The bromodomain protein BRD4 positively regulates necroptosis via modulating MLKL expression.
ABSTRACT: Necroptosis is a programmed form of necrotic cell death, which is tightly regulated by the necroptotic signaling pathway containing receptor-interacting protein (RIP)1, RIP3, and mixed-lineage kinase domain-like (MLKL) protein. In addition to the RIP1-RIP3-MLKL axis, other factors regulating necroptosis are still largely unknown. Here a cell-based small-molecule screening led to the finding that BET inhibitors protected cells from necroptosis in the TNF?/Smac-mimetic/Z-VAD-FMK (TSZ)-induced cell necroptosis model. Mechanistic studies revealed that BET inhibitors acted by downregulating MLKL expression. Further research demonstrated that BRD4, IRF1, P-TEFb, and RNA polymerase II formed a transcription complex to regulate the expression of MLKL, and BET inhibitors interfered with the transcription complex formation. In necroptosis-related disease model, the BET inhibitor JQ-1 showed promising therapeutic effects. Collectively, our studies establish, for the first time, BRD4 as a new epigenetic factor regulating necroptosis, and highlight the potential of BET inhibitors in the treatment of necroptosis-related diseases.
Project description:Chemotherapy can reinstate anticancer immunosurveillance through inducing tumor immunogenic cell death (ICD). Here, we show that anthracyclines and oxaliplatin can trigger necroptosis in murine cancer cell lines expressing receptor-interacting serine-threonine kinase 3 (RIP3) and mixed lineage kinase domain-like (MLKL). Necroptotic cells featured biochemical hallmarks of ICD and stimulated anticancer immune responses in vivo. Chemotherapy normally killed Rip3 (-/-) and Mlkl (-/-) tumor cells and normally induced caspase-3 activation in such cells, yet was unable to reduce their growth in vivo. RIP3 or MLKL deficiency abolished the capacity of dying cancer cells to elicit an immune response. This could be attributed to reduced release of ATP and high mobility group box 1 (HMGB1) by RIP3 and MLKL-deficient cells. Measures designed to compensate for deficient ATP and HMGB1 signaling restored the chemotherapeutic response of Rip3 (-/-) and Mlkl (-/-) cancers. Altogether, these results suggest that RIP3 and MLKL can contribute to ICD signaling and tumor immunogenicity.
Project description:BACKGROUND:Tumor cell repopulation after radiotherapy is a major cause for the tumor radioresistance and recurrence. This study aims to investigate the underlying mechanism of tumor repopulation after radiotherapy, with focus on whether and how necroptosis takes part in this process. METHODS:Necroptosis after irradiation were examined in vitro and in vivo. And the growth-promoting effect of necroptotic cells was investigated by chemical inhibitors or shRNA against necroptosis associated proteins and genes in in vitro and in vivo tumor repopulation models. Downstream relevance factors of necroptosis were identified by western blot and chemiluminescent immunoassays. Finally, the immunohistochemistry staining of identified necroptosis association growth stimulation factor was conducted in human colorectal tumor specimens to verify the relationship with clinical outcome. RESULTS:Radiation-induced necroptosis depended on activation of RIP1/RIP3/MLKL pathway, and the evidence in vitro and in vivo demonstrated that the inhibition of necroptosis attenuated growth-stimulating effects of irradiated tumor cells on living tumor reporter cells. The JNK/IL-8 were identified as downstream molecules of pMLKL during necroptosis, and inhibition of JNK, IL-8 or IL-8 receptor significantly reduced tumor repopulation after radiotherapy. Moreover, the high expression of IL-8 was associated with poor clinical prognosis in colorectal cancer patients. CONCLUSIONS:Necroptosis associated tumor repopulation after radiotherapy depended on activation of RIP1/RIP3/MLKL/JNK/IL-8 pathway. This novel pathway provided new insight into understanding the mechanism of tumor radioresistance and repopulation, and MLKL/JNK/IL-8 could be developed as promising targets for blocking tumor repopulation to enhance the efficacy of colorectal cancer radiotherapy.
Project description:Necroptosis, a form of regulated necrotic cell death, is governed by RIP1/RIP3-mediated activation of MLKL. However, the signaling process leading to necroptotic death remains to be elucidated. In this study, we found that PUMA, a proapoptotic BH3-only Bcl-2 family member, is transcriptionally activated in an RIP3/MLKL-dependent manner following induction of necroptosis. The induction of PUMA, which is mediated by autocrine TNF-? and enhanced NF-?B activity, contributes to necroptotic death in RIP3-expressing cells with caspases inhibited. On induction, PUMA promotes the cytosolic release of mitochondrial DNA and activation of the DNA sensors DAI/Zbp1 and STING, leading to enhanced RIP3 and MLKL phosphorylation in a positive feedback loop. Furthermore, deletion of PUMA partially rescues necroptosis-mediated developmental defects in FADD-deficient embryos. Collectively, our results reveal a signal amplification mechanism mediated by PUMA and cytosolic DNA sensors that is involved in TNF-driven necroptotic death in vitro and in vivo.
