanying necroptosis. Hitomi et al. [10] have recently reported that the induction of necroptosis by zVAD.fmk in L929 cells is associated with increased synthesis of TNFα, which potentiates cell death. Therefore, we examined whether Akt and its effectors contribute to TNFα synthesis. Consistent with a RIP1-dependent increase in TNFα protein (Fig. S6A, B), we found that TNFα mRNA levels increased during necroptosis in L929 cells in a RIP1 (Fig. S6C. Under serum free conditions, bFGF alone triggered some induction of TNFα mRNA, while its combination with zVAD.fmk (but not zVAD.fmk alone) caused a pronounced further increase (Fig. 6A). Conversely, PDGF caused a modest upregulation of TNFα mRNA, which was not further increased in the presence of zVAD.fmk (Fig. 6A), demonstrating that activation of necroptosis is specifically accompanied by a marked increase in autocrine TNFα synthesis.\n10.1371/journal.pone.0056576.g006 Figure 6 Akt and mTORC1 control autocrine TNFα synthesis and JNK activation during necroptosis.\n(A) Cells were treated under serum free conditions with bFGF or PDGF with or without zVAD.fmk for 9 hr, followed by qRT-PCR analysis of mTNFα. Data was normalized to mouse 18S RNA. (B) Necroptosis was induced by zVAD.fmk or TNFα in cells treated with Nec-1, rapamycin (rapa), or Akt inh. VIII inh. followed by qRT-PCR analysis of TNFα mRNA levels. (C-F) L929 cells with siRNA knockdown of Akt isoforms (C,E) or mTOR (D,F) were stimulated with zVAD.fmk or TNFα for 9 hr, followed by qRT-PCR analysis of mTNFα (C,D) or western blot (E,F). In all graphs, average±SD was plotted. Further analysis suggested that both Akt and mTORC1 contribute to the upregulation of TNFα mRNA during necroptosis as both small-molecule inhibition and siRNA knockdown of Akt and mTOR reduced TNFα mRNA levels in necroptotic cells (Fig. 6B,C,D). Notably, RIP1 and Akt inhibitors had no effect on the levels of TNFα mRNA in control cells or in the cells stimulated with bFGF alone (Fig. 6A,B, Fig. S6C), suggesting that these kinases specifically mediate necroptosis-dependent increase in TNFα synthesis."}
pmc-enju-pas
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Akt Signaling Pathway Contributes to the Regulation of Necroptosis\nWe 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.\n10.1371/journal.pone.0056576.g005 Figure 5 mTORC1 contributes to the regulation of necroptosis.\n(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. Akt kinase is considered to be a pro-survival protein that inhibits apoptosis through the control of multiple effectors including mTORC1, GSK-3 and others [28]. An important question is whether these same molecules reverse their pro-survival roles during necroptosis. We found that inhibition of mTORC1 by rapamycin, an inhibitor of the mTOR co-factor Raptor [29], protected cells from necroptosis (Fig. 5C). Similarly, the direct mTOR kinase inhibitor Torin1 [30] and the dual PI3K/mTOR inhibitor PI-103 [31] also efficiently inhibited necroptosis (Fig. 5C). Knockdown of mTOR using siRNA further validated the small-molecule inhibitor data indicating a role for mTOR in necroptosis by protecting cells from both zVAD.fmk and TNFα induced death (Fig. 5D).\nmTORC1 regulates translation through activation of p70S6 kinase and, subsequently, ribosomal protein S6 [32]. Notably, a genome-wide siRNA screen [10] suggested an important role for protein translation in necroptosis. Consistently, we found that the small molecule inhibitor of p70S6K PF-4708671 [33] attenuated necroptosis at the concentrations required to block S6 phosphorylation (Fig. S5C, D). Partial siRNA knockdown of S6 protein attenuated necroptosis as well (Fig. S5E), suggesting that translational control by p70S6K/S6 may play a role in necroptosis. Overall, while the full repertoire of Akt targets during necroptosis remains to be fully explored, our data provide evidence that the activity of an anti-apoptotic branch of Akt signaling can promote necroptosis.\nRIP1 kinase, Akt, mTORC1 and JNK control the upregulation of TNFα accompanying necroptosis. Hitomi et al. [10] have recently reported that the induction of necroptosis by zVAD.fmk in L929 cells is associated with increased synthesis of TNFα, which potentiates cell death. Therefore, we examined whether Akt and its effectors contribute to TNFα synthesis. Consistent with a RIP1-dependent increase in TNFα protein (Fig. S6A, B), we found that TNFα mRNA levels increased during necroptosis in L929 cells in a RIP1 (Fig. S6C. Under serum free conditions, bFGF alone triggered some induction of TNFα mRNA, while its combination with zVAD.fmk (but not zVAD.fmk alone) caused a pronounced further increase (Fig. 6A). Conversely, PDGF caused a modest upregulation of TNFα mRNA, which was not further increased in the presence of zVAD.fmk (Fig. 6A), demonstrating that activation of necroptosis is specifically accompanied by a marked increase in autocrine TNFα synthesis.\n10.1371/journal.pone.0056576.g006 Figure 6 Akt and mTORC1 control autocrine TNFα synthesis and JNK activation during necroptosis.\n(A) Cells were treated under serum free conditions with bFGF or PDGF with or without zVAD.fmk for 9 hr, followed by qRT-PCR analysis of mTNFα. Data was normalized to mouse 18S RNA. (B) Necroptosis was induced by zVAD.fmk or TNFα in cells treated with Nec-1, rapamycin (rapa), or Akt inh. VIII inh. followed by qRT-PCR analysis of TNFα mRNA levels. (C-F) L929 cells with siRNA knockdown of Akt isoforms (C,E) or mTOR (D,F) were stimulated with zVAD.fmk or TNFα for 9 hr, followed by qRT-PCR analysis of mTNFα (C,D) or western blot (E,F). In all graphs, average±SD was plotted. Further analysis suggested that both Akt and mTORC1 contribute to the upregulation of TNFα mRNA during necroptosis as both small-molecule inhibition and siRNA knockdown of Akt and mTOR reduced TNFα mRNA levels in necroptotic cells (Fig. 6B,C,D). Notably, RIP1 and Akt inhibitors had no effect on the levels of TNFα mRNA in control cells or in the cells stimulated with bFGF alone (Fig. 6A,B, Fig. S6C), suggesting that these kinases specifically mediate necroptosis-dependent increase in TNFα synthesis."}
bionlp-st-ge-2016-coref
{"project":"bionlp-st-ge-2016-coref","denotations":[{"id":"T9197","span":{"begin":5513,"end":5517},"obj":"Antecedent"},{"id":"T9196","span":{"begin":5677,"end":5690},"obj":"Anaphor"},{"id":"T9195","span":{"begin":4330,"end":4342},"obj":"Antecedent"},{"id":"T9194","span":{"begin":4357,"end":4362},"obj":"Anaphor"},{"id":"T9193","span":{"begin":596,"end":602},"obj":"Antecedent"},{"id":"T9192","span":{"begin":607,"end":610},"obj":"Anaphor"}],"relations":[{"id":"R5906","pred":"boundBy","subj":"T9192","obj":"T9193"},{"id":"R5907","pred":"boundBy","subj":"T9194","obj":"T9195"},{"id":"R5908","pred":"boundBy","subj":"T9196","obj":"T9197"}],"namespaces":[{"prefix":"_base","uri":"https://bionlp.dbcls.jp/ontology/ge.owl#"}],"text":"The Akt Signaling Pathway Contributes to the Regulation of Necroptosis\nWe 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.\n10.1371/journal.pone.0056576.g005 Figure 5 mTORC1 contributes to the regulation of necroptosis.\n(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. Akt kinase is considered to be a pro-survival protein that inhibits apoptosis through the control of multiple effectors including mTORC1, GSK-3 and others [28]. An important question is whether these same molecules reverse their pro-survival roles during necroptosis. We found that inhibition of mTORC1 by rapamycin, an inhibitor of the mTOR co-factor Raptor [29], protected cells from necroptosis (Fig. 5C). Similarly, the direct mTOR kinase inhibitor Torin1 [30] and the dual PI3K/mTOR inhibitor PI-103 [31] also efficiently inhibited necroptosis (Fig. 5C). Knockdown of mTOR using siRNA further validated the small-molecule inhibitor data indicating a role for mTOR in necroptosis by protecting cells from both zVAD.fmk and TNFα induced death (Fig. 5D).\nmTORC1 regulates translation through activation of p70S6 kinase and, subsequently, ribosomal protein S6 [32]. Notably, a genome-wide siRNA screen [10] suggested an important role for protein translation in necroptosis. Consistently, we found that the small molecule inhibitor of p70S6K PF-4708671 [33] attenuated necroptosis at the concentrations required to block S6 phosphorylation (Fig. S5C, D). Partial siRNA knockdown of S6 protein attenuated necroptosis as well (Fig. S5E), suggesting that translational control by p70S6K/S6 may play a role in necroptosis. Overall, while the full repertoire of Akt targets during necroptosis remains to be fully explored, our data provide evidence that the activity of an anti-apoptotic branch of Akt signaling can promote necroptosis.\nRIP1 kinase, Akt, mTORC1 and JNK control the upregulation of TNFα accompanying necroptosis. Hitomi et al. [10] have recently reported that the induction of necroptosis by zVAD.fmk in L929 cells is associated with increased synthesis of TNFα, which potentiates cell death. Therefore, we examined whether Akt and its effectors contribute to TNFα synthesis. Consistent with a RIP1-dependent increase in TNFα protein (Fig. S6A, B), we found that TNFα mRNA levels increased during necroptosis in L929 cells in a RIP1 (Fig. S6C. Under serum free conditions, bFGF alone triggered some induction of TNFα mRNA, while its combination with zVAD.fmk (but not zVAD.fmk alone) caused a pronounced further increase (Fig. 6A). Conversely, PDGF caused a modest upregulation of TNFα mRNA, which was not further increased in the presence of zVAD.fmk (Fig. 6A), demonstrating that activation of necroptosis is specifically accompanied by a marked increase in autocrine TNFα synthesis.\n10.1371/journal.pone.0056576.g006 Figure 6 Akt and mTORC1 control autocrine TNFα synthesis and JNK activation during necroptosis.\n(A) Cells were treated under serum free conditions with bFGF or PDGF with or without zVAD.fmk for 9 hr, followed by qRT-PCR analysis of mTNFα. Data was normalized to mouse 18S RNA. (B) Necroptosis was induced by zVAD.fmk or TNFα in cells treated with Nec-1, rapamycin (rapa), or Akt inh. VIII inh. followed by qRT-PCR analysis of TNFα mRNA levels. (C-F) L929 cells with siRNA knockdown of Akt isoforms (C,E) or mTOR (D,F) were stimulated with zVAD.fmk or TNFα for 9 hr, followed by qRT-PCR analysis of mTNFα (C,D) or western blot (E,F). In all graphs, average±SD was plotted. Further analysis suggested that both Akt and mTORC1 contribute to the upregulation of TNFα mRNA during necroptosis as both small-molecule inhibition and siRNA knockdown of Akt and mTOR reduced TNFα mRNA levels in necroptotic cells (Fig. 6B,C,D). Notably, RIP1 and Akt inhibitors had no effect on the levels of TNFα mRNA in control cells or in the cells stimulated with bFGF alone (Fig. 6A,B, Fig. S6C), suggesting that these kinases specifically mediate necroptosis-dependent increase in TNFα synthesis."}
bionlp-st-ge-2016-test-proteins
