RESUMO
The necrosome is a protein complex required for signaling in cells that results in necroptosis, which is also dependent on tumor necrosis factor receptor (TNF-R) signaling. TNFα promotes necroptosis, and its expression is facilitated by mitogen-activated protein (MAP) kinase-activated protein kinase 2 (MK2) but is inhibited by the RNA-binding protein tristetraprolin (TTP, encoded by the Zfp36 gene). We have stimulated murine macrophages from WT, MyD88-/-, Trif-/-, MyD88-/-Trif-/-, MK2-/-, and Zfp36-/- mice with graded doses of lipopolysaccharide (LPS) and various inhibitors to evaluate the role of various genes in Toll-like receptor 4 (TLR4)-induced necroptosis. Necrosome signaling, cytokine production, and cell death were evaluated by immunoblotting, ELISA, and cell death assays, respectively. We observed that during TLR4 signaling, necrosome activation is mediated through the adaptor proteins MyD88 and TRIF, and this is inhibited by MK2. In the absence of MK2-mediated necrosome activation, lipopolysaccharide-induced TNFα expression was drastically reduced, but MK2-deficient cells became highly sensitive to necroptosis even at low TNFα levels. In contrast, during tonic TLR4 signaling, WT cells did not undergo necroptosis, even when MK2 was disabled. Of note, necroptosis occurred only in the absence of TTP and was mediated by the expression of TNFα and activation of JUN N-terminal kinase (JNK). These results reveal that TTP plays an important role in inhibiting TNFα/JNK-induced necrosome signaling and resultant cytotoxicity.
Assuntos
Necroptose , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , Tristetraprolina/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Caspase 8/química , Caspase 8/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/deficiência , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Necroptose/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Tristetraprolina/deficiência , Tristetraprolina/genética , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Traditionally viewed as an autodigestive pathway, autophagy also facilitates cellular secretion; however, the mechanisms underlying these processes remain unclear. Here, we demonstrate that components of the autophagy machinery specify secretion within extracellular vesicles (EVs). Using a proximity-dependent biotinylation proteomics strategy, we identify 200 putative targets of LC3-dependent secretion. This secretome consists of a highly interconnected network enriched in RNA-binding proteins (RBPs) and EV cargoes. Proteomic and RNA profiling of EVs identifies diverse RBPs and small non-coding RNAs requiring the LC3-conjugation machinery for packaging and secretion. Focusing on two RBPs, heterogeneous nuclear ribonucleoprotein K (HNRNPK) and scaffold-attachment factor B (SAFB), we demonstrate that these proteins interact with LC3 and are secreted within EVs enriched with lipidated LC3. Furthermore, their secretion requires the LC3-conjugation machinery, neutral sphingomyelinase 2 (nSMase2) and LC3-dependent recruitment of factor associated with nSMase2 activity (FAN). Hence, the LC3-conjugation pathway controls EV cargo loading and secretion.
Assuntos
Autofagossomos/metabolismo , Autofagia/genética , Vesículas Extracelulares/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Ligação a RNA/genética , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Autofagossomos/química , Proteína 7 Relacionada à Autofagia/deficiência , Proteína 7 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/deficiência , Proteínas Relacionadas à Autofagia/genética , Transporte Biológico , Biotinilação , Vesículas Extracelulares/química , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/química , Lisossomos/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteômica/métodos , Células RAW 264.7 , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/classificação , Proteínas de Ligação a RNA/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismoRESUMO
Postoperative ileus (POI) is triggered by an innate immune response in the muscularis externa (ME) and is accompanied by bacterial translocation. Bacteria can trigger an innate immune response via toll-like receptor (TLR) activation, but the latter's contribution to POI has been disproved for several TLRs, including TLR2 and TLR4. Herein we investigated the role of double-stranded RNA detection via TLR3 and TIR-domain-containing adapter-inducing interferon-ß (TRIF) signaling pathway in POI. POI was induced by small bowel intestinal manipulation in wt, TRIF-/-, TLR3-/-, type I interferon receptor-/- and interferon-ß reporter mice, all on C57BL/6 background, and POI severity was quantified by gene expression analysis, gastrointestinal transit and leukocyte extravasation into the ME. TRIF/TLR3 deficiency reduced postoperative ME inflammation and prevented POI. With bone marrow transplantation, RNA-sequencing, flow cytometry and immunohistochemistry we revealed a distinct TLR3-expressing radio-resistant MHCIIhiCX3CR1- IBA-1+ resident macrophage population within the deep myenteric plexus. TLR3 deficiency in these cells, but not in MHCIIhiCX3CR1+ macrophages, reduced cytokine expression in POI. While this might not be an exclusive macrophage-privileged pathway, the TLR3/TRIF axis contributes to proinflammatory cytokine production in MHCIIhiCX3CR1- IBA-1+ macrophages during POI. Deficiency in TLR3/TRIF protects mice from POI. These data suggest that TLR3 antagonism may prevent POI in humans.
