PMC:3585731 / 34069-37013 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3585731","sourcedb":"PMC","sourceid":"3585731","source_url":"http://www.ncbi.nlm.nih.gov/pmc/3585731","text":"Akt Controls TNFα Production in Other Cell Types\nAfter establishing the role of RIP1 kinase-dependent signaling to Akt in L929 cells, we sought to expand our study to other cell types that are known to undergo necroptotic cell death. Fas-associated protein with death domain (FADD)-deficient Jurkat T lymphocytes and the macrophage cell lines (J774A.1 and RAW264.7) are other models of necroptosis, which can be induced by stimulation with TNFα or zVAD.fmk, respectively [17]. Similar to L929 cells, a RIP1 kinase dependent increase in the phosphorylation of Thr308 on Akt occurred during necroptosis (Fig. 8B,D,F) in these cell types. Furthermore, TNFα mRNA levels were increased in each of these cell types during necroptosis and efficiently inhibited by both RIP1 and Akt inhibitors (Fig. 8A,C,E). However, inhibition of Akt did not protect these cells from death (Fig. S9A,B,C). These results indicate that regulation of autocrine TNFα synthesis and necroptosis-associated inflammatory signaling may be a more important function of Akt pathway activation by RIP1 kinase in multiple cell types compared to its contribution to cell death.\n10.1371/journal.pone.0056576.g008 Figure 8 Akt signaling contributes to autocrine TNFα production in multiple cell types.\nFADD deficient Jurkat cells were treated with TNFα followed by measurement of (A) human TNFα mRNA levels by qRT-PCR and normalized using human 18S RNA or (B) western blot at 9 hr. RAW 264.7 or J774A.1 cells were treated with zVAD.fmk (100 uM or 50 uM respectively) followed by (C,E) measurement of TNFα mRNA levels by qRT-PCR or (D,F) western blot at 9 hr. (G) Akt null mouse lung fibroblasts expressing Myr-Akt or K179M were treated with zVAD.fmk and TNFα followed by measurement of TNFα mRNA levels by qRT-PCR at 9 hr. (H) Mouse lung fibroblasts expressing only endogenous Akt1 or Akt2 were treated with zVAD.fmk and TNFα followed by measurement of TNFα mRNA levels by qRT-PCR at 9 hr. We next chose to look at the role of Akt in necroptosis in mouse lung fibroblasts. Lung fibroblasts selected to survive after deletion of all three Akt isoforms [40] were resistant to cell death induced by the addition of TNFα and zVAD.fmk. Expression of catalytically active Akt (Myr-Akt) in these cells restored TNFα mRNA production in response to TNFα and zVAD.fmk (Fig. 8G) without re-establishing cell death (Fig. S9D). Consistent with our earlier Akt knockdown data, lung fibroblasts expressing endogenous Akt1 or Akt2 were phosphorylated on Thr308 in response to TNFα and zVAD.fmk (Fig. S9E) and in both cases robust RIP1-dependent TNFα mRNA upregulation occurred under necroptotic conditions (Fig. 8H). These data further support the notion that Akt activity is critical for autocrine TNFα synthesis, even in the absence of necroptotic cell death, indicating an unexpected differentiation between Akt-mediated inflammatory signaling under necroptotic conditions and cell death per se.","divisions":[{"label":"title","span":{"begin":0,"end":48}},{"label":"p","span":{"begin":49,"end":1140}},{"label":"figure 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