Mixed Lineage Kinase Domain-like Phosphorylation Research Articles

Oleanolic Acid Acetate Alleviates Cisplatin-Induced Nephrotoxicity via Inhibition of Apoptosis and Necroptosis In Vitro and In Vivo.

Cisplatin is a widely used anti-cancer drug for treating solid tumors, but it is associated with severe side effects, including nephrotoxicity. Various studies have suggested that the nephrotoxicity of cisplatin could be overcome; nonetheless, an effective adjuvant drug has not yet been established. Oleanolic acid acetate (OAA), a triterpenoid isolated from Vigna angularis, is commonly used to treat inflammatory and allergic diseases. This study aimed to investigate the protective effects of OAA against cisplatin-induced apoptosis and necroptosis using TCMK-1 cells and a mouse model. In cisplatin-treated TCMK-1 cells, OAA treatment significantly reduced Bax and cleaved-caspase3 expression, whereas it increased Bcl-2 expression. Moreover, in a cisplatin-induced kidney injury mouse model, OAA treatment alleviated weight loss in the body and major organs and also relieved cisplatin-induced nephrotoxicity symptoms. RNA sequencing analysis of kidney tissues identified lipocalin-2 as the most upregulated gene by cisplatin. Additionally, necroptosis-related genes such as receptor-interacting protein kinase (RIPK) and mixed lineage kinase domain-like (MLKL) were identified. In an in vitro study, the phosphorylation of RIPKs and MLKL was reduced by OAA pretreatment in both cisplatin-treated cells and cells boosted via co-treatment with z-VAD-FMK. In conclusion, OAA could protect the kidney from cisplatin-induced nephrotoxicity and may serve as an anti-cancer adjuvant.

RIP1/3-dependent programmed necrosis induces intestinal injury in septic rats.

The regulation of various types of cell death may help to restore the normal physiological function of cells and play a protective role in sepsis. In the current study, we explore the role of programmed cell necrosis in sepsis and the underlying mechanisms. The septic rat model is established by Cecal-ligation and perforation (CLP), and the in vitro model is established by LPS in IEC-6 cells. Our results demonstrate that receptor-interacting protein 1 (RIP1) is significantly upregulated in the ileum of septic rats and LPS-treated IEC-6 cells at both the mRNA and protein levels. Nec-1, an inhibitor of RIP1, reduces the protein levels of RIP1, p-RIP3, and phosphorylated mixed-lineage kinase domain-like (MLKL) (serine 358) and relieves intestinal injury in CLP-induced septic rats with decreased IL-6 and TNF-α levels. The in vitro experiments further reveal that LPS induces the colocalization of RIP1 and RIP3, resulting in the phosphorylation and translocation of MLKL to the plasma membrane in IEC-6 cells. LPS also facilitates ROS production in IEC-6 cells, but this effect is further reversed by Nec-1, si-RIP1 and si-RIP3. Furthermore, LPS-induced necrosis in IEC-6 cells is counteracted by NAC. Thus, we conclude that RIP1/RIP3-dependent programmed cell necrosis participates in intestinal injury in sepsis and may be associated with RIP1/RIP3-mediated ROS.

Necroptosis executioner MLKL plays pivotal roles in agonist-induced platelet prothrombotic responses and lytic cell death in a temporal order.

Necroptosis is a form of programmed cell death executed by receptor-interacting serine/threonine protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like (MLKL). Platelets are circulating cells that play central roles in haemostasis and pathological thrombosis. In this study we demonstrate seminal contribution of MLKL in transformation of agonist-stimulated platelets to active haemostatic units progressing eventually to necrotic death on a temporal scale, thus attributing a yet unrecognized fundamental role to MLKL in platelet biology. Physiological agonists like thrombin instigated phosphorylation and subsequent oligomerization of MLKL in platelets in a RIPK3-independent but phosphoinositide 3-kinase (PI3K)/AKT-dependent manner. Inhibition of MLKL significantly curbed agonist-induced haemostatic responses in platelets that included platelet aggregation, integrin activation, granule secretion, procoagulant surface generation, rise in intracellular calcium, shedding of extracellular vesicles, platelet-leukocyte interactions and thrombus formation under arterial shear. MLKL inhibition, too, prompted impairment in mitochondrial oxidative phosphorylation and aerobic glycolysis in stimulated platelets, accompanied with disruption in mitochondrial transmembrane potential, augmented proton leak and drop in both mitochondrial calcium as well as ROS. These findings underscore the key role of MLKL in sustaining OXPHOS and aerobic glycolysis that underlie energy-intensive platelet activation responses. Prolonged exposure to thrombin provoked oligomerization and translocation of MLKL to plasma membranes forming focal clusters that led to progressive membrane permeabilization and decline in platelet viability, which was prevented by inhibitors of PI3K/MLKL. In summary, MLKL plays vital role in transitioning of stimulated platelets from relatively quiescent cells to functionally/metabolically active prothrombotic units and their ensuing progression to necroptotic death.