Assuntos
Íleus/etiologia , Macrófagos/imunologia , Complicações Pós-Operatórias/etiologia , Receptor 3 Toll-Like/imunologia , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/imunologia , Animais , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/imunologia , Modelos Animais de Doenças , Feminino , Expressão Gênica , Íleus/imunologia , Íleus/patologia , Imunidade Inata , Macrófagos/classificação , Macrófagos/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Plexo Mientérico/imunologia , Complicações Pós-Operatórias/imunologia , Complicações Pós-Operatórias/patologia , Tolerância a Radiação/imunologia , Receptor de Interferon alfa e beta/deficiência , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/imunologia , Transdução de Sinais/imunologia , Receptor 3 Toll-Like/deficiência , Receptor 3 Toll-Like/genética , Quimeras de Transplante/imunologiaRESUMO
BACKGROUND: Sepsis is a life-threatening condition often manifested as marked inflammation and severe coagulopathy. Toll-like receptors (TLRs) play a pivotal role in inflammation, organ dysfunction and mortality in animal sepsis. OBJECTIVES: To investigate the role of TLR signaling in mediating sepsis-induced coagulopathy (SIC) in a mouse model. METHODS: Polymicrobial sepsis was created by cecal ligation and puncture (CLP) or fecal slurry peritoneal injection. To quantify global clotting function, two viscoelastic assays were performed with rotational thromboelastometry, and the results were presented as maximum clot firmness (MCF): (a) EXTEM to test tissue factor (TF)-initiated clot formation; and (b) FIBTEM to test EXTEM in the presence of a platelet inhibitor, cytochalasin D. Plasma coagulation factors were quantified with ELISA. TF gene expression and protein expression were determined with real-time quantitative reverse transcription PCR and flow cytometry, respectively. RESULTS: Between 4 and 24 hours after CLP surgery, wild-type mice showed significant MCF reduction in both EXTEM and FIBTEM tests. This was accompanied by marked thrombocytopenia and a significant increase in the levels of plasminogen activator inhibitor-1, plasma TF, and D-dimer. In comparison, TLR2-/- and TLR7-/- CLP mice showed preserved MCF and platelet counts, and near-normal plasma TF levels. Bone marrow-derived macrophages treated with a TLR2 agonist Pam3cys-Ser-(Lys)4 (Pam3cys) or a TLR7 agonist (R837) showed marked increases in TF gene expression and protein expression. MicroRNA-146a, a newly identified proinflammatory mediator that is upregulated during sepsis, induced TF production via a TLR7-dependent mechanism. CONCLUSIONS: Murine sepsis leads to an increased procoagulant response, thrombocytopenia, and global coagulopathy. TLR2 and TLR7 play an important role in procoagulant production and in SIC.
