TAK1 is required for survival of mouse fibroblasts treated with TRAIL, and does so by NF-kappaB dependent induction of cFLIPL - PubMed (original) (raw)

doi: 10.1371/journal.pone.0008620.

Ulrich Nachbur, Wendy Diane Cook, Ian Edward Gentle, Donia Moujalled, Maryline Moulin, Wendy Wei-Lynn Wong, Nufail Khan, Diep Chau, Bernard Andrew Callus, James Edward Vince, John Silke, David Lawrence Vaux

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TAK1 is required for survival of mouse fibroblasts treated with TRAIL, and does so by NF-kappaB dependent induction of cFLIPL

Josep Maria Lluis et al. PLoS One. 2010.

Erratum in

Abstract

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known as a "death ligand"-a member of the TNF superfamily that binds to receptors bearing death domains. As well as causing apoptosis of certain types of tumor cells, TRAIL can activate both NF-kappaB and JNK signalling pathways. To determine the role of TGF-beta-Activated Kinase-1 (TAK1) in TRAIL signalling, we analyzed the effects of adding TRAIL to mouse embryonic fibroblasts (MEFs) derived from TAK1 conditional knockout mice. TAK1-/- MEFs were significantly more sensitive to killing by TRAIL than wild-type MEFs, and failed to activate NF-kappaB or JNK. Overexpression of IKK2-EE, a constitutive activator of NF-kappaB, protected TAK1-/- MEFs against TRAIL killing, suggesting that TAK1 activation of NF-kappaB is critical for the viability of cells treated with TRAIL. Consistent with this model, TRAIL failed to induce the survival genes cIAP2 and cFlipL in the absence of TAK1, whereas activation of NF-kappaB by IKK2-EE restored the levels of both proteins. Moreover, ectopic expression of cFlipL, but not cIAP2, in TAK1-/- MEFs strongly inhibited TRAIL-induced cell death. These results indicate that cells that survive TRAIL treatment may do so by activation of a TAK1-NF-kappaB pathway that drives expression of cFlipL, and suggest that TAK1 may be a good target for overcoming TRAIL resistance.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. The kinase activity of TAK1 is required for TRAIL survival in MEFs.

(A) Immunoblot analysis of TAK1 levels before and after TAK1flox/flox MEFs were infected with lentivirus expressing Cre protein. (B) Polyclonal populations of wild-type (TAK1flox/flox) and TAK1 knock out (TAK1−/−) MEFs were treated with different concentrations of TRAIL for 48 h. (C,D). WT TAK1 but not mutant TAK1 (K63W, kinase null) complementation of TAK1−/− MEFs protects against TRAIL sensitivity. (C) Time course of TAK1 levels after induction with 10 nM of 4-hydroxytamoxifen (4HT). (D) Cells were treated with TRAIL 1 µg/ml for 48 h and viability was assessed by PI staining and flow cytometry. The mean and SEM of three independent experiments is shown.

Figure 2

Figure 2. TRAIL induced death of TAK1−/− MEFs is mediated by caspase 8.

(A) Time course of viability of TAK1flox/flox and TAK1−/− MEFs exposed to TRAIL 1 µg/ml. (B) Cleavage of caspase 8, 3 and PARP in TRAIL treated WT and TAK1−/− MEFs. (C) Wild-type, TAK1−/−, caspase 8 knock out (Casp8−/−), Bax and Bak double knock out (Bax−/−/Bak−/−) MEFs were coincubated with 30 nM 5Z-7-oxozeanol (an inhibitor of TAK1's catalytic activity) and TRAIL 1 µg/ml for 24 h. The mean and SEM of three independent experiments is shown.

Figure 3

Figure 3. In TAK1−/− MEFs, TRAIL fails to activate p65/RelA NF-κB and JNK signalling pathways.

MEFs were stimulated with 1 µg/ml TRAIL for the indicated times, and cell lysates were probed for (A), phospho p65, p65, and IκB-α to determine NF-κB activation, and (B), phospho c-jun, JNK and b-jun to determine JNK activation. (C) Viability of Nemo/IKKγ knock out MEFs (IKKγ−/−), c-jun knock out MEFs (c-jun−/−) and JNK knock out MEFs (JNK−/−) after TRAIL exposure (1 µg/ml, 48 h). The mean and SEM of three independent experiments is shown.

Figure 4

Figure 4. Activation of NF-κB by over-expression of IKK2-EE induces cFlip and rescues TAK1−/− MEFs from TRAIL induced cell death.

Inducible over-expression of dominant active IKK2-EE in TAK1−/− MEFs was accompanied by an increase in phosphorylation of p65 and IκB-α degradation (A), and blocked sensitivity to TRAIL (1 µg/ml, 48 h) (B). Expression of both cIAP2 and cFlipL genes was elevated as determined by real time RT-PCR, using the housekeeping gene 18S rRNA as internal control (C). Western blot for cFlip and cIAP1 in TAK1−/− MEFs stably infected with IKK2EE (TAK1−/−uasIKK2EE) after addition of 10 nM 4HT for different times (* indicates non-specific band).

Figure 5

Figure 5. c-FlipL over-expression is able to reduce killing of TAK1−/− MEFs by TRAIL, whereas Flipp43 and FlipR have little effect.

(A) Endogenous c-Flip levels after TRAIL treatment in both TAK1−/−, TAK1flox/flox MEFs and (B) TAK1−/− MEFs reconstituted with TAK1 WT or TAK1 (k63W). (C) Lentiviral-mediated inducible expression of the different murine forms of c-Flip (c-FlipL, c-Flipp43, c-FlipR) in TAK1−/− MEFs measured by Western blot. (D) The ability of the different forms of c-Flip to protect TAK1−/− MEFs against killing by TRAIL (1 µg/ml, 48 h) was evaluated by PI exclusion.

Figure 6

Figure 6. Cellular FlipL turnover is not affected by the presence or absence of TAK1, and cIAP2 over-expression in TAK1−/− cells fails to protect against TRAIL killing.

(A) TAK1flox/flox and TAK1−/− MEFs over-expressing cFlipL were incubated with 10 µg/ml cyclohexamide for different times and kinetics of cFlipL degradation was detected by immunoblot. (B,C) TAK1−/− MEFs were stably infected with cIAP2 using a lentiviral Tet-off system. After addition of 1 µg/ml-10 ng/ml doxycycline cIAP2 protein levels were determined by Western blot (B), and cell viability in response to TRAIL was measured by PI exclusion (C).

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