Celastrol inhibits necroptosis by attenuating the RIPK1/RIPK3/MLKL pathway and confers protection against acute pancreatitis in mice.

Necroptosis is a necrotic form of regulated cell death, which is primarily mediated by the receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage kinase domain-like (MLKL) pathway in a caspase-independent manner. Necroptosis has been found to occur in virtually all tissues and diseases evaluated, including pancreatitis. Celastrol, a pentacyclic triterpene extracted from the roots of Tripterygium wilfordii (thunder god vine), possesses potent anti-inflammatory and anti-oxidative activities. Yet, it is unclear whether celastrol has any effects on necroptosis and necroptotic-related diseases. Here we showed that celastrol significantly suppressed necroptosis induced by lipopolysaccharide (LPS) plus pan-caspase inhibitor (IDN-6556) or by tumor-necrosis factor-α in combination with LCL-161 (Smac mimetic) and IDN-6556 (TSI). In these in vitro cellular models, celastrol inhibited the phosphorylation of RIPK1, RIPK3, and MLKL and the formation of necrosome during necroptotic induction, suggesting its possible action on upstream signaling of the necroptotic pathway. Consistent with the known role of mitochondrial dysfunction in necroptosis, we found that celastrol significantly rescued TSI-induced loss of mitochondrial membrane potential. TSI-induced intracellular and mitochondrial reactive oxygen species (mtROS), which are involved in the autophosphorylation of RIPK1 and recruitment of RIPK3, were significantly attenuated by celastrol. Moreover, in a mouse model of acute pancreatitis that is associated with necroptosis, celastrol administration significantly reduced the severity of caerulein-induced acute pancreatitis accompanied by decreased phosphorylation of MLKL in pancreatic tissues. Collectively, celastrol can attenuate the activation of RIPK1/RIPK3/MLKL signaling likely by attenuating mtROS production, thereby inhibiting necroptosis and conferring protection against caerulein-induced pancreatitis in mice.

Tyramide Signal Amplification for the Immunofluorescent Staining of ZBP1-dependent Phosphorylation of RIPK3 and MLKL After HSV-1 Infection in Human Cells.

The kinase Receptor-interacting serine/threonine protein kinase 3 (RIPK3) and its substrate mixed lineage kinase domain-like (MLKL) are critical regulators of necroptosis, an inflammatory form of cell death with important antiviral functions. Autophosphorylation of RIPK3 induces phosphorylation and activation of the pore-forming executioner protein of necroptosis MLKL. Trafficking and oligomerization of phosphorylated MLKL at the cell membrane results in cell lysis, characteristic of necroptotic cell death. The nucleic acid sensor ZBP1 is activated by binding to left-handed Z-form double-stranded RNA (Z-RNA) after infection with RNA and DNA viruses. ZBP1 activation restricts virus infection by inducing regulated cell death, including necroptosis, of infected host cells. Immunofluorescence microscopy permits the visualization of different signaling steps downstream of ZBP1-mediated necroptosis on a per-cell basis. However, the sensitivity of standard fluorescence microscopy, using current commercially available phospho-specific antibodies against human RIPK3 and MLKL, precludes reproducible imaging of these markers. Here, we describe an optimized staining procedure for serine (S) phosphorylated RIPK3 (S227) and MLKL (S358) in human HT-29 cells infected with herpes simplex virus 1 (HSV-1). The inclusion of a tyramide signal amplification (TSA) step in the immunofluorescent staining protocol allows the specific detection of S227 phosphorylated RIPK3. Moreover, TSA greatly increases the sensitivity of the detection of S358 phosphorylated MLKL. Together, this method enables the visualization of these two critical signaling events during the induction of ZBP1-induced necroptosis.