{"project":"bionlp-st-ge-2016-test-proteins","denotations":[{"id":"T9239","span":{"begin":5746,"end":5750},"obj":"Protein"},{"id":"T30689","span":{"begin":4906,"end":4910},"obj":"Protein"},{"id":"T30688","span":{"begin":4854,"end":4857},"obj":"Protein"},{"id":"T30687","span":{"begin":4818,"end":4823},"obj":"Protein"},{"id":"T30686","span":{"begin":4738,"end":4742},"obj":"Protein"},{"id":"T30685","span":{"begin":4628,"end":4632},"obj":"Protein"},{"id":"T9238","span":{"begin":5627,"end":5631},"obj":"Protein"},{"id":"T9237","span":{"begin":5568,"end":5572},"obj":"Protein"},{"id":"T9236","span":{"begin":5513,"end":5517},"obj":"Protein"},{"id":"T9235","span":{"begin":5451,"end":5455},"obj":"Protein"},{"id":"T9234","span":{"begin":5344,"end":5348},"obj":"Protein"},{"id":"T9233","span":{"begin":4535,"end":4539},"obj":"Protein"},{"id":"T9232","span":{"begin":4346,"end":4350},"obj":"Protein"},{"id":"T9231","span":{"begin":4177,"end":4181},"obj":"Protein"},{"id":"T9230","span":{"begin":4138,"end":4142},"obj":"Protein"},{"id":"T9229","span":{"begin":4093,"end":4097},"obj":"Protein"},{"id":"T9228","span":{"begin":4028,"end":4032},"obj":"Protein"},{"id":"T9227","span":{"begin":3986,"end":3990},"obj":"Protein"},{"id":"T9226","span":{"begin":3959,"end":3963},"obj":"Protein"},{"id":"T9225","span":{"begin":3925,"end":3929},"obj":"Protein"},{"id":"T9224","span":{"begin":3822,"end":3826},"obj":"Protein"},{"id":"T9223","span":{"begin":3647,"end":3651},"obj":"Protein"},{"id":"T9222","span":{"begin":3586,"end":3590},"obj":"Protein"},{"id":"T9221","span":{"begin":3338,"end":3340},"obj":"Protein"},{"id":"T9220","span":{"begin":3331,"end":3337},"obj":"Protein"},{"id":"T9219","span":{"begin":3236,"end":3238},"obj":"Protein"},{"id":"T9218","span":{"begin":3175,"end":3177},"obj":"Protein"},{"id":"T9217","span":{"begin":3089,"end":3095},"obj":"Protein"},{"id":"T9216","span":{"begin":2911,"end":2913},"obj":"Protein"},{"id":"T9215","span":{"begin":2861,"end":2873},"obj":"Protein"},{"id":"T9214","span":{"begin":2780,"end":2784},"obj":"Protein"},{"id":"T9213","span":{"begin":2405,"end":2411},"obj":"Protein"},{"id":"T9212","span":{"begin":1288,"end":1292},"obj":"Protein"},{"id":"T9211","span":{"begin":793,"end":799},"obj":"Protein"},{"id":"T9210","span":{"begin":621,"end":623},"obj":"Protein"},{"id":"T9209","span":{"begin":596,"end":602},"obj":"Protein"},{"id":"T9208","span":{"begin":593,"end":594},"obj":"Protein"},{"id":"T9207","span":{"begin":586,"end":592},"obj":"Protein"},{"id":"T9206","span":{"begin":578,"end":584},"obj":"Protein"},{"id":"T9205","span":{"begin":523,"end":527},"obj":"Protein"},{"id":"T9204","span":{"begin":516,"end":521},"obj":"Protein"},{"id":"T9203","span":{"begin":509,"end":514},"obj":"Protein"},{"id":"T9202","span":{"begin":479,"end":481},"obj":"Protein"},{"id":"T9201","span":{"begin":470,"end":476},"obj":"Protein"},{"id":"T9200","span":{"begin":437,"end":468},"obj":"Protein"},{"id":"T9199","span":{"begin":397,"end":401},"obj":"Protein"},{"id":"T9198","span":{"begin":380,"end":395},"obj":"Protein"},{"id":"T30692","span":{"begin":5184,"end":5189},"obj":"Protein"},{"id":"T30691","span":{"begin":5137,"end":5141},"obj":"Protein"},{"id":"T30690","span":{"begin":5012,"end":5016},"obj":"Protein"},{"id":"T30248","span":{"begin":1928,"end":1932},"obj":"Protein"},{"id":"T30247","span":{"begin":1715,"end":1719},"obj":"Protein"},{"id":"T30246","span":{"begin":1562,"end":1566},"obj":"Protein"},{"id":"T30245","span":{"begin":1554,"end":1558},"obj":"Protein"},{"id":"T30244","span":{"begin":1454,"end":1458},"obj":"Protein"}],"namespaces":[{"prefix":"_base","uri":"http://bionlp.dbcls.jp/ontology/ge.owl#"}],"text":"The Akt Signaling Pathway Contributes to the Regulation of Necroptosis\nWe 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.\n10.1371/journal.pone.0056576.g005 Figure 5 mTORC1 contributes to the regulation of necroptosis.\n(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. Akt kinase is considered to be a pro-survival protein that inhibits apoptosis through the control of multiple effectors including mTORC1, GSK-3 and others [28]. An important question is whether these same molecules reverse their pro-survival roles during necroptosis. We found that inhibition of mTORC1 by rapamycin, an inhibitor of the mTOR co-factor Raptor [29], protected cells from necroptosis (Fig. 5C). Similarly, the direct mTOR kinase inhibitor Torin1 [30] and the dual PI3K/mTOR inhibitor PI-103 [31] also efficiently inhibited necroptosis (Fig. 5C). Knockdown of mTOR using siRNA further validated the small-molecule inhibitor data indicating a role for mTOR in necroptosis by protecting cells from both zVAD.fmk and TNFα induced death (Fig. 5D).\nmTORC1 regulates translation through activation of p70S6 kinase and, subsequently, ribosomal protein S6 [32]. Notably, a genome-wide siRNA screen [10] suggested an important role for protein translation in necroptosis. Consistently, we found that the small molecule inhibitor of p70S6K PF-4708671 [33] attenuated necroptosis at the concentrations required to block S6 phosphorylation (Fig. S5C, D). Partial siRNA knockdown of S6 protein attenuated necroptosis as well (Fig. S5E), suggesting that translational control by p70S6K/S6 may play a role in necroptosis. Overall, while the full repertoire of Akt targets during necroptosis remains to be fully explored, our data provide evidence that the activity of an anti-apoptotic branch of Akt signaling can promote necroptosis.\nRIP1 kinase, Akt, mTORC1 and JNK control the upregulation of TNFα accompanying necroptosis. Hitomi et al. [10] have recently reported that the induction of necroptosis by zVAD.fmk in L929 cells is associated with increased synthesis of TNFα, which potentiates cell death. Therefore, we examined whether Akt and its effectors contribute to TNFα synthesis. Consistent with a RIP1-dependent increase in TNFα protein (Fig. S6A, B), we found that TNFα mRNA levels increased during necroptosis in L929 cells in a RIP1 (Fig. S6C. Under serum free conditions, bFGF alone triggered some induction of TNFα mRNA, while its combination with zVAD.fmk (but not zVAD.fmk alone) caused a pronounced further increase (Fig. 6A). Conversely, PDGF caused a modest upregulation of TNFα mRNA, which was not further increased in the presence of zVAD.fmk (Fig. 6A), demonstrating that activation of necroptosis is specifically accompanied by a marked increase in autocrine TNFα synthesis.\n10.1371/journal.pone.0056576.g006 Figure 6 Akt and mTORC1 control autocrine TNFα synthesis and JNK activation during necroptosis.\n(A) Cells were treated under serum free conditions with bFGF or PDGF with or without zVAD.fmk for 9 hr, followed by qRT-PCR analysis of mTNFα. Data was normalized to mouse 18S RNA. (B) Necroptosis was induced by zVAD.fmk or TNFα in cells treated with Nec-1, rapamycin (rapa), or Akt inh. VIII inh. followed by qRT-PCR analysis of TNFα mRNA levels. (C-F) L929 cells with siRNA knockdown of Akt isoforms (C,E) or mTOR (D,F) were stimulated with zVAD.fmk or TNFα for 9 hr, followed by qRT-PCR analysis of mTNFα (C,D) or western blot (E,F). In all graphs, average±SD was plotted. Further analysis suggested that both Akt and mTORC1 contribute to the upregulation of TNFα mRNA during necroptosis as both small-molecule inhibition and siRNA knockdown of Akt and mTOR reduced TNFα mRNA levels in necroptotic cells (Fig. 6B,C,D). Notably, RIP1 and Akt inhibitors had no effect on the levels of TNFα mRNA in control cells or in the cells stimulated with bFGF alone (Fig. 6A,B, Fig. S6C), suggesting that these kinases specifically mediate necroptosis-dependent increase in TNFα synthesis."}
bionlp-st-ge-2016-uniprot
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Akt Signaling Pathway Contributes to the Regulation of Necroptosis\nWe 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.\n10.1371/journal.pone.0056576.g005 Figure 5 mTORC1 contributes to the regulation of necroptosis.\n(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. Akt kinase is considered to be a pro-survival protein that inhibits apoptosis through the control of multiple effectors including mTORC1, GSK-3 and others [28]. An important question is whether these same molecules reverse their pro-survival roles during necroptosis. We found that inhibition of mTORC1 by rapamycin, an inhibitor of the mTOR co-factor Raptor [29], protected cells from necroptosis (Fig. 5C). Similarly, the direct mTOR kinase inhibitor Torin1 [30] and the dual PI3K/mTOR inhibitor PI-103 [31] also efficiently inhibited necroptosis (Fig. 5C). Knockdown of mTOR using siRNA further validated the small-molecule inhibitor data indicating a role for mTOR in necroptosis by protecting cells from both zVAD.fmk and TNFα induced death (Fig. 5D).\nmTORC1 regulates translation through activation of p70S6 kinase and, subsequently, ribosomal protein S6 [32]. Notably, a genome-wide siRNA screen [10] suggested an important role for protein translation in necroptosis. Consistently, we found that the small molecule inhibitor of p70S6K PF-4708671 [33] attenuated necroptosis at the concentrations required to block S6 phosphorylation (Fig. S5C, D). Partial siRNA knockdown of S6 protein attenuated necroptosis as well (Fig. S5E), suggesting that translational control by p70S6K/S6 may play a role in necroptosis. Overall, while the full repertoire of Akt targets during necroptosis remains to be fully explored, our data provide evidence that the activity of an anti-apoptotic branch of Akt signaling can promote necroptosis.\nRIP1 kinase, Akt, mTORC1 and JNK control the upregulation of TNFα accompanying necroptosis. Hitomi et al. [10] have recently reported that the induction of necroptosis by zVAD.fmk in L929 cells is associated with increased synthesis of TNFα, which potentiates cell death. Therefore, we examined whether Akt and its effectors contribute to TNFα synthesis. Consistent with a RIP1-dependent increase in TNFα protein (Fig. S6A, B), we found that TNFα mRNA levels increased during necroptosis in L929 cells in a RIP1 (Fig. S6C. Under serum free conditions, bFGF alone triggered some induction of TNFα mRNA, while its combination with zVAD.fmk (but not zVAD.fmk alone) caused a pronounced further increase (Fig. 6A). Conversely, PDGF caused a modest upregulation of TNFα mRNA, which was not further increased in the presence of zVAD.fmk (Fig. 6A), demonstrating that activation of necroptosis is specifically accompanied by a marked increase in autocrine TNFα synthesis.\n10.1371/journal.pone.0056576.g006 Figure 6 Akt and mTORC1 control autocrine TNFα synthesis and JNK activation during necroptosis.\n(A) Cells were treated under serum free conditions with bFGF or PDGF with or without zVAD.fmk for 9 hr, followed by qRT-PCR analysis of mTNFα. Data was normalized to mouse 18S RNA. (B) Necroptosis was induced by zVAD.fmk or TNFα in cells treated with Nec-1, rapamycin (rapa), or Akt inh. VIII inh. followed by qRT-PCR analysis of TNFα mRNA levels. (C-F) L929 cells with siRNA knockdown of Akt isoforms (C,E) or mTOR (D,F) were stimulated with zVAD.fmk or TNFα for 9 hr, followed by qRT-PCR analysis of mTNFα (C,D) or western blot (E,F). In all graphs, average±SD was plotted. Further analysis suggested that both Akt and mTORC1 contribute to the upregulation of TNFα mRNA during necroptosis as both small-molecule inhibition and siRNA knockdown of Akt and mTOR reduced TNFα mRNA levels in necroptotic cells (Fig. 6B,C,D). Notably, RIP1 and Akt inhibitors had no effect on the levels of TNFα mRNA in control cells or in the cells stimulated with bFGF alone (Fig. 6A,B, Fig. S6C), suggesting that these kinases specifically mediate necroptosis-dependent increase in TNFα synthesis."}
