Adherent cells detach from cell culture plates during cell death. This characteristic can be used for the indirect quantification of cell death and to determine differences in proliferation upon ...stimulation with death-inducing agents. One simple method to detect maintained adherence of cells is the staining of attached cells with crystal violet dye, which binds to proteins and DNA. Cells that undergo cell death lose their adherence and are subsequently lost from the population of cells, reducing the amount of crystal violet staining in a culture. This protocol describes a quick and reliable screening method that is suitable for the examination of the impact of chemotherapeutics or other compounds on cell survival and growth inhibition. However, characterization of the cause of reduced crystal violet staining requires additional methods detailed elsewhere.
The intracellular regulation of cell death pathways by cIAPs has been enigmatic. Here we show that loss of cIAPs promotes the spontaneous formation of an intracellular platform that activates either ...apoptosis or necroptosis. This 2 MDa intracellular complex that we designate “Ripoptosome” is necessary but not sufficient for cell death. It contains RIP1, FADD, caspase-8, caspase-10, and caspase inhibitor cFLIP isoforms. cFLIPL prevents Ripoptosome formation, whereas, intriguingly, cFLIPS promotes Ripoptosome assembly. When cIAPs are absent, caspase activity is the “rheostat” that is controlled by cFLIP isoforms in the Ripoptosome and decides if cell death occurs by RIP3-dependent necroptosis or caspase-dependent apoptosis. RIP1 is the core component of the complex. As exemplified by our studies for TLR3 activation, our data argue that the Ripoptosome critically influences the outcome of membrane-bound receptor triggering. The differential quality of cell death mediated by the Ripoptosome may cause important pathophysiological consequences during inflammatory responses.
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► Formation of the Ripoptosome, an intracellular death platform, is inhibited by cIAPs ► This high MW platform can be recruited to TLR3, leading to apoptosis or necroptosis ► cFLIPS blocks caspase-8 activity in the complex required for disassembly/stability ► cFLIPS promotes necroptosis in the absence of cIAPs
The death-inducing signaling complex (DISC) initiates death receptor-induced apoptosis. DISC assembly and activation are controlled by c-FLIP isoforms, which function as pro-apoptotic (c-FLIPL only) ...or anti-apoptotic (c-FLIPL/c-FLIPS) regulators of procaspase-8 activation. Current models assume that c-FLIP directly competes with procaspase-8 for recruitment to FADD. Using a functional reconstituted DISC, structure-guided mutagenesis, and quantitative LC-MS/MS, we show that c-FLIPL/S binding to the DISC is instead a co-operative procaspase-8-dependent process. FADD initially recruits procaspase-8, which in turn recruits and heterodimerizes with c-FLIPL/S via a hierarchical binding mechanism. Procaspase-8 activation is regulated by the ratio of unbound c-FLIPL/S to procaspase-8, which determines composition of the procaspase-8:c-FLIPL/S heterodimer. Thus, procaspase-8:c-FLIPL exhibits localized enzymatic activity and is preferentially an activator, promoting DED-mediated procaspase-8 oligomer assembly, whereas procaspase-8:c-FLIPS lacks activity and potently blocks procaspase-8 activation. This co-operative hierarchical binding model explains the dual role of c-FLIPL and crucially defines how c-FLIP isoforms differentially control cell fate.
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•c-FLIP isoforms (L/S) do not directly compete with caspase-8 for binding to FADD•c-FLIP binds to the DISC via a co-operative hierarchical caspase-8-dependent process•Co-operative and hierarchical binding crucially explains the dual function of c-FLIPL•Our unified model defines how c-FLIP isoforms differentially direct cell fate
It is unclear how c-FLIP isoforms can differentially regulate caspase-8 activation to direct cell fate. Hughes et al. show that c-FLIPL/S recruitment to FADD is indirect and requires caspase-8. This co-operative hierarchical binding process explains the conundrum of the dual role of c-FLIPL and defines how c-FLIPL/S differentially control cell fate.
