RESUMEN
Protein ubiquitination regulates protein stability and modulates the composition of signaling complexes. A20 is a negative regulator of inflammatory signaling, but the molecular mechanisms involved are ill understood. Here, we generated Tnfaip3 gene-targeted A20 mutant mice bearing inactivating mutations in the zinc finger 7 (ZnF7) and ZnF4 ubiquitin-binding domains, revealing that binding to polyubiquitin is essential for A20 to suppress inflammatory disease. We demonstrate that a functional ZnF7 domain was required for recruiting A20 to the tumor necrosis factor receptor 1 (TNFR1) signaling complex and to suppress inflammatory signaling and cell death. The combined inactivation of ZnF4 and ZnF7 phenocopied the postnatal lethality and severe multiorgan inflammation of A20-deficient mice. Conditional tissue-specific expression of mutant A20 further revealed the key role of ubiquitin-binding in myeloid and intestinal epithelial cells. Collectively, these results demonstrate that the anti-inflammatory and cytoprotective functions of A20 are largely dependent on its ubiquitin-binding properties.
Asunto(s)
Inflamación/metabolismo , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/metabolismo , Animales , Células Epiteliales/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Células Mieloides/metabolismo , Poliubiquitina/metabolismo , Unión Proteica/fisiología , Transducción de Señal/fisiología , Factor de Necrosis Tumoral alfa/metabolismo , Ubiquitina/metabolismo , Ubiquitinación/fisiología , Dedos de Zinc/fisiologíaRESUMEN
Tumor necrosis factor (TNF) plays a central role in orchestrating mammalian inflammatory responses. It promotes inflammation either directly by inducing inflammatory gene expression or indirectly by triggering cell death. TNF-mediated cell death-driven inflammation can be beneficial during infection by providing cell-extrinsic signals that help to mount proper immune responses. Uncontrolled cell death caused by TNF is instead highly detrimental and is believed to cause several human autoimmune diseases. Death is not the default response to TNF sensing. Molecular brakes, or cell death checkpoints, actively repress TNF cytotoxicity to protect the organism from its detrimental consequences. These checkpoints therefore constitute essential safeguards against inflammatory diseases. Recent advances in the field have revealed the existence of several new and unexpected brakes against TNF cytotoxicity and pathogenicity.
Asunto(s)
Apoptosis , Transducción de Señal , Animales , Humanos , Necrosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores , Muerte Celular , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Inflamación/patología , MamíferosRESUMEN
The anti-inflammatory protein A20 serves as a critical brake on NF-κB signaling and NF-κB-dependent inflammation. In humans, polymorphisms in or near the TNFAIP3/A20 gene have been associated with several inflammatory disorders, including rheumatoid arthritis (RA), and experimental studies in mice have demonstrated that myeloid-specific A20 deficiency causes the development of a severe polyarthritis resembling human RA. Myeloid A20 deficiency also promotes osteoclastogenesis in mice, suggesting a role for A20 in the regulation of osteoclast differentiation and bone formation. We show here that osteoclast-specific A20 knockout mice develop severe osteoporosis, but not inflammatory arthritis. In vitro, osteoclast precursor cells from A20 deficient mice are hyper-responsive to RANKL-induced osteoclastogenesis. Mechanistically, we show that A20 is recruited to the RANK receptor complex within minutes of ligand binding, where it restrains NF-κB activation independently of its deubiquitinating activity but through its zinc finger (ZnF) 4 and 7 ubiquitin-binding functions. Together, these data demonstrate that A20 acts as a regulator of RANK-induced NF-κB signaling to control osteoclast differentiation, assuring proper bone development and turnover.
