Detection of Necroptosis in Ligand-Mediated and Hypoxia-Induced Injury of Hepatocytes Using a Novel Optic Probe-Detecting Receptor-Interacting Protein (RIP)1/RIP3 Binding

Liver injury is often observed in various pathological conditions including posthepatectomy state and cancer chemotherapy. It occurs mainly as a consequence of the combined necrotic and apoptotic types of cell death. In order to study liver/hepatocyte injury by the necrotic type of cell death, we studied signal-regulated necrosis (necroptosis) by developing a new optic probe for detecting receptor-interacting protein kinase 1 (RIP)/RIP3 binding, an essential process for necroptosis induction. In the mouse hepatocyte cell line, TIB-73 cells, TNF-α/cycloheximide (T/C) induced RIP1/3 binding only when caspase activity was suppressed by the caspase-specific inhibitor z-VAD-fmk (zVAD). T/C/zVAD-induced RIP1/3 binding was inhibited by necrostatin-1 (Nec-1), an allosteric inhibitor of RIP1. The reduced cell survival by T/C/zVAD was improved by Nec-1. These facts indicate that T/C induces necroptosis of hepatocytes when the apoptotic pathway is inhibited/unavailable. FasL also induced cell death, which was only partially inhibited by zVAD, indicating the possible involvement of necroptosis rather than apoptosis. FasL activated caspase 3 and, similarly, induced RIP1/3 binding when the caspases were inactivated. Interestingly, FasL-induced RIP1/3 binding was significantly suppressed by the antioxidants Trolox and N-acetyl cysteine (NAC), suggesting the involvement of reactive oxygen species (ROS) in FasL-induced necroptotic cellular processes. H2O2, by itself, induced RIP1/3 binding that was suppressed by Nec-1, but not by zVAD. Hypoxia induced RIP1/3 binding after reoxygenation, which was suppressed by Nec-1 or by the antioxidants. Cell death induced by hypoxia/reoxygenation (H/R) was also improved by Nec-1. Similar to H2O2, H/R did not require caspase inhibition for RIP1/3 binding, suggesting the involvement of a caspase-independent mechanism for non-ligand-induced and/or redox-mediated necroptosis. These data indicate that ROS can induce necroptosis and mediate the FasL- and hypoxia-induced necroptosis via a molecular mechanism that differs from a conventional caspase-dependent pathway. In conclusion, necroptosis is potentially involved in liver/hepatocyte injury induced by oxidative stress and FasL in the absence of apoptosis.

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TNF-α/Fas-RIP-1–induced cell death signaling separates murine hematopoietic stem cells/progenitors into 2 distinct populations

Blood ◽

10.1182/blood-2011-06-359448 ◽

2011 ◽

Vol 118 (23) ◽

pp. 6057-6067 ◽

Cited By ~ 28

Author(s):

Yechen Xiao ◽

Hongling Li ◽

Jun Zhang ◽

Andrew Volk ◽

Shubin Zhang ◽

...

Keyword(s):

Bone Marrow ◽

Cell Death ◽

Bone Marrow Failure ◽

Caspase 8 ◽

Apoptotic Pathway ◽

Interacting Protein ◽

Hematopoietic Stem ◽

Complete Inactivation ◽

Bone Marrow Failure Syndromes ◽

Tnf Α

AbstractWe studied the effects of TNF-α and Fas-induced death signaling in hematopoietic stem and progenitor cells (HSPCs) by examining their contributions to the development of bone marrow failure syndromes in Tak1-knockout mice (Tak1−/−). We found that complete inactivation of TNF-α signaling by deleting both of its receptors, 1 and 2 (Tnfr1−/−r2−/−), can prevent the death of 30% to 40% of Tak1−/− HSPCs and partially repress the bone marrow failure phenotype of Tak1−/− mice. Fas deletion can prevent the death of 5% to 10% of Tak1−/− HSPCs but fails to further improve the survival of Tak1−/−Tnfr1−/−r2−/− HSPCs, suggesting that Fas might induce death within a subset of TNF-α-sensitive HSPCs. This TNF-α/Fas-induced cell death is a type of receptor-interacting protein-1 (RIP-1)–dependent programmed necrosis called necroptosis, which can be prevented by necrostatin-1, a specific RIP-1 inhibitor. In addition, we found that the remaining Tak1−/− HSPCs died of apoptosis mediated by the caspase-8–dependent extrinsic apoptotic pathway. This apoptosis can be converted into necroptosis by the inhibition of caspase-8 and prevented by inhibiting both caspase-8 and RIP-1 activities. We concluded that HSPCs are heterogeneous populations in response to death signaling stimulation. Tak1 mediates a critical survival signal, which protects against both TNF-α/Fas-RIP-1–dependent necroptosis and TNF-α/Fas-independent apoptosis in HSPCs.

