TNF can activate RIPK3 and cause programmed necrosis in the absence of RIPK1

Abstract

Ligation of tumor necrosis factor receptor 1 (TNFR1) can cause cell death by caspase 8 or receptor-interacting protein kinase 1 (RIPK1)- and RIPK3-dependent mechanisms. It has been assumed that because RIPK1 bears a death domain (DD), but RIPK3 does not, RIPK1 is necessary for recruitment of RIPK3 into signaling and death-inducing complexes. To test this assumption, we expressed elevated levels of RIPK3 in murine embryonic fibroblasts (MEFs) from wild-type (WT) and gene-deleted mice, and exposed them to TNF. Neither treatment with TNF nor overexpression of RIPK3 alone caused MEFs to die, but when levels of RIPK3 were increased, addition of TNF killed WT, Ripk1(-/-), caspase 8(-/-), and Bax(-/-)/Bak(-/-) MEFs, even in the presence of the broad-spectrum caspase inhibitor Q-VD-OPh. In contrast, Tnfr1(-/-) and Tradd(-/-) MEFs did not die. These results show for the first time that in the absence of RIPK1, TNF can activate RIPK3 to induce cell death both by a caspase 8-dependent mechanism and by a separate Bax/Bak- and caspase-independent mechanism. RIPK1 is therefore not essential for TNF to activate RIPK3 to induce necroptosis nor for the formation of a functional ripoptosome/necrosome.

Figure 1

TNF causes death of cells with elevated RIPK3. (a) WT MEFs were infected with a lentiviral vector-expressing FLAG RIPK3. Cells were induced with 10 nM 4HT for 24 h, and lysates were probed for FLAG. β-Actin was used as a loading control. *, indicates a nonspecific band. (b) Expression of FLAG-RIPK3 in MEFs was induced with 10 nM 4HT for 24 h (lower panels), and cells were then treated with 100 ng/ml TNF or TNF plus 500 nM smac-mimetic for a further 24 h. Cells were stained with PI and analyzed by flow cytometry to detect loss of plasma membrane integrity

Figure 2

TNFR1 and TRADD are necessary for TNF to cause death of MEFs treated with smac-mimetic or with elevated RIPK3. FLAG-RIPK3 was induced in WT, Tnfr1^−/−, Tnfr2^−/−, and Tradd^−/− MEFs with 10 nM 4HT for 24 h. Cells were then treated with 100 ng/ml TNF or TNF plus 500 nM smac-mimetic for a further 24 h, stained with PI, and analyzed by flow cytometry. Columns show mean±S.E.M., where n=3 independently performed experiments

Figure 3

Both caspase 8 and RIPK1 are required for TNF plus smac-mimetic to cause cell death, but when levels of RIPK3 are elevated, TNF alone can induce cell death in the absence of caspase 8 or RIPK1. (a) MEF lines were untreated (white bars) or treated with 100 ng/ml TNF plus 500 nM smac-mimetic (black bars) for 24 h. Cell viability was determined by PI exclusion using a flow cytometer. Columns show mean±S.E.M., where n=3 independently performed experiments. (b) WT, caspase 8^−/−, and Ripk1^−/− MEFs bearing the inducible FLAG RIPK3 vector were untreated or treated with 10 nM 4HT for 24 h, and lysates were probed with antibodies to reveal levels of endogenous or induced FLAG-tagged proteins. (c) Cell lines were induced with 10 nM 4HT for 24 h, and then treated, where indicated, with 100 ng/ml TNF and 500 nM smac-mimetic (SM) for a further 24 h, and viability determined by flow cytometric analysis of PI exclusion. Columns show mean±S.E.M., where n=3 independently performed experiments. (d) Cell lines were induced with 10 nM 4HT for 24 h and cultured with 100 ng/ml TNF alone or TNF plus 500 nM smac-mimetic for 48 h. Cells were resuspended using trypsin, re-plated, and after 5 days stained with crystal violet to assess clonogenicity

Figure 4

Death induced by TNF and activated RIPK3 is independent of proapoptotic Bcl-2 family proteins Bax and Bak. WT and Bax^−/−/Bak^−/− MEFs bearing the inducible FLAG-RIPK3 vector were induced with 10 nM 4HT for 24 h. Where indicated, cells were pretreated with 10 μM Q-VD-OPh for 1 h and subsequently treated with 100 ng/ml TNF or TNF plus 500 nM smac-mimetic (SM) for 24 h. Cells were stained with PI and cell viability determined by flow cytometry. Columns show mean±S.E.M., where n=3 independently performed experiments

Figure 5

TNF treatment of cells overexpressing RIPK3 activates caspases, but this is not necessary for cell death. (a) WT and Ripk1^−/− MEFs were induced with 10 nM 4HT for 24 h and treated with 100 ng/ml TNF. After 4 h, cells were harvested, lysed, and analyzed by western blotting, and probed for poly (ADP-ribose) polymerase (PARP) and both full-length and cleaved caspase 8. β-Actin was used as a loading control. (b, c) WT (caspase 8^lox/lox), caspase 8^−/−, and Ripk1^−/− MEFs in duplicate wells were induced with 10 nM 4HT for 24 h and treated with 100 ng/ml TNF. (b) After 4 h, cells from one set of wells were harvested, lysed, and analyzed by western blot probed for PARP and cleaved caspase 3. (c) After 4 h in culture, the cells in the second set of wells were re-suspended, stained with 100 ng/ml PI, and cell viability determined by flow cytometry. Columns show mean±S.E.M., where n=3 independently performed experiments

Figure 6

Elevated levels of MLKL in Fadd^−/− MEFs allows cell death by TNF-induced activation of RIPK3. (a) Fadd^−/− MEFs were infected with a doxycycline (dox)-inducible MLKL expression vector. Cells were treated with 1 μg/ml of dox for 24 h. Cell lysates were harvested and probed for MLKL as indicated and β-actin was used as a loading control. (b) Fadd^−/− MEFs bearing vectors for dox-inducible MLKL and 4HT-inducible RIPK3 were treated with 1 μg/ml dox, 10 nM 4HT, and 100 ng/ml TNF for 24 h. Cells were re-suspended using trypsin, stained with 100 ng/ml PI, and analyzed by flow cytometry. (c) WT MEFs bearing a 4HT-inducible RIPK3 vector and Fadd^−/− MEFs bearing vectors for 4HT-inducible RIPK3 and dox-inducible MLKL were treated with 1 μg/ml dox and 10 nM 4HT for 24 h and treated with 100 ng/ml TNF for 4 h. Cell lysates were harvested and probed for caspase 8 (full-length), cleaved caspase 8 and MLKL as indicated, and β-actin was used as a loading control

Figure 7

Model for activation of caspase-dependent and -independent cell death mechanisms by TNF. Under normal conditions, TNF treatment of WT MEFs does not induce cell death. When IAPs are depleted with smac-mimetic, TNF can activate caspase 8 by a pathway requiring RIPK1 to cause cell death that can be blocked by the caspase inhibitor Q-VD-OPh. When levels of RIPK3 are elevated, TNF not only activates caspase 8 but also activates a caspase-independent death mechanism, but it does not require RIPK1 for either