Caspase-8 (Casp8) suppresses receptor-interacting protein kinase-3 (RIPK3)/mixed lineage kinase domain-like protein (MLKL)-dependent necroptosis, demonstrated by the genetic evidence that deletion of Ripk3 or Mlkl prevented embryonic lethality of Casp8-deficient mice. However, the detailed mechanisms by which Casp8 deficiency triggers necroptosis during embryonic development remain unclear. In this article, we show that Casp8 deletion caused formation of the RIPK1-RIPK3 necrosome in the yolk sac, leading to vascularization defects, prevented by MLKL and RIPK3 deficiency, or RIPK3 RHIM mutant (RIPK3 V448P), but not by the RIPK1 kinase-dead mutant (RIPK1 K45A). In addition, Ripk1K45A/K45ACasp8−/− mice died on embryonic day 14.5, which was delayed to embryonic day 17.5 by ablation of one allele in Ripk1 and was completely rescued by ablation of Mlkl. Our results revealed an in vivo role of RIPK3 RHIM and RIPK1K45A scaffold-mediated necroptosis in Casp8 deficiency embryonic development and suggested that the Casp8-deficient yolk sac might be implicated in identifying novel regulators as an in vivo necroptotic model.

Necroptosis is a form of programmed cell death that has been implicated in embryonic development, tissue homeostasis, immunity, and inflammation (13). Necroptotic cell death in murine L929 fibrosarcoma (4) or human primary T cells (5) induced by TNF-α depends on receptor-interacting protein kinase-1 (RIPK1) kinase activation, which can be blocked by an inhibitor such as necrostatin-1 (Nec-1) or kinase-dead mutant (Ripk1K45A or Ripk1D138N) (612). When caspase-8 (Casp8) is absent or its catalytic activity is blocked by inhibitors, RIPK1 is in its kinase-active form to recruit RIPK3 (1316) and phosphorylate the mixed lineage kinase domain-like protein (MLKL) (17, 18). The RIP homotypic interaction motif (RHIM) mediates the RIPK1-RIPK3 heterointeraction to form necrosomes, leading to MLKL phosphorylation and translocation to the plasma membrane, which can trigger membrane leakage (1923). Thus, the kinase activity of RIPK1 plays a central role in necroptosis.

Casp8 not only inhibits necroptosis triggered by the death receptor in vitro but also blocks in vivo necroptosis through its catalytic activity. Although the absence of Casp8 leads to embryonic lethality, mice may survive past weaning if either of the necroptosis-mediating genes, Ripk3 or Mlkl, is coablated (2427). Mutation in the catalytic site of Casp8 (C362S or C362A), similar to disruption of Casp8, leads to embryonic lethality (28, 29), whereas mice harboring oligomerization-deficient (F122G and L123G) and noncleavable mutants (D387A or D212A/D218A/D225A/D387A) are viable (28, 3032). In the specific absence of catalytic activity of Casp8 or the Casp8 scaffold in endothelial cells, Casp8fl/flTie1cre, Casp8C362S/flTie2cre, and Casp8fl/flTie2cre embryos showed a similar gross pathology associated with a defect in yolk sac vascularization, which causes embryonic lethality at the same developmental stage as Casp8 deficiency (28, 33). However, the mechanisms of necroptosis in yolk sac triggered by Casp8 deficiency during embryogenesis remain to be established.

In this study, we generated Casp8 null mutation mice and showed embryonic lethality of Casp8-deficient mice by inducing RIPK1-RIPK3–mediated necroptosis in yolk sac vascularization, which is prevented by coablation of Ripk3, Mlkl, or RIPK3 RHIM mutant (RIPK3 V448P), but not by the RIPK1 kinase–dead mutant (RIPK1 K45A), indicating that Casp8 deficiency triggers RIPK1 kinase–independent necroptosis during embryonic development. Furthermore, the death of Ripk1K45A/K45ACasp8−/− on embryonic day 14.5 (E14.5) was delayed to E17.5 by ablation of one allele in Ripk1 or was fully rescued by codeletion of Mlkl, suggesting that RIPK1 scaffold-dependent necrosome formation in yolk sac mediated by the RHIM interaction of RIPK1-RIPK3 is a mechanism of embryonic lethality in Casp8-deficient mice.

Casp8+/− knockin mice were generated by mutating “TT” to “A” in exon 8 of mouse Casp8 locus via the CRISPR/Cas9 system (Bioray Laboratories, Shanghai, China). The Casp8+/− mice were backcrossed with C57BL/6J for eight generations. Genotyping of Casp8+/− mice was conducted with mouse-tail DNAs by PCR (95°C, 4 min; 95°C, 30 s; 58°C, 30 s; 72°C, 30 s; 72°C, 5 min; 35 cycles) and confirmed by sequencing analysis. The PCR primers used for genotyping were the following: forward primer 5′-CAGAGGCTCTGAGTAAGACC-3′; reverse primer 5′-CTGAGGACATCTTTCCCTCAG-3′; and sequencing primer 5′-CAGAGGCTCTGAGTAAGACC-3′. Mlkl−/−, Ripk1+/−, Ripk1K45A/K45A, Ripk3−/−, and Ripk3V448P/V448P mice have been previously described and maintained on C57BL/6 genetic background (9, 14, 34, 35). All mice were housed and cared for in a specific pathogen-free environment. Animal experiments were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of the Institute of Nutrition and Health, Shanghai Institutes for Biological Sciences, University of Chinese Academy of Sciences.