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TWEAK and RIPK1 mediate a second wave of cell death during AKI

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1716578115 ◽

2018 ◽

Vol 115 (16) ◽

pp. 4182-4187 ◽

Cited By ~ 44

Author(s):

Diego Martin-Sanchez ◽

Miguel Fontecha-Barriuso ◽

Susana Carrasco ◽

Maria Dolores Sanchez-Niño ◽

Anne von Mässenhausen ◽

...

Keyword(s):

Cell Death ◽

Kidney Injury ◽

Early Time ◽

Therapeutic Window ◽

Kidney Dysfunction ◽

Interacting Protein ◽

Cell Death Pathways ◽

Caspase Inhibition ◽

Regulated Necrosis ◽

Second Wave

Acute kidney injury (AKI) is characterized by necrotic tubular cell death and inflammation. The TWEAK/Fn14 axis is a mediator of renal injury. Diverse pathways of regulated necrosis have recently been reported to contribute to AKI, but there are ongoing discussions on the timing or molecular regulators involved. We have now explored the cell death pathways induced by TWEAK/Fn14 activation and their relevance during AKI. In cultured tubular cells, the inflammatory cytokine TWEAK induces apoptosis in a proinflammatory environment. The default inhibitor of necroptosis [necrostatin-1 (Nec-1)] was protective, while caspase inhibition switched cell death to necroptosis. Additionally, folic acid-induced AKI in mice resulted in increased expression of Fn14 and necroptosis mediators, such as receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed lineage domain-like protein (MLKL). Targeting necroptosis with Nec-1 or by genetic RIPK3 deficiency and genetic Fn14 ablation failed to be protective at early time points (48 h). However, a persistently high cell death rate and kidney dysfunction (72–96 h) were dependent on an intact TWEAK/Fn14 axis driving necroptosis. This was prevented by Nec-1, or MLKL, or RIPK3 deficiency and by Nec-1 stable (Nec-1s) administered before or after induction of AKI. These data suggest that initial kidney damage and cell death are amplified through recruitment of inflammation-dependent necroptosis, opening a therapeutic window to treat AKI once it is established. This may be relevant for clinical AKI, since using current diagnostic criteria, severe injury had already led to loss of renal function at diagnosis.

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Posttranslational Modification of Bcl-2 Facilitates Its Proteasome-Dependent Degradation: Molecular Characterization of the Involved Signaling Pathway

Molecular and Cellular Biology ◽

10.1128/mcb.20.5.1886-1896.2000 ◽

2000 ◽

Vol 20 (5) ◽

pp. 1886-1896 ◽

Cited By ~ 245

Author(s):

Kristin Breitschopf ◽

Judith Haendeler ◽

Philipp Malchow ◽

Andreas M. Zeiher ◽

Stefanie Dimmeler

Keyword(s):

Endothelial Cells ◽

Protein Kinase ◽

Map Kinase ◽

Specific Inhibitor ◽

26S Proteasome ◽

Apoptotic Pathway ◽

Critical Determinant ◽

Intact Cells ◽

Antiapoptotic Protein ◽

Tnf Α

ABSTRACT The ratio of proapoptotic versus antiapoptotic Bcl-2 members is a critical determinant that plays a significant role in altering susceptibility to apoptosis. Therefore, a reduction of antiapoptotic protein levels in response to proximal signal transduction events may switch on the apoptotic pathway. In endothelial cells, tumor necrosis factor alpha (TNF-α) induces dephosphorylation and subsequent ubiquitin-dependent degradation of the antiapoptotic protein Bcl-2. Here, we investigate the role of different putative phosphorylation sites to facilitate Bcl-2 degradation. Mutation of the consensus protein kinase B/Akt site or of potential protein kinase C or cyclic AMP-dependent protein kinase sites does not affect Bcl-2 stability. In contrast, inactivation of the three consensus mitogen-activated protein (MAP) kinase sites leads to a Bcl-2 protein that is ubiquitinated and subsequently degraded by the 26S proteasome. Inactivation of these sites within Bcl-2 revealed that dephosphorylation of Ser87 appears to play a major role. A Ser-to-Ala substitution at this position results in 50% degradation, whereas replacement of Thr74 with Ala leads to 25% degradation, as assessed by pulse-chase studies. We further demonstrated that incubation with TNF-α induces dephosphorylation of Ser87 of Bcl-2 in intact cells. Furthermore, MAP kinase triggers phosphorylation of Bcl-2, whereas a reduction in Bcl-2 phosphorylation was observed in the presence of MAP kinase-specific phosphatases or the MAP kinase-specific inhibitor PD98059. Moreover, we show that oxidative stress mediates TNF-α-stimulated proteolytic degradation of Bcl-2 by reducing MAP kinase activity. Taken together, these results demonstrate a direct protective role for Bcl-2 phosphorylation by MAP kinase against apoptotic challenges to endothelial cells and other cells.