Asunto(s)
FN-kappa B , Humanos , Animales , RatonesRESUMEN
BACKGROUND: Receptor-interacting protein kinase 1 (RIPK1) is a key mediator of regulated cell death (including apoptosis and necroptosis) and inflammation, both drivers of COPD pathogenesis. We aimed to define the contribution of RIPK1 kinase-dependent cell death and inflammation in the pathogenesis of COPD. METHODS: We assessed RIPK1 expression in single-cell RNA sequencing (RNA-seq) data from human and mouse lungs, and validated RIPK1 levels in lung tissue of COPD patients via immunohistochemistry. Next, we assessed the consequences of genetic and pharmacological inhibition of RIPK1 kinase activity in experimental COPD, using Ripk1 S25D/S25D kinase-deficient mice and the RIPK1 kinase inhibitor GSK'547. RESULTS: RIPK1 expression increased in alveolar type 1 (AT1), AT2, ciliated and neuroendocrine cells in human COPD. RIPK1 protein levels were significantly increased in airway epithelium of COPD patients compared with never-smokers and smokers without airflow limitation. In mice, exposure to cigarette smoke (CS) increased Ripk1 expression similarly in AT2 cells, and further in alveolar macrophages and T-cells. Genetic and/or pharmacological inhibition of RIPK1 kinase activity significantly attenuated airway inflammation upon acute and subacute CS exposure, as well as airway remodelling, emphysema, and apoptotic and necroptotic cell death upon chronic CS exposure. Similarly, pharmacological RIPK1 kinase inhibition significantly attenuated elastase-induced emphysema and lung function decline. Finally, RNA-seq on lung tissue of CS-exposed mice revealed downregulation of cell death and inflammatory pathways upon pharmacological RIPK1 kinase inhibition. CONCLUSIONS: RIPK1 kinase inhibition is protective in experimental models of COPD and may represent a novel promising therapeutic approach.
Asunto(s)
Enfisema , Enfermedad Pulmonar Obstructiva Crónica , Enfisema Pulmonar , Humanos , Ratones , Animales , Pulmón , Muerte Celular , Inflamación/metabolismo , Ratones Endogámicos C57BL , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismoRESUMEN
A20 is a potent anti-inflammatory molecule, and mutations in TNFAIP3, the gene encoding A20, are associated with a wide panel of inflammatory pathologies, both in human and mouse. The anti-inflammatory properties of A20 are commonly attributed to its ability to suppress inflammatory NF-κB signaling by functioning as a ubiquitin-editing enzyme. However, A20 also protects cells from death, independently of NF-κB regulation, and recent work has demonstrated that cell death may drive some of the inflammatory conditions caused by A20 deficiency. Adding to the fact that the protective role of A20 does not primarily rely on its catalytic activities, these findings shed new light on A20 biology.
Asunto(s)
Inflamación , FN-kappa B , Humanos , Animales , Ratones , FN-kappa B/metabolismo , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/metabolismo , Inflamación/metabolismo , Transducción de Señal , Muerte Celular , AntiinflamatoriosRESUMEN
TNF is a master pro-inflammatory cytokine. Activation of TNFR1 by TNF can result in both RIPK1-independent apoptosis and RIPK1 kinase-dependent apoptosis or necroptosis. These cell death outcomes are regulated by two distinct checkpoints during TNFR1 signaling. TNF-mediated NF-κB-dependent induction of pro-survival or anti-apoptotic molecules is a well-known late checkpoint in the pathway, protecting cells from RIPK1-independent death. On the other hand, the molecular mechanism regulating the contribution of RIPK1 to cell death is far less understood. We demonstrate here that the IKK complex phosphorylates RIPK1 at TNFR1 complex I and protects cells from RIPK1 kinase-dependent death, independent of its function in NF-κB activation. We provide in vitro and in vivo evidence that inhibition of IKKα/IKKß or its upstream activators sensitizes cells to death by inducing RIPK1 kinase-dependent apoptosis or necroptosis. We therefore report on an unexpected, NF-κB-independent role for the IKK complex in protecting cells from RIPK1-dependent death downstream of TNFR1.
