Literatura científica selecionada sobre o tema "RIPK3-MLKL-necroptotic pathway"

The necrosome is a large-molecular-weight complex in which the terminal effector of the necroptotic pathway, Mixed Lineage Kinase Domain-Like protein (MLKL), is activated to induce necroptotic cell death. The precise mechanism of MLKL activation by the upstream kinase, Receptor Interacting Serine/Threonine Protein Kinase 3 (RIPK3) and the role of Receptor Interacting Serine/Threonine Protein Kinase 1 (RIPK1) in mediating MLKL activation remain incompletely understood. Here, we reconstituted human necrosome interactions in yeast by inducible expression of these necrosome effectors. Functional interactions were reflected by the detection of phosphorylated MLKL, plasma membrane permeabilization, and reduced proliferative potential. Following overexpression of human necrosome effectors in yeast, MLKL aggregated in the periphery of the cell, permeabilized the plasma membrane and compromised clonogenic potential. RIPK1 had little impact on RIPK3/MLKL-mediated yeast lethality; however, it exacerbated the toxicity provoked by co-expression of MLKL with a RIPK3 variant bearing a mutated RHIM-domain. Small molecule necroptotic inhibitors necrostatin-1 and TC13172, and viral inhibitors M45 (residues 1–90) and BAV_Rmil, abated the yeast toxicity triggered by the reconstituted necrosome. This yeast model provides a convenient tool to study necrosome protein interactions and to screen for and characterize potential necroptotic inhibitors.

The necroptosis cell death pathway has been implicated in host defense and in the pathology of inflammatory diseases. While phosphorylation of the necroptotic effector pseudokinase Mixed Lineage Kinase Domain-Like (MLKL) by the upstream protein kinase RIPK3 is a hallmark of pathway activation, the precise checkpoints in necroptosis signaling are still unclear. Here we have developed monobodies, synthetic binding proteins, that bind the N-terminal four-helix bundle (4HB) “killer” domain and neighboring first brace helix of human MLKL with nanomolar affinity. When expressed as genetically encoded reagents in cells, these monobodies potently block necroptotic cell death. However, they did not prevent MLKL recruitment to the “necrosome” and phosphorylation by RIPK3, nor the assembly of MLKL into oligomers, but did block MLKL translocation to membranes where activated MLKL normally disrupts membranes to kill cells. An X-ray crystal structure revealed a monobody-binding site centered on the α4 helix of the MLKL 4HB domain, which mutational analyses showed was crucial for reconstitution of necroptosis signaling. These data implicate the α4 helix of its 4HB domain as a crucial site for recruitment of adaptor proteins that mediate membrane translocation, distinct from known phospholipid binding sites.

Several programmed lytic and necrotic-like cell death mechanisms have now been uncovered, including the recently described receptor interacting protein kinase-3 (RIPK3)-mixed lineage kinase domain-like (MLKL)-dependent necroptosis pathway. Genetic experiments have shown that programmed necrosis, including necroptosis, can play a pivotal role in regulating host-resistance against microbial infections. Alternatively, excess or unwarranted necroptosis may be pathological in autoimmune and autoinflammatory diseases. This review highlights the recent advances in our understanding of the post-translational control of RIPK3-MLKL necroptotic signaling. We discuss the critical function of phosphorylation in the execution of necroptosis, and highlight the emerging regulatory roles for several ubiquitin ligases and deubiquitinating enzymes. Finally, based on current evidence, we discuss the potential mechanisms by which the essential, and possibly terminal, necroptotic effector, MLKL, triggers the disruption of cellular membranes to cause cell lysis.

