Mixed lineage kinase domain-like is a key receptor interacting protein 3 downstream component of TNF-induced necrosis

Abstract

Tumor necrosis factor (TNF) is an important inflammatory cytokine and induces many cellular responses, including inflammation, cell proliferation, apoptosis, and necrosis. It is known that receptor interacting protein (RIP) kinases, RIP1 and RIP3, are key effectors of TNF-induced necrosis, but little is known about how these two RIP kinases mediate this process, although reactive oxygen species (ROS) generation and JNK activation have been suggested to be two downstream events of RIP kinases. Here we report the identification of mixed lineage kinase domain-like, MLKL, as a key RIP3 downstream component of TNF-induced necrosis. Through screening a kinase/phosphatase shRNA library in human colon adenocarcinoma HT-29 cells, we found that knockdown of MLKL blocked TNF-induced necrosis. Our data suggest that MLKL functions downstream of RIP1 and RIP3 and is recruited to the necrosome through its interaction with RIP3. Finally, we found that MLKL is required for the generation of ROS and the late-phase activation of JNK during TNF-induced necrosis. However, because these two events are not involved in TNF-induced necrosis in HT-29 cells, the target of MLKL during TNF-induced necrosis remains elusive. Taken together, our study suggests that MLKL is a key RIP3 downstream component of TNF-induced necrotic cell death.T umor necrosis factor (TNF) is a pleiotropic inflammatory cytokine and plays a critical role in diverse cellular events, including cell proliferation, differentiation, apoptosis, and necrosis (1, 2). TNF is also a major mediator of both inflammation and immunity and is involved in many pathological conditions and autoimmune diseases, such as rheumatoid arthritis and Crohn disease (3). Since its tumoricidal activity was discovered, the TNF pathway has been one of the most studied signaling pathways (4). In almost all types of cells treated with TNF, the transcription factor NF-κB and three MAP kinases, ERK, JNK, and p38, are activated and, occasionally, apoptotic or necrotic cell death can be induced as well (5, 6).The molecular mechanisms of TNF signaling have been significantly worked out. It is known that the binding of TNF homotrimer to TNF-receptor 1 (TNF-R1) initiates the formation of TNF-R1 signaling complex by recruiting several adaptor/ effector proteins. TRADD (TNF-R1-associated death domain protein) is the first protein to interact with the receptor through its death domain and recruits other effector proteins, such as RIP1 (receptor interacting protein) and TRAF2 (TNFR-associated factor 2) to form the TNF-R1 signaling complex leading to the activation of several pathways, including NF-κB and MAP kinases (1, 2). Both TRAF2 and RIP1 are necessary for the activation of NF-κB and MAP kinase pathways through recruiting IKK (IκB kinase) and MAP3Ks to the complex (7). Under certain conditions, the complex of TRADD, RIP1, and TRAF2 proteins dissociates from the receptor and recruits other proteins to form different secondary complexes to mediate apoptosis and necrosis (8, 9). Apoptosis is primarily initia...

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

“…[88][89][90][91] MLKL also promotes the generation of reactive oxygen species (ROS) and late phase activation of JNK. 92 The increased production of ROS, especially by the mitochondria, has been strongly linked with mediating TNFinduced necrosis. [93][94][95] Indeed cIAP1 and TAK1 has been shown to block TNF-induced necrosis by inhibiting RIP1/ RIP3-mediated production of ROS.…”

The phospholipid code: a key component of dying cell recognition, tumor progression and host–microbe interactions

“…When apoptosis is blocked, for example, using the pan caspase inhibitor zVAD-fmk, assembly of the 'necrosome' is triggered within the cytosol, a complex formed by receptor-interacting serine/threonine-protein kinase-1 and -3 (RIPK1 and RIPK3, respectively), 24 followed by recruitment and activation of mixed lineage kinase domain-like (MLKL) protein. 25,26 Recently, it has been proposed that during necroptosis, MLKL is translocated to the plasma membrane where it oligomerizes upon interaction with PI(4,5)P 2 and PI(5)P via its N-terminal four helical bundle domain to induce membrane permeabilization 27 (Figure 2c). Interestingly, whether necroptotic, primary necrotic and secondary necrotic cells are recognized by the same phospholipid detector(s) to mediate their removal is unclear.…”