Project description:Mixed lineage kinase domain-like (MLKL) is an essential molecule of necroptosis, a cell death process that is initiated by direct disruption of the plasma membrane. During necroptosis, MLKL is phosphorylated by receptor interacting protein kinase-3 (RIPK3 or RIP3), and then translocates to the plasma membrane to disrupt membrane integrity. Recent data suggest that MLKL also has a RIP3-indendent function in the generation of intraluminal and extracellular vesicles (EVs), as well as in myelin sheath breakdown when promoting sciatic nerve regeneration. Here we show that depletion of MLKL enhances TRAIL-induced cell death in a RIP3-independent manner. Depletion of MLKL leads to prolonged cytotoxic signals that increase TRAIL-induced cell death. Initially, TRAIL binds to DR5 at the cell surface and is endocytosed at similar rates in MLKL-expressing and MLKL-depleted cells, eventual degradation of intracellular TRAIL by the lysosome is delayed in MLKL-depleted cells, corresponding with prolonged/enhanced intracellular signals such as p-ERK and p-p38 in these cells. Colocalization of TRAIL with the marker of early endosomes, EEA1 suggests that TRAIL is accumulated in early endosomes in MLKL-depleted cells compared to MLKL-expressing cells. This indicates that depletion of MLKL reduces receptor-ligand endosomal trafficking leading to increased TRAIL-cytotoxicity. An MLKL mutant that compromises its necroptotic function and its function in the generation of EVs was sufficient to rescue MLKL deficiency, suggesting that the N-terminal structural elements necessary for these functions are not required for the function of MLKL in the intracellular trafficking associated with regulating death receptor cytotoxicity. A reduction in MLKL expression in cancer cells would therefore be expected to result in enhanced TRAIL-induced therapeutic efficacy.
Project description:Necroptosis, a form of cell loss involving the RIP1-RIP3-MLKL axis, has been identified in cardiac pathologies while its inhibition is cardioprotective. We investigated whether the improvement of heart function because of ischaemic preconditioning is associated with mitigation of necroptotic signaling, and these effects were compared with a pharmacological antinecroptotic approach targeting RIP1. Langendorff-perfused rat hearts were subjected to ischaemic preconditioning with or without a RIP1 inhibitor (Nec-1s). Necroptotic signaling and the assessment of oxidative damage and a putative involvement of CaMKII in this process were analysed in whole tissue and subcellular fractions. Ischaemic preconditioning, Nec-1s and their combination improved postischaemic heart function recovery and reduced infarct size to a similar degree what was in line with the prevention of MLKL oligomerization and translocation to the membrane. On the other hand, membrane peroxidation and apoptosis were unchanged by either approach. Ischaemic preconditioning failed to ameliorate ischaemia-reperfusion-induced increase in RIP1 and RIP3 while pSer229-RIP3 levels were reduced only by Nec-1s. In spite of the additive phosphorylation of CaMKII and PLN because of ditherapy, the postischaemic contractile force and relaxation was comparably improved in all the intervention groups while antiarrhythmic effects were observed in the ischaemic preconditioning group only. Necroptosis inhibition seems to be involved in cardioprotection of ischaemic preconditioning and is comparable but not intensified by an anti-RIP1 agent. Changes in oxidative stress nor CaMKII signaling are unlikely to explain the beneficial effects.
Project description:Necroptosis is a form of programmed necrotic cell death mediated by the kinase RIPK3 and its substrate MLKL. MLKL, which displays plasma membrane (PM) pore-forming activity upon phosphorylation, functions as the executioner during necroptosis. Thus, it was previously assumed that MLKL phosphorylation is the endpoint of the necroptotic signaling pathway. Here, we summarize several events that characterize the dying necroptotic cells after MLKL phosphorylation, including Ca2+ influx, phosphatidylserine (PS) externalization, PM repair by ESCRT-III activation, and the final compromise of PM integrity. These processes add several unexpected regulatory events downstream of MLKL signaling. We have also observed that CoCl2, which may mimic hypoxia, can induce necroptosis, which suggests that in vivo triggers of necroptosis might include a transient lack of O2.
Project description:The pseudokinase mixed lineage kinase domain-like protein (MLKL) is a key component of tumor necrosis factor (TNF)-induced necroptosis and plays a crucial role in necroptosis execution. However, the mechanisms that control MLKL activity are not completely understood. Here, we identify the molecular chaperone Hsp90 as a novel MLKL-interacting protein. We show that Hsp90 associates with MLKL and is required for MLKL stability. Moreover, we find that Hsp90 also regulates the stability of the upstream RIP3 kinase. Interference with Hsp90 function with the 17AAG inhibitor destabilizes MLKL and RIP3, resulting in their degradation by the proteasome pathway. Furthermore, we find that Hsp90 is required for TNF-stimulated necrosome assembly. Disruption of Hsp90 function prevents necrosome formation and strongly reduces MLKL phosphorylation and inhibits TNF-induced necroptosis. Consistent with a positive role of Hsp90 in necroptosis, coexpression of Hsp90 increases MLKL oligomerization and plasma membrane translocation and enhances MLKL-mediated necroptosis. Our findings demonstrate that an efficient necrotic response requires a functional Hsp90.