Necroptosis is associated with Rab27-independent expulsion of extracellular vesicles containing RIPK3 and MLKL.

Extracellular vesicle (EV) secretion is an important mechanism used by cells to release biomolecules. A common necroptosis effector—mixed lineage kinase domain like (MLKL)—was recently found to participate in the biogenesis of small and large EVs independent of its function in necroptosis. The objective of the current study is to gain mechanistic insights into EV biogenesis during necroptosis. Assessing EV number by nanoparticle tracking analysis revealed an increased number of EVs released during necroptosis. To evaluate the nature of such vesicles, we performed a newly adapted, highly sensitive mass spectrometry‐based proteomics on EVs released by healthy or necroptotic cells. Compared to EVs released by healthy cells, EVs released during necroptosis contained a markedly higher number of unique proteins. Receptor interacting protein kinase‐3 (RIPK3) and MLKL were among the proteins enriched in EVs released during necroptosis. Further, mouse embryonic fibroblasts (MEFs) derived from mice deficient of Rab27a and Rab27b showed diminished basal EV release but responded to necroptosis with enhanced EV biogenesis as the wildtype MEFs. In contrast, necroptosis‐associated EVs were sensitive to Ca2+ depletion or lysosomal disruption. Neither treatment affected the RIPK3‐mediated MLKL phosphorylation. An unbiased screen using RIPK3 immunoprecipitation‐mass spectrometry on necroptotic EVs led to the identification of Rab11b in RIPK3 immune‐complexes. Our data suggests that necroptosis switches EV biogenesis from a Rab27a/b dependent mechanism to a lysosomal mediated mechanism.

Cell Death Triggers Induce MLKL Cleavage in Multiple Myeloma Cells, Which may Promote Cell Death.

Necroptosis is a type of caspase-independent programmed cell death that has been implicated in cancer development. Activation of the canonical necroptotic pathway is often characterized with successive signaling events as the phosphorylation of mixed lineage kinase domain-like (MLKL) by receptor-interacting protein kinase-3 (RIPK3), followed by MLKL oligomerization and plasma membrane rupture. Here, we demonstrate that omega-3 polyunsaturated fatty acids DHA/EPA and the proteasome inhibitor bortezomib induce necroptosis in human multiple myeloma (MM) cells in a RIPK3 independent manner. In addition, it seemed to be that phosphorylation of MLKL was not essential for necroptosis induction in MM cells. We show that treatment of MM cells with these cytotoxic compounds induced cleavage of MLKL into a 35 kDa protein. Furthermore, proteolytic cleavage of MLKL was triggered by activated caspase-3/8/10, and mutation of Asp140Ala in MLKL blocked this cleavage. The pan-caspase inhibitor ZVAD-FMK efficiently prevented DHA/EPA and bortezomib induced cell death. In addition, nuclear translocation of total MLKL and the C-terminus were detected in treated MM cells. Collectively, this present study suggests that caspase-mediated necroptosis may occur under (patho)physiological conditions, delineating a novel regulatory mechanism of necroptosis in RIPK3-deficient cancer cells.

PANoptosis: new insights in regulated cell death in ischemia/reperfusion models.

PANoptosis: new insights in regulated cell death in ischemia/reperfusion models.

Excess fructose enhances oleatic cytotoxicity via reactive oxygen species production and causes necroptosis in hepatocytes