Asunto(s)
Quinasa I-kappa B/metabolismo , FN-kappa B/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Caspasa 8/metabolismo , Muerte Celular , Línea Celular , Embrión de Mamíferos/citología , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Ratones , Fosforilación , Transducción de Señal , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Immunogenic cell death (ICD) occurs when a dying cell releases cytokines and damage-associated molecular patterns, acting as adjuvants, and expresses Ags that induce a specific antitumor immune response. ICD is studied mainly in the context of regulated cell death pathways, especially caspase-mediated apoptosis marked by endoplasmic reticulum stress and calreticulin exposure and, more recently, also in relation to receptor-interacting protein kinase-driven necroptosis, whereas unregulated cell death like accidental necrosis is nonimmunogenic. Importantly, the murine cancer cell lines used in ICD studies often express virally derived peptides that are recognized by the immune system as tumor-associated Ags. However, it is unknown how different cell death pathways may affect neoepitope cross-presentation and Ag recognition of cancer cells. We used a prophylactic tumor vaccination model and observed that both apoptotic and necroptotic colon carcinoma CT26 cells efficiently immunized mice against challenge with a breast cancer cell line that expresses the same immunodominant tumor Ag, AH1, but only necroptotic CT26 cells would mount an immune response against CT26-specific neoepitopes. By CRISPR/Cas9 genome editing, we knocked out AH1 and saw that only necroptotic CT26 cells were still able to protect mice against tumor challenge. Hence, in this study, we show that endogenous AH1 tumor Ag expression can mask the strength of immunogenicity induced by different cell death pathways and that upon knockout of AH1, necroptosis was more immunogenic than apoptosis in a prophylactic tumor vaccination model. This work highlights necroptosis as a possible preferred ICD form over apoptosis in the treatment of cancer.
Asunto(s)
Antígenos de Neoplasias/inmunología , Apoptosis/inmunología , Epítopos Inmunodominantes/inmunología , Necroptosis/inmunología , Neoplasias Experimentales/inmunología , Animales , Línea Celular Tumoral , Femenino , Ratones , Ratones Endogámicos BALB CRESUMEN
Rationale: Respiratory syncytial virus (RSV) bronchiolitis causes significant infant mortality. Bronchiolitis is characterized by airway epithelial cell (AEC) death; however, the mode of death remains unknown.Objectives: To determine whether necroptosis contributes to RSV bronchiolitis pathogenesis via HMGB1 (high mobility group box 1) release.Methods: Nasopharyngeal samples were collected from children presenting to the hospital with acute respiratory infection. Primary human AECs and neonatal mice were inoculated with RSV and murine Pneumovirus, respectively. Necroptosis was determined via viability assays and immunohistochemistry for RIPK1 (receptor-interacting protein kinase-1), MLKL (mixed lineage kinase domain-like pseudokinase) protein, and caspase-3. Necroptosis was blocked using pharmacological inhibitors and RIPK1 kinase-dead knockin mice.Measurements and Main Results: HMGB1 levels were elevated in nasopharyngeal samples of children with acute RSV infection. RSV-induced epithelial cell death was associated with increased phosphorylated RIPK1 and phosphorylated MLKL but not active caspase-3 expression. Inhibition of RIPK1 or MLKL attenuated RSV-induced HMGB1 translocation and release, and lowered viral load. MLKL inhibition increased active caspase-3 expression in a caspase-8/9-dependent manner. In susceptible mice, Pneumovirus infection upregulated RIPK1 and MLKL expression in the airway epithelium at 8 to 10 days after infection, coinciding with AEC sloughing, HMGB1 release, and neutrophilic inflammation. Genetic or pharmacological inhibition of RIPK1 or MLKL attenuated these pathologies, lowered viral load, and prevented type 2 inflammation and airway remodeling. Necroptosis inhibition in early life ameliorated asthma progression induced by viral or allergen challenge in later life.Conclusions: Pneumovirus infection induces AEC necroptosis. Inhibition of necroptosis may be a viable strategy to limit the severity of viral bronchiolitis and break its nexus with asthma.