Assuntos
Transtornos da Coagulação Sanguínea/etiologia , Coagulação Sanguínea , Macrófagos/metabolismo , Sepse/complicações , Receptor 2 Toll-Like/metabolismo , Receptor 3 Toll-Like/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Transtornos da Coagulação Sanguínea/sangue , Transtornos da Coagulação Sanguínea/metabolismo , Células Cultivadas , Citocinas/sangue , Modelos Animais de Doenças , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/deficiência , Fator 88 de Diferenciação Mieloide/genética , Sepse/sangue , Sepse/metabolismo , Transdução de Sinais , Trombocitopenia/sangue , Trombocitopenia/etiologia , Trombocitopenia/metabolismo , Receptor 2 Toll-Like/deficiência , Receptor 2 Toll-Like/genética , Receptor 3 Toll-Like/deficiência , Receptor 3 Toll-Like/genéticaRESUMO
Sortilin 1 (Sort1) is a member of the Vps10p domain intracellular trafficking receptor family. Genetic variations of the SORT1 gene are strongly associated with plasma cholesterol levels in humans. Recent studies have linked Sort1 to regulation of cholesterol metabolism in hepatocytes and pro-inflammatory response in macrophages, but the tissue-specific roles of Sort1 in lipid metabolism have not been well defined. We developed Sort1 floxed mice and investigated the development of Western diet (WD)-induced steatosis, hepatic inflammatory response, and hyperlipidemia in hepatocyte Sort1 KO mice and myeloid cell Sort1 KO mice. Our findings suggest that hepatocyte Sort1 deficiency attenuated diet-induced hepatic steatosis and hypercholesterolemia in mice. In contrast, myeloid Sort1 deficiency did not reduce hepatic cytokine expression or plasma cholesterol levels, but exacerbated hepatic triglyceride accumulation in WD-fed mice. Finally, we showed that treating WD-fed mice with an orally bioavailable Sort1 inhibitor, AF38469, decreased plasma cholesterol and hepatic cytokine expression. AF38469 treatment did not affect diet-induced obesity or insulin resistance, but was associated with reduced hepatic VLDL secretion and higher hepatic cholesterol 7α-hydrolase expression in WD-fed mice. In conclusion, findings from this study suggest that Sort1 loss-of-function in hepatocytes contributes to lower plasma cholesterol, and pharmacological inhibition of Sort1 attenuates diet-induced hypercholesterolemia in mice.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/antagonistas & inibidores , Proteínas Adaptadoras de Transporte Vesicular/genética , Colesterol/sangue , Dieta Ocidental/efeitos adversos , Técnicas de Inativação de Genes , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Animais , Glicemia/metabolismo , Citocinas/metabolismo , Jejum/sangue , Fígado Gorduroso/sangue , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Impaired lymphangiogenesis is a complication of chronic complex diseases, including diabetes. VEGF-C/VEGFR3 signaling promotes lymphangiogenesis, but how this pathway is affected in diabetes remains poorly understood. We previously demonstrated that loss of epsins 1 and 2 in lymphatic endothelial cells (LECs) prevented VEGF-C-induced VEGFR3 from endocytosis and degradation. Here, we report that diabetes attenuated VEGF-C-induced lymphangiogenesis in corneal micropocket and Matrigel plug assays in WT mice but not in mice with inducible lymphatic-specific deficiency of epsins 1 and 2 (LEC-iDKO). Consistently, LECs isolated from diabetic LEC-iDKO mice elevated in vitro proliferation, migration, and tube formation in response to VEGF-C over diabetic WT mice. Mechanistically, ROS produced in diabetes induced c-Src-dependent but VEGF-C-independent VEGFR3 phosphorylation, and upregulated epsins through the activation of transcription factor AP-1. Augmented epsins bound to and promoted degradation of newly synthesized VEGFR3 in the Golgi, resulting in reduced availability of VEGFR3 at the cell surface. Preclinically, the loss of lymphatic-specific epsins alleviated insufficient lymphangiogenesis and accelerated the resolution of tail edema in diabetic mice. Collectively, our studies indicate that inhibiting expression of epsins in diabetes protects VEGFR3 against degradation and ameliorates diabetes-triggered inhibition of lymphangiogenesis, thereby providing a novel potential therapeutic strategy to treat diabetic complications.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/deficiência , Diabetes Mellitus Experimental/metabolismo , Linfangiogênese/fisiologia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Proteína Tirosina Quinase CSK , Diabetes Mellitus Experimental/patologia , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Humanos , Camundongos , Camundongos Knockout , Modelos Biológicos , Proteólise , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Fator de Transcrição AP-1/metabolismo , Fator C de Crescimento do Endotélio Vascular/metabolismo , Quinases da Família src/metabolismoRESUMO
