Developmental checkpoints guarded by regulated necrosis

Abstract

The process of embryonic development is highly regulated through the symbiotic control of differentiation and programmed cell death pathways, which together sculpt tissues and organs. The importance of programmed necrotic (RIPK-dependent necroptosis) cell death during development has recently been recognized as important and has largely been characterized using genetically engineered animals. Suppression of necroptosis appears to be essential for murine development and occurs at three distinct checkpoints, E10.5, E16.5, and P1. These distinct time points have helped delineate the molecular pathways and regulation of necroptosis. The embryonic lethality at E10.5 seen in knockouts of caspase-8, FADD, or FLIP (cflar), components of the extrinsic apoptosis pathway, resulted in pallid embryos that did not exhibit the expected cellular expansions. This was the first suggestion that these factors play an important role in the inhibition of necroptotic cell death. The embryonic lethality at E16.5 highlighted the importance of TNF engaging necroptosis in vivo, since elimination of TNFR1 from casp8 (-/-), fadd (-/-), or cflar (-/-), ripk3 (-/-) embryos delayed embryonic lethality from E10.5 until E16.5. The P1 checkpoint demonstrates the dual role of RIPK1 in both the induction and inhibition of necroptosis, depending on the upstream signal. This review summarizes the role of necroptosis in development and the genetic evidence that helped detail the molecular mechanisms of this novel pathway of programmed cell death.

Keywords: Caspase-8; Development; Necroptosis; RIPK3.

Fig. 1

Pathways leading to extrinsic apoptosis and necroptosis. Binding of TNF to TNFR1 leads to the recruitment of TRADD, FADD and RIPK1 to form the proinflammatory complex I. TRAF2 binds to TRADD and recruits the E3 ligases cIAP1 and cIAP2 that mediate the ubiquitylation of RIPK1. This leads to the activation of the IKK complex (consisting of NEMO, IKKα and IKKβ) through phosphorylation by TAK1 and the subsequent degradation of IκBα and the release and nuclear translocation of the NF-κB1 complex. In case of RIPK1 deubiquitylation, through cIAP inhibition or ubiquitin removal by the deubiquitylase CYLD, or binding of FasL to CD95, RIPK1 can associate with FADD and recruit procaspase-8 and FLIP in complex IIa. Homodimerization of caspase-8 on FADD can fully activate caspase-8 and leads to apoptosis. However, when FLIP heterodimerizes with caspase-8, the full activation is inhibited and apoptosis is blocked. RIPK1 binds and activates RIPK3, resulting in formation of the necrosome and the activation the pseudokinase MLKL, leading to necroptosis. The caspase-8/FLIP heterodimer also inhibits necroptosis. Upon ligation of TLR3 and 4, necroptosis can be engaged through TRIF-mediated RIPK3 activation. Binding of IFNα/β or IFNγ to their respective receptors also results in activation of necroptosis. Necroptosis induced via either TRIF or IFN receptors can be blocked by RIPK1

Fig. 2

Developmental checkpoints of mice deficient for components of extrinsic apoptosis and necroptosis. Lifespan of mice harboring single (a), double (b) or triple (c) deletion of genes involved in extrinsic apoptosis and necroptosis. The embryonic dates listed are for RelA-, HOIP-, and TAK1-deficient mice, rather than animals deficient for other components of these complexes (p50, Sharpin, HOIL1, TAB1, and TAB2)