Asunto(s)
Bronquiolitis/virología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Proteína HMGB1/metabolismo , Necroptosis , Mucosa Respiratoria/citología , Infecciones por Virus Sincitial Respiratorio/metabolismo , Animales , Preescolar , Humanos , Lactante , Ratones , Estudios ProspectivosRESUMEN
Receptor interacting protein kinase 1 (RIPK1) has an essential role in the signalling triggered by death receptors and pattern recognition receptors. RIPK1 is believed to function as a node driving NF-κB-mediated cell survival and inflammation as well as caspase-8 (CASP8)-dependent apoptotic or RIPK3/MLKL-dependent necroptotic cell death. The physiological relevance of this dual function has remained elusive because of the perinatal death of RIPK1 full knockout mice. To circumvent this problem, we generated RIPK1 conditional knockout mice, and show that mice lacking RIPK1 in intestinal epithelial cells (IECs) spontaneously develop severe intestinal inflammation associated with IEC apoptosis leading to early death. This early lethality was rescued by antibiotic treatment, MYD88 deficiency or tumour-necrosis factor (TNF) receptor 1 deficiency, demonstrating the importance of commensal bacteria and TNF in the IEC Ripk1 knockout phenotype. CASP8 deficiency, but not RIPK3 deficiency, rescued the inflammatory phenotype completely, indicating the indispensable role of RIPK1 in suppressing CASP8-dependent apoptosis but not RIPK3-dependent necroptosis in the intestine. RIPK1 kinase-dead knock-in mice did not exhibit any sign of inflammation, suggesting that RIPK1-mediated protection resides in its kinase-independent platform function. Depletion of RIPK1 in intestinal organoid cultures sensitized them to TNF-induced apoptosis, confirming the in vivo observations. Unexpectedly, TNF-mediated NF-κB activation remained intact in these organoids. Our results demonstrate that RIPK1 is essential for survival of IECs, ensuring epithelial homeostasis by protecting the epithelium from CASP8-mediated IEC apoptosis independently of its kinase activity and NF-κB activation.
Asunto(s)
Apoptosis , Células Epiteliales/citología , Células Epiteliales/metabolismo , Epitelio/metabolismo , Homeostasis , Mucosa Intestinal/metabolismo , Intestinos/citología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Animales , Antibacterianos/farmacología , Apoptosis/efectos de los fármacos , Caspasa 8/genética , Caspasa 8/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/patología , Epitelio/efectos de los fármacos , Epitelio/patología , Femenino , Eliminación de Gen , Homeostasis/efectos de los fármacos , Inflamación/metabolismo , Inflamación/patología , Intestinos/efectos de los fármacos , Intestinos/patología , Masculino , Ratones , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/deficiencia , FN-kappa B/metabolismo , Necrosis , Organoides/citología , Organoides/efectos de los fármacos , Organoides/enzimología , Organoides/metabolismo , Proteínas Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/deficiencia , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Receptores Tipo I de Factores de Necrosis Tumoral/deficiencia , Análisis de Supervivencia , Factores de Necrosis Tumoral/farmacologíaRESUMEN
BACKGROUND: Three pregnancies with male offspring in one family were complicated by severe polyhydramnios and prematurity. One fetus died; the other two had transient massive salt-wasting and polyuria reminiscent of antenatal Bartter's syndrome. METHODS: To uncover the molecular cause of this possibly X-linked disease, we performed whole-exome sequencing of DNA from two members of the index family and targeted gene analysis of other members of this family and of six additional families with affected male fetuses. We also evaluated a series of women with idiopathic polyhydramnios who were pregnant with male fetuses. We performed immunohistochemical analysis, knockdown and overexpression experiments, and protein-protein interaction studies. RESULTS: We identified a mutation in MAGED2 in each of the 13 infants in our analysis who had transient antenatal Bartter's syndrome. MAGED2 encodes melanoma-associated antigen D2 (MAGE-D2) and maps to the X chromosome. We also identified two different MAGED2 mutations in two families with idiopathic polyhydramnios. Four patients died perinatally, and 11 survived. The initial presentation was more severe than in known types of antenatal Bartter's syndrome, as reflected by an earlier onset of polyhydramnios and labor. All symptoms disappeared spontaneously during follow-up in the infants who survived. We showed that MAGE-D2 affects the expression and function of the sodium chloride cotransporters NKCC2 and NCC (key components of salt reabsorption in the distal renal tubule), possibly through adenylate cyclase and cyclic AMP signaling and a cytoplasmic heat-shock protein. CONCLUSIONS: We found that MAGED2 mutations caused X-linked polyhydramnios with prematurity and a severe but transient form of antenatal Bartter's syndrome. MAGE-D2 is essential for fetal renal salt reabsorption, amniotic fluid homeostasis, and the maintenance of pregnancy. (Funded by the University of Groningen and others.).