The sorting receptor Sortilin functions in the regulation of glucose and lipid metabolism. Dysfunctional lipid uptake, storage, and metabolism contribute to several major human diseases including atherosclerosis and obesity. Sortilin associates with cardiovascular disease; however, the role of Sortilin in adipose tissue and lipid metabolism remains unclear. Here we show that in the low-density lipoprotein receptor-deficient (Ldlr-/-) atherosclerosis model, Sortilin deficiency (Sort1-/-) in female mice suppresses Niemann-Pick type C1-Like 1 (Npc1l1) mRNA levels, reduces body and white adipose tissue weight, and improves brown adipose tissue function partially via transcriptional downregulation of Krüppel-like factor 4 and Liver X receptor. Female Ldlr-/-Sort1-/- mice on a high-fat/cholesterol diet had elevated plasma Fibroblast growth factor 21 and Adiponectin, an adipokine that when reduced is associated with obesity and cardiovascular disease-related factors. Additionally, Sort1 deficiency suppressed cholesterol absorption in both female mice ex vivo intestinal tissue and human colon Caco-2 cells in a similar manner to treatment with the NPC1L1 inhibitor ezetimibe. Together our findings support a novel role of Sortilin in energy regulation and lipid homeostasis in female mice, which may be a potential therapeutic target for obesity and cardiovascular disease.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/deficiência , Tecido Adiposo/metabolismo , Colesterol/metabolismo , Metabolismo Energético , Regulação da Expressão Gênica , Metabolismo dos Lipídeos , Proteínas Adaptadoras de Transporte Vesicular/genética , Adiponectina/sangue , Animais , Aterosclerose/sangue , Aterosclerose/genética , Aterosclerose/metabolismo , Células CACO-2 , Colesterol/farmacocinética , Dieta Hiperlipídica , Feminino , Células HEK293 , Células Hep G2 , Humanos , Absorção Intestinal , Fator 4 Semelhante a Kruppel , Masculino , Camundongos Knockout , Obesidade/sangue , Obesidade/genética , Obesidade/metabolismo , Receptores de LDL/deficiência , Receptores de LDL/genéticaRESUMO
Metabolic acidosis is the most common acid-base disorder in septic patients and is associated with increased mortality. Previously, we demonstrated that sepsis induced by cecal ligation and puncture (CLP) impairs [Formula: see text] absorption in the medullary thick ascending limb (MTAL) by 1) decreasing the intrinsic [Formula: see text] absorptive capacity and 2) enhancing inhibition of [Formula: see text] absorption by LPS through upregulation of Toll-like receptor (TLR) 4 signaling. Both effects depend on ERK activation. Monophosphoryl lipid A (MPLA) is a detoxified TLR4 agonist that enhances innate antimicrobial immunity and improves survival following sepsis. Pretreatment of MTALs with MPLA in vitro prevents LPS inhibition of [Formula: see text] absorption. Here we examined whether pretreatment with MPLA would protect the MTAL against sepsis. Vehicle or MPLA was administered to mice 48 h before sham or CLP surgery, and MTALs were studied in vitro 18 h postsurgery. Pretreatment with MPLA prevented the effects of sepsis to decrease the basal [Formula: see text] absorption rate and enhance inhibition by LPS. These protective effects were mediated through MPLA stimulation of a Toll/IL-1 receptor domain-containing adaptor-inducing IFN-ß-(TRIF)-dependent phosphatidylinositol 3-kinase-Akt pathway that prevents sepsis- and LPS-induced ERK activation. The effects of MPLA to improve MTAL [Formula: see text] absorption were associated with marked improvement in plasma [Formula: see text] concentration, supporting a role for the kidneys in the pathogenesis of sepsis-induced metabolic acidosis. These studies support detoxified TLR4-based immunomodulators, such as MPLA, that enhance antimicrobial responses as a safe and effective approach to prevent or treat sepsis-induced renal tubule dysfunction and identify cell signaling pathways that can be targeted to preserve MTAL [Formula: see text] absorption and attenuate metabolic acidosis during sepsis.