TNF is a proinflammatory cytokine that is critical for the coordination of tissue homeostasis. RIPK1 and TRADD are the main participants in the transduction of TNF signaling. However, data on the ...cell fate-controlling functions of both molecules are quite controversial. Here, we address the functions of RIPK1 and TRADD in TNF signaling by generating RIPK1- or TRADD-deficient human cell lines. We demonstrate that RIPK1 is relevant for TNF-induced apoptosis and necroptosis in conditions with depleted IAPs. In addition, TRADD is dispensable for necroptosis but required for apoptosis. We reveal a new possible function of TRADD as a negative regulator of NIK stabilization and subsequent ripoptosome formation. Furthermore, we show that RIPK1 and TRADD do not appear to be essential for the activation of MAPK signaling. Moreover, partially repressing NF-κB activation in both RIPK1 and TRADD KO cells does not result in sensitization to TNF alone due to the absence of NIK stabilization. Importantly, we demonstrate that RIPK1 is essential for preventing TRADD from undergoing TNF-induced ubiquitination and degradation. Taken together, our findings provide further insights into the specific functions of RIPK1 and TRADD in the regulation of TNF-dependent signaling, which controls the balance between cell death and survival.
Apoptosis is commonly thought to represent an immunologically silent or even anti-inflammatory mode of cell death, resulting in cell clearance in the absence of explicit activation of the immune ...system. However, here we show that Fas/CD95-induced apoptosis is associated with the production of an array of cytokines and chemokines, including IL-6, IL-8, CXCL1, MCP-1, and GMCSF. Fas-induced production of MCP-1 and IL-8 promoted chemotaxis of phagocytes toward apoptotic cells, suggesting that these factors serve as “find-me” signals in this context. We also show that RIPK1 and IAPs are required for optimal production of cytokines and chemokines in response to Fas receptor stimulation. Consequently, a synthetic IAP antagonist potently suppressed Fas-dependent expression of multiple proinflammatory mediators and inhibited Fas-induced chemotaxis. Thus, in addition to provoking apoptosis, Fas receptor stimulation can trigger the secretion of chemotactic factors and other immunologically active proteins that can influence immune responsiveness toward dying cells.
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► Fas-induced apoptosis is associated with secretion of cytokines and chemokines ► Fas-driven MCP-1 and IL-8 were chemotactic for monocytes and neutrophils, respectively ► IL-8 and MCP-1 can act as “find-me” signals for apoptotic cells ► Apoptotic cells can actively communicate with cells of the immune system
A role for cellular inhibitors of apoptosis (IAPs cIAPs) in preventing CD95 death has been suspected but not previously explained mechanistically. In this study, we find that the loss of cIAPs leads ...to a dramatic sensitization to CD95 ligand (CD95L) killing. Surprisingly, this form of cell death can only be blocked by a combination of RIP1 (receptor-interacting protein 1) kinase and caspase inhibitors. Consistently, we detect a large increase in RIP1 levels in the CD95 death-inducing signaling complex (DISC) and in a secondary cytoplasmic complex (complex II) in the presence of IAP antagonists and loss of RIP1-protected cells from CD95L/IAP antagonist-induced death. Cells resistant to CD95L/IAP antagonist treatment could be sensitized by short hairpin RNA-mediated knockdown of cellular FLICE-inhibitory protein (cFLIP). However, only cFLIPL and not cFLIPS interfered with RIP1 recruitment to the DISC and complex II and protected cells from death. These results demonstrate a fundamental role for RIP1 in CD95 signaling and provide support for a physiological role of caspase-independent death receptor-mediated cell death.