The primary Abs used were PARP1 (catalog number [Cat#] 9542s; CST), Casp3 (Cat#9662S; CST), Casp8 (Cat#8592S; CST), RIP1 (Cat#610459; BD), P-RIP1 (Ser166) (Cat#31122S; CST), RIP3 (Cat#ab72106; Abcam), P-RIP3 (Ser232) (Cat#ab195117; Abcam), MLKL (Cat#AP14272b; Abgent), P-MLKL (Ser345) (Cat#ab196436; Abcam), GAPDH (Cat#G9545; Sigma), Z-DNA–binding protein 1 (ZBP1; Cat#AG-20B-0010; AdipoGen), CD3-FITC (Cat#11-0031-82; eBioscience), and B220-allophycocyanin (Cat#17-0452-83; eBioscience). The compounds used were Z-VAD-FMK (Cat#HY-16658; MCE), Nec-1 (Cat#HY-15760; MCE), GSK’872 (Cat#HY-101872; MCE), and Mouse TNF-α (Cat#410-MT-050; R&D).

Mouse embryonic fibroblast (MEF) cells were maintained in high-glucose DMEM (Cat#SH30243; Hyclone) supplemented with 10% FBS (Cat#04-001-1A; Bioind) and 100 U penicillin/streptomycin (Cat#15140122; Life Technologies). Cells were maintained at 37°C and 5% CO2. Ripk3−/−Casp8−/−, Ripk3V448P/V448PCasp8−/−, Mlkl−/−Casp8−/−, and Ripk1K45A/K45ACasp8−/− MEFs were isolated from E12.5 embryos. Casp8−/− MEFs were isolated from E10.5 embryos, head and visceral tissues were dissected, and remaining bodies were incubated with 4 ml trypsin/EDTA solution (Life Technologies) per embryo at 37°C for 1 h. After trypsinization, an equal amount of medium was mixed and pipetted up and down a few times cultured in DMEM medium (10% FBS, 1% penicillin/streptomycin) and transformed with an SV40 large T Ag-expressing lentivirus.

Male and female mice at 8 wk old were crossed in one box; mice were designated E0.5 on the morning a vaginal plug was detected. Embryos ranging in age from E9.5 to E17.5 were analyzed. Yolk sacs were washed multiple times with PBS and then Nonidet P-40 (NP-40) buffer lysis for 30 min at 4°C. For Casp8 expression test, ∼E10.5–E12.5 embryos were harvested in 6 M urea lysis buffer (6 M urea, 20 mM Tris–HCl [pH 7.5], 135 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 1% Triton X-100) supplemented with 1 mM PMSF and 1× protease inhibitor mixture (Cat#4693132001; Roche). Lysates were centrifuged at 16,000 × g for 30 min at 4°C, and supernatants were diluted with 4× SDS-PAGE sample loading buffer (240 mM Tris–HCl [pH 6.8], 40% [v/v] glycerol, 8% [v/v] SDS, 0.04% bromophenol blue, and 5% [v/v] 2-ME).

E11.5 or E15.5 embryos were fixed in 4% paraformaldehyde; paraffin-embedded tissue sections were stained with 0.05 μg/ml anti-cleaved Casp3 Ab (Cell Signaling Technology). TUNEL staining was performed using the Roche Kit with Proteinase K digestion. Stained slides were digitized using a Nanozoomer digital slide scanner.

Embryo tissue, yolk sac, and MEF cells were lysed with NP-40 buffer (120 mM NaCl, 10 mM Tris–HCl [pH 7.4], 1 mM EDTA, 0.2% NP-40, 10% glycerol) supplemented with 1 mM PMSF and 1× protease inhibitor mixture (Cat#4693132001; Roche). For immunoprecipitation (IP) of RIPK1-RIPK3–associated complexes, the lysate was incubated with anti-RIPK1 overnight at 4°C. The immunocomplex was captured by protein A/G agarose (Cat#16-125; Millipore). Beads were washed four times, and the immunocomplex was eluted from beads by loading buffer. Immune complexes were eluted by boiling in reducing Western blot loading buffer and resolved by Western blot using the Abs described.

Lymphocytes were isolated from the spleen, lymph nodes, and blood of mice. Abs against mouse CD3 (Cat#11-0031-82; eBioscience) and B220 (Cat#17-0452-83; eBioscience) were fluorescence conjugated and used for flow cytometry analysis. Single-cell suspensions of lymphocytes were stained on ice for half an hour with fluorescence-conjugated Abs in the staining buffer. After staining, cells were immediately analyzed by flow cytometry (FACSAria III; BD Biosciences).

Data in this study are representative results of at least three independent experiments. The in vitro results were presented as the mean ± SD of triplicate wells. The statistical significance of data was evaluated by Student t test in which p < 0.01 was considered significant and p < 0.001 was highly significant. The statistical calculations were performed with GraphPad Prism software.

Both tie-1-Cre– and tie-2-Cre–specific deletion of Casp8 in endothelial cells caused embryonic lethality at around E11.5, coinciding with formation of an abnormal yolk sac vasculature, which resembles the phenotype of Casp8−/− embryos (28, 33). When the necroptosis-mediating gene Ripk3 or Mlkl was coablated, Ripk3−/−Casp8−/− or Mlkl−/−Casp8−/− mice survived past weaning (2426). To determine the mechanism of necroptosis in vivo, we generated a Casp8 null allele by replacing AA with T in exon 8 using the CRISPR-Cas9 system (Supplemental Fig. 1A, 1B). Consistent with previous reports, we crossed Casp8+/− mice to generate Casp8−/− mutant animals and found that Casp8−/− mice died during embryogenesis, because intercrossing of heterozygous mice generated only Casp8+/+ and Casp8+/− offspring (Supplemental Fig. 1C). We confirmed the absence of Casp8 protein using E10.5, E11.5, and E12.5 embryo blots with anti-Casp8, which revealed loss of Casp8 expression in Casp8−/− deletion embryos (Supplemental Fig. 1D).