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MLKL forms disulfide bond-dependent amyloid-like polymers to induce necroptosis

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1707531114 ◽

2017 ◽

Vol 114 (36) ◽

pp. E7450-E7459 ◽

Cited By ~ 52

Author(s):

Shuzhen Liu ◽

Hua Liu ◽

Andrea Johnston ◽

Sarah Hanna-Addams ◽

Eduardo Reynoso ◽

...

Keyword(s):

Cell Death ◽

Disulfide Bond ◽

Kinase Domain ◽

Proteinase K ◽

Interacting Protein ◽

Tnf Α ◽

Mouse Cells ◽

Red Dye ◽

Necessary And Sufficient ◽

Human And Mouse

Mixed-lineage kinase domain-like protein (MLKL) is essential for TNF-α–induced necroptosis. How MLKL promotes cell death is still under debate. Here we report that MLKL forms SDS-resistant, disulfide bond-dependent polymers during necroptosis in both human and mouse cells. MLKL polymers are independent of receptor-interacting protein kinase 1 and 3 (RIPK1/RIPK3) fibers. Large MLKL polymers are more than 2 million Da and are resistant to proteinase K digestion. MLKL polymers are fibers 5 nm in diameter under electron microscopy. Furthermore, the recombinant N-terminal domain of MLKL forms amyloid-like fibers and binds Congo red dye. MLKL mutants that cannot form polymers also fail to induce necroptosis efficiently. Finally, the compound necrosulfonamide conjugates cysteine 86 of human MLKL and blocks MLKL polymer formation and subsequent cell death. These results demonstrate that disulfide bond-dependent, amyloid-like MLKL polymers are necessary and sufficient to induce necroptosis.

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The molecular mechanisms of MLKL-dependent and MLKL-independent necrosis

Journal of Molecular Cell Biology ◽

10.1093/jmcb/mjaa055 ◽

2020 ◽

Author(s):

Lu Li ◽

An Tong ◽

Qiangsheng Zhang ◽

Yuquan Wei ◽

Xiawei Wei

Keyword(s):

Molecular Mechanisms ◽

Death Receptor ◽

Kinase Domain ◽

External Stress ◽

Programmed Necrosis ◽

Interacting Protein ◽

Mixed Lineage Kinase ◽

Caspase Inhibition ◽

Regulated Necrosis ◽

Receptor Family

Abstract Necrosis, a type of unwanted and passive cell demise, usually occurs under the excessive external stress and is considered to be unregulated. However, under some special conditions such as caspase inhibition, necrosis is regulable in a well-orchestrated way. The term ‘regulated necrosis’ has been proposed to describe such programmed necrosis. Recently, several forms of necrosis, including necroptosis, pyroptosis, ferroptosis, parthanatos, oxytosis, NETosis, and Na+/K+-ATPase-mediated necrosis, have been identified, and some crucial regulators governing regulated necrosis have also been discovered. Mixed lineage kinase domain-like pseudokinase (MLKL), a core regulator in necroptosis, acts as an executioner in response to ligands of death receptor family. Its activation requires the receptor-interacting protein kinases, RIP1 and RIP3. However, MLKL is only involved in necroptosis, that is, MLKL is dispensable for necrosis. Therefore, this review is aimed at summarizing the molecular mechanisms of MLKL-dependent and MLKL-independent necrosis.