UBERON-AE
{"project":"UBERON-AE","denotations":[{"id":"T30669","span":{"begin":4711,"end":4716},"obj":"http://purl.obolibrary.org/obo/UBERON_0001977"},{"id":"T30233","span":{"begin":1515,"end":1520},"obj":"http://purl.obolibrary.org/obo/UBERON_0001977"},{"id":"T9084","span":{"begin":4115,"end":4120},"obj":"http://purl.obolibrary.org/obo/UBERON_0001977"}],"text":"The Akt Signaling Pathway Contributes to the Regulation of Necroptosis\nWe 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.\n10.1371/journal.pone.0056576.g005 Figure 5 mTORC1 contributes to the regulation of necroptosis.\n(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. Akt kinase is considered to be a pro-survival protein that inhibits apoptosis through the control of multiple effectors including mTORC1, GSK-3 and others [28]. An important question is whether these same molecules reverse their pro-survival roles during necroptosis. We found that inhibition of mTORC1 by rapamycin, an inhibitor of the mTOR co-factor Raptor [29], protected cells from necroptosis (Fig. 5C). Similarly, the direct mTOR kinase inhibitor Torin1 [30] and the dual PI3K/mTOR inhibitor PI-103 [31] also efficiently inhibited necroptosis (Fig. 5C). Knockdown of mTOR using siRNA further validated the small-molecule inhibitor data indicating a role for mTOR in necroptosis by protecting cells from both zVAD.fmk and TNFα induced death (Fig. 5D).\nmTORC1 regulates translation through activation of p70S6 kinase and, subsequently, ribosomal protein S6 [32]. Notably, a genome-wide siRNA screen [10] suggested an important role for protein translation in necroptosis. Consistently, we found that the small molecule inhibitor of p70S6K PF-4708671 [33] attenuated necroptosis at the concentrations required to block S6 phosphorylation (Fig. S5C, D). Partial siRNA knockdown of S6 protein attenuated necroptosis as well (Fig. S5E), suggesting that translational control by p70S6K/S6 may play a role in necroptosis. Overall, while the full repertoire of Akt targets during necroptosis remains to be fully explored, our data provide evidence that the activity of an anti-apoptotic branch of Akt signaling can promote necroptosis.\nRIP1 kinase, Akt, mTORC1 and JNK control the upregulation of TNFα accompanying necroptosis. Hitomi et al. [10] have recently reported that the induction of necroptosis by zVAD.fmk in L929 cells is associated with increased synthesis of TNFα, which potentiates cell death. Therefore, we examined whether Akt and its effectors contribute to TNFα synthesis. Consistent with a RIP1-dependent increase in TNFα protein (Fig. S6A, B), we found that TNFα mRNA levels increased during necroptosis in L929 cells in a RIP1 (Fig. S6C. Under serum free conditions, bFGF alone triggered some induction of TNFα mRNA, while its combination with zVAD.fmk (but not zVAD.fmk alone) caused a pronounced further increase (Fig. 6A). Conversely, PDGF caused a modest upregulation of TNFα mRNA, which was not further increased in the presence of zVAD.fmk (Fig. 6A), demonstrating that activation of necroptosis is specifically accompanied by a marked increase in autocrine TNFα synthesis.\n10.1371/journal.pone.0056576.g006 Figure 6 Akt and mTORC1 control autocrine TNFα synthesis and JNK activation during necroptosis.\n(A) Cells were treated under serum free conditions with bFGF or PDGF with or without zVAD.fmk for 9 hr, followed by qRT-PCR analysis of mTNFα. Data was normalized to mouse 18S RNA. (B) Necroptosis was induced by zVAD.fmk or TNFα in cells treated with Nec-1, rapamycin (rapa), or Akt inh. VIII inh. followed by qRT-PCR analysis of TNFα mRNA levels. (C-F) L929 cells with siRNA knockdown of Akt isoforms (C,E) or mTOR (D,F) were stimulated with zVAD.fmk or TNFα for 9 hr, followed by qRT-PCR analysis of mTNFα (C,D) or western blot (E,F). In all graphs, average±SD was plotted. Further analysis suggested that both Akt and mTORC1 contribute to the upregulation of TNFα mRNA during necroptosis as both small-molecule inhibition and siRNA knockdown of Akt and mTOR reduced TNFα mRNA levels in necroptotic cells (Fig. 6B,C,D). Notably, RIP1 and Akt inhibitors had no effect on the levels of TNFα mRNA in control cells or in the cells stimulated with bFGF alone (Fig. 6A,B, Fig. S6C), suggesting that these kinases specifically mediate necroptosis-dependent increase in TNFα synthesis."}
GO-BP
{"project":"GO-BP","denotations":[{"id":"T30698","span":{"begin":4867,"end":4878},"obj":"http://purl.obolibrary.org/obo/GO_0097528"},{"id":"T30697","span":{"begin":4669,"end":4680},"obj":"http://purl.obolibrary.org/obo/GO_0097528"},{"id":"T30696","span":{"begin":4867,"end":4878},"obj":"http://purl.obolibrary.org/obo/GO_0070266"},{"id":"T30695","span":{"begin":4669,"end":4680},"obj":"http://purl.obolibrary.org/obo/GO_0070266"},{"id":"T30694","span":{"begin":4647,"end":4650},"obj":"http://purl.obolibrary.org/obo/GO_0004705"},{"id":"T30693","span":{"begin":4633,"end":4642},"obj":"http://purl.obolibrary.org/obo/GO_0009058"},{"id":"T30254","span":{"begin":2001,"end":2012},"obj":"http://purl.obolibrary.org/obo/GO_0097528"},{"id":"T30253","span":{"begin":1396,"end":1407},"obj":"http://purl.obolibrary.org/obo/GO_0097528"},{"id":"T30252","span":{"begin":1987,"end":2012},"obj":"http://purl.obolibrary.org/obo/GO_0070266"},{"id":"T30251","span":{"begin":1396,"end":1407},"obj":"http://purl.obolibrary.org/obo/GO_0070266"},{"id":"T30250","span":{"begin":1382,"end":1407},"obj":"http://purl.obolibrary.org/obo/GO_0060544"},{"id":"T30249","span":{"begin":1382,"end":1392},"obj":"http://purl.obolibrary.org/obo/GO_0065007"},{"id":"T9312","span":{"begin":5751,"end":5760},"obj":"http://purl.obolibrary.org/obo/GO_0009058"},{"id":"T9311","span":{"begin":4540,"end":4549},"obj":"http://purl.obolibrary.org/obo/GO_0009058"},{"id":"T9310","span":{"begin":3930,"end":3939},"obj":"http://purl.obolibrary.org/obo/GO_0009058"},{"id":"T9309","span":{"begin":3809,"end":3818},"obj":"http://purl.obolibrary.org/obo/GO_0009058"},{"id":"T9308","span":{"begin":3615,"end":3618},"obj":"http://purl.obolibrary.org/obo/GO_0004705"},{"id":"T9307","span":{"begin":2993,"end":3012},"obj":"http://purl.obolibrary.org/obo/GO_0006412"},{"id":"T9306","span":{"begin":2827,"end":2838},"obj":"http://purl.obolibrary.org/obo/GO_0006412"},{"id":"T9305","span":{"begin":2531,"end":2535},"obj":"http://purl.obolibrary.org/obo/GO_0016303"},{"id":"T9304","span":{"begin":2489,"end":2505},"obj":"http://purl.obolibrary.org/obo/GO_0033673"},{"id":"T9303","span":{"begin":2121,"end":2130},"obj":"http://purl.obolibrary.org/obo/GO_0097194"},{"id":"T9302","span":{"begin":2121,"end":2130},"obj":"http://purl.obolibrary.org/obo/GO_0006915"},{"id":"T9301","span":{"begin":2282,"end":2294},"obj":"http://purl.obolibrary.org/obo/GO_0043066"},{"id":"T9300","span":{"begin":2086,"end":2098},"obj":"http://purl.obolibrary.org/obo/GO_0043066"},{"id":"T9299","span":{"begin":3851,"end":3856},"obj":"http://purl.obolibrary.org/obo/GO_0016265"},{"id":"T9298","span":{"begin":2793,"end":2798},"obj":"http://purl.obolibrary.org/obo/GO_0016265"},{"id":"T9297","span":{"begin":1277,"end":1282},"obj":"http://purl.obolibrary.org/obo/GO_0016265"},{"id":"T9296","span":{"begin":3846,"end":3856},"obj":"http://purl.obolibrary.org/obo/GO_0008219"},{"id":"T9295","span":{"begin":1272,"end":1282},"obj":"http://purl.obolibrary.org/obo/GO_0008219"},{"id":"T9294","span":{"begin":3551,"end":3560},"obj":"http://purl.obolibrary.org/obo/GO_0023052"},{"id":"T9293","span":{"begin":1213,"end":1222},"obj":"http://purl.obolibrary.org/obo/GO_0023052"},{"id":"T9292","span":{"begin":2191,"end":2194},"obj":"http://purl.obolibrary.org/obo/GO_0050321"},{"id":"T9291","span":{"begin":586,"end":589},"obj":"http://purl.obolibrary.org/obo/GO_0050321"},{"id":"T9290","span":{"begin":356,"end":359},"obj":"http://purl.obolibrary.org/obo/GO_0050321"},{"id":"T9289","span":{"begin":190,"end":205},"obj":"http://purl.obolibrary.org/obo/GO_0016301"},{"id":"T9288","span":{"begin":3178,"end":3193},"obj":"http://purl.obolibrary.org/obo/GO_0016310"},{"id":"T9287","span":{"begin":936,"end":951},"obj":"http://purl.obolibrary.org/obo/GO_0016310"},{"id":"T9286","span":{"begin":743,"end":758},"obj":"http://purl.obolibrary.org/obo/GO_0016310"},{"id":"T9285","span":{"begin":688,"end":703},"obj":"http://purl.obolibrary.org/obo/GO_0016310"},{"id":"T9284","span":{"begin":268,"end":283},"obj":"http://purl.obolibrary.org/obo/GO_0016310"},{"id":"T9283","span":{"begin":144,"end":159},"obj":"http://purl.obolibrary.org/obo/GO_0016310"},{"id":"T9282","span":{"begin":5712,"end":5723},"obj":"http://purl.obolibrary.org/obo/GO_0097528"},{"id":"T9281","span":{"begin":5361,"end":5372},"obj":"http://purl.obolibrary.org/obo/GO_0097528"},{"id":"T9280","span":{"begin":4461,"end":4472},"obj":"http://purl.obolibrary.org/obo/GO_0097528"},{"id":"T9279","span":{"begin":4062,"end":4073},"obj":"http://purl.obolibrary.org/obo/GO_0097528"},{"id":"T9278","span":{"begin":3742,"end":3753},"obj":"http://purl.obolibrary.org/obo/GO_0097528"},{"id":"T9277","span":{"begin":3665,"end":3676},"obj":"http://purl.obolibrary.org/obo/GO_0097528"},{"id":"T9276","span":{"begin":3573,"end":3584},"obj":"http://purl.obolibrary.org/obo/GO_0097528"},{"id":"T9275","span":{"begin":3430,"end":3441},"obj":"http://purl.obolibrary.org/obo/GO_0097528"},{"id":"T9274","span":{"begin":3360,"end":3371},"obj":"http://purl.obolibrary.org/obo/GO_0097528"},{"id":"T9273","span":{"begin":3258,"end":3269},"obj":"http://purl.obolibrary.org/obo/GO_0097528"},{"id":"T9272","span":{"begin":3123,"end":3134},"obj":"http://purl.obolibrary.org/obo/GO_0097528"},{"id":"T9271","span":{"begin":3016,"end":3027},"obj":"http://purl.obolibrary.org/obo/GO_0097528"},{"id":"T9270","span":{"begin":2725,"end":2736},"obj":"http://purl.obolibrary.org/obo/GO_0097528"},{"id":"T9269","span":{"begin":2590,"end":2601},"obj":"http://purl.obolibrary.org/obo/GO_0097528"},{"id":"T9268","span":{"begin":2439,"end":2450},"obj":"http://purl.obolibrary.org/obo/GO_0097528"},{"id":"T9267","span":{"begin":2308,"end":2319},"obj":"http://purl.obolibrary.org/obo/GO_0097528"},{"id":"T9266","span":{"begin":1037,"end":1048},"obj":"http://purl.obolibrary.org/obo/GO_0097528"},{"id":"T9265","span":{"begin":998,"end":1009},"obj":"http://purl.obolibrary.org/obo/GO_0097528"},{"id":"T9264","span":{"begin":102,"end":113},"obj":"http://purl.obolibrary.org/obo/GO_0097528"},{"id":"T9263","span":{"begin":59,"end":70},"obj":"http://purl.obolibrary.org/obo/GO_0097528"},{"id":"T9262","span":{"begin":4447,"end":4472},"obj":"http://purl.obolibrary.org/obo/GO_0070266"},{"id":"T9261","span":{"begin":3729,"end":3753},"obj":"http://purl.obolibrary.org/obo/GO_0070266"},{"id":"T9260","span":{"begin":5361,"end":5372},"obj":"http://purl.obolibrary.org/obo/GO_0070266"},{"id":"T9259","span":{"begin":4461,"end":4472},"obj":"http://purl.obolibrary.org/obo/GO_0070266"},{"id":"T9258","span":{"begin":4062,"end":4073},"obj":"http://purl.obolibrary.org/obo/GO_0070266"},{"id":"T9257","span":{"begin":3742,"end":3753},"obj":"http://purl.obolibrary.org/obo/GO_0070266"},{"id":"T9256","span":{"begin":3665,"end":3676},"obj":"http://purl.obolibrary.org/obo/GO_0070266"},{"id":"T9255","span":{"begin":3573,"end":3584},"obj":"http://purl.obolibrary.org/obo/GO_0070266"},{"id":"T9254","span":{"begin":3430,"end":3441},"obj":"http://purl.obolibrary.org/obo/GO_0070266"},{"id":"T9253","span":{"begin":3360,"end":3371},"obj":"http://purl.obolibrary.org/obo/GO_0070266"},{"id":"T9252","span":{"begin":3258,"end":3269},"obj":"http://purl.obolibrary.org/obo/GO_0070266"},{"id":"T9251","span":{"begin":3123,"end":3134},"obj":"http://purl.obolibrary.org/obo/GO_0070266"},{"id":"T9250","span":{"begin":3016,"end":3027},"obj":"http://purl.obolibrary.org/obo/GO_0070266"},{"id":"T9249","span":{"begin":2725,"end":2736},"obj":"http://purl.obolibrary.org/obo/GO_0070266"},{"id":"T9248","span":{"begin":2590,"end":2601},"obj":"http://purl.obolibrary.org/obo/GO_0070266"},{"id":"T9247","span":{"begin":2439,"end":2450},"obj":"http://purl.obolibrary.org/obo/GO_0070266"},{"id":"T9246","span":{"begin":2308,"end":2319},"obj":"http://purl.obolibrary.org/obo/GO_0070266"},{"id":"T9245","span":{"begin":1037,"end":1048},"obj":"http://purl.obolibrary.org/obo/GO_0070266"},{"id":"T9244","span":{"begin":998,"end":1009},"obj":"http://purl.obolibrary.org/obo/GO_0070266"},{"id":"T9243","span":{"begin":102,"end":113},"obj":"http://purl.obolibrary.org/obo/GO_0070266"},{"id":"T9242","span":{"begin":59,"end":70},"obj":"http://purl.obolibrary.org/obo/GO_0070266"},{"id":"T9241","span":{"begin":45,"end":70},"obj":"http://purl.obolibrary.org/obo/GO_0060544"},{"id":"T9240","span":{"begin":45,"end":55},"obj":"http://purl.obolibrary.org/obo/GO_0065007"}],"text":"The Akt Signaling Pathway Contributes to the Regulation of Necroptosis\nWe 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.\n10.1371/journal.pone.0056576.g005 Figure 5 mTORC1 contributes to the regulation of necroptosis.\n(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. Akt kinase is considered to be a pro-survival protein that inhibits apoptosis through the control of multiple effectors including mTORC1, GSK-3 and others [28]. An important question is whether these same molecules reverse their pro-survival roles during necroptosis. We found that inhibition of mTORC1 by rapamycin, an inhibitor of the mTOR co-factor Raptor [29], protected cells from necroptosis (Fig. 5C). Similarly, the direct mTOR kinase inhibitor Torin1 [30] and the dual PI3K/mTOR inhibitor PI-103 [31] also efficiently inhibited necroptosis (Fig. 5C). Knockdown of mTOR using siRNA further validated the small-molecule inhibitor data indicating a role for mTOR in necroptosis by protecting cells from both zVAD.fmk and TNFα induced death (Fig. 5D).\nmTORC1 regulates translation through activation of p70S6 kinase and, subsequently, ribosomal protein S6 [32]. Notably, a genome-wide siRNA screen [10] suggested an important role for protein translation in necroptosis. Consistently, we found that the small molecule inhibitor of p70S6K PF-4708671 [33] attenuated necroptosis at the concentrations required to block S6 phosphorylation (Fig. S5C, D). Partial siRNA knockdown of S6 protein attenuated necroptosis as well (Fig. S5E), suggesting that translational control by p70S6K/S6 may play a role in necroptosis. Overall, while the full repertoire of Akt targets during necroptosis remains to be fully explored, our data provide evidence that the activity of an anti-apoptotic branch of Akt signaling can promote necroptosis.\nRIP1 kinase, Akt, mTORC1 and JNK control the upregulation of TNFα accompanying necroptosis. Hitomi et al. [10] have recently reported that the induction of necroptosis by zVAD.fmk in L929 cells is associated with increased synthesis of TNFα, which potentiates cell death. Therefore, we examined whether Akt and its effectors contribute to TNFα synthesis. Consistent with a RIP1-dependent increase in TNFα protein (Fig. S6A, B), we found that TNFα mRNA levels increased during necroptosis in L929 cells in a RIP1 (Fig. S6C. Under serum free conditions, bFGF alone triggered some induction of TNFα mRNA, while its combination with zVAD.fmk (but not zVAD.fmk alone) caused a pronounced further increase (Fig. 6A). Conversely, PDGF caused a modest upregulation of TNFα mRNA, which was not further increased in the presence of zVAD.fmk (Fig. 6A), demonstrating that activation of necroptosis is specifically accompanied by a marked increase in autocrine TNFα synthesis.\n10.1371/journal.pone.0056576.g006 Figure 6 Akt and mTORC1 control autocrine TNFα synthesis and JNK activation during necroptosis.\n(A) Cells were treated under serum free conditions with bFGF or PDGF with or without zVAD.fmk for 9 hr, followed by qRT-PCR analysis of mTNFα. Data was normalized to mouse 18S RNA. (B) Necroptosis was induced by zVAD.fmk or TNFα in cells treated with Nec-1, rapamycin (rapa), or Akt inh. VIII inh. followed by qRT-PCR analysis of TNFα mRNA levels. (C-F) L929 cells with siRNA knockdown of Akt isoforms (C,E) or mTOR (D,F) were stimulated with zVAD.fmk or TNFα for 9 hr, followed by qRT-PCR analysis of mTNFα (C,D) or western blot (E,F). In all graphs, average±SD was plotted. Further analysis suggested that both Akt and mTORC1 contribute to the upregulation of TNFα mRNA during necroptosis as both small-molecule inhibition and siRNA knockdown of Akt and mTOR reduced TNFα mRNA levels in necroptotic cells (Fig. 6B,C,D). Notably, RIP1 and Akt inhibitors had no effect on the levels of TNFα mRNA in control cells or in the cells stimulated with bFGF alone (Fig. 6A,B, Fig. S6C), suggesting that these kinases specifically mediate necroptosis-dependent increase in TNFα synthesis."}
GO-MF
{"project":"GO-MF","denotations":[{"id":"T9321","span":{"begin":4309,"end":4313},"obj":"http://purl.obolibrary.org/obo/GO_0005161"},{"id":"T9320","span":{"begin":3615,"end":3618},"obj":"http://purl.obolibrary.org/obo/GO_0004705"},{"id":"T9319","span":{"begin":2531,"end":2535},"obj":"http://purl.obolibrary.org/obo/GO_0016303"},{"id":"T9318","span":{"begin":2893,"end":2910},"obj":"http://purl.obolibrary.org/obo/GO_0003735"},{"id":"T9317","span":{"begin":441,"end":458},"obj":"http://purl.obolibrary.org/obo/GO_0003735"},{"id":"T9316","span":{"begin":2191,"end":2194},"obj":"http://purl.obolibrary.org/obo/GO_0050321"},{"id":"T9315","span":{"begin":586,"end":589},"obj":"http://purl.obolibrary.org/obo/GO_0050321"},{"id":"T9314","span":{"begin":356,"end":359},"obj":"http://purl.obolibrary.org/obo/GO_0050321"},{"id":"T9313","span":{"begin":190,"end":205},"obj":"http://purl.obolibrary.org/obo/GO_0016301"},{"id":"T30700","span":{"begin":4746,"end":4750},"obj":"http://purl.obolibrary.org/obo/GO_0005161"},{"id":"T30699","span":{"begin":4647,"end":4650},"obj":"http://purl.obolibrary.org/obo/GO_0004705"},{"id":"T30255","span":{"begin":1660,"end":1670},"obj":"http://purl.obolibrary.org/obo/GO_0003823"}],"text":"The Akt Signaling Pathway Contributes to the Regulation of Necroptosis\nWe 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.\n10.1371/journal.pone.0056576.g005 Figure 5 mTORC1 contributes to the regulation of necroptosis.\n(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. Akt kinase is considered to be a pro-survival protein that inhibits apoptosis through the control of multiple effectors including mTORC1, GSK-3 and others [28]. An important question is whether these same molecules reverse their pro-survival roles during necroptosis. We found that inhibition of mTORC1 by rapamycin, an inhibitor of the mTOR co-factor Raptor [29], protected cells from necroptosis (Fig. 5C). Similarly, the direct mTOR kinase inhibitor Torin1 [30] and the dual PI3K/mTOR inhibitor PI-103 [31] also efficiently inhibited necroptosis (Fig. 5C). Knockdown of mTOR using siRNA further validated the small-molecule inhibitor data indicating a role for mTOR in necroptosis by protecting cells from both zVAD.fmk and TNFα induced death (Fig. 5D).\nmTORC1 regulates translation through activation of p70S6 kinase and, subsequently, ribosomal protein S6 [32]. Notably, a genome-wide siRNA screen [10] suggested an important role for protein translation in necroptosis. Consistently, we found that the small molecule inhibitor of p70S6K PF-4708671 [33] attenuated necroptosis at the concentrations required to block S6 phosphorylation (Fig. S5C, D). Partial siRNA knockdown of S6 protein attenuated necroptosis as well (Fig. S5E), suggesting that translational control by p70S6K/S6 may play a role in necroptosis. Overall, while the full repertoire of Akt targets during necroptosis remains to be fully explored, our data provide evidence that the activity of an anti-apoptotic branch of Akt signaling can promote necroptosis.\nRIP1 kinase, Akt, mTORC1 and JNK control the upregulation of TNFα accompanying necroptosis. Hitomi et al. [10] have recently reported that the induction of necroptosis by zVAD.fmk in L929 cells is associated with increased synthesis of TNFα, which potentiates cell death. Therefore, we examined whether Akt and its effectors contribute to TNFα synthesis. Consistent with a RIP1-dependent increase in TNFα protein (Fig. S6A, B), we found that TNFα mRNA levels increased during necroptosis in L929 cells in a RIP1 (Fig. S6C. Under serum free conditions, bFGF alone triggered some induction of TNFα mRNA, while its combination with zVAD.fmk (but not zVAD.fmk alone) caused a pronounced further increase (Fig. 6A). Conversely, PDGF caused a modest upregulation of TNFα mRNA, which was not further increased in the presence of zVAD.fmk (Fig. 6A), demonstrating that activation of necroptosis is specifically accompanied by a marked increase in autocrine TNFα synthesis.\n10.1371/journal.pone.0056576.g006 Figure 6 Akt and mTORC1 control autocrine TNFα synthesis and JNK activation during necroptosis.\n(A) Cells were treated under serum free conditions with bFGF or PDGF with or without zVAD.fmk for 9 hr, followed by qRT-PCR analysis of mTNFα. Data was normalized to mouse 18S RNA. (B) Necroptosis was induced by zVAD.fmk or TNFα in cells treated with Nec-1, rapamycin (rapa), or Akt inh. VIII inh. followed by qRT-PCR analysis of TNFα mRNA levels. (C-F) L929 cells with siRNA knockdown of Akt isoforms (C,E) or mTOR (D,F) were stimulated with zVAD.fmk or TNFα for 9 hr, followed by qRT-PCR analysis of mTNFα (C,D) or western blot (E,F). In all graphs, average±SD was plotted. Further analysis suggested that both Akt and mTORC1 contribute to the upregulation of TNFα mRNA during necroptosis as both small-molecule inhibition and siRNA knockdown of Akt and mTOR reduced TNFα mRNA levels in necroptotic cells (Fig. 6B,C,D). Notably, RIP1 and Akt inhibitors had no effect on the levels of TNFα mRNA in control cells or in the cells stimulated with bFGF alone (Fig. 6A,B, Fig. S6C), suggesting that these kinases specifically mediate necroptosis-dependent increase in TNFα synthesis."}