Non-alcoholic fatty liver disease (NAFLD), the hepatic phenotype of metabolic syndrome, has been identified as a major health concern as the number of cirrhosis and deaths associated with NAFLD is expected to increase. Although fructose intake has been considered to be a progressive factor in the pathophysiology of NAFLD, it remains unclear how fructose contributes to hepatocellular damage during lipotoxicity. In the present study, we aimed to analyze the hepatotoxicity of fructose in steatosis. Fructose effects on lipotoxicity were evaluated in HepG2 cells, primary mouse hepatocytes, and in mice fed a high-fat diet with or without sucrose (HFDS/HFD). Oleate induced caspase 3-independent cell death in HepG2 cells and primary mouse hepatocytes cultured in fructose-supplemented medium, and induced cleavage of caspase-1 in primary mouse hepatocytes. In addition, the number of cells stained positive for reactive oxygen species (ROS) was significantly increased, and N-acetyl cysteine was found to inhibit ROS production and cell death. Cell death was confirmed to be through necrotic cell death, and phosphorylation of mixed lineage kinase domain-like (MLKL) protein was observed. Taken together, hepatocyte cytotoxicity was due to excess fructose with oleate-induced ROS-mediated necroptosis. HFDS mice showed progressive hepatic fibrosis and inflammation and a higher NAS score than HFD mice or mice fed a control diet. The expression of hemoxygenase-1, phosphorylation of MLKL, cleavage of caspase1, and apoptosis were significantly increased in the livers of mice fed a HFDS. Overall, excess fructose intake induces necroptosis through the production of ROS and enhances the toxicity of oleatic cytotoxicity.

Impairment of Mitochondrial ATP Synthesis Induces RIPK3-dependent Necroptosis in Lung Epithelial Cells During Lung Injury by Lung Inflammation.

Dysfunction of mitochondrial metabolism is implicated in cellular injury and cell death. While mitochondrial dysfunction is associated with lung injury by lung inflammation, the mechanism by which the impairment of mitochondrial ATP synthesis regulates necroptosis during acute lung injury (ALI) by lung inflammation is unclear. Here, we showed that the impairment of mitochondrial ATP synthesis induces receptor interacting serine/threonine kinase 3 (RIPK3)-dependent necroptosis during lung injury by lung inflammation. We found that the impairment of mitochondrial ATP synthesis by oligomycin, an inhibitor of ATP synthase, resulted in increased lung injury and RIPK3 levels in lung tissues during lung inflammation by LPS in mice. The elevated RIPK3 and RIPK3 phosphorylation levels by oligomycin resulted in high mixed lineage kinase domain-like (MLKL) phosphorylation, the terminal molecule in necroptotic cell death pathway, in lung epithelial cells during lung inflammation. Moreover, the levels of protein in bronchoalveolar lavage fluid (BALF) were increased by the activation of necroptosis via oligomycin during lung inflammation. Furthermore, the levels of ATP5A, a catalytic subunit of the mitochondrial ATP synthase complex for ATP synthesis, were reduced in lung epithelial cells of lung tissues from patients with acute respiratory distress syndrome (ARDS), the most severe form of ALI. The levels of RIPK3, RIPK3 phosphorylation and MLKL phosphorylation were elevated in lung epithelial cells in patients with ARDS. Our results suggest that the impairment of mitochondrial ATP synthesis induces RIPK3-dependent necroptosis in lung epithelial cells during lung injury by lung inflammation.

CAMK2/CaMKII activates MLKL in short-term starvation to facilitate autophagic flux