Project description:BACKGROUND:Necroptosis is a form of programmed cell death that is accompanied by release of intracellular contents, and reportedly contributes to various diseases. Here, we investigate the significance of necroptosis in pancreatic cancer. METHODS:We used immunohistochemistry and western blot analysis to evaluate expression of the key mediators of necroptosis-receptor-interacting serine/threonine protein kinase 3 (RIP3) and mixed lineage kinase domain-like (MLKL)-in human pancreatic cancer. We also tested the effects of conditioned media (CM) from necroptotic cells on pancreatic cancer cells in Transwell migration and Matrigel invasion assays. Protein array analysis was used to investigate possible mediators derived from necroptotic cells. RESULTS:RIP3 and MLKL are highly expressed in human pancreatic cancer tissues compared with normal pancreas. MLKL expression was particularly intense at the tumor invasion front. CM derived from necroptotic cells promoted cancer cell migration and invasion, but not CM derived from apoptotic cells. C-X-C motif chemokine 5 (CXCL5) was upregulated in CM derived from necroptotic cells compared with CM derived from control or apoptotic cells. Moreover, expression of the receptor for CXCL5, C-X-C-motif chemokine receptor-2 (CXCR2), was upregulated in pancreatic cancer cells. Inhibition of CXCR2 suppressed cancer cell migratory and invasive behavior enhanced by necroptosis. CONCLUSION:These findings indicate that necroptosis at the pancreatic cancer invasion front can promote cancer cell migration and invasion via the CXCL5-CXCR2 axis.
Project description:Our earlier studies proved that RIPK3-mediated necroptosis might be an important mode of renal tubular cell death in rats with chronic renal injury and the necroptotic cell death can be triggered by tumor necrosis factor-? (TNF-?) in vitro, but the triggering role of angiotensin II (AngII), which exerts notable effects on renal cells for the initiation and progression of renal tubulointerstitial fibrosis, is largely unknown. Here, we identified the presence of necroptotic cell death in the tubular cells of AngII-induced chronic renal injury and fibrosis mice and assessed the percentage of necroptotic renal tubular cell death with the disruption of this necroptosis by the addition of necrostatin-1 (Nec-1). Furthermore, the observation was further confirmed in HK-2 cells treated with AngII and RIPK1/3 or MLKL inhibitors. The detection of Fas and FasL proteins led us to investigate the contribution of the Fas/FasL signaling pathway to AngII-induced necroptosis. Disruption of FasL decreased the percentage of necroptotic cells, suggesting that Fas and FasL are likely key signal molecules in the necroptosis of HK-2 cells induced by AngII. Our data suggest that AngII exposure might trigger RIPK3-MLKL-mediated necroptosis in renal tubular epithelial cells by activating the Fas/FasL signaling pathway in vivo and in vitro.
Project description:Necroptosis has been recognized in heart failure (HF). In this study, we investigated detailed necroptotic signalling in infarcted and non-infarcted areas separately and its mechanistic link with main features of HF. Post-infarction HF in rats was induced by left coronary occlusion (60 minutes) followed by 42-day reperfusion. Heart function was assessed echocardiographically. Molecular signalling and proposed mechanisms (oxidative stress, collagen deposition and inflammation) were investigated in whole hearts and in subcellular fractions when appropriate. In post-infarction failing hearts, TNF and pSer229-RIP3 levels were comparably increased in both infarcted and non-infarcted areas. Its cytotoxic downstream molecule p-MLKL, indicating necroptosis execution, was detected in infarcted area. In non-infarcted area, despite increased pSer229-RIP3, p-MLKL was present in neither whole cells nor the cell membrane known to be associated with necroptosis execution. Likewise, increased membrane lipoperoxidation and NOX2 levels unlikely promoted pro-necroptotic environment in non-infarcted area. Collagen deposition and the inflammatory csp-1-IL-1? axis were active in both areas of failing hearts, while being more pronounced in infarcted tissue. Although apoptotic proteins were differently expressed in infarcted and non-infarcted tissue, apoptosis was found to play an insignificant role. p-MLKL-driven necroptosis and inflammation while inflammation only (without necroptotic cell death) seem to underlie fibrotic healing and progressive injury in infarcted and non-infarcted areas of failing hearts, respectively. Upregulation of pSer229-RIP3 in both HF areas suggests that this kinase, associated with both necroptosis and inflammation, is likely to play a dual role in HF progression.