Clustering of death receptors in lipid rafts initiates neutrophil spontaneous apoptosis

Neutrophils die by apoptosis spontaneously within 12–24 h of their release from the bone marrow. The mechanism regulating entry of neutrophils into apoptosis at the end of their life-span is currently under debate. Our data suggest that neutrophil apoptosis involves a novel mechanism of caspase 8 activation that is indirectly regulated by accumulation of reactive oxygen species. We detected early activation of caspase 8 upstream of caspase 3 activation, suggesting death receptor signalling. The CD95 DISC (death-inducing signalling complex) was detected in neutrophils, but blocking antibodies to death receptors did not inhibit apoptosis, suggesting a novel mechanism for caspase 8 activation. Death receptor clustering in ceramide-rich lipid rafts is thought to be an early event in their signalling, so we investigated the role of ceramide generated by ASM (acid sphingomyelinase) in neutrophil apoptosis. Ceramide was generated early in neutrophil apoptosis, and ASM activity was required for neutrophil apoptosis. Moreover, neutrophil apoptosis was significantly delayed in ASM−/− mice compared with their wild-type littermates. CD95 DISC components were present in lipid rafts in neutrophils, and were progressively clustered in cultured neutrophils. Generation of ceramide was blocked by desferrioxamine, suggesting that hydroxyl radicals are important for the activation of ASM. This observation was in line with our earlier observation of a precipitous drop in reduced glutathione in the aging neutrophil.

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Early events in spontaneous neutrophil apoptosis

Biochemical Society Transactions ◽

10.1042/bst0320461 ◽

2004 ◽

Vol 32 (3) ◽

pp. 461-464 ◽

Cited By ~ 53

Author(s):

D. Scheel-Toellner ◽

K.-Q. Wang ◽

P.R. Webb ◽

S.H. Wong ◽

R. Craddock ◽

...

Keyword(s):

Mitochondrial Membrane ◽

Death Receptor ◽

Current Evidence ◽

Caspase 8 ◽

Neutrophil Apoptosis ◽

Spontaneous Apoptosis ◽

Resolution Of Inflammation ◽

Receptor Signalling ◽

Apoptotic Protein

Neutrophils are very abundant, short-lived leucocytes and their death by apoptosis is central to homoeostasis and the resolution of inflammation, yet the trigger for apoptosis is still a topic of debate. Depolarization of the mitochondrial membrane has been supposed to initiate neutrophil spontaneous apoptosis, as neutrophils gradually lose the anti-apoptotic protein Mcl-1 and Bax translocates and inserts into the mitochondrial membrane. However, other reports show that caspase 8 is required for neutrophil apoptosis, suggesting the involvement of DR (death receptor) signalling. As DR ligation is not required for neutrophil apoptosis, this raises the intriguing possibility that activation of caspase 8 during neutrophil apoptosis occurs via a novel mechanism. In the present paper, we discuss the current evidence for mechanisms occurring in neutrophil apoptosis, which could trigger DR signalling in the absence of DR ligation.

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Reactive oxygen species limit neutrophil life span by activating death receptor signaling

Blood ◽

10.1182/blood-2004-01-0191 ◽

2004 ◽

Vol 104 (8) ◽

pp. 2557-2564 ◽

Cited By ~ 132

Author(s):

Dagmar Scheel-Toellner ◽

Keqing Wang ◽

Rachel Craddock ◽

Paul R. Webb ◽

Helen M. McGettrick ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Life Span ◽