Excess iron has been reported to lead to osteoblastic cell damage, which is a crucial pathogenesis of iron overload-related osteoporosis. However, the cytotoxic mechanisms have not been fully documented. In the present study, we focused on whether necroptosis contributes to iron overload-induced osteoblastic cell death and related underlying mechanisms. Here, we showed that the cytotoxicity of iron overload in osteoblastic cells was mainly due to necrosis, as evidenced by the Hoechst 33258/PI staining, Annexin-V/PI staining, and transmission electronic microscopy. Furthermore, we revealed that iron overload-induced osteoblastic necrosis might be mediated via the RIPK1/RIPK3/MLKL necroptotic pathway. In addition, we also found that iron overload was able to trigger mitochondrial permeability transition pore (mPTP) opening, which is a critical downstream event in the execution of necroptosis. The key finding of our experiment was that iron overload-induced necroptotic cell death might depend on reactive oxygen species (ROS) generation, as N-acetylcysteine effectively rescued mPTP opening and necroptotic cell death. ROS induced by iron overload promote necroptosis via a positive feedback mechanism, as on the one hand N-acetylcysteine attenuates the upregulation of RIPK1 and RIPK3 and phosphorylation of RIPK1, RIPK3, and MLKL and on the other hand Nec-1, siRIPK1, or siRIPK3 reduced ROS generation. In summary, iron overload induced necroptosis of osteoblastic cells in vitro, which is mediated, at least in part, through the RIPK1/RIPK3/MLKL pathway. We also highlight the critical role of ROS in the regulation of iron overload-induced necroptosis in osteoblastic cells.

Necroptosis is a lytic, proinflammatory cell death pathway, which has been implicated in host defense and, when dysregulated, the pathology of many human diseases. The central mediators of this pathway are the receptor-interacting serine/threonine protein kinases RIPK1 and RIPK3 and the terminal executioner, the pseudokinase mixed lineage kinase domain–like (MLKL). Here, we review the chronology of signaling along the RIPK1-RIPK3-MLKL axis and highlight how the subcellular compartmentalization of signaling events controls the initiation and execution of necroptosis. We propose that a network of modulators surrounds the necroptotic signaling core and that this network, rather than acting universally, tunes necroptosis in a context-, cell type–, and species-dependent manner. Such a high degree of mechanistic flexibility is likely an important property that helps necroptosis operate as a robust, emergency form of cell death.

Abstract Neutrophilic dermatoses are a group of inflammatory skin disorders characterized by sterile infiltrates of neutrophils. These syndromes include pyoderma gangrenosum (PG) and Sweet's syndrome (SS), and they are associated with an increased risk of inflammatory bowel disease, rheumatoid arthritis, and hematologic malignancy, particularly monocytic or myelomonocytic leukemia (AML). IL-1 was first proposed in 1987 as a factor in SS, and the presence of "fragmented neutrophil nuclei" was hypothesized to contribute to disease. IL-1 has subsequently been reported at high levels in lesions of SS, and IL-1 neutralizing therapies have met with success in some SS patients. PG is also reported to be responsive to IL-1 neutralizing therapy, including PG patients with psoriatic arthritis. Splicing variants and promoter region deletions of protein tyrosine phosphatase-6 (PTPN6, or Src homology region 2 domain-containing phosphatase-1, SHP1) are a feature of SS and PG, consistent with the findings that mutations in Ptpn6 drive spontaneous IL-1R-dependent skin inflammation in mice. Mice lacking Ptpn6 develop a cutaneous inflammatory disease that is dependent on the IL-1 receptor (IL-1R), G-CSF, and neutrophils. We hypothesized that production of IL-1 by dying neutrophils drives skin inflammation in the setting of Ptpn6 deficiency. To investigate the mechanisms controlling pathogenic IL-1 release in mice lacking Ptpn6 specifically in neutrophils (Ptpn6∆PMN), we looked directly at cytokine production from neutrophils undergoing inflammatory forms of cell death. We found that stimuli engaging Ripk3/Mlkl-dependent necroptotic forms of cell death result in transcription, processing, and release of bioactive IL-1α and IL-1β from neutrophils. Production of IL-1α and IL-1β were increased in Ptpn6∆PMN neutrophils treated to undergo necroptosis. Ptpn6∆PMN neutrophils displayed increased rates of spontaneous cell death in the presence of G-CSF or IFN-γ alone, and were hypersensitive to necroptotic stimuli. These cell death abnormalities were absent in Ptpn6∆PMN Casp8∆PMN Mlkl-/- neutrophils, demonstrating that Ptpn6 regulates both apoptosis and necroptosis to prevent IL-1 release. We next examined the contribution of apoptosis and necroptosis in the development of spontaneous cutaneous inflammatory disease in Ptpn6∆PMN mice. Loss of either the Caspase-8-dependent apoptotic pathway or the Ripk3/Mlkl-dependent necroptotic pathway was not sufficient to prevent inflammation in mice. However, combined deletion of Caspase-8 and Ripk3/Mlkl protected Ptpn6∆PMN mice (Ptpn6∆PMN Casp8∆PMN Ripk3-/- and Ptpn6∆PMN Casp8∆PMN Mlkl-/- mice). Ripk1 is known to act as a physiological negative regulator of both Caspase-8-dependent apoptosis and Ripk3/Mlkl-dependent necroptosis. We found that the absence of Ripk1 in neutrophils in Ptpn6∆PMN Ripk1∆PMN mice resulted in heightened sensitivity of neutrophils to cell death and accelerated onset of cutaneous inflammatory disease. These results reveal Ripk1 as a critical physiological negative regulator of neutrophil inflammatory cell death and IL-1 production, and cutaneous inflammation. Together, these data emphasize dual functions for Ptpn6 in negative regulation of IL-1α/β transcription, and to prevent Caspase-8- and Ripk3/Mlkl-dependent cell death and concomitant IL-1α/β processing and release. These findings implicate neutrophils as the dominant producers of IL-1 in neutrophilic dermatoses, and identify novel therapeutic targets that could be exploited to control inflammatory forms of cell death in these skin disorders. Disclosures No relevant conflicts of interest to declare.