ABSTRACT MLKL (mixed lineage kinase domain like pseudokinase) is a well-known core component of necrosome that executes necroptotic cell death upon phosphorylation by RIPK3 (receptor interacting serine/threonine kinase 3). Recent studies also implicate a role of MLKL in endosomal trafficking, which is not always dependent on RIPK3. Using mouse Neuro-2a and L929 as well as human HEK293 and HT29 cells, we show here that MLKL is phosphorylated in response to serum and amino acid deprivation from the culture medium, in a manner that depends on CAMK2/CaMKII (calcium/calmodulin dependent protein kinase II) but not RIPK3. The starvation-induced increase in MLKL phosphorylation was accompanied by decreases in levels of lipidated MAP1LC3B/LC3B (microtubule associated protein 1 light chain 3 beta; LC3-II) and SQSTM1/p62 (sequestosome 1), markers of autophagosomes. These changes were prevented by disrupting either MLKL or CAMK2 by pharmacology and genetic manipulations. Moreover, disrupting MLKL or CAMK2 also inhibited the incorporation of LC3-II into autolysosomes, demonstrating a role of the CAMK2-MLKL pathway in facilitating autophagic flux during short-term starvation, in contrast to necroptosis which suppressed autophagic flux. Furthermore, unlike the necroptotic pathway, the starvation-evoked CAMK2-mediated MLKL phosphorylation protected cells from starvation-induced death. We propose that upon nutrient deprivation, MLKL is activated by CAMK2, which in turn facilitates membrane scission needed for autophagosome maturation, allowing the proper fusion of the autophagosome with lysosome and the subsequent substance degradation. This novel function is independent of RIPK3 and is not involved in necroptosis, implicating new roles for this pseudokinase in cell survival, signaling and metabolism. Abbreviations: CAMK2/CaMKII: calcium/calmodulin dependent protein kinase II; DIABLO/SMAC: direct inhibitor of apoptosis-binding protein with low pI/second mitochondria-derived activator of caspase; ECS: extracellular solution; ESCRT: endosomal sorting complexes required for transport; FBS: fetal bovine serum; GSK3B: glycogen synthase kinase 3 beta; HBSS: Hanks’ balanced salt solution; KO: knockout; LC3-II: lipidated microtubule associated protein 1 light chain 3 beta; LDH: lactate dehydrogenase; MLKL: mixed lineage kinase domain like pseudokinase; MTOR: mechanistic target of rapamycin kinase; MTORC1: MTOR complex 1; N2a: Neuro-2a neuroblastoma; Nec-1: necrostatin-1; NSA: necrosulfonamide; PBS: phosphate-buffered saline; PI: propidium iodide; PK-hLC3: pHluorin-mKate2-human LC3; RIPK1: receptor interacting serine/threonine kinase 1; RIPK3: receptor interacting serine/threonine kinase 3; ROS: reactive oxygen species; RPS6KB1/S6K: ribosomal protein S6 kinase B1; shRNA: short hairpin RNA; siRNA: small interference RNA; SQSTM1/p62: sequestosome 1; TBS: Tris-buffered saline; TNF/TNF-α: tumor necrosis factor; TSZ, treatment with TNF + DIABLO mimetics + z-VAD-FMK.

Inhibition of MLKL-dependent necroptosis via downregulating interleukin-1R1 contributes to neuroprotection of hypoxic preconditioning in transient global cerebral ischemic rats

BackgroundOur previous study indicated that hypoxic preconditioning reduced receptor interacting protein (RIP) 3-mediated necroptotic neuronal death in hippocampal CA1 of adult rats after transient global cerebral ischemia (tGCI). Although mixed lineage kinase domain-like (MLKL) has emerged as a crucial molecule for necroptosis induction downstream of RIP3, how MLKL executes necroptosis is not yet well understood. In this study, we aim to elucidate the molecular mechanism underlying hypoxic preconditioning that inactivates MLKL-dependent neuronal necroptosis after tGCI.MethodsTransient global cerebral ischemia was induced by the four-vessel occlusion method. Twenty-four hours before ischemia, rats were exposed to systemic hypoxia with 8% O2 for 30 min. Western blotting was used to detect the expression of MLKL and interleukin-1 type 1 receptor (IL-1R1) in CA1. Immunoprecipitation was used to assess the interactions among IL-1R1, RIP3, and phosphorylated MLKL (p-MLKL). The concentration of intracellular free calcium ion (Ca2+) was measured using Fluo-4 AM. Silencing and overexpression studies were used to study the role of p-MLKL in tGCI-induced neuronal death.ResultsHypoxic preconditioning decreased the phosphorylation of MLKL both in neurons and microglia of CA1 after tGCI. The knockdown of MLKL with siRNA decreased the expression of p-MLKL and exerted neuroprotective effects after tGCI, whereas treatment with lentiviral delivery of MLKL showed opposite results. Mechanistically, hypoxic preconditioning or MLKL siRNA attenuated the RIP3-p-MLKL interaction, reduced the plasma membrane translocation of p-MLKL, and blocked Ca2+ influx after tGCI. Furthermore, hypoxic preconditioning downregulated the expression of IL-1R1 in CA1 after tGCI. Additionally, neutralizing IL-1R1 with its antagonist disrupted the interaction between IL-1R1 and the necrosome, attenuated the expression and the plasma membrane translocation of p-MLKL, thus alleviating neuronal death after tGCI.ConclusionsThese data support that the inhibition of MLKL-dependent neuronal necroptosis through downregulating IL-1R1 contributes to neuroprotection of hypoxic preconditioning against tGCI.