Casp8−/− mice died at around E11.5 because of hyperemia in the abdominal areas and an abnormal yolk sac vasculature (Fig. 1A). Immunoblotting of cleaved PARP1 and cleaved Casp3, markers of apoptosis, revealed significantly increased levels of apoptosis in Casp8−/− embryos (Fig. 1C, Supplemental Fig. 1D). Immunolabeling of cleaved Casp3 also suggested that apoptosis occurred in the abdominal areas of the Casp8−/− embryo (Fig. 1B). In addition, the Casp8−/− yolk sac did not show increased apoptosis with detectable levels of markers of apoptosis (data not shown). RIPK1 or RIPK3 phosphorylation and MLKL phosphorylation are hallmarks of necroptosis. Notably, the Casp8−/− yolk sac exhibited RIPK1 or RIPK3 phosphorylation as detected by an upshift in the anti-RIPK3 blotting band and MLKL phosphorylation (Supplemental Fig. 2G), coinciding with vascular defects (Fig. 1D). To confirm necroptosis occurred in the yolk sac, we lysed the yolk sac of individual embryos. Immunoblot analysis of RIPK1 immunoprecipitates revealed that RIPK3 strongly interacted with RIPK1 in the Casp8−/− yolk sac (Fig. 1E), indicating that necrosome formation through RIPK1-RIPK3 interaction was involved in triggering necroptosis in the Casp8−/− yolk sac. Thus, these data demonstrated that RIPK1-RIPK3-MLKL signaling in the yolk sac drove the embryonic lethality of Casp8−/− mice.

FIGURE 1.

Casp8 prevents yolk sacs necroptosis during mouse embryogenesis.

(A) E11.5 yolk sacs and embryos representative of WT (n = 7) and Casp8−/− (n = 5). Arrows denote sites of hemorrhage. (B) Hematoxylin and eosin (H&E)-stained section of E11.5 embryos, representative of three embryos per genotype. E11.5 embryos stained with anti-cleaved Casp3 (green, C-Casp3) Ab and DAPI (blue) for nuclei. Images with disrupted fetal liver are shown. Scale bars, 200 μm (top); 50 μm (bottom). Images are representative of two embryos per genotype. (C) Western blots of E11.5 embryos (Casp8+/+, Casp8+/−, Casp8−/−). GAPDH was used as a loading control. Lanes, individual mice. Results are representative of three independent experiments. (D) Western blots of E11.5 yolk sacs (Casp8+/+, Casp8+/−, Casp8−/−). GAPDH was used as a loading control. Lanes, individual mice. Results are representative of three independent experiments. (E) Western blots of E11.5 yolk sac lysates from mice of the indicated genotypes (Casp8+/+, Casp8+/−, Casp8−/−) before (Input) and after IP with anti-RIPK1 Ab. GAPDH was used as a loading control. Lanes, individual mice. Results are representative of three independent experiments.

FIGURE 1.

Casp8 prevents yolk sacs necroptosis during mouse embryogenesis.

(A) E11.5 yolk sacs and embryos representative of WT (n = 7) and Casp8−/− (n = 5). Arrows denote sites of hemorrhage. (B) Hematoxylin and eosin (H&E)-stained section of E11.5 embryos, representative of three embryos per genotype. E11.5 embryos stained with anti-cleaved Casp3 (green, C-Casp3) Ab and DAPI (blue) for nuclei. Images with disrupted fetal liver are shown. Scale bars, 200 μm (top); 50 μm (bottom). Images are representative of two embryos per genotype. (C) Western blots of E11.5 embryos (Casp8+/+, Casp8+/−, Casp8−/−). GAPDH was used as a loading control. Lanes, individual mice. Results are representative of three independent experiments. (D) Western blots of E11.5 yolk sacs (Casp8+/+, Casp8+/−, Casp8−/−). GAPDH was used as a loading control. Lanes, individual mice. Results are representative of three independent experiments. (E) Western blots of E11.5 yolk sac lysates from mice of the indicated genotypes (Casp8+/+, Casp8+/−, Casp8−/−) before (Input) and after IP with anti-RIPK1 Ab. GAPDH was used as a loading control. Lanes, individual mice. Results are representative of three independent experiments.

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Next, we examined necroptotic signaling in primary MEFs by comparing Casp8+/+, Casp8+/−, and Casp8−/− MEFs treated with TNF-α plus Smac mimic and the pan-caspase inhibitor Z-VAD-FMK. Casp8−/− MEFs exhibited RIPK1 and RIPK3 phosphorylation in medium alone, whereas the level of RIPK3 and MLKL proteins decreased compared with Casp8+/+ or Casp8+/− MEFs (Fig. 2A), suggesting that some cells spontaneously died because of the loss of Casp8. Casp8+/− MEFs were more sensitive than Casp8+/+ MEFs to TNF-α plus Smac mimic and the pan-caspase inhibitor Z-VAD-FMK treatment, whereas phosphorylation of RIPK3 and MLKL was increased (Fig. 2A). Embryonic lethality of Casp8−/− mice was notably prevented by either Ripk3 (Fig. 2B, Supplemental Fig. 2A, 2B) or Mlkl (Fig. 2B, Supplemental Fig. 2C, 2D) deletion, suggesting that increased apoptosis in Casp8−/− embryos was a secondary consequence and drove the pathology. Notably, Casp8−/− mice expressing RIPK3 with an RHIM mutation (Ripk3V448P/V448PCasp8−/−) also survived beyond weaning (Fig. 2B, Supplemental Fig. 2E, 2F). Ripk3−/−Casp8−/−, Ripk3V448P/V448PCasp8−/− (Supplemental Fig. 3A, 3D), and Mlkl−/−Casp8−/− (Supplemental Fig. 4A–E) mice were viable but developed lymphadenopathy and splenomegaly similar to Ripk3−/−Fadd−/−, Ripk3V448P/V448PFadd−/−, and Mlkl−/−Fadd−/− mice (24, 34, 35). The data show that necrosomes formation is the primary driver of necroptosis in Casp8−/− mice because RHIM of RIPK3 is critical for RIPK1-RIPK3–mediated necrosome formation.