Lipid Rafts ◽

Death Receptor ◽

Caspase 8 ◽

Neutrophil Apoptosis ◽

Oxygen Species ◽

Receptor Signaling ◽

Death Receptor Signaling ◽

Ceramide Generation

Abstract Neutrophils are abundant, short-lived leukocytes, and their death by apoptosis is central to hemostasis and the resolution of inflammation, yet the trigger for their entry into apoptosis is unknown. We show here that death receptor signaling, including CD95 death-inducing signaling complex (DISC) formation and caspase 8 activation, occurred early in neutrophil apoptosis. However, death receptor ligation was not required for apoptosis, suggesting a novel mechanism for caspase 8 activation. We detected ceramide generation and clustering of CD95 in lipid rafts early in neutrophil apoptosis, and neutrophil apoptosis and ceramide generation were both significantly inhibited in acid sphingomyelinase knockout (ASM–/–) mice compared to wild-type littermates. Further studies revealed that ceramide generation, CD95 clustering, and neutrophil apoptosis were dependent on reactive oxygen species (ROSs) and were preceded by a fall in reduced glutathione levels. We propose that accumulation of ROSs, as a consequence of altered redox status, initiates ligand-independent death receptor signaling via activation of ASM and clustering of preformed DISC components in lipid rafts and is therefore a primary factor limiting neutrophil life span.

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TVB Receptors for Cytopathic and Noncytopathic Subgroups of Avian Leukosis Viruses Are Functional Death Receptors

Journal of Virology ◽

10.1128/jvi.74.24.11490-11494.2000 ◽

2000 ◽

Vol 74 (24) ◽

pp. 11490-11494 ◽

Cited By ~ 32

Author(s):

Jürgen Brojatsch ◽

John Naughton ◽

Heather B. Adkins ◽

John A. T. Young

Keyword(s):

Cell Death ◽

Viral Infections ◽

Tumor Necrosis Factor Receptor ◽

Death Receptor ◽

Death Receptors ◽

Death Domain ◽

Cytopathic Effects ◽

Receptor Interactions ◽

Surface Envelope

ABSTRACT The identification of TVBS3, a cellular receptor for the cytopathic subgroups B and D of avian leukosis virus (ALV-B and ALV-D), as a tumor necrosis factor receptor-related death receptor with a cytoplasmic death domain, provides a compelling argument that viral Env-receptor interactions are linked to cell death (4). However, other TVB proteins have been described that appear to have similar death domains but are cellular receptors for the noncytopathic subgroup E of ALV (ALV-E): TVBT, a turkey subgroup E-specific ALV receptor, and TVBS1, a chicken receptor for subgroups B, D, and E ALV. To begin to understand the role of TVB receptors in the cytopathic effects associated with infection by specific ALV subgroups, we asked whether binding of a soluble ALV-E surface envelope protein (SU) to its receptor can lead to cell death. Here we report that ALV-E SU-receptor interactions can induce apoptosis in quail or turkey cells. We also show directly that TVBS1and TVBT are functional death receptors that can trigger cell death by apoptosis via a mechanism involving their cytoplasmic death domains and activation of the caspase pathway. These data demonstrate that ALV-B and ALV-E use functional death receptors to enter cells, and it remains to be determined why only subgroups B and D viral infections lead specifically to cell death.

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Model-based dissection of CD95 signaling dynamics reveals both a pro- and antiapoptotic role of c-FLIPL

The Journal of Cell Biology ◽

10.1083/jcb.201002060 ◽

2010 ◽

Vol 190 (3) ◽

pp. 377-389 ◽

Cited By ~ 101

Author(s):

Nicolai Fricker ◽

Joel Beaudouin ◽

Petra Richter ◽

Roland Eils ◽

Peter H. Krammer ◽

...

Keyword(s):