GO-CC
{"project":"GO-CC","denotations":[{"id":"T30703","span":{"begin":5041,"end":5046},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T30702","span":{"begin":4914,"end":4919},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T30701","span":{"begin":4603,"end":4609},"obj":"http://purl.obolibrary.org/obo/GO_0031931"},{"id":"T30260","span":{"begin":1660,"end":1670},"obj":"http://purl.obolibrary.org/obo/GO_0019815"},{"id":"T30259","span":{"begin":1945,"end":1949},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T30258","span":{"begin":1836,"end":1841},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T30257","span":{"begin":1418,"end":1423},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T30256","span":{"begin":1356,"end":1362},"obj":"http://purl.obolibrary.org/obo/GO_0031931"},{"id":"T9333","span":{"begin":3774,"end":3793},"obj":"http://purl.obolibrary.org/obo/GO_0009986"},{"id":"T9332","span":{"begin":5303,"end":5309},"obj":"http://purl.obolibrary.org/obo/GO_0031931"},{"id":"T9331","span":{"begin":3604,"end":3610},"obj":"http://purl.obolibrary.org/obo/GO_0031931"},{"id":"T9330","span":{"begin":2810,"end":2816},"obj":"http://purl.obolibrary.org/obo/GO_0031931"},{"id":"T9329","span":{"begin":2349,"end":2355},"obj":"http://purl.obolibrary.org/obo/GO_0031931"},{"id":"T9328","span":{"begin":2183,"end":2189},"obj":"http://purl.obolibrary.org/obo/GO_0031931"},{"id":"T9327","span":{"begin":5605,"end":5610},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T9326","span":{"begin":5589,"end":5594},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T9325","span":{"begin":5483,"end":5488},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T9324","span":{"begin":4082,"end":4087},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T9323","span":{"begin":3846,"end":3850},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T9322","span":{"begin":1272,"end":1276},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T30261","span":{"begin":1660,"end":1670},"obj":"http://purl.obolibrary.org/obo/GO_0042571"}],"text":"The Akt Signaling Pathway Contributes to the Regulation of Necroptosis\nWe 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.\n10.1371/journal.pone.0056576.g005 Figure 5 mTORC1 contributes to the regulation of necroptosis.\n(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. Akt kinase is considered to be a pro-survival protein that inhibits apoptosis through the control of multiple effectors including mTORC1, GSK-3 and others [28]. An important question is whether these same molecules reverse their pro-survival roles during necroptosis. We found that inhibition of mTORC1 by rapamycin, an inhibitor of the mTOR co-factor Raptor [29], protected cells from necroptosis (Fig. 5C). Similarly, the direct mTOR kinase inhibitor Torin1 [30] and the dual PI3K/mTOR inhibitor PI-103 [31] also efficiently inhibited necroptosis (Fig. 5C). Knockdown of mTOR using siRNA further validated the small-molecule inhibitor data indicating a role for mTOR in necroptosis by protecting cells from both zVAD.fmk and TNFα induced death (Fig. 5D).\nmTORC1 regulates translation through activation of p70S6 kinase and, subsequently, ribosomal protein S6 [32]. Notably, a genome-wide siRNA screen [10] suggested an important role for protein translation in necroptosis. Consistently, we found that the small molecule inhibitor of p70S6K PF-4708671 [33] attenuated necroptosis at the concentrations required to block S6 phosphorylation (Fig. S5C, D). Partial siRNA knockdown of S6 protein attenuated necroptosis as well (Fig. S5E), suggesting that translational control by p70S6K/S6 may play a role in necroptosis. Overall, while the full repertoire of Akt targets during necroptosis remains to be fully explored, our data provide evidence that the activity of an anti-apoptotic branch of Akt signaling can promote necroptosis.\nRIP1 kinase, Akt, mTORC1 and JNK control the upregulation of TNFα accompanying necroptosis. Hitomi et al. [10] have recently reported that the induction of necroptosis by zVAD.fmk in L929 cells is associated with increased synthesis of TNFα, which potentiates cell death. Therefore, we examined whether Akt and its effectors contribute to TNFα synthesis. Consistent with a RIP1-dependent increase in TNFα protein (Fig. S6A, B), we found that TNFα mRNA levels increased during necroptosis in L929 cells in a RIP1 (Fig. S6C. Under serum free conditions, bFGF alone triggered some induction of TNFα mRNA, while its combination with zVAD.fmk (but not zVAD.fmk alone) caused a pronounced further increase (Fig. 6A). Conversely, PDGF caused a modest upregulation of TNFα mRNA, which was not further increased in the presence of zVAD.fmk (Fig. 6A), demonstrating that activation of necroptosis is specifically accompanied by a marked increase in autocrine TNFα synthesis.\n10.1371/journal.pone.0056576.g006 Figure 6 Akt and mTORC1 control autocrine TNFα synthesis and JNK activation during necroptosis.\n(A) Cells were treated under serum free conditions with bFGF or PDGF with or without zVAD.fmk for 9 hr, followed by qRT-PCR analysis of mTNFα. Data was normalized to mouse 18S RNA. (B) Necroptosis was induced by zVAD.fmk or TNFα in cells treated with Nec-1, rapamycin (rapa), or Akt inh. VIII inh. followed by qRT-PCR analysis of TNFα mRNA levels. (C-F) L929 cells with siRNA knockdown of Akt isoforms (C,E) or mTOR (D,F) were stimulated with zVAD.fmk or TNFα for 9 hr, followed by qRT-PCR analysis of mTNFα (C,D) or western blot (E,F). In all graphs, average±SD was plotted. Further analysis suggested that both Akt and mTORC1 contribute to the upregulation of TNFα mRNA during necroptosis as both small-molecule inhibition and siRNA knockdown of Akt and mTOR reduced TNFα mRNA levels in necroptotic cells (Fig. 6B,C,D). Notably, RIP1 and Akt inhibitors had no effect on the levels of TNFα mRNA in control cells or in the cells stimulated with bFGF alone (Fig. 6A,B, Fig. S6C), suggesting that these kinases specifically mediate necroptosis-dependent increase in TNFα synthesis."}
sentences
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Akt Signaling Pathway Contributes to the Regulation of Necroptosis\nWe 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.\n10.1371/journal.pone.0056576.g005 Figure 5 mTORC1 contributes to the regulation of necroptosis.\n(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. Akt kinase is considered to be a pro-survival protein that inhibits apoptosis through the control of multiple effectors including mTORC1, GSK-3 and others [28]. An important question is whether these same molecules reverse their pro-survival roles during necroptosis. We found that inhibition of mTORC1 by rapamycin, an inhibitor of the mTOR co-factor Raptor [29], protected cells from necroptosis (Fig. 5C). Similarly, the direct mTOR kinase inhibitor Torin1 [30] and the dual PI3K/mTOR inhibitor PI-103 [31] also efficiently inhibited necroptosis (Fig. 5C). Knockdown of mTOR using siRNA further validated the small-molecule inhibitor data indicating a role for mTOR in necroptosis by protecting cells from both zVAD.fmk and TNFα induced death (Fig. 5D).\nmTORC1 regulates translation through activation of p70S6 kinase and, subsequently, ribosomal protein S6 [32]. Notably, a genome-wide siRNA screen [10] suggested an important role for protein translation in necroptosis. Consistently, we found that the small molecule inhibitor of p70S6K PF-4708671 [33] attenuated necroptosis at the concentrations required to block S6 phosphorylation (Fig. S5C, D). Partial siRNA knockdown of S6 protein attenuated necroptosis as well (Fig. S5E), suggesting that translational control by p70S6K/S6 may play a role in necroptosis. Overall, while the full repertoire of Akt targets during necroptosis remains to be fully explored, our data provide evidence that the activity of an anti-apoptotic branch of Akt signaling can promote necroptosis.\nRIP1 kinase, Akt, mTORC1 and JNK control the upregulation of TNFα accompanying necroptosis. Hitomi et al. [10] have recently reported that the induction of necroptosis by zVAD.fmk in L929 cells is associated with increased synthesis of TNFα, which potentiates cell death. Therefore, we examined whether Akt and its effectors contribute to TNFα synthesis. Consistent with a RIP1-dependent increase in TNFα protein (Fig. S6A, B), we found that TNFα mRNA levels increased during necroptosis in L929 cells in a RIP1 (Fig. S6C. Under serum free conditions, bFGF alone triggered some induction of TNFα mRNA, while its combination with zVAD.fmk (but not zVAD.fmk alone) caused a pronounced further increase (Fig. 6A). Conversely, PDGF caused a modest upregulation of TNFα mRNA, which was not further increased in the presence of zVAD.fmk (Fig. 6A), demonstrating that activation of necroptosis is specifically accompanied by a marked increase in autocrine TNFα synthesis.\n10.1371/journal.pone.0056576.g006 Figure 6 Akt and mTORC1 control autocrine TNFα synthesis and JNK activation during necroptosis.\n(A) Cells were treated under serum free conditions with bFGF or PDGF with or without zVAD.fmk for 9 hr, followed by qRT-PCR analysis of mTNFα. Data was normalized to mouse 18S RNA. (B) Necroptosis was induced by zVAD.fmk or TNFα in cells treated with Nec-1, rapamycin (rapa), or Akt inh. VIII inh. followed by qRT-PCR analysis of TNFα mRNA levels. (C-F) L929 cells with siRNA knockdown of Akt isoforms (C,E) or mTOR (D,F) were stimulated with zVAD.fmk or TNFα for 9 hr, followed by qRT-PCR analysis of mTNFα (C,D) or western blot (E,F). In all graphs, average±SD was plotted. Further analysis suggested that both Akt and mTORC1 contribute to the upregulation of TNFα mRNA during necroptosis as both small-molecule inhibition and siRNA knockdown of Akt and mTOR reduced TNFα mRNA levels in necroptotic cells (Fig. 6B,C,D). Notably, RIP1 and Akt inhibitors had no effect on the levels of TNFα mRNA in control cells or in the cells stimulated with bFGF alone (Fig. 6A,B, Fig. S6C), suggesting that these kinases specifically mediate necroptosis-dependent increase in TNFα synthesis."}
simple1