BackgroundNecroptosis is a newly discovered cell death pathway that plays a critical role in AKI. The involvement of integrin-linked kinase (ILK) in necroptosis has not been studied.MethodsWe performed experiments in mice with an Ilk deletion in collecting duct (CD) principal cells (PCs), and cultured tubular epithelial cells treated with an ILK inhibitor or ILK siRNA knockdown.ResultsIlk deletion in CD PCs resulted in acute tubular injury and early mortality in mice. Progressive interstitial fibrosis and inflammation associated with the activation of the canonical TGF-β signaling cascade were detected in the kidneys of the mice lacking ILK in the CD PCs. In contrast to the minimal apoptosis detected in the animals’ injured CDs, widespread necroptosis was present in ILK-deficient PCs, characterized by cell swelling, deformed mitochondria, and rupture of plasma membrane. In addition, ILK deficiency resulted in increased expression and activation of necroptotic proteins MLKL and RIPK3, and membrane translocation of MLKL in CD PCs. ILK inhibition and siRNA knockdown reduced cell survival in cultured tubular cells, concomitant with increased membrane accumulation of MLKL and/or phospho-MLKL. Administration of a necroptosis inhibitor, necrostatin-1, blocked cell death in vitro and significantly attenuated inflammation, interstitial fibrosis, and renal failure in ILK-deficient mice.ConclusionsThe study demonstrates the critical involvement of ILK in necroptosis through modulation of the RIPK3 and MLKL pathway and highlights the contribution of CD PC injury to the development of inflammation and interstitial fibrosis of the kidney.

AbstractSustained exposure to pro-inflammatory cytokines in the leptomeninges is thought to play a major role in the pathogenetic mechanisms leading to cortical pathology in multiple sclerosis (MS). Although the molecular mechanisms underlying neurodegeneration in the grey matter remain unclear, several lines of evidence suggest a prominent role for tumour necrosis factor (TNF). Using cortical grey matter tissue blocks from post-mortem brains from 28 secondary progressive MS subjects and ten non-neurological controls, we describe an increase in expression of multiple steps in the TNF/TNF receptor 1 signaling pathway leading to necroptosis, including the key proteins TNFR1, FADD, RIPK1, RIPK3 and MLKL. Activation of this pathway was indicated by the phosphorylation of RIPK3 and MLKL and the formation of protein oligomers characteristic of necrosomes. In contrast, caspase-8 dependent apoptotic signaling was decreased. Upregulation of necroptotic signaling occurred predominantly in macroneurons in cortical layers II–III, with little expression in other cell types. The presence of activated necroptotic proteins in neurons was increased in MS cases with prominent meningeal inflammation, with a 30-fold increase in phosphoMLKL+ neurons in layers I–III. The density of phosphoMLKL+ neurons correlated inversely with age at death, age at progression and disease duration. In vivo induction of chronically elevated TNF and INFγ levels in the CSF in a rat model via lentiviral transduction in the meninges, triggered inflammation and neurodegeneration in the underlying cortical grey matter that was associated with increased neuronal expression of TNFR1 and activated necroptotic signaling proteins. Exposure of cultured primary rat cortical neurons to TNF induced necroptosis when apoptosis was inhibited. Our data suggest that neurons in the MS cortex are dying via TNF/TNFR1 stimulated necroptosis rather than apoptosis, possibly initiated in part by chronic meningeal inflammation. Neuronal necroptosis represents a pathogenetic mechanism that is amenable to therapeutic intervention at several points in the signaling pathway.