RIPK3-Dependent Necroptosis Is Induced and Restricts Viral Replication in Human Astrocytes Infected With Zika Virus.

Apoptosis, pyroptosis and necroptosis are regulated processes of cell death which can be crucial for viral disease outcomes in hosts because of their effects on viral pathogenicity and host resistance. Zika virus (ZIKV) is a mosquito-borne flavivirus, which infects humans and can cause neurological disorders. Neural developmental disorders and microcephaly could occur in infected fetuses. Several types of nervous cells have been reported to be susceptible to ZIKV infection. Human astrocytes play important roles in the nutritional support and defense of neurons. In this study, we show that human astrocytes are susceptible to ZIKV infection and undergo progressive cell death after infection. In infected astrocytes we detected no cleavage or activation of pro-caspase-3 and pro-caspase-1. Apoptotic substrates and increased secretion of interleukin (IL)-1β or IL-18 were not detected, either. These ruled out the occurrence of apoptosis or pyroptosis in ZIKV-infected astrocytes. We detected, however, an increase of phosphorylated receptor-interacting serine/threonine-protein kinase (RIPK)1, RIPK3, and mixed lineage kinase domain-like (MLKL) protein, indicating that programmed necrosis, or necroptosis, was induced in infected astrocytes. The phosphorylation and cell death were inhibited in cells pre-treated with GSK’872, an inhibitor of RIPK3, while inhibition of RIPK1 with an inhibitor, Necrostatin-1, had no effect, suggesting that ZIKV-induced necroptosis was RIPK1-independent in astrocytes. Consistent with this finding, the inhibition of RIPK1 had no effect on the phosphorylation of MLKL. We showed evidence that MLKL phosphorylation was RIPK3-dependent and ZBP-1, which could stimulate RIPK3, was upregulated in ZIKV-infected astrocytes. Finally, we demonstrated that in GSK’872-pre-treated astrocytes, viral replication increased significantly, which indicates that necroptosis may be protective against viral replication in astrocytes. Our finding that astrocytes uniquely underwent necroptosis in response to ZIKV infection provides insight and helps us better understand the viral pathogenesis in the ZIKV-infected central nervous system.

Neuroprotective effect of ketamine against TNF-α-induced necroptosis in hippocampal neurons.

Tumour necrosis factor‐α (TNF‐α), a crucial cytokine, has various homeostatic and pathogenic bioactivities. The aim of this study was to assess the neuroprotective effect of ketamine against TNF‐α‐induced motor dysfunction and neuronal necroptosis in male C57BL/6J mice in vivo and HT‐22 cell lines in vitro. The behavioural testing results of the present study indicate that ketamine ameliorated TNF‐α‐induced neurological dysfunction. Moreover, immunohistochemical staining results showed that TNF‐α‐induced brain dysfunction was caused by necroptosis and microglial activation, which could be attenuated by ketamine pre‐treatment inhibiting reactive oxygen species production and mixed lineage kinase domain‐like phosphorylation in hippocampal neurons. Therefore, we concluded that ketamine may have neuroprotective effects as a potent inhibitor of necroptosis, which provides a new theoretical and experimental basis for the application of ketamine in TNF‐α‐induced necroptosis‐associated diseases.

The necroptotic cell death pathway operates in megakaryocytes, but not in platelet synthesis

Necroptosis is a pro-inflammatory cell death program executed by the terminal effector, mixed lineage kinase domain-like (MLKL). Previous studies suggested a role for the necroptotic machinery in platelets, where loss of MLKL or its upstream regulator, RIPK3 kinase, impacted thrombosis and haemostasis. However, it remains unknown whether necroptosis operates within megakaryocytes, the progenitors of platelets, and whether necroptotic cell death might contribute to or diminish platelet production. Here, we demonstrate that megakaryocytes possess a functional necroptosis signalling cascade. Necroptosis activation leads to phosphorylation of MLKL, loss of viability and cell swelling. Analyses at steady state and post antibody-mediated thrombocytopenia revealed that platelet production was normal in the absence of MLKL, however, platelet activation and haemostasis were impaired with prolonged tail re-bleeding times. We conclude that MLKL plays a role in regulating platelet function and haemostasis and that necroptosis signalling in megakaryocytes is dispensable for platelet production.