FIGURE 2.

Caspase-8 prevents the formation of the RIPK1-RIPK3 complex that was dependent on RIPK3 RHIM dependent but not RIPK1 kinase activity.

(A) Western blots of Casp8+/+, Casp8+/−, and Casp8−/− immortalized MEFs treated with 1 μM Smac mimic, 20 ng/ml TNF-α plus 20 μM zVAD for 6 h (+TSZ) or 0 h (−TSZ) as indicated. Results are representative of three independent experiments. (B) E12.5 yolk sacs and embryos representative of WT (n = 2), Casp8−/− (n = 3), Ripk3−/−Casp8−/− (n = 6), Mlkl−/−Casp8−/− (n = 2), and Ripk3v448p/v448pCasp8−/− (n = 2). Diagram depicting the extent of viability of different strains of Casp8−/− mice was shown below. (C) Western blots of Casp8+/+, Casp8−/−, Ripk3−/−Casp8−/−, Mlkl−/−Casp8−/−, and Ripk3v448p/v448pCasp8−/− immortalized MEFs after 24-h Nec-1 treatment. Results are representative of three independent experiments. Nec-1, 30 μM (−, untreated; +, treated). (D) Western blots of Casp8+/+, Ripk3−/−, and Mlkl−/−Casp8−/− immortalized MEFs after 24-h treatment. Nec-1 (iRIP1), 30 μM. GSK’872 (iRIP3), 10 nM (−, untreated). Results are representative of three independent experiments. (E) Western blots of Mlkl−/−Casp8−/− immortalized MEFs lysates before (Input) and after IP with anti-RIPK1 Ab. GAPDH was used as a loading control. Results are representative of three independent experiments.

FIGURE 2.

Caspase-8 prevents the formation of the RIPK1-RIPK3 complex that was dependent on RIPK3 RHIM dependent but not RIPK1 kinase activity.

(A) Western blots of Casp8+/+, Casp8+/−, and Casp8−/− immortalized MEFs treated with 1 μM Smac mimic, 20 ng/ml TNF-α plus 20 μM zVAD for 6 h (+TSZ) or 0 h (−TSZ) as indicated. Results are representative of three independent experiments. (B) E12.5 yolk sacs and embryos representative of WT (n = 2), Casp8−/− (n = 3), Ripk3−/−Casp8−/− (n = 6), Mlkl−/−Casp8−/− (n = 2), and Ripk3v448p/v448pCasp8−/− (n = 2). Diagram depicting the extent of viability of different strains of Casp8−/− mice was shown below. (C) Western blots of Casp8+/+, Casp8−/−, Ripk3−/−Casp8−/−, Mlkl−/−Casp8−/−, and Ripk3v448p/v448pCasp8−/− immortalized MEFs after 24-h Nec-1 treatment. Results are representative of three independent experiments. Nec-1, 30 μM (−, untreated; +, treated). (D) Western blots of Casp8+/+, Ripk3−/−, and Mlkl−/−Casp8−/− immortalized MEFs after 24-h treatment. Nec-1 (iRIP1), 30 μM. GSK’872 (iRIP3), 10 nM (−, untreated). Results are representative of three independent experiments. (E) Western blots of Mlkl−/−Casp8−/− immortalized MEFs lysates before (Input) and after IP with anti-RIPK1 Ab. GAPDH was used as a loading control. Results are representative of three independent experiments.

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Next, we investigated the effects of RIPK kinase activity on necroptotic signaling in primary Ripk3−/−Casp8−/−, Ripk3V448P/V448PCasp8−/−, and Mlkl−/−Casp8−/− MEFs in the presence or absence of the RIPK1 kinase inhibitor Nec-1. Although RIPK3 deficiency or RHIM mutation compromised RIPK1 phosphorylation, it occurred normally in Mlkl−/−Casp8−/− MEFs (Fig. 2C), suggesting that RIPK1 and RIPK3 phosphorylation in Casp8−/− MEFs does not require MLKL. Mlkl−/−Casp8−/− MEFs treated with Nec-1 also maintained normal levels of RIPK3 phosphorylation, whereas RIPK1 phosphorylation was blocked (Fig. 2C). We examined RIPK1 phosphorylation in Mlkl−/−Casp8−/− MEFs treated with the RIPK3 kinase inhibitor GSK’872 (36), which revealed that RIPK1 phosphorylation occurred after GSK’872 treatment (Fig. 2D). Immunoblot analysis of RIPK1 immunoprecipitates revealed that RIPK3 strongly interacted with RIPK1 in Mlkl−/−Casp8−/− MEFs, and GSK’872, but not Nec-1, treatment reduced the RIPK1-RIPK3 interaction (Fig. 2E). Thus, Casp8 deficiency can trigger the RIPK1 kinase–independent interaction of RIPK1 with RIPK3 to form necrosomes in vitro. Notably, considering the mechanisms here that Casp8 deficiency can trigger the kinase activity–independent interaction between RIPK1 and RIPK3 to form necrosomes was revealed in MEFs, we agree that these mechanisms might not be applied to the cases in embryos. Therefore, we need to further investigate the contribution of RIPK1 kinase activity–independent function on Casp8−/− mice by utility of more in vivo mouse models.