Necroptosis, or regulated necrosis, is an important type of programmed cell death in addition to apoptosis. Necroptosis induction leads to cell membrane disruption, inflammation and vascularization. It plays important roles in various pathological processes, including neurodegeneration, inflammatory diseases, multiple cancers, and kidney injury. The molecular regulation of necroptotic pathway has been intensively studied in recent years. Necroptosis can be triggered by multiple stimuli and this pathway is regulated through activation of receptor-interacting protein kinase 1 (RIPK1), RIPK3 and pseudokinase mixed lineage kinase domain-like (MLKL). A better understanding of the mechanism of regulation of necroptosis will further aid to the development of novel drugs for necroptosis-associated human diseases. In this review, we focus on new insights in the regulatory machinery of necroptosis. We further discuss the role of necroptosis in different pathologies, its potential as a therapeutic target and the current status of clinical development of drugs interfering in the necroptotic pathway.

Background: The purinergic P2X7 receptor (P2X7R) is a nucleotide-gated ionotropic channel chiefly involved in the inflammatory response triggered by release of ATP from damaged cells. It is largely expressed in inflammatory cells and plays a key role in promoting the release of pro-inflammatory cytokines such as IL-1β. Increasing evidence suggest that it is also expressed in fibroblasts and may play a pivotal role in the development of tissue fibrosis in different body districts. Therefore, we hypothesized a possible P2X7R involvement in the pathogenesis of two different diseases, Systemic sclerosis (SSc; scleroderma) and Chronic Heart Failure (CHF), that include the development of tissue fibrosis among their hallmarks. SSc is a connective tissue disease characterized by generalized fibrosis of the skin and internal organs, for which no effective treatments are currently available. CHF is a complex multistep disorder representing the final result of a number of cardiovascular diseases characterized by cardiac remodelling. Objective and methods: The aims of this study are to investigate the expression and the function of P2X7R in cultured human dermal fibroblasts from SSc patients in comparison with healthy dermal fibroblasts as control or in cardiac fibroblasts from CHF patients in comparison with human atrial fibroblast cell line as control, and also to analyze putative underlying mechanistic pathways involved in the fibrotic process. SSc fibroblasts were isolated from the skin biopsies of SSc patients and healthy volunteers; while CHF fibroblasts were isolated from human atrial fragments obtained from patients undergoing cardiac surgical intervention and the cell line Normal Human Cardiac Fibroblasts-Atrium (NHCF-A) was used as control. In these cells, P2X7R expression were evaluated by flow cytometry and RT q-PCR. P2X7R function and the ensuing effects were assessed by measuring: -cytosolic Ca2+ entry, by single cell fluorescence microscopy; -collagen production, by EIA assay; -α-SMA expression, by immunofluorescence; -fibroblasts migration, by using a wound healing scratch assay. Intracellular pathways potentially involved in fibrogenic process were investigated by measuring: -the effect of P2X7R and/or ERK1/2 inhibitors in different experiments; -the expression of ERK 1/2 by Western Blot analysis; -pro-inflammatory cytokines/growth factor release by ELISA. Results: P2X7R was overexpressed in SSc fibroblasts, compared to control fibroblast, and its stimulation correspond to a markedly higher Ca2+ permeability and collagen production. Moreover, increased α-SMA expression/organization and cell migration were observed in lipopolysaccharides (LPS)-primed SSc fibroblasts after P2X7R stimulation with respect to control fibroblasts. P2X7R-stimulation with LPS plus 2,3-O-(4-benzoylbenzoyl)-ATP (BzATP) induced IL-6 release in SSc fibroblasts, however IL-6 stimulation did not affected collagen production. Instead, collagen secretion was completely abrogated by P2X7R-inhibition with periodate-oxidized ATP (oATP) or by ERK 1/2-inhibition with FR-180204. Cardiac fibroblasts from CHF patients showed, with respect to control cardiac fibroblasts, a