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Antígenos de Neoplasias/genética , Síndrome de Bartter/genética , Enfermedades Genéticas Ligadas al Cromosoma X , Mutación , Polihidramnios/genética , Femenino , Muerte Fetal , Enfermedades Fetales/genética , Feto/metabolismo , Humanos , Riñón/metabolismo , Masculino , Linaje , Embarazo , Nacimiento Prematuro/genética , Análisis de Secuencia de ADN , Simportadores del Cloruro de Sodio/metabolismo , Miembro 1 de la Familia de Transportadores de Soluto 12/metabolismoRESUMEN
TNF is a master proinflammatory cytokine whose pathogenic role in inflammatory disorders has long been attributed to induction of proinflammatory mediators. TNF also activates cell survival and death pathways, and recent studies demonstrated that TNF also causes inflammation by inducing cell death. The default response of most cells to TNF is survival and NF-κB-mediated upregulation of prosurvival molecules is a well-documented protective mechanism downstream of TNFR1. Recent studies revealed the existence of an NF-κB-independent cell death checkpoint that restricts cell demise by inactivating RIPK1. Disruption of this checkpoint leads to RIPK1 kinase-dependent death and causes inflammation in vivo. These revelations bring complexity to the control of TNF-induced cell death, and suggest clinical benefit of RIPK1 inhibitors in TNF-driven human inflammatory disorders.
Asunto(s)
FN-kappa B/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Transducción de Señal , Animales , Biomarcadores , Muerte Celular , Citocinas/metabolismo , Regulación de la Expresión Génica , Humanos , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Mediadores de Inflamación/metabolismo , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/metabolismo , Unión Proteica , Procesamiento Proteico-PostraduccionalRESUMEN
In this issue of Molecular Cell, Tenev et al. and Feoktistova et al. describe the Ripoptosome, a cytosolic death-inducing RIP1-, FADD-, and caspase-8-containing complex that spontaneously assembles upon cIAP depletion, challenging the view that such complexes exclusively originate from receptor activation.
RESUMEN
Cellular inhibitor of apoptosis proteins (cIAPs) block apoptosis, but their physiological functions are still under investigation. Here, we report that cIAP1 and cIAP2 are E3 ubiquitin ligases that are required for receptor-interacting protein 2 (RIP2) ubiquitination and for nucleotide-binding and oligomerization (NOD) signaling. Macrophages derived from Birc2(-/-) or Birc3(-/-) mice, or colonocytes depleted of cIAP1 or cIAP2 by RNAi, were defective in NOD signaling and displayed sharp attenuation of cytokine and chemokine production. This blunted response was observed in vivo when Birc2(-/-) and Birc3(-/-) mice were challenged with NOD agonists. Defects in NOD2 signaling are associated with Crohn's disease, and muramyl dipeptide (MDP) activation of NOD2 signaling protects mice from experimental colitis. Here, we show that administration of MDP protected wild-type but not Ripk2(-/-) or Birc3(-/-) mice from colitis, confirming the role of the cIAPs in NOD2 signaling in vivo. This discovery provides therapeutic opportunities in the treatment of NOD-dependent immunologic and inflammatory diseases.