Because RIPK1 kinase activity has a critical role in regulating necroptosis in vitro and in vivo, we examined whether RIPK1 kinase activity contributes to necroptosis in Casp8−/− mice. To examine RIPK1 catalytic activity, autophosphorylation site Ab (anti–p-S166-RIPK1, p-RIPK1) was blotted in wild-type (WT), Ripk3−/−, Mlkl−/−Casp8−/−, and Ripk1K45A/K45ACasp8−/− immortalized MEFs. We found that RIPK1 autophosphorylation (S166) was induced in Mlkl−/−Casp8−/− MEFs, which was caused by the deficiency of Casp8, while p–S166-RIPK1 was completely blocked by the RIPK1 K45A mutation in Ripk1K45A/K45ACasp8−/− MEFs (Supplemental Fig. 4F). These results confirmed that the K45A mutation indeed impaired RIPK1 kinase activity, which is further consistent with previous reports from us and others (6, 9, 37). We generated Ripk1K45A/K45ACasp8−/− embryos by Ripk1K45A/K45ACasp8+/− intercrossing. Notably, Ripk1K45A/K45ACasp8−/− mice died during embryogenesis as intercrossing of heterozygous mice produced only Ripk1K45A/K45ACasp8+/+ and Ripk1K45A/K45ACasp8+/− offspring (Fig. 3A). RIPK1 kinase inactive (K45A) resulted in delayed lethality in Casp8−/− embryos only at around E14.5 with an abnormal yolk sac vasculature (Fig. 3B). This was similar to the effects of inactive RIPK1 (D138N) causing delayed embryo lethality of Casp8−/− mice (31). TUNEL was performed to detect cell death in the skin and fetal liver of E15.5 embryos. The results showed that massive cell death persisted in Ripk1K45A/K45ACasp8−/− embryos; however, this trend was not consistent in control embryos at E15.5 (Fig. 3C) and suggests that RIPK1 kinase activity is independent of embryonic lethality of Casp8−/− mice.

FIGURE 3.

Kinase-dead mutant RIPK1K45A cannot rescue Casp8−/− lethality.

(A) Observed numbers of offspring from Ripk1K45A/K45ACasp8+/− intercrosses and numbers expected from Mendelian ratios at the indicated stage of development. (B) E11.5 yolk sacs and embryos representative of Ripk1K45A/K45ACasp8+/+ (n = 1), Ripk1K45A/K45ACasp8+/− (n = 5), and Ripk1K45A/K45ACasp8−/− (n = 3); E12.5 yolk sacs and embryos representative of Ripk1K45A/K45ACasp8+/+ (n = 2), Ripk1K45A/K45ACasp8+/− (n = 9), and Ripk1K45A/K45ACasp8−/− (n = 4); E13.5 yolk sacs and embryos representative of Ripk1K45A/K45ACasp8+/+(n = 1), Ripk1K45A/K45ACasp8+/− (n = 4), and Ripk1K45A/K45ACasp8−/− (n = 3); E14.5 yolk sacs and embryos representative of Ripk1K45A/K45ACasp8+/+ (n = 4), Ripk1K45A/K45ACasp8+/− (n = 13), and Ripk1K45A/K45ACasp8−/− (n = 4); E15.5 yolk sacs and embryos representative of Ripk1K45A/K45ACasp8+/+ (n = 3), Ripk1K45A/K45ACasp8+/− (n = 4), and Ripk1K45A/K45ACasp8−/− (n = 2). (C) Hematoxylin and eosin (H&E)-stained section of E15.5 embryos, representative of three embryos per genotype. Scale bars, 200 μm. E11.5 embryos stained with TUNEL (green) and DAPI (blue) for nuclei. Images with disrupted spin and fetal liver are shown. Scale bars, 50 μm. Images are representative of two embryos per genotype.

FIGURE 3.

Kinase-dead mutant RIPK1K45A cannot rescue Casp8−/− lethality.

(A) Observed numbers of offspring from Ripk1K45A/K45ACasp8+/− intercrosses and numbers expected from Mendelian ratios at the indicated stage of development. (B) E11.5 yolk sacs and embryos representative of Ripk1K45A/K45ACasp8+/+ (n = 1), Ripk1K45A/K45ACasp8+/− (n = 5), and Ripk1K45A/K45ACasp8−/− (n = 3); E12.5 yolk sacs and embryos representative of Ripk1K45A/K45ACasp8+/+ (n = 2), Ripk1K45A/K45ACasp8+/− (n = 9), and Ripk1K45A/K45ACasp8−/− (n = 4); E13.5 yolk sacs and embryos representative of Ripk1K45A/K45ACasp8+/+(n = 1), Ripk1K45A/K45ACasp8+/− (n = 4), and Ripk1K45A/K45ACasp8−/− (n = 3); E14.5 yolk sacs and embryos representative of Ripk1K45A/K45ACasp8+/+ (n = 4), Ripk1K45A/K45ACasp8+/− (n = 13), and Ripk1K45A/K45ACasp8−/− (n = 4); E15.5 yolk sacs and embryos representative of Ripk1K45A/K45ACasp8+/+ (n = 3), Ripk1K45A/K45ACasp8+/− (n = 4), and Ripk1K45A/K45ACasp8−/− (n = 2). (C) Hematoxylin and eosin (H&E)-stained section of E15.5 embryos, representative of three embryos per genotype. Scale bars, 200 μm. E11.5 embryos stained with TUNEL (green) and DAPI (blue) for nuclei. Images with disrupted spin and fetal liver are shown. Scale bars, 50 μm. Images are representative of two embryos per genotype.

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To further characterize the mechanisms underlying the RIPK1 kinase–independent embryonic lethality observed in Ripk1K45A/K45ACasp8−/− mice, we isolated primary MEFs from Ripk1K45A/K45ACasp8+/+, Ripk1K45A/K45ACasp8+/−, and Ripk1K45A/K45ACasp8−/− embryos. Ripk1K45A/K45ACasp8−/− MEFs also exhibited RIPK3 phosphorylation in medium alone, whereas MLKL expression decreased compared with expression levels in Ripk1K45A/K45ACasp8+/+ or Ripk1K45A/K45ACasp8+/− MEFs (Fig. 4A). Immunoblot analysis of RIPK1 immunoprecipitates revealed that RIPK3 strongly interacted with RIPK1K45A in the Ripk1K45A/K45ACasp8−/− MEFs (Fig. 4A). Because Ripk1−/−Casp8−/− mice survived until birth, we hypothesized that RIPK1 scaffold-dependent and kinase activity–independent function promoted necroptosis in Casp8−/− mice.

