Abstract
Necroptosis, initially identified as a backup cell death program when apoptosis is hindered, is a prominent feature in the etiology and progression of many human diseases, such as ischemic injury and sepsis. Receptor-interacting protein kinase 3 (RIPK3) is the cardinal regulator of this cell death modality, recruiting and phosphorylating the executioner mixed lineage kinase domain-like protein (MLKL) to signal necroptosis, which is terminated by a cellular plasma membrane rupture and the leakage of intracellular contents from dying cells. Experimental data to date indicate that RIPK3 and MLKL is the core machinery essential for all necroptotic cell death responses. By using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) technology, we showed that Ripk3 and Mlkl knockout and Ripk3/Mlkl double-knockout in necroptosis-sensitive cell lines extensively block susceptibility to necroptosis, in each case to an indistinguishable degree. In vivo studies using Ripk3- or Mlkl-deficient mice validated kidney ischemia reperfusion injury and high-dose tumor necrosis factor (TNF) availability, as druggable targets in necroptotic-mediated pathologies. Here, we demonstrated that Ripk3 or Mlkl-deficient mice are protected to a similar extent from kidney ischemia reperfusion injury and TNF-induced toxicity. Remarkably, in contrast to each single knockout, Ripk3/Mlkl double-deficient mice did not have appreciable protection from either of the above necroptotic-mediated pathologies. Paradoxically, the double-knockout mice resembled, in each case, the vulnerable wild-type mice, revealing novel complexities in the mechanisms of inflammation-driven diseases, due to aberrant cell death.
Highlights
Necroptosis is a caspase-independent programmed cell death mediated by receptor-interacting protein kinase 3 (RIPK3) activation (Cho et al, 2009; He et al, 2009; Zhang et al, 2009) and the pursuant RIPK3-mediated phosphorylation of its pseudokinase substrate mixed lineage kinase domain-like protein (MLKL) (Sun et al, 2012)
We recently discovered that necroptosis and ferroptosis, a caspase-independent regulated cell death modality characterized by the accumulation of lethal lipid reactive oxygen species (ROS) which is produced through iron-dependent lipid peroxidation, are alternative cell death pathways that operate in acute kidney failure, where each death modality can compensate for another when one is compromised (Müller et al, 2017)
We, along with others have reported that Ripk3 deficiency and catalytically inactive RIPK1 are beneficial in renal ischemia-reperfusion injury (IRI), Gaucher’s disease, myocardial infarction, and the highdose tumor necrosis factor (TNF) shock model (Linkermann et al, 2013; Polykratis et al, 2014; Vitner et al, 2014; Zhang et al, 2016)
Summary
Necroptosis is a caspase-independent programmed cell death mediated by receptor-interacting protein kinase 3 (RIPK3) activation (Cho et al, 2009; He et al, 2009; Zhang et al, 2009) and the pursuant RIPK3-mediated phosphorylation of its pseudokinase substrate mixed lineage kinase domain-like protein (MLKL) (Sun et al, 2012) This initial stimulus prompts a conformational change that results in MLKL oligomerization, plasma membrane translocation, and lethal permeation of the lipid bilayer, leading to the release of cellular content, which triggers an inflammatory response (Rickard et al, 2014b). The role of natural necroptosis in human diseases remains controversial, and the potential off-target effects of the applied inhibitors besides kinase activity and existing scaffold functions of the involved proteins often complicate the interpretation of findings In this context, our previously published data verify that pharmacologically blocking necroptosis may worsen diseases such as acute pancreatitis or vascular leakage syndrome, which is triggered by a high-dose tumor necrosis factor (TNF). Our findings describe for the first time that combined knockout of the necrosome members Ripk and Mlkl in an entire organism antagonizes the beneficial effect of the respective single knockouts in necroptotic cell death processes of severe IRI and TNFinduced shock
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