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Role of Cell Death in Cellular Processes During Odontogenesis

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.671475 ◽

2021 ◽

Vol 9 ◽

Author(s):

John Abramyan ◽

Poongodi Geetha-Loganathan ◽

Marie Šulcová ◽

Marcela Buchtová

Keyword(s):

Cell Death ◽

Tooth Development ◽

Apoptotic Cell ◽

Tooth Germ ◽

Oral Epithelium ◽

Special Focus ◽

Apoptotic Cells ◽

Molecular Signaling ◽

Apoptotic Pathway ◽

Cellular Processes

The development of a tooth germ in a precise size, shape, and position in the jaw, involves meticulous regulation of cell proliferation and cell death. Apoptosis, as the most common type of programmed cell death during embryonic development, plays a number of key roles during odontogenesis, ranging from the budding of the oral epithelium during tooth initiation, to later tooth germ morphogenesis and removal of enamel knot signaling center. Here, we summarize recent knowledge about the distribution and function of apoptotic cells during odontogenesis in several vertebrate lineages, with a special focus on amniotes (mammals and reptiles). We discuss the regulatory roles that apoptosis plays on various cellular processes during odontogenesis. We also review apoptosis-associated molecular signaling during tooth development, including its relationship with the autophagic pathway. Lastly, we cover apoptotic pathway disruption, and alterations in apoptotic cell distribution in transgenic mouse models. These studies foster a deeper understanding how apoptotic cells affect cellular processes during normal odontogenesis, and how they contribute to dental disorders, which could lead to new avenues of treatment in the future.

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Combined inhibition of apoptosis and necrosis promotes transient neuroprotection of retinal ganglion cells and partial-axon regeneration after optic nerve damage

10.1101/357566 ◽

2018 ◽

Cited By ~ 2

Author(s):

Maki Kayama ◽

Kumiko Omura ◽

Yusuke Murakami ◽

Edith Reshef ◽

Aristomenis Thanos ◽

...

Keyword(s):

Cell Death ◽

Optic Nerve ◽

Axonal Regeneration ◽

Axon Regeneration ◽

Ganglion Cells ◽

Retinal Ganglion ◽

Interacting Protein ◽

Caspase Inhibition ◽

Apoptosis And Necrosis

SUMMARYRetinal ganglion cell (RGC) death is the hallmark of glaucoma. Axonal injury is thought to precede RGC loss in glaucoma, and thus studies using an optic nerve (ON) crush model have been widely used to investigate mechanisms of cell death that are common to both conditions. Prior work has focused on the involvement of caspases in RGC death, but little is known about the contribution of other forms of cell death such as necrosis. In this study we show that receptor interacting protein (RIP) kinase-mediated necrosis normally plays a role in RGC death and acts in concert with caspase-dependent apoptosis. The expression of RIP3, a key activator of RIP1 kinase, as well as caspase activity, increased following ON injury. Caspase inhibition alone failed to provide substantial protection to injured RGCs and unexpectedly exacerbated necrosis. In contrast, pharmacologic or genetic inhibition of RIP kinases in combination with caspase blockade delayed both apoptotic and necrotic RGC death, although RGCs still continued to die. Furthermore, inhibition of RIP1 kinase promoted a moderate level of axon regeneration that was only minimal affected by caspase inhibition. In conclusion, multiple approaches are required for effective RGC death prevention and axonal regeneration. Further studies are needed to elucidate more effective long term strategies that can lead to sustained neuroprotection and regeneration.

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Elevated ZIPK is required for TNF-α-induced cell adhesion molecule expression and leucocyte adhesion in endothelial cells

Acta Biochimica et Biophysica Sinica ◽

10.1093/abbs/gmab019 ◽

2021 ◽

Vol 53 (5) ◽

pp. 567-574

Author(s):

Weiwei Zeng ◽

Zhiyuan Sun ◽

Tengxiang Ma ◽

Xiaobin Song ◽

Shuai Li ◽

...

Keyword(s):

Endothelial Cells ◽

Cell Adhesion ◽

Umbilical Vein ◽

Specific Inhibitor ◽

Critical Event ◽

Necrosis Factor Alpha ◽

Interacting Protein ◽

Multiple Substrates ◽

Leucocyte Adhesion ◽

Tnf Α

Abstract Leucocyte adhesion to the vascular endothelium is a critical event in the early inflammatory response to infection and injury. This process is primarily regulated by the expression of cell adhesion molecules (CAMs) in endothelial cells. It has been well documented that tumor necrosis factor alpha (TNF-α) is a key regulator of CAM expression within this process, but its regulatory mechanism remains controversial. To investigate the scenario within this process, we assessed the role of zipper-interacting protein kinase (ZIPK), a serine/threonine kinase with multiple substrates, in CAM expression. We used TNF-α as inflammatory stimulator and found that ZIPK was integrated into the signaling regulation of TNF-α-mediated CAM expression. In human umbilical vein endothelial cells (HUVECs), TNF-α exposure led to significantly increased expression of both intercellular CAM-1 (ICAM-1) and vascular CAM-1 (VCAM-1), along with an increase in the adhesion of THP-1 monocytes to HUVECs. Simultaneously, ZIPK gene was also up-regulated at the transcription level. These effects were clearly inhibited by the ZIPK-specific inhibitor Tc-DAPK6 or small interfering RNA (siRNA) capable of specifically inhibiting ZIPK expression. We thus suggest that both ZIPK activation and ZIPK gene expression are necessary for TNF-α-mediated CAM expression and leucocyte adhesion. Interestingly, ZIPK inhibition also significantly suppressed TNF-α-induced nuclear factor kappa B (NF-κB) activation, indicating that TNF-α-mediated ZIPK expression functions upstream of NF-κB and CAM expression. We thus propose a TNF-α/ZIPK/NF-κB signaling axis for CAM expression that is necessary for leucocyte adhesion to endothelial cells. Our data in this study revealed a potential molecular target for exploring anti-inflammation drugs.