FIGURE 4.

RIPK1 interacts with RIPK3 to trigger necroptosis in Ripk1K45A/K45ACasp8−/− mice.

(A) The cell lysates of immortalized MEFs derived from mice of the indicated genotypes (Ripk1K45A/K45ACasp8+/+, Ripk1K45A/K45ACasp8+/−, Ripk1K45A/K45ACasp8−/−) were immunoprecipitated using anti-RIPK1 and were analyzed by Western blotting using the indicated Abs. GAPDH was used as a loading control. Results are representative of three independent experiments. (B) E14.5 yolk sacs and embryos representative of Ripk1K45A/K45ACasp8+/+ (n = 2), Ripk1K45A/K45ACasp8−/− (n = 3), Ripk1K45A/−Casp8−/− (n = 5), and Ripk1−/−Casp8−/− (n = 2). (C) The E14.5 yolk sac lysates from embryos of the indicated genotypes (Ripk1K45A/−Casp8−/−, Ripk1K45A/K45ACasp8+/+, Ripk1−/−Casp8−/−, Ripk1K45A/−Casp8+/+, Ripk1K45A/K45ACasp8−/−, Ripk1K45A/−Casp8+/−) were immunoprecipitated using anti-RIPK1 and were analyzed by Western blotting using the indicated Abs. GAPDH was used as a loading control. Lanes, individual mice.

FIGURE 4.

RIPK1 interacts with RIPK3 to trigger necroptosis in Ripk1K45A/K45ACasp8−/− mice.

(A) The cell lysates of immortalized MEFs derived from mice of the indicated genotypes (Ripk1K45A/K45ACasp8+/+, Ripk1K45A/K45ACasp8+/−, Ripk1K45A/K45ACasp8−/−) were immunoprecipitated using anti-RIPK1 and were analyzed by Western blotting using the indicated Abs. GAPDH was used as a loading control. Results are representative of three independent experiments. (B) E14.5 yolk sacs and embryos representative of Ripk1K45A/K45ACasp8+/+ (n = 2), Ripk1K45A/K45ACasp8−/− (n = 3), Ripk1K45A/−Casp8−/− (n = 5), and Ripk1−/−Casp8−/− (n = 2). (C) The E14.5 yolk sac lysates from embryos of the indicated genotypes (Ripk1K45A/−Casp8−/−, Ripk1K45A/K45ACasp8+/+, Ripk1−/−Casp8−/−, Ripk1K45A/−Casp8+/+, Ripk1K45A/K45ACasp8−/−, Ripk1K45A/−Casp8+/−) were immunoprecipitated using anti-RIPK1 and were analyzed by Western blotting using the indicated Abs. GAPDH was used as a loading control. Lanes, individual mice.

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To determine the impact of RIPK1 scaffold on necrosome formation of yolk sac of Ripk1K45A/K45ACasp8−/− mice, we deleted an allele of RIPK1 (Ripk1+/−) in the background of Ripk1K45A/K45ACasp8−/− mice through intercrossing. Ripk1K45A/−Casp8−/− mice were viable at E14.5 with normal-appearing embryos and yolk sac vasculature as Ripk1−/−Casp8−/− embryos (Fig. 4B). Furthermore, immunoblot analysis of RIPK1 immunoprecipitates revealed that the RIPK3-RIPK1K45A interaction was slightly decreased by the absence of one allele of RIPK1 in Ripk1K45A/−Casp8−/− mice compared with Ripk1K45A/K45ACasp8−/− mice (Fig. 4C). Therefore, the results suggest that RIPK1 scaffolds contribute to lethality of Ripk1K45A/K45ACasp8−/− embryos at midgestation rather than RIPK1 kinase function.

Further, we observed that Ripk1K45A/−Casp8−/− mice did not survive until birth and had an abnormal yolk sac at E17.5 (Fig. 5A). Immunoblot analysis of RIPK1 immunoprecipitates revealed that RIPK3 still interacted with RIPK1K45A in the Ripk1K45A/−Casp8−/− yolk sac lysate (Fig. 5B), indicating that one allele of RIPK1K45A is sufficient to promote necrosome formation at later stages of embryonic development. ZBP1, also identified as DAI/DLM-1, is known to function upstream of RIPK3 and interacts with RIPK3 through their RIP homotypic interaction motif (RHIM) domains. These domains assist in the formation of the RIPK1-RIPK3-ZBP1 complex (38). Interestingly, immunoblot analysis of yolk sac extracts from E11.5 Casp8−/− (Fig. 1D), E14.5 Ripk1K45A/K45ACasp8−/− (Fig. 4C), and E17.5 Ripk1K45A/−Casp8−/− (Fig. 5B) mice showed that ZBP1 expression was significantly increased. We determined whether Mlkl deficiency rescues the lethality of Ripk1K45A/K45ACasp8−/− mice to confirm necroptotic-dependent embryonic lethality in Ripk1K45A/K45ACasp8−/− mice. Mlkl−/−Ripk1K45A/K45ACasp8−/− mice survived normally past weaning (Fig. 5C, 5D), which was similar to trends in Mlkl−/−Casp8−/− mice (data not shown). Casp8−/− caused RIPK1-RIPK3 complex formation in yolk sac, while ablation of MLKL had no impact on the formation of the RIPK1-RIPK3 complex (Fig. 2E). We also observed the RIPK1K45A-RIPK3 complex in Ripk1K45A/K45ACasp8−/−Mlkl−/− mice (data not shown) where the formation of the RIPK1-RIPK3 complex is dependent on the RIPK1/RIPK1K45A scaffold, but not MLKL. Therefore, embryonic lethality of Ripk1K45A/K45ACasp8−/− mice continued to be attributable to MLKL-mediated necroptosis in the yolk sac. This suggests that kinase activity of RIPK1 was not required for necrosome formation–dependent necroptosis in the yolk sac vascularization of Casp8−/− mice (Fig. 5E).