Death Receptor ◽

Caspase 8 ◽

Converting Enzyme ◽

Receptor Stimulation ◽

Western Blots ◽

Antiapoptotic Protein ◽

Signaling Complex ◽

Signaling Dynamics ◽

Induced Apoptosis

Cellular FADD-like interleukin-1β–converting enzyme inhibitory proteins (c-FLIPs; isoforms c-FLIP long [c-FLIPL], c-FLIP short [c-FLIPS], and c-FLIP Raji [c-FLIPR]) regulate caspase-8 activation and death receptor (DR)–induced apoptosis. In this study, using a combination of mathematical modeling, imaging, and quantitative Western blots, we present a new mathematical model describing caspase-8 activation in quantitative terms, which highlights the influence of c-FLIP proteins on this process directly at the CD95 death-inducing signaling complex. We quantitatively define how the stoichiometry of c-FLIP proteins determines sensitivity toward CD95-induced apoptosis. We show that c-FLIPL has a proapoptotic role only upon moderate expression in combination with strong receptor stimulation or in the presence of high amounts of one of the short c-FLIP isoforms, c-FLIPS or c-FLIPR. Our findings resolve the present controversial discussion on the function of c-FLIPL as a pro- or antiapoptotic protein in DR-mediated apoptosis and are important for understanding the regulation of CD95-induced apoptosis, where subtle differences in c-FLIP concentrations determine life or death of the cells.

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Sweet modification and regulation of death receptor signalling pathway

The Journal of Biochemistry ◽

10.1093/jb/mvab034 ◽

2021 ◽

Author(s):

Kenta Moriwaki ◽

Francis K M Chan ◽

Eiji Miyoshi

Keyword(s):

Critical Role ◽

Immune Surveillance ◽

Death Receptor ◽

Death Receptors ◽

Receptor Activation ◽

Intracellular Signalling ◽

Human Diseases ◽

Tumour Necrosis Factor Receptor ◽

Death Domain ◽

Receptor Signalling

Abstract Death receptors, members of the tumour necrosis factor receptor (TNFR) superfamily, are characterized by the presence of a death domain in the cytosolic region. TNFR1, Fas and TNF-related apoptosis-inducing ligand receptors, which are prototypical death receptors, exert pleiotropic functions in cell death, inflammation and immune surveillance. Hence, they are involved in several human diseases. The activation of death receptors and downstream intracellular signalling is regulated by various posttranslational modifications, such as phosphorylation, ubiquitination and glycosylation. Glycosylation is one of the most abundant and versatile modifications to proteins and lipids, and it plays a critical role in the development and physiology of organisms, as well as the pathology of many human diseases. Glycans control a number of cellular events, such as receptor activation, signal transduction, endocytosis, cell recognition and cell adhesion. It has been demonstrated that oligo- and monosaccharides modify death receptors and intracellular signalling proteins and regulate their functions. Here, we review the current understanding of glycan modifications of death receptor signalling and their impact on signalling activity.

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Death receptors

Essays in Biochemistry ◽

10.1042/bse0390053 ◽

2003 ◽

Vol 39 ◽

pp. 53-71 ◽

Cited By ~ 95

Author(s):

Harald Wajant

Keyword(s):

Molecular Mechanisms ◽

Ectodermal Dysplasia ◽

Death Receptor ◽

Growth Factor Receptor ◽

Death Receptors ◽

Tnf Receptor ◽

Death Domain ◽

Receptor Signalling ◽

Death Receptor 3 ◽

Necrosis Factor

Death receptors [Fas/Apo-1/CD95, TNF-R1 [tumour necrosis factor (TNF) receptor 1], DR3 [death receptor 3], TRAIL-R1 [TNF-related apoptosis-inducing ligand receptor 1], TRAIL-R2, DR6, p75-NGFR [p75-nerve growth factor receptor], EDAR [ectodermal dysplasia receptor]] form a subgroup of the TNF-R superfamily that can induce apoptosis (programmed cell death) via a conserved cytoplasmic signalling module termed the death domain. Although death receptors have been recognized mainly as apoptosis inducers, there is growing evidence that these receptors also fulfil a variety of nonapoptotic functions. This review is focused on the molecular mechanisms of apoptotic and non-apoptotic death receptor signalling in light of the phenotype of mice deficient in the various death receptors.

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Balance between short and long isoforms of cFLIP regulates Fas-mediated apoptosis in vivo

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1517562113 ◽

2016 ◽

Vol 113 (6) ◽

pp. 1606-1611 ◽

Cited By ~ 23

Author(s):

Daniel R. Ram ◽

Vladimir Ilyukha ◽

Tatyana Volkova ◽

Anton Buzdin ◽

Albert Tai ◽

...