Assuntos
Acidose/prevenção & controle , Bicarbonatos/metabolismo , Lipídeo A/análogos & derivados , Alça do Néfron/efeitos dos fármacos , Reabsorção Renal/efeitos dos fármacos , Sepse/tratamento farmacológico , Equilíbrio Ácido-Base/efeitos dos fármacos , Acidose/metabolismo , Acidose/fisiopatologia , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Bicarbonatos/sangue , Bicarbonatos/urina , Modelos Animais de Doenças , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Lipídeo A/farmacologia , Alça do Néfron/metabolismo , Alça do Néfron/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sepse/metabolismo , Sepse/fisiopatologia , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/agonistasRESUMO
OBJECTIVE: Inflammatory macrophages promote the development of atherosclerosis. We have identified the adaptor protein Dab2 (disabled homolog 2) as a regulator of phenotypic polarization in macrophages. The absence of Dab2 in myeloid cells promotes an inflammatory phenotype, but the impact of myeloid Dab2 deficiency on atherosclerosis has not been shown. APPROACH AND RESULTS: To determine the role of myeloid Dab2 in atherosclerosis, Ldlr-/- mice were reconstituted with either Dab2-positive or Dab2-deficient bone marrow and fed a western diet. Consistent with our previous finding that Dab2 inhibits NFκB (nuclear factor κ-light-chain-enhancer of activated B cells) signaling in macrophages, Ldlr-/- mice reconstituted with Dab2-deficient bone marrow had increased systemic inflammation as evidenced by increased serum IL-6 (interleukin-6) levels and increased inflammatory cytokine expression levels in liver. Serum lipid levels were significantly lower in Ldlr-/- mice reconstituted with Dab2-deficient bone marrow, and further examination of livers from these mice revealed drastically increased inflammatory tissue damage and massive infiltration of immune cells. Surprisingly, the atherosclerotic lesion burden in Ldlr-/- mice reconstituted with Dab2-deficient bone marrow was decreased compared with Ldlr-/- mice reconstituted with wild-type bone marrow. Further analysis of aortic root sections revealed increased macrophage content and evidence of increased apoptosis in lesions from Ldlr-/- mice reconstituted with Dab2-deficient bone marrow but no difference in collagen or α-smooth muscle actin content. CONCLUSIONS: Dab2 deficiency in myeloid cells promotes inflammation in livers and atherosclerotic plaques in a mouse model of atherosclerosis. Nevertheless, decreased serum lipids as a result of massive inflammatory liver damage may preclude an appreciable increase in atherosclerotic lesion burden in mice reconstituted with Dab2-deficient bone marrow.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/deficiência , Aorta/metabolismo , Doenças da Aorta/metabolismo , Aterosclerose/metabolismo , Hepatite/metabolismo , Fígado/metabolismo , Macrófagos/metabolismo , Placa Aterosclerótica , Receptores de LDL/deficiência , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/patologia , Proteínas Reguladoras de Apoptose , Aterosclerose/genética , Aterosclerose/patologia , Caspases/metabolismo , Dieta Hiperlipídica , Modelos Animais de Doenças , Progressão da Doença , Feminino , Hepatite/genética , Hepatite/patologia , Humanos , Interleucina-6/sangue , Células Jurkat , Lipídeos/sangue , Fígado/patologia , Macrófagos/patologia , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Fenótipo , Receptores de LDL/genética , Transdução de Sinais , Triglicerídeos/metabolismoRESUMO
BACKGROUND: Human induced pluripotent stem cells (iPSCs) have been verified as a powerful cell model for the study of pathogenesis in hereditary disease. Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations of PKD or non-PKD genes. The pathogenesis of ADPKD remains unexplored because of the lack of a true human cell model. METHODS: Six ADPKD patients and four healthy individuals were recruited as donors of somatic cells from a Chinese ADPKD family without mutations of the PKD genes but carrying SAMSN1 gene deletion. The ADPKD-iPSCs were generated from somatic cells and were induced into kidney-like cells (KLCs) by a novel three-step method involving cytokines and renal epithelium growth medium. Furthermore, we analyzed functional properties of these KLCs by water transportation and albumin absorption assays. RESULTS: We successfully generated iPSCs from ADPKD patients and differentiated them into KLCs that showed morphological and functional characteristics of human kidney cells. Further, we also found that ADPKD-iPSC-KLCs had a significantly higher rate of apoptosis and a significantly lower capacity for water transportation and albumin absorption compared to healthy sibling-derived differentiated KLCs. Furthermore, knockdown of SAMSN1 in control iPSCs may attenuate differentiation and/or function of KLCs. CONCLUSIONS: These data show that we have created the first iPSCs established from ADPKD patients without mutations in the PKD genes, and suggest that the deletion mutation of SAMSN1 might be involved in the differentiation and/or function of KLCs. ADPKD-iPSC-KLCs can be used as a versatile model system for the study of kidney disease.