MLKL-dependent signaling regulates autophagic flux in a murine model of non-alcohol-associated fatty liver and steatohepatitis

Autophagy maintains cellular homeostasis and plays a critical role in the development of non-alcoholic fatty liver and steatohepatitis. The pseudokinase mixed lineage kinase domain-like (MLKL) is a key downstream effector of receptor interacting protein kinase 3 (RIP3) in the necroptotic pathway of programmed cell death. However, recent data reveal that MLKL also regulates autophagy. Herein, we tested the hypothesis that MLKL contributes to the progression of Western diet-induced liver injury in mice by regulating autophagy. Rip3+/+, Rip3-/-, Mlkl+/+ and Mlkl-/- mice were fed a Western diet (FFC diet, high in fat, fructose and cholesterol) or chow for 12 weeks. AML12 and primary mouse hepatocytes were exposed to palmitic acid (PA). The FFC diet increased expression, phosphorylation and oligomerization of MLKL in the liver. Mlkl, but not Rip3, deficiency protected mice from FFC diet-induced liver injury. The FFC dietalso induced accumulation of p62 and LC3-II, as well as markers of endoplasmic reticulum stress, in Mlkl+/+ but not Mlkl-/- mice. Mlkl deficiency in mice also prevented the inhibition of autophagy by a protease inhibitor, leupeptin. Using an mRFP-GFP-LC3 reporter in cultured hepatocytes revealed that PA blocked the fusion of autophagosomes with lysosomes. PA triggered MLKL expression and translocation, first to autophagosomes and then to the plasma membrane, independently of Rip3. Mlkl, but not Rip3, deficiency prevented inhibition of autophagy in PA-treated hepatocytes. Overexpression of Mlkl blocked autophagy independently of PA. Additionally, pharmacologic inhibition of autophagy induced MLKL expression and translocation to the plasma membrane in hepatocytes. Taken together, these data indicate that MLKL-dependent, but RIP3-independent, signaling contributes to FFC diet-induced liver injury by inhibiting autophagy. Autophagy is a regulated process that maintains cellular homeostasis. Impaired autophagy contributes to cell injury and death, thus playing a critical role in the pathogenesis of a number of diseases, including non-alcohol-associated fatty liver and steatohepatitis. Herein, we show that Mlkl-dependent, but Rip3-independent, signaling contributed to diet-induced liver injury and inflammatory responses by inhibiting autophagy. These data identify a novel co-regulatory mechanism between necroptotic and autophagic signaling pathways in non-alcoholic fatty liver disease.

Bcl-2 regulates pyroptosis and necroptosis by targeting BH3-like domains in GSDMD and MLKL

Apoptosis is a form of programmed cell death in multicellular organisms. Bcl-2 prevents apoptosis and promotes cellular survival by neutralizing BH3 domain-containing proteins, which directly activate the pore-forming proteins BAX and BAK. However, Bcl-2 is not known to regulate other cell death effectors such as gasdermin D (GSDMD) or mixed lineage kinase domain-like (MLKL), whose activation causes pyroptosis and necroptosis, respectively. Here, we identify a BH3-like domain in both GSDMD and MLKL that mediates an interaction with B-cell lymphoma 2 (Bcl-2). The presence of Bcl-2 reduced GSDMD cleavage at D275 by caspase-1, 4 or 5, and enhanced the GSDMD cleavage at D87. The GSDMD D87 cleavage inactivates the pyroptotic execution program. The presence of Bcl-2 also limited RIP3 mediated phosphorylation of MLKL, which reduced MLKL oligomerization and tempered the induction of necroptosis. Our observations suggest that the presence of Bcl-2 limits the induction of three forms of cell death apoptosis, pyroptosis, and necroptosis.