Asunto(s)
Inmunidad Innata , Proteínas Inhibidoras de la Apoptosis/metabolismo , Proteína Adaptadora de Señalización NOD1/metabolismo , Proteína Adaptadora de Señalización NOD2/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Acetilmuramil-Alanil-Isoglutamina/farmacología , Animales , Apoptosis/inmunología , Proteína 3 que Contiene Repeticiones IAP de Baculovirus , Colitis/enzimología , Colitis/inmunología , Colitis/patología , Citocinas/inmunología , Citocinas/metabolismo , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Adaptadora de Señalización NOD1/agonistas , Proteína Adaptadora de Señalización NOD1/inmunología , Proteína Adaptadora de Señalización NOD2/agonistas , Proteína Adaptadora de Señalización NOD2/inmunología , Proteína Serina-Treonina Quinasa 2 de Interacción con Receptor , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/inmunología , Receptores de Reconocimiento de Patrones/agonistas , Receptores de Reconocimiento de Patrones/inmunología , Receptores de Reconocimiento de Patrones/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/inmunología , Ubiquitina-Proteína Ligasas , Ubiquitinación/inmunologíaRESUMEN
As indicated by their name, members of the Inhibitor of APoptosis (IAP) family were first believed to be functionally restricted to apoptosis inhibition. It is now clear that IAPs have a much wider spectrum of action, and recent studies even suggest that some of its members primarily regulate inflammatory responses. Inflammation, the first response of the immune system to infection or tissue injury, is highly regulated by ubiquitylation - a posttranslational modification of proteins with various consequences. In this review, we focus on the recently reported functions of XIAP, cIAP1 and cIAP2 as ubiquitin ligases regulating innate immunity and inflammation.
Asunto(s)
Inmunidad Innata , Inflamación/inmunología , Proteínas Inhibidoras de la Apoptosis/inmunología , Ubiquitina-Proteína Ligasas/inmunología , Animales , Humanos , Transducción de SeñalRESUMEN
BACKGROUND & AIMS: The severity of liver diseases is exacerbated by the death of hepatocytes, which can be induced by the sensing of pathogen associated molecular patterns (PAMPs) derived from the gut microbiota. The molecular mechanisms regulating these cell death pathways are poorly documented. In this study, we investigated the role of the receptor interacting protein kinase 1 (RIPK1), a protein known to regulate cell fate decisions, in the death of hepatocytes using two in vivo models of PAMP-induced hepatitis. METHODS: Hepatitis was induced in mice by independent injections of two different bacterial PAMPs: lipopolysaccharide (LPS) and unmethylated CpG oligodeoxynucleotide (CpG-DNA) motifs. The role of RIPK1 was evaluated by using mice specifically lacking RIPK1 in liver parenchymal cells (Ripk1LPC-KO). Administration of liposome-encapsulated clodronate served to investigate the role of Kupffer cells in the establishment of the disease. Etanercept, a tumor necrosis factor (TNF)-decoy receptor, was used to study the contribution of TNF-α during LPS-mediated liver injury. RESULTS: Whereas RIPK1 deficiency in liver parenchymal cells did not trigger basal hepatolysis, it greatly sensitized hepatocytes to apoptosis and liver damage following a single injection of LPS or CpG-DNA. Importantly, hepatocyte death was prevented by previous macrophage depletion or by TNF inhibition. CONCLUSIONS: Our data highlight the pivotal function of RIPK1 in maintaining liver homeostasis in conditions of macrophage-induced TNF burst in response to PAMPs sensing. LAY SUMMARY: Excessive death of hepatocytes is a characteristic of liver injury. A new programmed cell death pathway has been described involving upstream death ligands such as TNF and downstream kinases such as RIPK1. Here, we show that in the presence of LPS liver induced hepatic injury was due to secretion of TNF by liver macrophages, and that RIPK1 acts as a powerful protector of hepatocyte death. This newly identified pathway in the liver may be helpful in the management of patients to predict their risk of developing acute liver failure.
