Survival function of the FADD-CASPASE-8-cFLIP(L) complex

Abstract

Caspase-8, the initiator caspase of the death receptor pathway of apoptosis, its adapter molecule, FADD, required for caspase-8 activation, and cFLIPL, a caspase-8-like protein that lacks a catalytic site and blocks caspase-8-mediated apoptosis, are each essential for embryonic development. Animals deficient in any of these genes present with E10.5 embryonic lethality. Recent studies have shown that development in caspase-8-deficient mice is rescued by ablation of RIPK3, a kinase that promotes a form of programmed, necrotic cell death. Here, we show that FADD, RIPK3 double-knockout mice develop normally but that the lethal effects of cFLIP deletion are not rescued by RIPK3 deficiency. Remarkably, in mice lacking FADD, cFLIP, and RIPK3, embryonic development is normal. This can be explained by the convergence of two cell processes: the enzymatic activity of the FADD-caspase-8-cFLIPL complex blocks RIPK3-dependent signaling (including necrosis), whereas cFLIPL blocks RIPK3-independent apoptosis promoted by the FADD-caspase-8 complex.

Figure 1. FADD^−/−, RIPK3^−/− mice are viable and overtly normal, functionally deficient for FADD and display severe progressive lymphoaccumulation

(A) Expected and observed frequency of FADD status in offspring from crosses of FADD^+/−, RIPK3^−/− animals. The resulting offspring were genotyped at weaning (p=0.4972). (B) Plot of weight in grams of littermate FADD^+/+, RIPK3^−/−, FADD^+/−, RIPK3^−/−, and FADD^−/−, RIPK3^−/− animals. (C) Effect of anti-CD95 in vivo. 15 µg of agonist anti-CD95 antibody Jo2 was injected intravenously into FADD^+/−, RIPK3^−/− or FADD^−/−, RIPK3^−/− animals. Animals were monitored and euthanized when moribund. (D) Lymphoid organs removed from young (4 wk) and old (20 wk) littermate mice of the indicated genotypes. Scale bar is 1 cm. See also Figure S1.

Figure 2. FLIP^−/−, RIPK3^−/− mice are embryonic lethal

(A) Expected and observed frequency of FLIP status in offspring from crosses of FLIP^+/−, RIPK3^−/− animals. The resulting offspring were genotyped at the indicated developmental time points or at weaning. Asterisks reflect malformed embryos. (B) Embryos (left) and H&E stained sections of fixed embryos (right) of the indicated genotypes at the indicated timepoints. Representative images are presented, n ≥ 3 for each genotype. Scale bars for images on left are 1 mm and for sections on right are 500 µm. (C) Embryos of the indicated genotypes at E13.5 (left and middle) showing vascularization of the embryos and yolk sacs. Left scale bar is 1 mm, middle scale bar is 333 µm. Right panels show PECAM-1 immunostaining on sections from E11.5 embryos of the indicated genotypes. Representative images are presented, n ≥ 3 for each genotype. Scale bar is 250 µm.

Figure 3. FLIP-deficient cells and embryos undergo apoptosis in the absence of RIPK3

(A) Immunoprecipitation of a FADD containing complex from SVEC4–10 cells treated with or without 20 ng/mL TNF and 50 µM zVAD-fmk. NIH3T3 cells, with (B) or without (C) stably expressed RIPK3 were transfected with the indicated siRNAs for 48 hr, followed by treatment with TNF for 9 hr. At harvest, cultures were split and cell death was assessed by AnnexinV-APC and propidium iodide (PI) staining (with AnnexinV⁺,propidium iodide⁻ as apoptotic [pink] and AnnexinV⁺,propidium iodide⁺ as necrotic or late apoptotic [blue]), while the presence of cleaved caspase-3 (green) was assessed by intracellular staining. (D) Cleaved caspase-3 immunostaining in sections from E9.5–10 embryos of the indicated genotypes. Arrowheads mark areas of focal cleaved caspase-3 staining. Scale bars are 500 µm. (E) Cleaved caspase-3 immunostaining in heart section from an E9.5 FLIP^−/−, RIPK3^−/− embryo. Scale bar is 100 µm. For D and E, representative images are presented, n ≥ 3 for each genotype. See also Figure S2.

Figure 4. FLIP^−/−, FADD^−/−, RIPK3^−/− mice are viable with overtly normal development, but display severe progressive lymphoaccumulation

(A) Expected and observed frequency of FLIP and FADD status in offspring from crosses of FLIP^+/−, FADD^−/−, RIPK3^−/− animals. The resulting offspring were genotyped for FLIP status at weaning (p=0.3828). (B) Plot of weight in grams of littermate animals of the indicated genotypes at different ages. (C) Lymphoid organs removed from young (6 wk) and old (16 wk) littermate mice of the indicated genotypes. Scale bar is 1cm. (D) Expected and observed frequency of caspase-8 status in offspring from crossing of αMyHC-Cre⁺Casp8^+/− with Casp8^flox/flox animals. See also Figures S3 and S4.