RESUMO
The linear ubiquitin chain assembly complex (LUBAC) is required for optimal gene activation and prevention of cell death upon activation of immune receptors, including TNFR1 1 . Deficiency in the LUBAC components SHARPIN or HOIP in mice results in severe inflammation in adulthood or embryonic lethality, respectively, owing to deregulation of TNFR1-mediated cell death2-8. In humans, deficiency in the third LUBAC component HOIL-1 causes autoimmunity and inflammatory disease, similar to HOIP deficiency, whereas HOIL-1 deficiency in mice was reported to cause no overt phenotype9-11. Here we show, by creating HOIL-1-deficient mice, that HOIL-1 is as essential for LUBAC function as HOIP, albeit for different reasons: whereas HOIP is the catalytically active component of LUBAC, HOIL-1 is required for LUBAC assembly, stability and optimal retention in the TNFR1 signalling complex, thereby preventing aberrant cell death. Both HOIL-1 and HOIP prevent embryonic lethality at mid-gestation by interfering with aberrant TNFR1-mediated endothelial cell death, which only partially depends on RIPK1 kinase activity. Co-deletion of caspase-8 with RIPK3 or MLKL prevents cell death in Hoil-1-/- (also known as Rbck1-/-) embryos, yet only the combined loss of caspase-8 with MLKL results in viable HOIL-1-deficient mice. Notably, triple-knockout Ripk3-/-Casp8-/-Hoil-1-/- embryos die at late gestation owing to haematopoietic defects that are rescued by co-deletion of RIPK1 but not MLKL. Collectively, these results demonstrate that both HOIP and HOIL-1 are essential LUBAC components and are required for embryogenesis by preventing aberrant cell death. Furthermore, they reveal that when LUBAC and caspase-8 are absent, RIPK3 prevents RIPK1 from inducing embryonic lethality by causing defects in fetal haematopoiesis.
Assuntos
Proteínas de Transporte/metabolismo , Morte Celular , Desenvolvimento Embrionário , Hematopoese , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Animais , Proteínas de Transporte/química , Proteínas de Transporte/genética , Caspase 8/genética , Caspase 8/metabolismo , Morte Celular/genética , Perda do Embrião/genética , Desenvolvimento Embrionário/genética , Células Endoteliais/citologia , Feminino , Hematopoese/genética , Camundongos , Camundongos Endogâmicos C57BL , Domínios Proteicos , Proteínas Quinases/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/deficiência , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genéticaRESUMO
Linear ubiquitination is a key posttranslational modification that regulates immune signaling and cell death pathways, notably tumor necrosis factor receptor 1 (TNFR1) signaling. The only known enzyme complex capable of forming linear ubiquitin chains under native conditions to date is the linear ubiquitin chain assembly complex, of which the catalytic core component is heme-oxidized iron regulatory protein 2 ubiquitin ligase-1-interacting protein (HOIP). To understand the underlying mechanisms of maintenance of liver homeostasis and the role of linear ubiquitination specifically in liver parenchymal cells, we investigated the physiological role of HOIP in the liver parenchyma. To do so, we created mice harboring liver parenchymal cell-specific deletion of HOIP (HoipΔhep mice) by crossing Hoip-floxed mice with albumin-Cre mice. HOIP deficiency in liver parenchymal cells triggered tumorigenesis at 18 months of age preceded by spontaneous hepatocyte apoptosis and liver inflammation within the first month of life. In line with the emergence of inflammation, HoipΔhep mice displayed enhanced liver regeneration and DNA damage. In addition, consistent with increased apoptosis, HOIP-deficient hepatocytes showed enhanced caspase activation and endogenous formation of a death-inducing signaling complex which activated caspase-8. Unexpectedly, exacerbated caspase activation and apoptosis were not dependent on TNFR1, whereas ensuing liver inflammation and tumorigenesis were promoted by TNFR1 signaling. CONCLUSION: The linear ubiquitin chain assembly complex serves as a previously undescribed tumor suppressor in the liver, restraining TNFR1-independent apoptosis in hepatocytes which, in its absence, is causative of TNFR1-mediated inflammation, resulting in hepatocarcinogenesis. (Hepatology 2017;65:1963-1978).
Assuntos
Apoptose/efeitos dos fármacos , Hepatite/imunologia , Neoplasias Hepáticas/patologia , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/farmacologia , Animais , Apoptose/fisiologia , Carcinogênese/patologia , Caspase 8/metabolismo , Morte Celular/efeitos dos fármacos , Modelos Animais de Doenças , Hepatite/patologia , Hepatócitos/metabolismo , Hepatócitos/patologia , Neoplasias Hepáticas/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Distribuição Aleatória , Valores de Referência , Células Tumorais Cultivadas , Ubiquitina-Proteína Ligases/deficiência , Ubiquitinação/efeitos dos fármacosRESUMO
The linear ubiquitin chain assembly complex (LUBAC), composed of HOIP, HOIL-1 and SHARPIN, is required for optimal TNF-mediated gene activation and to prevent cell death induced by TNF. Here, we demonstrate that keratinocyte-specific deletion of HOIP or HOIL-1 (E-KO) results in severe dermatitis causing postnatal lethality. We provide genetic and pharmacological evidence that the postnatal lethal dermatitis in HoipE-KO and Hoil-1E-KO mice is caused by TNFR1-induced, caspase-8-mediated apoptosis that occurs independently of the kinase activity of RIPK1. In the absence of TNFR1, however, dermatitis develops in adulthood, triggered by RIPK1-kinase-activity-dependent apoptosis and necroptosis. Strikingly, TRAIL or CD95L can redundantly induce this disease-causing cell death, as combined loss of their respective receptors is required to prevent TNFR1-independent dermatitis. These findings may have implications for the treatment of patients with mutations that perturb linear ubiquitination and potentially also for patients with inflammation-associated disorders that are refractory to inhibition of TNF alone.
Assuntos
Proteínas de Transporte/metabolismo , Dermatite/metabolismo , Proteína Ligante Fas/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Animais Recém-Nascidos , Proteínas de Transporte/genética , Morte Celular/efeitos dos fármacos , Morte Celular/genética , Células Cultivadas , Dermatite/genética , Peptídeos e Proteínas de Sinalização Intracelular , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/patologia , Ubiquitina-Proteína Ligases/genéticaRESUMO
Linear ubiquitination is crucial for innate and adaptive immunity. The linear ubiquitin chain assembly complex (LUBAC), consisting of HOIL-1, HOIP, and SHARPIN, is the only known ubiquitin ligase that generates linear ubiquitin linkages. HOIP is the catalytically active LUBAC component. Here, we show that both constitutive and Tie2-Cre-driven HOIP deletion lead to aberrant endothelial cell death, resulting in defective vascularization and embryonic lethality at midgestation. Ablation of tumor necrosis factor receptor 1 (TNFR1) prevents cell death, vascularization defects, and death at midgestation. HOIP-deficient cells are more sensitive to death induction by both tumor necrosis factor (TNF) and lymphotoxin-α (LT-α), and aberrant complex-II formation is responsible for sensitization to TNFR1-mediated cell death in the absence of HOIP. Finally, we show that HOIP's catalytic activity is necessary for preventing TNF-induced cell death. Hence, LUBAC and its linear-ubiquitin-forming activity are required for maintaining vascular integrity during embryogenesis by preventing TNFR1-mediated endothelial cell death.