Asunto(s)
Hepatitis Animal/metabolismo , Hepatitis Animal/patología , Hepatocitos/metabolismo , Hepatocitos/patología , Macrófagos del Hígado/metabolismo , Macrófagos del Hígado/patología , Moléculas de Patrón Molecular Asociado a Patógenos/toxicidad , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Apoptosis/efectos de los fármacos , Hepatitis Animal/etiología , Hepatocitos/efectos de los fármacos , Macrófagos del Hígado/efectos de los fármacos , Lipopolisacáridos/toxicidad , Ratones , Ratones Noqueados , Proteína Serina-Treonina Quinasas de Interacción con Receptores/deficiencia , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genéticaRESUMEN
Tumor necrosis factor (TNF) is a master pro-inflammatory cytokine, and inappropriate TNF signaling is implicated in the pathology of many inflammatory diseases. Ligation of TNF to its receptor TNFR1 induces the transient formation of a primary membrane-bound signaling complex, known as complex I, that drives expression of pro-survival genes. Defective complex I activation results in induction of cell death, in the form of apoptosis or necroptosis. This switch occurs via internalization of complex I components and assembly and activation of secondary cytoplasmic death complexes, respectively known as complex II and necrosome. In this review, we discuss the crucial regulatory functions of ubiquitination-a post-translational protein modification consisting of the covalent attachment of ubiquitin, and multiples thereof, to target proteins-to the various steps of TNFR1 signaling leading to necroptosis.
Asunto(s)
Apoptosis/fisiología , Necrosis/patología , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Ubiquitinación/fisiología , Animales , Ratones , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Transducción de SeñalRESUMEN
The inhibitor of apoptosis (IAP) family of proteins enhances cell survival through mechanisms that remain uncertain. In this report, we show that cIAP1 and cIAP2 promote cancer cell survival by functioning as E3 ubiquitin ligases that maintain constitutive ubiquitination of the RIP1 adaptor protein. We demonstrate that AEG40730, a compound modeled on BIR-binding tetrapeptides, binds to cIAP1 and cIAP2, facilitates their autoubiquitination and proteosomal degradation, and causes a dramatic reduction in RIP1 ubiquitination. We show that cIAP1 and cIAP2 directly ubiquitinate RIP1 and induce constitutive RIP1 ubiquitination in cancer cells and demonstrate that constitutively ubiquitinated RIP1 associates with the prosurvival kinase TAK1. When deubiquitinated by AEG40730 treatment, RIP1 binds caspase-8 and induces apoptosis. These findings provide insights into the function of the IAPs and provide new therapeutic opportunities in the treatment of cancer.
Asunto(s)
Proteínas Inhibidoras de la Apoptosis/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Proteínas de Unión al ARN/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Proteína 3 que Contiene Repeticiones IAP de Baculovirus , Caspasa 8/metabolismo , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Femenino , Humanos , Neoplasias Ováricas , Sulfonamidas/farmacologíaRESUMEN
MAGED1, NECDIN and MAGEL2 are members of the MAGE gene family. The latter two of these genes have been involved in Prader-Willi syndrome (PWS), which includes hyperphagia, repetitive and compulsive behaviors, and cognitive impairment. Here, we show that Maged1-deficient mice develop progressive obesity associated with hyperphagia and reduced motor activity. Loss of Maged1 also results in a complex behavioral syndrome that includes reduced social interactions and memory, deficient sexual behavior, as well as increased anxiety and self-grooming. Oxytocin (OT), which is produced in the hypothalamus, can act as a neurotransmitter that reduces anxiety, promotes social behaviors and regulates food intake. Growing evidences indicate that OT is involved in autism. We found that Maged1 mutants showed a severe reduction in the levels of mature OT, but not of its precursors, in the hypothalamus. Moreover, the administration of OT rescued the deficit in social memory of these mice. We conclude that Maged1 is required for OT processing or stability. A decrease in mature OT levels in Maged1 mutants affects social interactions and possibly other behavioral processes. Our observations suggest that, in human, MAGED1 could play a role in autism or cause a neurodevelopmental condition that is reminiscent of the PWS.