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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.\n10.1371/journal.pone.0056576.g005 Figure 5 mTORC1 contributes to the regulation of necroptosis.\n(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. Akt kinase is considered to be a pro-survival protein that inhibits apoptosis through the control of multiple effectors including mTORC1, GSK-3 and others [28]. An important question is whether these same molecules reverse their pro-survival roles during necroptosis. We found that inhibition of mTORC1 by rapamycin, an inhibitor of the mTOR co-factor Raptor [29], protected cells from necroptosis (Fig. 5C). Similarly, the direct mTOR kinase inhibitor Torin1 [30] and the dual PI3K/mTOR inhibitor PI-103 [31] also efficiently inhibited necroptosis (Fig. 5C). Knockdown of mTOR using siRNA further validated the small-molecule inhibitor data indicating a role for mTOR in necroptosis by protecting cells from both zVAD.fmk and TNFα induced death (Fig. 5D).\nmTORC1 regulates translation through activation of p70S6 kinase and, subsequently, ribosomal protein S6 [32]. Notably, a genome-wide siRNA screen [10] suggested an important role for protein translation in necroptosis. Consistently, we found that the small molecule inhibitor of p70S6K PF-4708671 [33] attenuated necroptosis at the concentrations required to block S6 phosphorylation (Fig. S5C, D). Partial siRNA knockdown of S6 protein attenuated necroptosis as well (Fig. S5E), suggesting that translational control by p70S6K/S6 may play a role in necroptosis. Overall, while the full repertoire of Akt targets during necroptosis remains to be fully explored, our data provide evidence that the activity of an anti-apoptotic branch of Akt signaling can promote necroptosis.\nRIP1 kinase, Akt, mTORC1 and JNK control the upregulation of TNFα accompanying necroptosis. Hitomi et al. [10] have recently reported that the induction of necroptosis by zVAD.fmk in L929 cells is associated with increased synthesis of TNFα, which potentiates cell death. Therefore, we examined whether Akt and its effectors contribute to TNFα synthesis. Consistent with a RIP1-dependent increase in TNFα protein (Fig. S6A, B), we found that TNFα mRNA levels increased during necroptosis in L929 cells in a RIP1 (Fig. S6C. Under serum free conditions, bFGF alone triggered some induction of TNFα mRNA, while its combination with zVAD.fmk (but not zVAD.fmk alone) caused a pronounced further increase (Fig. 6A). Conversely, PDGF caused a modest upregulation of TNFα mRNA, which was not further increased in the presence of zVAD.fmk (Fig. 6A), demonstrating that activation of necroptosis is specifically accompanied by a marked increase in autocrine TNFα synthesis.\n10.1371/journal.pone.0056576.g006 Figure 6 Akt and mTORC1 control autocrine TNFα synthesis and JNK activation during necroptosis.\n(A) Cells were treated under serum free conditions with bFGF or PDGF with or without zVAD.fmk for 9 hr, followed by qRT-PCR analysis of mTNFα. Data was normalized to mouse 18S RNA. (B) Necroptosis was induced by zVAD.fmk or TNFα in cells treated with Nec-1, rapamycin (rapa), or Akt inh. VIII inh. followed by qRT-PCR analysis of TNFα mRNA levels. (C-F) L929 cells with siRNA knockdown of Akt isoforms (C,E) or mTOR (D,F) were stimulated with zVAD.fmk or TNFα for 9 hr, followed by qRT-PCR analysis of mTNFα (C,D) or western blot (E,F). In all graphs, average±SD was plotted. Further analysis suggested that both Akt and mTORC1 contribute to the upregulation of TNFα mRNA during necroptosis as both small-molecule inhibition and siRNA knockdown of Akt and mTOR reduced TNFα mRNA levels in necroptotic cells (Fig. 6B,C,D). Notably, RIP1 and Akt inhibitors had no effect on the levels of TNFα mRNA in control cells or in the cells stimulated with bFGF alone (Fig. 6A,B, Fig. S6C), suggesting that these kinases specifically mediate necroptosis-dependent increase in TNFα synthesis."}
BioNLP16_DUT
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Akt Signaling Pathway Contributes to the Regulation of Necroptosis\nWe 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.\n10.1371/journal.pone.0056576.g005 Figure 5 mTORC1 contributes to the regulation of necroptosis.\n(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. Akt kinase is considered to be a pro-survival protein that inhibits apoptosis through the control of multiple effectors including mTORC1, GSK-3 and others [28]. An important question is whether these same molecules reverse their pro-survival roles during necroptosis. We found that inhibition of mTORC1 by rapamycin, an inhibitor of the mTOR co-factor Raptor [29], protected cells from necroptosis (Fig. 5C). Similarly, the direct mTOR kinase inhibitor Torin1 [30] and the dual PI3K/mTOR inhibitor PI-103 [31] also efficiently inhibited necroptosis (Fig. 5C). Knockdown of mTOR using siRNA further validated the small-molecule inhibitor data indicating a role for mTOR in necroptosis by protecting cells from both zVAD.fmk and TNFα induced death (Fig. 5D).\nmTORC1 regulates translation through activation of p70S6 kinase and, subsequently, ribosomal protein S6 [32]. Notably, a genome-wide siRNA screen [10] suggested an important role for protein translation in necroptosis. Consistently, we found that the small molecule inhibitor of p70S6K PF-4708671 [33] attenuated necroptosis at the concentrations required to block S6 phosphorylation (Fig. S5C, D). Partial siRNA knockdown of S6 protein attenuated necroptosis as well (Fig. S5E), suggesting that translational control by p70S6K/S6 may play a role in necroptosis. Overall, while the full repertoire of Akt targets during necroptosis remains to be fully explored, our data provide evidence that the activity of an anti-apoptotic branch of Akt signaling can promote necroptosis.\nRIP1 kinase, Akt, mTORC1 and JNK control the upregulation of TNFα accompanying necroptosis. Hitomi et al. [10] have recently reported that the induction of necroptosis by zVAD.fmk in L929 cells is associated with increased synthesis of TNFα, which potentiates cell death. Therefore, we examined whether Akt and its effectors contribute to TNFα synthesis. Consistent with a RIP1-dependent increase in TNFα protein (Fig. S6A, B), we found that TNFα mRNA levels increased during necroptosis in L929 cells in a RIP1 (Fig. S6C. Under serum free conditions, bFGF alone triggered some induction of TNFα mRNA, while its combination with zVAD.fmk (but not zVAD.fmk alone) caused a pronounced further increase (Fig. 6A). Conversely, PDGF caused a modest upregulation of TNFα mRNA, which was not further increased in the presence of zVAD.fmk (Fig. 6A), demonstrating that activation of necroptosis is specifically accompanied by a marked increase in autocrine TNFα synthesis.\n10.1371/journal.pone.0056576.g006 Figure 6 Akt and mTORC1 control autocrine TNFα synthesis and JNK activation during necroptosis.\n(A) Cells were treated under serum free conditions with bFGF or PDGF with or without zVAD.fmk for 9 hr, followed by qRT-PCR analysis of mTNFα. Data was normalized to mouse 18S RNA. (B) Necroptosis was induced by zVAD.fmk or TNFα in cells treated with Nec-1, rapamycin (rapa), or Akt inh. VIII inh. followed by qRT-PCR analysis of TNFα mRNA levels. (C-F) L929 cells with siRNA knockdown of Akt isoforms (C,E) or mTOR (D,F) were stimulated with zVAD.fmk or TNFα for 9 hr, followed by qRT-PCR analysis of mTNFα (C,D) or western blot (E,F). In all graphs, average±SD was plotted. Further analysis suggested that both Akt and mTORC1 contribute to the upregulation of TNFα mRNA during necroptosis as both small-molecule inhibition and siRNA knockdown of Akt and mTOR reduced TNFα mRNA levels in necroptotic cells (Fig. 6B,C,D). Notably, RIP1 and Akt inhibitors had no effect on the levels of TNFα mRNA in control cells or in the cells stimulated with bFGF alone (Fig. 6A,B, Fig. S6C), suggesting that these kinases specifically mediate necroptosis-dependent increase in TNFα synthesis."}
BioNLP16_Messiy
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Akt Signaling Pathway Contributes to the Regulation of Necroptosis\nWe 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.\n10.1371/journal.pone.0056576.g005 Figure 5 mTORC1 contributes to the regulation of necroptosis.\n(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. Akt kinase is considered to be a pro-survival protein that inhibits apoptosis through the control of multiple effectors including mTORC1, GSK-3 and others [28]. An important question is whether these same molecules reverse their pro-survival roles during necroptosis. We found that inhibition of mTORC1 by rapamycin, an inhibitor of the mTOR co-factor Raptor [29], protected cells from necroptosis (Fig. 5C). Similarly, the direct mTOR kinase inhibitor Torin1 [30] and the dual PI3K/mTOR inhibitor PI-103 [31] also efficiently inhibited necroptosis (Fig. 5C). Knockdown of mTOR using siRNA further validated the small-molecule inhibitor data indicating a role for mTOR in necroptosis by protecting cells from both zVAD.fmk and TNFα induced death (Fig. 5D).\nmTORC1 regulates translation through activation of p70S6 kinase and, subsequently, ribosomal protein S6 [32]. Notably, a genome-wide siRNA screen [10] suggested an important role for protein translation in necroptosis. Consistently, we found that the small molecule inhibitor of p70S6K PF-4708671 [33] attenuated necroptosis at the concentrations required to block S6 phosphorylation (Fig. S5C, D). Partial siRNA knockdown of S6 protein attenuated necroptosis as well (Fig. S5E), suggesting that translational control by p70S6K/S6 may play a role in necroptosis. Overall, while the full repertoire of Akt targets during necroptosis remains to be fully explored, our data provide evidence that the activity of an anti-apoptotic branch of Akt signaling can promote necroptosis.\nRIP1 kinase, Akt, mTORC1 and JNK control the upregulation of TNFα accompanying necroptosis. Hitomi et al. [10] have recently reported that the induction of necroptosis by zVAD.fmk in L929 cells is associated with increased synthesis of TNFα, which potentiates cell death. Therefore, we examined whether Akt and its effectors contribute to TNFα synthesis. Consistent with a RIP1-dependent increase in TNFα protein (Fig. S6A, B), we found that TNFα mRNA levels increased during necroptosis in L929 cells in a RIP1 (Fig. S6C. Under serum free conditions, bFGF alone triggered some induction of TNFα mRNA, while its combination with zVAD.fmk (but not zVAD.fmk alone) caused a pronounced further increase (Fig. 6A). Conversely, PDGF caused a modest upregulation of TNFα mRNA, which was not further increased in the presence of zVAD.fmk (Fig. 6A), demonstrating that activation of necroptosis is specifically accompanied by a marked increase in autocrine TNFα synthesis.\n10.1371/journal.pone.0056576.g006 Figure 6 Akt and mTORC1 control autocrine TNFα synthesis and JNK activation during necroptosis.\n(A) Cells were treated under serum free conditions with bFGF or PDGF with or without zVAD.fmk for 9 hr, followed by qRT-PCR analysis of mTNFα. Data was normalized to mouse 18S RNA. (B) Necroptosis was induced by zVAD.fmk or TNFα in cells treated with Nec-1, rapamycin (rapa), or Akt inh. VIII inh. followed by qRT-PCR analysis of TNFα mRNA levels. (C-F) L929 cells with siRNA knockdown of Akt isoforms (C,E) or mTOR (D,F) were stimulated with zVAD.fmk or TNFα for 9 hr, followed by qRT-PCR analysis of mTNFα (C,D) or western blot (E,F). In all graphs, average±SD was plotted. Further analysis suggested that both Akt and mTORC1 contribute to the upregulation of TNFα mRNA during necroptosis as both small-molecule inhibition and siRNA knockdown of Akt and mTOR reduced TNFα mRNA levels in necroptotic cells (Fig. 6B,C,D). Notably, RIP1 and Akt inhibitors had no effect on the levels of TNFα mRNA in control cells or in the cells stimulated with bFGF alone (Fig. 6A,B, Fig. S6C), suggesting that these kinases specifically mediate necroptosis-dependent increase in TNFα synthesis."}
