Mlkl knockout mice demonstrate the indispensable role of Mlkl in necroptosis

Abstract

Mixed lineage kinase domain-like protein (Mlkl) was recently found to interact with receptor interacting protein 3 (Rip3) and to be essential for tumor necrosis factor (TNF)-induced programmed necrosis (necroptosis) in cultured cell lines. We have generated Mlkl-deficient mice by transcription activator-like effector nucleases (TALENs)-mediated gene disruption and found Mlkl to be dispensable for normal mouse development as well as immune cell development. Mlkl-deficient mouse embryonic fibroblasts (MEFs) and macrophages both showed resistance to necrotic but not apoptotic stimuli. Mlkl-deficient MEFs and macrophages were indistinguishable from wild-type cells in their ability to activate NF-κB, ERK, JNK, and p38 in response to TNF and lipopolysaccharides (LPS), respectively. Consistently, Mlkl-deficient macrophages and mice exhibited normal interleukin-1β (IL-1β), IL-6, and TNF production after LPS treatment. Mlkl deficiency protects mice from cerulean-induced acute pancreatitis, a necrosis-related disease, but has no effect on polymicrobial septic shock-induced animal death. Our results provide genetic evidence for the role of Mlkl in necroptosis.

Necroptosis is a key mediator of enterocytes loss in intestinal ischaemia/reperfusion injury

“…In addition, RIP3 controls necroptosis by initiating the pro‐necrotic kinase cascade and assembling of the RIP1/RIP3 complex, thus leading to the regulation of programmed necrosis 39. Mixed lineage kinase domain‐like protein, the direct substrate of RIP3, is essential for the TNFR‐mediated formation of necrosome and targeting appropriate downstream effectors in the necroptosis‐inducing process 40, 41, 42. In our current study, RIP1 and RIP3 expression were increased in the intestinal response to I/R (Fig.…”

RIP kinases: key decision makers in cell death and innate immunity

“…84 The recruitment of MLKL to the necrosome leads to RIP3-mediated phosphorylation of MLKL with the RIP3 inhibitor, necrosulfonamide, blocking necrosis downstream of RIP3 activation 85 and MLKL-deficient mice being resistant to necroptosis. 86,87 Various downstream effector mechanisms have been proposed to mediate necroptosis. Phosphorylation of MLKL promotes its oligomerization and translocation to the plasma membrane where it interacts with phospholipids and compromises membrane integrity ultimately resulting in cell rupture.…”

Evolutionary divergence of the necroptosis effector MLKL

“…The key effectors in the pathway are the protein kinases, receptor interacting protein kinase (RIPK)-1 and RIPK3, [1][2][3][4] and the mixed-lineage kinase domain-like (MLKL) pseudokinase. [5][6][7][8] RIPK3 phosphorylates the pseudokinase domain of MLKL, the most terminal known essential component of the pathway, 5,6 which is believed to induce a conformational change and unleash the N-terminal four-helix bundle (4HB) domain of MLKL: an executioner domain. 5,9,10 Several models have been proposed for how this 4HB domain might induce cell death, including activation of downstream effectors, such as ion channels, 11,12 direct permeabilization of membranes and/or formation of a transmembrane pore, 13,14 all of which remain the subject of debate.…”