higher surface expression of P2X7R and an enhanced receptor function in terms of Ca2+ influx and collagen production. Unlike from SSc fibroblasts, P2X7R activation did not induce cytokine changes. Moreover, TNF-α induced a further increase of P2X7R expression. Finally, phosphorylated-ERK was more expressed in CHF fibroblasts compared to control cell and strictly correlated with the collagen production. Conclusions: In conclusions, our data provide evidence that both in dermal fibroblasts from SSc patients and in cardiac fibroblasts from CHF patients the expression and the function of the purinergic P2X7R are increased compared to healthy controls and are related to an increased collagen secretion. All together these results suggest a causative role of the P2X7R in the pathophysiology of the SSc and CHF and suggest P2X7R as a new attractive target for pharmacological modulation under these pathological conditions.
Background: A novel neutrophils defense mechanism discovered in recent years consists in the extracellular release of network consisting of DNA associated with histones and neutrophils granule enzymes in the form of Neutrophil Extracellular Traps (NETs). Although NETs were originally recognized as a host defence mechanism in which neutrophils release their nuclear and granular contents to kill pathogens, today it is know that NETs are also involved in the pathogenesis of autoimmune and inflammatory diseases, including microcrystalline arthropathies such as gout and pseudogout. Objective and Methods: The aim of this study is to characterize NETs formation in synovial fluid of patients affected by microcrystal arthritis (gout and pseudogout caused, respectively, by MSU or CPPD crystals) compared to that of arthritis not induced by microcrystals (rheumatoid arthritis, psoriatic arthritis). Our first step was to separate neutrophils from other cells present in synovial fluid of gout, pseudogout and non-microcrystal arthritis samples obtained through arthrocentesis. In order to pursue our goal in these samples we evaluated: -the presence of NETs using an immunofluorescence technique; -the amount of NETs released through a fluorimetric assay measuring extracellular DNA; -the levels of pro-inflammatory cytokines and Neutrophil Elastase (NE) by ELISA test. The potential involvement of RIPK3-MLKL-activated necroptotic pathway in NETs formation were also investigated through the analysis of phosphorylated (p)-MLKL, measured by Western Blot technique. In vitro experiments were also performed to evaluate how neutrophils separated from peripheral blood of healthy donors undergo NETs formation when incubated with MSU or CPPD crystals. Results: The experiments performed in this study showed: -microscope images of NETs structures released by neutrophils obtained from synovial fluid of both microcrystal-induced arthritis and non-microcrystal arthritis; -fluorimetric measurement of NETs released directly correlated to microcrystals concentration present in the synovial fluid; -elevated level of IL-6 in both microcrystal and non-microcrystal arthritis; higher IL-1β, IL-8 and IL-10 concentrations in samples from microcrystal-induced arthritis containing a huge amounts of NETs, respect to microcrystal arthritis samples containing low amounts of NETs and non-microcrystal arthritis; -phosphorylation of MLKL, as an index of necroptotic pathway activation in both microcrystal e non-microcrystal arthritis. In vitro experiments confirmed ex vivo data: -increasing concentrations of MSU and CPPD crystals induce NETs release and necroptosis activation in a dose-dependent manner. Conclusions: In conclusions our data provide evidence of NETs formation in synovial fluid of patients affected by gout, pseudogout and non-microcrystal arthritis. Interestingly, NETs formation appears higher in microcrystal arthritis samples and directly correlates with microcrystals (MSU or CPPD) present in synovial fluid of gout and pseudogout patients. Moreover, activation of RIPK3-MLKL necroptotic pathway seems to be involved in NETs production in all these diseases. Therefore, our study pointed out the importance of RIPK3-MLKL activation in NETs release suggesting this pathway as a potential target to regulate NETs cascade in microcrystal and non-microcrystal arthritis.