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Akt Signaling Pathway Contributes to the Regulation of Necroptosis\nWe 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.\n10.1371/journal.pone.0056576.g005 Figure 5 mTORC1 contributes to the regulation of necroptosis.\n(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. Akt kinase is considered to be a pro-survival protein that inhibits apoptosis through the control of multiple effectors including mTORC1, GSK-3 and others [28]. An important question is whether these same molecules reverse their pro-survival roles during necroptosis. We found that inhibition of mTORC1 by rapamycin, an inhibitor of the mTOR co-factor Raptor [29], protected cells from necroptosis (Fig. 5C). Similarly, the direct mTOR kinase inhibitor Torin1 [30] and the dual PI3K/mTOR inhibitor PI-103 [31] also efficiently inhibited necroptosis (Fig. 5C). Knockdown of mTOR using siRNA further validated the small-molecule inhibitor data indicating a role for mTOR in necroptosis by protecting cells from both zVAD.fmk and TNFα induced death (Fig. 5D).\nmTORC1 regulates translation through activation of p70S6 kinase and, subsequently, ribosomal protein S6 [32]. Notably, a genome-wide siRNA screen [10] suggested an important role for protein translation in necroptosis. Consistently, we found that the small molecule inhibitor of p70S6K PF-4708671 [33] attenuated necroptosis at the concentrations required to block S6 phosphorylation (Fig. S5C, D). Partial siRNA knockdown of S6 protein attenuated necroptosis as well (Fig. S5E), suggesting that translational control by p70S6K/S6 may play a role in necroptosis. Overall, while the full repertoire of Akt targets during necroptosis remains to be fully explored, our data provide evidence that the activity of an anti-apoptotic branch of Akt signaling can promote necroptosis.\nRIP1 kinase, Akt, mTORC1 and JNK control the upregulation of TNFα accompanying necroptosis. Hitomi et al. [10] have recently reported that the induction of necroptosis by zVAD.fmk in L929 cells is associated with increased synthesis of TNFα, which potentiates cell death. Therefore, we examined whether Akt and its effectors contribute to TNFα synthesis. Consistent with a RIP1-dependent increase in TNFα protein (Fig. S6A, B), we found that TNFα mRNA levels increased during necroptosis in L929 cells in a RIP1 (Fig. S6C. Under serum free conditions, bFGF alone triggered some induction of TNFα mRNA, while its combination with zVAD.fmk (but not zVAD.fmk alone) caused a pronounced further increase (Fig. 6A). Conversely, PDGF caused a modest upregulation of TNFα mRNA, which was not further increased in the presence of zVAD.fmk (Fig. 6A), demonstrating that activation of necroptosis is specifically accompanied by a marked increase in autocrine TNFα synthesis.\n10.1371/journal.pone.0056576.g006 Figure 6 Akt and mTORC1 control autocrine TNFα synthesis and JNK activation during necroptosis.\n(A) Cells were treated under serum free conditions with bFGF or PDGF with or without zVAD.fmk for 9 hr, followed by qRT-PCR analysis of mTNFα. Data was normalized to mouse 18S RNA. (B) Necroptosis was induced by zVAD.fmk or TNFα in cells treated with Nec-1, rapamycin (rapa), or Akt inh. VIII inh. followed by qRT-PCR analysis of TNFα mRNA levels. (C-F) L929 cells with siRNA knockdown of Akt isoforms (C,E) or mTOR (D,F) were stimulated with zVAD.fmk or TNFα for 9 hr, followed by qRT-PCR analysis of mTNFα (C,D) or western blot (E,F). In all graphs, average±SD was plotted. Further analysis suggested that both Akt and mTORC1 contribute to the upregulation of TNFα mRNA during necroptosis as both small-molecule inhibition and siRNA knockdown of Akt and mTOR reduced TNFα mRNA levels in necroptotic cells (Fig. 6B,C,D). Notably, RIP1 and Akt inhibitors had no effect on the levels of TNFα mRNA in control cells or in the cells stimulated with bFGF alone (Fig. 6A,B, Fig. S6C), suggesting that these kinases specifically mediate necroptosis-dependent increase in TNFα synthesis."}
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Akt Signaling Pathway Contributes to the Regulation of Necroptosis\nWe 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.\n10.1371/journal.pone.0056576.g005 Figure 5 mTORC1 contributes to the regulation of necroptosis.\n(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. Akt kinase is considered to be a pro-survival protein that inhibits apoptosis through the control of multiple effectors including mTORC1, GSK-3 and others [28]. An important question is whether these same molecules reverse their pro-survival roles during necroptosis. We found that inhibition of mTORC1 by rapamycin, an inhibitor of the mTOR co-factor Raptor [29], protected cells from necroptosis (Fig. 5C). Similarly, the direct mTOR kinase inhibitor Torin1 [30] and the dual PI3K/mTOR inhibitor PI-103 [31] also efficiently inhibited necroptosis (Fig. 5C). Knockdown of mTOR using siRNA further validated the small-molecule inhibitor data indicating a role for mTOR in necroptosis by protecting cells from both zVAD.fmk and TNFα induced death (Fig. 5D).\nmTORC1 regulates translation through activation of p70S6 kinase and, subsequently, ribosomal protein S6 [32]. Notably, a genome-wide siRNA screen [10] suggested an important role for protein translation in necroptosis. Consistently, we found that the small molecule inhibitor of p70S6K PF-4708671 [33] attenuated necroptosis at the concentrations required to block S6 phosphorylation (Fig. S5C, D). Partial siRNA knockdown of S6 protein attenuated necroptosis as well (Fig. S5E), suggesting that translational control by p70S6K/S6 may play a role in necroptosis. Overall, while the full repertoire of Akt targets during necroptosis remains to be fully explored, our data provide evidence that the activity of an anti-apoptotic branch of Akt signaling can promote necroptosis.\nRIP1 kinase, Akt, mTORC1 and JNK control the upregulation of TNFα accompanying necroptosis. Hitomi et al. [10] have recently reported that the induction of necroptosis by zVAD.fmk in L929 cells is associated with increased synthesis of TNFα, which potentiates cell death. Therefore, we examined whether Akt and its effectors contribute to TNFα synthesis. Consistent with a RIP1-dependent increase in TNFα protein (Fig. S6A, B), we found that TNFα mRNA levels increased during necroptosis in L929 cells in a RIP1 (Fig. S6C. Under serum free conditions, bFGF alone triggered some induction of TNFα mRNA, while its combination with zVAD.fmk (but not zVAD.fmk alone) caused a pronounced further increase (Fig. 6A). Conversely, PDGF caused a modest upregulation of TNFα mRNA, which was not further increased in the presence of zVAD.fmk (Fig. 6A), demonstrating that activation of necroptosis is specifically accompanied by a marked increase in autocrine TNFα synthesis.\n10.1371/journal.pone.0056576.g006 Figure 6 Akt and mTORC1 control autocrine TNFα synthesis and JNK activation during necroptosis.\n(A) Cells were treated under serum free conditions with bFGF or PDGF with or without zVAD.fmk for 9 hr, followed by qRT-PCR analysis of mTNFα. Data was normalized to mouse 18S RNA. (B) Necroptosis was induced by zVAD.fmk or TNFα in cells treated with Nec-1, rapamycin (rapa), or Akt inh. VIII inh. followed by qRT-PCR analysis of TNFα mRNA levels. (C-F) L929 cells with siRNA knockdown of Akt isoforms (C,E) or mTOR (D,F) were stimulated with zVAD.fmk or TNFα for 9 hr, followed by qRT-PCR analysis of mTNFα (C,D) or western blot (E,F). In all graphs, average±SD was plotted. Further analysis suggested that both Akt and mTORC1 contribute to the upregulation of TNFα mRNA during necroptosis as both small-molecule inhibition and siRNA knockdown of Akt and mTOR reduced TNFα mRNA levels in necroptotic cells (Fig. 6B,C,D). Notably, RIP1 and Akt inhibitors had no effect on the levels of TNFα mRNA in control cells or in the cells stimulated with bFGF alone (Fig. 6A,B, Fig. S6C), suggesting that these kinases specifically mediate necroptosis-dependent increase in TNFα synthesis."}
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,"pred":"pobj","subj":"T11416","obj":"T11415"},{"id":"R7529","pred":"aux","subj":"T11417","obj":"T11418"},{"id":"R7534","pred":"ROOT","subj":"T11418","obj":"T11418"},{"id":"R7539","pred":"det","subj":"T11419","obj":"T11420"},{"id":"R7544","pred":"prep","subj":"T11421","obj":"T11420"},{"id":"R7292","pred":"prep","subj":"T11255","obj":"T11254"},{"id":"R7293","pred":"pobj","subj":"T11256","obj":"T11255"},{"id":"R7549","pred":"pobj","subj":"T11422","obj":"T11421"},{"id":"R7695","pred":"conj","subj":"T11658","obj":"T11655"},{"id":"R6977","pred":"det","subj":"T10940","obj":"T10943"},{"id":"R7134","pred":"pobj","subj":"T11097","obj":"T11096"},{"id":"R7508","pred":"amod","subj":"T11413","obj":"T11414"}],"text":"The Akt Signaling Pathway Contributes to the Regulation of Necroptosis\nWe 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.\n10.1371/journal.pone.0056576.g005 Figure 5 mTORC1 contributes to the regulation of necroptosis.\n(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. Akt kinase is considered to be a pro-survival protein that inhibits apoptosis through the control of multiple effectors including mTORC1, GSK-3 and others [28]. An important question is whether these same molecules reverse their pro-survival roles during necroptosis. We found that inhibition of mTORC1 by rapamycin, an inhibitor of the mTOR co-factor Raptor [29], protected cells from necroptosis (Fig. 5C). Similarly, the direct mTOR kinase inhibitor Torin1 [30] and the dual PI3K/mTOR inhibitor PI-103 [31] also efficiently inhibited necroptosis (Fig. 5C). Knockdown of mTOR using siRNA further validated the small-molecule inhibitor data indicating a role for mTOR in necroptosis by protecting cells from both zVAD.fmk and TNFα induced death (Fig. 5D).