Keyword(s):

Splice Variants ◽

Death Receptor ◽

Liver Cells ◽

Caspase 8 ◽

Differential Splicing ◽

Genome Wide ◽

In The Wild ◽

Induced Apoptosis

cFLIP, an inhibitor of apoptosis, is a crucial regulator of cellular death by apoptosis and necroptosis; its importance in development is exemplified by the embryonic lethality in cFLIP–deficient animals. A homolog of caspase 8 (CASP8), cFLIP exists in two main isoforms: cFLIPL (long) and cFLIPR (short). Although both splice variants regulate death receptor (DR)-induced apoptosis by CASP8, the specific role of each isoform is poorly understood. Here, we report a previously unidentified model of resistance to Fas receptor-mediated liver failure in the wild-derived MSM strain, compared with susceptibility in C57BL/6 (B6) mice. Linkage analysis in F2 intercross (B6 x MSM) progeny identified several MSM loci controlling resistance to Fas-mediated death, including the caspase 8- and FADD-like apoptosis regulator (Cflar) locus encoding cFLIP. Furthermore, we identified a 21-bp insertion in the 3′ UTR of the fifth exon of Cflar in MSM that influences differential splicing of cFLIP mRNA. Intriguingly, we observed that MSM liver cells predominantly express the FLIPL variant, in contrast to B6 liver cells, which have higher levels of cFLIPR. In keeping with this finding, genome-wide RNA sequencing revealed a relative abundance of FLIPL transcripts in MSM hepatocytes whereas B6 liver cells had significantly more FLIPR mRNA. Importantly, we show that, in the MSM liver, CASP8 is present exclusively as its cleaved p43 product, bound to cFLIPL. Because of partial enzymatic activity of the heterodimer, it might prevent necroptosis. On the other hand, it prevents cleavage of CASP8 to p10/20 necessary for cleavage of caspase 3 and, thus, apoptosis induction. Therefore, MSM hepatocytes are predisposed for protection from DR-mediated cell death.

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Oxaliplatin resistance in colorectal cancer enhances TRAIL sensitivity via death receptor 4 upregulation and lipid raft localization

eLife ◽

10.7554/elife.67750 ◽

2021 ◽

Vol 10 ◽

Author(s):

Joshua D Greenlee ◽

Maria Lopez-Cavestany ◽

Nerymar Ortiz-Otero ◽

Kevin Liu ◽

Tejas Subramanian ◽

...

Keyword(s):

Colorectal Cancer ◽

Lipid Rafts ◽

Lipid Raft ◽

Death Receptor ◽

Cancer Death ◽

Blood Samples ◽

Death Receptor 4 ◽

Trail Sensitivity ◽

Resistant Cells

Colorectal cancer (CRC) remains a leading cause of cancer death, and its mortality is associated with metastasis and chemoresistance. We demonstrate that oxaliplatin-resistant CRC cells are sensitized to TRAIL-mediated apoptosis. Oxaliplatin-resistant cells exhibited transcriptional downregulation of caspase-10, but this had minimal effects on TRAIL sensitivity following CRISPR-Cas9 deletion of caspase-10 in parental cells. Sensitization effects in oxaliplatin-resistant cells were found to be a result of increased DR4, as well as significantly enhanced DR4 palmitoylation and translocation into lipid rafts. Raft perturbation via nystatin and resveratrol significantly altered DR4/raft colocalization and TRAIL sensitivity. Blood samples from metastatic CRC patients were treated with TRAIL liposomes, and a 57% reduction of viable circulating tumor cells (CTCs) was observed. Increased DR4/lipid raft colocalization in CTCs was found to correspond with increased oxaliplatin resistance and increased efficacy of TRAIL liposomes. To our knowledge, this is the first study to investigate the role of lipid rafts in primary CTCs.

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