Wendy D. Cook

IAPs limit activation of RIP kinases by TNF receptor 1 during development

Inhibitor of apoptosis (IAP) proteins cIAP1, cIAP2, and XIAP (X‐linked IAP) regulate apoptosis and cytokine receptor signalling, but their overlapping functions make it difficult to distinguish their individual roles. To do so, we deleted the genes for IAPs separately and in combination. While lack of any one of the IAPs produced no overt phenotype in mice, deletion of cIap1 with cIap2 or Xiap resulted in mid‐embryonic lethality. In contrast, Xiap−/−cIap2−/− mice were viable. The death of cIap2−/−cIap1−/− double mutants was rescued to birth by deletion of tumour necrosis factor (TNF) receptor 1, but not TNFR2 genes. Remarkably, hemizygosity for receptor‐interacting protein kinase 1 (Ripk1) allowed Xiap−/−cIap1−/− double mutants to survive past birth, and prolonged cIap2−/−cIap1−/− embryonic survival. Similarly, deletion of Ripk3 was able to rescue the mid‐gestation defect of cIap2−/−cIap1−/− embryos, as these embryos survived to E15.5. cIAPs are therefore required during development to limit activity of RIP kinases in the TNF receptor 1 signalling pathway.

IAPs must limit activation of RIP kinases by TNF Receptor 1 to prevent embryonic lethality

Inhibitor of apoptosis (IAP) proteins cIAP1, cIAP2, and XIAP (X‐linked IAP) regulate apoptosis and cytokine receptor signalling, but their overlapping functions make it difficult to distinguish their individual roles. To do so, we deleted the genes for IAPs separately and in combination. While lack of any one of the IAPs produced no overt phenotype in mice, deletion of cIap1 with cIap2 or Xiap resulted in mid‐embryonic lethality. In contrast, Xiap−/−cIap2−/− mice were viable. The death of cIap2−/−cIap1−/− double mutants was rescued to birth by deletion of tumour necrosis factor (TNF) receptor 1, but not TNFR2 genes. Remarkably, hemizygosity for receptor‐interacting protein kinase 1 (Ripk1) allowed Xiap−/−cIap1−/− double mutants to survive past birth, and prolonged cIap2−/−cIap1−/− embryonic survival. Similarly, deletion of Ripk3 was able to rescue the mid‐gestation defect of cIap2−/−cIap1−/− embryos, as these embryos survived to E15.5. cIAPs are therefore required during development to limit activity of RIP kinases in the TNF receptor 1 signalling pathway.

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

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.

In TNF-stimulated cells, RIPK1 promotes cell survival by stabilizing TRAF2 and cIAP1, which limits induction of non-canonical NF-κB and activation of caspase-8.

RIPK1 is involved in signaling from TNF and TLR family receptors. After receptor ligation, RIPK1 not only modulates activation of both canonical and NIK-dependent NF-κB, but also regulates caspase-8 activation and cell death. Although overexpression of RIPK1 can cause caspase-8-dependent cell death, when RIPK1−/− cells are exposed to TNF and low doses of cycloheximide, they die more readily than wild-type cells, indicating RIPK1 has pro-survival as well as pro-apoptotic activities (1, 2). To determine how RIPK1 promotes cell survival, we compared wild-type and RIPK1−/− cells treated with TNF. Although TRAF2 levels remained constant in TNF-treated wild-type cells, TNF stimulation of RIPK1−/− cells caused TRAF2 and cIAP1 to be rapidly degraded by the proteasome, which led to an increase in NIK levels. This resulted in processing of p100 NF-κB2 to p52, a decrease in levels of cFLIPL, and activation of caspase-8, culminating in cell death. Therefore, the pro-survival effect of RIPK1 is mediated by stabilization of TRAF2 and cIAP1.

RIPK1- and RIPK3-induced cell death mode is determined by target availability.

Both receptor-interacting protein kinase 1 (RIPK1) and RIPK3 can signal cell death following death receptor ligation. To study the requirements for RIPK-triggered cell death in the absence of death receptor signaling, we engineered inducible versions of RIPK1 and RIPK3 that can be activated by dimerization with the antibiotic coumermycin. In the absence of TNF or other death ligands, expression and dimerization of RIPK1 was sufficient to cause cell death by caspase- or RIPK3-dependent mechanisms. Dimerized RIPK3 induced cell death by an MLKL-dependent mechanism but, surprisingly, also induced death mediated by FADD, caspase 8 and RIPK1. Catalytically active RIPK3 kinase domains were essential for MLKL-dependent but not for caspase 8-dependent death. When RIPK1 or RIPK3 proteins were dimerized, the mode of cell death was determined by the availability of downstream molecules such as FADD, caspase 8 and MLKL. These observations imply that rather than a ‘switch’ operating between the two modes of cell death, the final mechanism depends on levels of the respective signaling and effector proteins.

Necroptosis induced by RIPK3 requires MLKL but not Drp1.