Programmed necrosis and necroptosis signalling Feoktistova, Maria; Leverkus, Martin
The FEBS journal,
January 2015, 2015-Jan, 2015-01-00, 20150101, Letnik:
282, Številka:
1
Journal Article
Recenzirano
In recent years, the paradigm of cell death regulation has changed. Nowadays, not only apoptosis but also several forms of necrosis (e.g. necroptosis) are considered to be regulated. The central ...roles of receptor‐interacting serine/threonine protein kinase1 (RIPK1), RIPK3, and mixed‐lineage kinase domain‐like protein, and the molecular signalling platforms in which these molecules participate, are being intensively studied. In particular, the role of RIPK1, being both a kinase and a scaffold molecule, in different cell death regulatory complexes is of great relevance for the field. This minireview aims to introduce the emerging and dynamic field of necroptosis to the reader, with a specific focus on intracellular signalling pathways involved in this process.
The understanding of cell death has dramatically changed. Necrosis (e.g. RIPK‐dependent necroptosis) is also executed in a genetically controlled manner. This minireview aims to introduce the process of necroptosis with a specific focus on intracellular signalling pathways. The central role of RIPK1/RIPK3/MLKL, the molecular signalling platforms where these molecules are activated, and recent in vivo evidence are described.
Toxic epidermal necrolysis (TEN) is a rare but potentially fatal drug hypersensitivity reaction. Although a number of pathophysiological hints have been identified over the past decade, details of ...the effector mechanisms within the skin remain obscure. A novel study by Kim et al. now sheds light on its pathophysiology. The investigators demonstrate convincingly that receptor-interacting kinase 3 (RIPK3) levels are upregulated substantially in the lesional skin of patients with TEN and that this is followed by the generation of reactive oxygen species, activation of mixed lineage kinase-like protein, and subsequent necroptotic cell death of keratinocytes. These data suggest that therapies that interfere with RIPK3 activation and necroptosis induction could benefit patients with TEN.
At an unbelievable pace, recent evidence has emerged that demonstrates the importance of a programmed form of necrosis (necroptosis) in physiology, pathophysiology and embryonic development. It is ...clear that the understanding of the intracellular control of necroptosis as compared to caspase-dependent apoptosis is of paramount importance. Tumorigenesis, immune surveillance of cancer and pathogen-induced disease, to name only a few, appear to be affected by the mode of cell death in vivo. Here, we discuss the Ripoptosome, a newly defined 2 MDa intracellular signalling complex that can be formed upon genotoxic stress or loss of inhibitor-of apoptosis proteins (IAPs). The Ripoptosome is a signaling platform that can switch modes between apoptotic and necroptotic cell death. In this report, we extend our recent studies and further the notion that the stoichiometric balance between RIP1 and cIAPs is critical for Ripoptosome formation. Furthermore, we demonstrate the critical relevance of the balance of expression levels of short (cFLIPS) or viral (vFLIP) forms of FLIP and RIP3 kinase for the spontaneous execution of necroptosis whenever cIAPs are absent in the cells. Our study thus supports and extends the intriguing role of the Ripoptosome for the regulation of apoptosis and necroptosis.
cFLIP is required for epidermal integrity and skin inflammation silencing via protection from TNF-induced keratinocyte apoptosis. Here, we generated and analyzed cFLIP epidermal KO mice with ...additional TNF deficiency. Intriguingly, the ablation of TNF rescued the pathological phenotype of epidermal cFLIP KO from characteristic weight loss and increased mortality. Moreover, the lack of TNF in these animals strongly reduced and delayed the epidermal hyperkeratosis and the increased apoptosis in keratinocytes. Our data demonstrate that TNF signaling in cFLIP-deficient keratinocytes is the critical factor for the regulation of skin inflammation via modulated cytokine and chemokine expression and, thus, the attraction of immune cells. Our data suggest that autocrine TNF loop activation upon cFLIP deletion is dispensable for T cells, but is critical for neutrophil attraction. Our findings provide evidence for a negative regulatory role of cFLIP for TNF-dependent apoptosis and partially for epidermal inflammation. However, alternative signaling pathways may contribute to the development of the dramatic skin disease upon cFLIP deletion. Our data warrant future studies of the regulatory mechanism controlling the development of skin disease upon cFLIP deficiency and the role of cFLIP/TNF in a number of inflammatory skin diseases, including toxic epidermal necrolysis (TEN).
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