Asunto(s)
Proteínas de Neoplasias , Oxitocina , Síndrome de Prader-Willi , Conducta Sexual Animal , Animales , Ansiedad/genética , Ansiedad/metabolismo , Ansiedad/fisiopatología , Trastorno Autístico/metabolismo , Trastorno Autístico/fisiopatología , Humanos , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Relaciones Interpersonales , Ratones , Proteínas de Neoplasias/deficiencia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Obesidad/genética , Obesidad/metabolismo , Obesidad/fisiopatología , Oxitocina/administración & dosificación , Oxitocina/biosíntesis , Oxitocina/metabolismo , Síndrome de Prader-Willi/genética , Síndrome de Prader-Willi/metabolismo , Síndrome de Prader-Willi/fisiopatología , Conducta Sexual Animal/efectos de los fármacos , Conducta Sexual Animal/fisiologíaRESUMEN
OBJECTIVE: Receptor-interacting protein kinase 1 (RIPK1) orchestrates the decision between cell survival and cell death in response to tumor necrosis factor (TNF) and other cytokines. Whereas the scaffolding function of RIPK1 is crucial to prevent TNF-induced apoptosis and necroptosis, its kinase activity is required for necroptosis and partially for apoptosis. Although TNF is a proinflammatory cytokine associated with ß-cell loss in diabetes, the mechanism by which TNF induces ß-cell demise remains unclear. METHODS: Here, we dissected the contribution of RIPK1 scaffold versus kinase functions to ß-cell death regulation using mice lacking RIPK1 specifically in ß-cells (Ripk1ß-KO mice) or expressing a kinase-dead version of RIPK1 (Ripk1D138N mice), respectively. These mice were challenged with streptozotocin, a model of autoimmune diabetes. Moreover, Ripk1ß-KO mice were further challenged with a high-fat diet to induce hyperglycemia. For mechanistic studies, pancreatic islets were subjected to various killing and sensitising agents. RESULTS: Inhibition of RIPK1 kinase activity (Ripk1D138N mice) did not affect the onset and progression of hyperglycemia in a type 1 diabetes model. Moreover, the absence of RIPK1 expression in ß-cells did not affect normoglycemia under basal conditions or hyperglycemia under diabetic challenges. Ex vivo, primary pancreatic islets are not sensitised to TNF-induced apoptosis and necroptosis in the absence of RIPK1. Intriguingly, we found that pancreatic islets display high levels of the antiapoptotic cellular FLICE-inhibitory protein (cFLIP) and low levels of apoptosis (Caspase-8) and necroptosis (RIPK3) components. Cycloheximide treatment, which led to a reduction in cFLIP levels, rendered primary islets sensitive to TNF-induced cell death which was fully blocked by caspase inhibition. CONCLUSIONS: Unlike in many other cell types (e.g., epithelial, and immune), RIPK1 is not required for cell death regulation in ß-cells under physiological conditions or diabetic challenges. Moreover, in vivo and in vitro evidence suggest that pancreatic ß-cells do not undergo necroptosis but mainly caspase-dependent death in response to TNF. Last, our results show that ß-cells have a distinct mode of regulation of TNF-cytotoxicity that is independent of RIPK1 and that may be highly dependent on cFLIP.