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The lncRNA H19-Derived MicroRNA-675 Promotes Liver Necroptosis by Targeting FADD

Cancers ◽

10.3390/cancers13030411 ◽

2021 ◽

Vol 13 (3) ◽

pp. 411

Author(s):

Rona Harari-Steinfeld ◽

Maytal Gefen ◽

Alina Simerzin ◽

Elina Zorde-Khvalevsky ◽

Mila Rivkin ◽

...

Keyword(s):

Cell Death ◽

Apoptotic Cell ◽

Specific Inhibitor ◽

Kinase Domain ◽

Tumor Promoter ◽

Death Domain ◽

Liver Inflammation ◽

Interacting Protein ◽

Important Regulator ◽

Cell Death Signaling

The H19-derived microRNA-675 (miR-675) has been implicated as both tumor promoter and tumor suppressor and also plays a role in liver inflammation. We found that miR-675 promotes cell death in human hepatocellular carcinoma (HCC) cell lines. We show that Fas-associated protein with death domain (FADD), a mediator of apoptotic cell death signaling, is downregulated by miR-675 and a negative correlation exists between miR-675 and FADD expression in mouse models of HCC (p = 0.014) as well as in human samples (p = 0.017). We demonstrate in a mouse model of liver inflammation that overexpression of miR-675 promotes necroptosis, which can be inhibited by the necroptosis-specific inhibitor Nec-1/Nec-1s. miR-675 induces the level of both p-MLKL (Mixed Lineage Kinase Domain-Like Pseudokinase) and RIP3 (receptor-interacting protein 3), which are key signaling molecules in necroptosis, and enhances MLKL binding to RIP3. miR-675 also inhibits the levels of cleaved caspases 8 and 3, suggesting that miR-675 induces a shift from apoptosis to a necroptotic cellular pathway. In conclusion, downregulation of FADD by miR-675 promotes liver necroptosis in response to inflammatory signals. We propose that this regulation cascade can stimulate and enhance the inflammatory response in the liver, making miR-675 an important regulator in liver inflammation and potentially also in HCC.

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Ferroptosis in Cancer Cell Biology

Cancers ◽

10.3390/cancers12010164 ◽

2020 ◽

Vol 12 (1) ◽

pp. 164 ◽

Cited By ~ 23

Author(s):

Christina M. Bebber ◽

Fabienne Müller ◽

Laura Prieto Clemente ◽

Josephine Weber ◽

Silvia von Karstedt

Keyword(s):

Cell Death ◽

Cell Biology ◽

Potential Role ◽

Kinase Activity ◽

Systemic Treatment ◽

Interacting Protein ◽

Tumour Immunology ◽

Tumour Suppression ◽

Regulated Necrosis ◽

Review Current

A major hallmark of cancer is successful evasion of regulated forms of cell death. Ferroptosis is a recently discovered type of regulated necrosis which, unlike apoptosis or necroptosis, is independent of caspase activity and receptor-interacting protein 1 (RIPK1) kinase activity. Instead, ferroptotic cells die following iron-dependent lipid peroxidation, a process which is antagonised by glutathione peroxidase 4 (GPX4) and ferroptosis suppressor protein 1 (FSP1). Importantly, tumour cells escaping other forms of cell death have been suggested to maintain or acquire sensitivity to ferroptosis. Therefore, therapeutic exploitation of ferroptosis in cancer has received increasing attention. Here, we systematically review current literature on ferroptosis signalling, cross-signalling to cellular metabolism in cancer and a potential role for ferroptosis in tumour suppression and tumour immunology. By summarising current findings on cell biology relevant to ferroptosis in cancer, we aim to point out new conceptual avenues for utilising ferroptosis in systemic treatment approaches for cancer.

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