\nmTORC1 regulates translation through activation of p70S6 kinase and, subsequently, ribosomal protein S6 [32]. Notably, a genome-wide siRNA screen [10] suggested an important role for protein translation in necroptosis. Consistently, we found that the small molecule inhibitor of p70S6K PF-4708671 [33] attenuated necroptosis at the concentrations required to block S6 phosphorylation (Fig. S5C, D). Partial siRNA knockdown of S6 protein attenuated necroptosis as well (Fig. S5E), suggesting that translational control by p70S6K/S6 may play a role in necroptosis. Overall, while the full repertoire of Akt targets during necroptosis remains to be fully explored, our data provide evidence that the activity of an anti-apoptotic branch of Akt signaling can promote necroptosis.\nRIP1 kinase, Akt, mTORC1 and JNK control the upregulation of TNFα accompanying necroptosis. Hitomi et al. [10] have recently reported that the induction of necroptosis by zVAD.fmk in L929 cells is associated with increased synthesis of TNFα, which potentiates cell death. Therefore, we examined whether Akt and its effectors contribute to TNFα synthesis. Consistent with a RIP1-dependent increase in TNFα protein (Fig. S6A, B), we found that TNFα mRNA levels increased during necroptosis in L929 cells in a RIP1 (Fig. S6C. Under serum free conditions, bFGF alone triggered some induction of TNFα mRNA, while its combination with zVAD.fmk (but not zVAD.fmk alone) caused a pronounced further increase (Fig. 6A). Conversely, PDGF caused a modest upregulation of TNFα mRNA, which was not further increased in the presence of zVAD.fmk (Fig. 6A), demonstrating that activation of necroptosis is specifically accompanied by a marked increase in autocrine TNFα synthesis.\n10.1371/journal.pone.0056576.g006 Figure 6 Akt and mTORC1 control autocrine TNFα synthesis and JNK activation during necroptosis.\n(A) Cells were treated under serum free conditions with bFGF or PDGF with or without zVAD.fmk for 9 hr, followed by qRT-PCR analysis of mTNFα. Data was normalized to mouse 18S RNA. (B) Necroptosis was induced by zVAD.fmk or TNFα in cells treated with Nec-1, rapamycin (rapa), or Akt inh. VIII inh. followed by qRT-PCR analysis of TNFα mRNA levels. (C-F) L929 cells with siRNA knockdown of Akt isoforms (C,E) or mTOR (D,F) were stimulated with zVAD.fmk or TNFα for 9 hr, followed by qRT-PCR analysis of mTNFα (C,D) or western blot (E,F). In all graphs, average±SD was plotted. Further analysis suggested that both Akt and mTORC1 contribute to the upregulation of TNFα mRNA during necroptosis as both small-molecule inhibition and siRNA knockdown of Akt and mTOR reduced TNFα mRNA levels in necroptotic cells (Fig. 6B,C,D). Notably, RIP1 and Akt inhibitors had no effect on the levels of TNFα mRNA in control cells or in the cells stimulated with bFGF alone (Fig. 6A,B, Fig. S6C), suggesting that these kinases specifically mediate necroptosis-dependent increase in TNFα synthesis."}
bionlp-st-ge-2016-test-tees
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Akt Signaling Pathway Contributes to the Regulation of Necroptosis\nWe 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.\n10.1371/journal.pone.0056576.g005 Figure 5 mTORC1 contributes to the regulation of necroptosis.\n(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. Akt kinase is considered to be a pro-survival protein that inhibits apoptosis through the control of multiple effectors including mTORC1, GSK-3 and others [28]. An important question is whether these same molecules reverse their pro-survival roles during necroptosis. We found that inhibition of mTORC1 by rapamycin, an inhibitor of the mTOR co-factor Raptor [29], protected cells from necroptosis (Fig. 5C). Similarly, the direct mTOR kinase inhibitor Torin1 [30] and the dual PI3K/mTOR inhibitor PI-103 [31] also efficiently inhibited necroptosis (Fig. 5C). Knockdown of mTOR using siRNA further validated the small-molecule inhibitor data indicating a role for mTOR in necroptosis by protecting cells from both zVAD.fmk and TNFα induced death (Fig. 5D).\nmTORC1 regulates translation through activation of p70S6 kinase and, subsequently, ribosomal protein S6 [32]. Notably, a genome-wide siRNA screen [10] suggested an important role for protein translation in necroptosis. Consistently, we found that the small molecule inhibitor of p70S6K PF-4708671 [33] attenuated necroptosis at the concentrations required to block S6 phosphorylation (Fig. S5C, D). Partial siRNA knockdown of S6 protein attenuated necroptosis as well (Fig. S5E), suggesting that translational control by p70S6K/S6 may play a role in necroptosis. Overall, while the full repertoire of Akt targets during necroptosis remains to be fully explored, our data provide evidence that the activity of an anti-apoptotic branch of Akt signaling can promote necroptosis.\nRIP1 kinase, Akt, mTORC1 and JNK control the upregulation of TNFα accompanying necroptosis. Hitomi et al. [10] have recently reported that the induction of necroptosis by zVAD.fmk in L929 cells is associated with increased synthesis of TNFα, which potentiates cell death. Therefore, we examined whether Akt and its effectors contribute to TNFα synthesis. Consistent with a RIP1-dependent increase in TNFα protein (Fig. S6A, B), we found that TNFα mRNA levels increased during necroptosis in L929 cells in a RIP1 (Fig. S6C. Under serum free conditions, bFGF alone triggered some induction of TNFα mRNA, while its combination with zVAD.fmk (but not zVAD.fmk alone) caused a pronounced further increase (Fig. 6A). Conversely, PDGF caused a modest upregulation of TNFα mRNA, which was not further increased in the presence of zVAD.fmk (Fig. 6A), demonstrating that activation of necroptosis is specifically accompanied by a marked increase in autocrine TNFα synthesis.\n10.1371/journal.pone.0056576.g006 Figure 6 Akt and mTORC1 control autocrine TNFα synthesis and JNK activation during necroptosis.\n(A) Cells were treated under serum free conditions with bFGF or PDGF with or without zVAD.fmk for 9 hr, followed by qRT-PCR analysis of mTNFα. Data was normalized to mouse 18S RNA. (B) Necroptosis was induced by zVAD.fmk or TNFα in cells treated with Nec-1, rapamycin (rapa), or Akt inh. VIII inh. followed by qRT-PCR analysis of TNFα mRNA levels. (C-F) L929 cells with siRNA knockdown of Akt isoforms (C,E) or mTOR (D,F) were stimulated with zVAD.fmk or TNFα for 9 hr, followed by qRT-PCR analysis of mTNFα (C,D) or western blot (E,F). In all graphs, average±SD was plotted. Further analysis suggested that both Akt and mTORC1 contribute to the upregulation of TNFα mRNA during necroptosis as both small-molecule inhibition and siRNA knockdown of Akt and mTOR reduced TNFα mRNA levels in necroptotic cells (Fig. 6B,C,D). Notably, RIP1 and Akt inhibitors had no effect on the levels of TNFα mRNA in control cells or in the cells stimulated with bFGF alone (Fig. 6A,B, Fig. S6C), suggesting that these kinases specifically mediate necroptosis-dependent increase in TNFα synthesis."}
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Akt Signaling Pathway Contributes to the Regulation of Necroptosis\nWe 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.\n10.1371/journal.pone.0056576.g005 Figure 5 mTORC1 contributes to the regulation of necroptosis.\n(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. Akt kinase is considered to be a pro-survival protein that inhibits apoptosis through the control of multiple effectors including mTORC1, GSK-3 and others [28]. An important question is whether these same molecules reverse their pro-survival roles during necroptosis. We found that inhibition of mTORC1 by rapamycin, an inhibitor of the mTOR co-factor Raptor [29], protected cells from necroptosis (Fig. 5C). Similarly, the direct mTOR kinase inhibitor Torin1 [30] and the dual PI3K/mTOR inhibitor PI-103 [31] also efficiently inhibited necroptosis (Fig. 5C). Knockdown of mTOR using siRNA further validated the small-molecule inhibitor data indicating a role for mTOR in necroptosis by protecting cells from both zVAD.fmk and TNFα induced death (Fig. 5D).\nmTORC1 regulates translation through activation of p70S6 kinase and, subsequently, ribosomal protein S6 [32]. Notably, a genome-wide siRNA screen [10] suggested an important role for protein translation in necroptosis. Consistently, we found that the small molecule inhibitor of p70S6K PF-4708671 [33] attenuated necroptosis at the concentrations required to block S6 phosphorylation (Fig. S5C, D). Partial siRNA knockdown of S6 protein attenuated necroptosis as well (Fig. S5E), suggesting that translational control by p70S6K/S6 may play a role in necroptosis. Overall, while the full repertoire of Akt targets during necroptosis remains to be fully explored, our data provide evidence that the activity of an anti-apoptotic branch of Akt signaling can promote necroptosis.\nRIP1 kinase, Akt, mTORC1 and JNK control the upregulation of TNFα accompanying necroptosis. Hitomi et al. [10] have recently reported that the induction of necroptosis by zVAD.fmk in L929 cells is associated with increased synthesis of TNFα, which potentiates cell death. Therefore, we examined whether Akt and its effectors contribute to TNFα synthesis. Consistent with a RIP1-dependent increase in TNFα protein (Fig. S6A, B), we found that TNFα mRNA levels increased during necroptosis in L929 cells in a RIP1 (Fig. S6C. Under serum free conditions, bFGF alone triggered some induction of TNFα mRNA, while its combination with zVAD.fmk (but not zVAD.fmk alone) caused a pronounced further increase (Fig. 6A). Conversely, PDGF caused a modest upregulation of TNFα mRNA, which was not further increased in the presence of zVAD.fmk (Fig. 6A), demonstrating that activation of necroptosis is specifically accompanied by a marked increase in autocrine TNFα synthesis.\n10.1371/journal.pone.0056576.g006 Figure 6 Akt and mTORC1 control autocrine TNFα synthesis and JNK activation during necroptosis.\n(A) Cells were treated under serum free conditions with bFGF or PDGF with or without zVAD.fmk for 9 hr, followed by qRT-PCR analysis of mTNFα. Data was normalized to mouse 18S RNA. (B) Necroptosis was induced by zVAD.fmk or TNFα in cells treated with Nec-1, rapamycin (rapa), or Akt inh. VIII inh. followed by qRT-PCR analysis of TNFα mRNA levels. (C-F) L929 cells with siRNA knockdown of Akt isoforms (C,E) or mTOR (D,F) were stimulated with zVAD.fmk or TNFα for 9 hr, followed by qRT-PCR analysis of mTNFα (C,D) or western blot (E,F). In all graphs, average±SD was plotted. Further analysis suggested that both Akt and mTORC1 contribute to the upregulation of TNFα mRNA during necroptosis as both small-molecule inhibition and siRNA knockdown of Akt and mTOR reduced TNFα mRNA levels in necroptotic cells (Fig. 6B,C,D). Notably, RIP1 and Akt inhibitors had no effect on the levels of TNFα mRNA in control cells or in the cells stimulated with bFGF alone (Fig. 6A,B, Fig. S6C), suggesting that these kinases specifically mediate necroptosis-dependent increase in TNFα synthesis."}