FIGURE 5.

MLKL deficiency rescues lethality in Ripk1K45A/K45ACasp8−/− mice.

(A) E15.5 yolk sacs and embryos representative of Ripk1K45A/K45ACasp8+/+ (n = 2), Ripk1K45A/K45ACasp8−/− (n = 6), Ripk1K45A/−Casp8−/− (n = 3); E16.5 yolk sacs and embryos representative of Ripk1K45A/K45ACasp8+/+ (n = 3), Ripk1K45A/K45ACasp8−/− (n = 2), Ripk1K45A/−Casp8−/− (n = 2); E17.5 yolk sacs and embryos representative of Ripk1K45A/K45ACasp8+/+ (n = 2), Ripk1K45A/K45ACasp8−/− (n = 2), Ripk1K45A/−Casp8−/− (n = 3). (B) Western blots of E17.5 yolk sac lysates from mice of the indicated genotypes (Ripk1K45A/K45ACasp8+/+, Ripk1K45A/K45ACasp8+/−, Ripk1K45A/−Casp8+/−, Ripk1K45A/−Casp8−/−) before (Input) and after IP with anti-RIPK1 Ab. GAPDH was used as a loading control. Lanes, individual mice. (C) Observed numbers of offspring from Mlkl−/−Ripk1K45A/K45ACasp8+/− intercrosses and numbers expected from Mendelian ratios at weaning. (D) Diagram depicting the extent of viability of different strains of Casp8−/− mice. (E) Proposed model for Casp8 deficiency–triggered necroptosis in vivo. (Left) Lack of Casp8 could result in necroptosis in yolk sac during embryogenesis. RIPK1 can recruit RIPK3 through the RHIM motif to form necrosomes, resulting in autophosphorylation of RIPK1 and autophosphorylation of RIPK3 leading to MLKL-dependent necroptosis. (Right) When RIPK1 kinase activity is disrupted in Casp8-deficient mice, RIPK1K45A is not autophosphorylatable, which could trigger RIPK1 kinase–independent recruitment and autophosphorylation of RIPK3. This suggests that the recruitment of RIPK3 to RIPK1 is independent of RIPK1 kinase activity, but scaffold function is dependent on Casp8 deficiency–triggered necroptosis in yolk sac during embryonic development.

FIGURE 5.

MLKL deficiency rescues lethality in Ripk1K45A/K45ACasp8−/− mice.

(A) E15.5 yolk sacs and embryos representative of Ripk1K45A/K45ACasp8+/+ (n = 2), Ripk1K45A/K45ACasp8−/− (n = 6), Ripk1K45A/−Casp8−/− (n = 3); E16.5 yolk sacs and embryos representative of Ripk1K45A/K45ACasp8+/+ (n = 3), Ripk1K45A/K45ACasp8−/− (n = 2), Ripk1K45A/−Casp8−/− (n = 2); E17.5 yolk sacs and embryos representative of Ripk1K45A/K45ACasp8+/+ (n = 2), Ripk1K45A/K45ACasp8−/− (n = 2), Ripk1K45A/−Casp8−/− (n = 3). (B) Western blots of E17.5 yolk sac lysates from mice of the indicated genotypes (Ripk1K45A/K45ACasp8+/+, Ripk1K45A/K45ACasp8+/−, Ripk1K45A/−Casp8+/−, Ripk1K45A/−Casp8−/−) before (Input) and after IP with anti-RIPK1 Ab. GAPDH was used as a loading control. Lanes, individual mice. (C) Observed numbers of offspring from Mlkl−/−Ripk1K45A/K45ACasp8+/− intercrosses and numbers expected from Mendelian ratios at weaning. (D) Diagram depicting the extent of viability of different strains of Casp8−/− mice. (E) Proposed model for Casp8 deficiency–triggered necroptosis in vivo. (Left) Lack of Casp8 could result in necroptosis in yolk sac during embryogenesis. RIPK1 can recruit RIPK3 through the RHIM motif to form necrosomes, resulting in autophosphorylation of RIPK1 and autophosphorylation of RIPK3 leading to MLKL-dependent necroptosis. (Right) When RIPK1 kinase activity is disrupted in Casp8-deficient mice, RIPK1K45A is not autophosphorylatable, which could trigger RIPK1 kinase–independent recruitment and autophosphorylation of RIPK3. This suggests that the recruitment of RIPK3 to RIPK1 is independent of RIPK1 kinase activity, but scaffold function is dependent on Casp8 deficiency–triggered necroptosis in yolk sac during embryonic development.

Close modal

Necroptosis is a form of regulated cell death defined morphologically by cell lysis with release of intracellular contents into the extracellular space. It has been implicated in embryonic development (39) and many human diseases such as Alzheimer's disease (40), multiple sclerosis (41), acute kidney injury (42), and others (3). Mechanism studies showed that when cells fail to activate the apical apoptotic mediator Casp8, RIPK1 kinase activation promotes formation of the cytosolic amyloid-like necrosome complex, also known as complex IIb, which causes RIPK3 phosphorylation and eventually leads to phosphorylation of MLKL. This series of events results in lysis of the plasma membrane to execute cell death (14, 17, 23). Genetic evidence shows that the embryonic mortality of Fadd−/−or Casp8−/− mice can be rescued by codeletion of Ripk1, Ripk3, or Mlkl, supporting the notion that the FADD-Casp8 complex inhibits RIPK1-RIPK3-MLKL–mediated necroptosis during development (2426, 35). The yolk sac of Fadd−/− or Casp8−/− embryos shows vascularization defects. However, it remains unclear whether these defects are attributable to excess necroptosis and its underlying mechanisms. In this study, our results show that a mutation affecting the kinase activity of RIPK1 (K45A) has little impact on embryonic lethality of Casp8−/− embryos, which is consistent with observations in a previous report for RIPK1D138N (31). In contrast, Ripk1K45A/−Casp8−/− mice can survive until E17.5 with normal yolk sac, suggesting that the loss of half of the RIPK1 scaffold function can effectively inhibit necroptosis in Casp8−/− embryos. More importantly, we first detected necrosome formation characterized with strong RIPK1-RIPK3 interaction in yolk sac tissue lysate of Casp8−/− embryos by coimmunoprecipitate analysis. We found that the necrosome formation mediated by RIPK1-RIPK3 interaction was not dependent on RIPK1 kinase activity because RIPK3 still strongly interacted with RIPK1K45A in the Ripk1K45A/K45ACasp8−/− yolk sac lysate. These results are consistent with embryonic death in Ripk1K45A/K45ACasp8−/− mice.