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/genética , Células Epiteliais/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Rim/metabolismo , Proteínas do Tecido Nervoso/genética , Rim Policístico Autossômico Dominante/genética , Receptores de Superfície Celular/genética , Canais de Cátion TRPP/genética , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Adolescente , Albuminas/metabolismo , Transporte Biológico , Diferenciação Celular , Hibridização Genômica Comparativa , Análise Mutacional de DNA , Células Epiteliais/patologia , Feminino , Deleção de Genes , Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Rim/patologia , Masculino , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/deficiência , Linhagem , Rim Policístico Autossômico Dominante/metabolismo , Rim Policístico Autossômico Dominante/patologia , Cultura Primária de Células , Receptores de Superfície Celular/deficiência , Canais de Cátion TRPP/metabolismo , Água/metabolismoRESUMO
Intersectin-1s (ITSN-1s), a multidomain adaptor protein, plays a vital role in endocytosis, cytoskeleton rearrangement and cell signaling. Recent studies have demonstrated that deficiency of ITSN-1s is a crucial early event in pulmonary pathogenesis. In lung cancer, ITSN-1s deficiency impairs Eps8 ubiquitination and favors Eps8-mSos1 interaction which activates Rac1 leading to enhanced lung cancer cell proliferation, migration and metastasis. Restoring ITSN-1s deficiency in lung cancer cells facilitates cytoskeleton changes favoring mesenchymal to epithelial transformation and impairs lung cancer progression. ITSN-1s deficiency in acute lung injury leads to impaired endocytosis which leads to ubiquitination and degradation of growth factor receptors such as Alk5. This deficiency is counterbalanced by microparticles which, via paracrine effects, transfer Alk5/TGFßRII complex to non-apoptotic cells. In the presence of ITSN-1s deficiency, Alk5-restored cells signal via Erk1/2 MAPK pathway leading to restoration and repair of lung architecture. In inflammatory conditions such as pulmonary artery hypertension, ITSN-1s full length protein is cleaved by granzyme B into EHITSN and SH3A-EITSN fragments. The EHITSN fragment leads to pulmonary cell proliferation via activation of p38 MAPK and Elk-1/c-Fos signaling. In vivo, ITSN-1s deficient mice transduced with EHITSN plasmid develop pulmonary vascular obliteration and plexiform lesions consistent with pathological findings seen in severe pulmonary arterial hypertension. These novel findings have significantly contributed to understanding the mechanisms and pathogenesis involved in pulmonary pathology. As demonstrated in these studies, genetically modified ITSN-1s expression mouse models will be a valuable tool to further advance our understanding of pulmonary pathology and lead to novel targets for treating these conditions.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/deficiência , Proteínas Adaptadoras de Transporte Vesicular/genética , Pneumopatias/genética , Pneumopatias/metabolismo , Animais , Humanos , Hipertensão Pulmonar/etiologia , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/metabolismo , Pneumopatias/etiologia , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismoRESUMO
Nucleic acids carrying pathogen-associated molecular patterns trigger innate immune responses and are used to activate host immunity. Although synthetic nucleic acids have been used for that purpose, they have shown limitations for in vivo and clinical applications. To address this issue, we tested a naturally occurring dsRNA extracted from rice bran (rb-dsRNA) and characterized it as a potent ligand of TLR3 and MDA5. In this study, intranasal administration of rb-dsRNA induced production of type I IFNs by alveolar macrophages and protected mice from morbidity and mortality resulting from respiratory virus infection, such as influenza A virus. This protection was completely absent in mice lacking both TRIF and MDA5, indicating the essential role of TLR3- and MDA5-dependent pathways. Interestingly, IFNAR1-deficient mice retained residual antiviral protection, which was abolished by pharmacological inhibition of caspase 1, but not IL-1ß signaling. In fact, rb-dsRNA activated caspase 1 via TRIF, resulting in the release of IL-1ß and LDH. In addition to the direct antiviral activity, rb-dsRNA modulated the immune cell population in the lungs by repopulating virus-depleted alveolar macrophages. Our data demonstrate that rb-dsRNA orchestrates IFN-dependent and -independent direct antiviral protection and that it is a potent immune stimulator modulating antiviral immunity in the lungs. These findings open doors to a range of precise immune-modulating studies and therapeutic options.