Necroptosis is a mechanism by which cells can kill themselves that does not require caspase activity or the presence of the pro-apoptotic Bcl-2 family members Bax or Bak. It has been reported that RIPK3 (receptor interacting protein kinase 3) activates MLKL (mixed lineage kinase domain-like) to cause cell death that requires dynamin-related protein 1 (Drp1), because survival was increased in cells depleted of Drp1 or treated with the Drp1 inhibitor mdivi-1. To analyze necroptosis in a system that does not require addition of tumor necrosis factor (TNF), we used a construct that allows RIPK3 to be induced in cells, and then dimerized via an E. coli gyrase domain fused to its carboxyl-terminus, using the dimeric gyrase binding antibiotic coumermycin. We have previously shown elsewhere that RIPK3 dimerized in this manner not only induces necroptosis but also apoptosis, which can be inhibited by the broad-spectrum caspase inhibitor Q-VD-OPh (QVD). In response to RIPK3 dimerization, wild-type mouse embryonic fibroblasts (MEFs) underwent cell death that was reduced but not completely blocked by QVD. In contrast, death upon dimerization of RIPK3 in Mlkl−/− MEFs was completely inhibited with QVD, confirming that MLKL is required for necroptosis. Similar to wild-type MEFs, most Drp1−/− MEFs died when RIPK3 was activated, even in the presence of QVD. Furthermore, overexpression of wild-type MLKL or dominant active mutants of MLKL (Q343A or S345E/S347E) caused death of wild-type and Drp1−/− MEFs that was not inhibited with QVD. These results indicate that necroptosis caused by RIPK3 requires MLKL but not Drp1.

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

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-κB and JNK signalling pathways. To determine the role of TGF-β-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-κB or JNK. Overexpression of IKK2-EE, a constitutive activator of NF-κB, protected TAK1−/− MEFs against TRAIL killing, suggesting that TAK1 activation of NF-κB 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-κB 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-κB pathway that drives expression of cFlipL, and suggest that TAK1 may be a good target for overcoming TRAIL resistance.

In mouse embryonic fibroblasts, neither caspase-8 nor cellular FLICE-inhibitory protein (FLIP) is necessary for TNF to activate NF-κB, but caspase-8 is required for TNF to cause cell death, and induction of FLIP by NF-κB is required to prevent it

Binding of TNF to TNF receptor-1 can give a pro-survival signal through activation of p65/RelA NF-κB, but also signals cell death. To determine the roles of FLICE-inhibitory protein (FLIP) and caspase-8 in TNF-induced activation of NF-κB and apoptosis, we used mouse embryonic fibroblasts derived from FLIP and caspase-8 gene-deleted mice, and treated them with TNF and a smac-mimetic compound that causes degradation of cellular inhibitor of apoptosis proteins (cIAPs). In cells treated with smac mimetic, TNF and Fas Ligand caused wild-type and FLIP−/− MEFs to die, whereas caspase-8−/− MEFs survived, indicating that caspase-8 is necessary for death of MEFs triggered by these ligands when IAPs are degraded. By contrast, neither caspase-8 nor FLIP was required for TNF to activate p65/RelA NF-κB, because IκB was degraded, p65 translocated to the nucleus, and an NF-κB reporter gene activated normally in caspase-8−/− or FLIP−/− MEFs. Reconstitution of FLIP−/− MEFs with the FLIP isoforms FLIP-L, FLIP-R, or FLIP-p43 protected these cells from dying when treated with TNF or FasL, whether or not cIAPs were depleted. These results show that in MEFs, caspase-8 is necessary for TNF- and FasL-induced death, and FLIP is needed to prevent it, but neither caspase-8 nor FLIP is required for TNF to activate NF-κB.

Erratum: TNF-stimulated cells, RIPK1 promotes cell survival by stabilizing TRAF2 and cIAP1, which limits induction of non-canonical NF-κB and activation of caspase-8 (Journal of Biological Chemistry (2016) 291 (2547))

The figure legend of Fig. 1B incorrectly stated that 25 mg of protein was loaded. The correct amount is 25 g. The description of Fig. 1C incorrectly stated that the concentration of chloroquine used was 200 mM. The correct concentration is 200 M. The description of Fig. 1D applies to Fig. 1E, and the description for Fig. 1D should instead read “wild-type and three different RIPK1 / MEF lines were treated with 100 ng/ml Fc-TNF for 0, 5, and 15 min. Cells were then lysed in DISC lysis buffer and split into soluble and insoluble fractions. 25 g of protein from both fractions were run on SDS-PAGE and analyzed for levels of indicated proteins by Western blotting analysis.” An asterisk denotes a nonspecific band.

Identification of an Xiap-like pseudogene on mouse chromosome 7.

The most thoroughly characterized mammalian IAP is XIAP/BIRC4, which can inhibit caspases 9, 3 and 7, but may also regulate apoptosis through interactions with other proteins such as Smac/DIABLO, HtrA2/Omi, XAF1, TAK1, cIAP1, and cIAP2. High throughput sequencing of the mouse genome revealed the existence of a gene resembling Xiap/Birc4 on mouse chromosome 7. To confirm the existence of this gene, and to determine its functional significance, we performed Southern and Northern blot analysis. This showed the presence of the Xiap-like gene in both wild-type and Xiap gene knock-out mice, but the corresponding mRNA was not detected in any tissues examined by Northern blot. Analysis of the gene sequence in all three possible reading frames predicts that expression of this gene would not give rise to a full-length protein, but only non-functional truncated polypeptides. Because its nucleotide sequence is 92% identical to Xiap, but it has no introns corresponding to those of Xiap, we conclude that Xiap-ps1 is a pseudogene generated by retro-transposition of a spliced Xiap message to chromosome 7.