A previous study demonstrated that elimination of TNFR1 from Casp8−/− embryos delayed embryonic lethality from E10.5 until E16.5 (43). Furthermore, embryonic lethality of Ripk1K45A/K45ACasp8−/− embryos can be fully rescued by Mlkl ablation. Together, we speculated that the death of Casp8−/− mice at E10.5 was caused by TNF-α–triggered RIPK1 kinase activity–dependent cell death, but the death of Casp8−/− mice at E14.5 was due to RIPK1 kinase activity–independent cell death. Although there has always been a lack of research in definitively necroptotic in vivo models, obvious necrosome formation in the Casp8−/− yolk sac might be implicated in identifying novel regulators as an in vivo necroptotic model.

Previous studies showed that the RIPK3 kinase–dead mutants D161N and K51A rescue Casp8−/− mice (36, 44), and thus demonstrate that RIPK3 enzymatic activity is involved in necrosome formation. RIPK1 is believed to be the upstream kinase to phosphorylate RIPK3 to promote necroptotic signaling (13, 14). RIPK1 kinase inhibitor Nec-1 can not only block RIPK1 phosphorylation but also downstream RIPK3 phosphorylation (8, 45). However, in this study, we found that neither RIPK1 nor RIPK3 was phosphorylated under RIPK3 inhibitor treatment, which is consistent with previous reports that the phosphorylation of both RIPK1 and RIPK3 was prevented on necroptotic induction in Ripk3-deficient cells (13), raising another possibility that RIPK3 might act as upstream kinase to active RIPK1 to promote heteroamyloid formation during necroptosis under certain conditions. Meanwhile, the RHIM-mediated RIPK1-RIPK3 interaction has been a critical function for MLKL-independent necrosome activation (34, 4648). In this study, we identified that a mutation in the RHIM motif of RIPK3 can also block necrosome formation in the Casp8−/− yolk sac, which also leads to inhibition of embryonic lethality in Casp8−/− mice. ZBP1 and TRIF are RHIM-containing proteins that also interact with RIPK3 (49, 50), and we found that short hairpin RNA knockdown of ZBP1 or TRIF repaired necrosome formation in MEFs (data not shown). Loss of both ZBP1 and TRIF was reported to rescue perinatal lethality in Ripk1−/−Casp8−/− mice, suggesting that TRIF and ZBP1 contribute to RIPK3 activation, which drives inflammation and perinatal lethality (46). Recently, ZBP1 was shown to induce necroptosis as an innate sensor of nucleic acids (5154). Therefore, the RIPK1-RIPK3 complex may have caused necroptosis during embryogenesis, and the TRIF-ZBP1-RIPK3 complex resulted in death because of RIPK1 knockdown or knockout in Casp8−/− mice. However, whether ZBP1 and TRIF coablation can rescue the death of Ripk1K45A/K45ACasp8−/− or Ripk1K45A/−Casp8−/− mice remains to be determined.

Collectively, our study shows that Casp8 protein represses necrosome formation in the yolk sac of embryos, ultimately leading to RIPK1 kinase enzymatic activity–independent but RIPK1 scaffold function–dependent necroptosis, which further leads to embryonic lethality of Casp8−/− mice. These results reveal a different role for the kinase activity and scaffold function of RIPK1 in the Casp8−/− yolk sac, which both are essential for activation of the necrosome and induction of necroptosis in most other necroptotic settings. More importantly, necrosome formation in the Casp8−/− yolk sac, which is easily detected by coimmunoprecipitate analysis, might provide an in vivo necroptotic model as a tool to study molecular mechanisms in the future.

We thank Lin Qiu (Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences) for flow cytometry technical support.

This work was supported by the Strategic Priority Research Program of Chinese Academy of Sciences (Grant XDA26040306); the National Natural Science Foundation of China (31970688); Open Research Fund Program of CAS Key Laboratory of Nutrition, Metabolism and Food Safety; and Shanghai Frontiers Science Center of Cellular Homeostasis and Human Diseases. Q.X. was supported by a grant from the National Natural Science Foundation of China (81801884).

The online version of this article contains supplemental material.

Abbreviations used in this article

     
  • Casp

    caspase

  •  
  • Cat#

    catalog number

  •  
  • E

    embryonic day

  •  
  • IP

    immunoprecipitation

  •  
  • MEF

    mouse embryonic fibroblast

  •  
  • MLKL

    mixed lineage kinase domain-like protein

  •  
  • Nec-1

    necrostatin-1

  •  
  • NP-40

    Nonidet P-40

  •  
  • RHIM

    RIP homotypic interaction motif

  •  
  • RIPK

    receptor-interacting protein kinase

  •  
  • WT

    wild-type

  •  
  • ZBP1

    Z-DNA–binding protein 1

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The authors have no financial conflicts of interest.

This article is distributed under the terms of the CC BY-NC 4.0 Unported license.

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