Viral MLKL Homologs Subvert Necroptotic Cell Death by Sequestering Cellular RIPK3

Necroptotic cell death has been implicated in many human pathologies and is thought to have evolved as an innate immunity mechanism. The pathway relies on two key effectors: the kinase receptor-interacting protein kinase 3 (RIPK3) and the terminal effector, the pseudokinase mixed-lineage kinase-domain-like (MLKL). We identify proteins with high sequence similarity to the pseudokinase domain of MLKL in poxvirus genomes. Expression of these proteins from the BeAn 58058 and Cotia poxviruses, but not swinepox, in human and mouse cells blocks cellular MLKL activation and necroptotic cell death. We show that viral MLKL-like proteins function as dominant-negative mimics of host MLKL, which inhibit necroptosis by sequestering RIPK3 via its kinase domain to thwart MLKL engagement and phosphorylation. These data support an ancestral role for necroptosis in defense against pathogens. Furthermore, mimicry of a cellular pseudokinase by a pathogen adds to the growing repertoire of functions performed by pseudokinases in signal transduction.

C-Jun N-terminal kinases differentially regulate TNF- and TLRs-mediated necroptosis through their kinase-dependent and -independent activities

Tumor necrosis factor (TNF) and Toll-like receptor (TLR)3/TLR4 activation trigger necroptotic cell death through downstream signaling complex containing receptor-interacting protein kinase 1 (RIPK1), RIPK3, and pseudokinase mixed lineage kinase-domain-like (MLKL). However, the regulation of necroptotic signaling pathway is far less investigated. Here we showed that c-Jun N-terminal kinases (JNK1 and JNK2) displayed kinase-dependent and -independent functions in regulating TNF- and TLRs-mediated necroptosis. We found that RIPK1 and RIPK3 promoted cell-death-independent JNK activation in macrophages, which contributed to pro-inflammatory cytokines production. Meanwhile, blocking the kinase activity of JNK dramatically reduced TNF and TLRs-induced necroptotic cell death. Consistently, inhibition of JNK activity protected mice from TNF-induced death and Staphylococcus aureus-mediated lung damage. However, depletion of JNK protein using siRNA sensitized macrophages to necroptosis that was triggered by LPS or poly I:C but still inhibited TNF-induced necroptosis. Mechanistic studies revealed that RIPK1 recruited JNK to the necrosome complex and their kinase activity was required for necrosome formation and the phosphorylation of MLKL in TNF- and TLRs-induced necroptosis. Loss of JNK protein consistently suppressed the phosphorylation of MLKL and necrosome formation in TNF-triggered necroptosis, but differentially promoted the phosphorylation of MLKL and necrosome formation in poly I:C-triggered necroptosis by promoting the oligomeration of TRIF. In conclusion, our findings define a differential role for JNK in regulating TNF- and TLRs-mediated necroptosis by their kinase or scaffolding activities.

Ischemia-reperfusion induces death receptor-independent necroptosis via calpain-STAT3 activation in a lung transplant setting.

Ischemia-reperfusion (I/R)-induced lung injury undermines lung transplantation (LTx) outcomes by predisposing lung grafts to primary graft dysfunction (PGD). Necrosis is a feature of I/R lung injury. However, regulated necrosis (RN) with specific signaling pathways has not been explored in an LTx setting. In this study, we investigated the role of RN in I/R-induced lung injury. To study I/R-induced cell death, we simulated an LTx procedure using our cell culture model with human lung epithelial (BEAS-2B) cells. After 18 h of cold ischemic time (CIT) followed by reperfusion, caspase-independent cell death, mitochondrial reactive oxygen species production, and mitochondrial membrane permeability were significantly increased. N-acetyl-Leu-Leu-norleucinal (ALLN) (calpain inhibitor) or necrostatin-1 (Nec-1) [receptor interacting serine/threonine kinase 1 (RIPK1) inhibitor] reduced these changes. ALLN altered RIPK1/RIPK3 expression and mixed lineage kinase domain-like (MLKL) phosphorylation, whereas Nec-1 did not change calpain/calpastatin expression. Furthermore, signal transducer and activator of transcription 3 (STAT3) was demonstrated to be downstream of calpain and regulate RIPK3 expression and MLKL phosphorylation during I/R. This calpain-STAT3-RIPK axis induces endoplasmic reticulum stress and mitochondrial calcium dysregulation. LTx patients' samples demonstrate that RIPK1, MLKL, and STAT3 mRNA expression increased from CIT to reperfusion. Moreover, the expressions of the key proteins are higher in PGD samples than in non-PGD samples. Cell death associated with prolonged lung preservation is mediated by the calpain-STAT3-RIPK axis. Inhibition of RIPK and/or calpain pathways could be an effective therapy in LTx.