Assuntos
Antivirais/isolamento & purificação , Vírus da Influenza A/imunologia , Interferon Tipo I/imunologia , Infecções por Orthomyxoviridae/imunologia , Oryza/genética , RNA de Cadeia Dupla/imunologia , RNA de Cadeia Dupla/isolamento & purificação , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Antivirais/imunologia , Inibidores de Caspase/administração & dosagem , Imunidade Inata , Interferon Tipo I/biossíntese , Helicase IFIH1 Induzida por Interferon/química , Helicase IFIH1 Induzida por Interferon/deficiência , Helicase IFIH1 Induzida por Interferon/genética , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/metabolismo , Ligantes , Pulmão/imunologia , Pulmão/virologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/imunologia , Camundongos , Infecções por Orthomyxoviridae/prevenção & controle , Oryza/química , Plantas/química , Plantas/genética , RNA de Cadeia Dupla/administração & dosagem , RNA de Cadeia Dupla/farmacologia , Receptor de Interferon alfa e beta/deficiência , Transdução de Sinais/efeitos dos fármacos , Receptor 3 Toll-Like/químicaRESUMO
Monophosphoryl lipid A (MPLA) is a detoxified derivative of LPS that induces tolerance to LPS and augments host resistance to bacterial infections. Previously, we demonstrated that LPS inhibits [Formula: see text] absorption in the medullary thick ascending limb (MTAL) through a basolateral Toll-like receptor 4 (TLR4)-myeloid differentiation factor 88 (MyD88)-ERK pathway. Here we examined whether pretreatment with MPLA would attenuate LPS inhibition. MTALs from rats were perfused in vitro with MPLA (1 µg/ml) in bath and lumen or bath alone for 2 h, and then LPS was added to (and MPLA removed from) the bath solution. Pretreatment with MPLA eliminated LPS-induced inhibition of [Formula: see text] absorption. In MTALs pretreated with MPLA plus a phosphatidylinositol 3-kinase (PI3K) or Akt inhibitor, LPS decreased [Formula: see text] absorption. MPLA increased Akt phosphorylation in dissected MTALs. The Akt activation was eliminated by a PI3K inhibitor and in MTALs from TLR4-/- or Toll/IL-1 receptor domain-containing adaptor-inducing IFN-ß (TRIF)-/- mice. The effect of MPLA to prevent LPS inhibition of [Formula: see text] absorption also was TRIF dependent. Pretreatment with MPLA prevented LPS-induced ERK activation; this effect was dependent on PI3K. MPLA alone had no effect on [Formula: see text] absorption, and MPLA pretreatment did not prevent ERK-mediated inhibition of [Formula: see text] absorption by aldosterone, consistent with MPLA's low toxicity profile. These results demonstrate that pretreatment with MPLA prevents the effect of LPS to inhibit [Formula: see text] absorption in the MTAL. This protective effect is mediated directly through MPLA stimulation of a TLR4-TRIF-PI3K-Akt pathway that prevents LPS-induced ERK activation. These studies identify detoxified TLR4-based immunomodulators as novel potential therapeutic agents to prevent or treat renal tubule dysfunction in response to bacterial infections.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Lipídeo A/análogos & derivados , Lipopolissacarídeos/toxicidade , Alça do Néfron/efeitos dos fármacos , Fosfatidilinositol 3-Quinase/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/efeitos dos fármacos , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Bicarbonatos/metabolismo , Citoproteção , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Técnicas In Vitro , Lipídeo A/farmacologia , Alça do Néfron/enzimologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Perfusão , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-Dawley , Reabsorção Renal/efeitos dos fármacos , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
Murine models of pulmonary arterial hypertension (PAH) that recapitulate the plexiform and obliterative arteriopathy seen in PAH patients and help in defining the molecular mechanisms involved are missing. Herein, we investigated whether intersectin-1s (ITSN) deficiency and prolonged lung expression of an ITSN fragment with endothelial cell (EC) proliferative potential (EHITSN), present in the lungs of PAH animal models and human patients, induce formation of plexiform/obliterative lesions and defined the molecular mechanisms involved. ITSN-deficient mice (knockout/heterozygous and knockdown) were subjected to targeted lung delivery of EHITSN via liposomes for 20 days. Immunohistochemistry and histological and morphometric analyses revealed a twofold increase in proliferative ECs and a 1.35-fold increase in proliferative α-smooth muscle actin-positive cells in the lungs of ITSN-deficient mice, transduced with the EHITSN relative to wild-type littermates. Treated mice developed severe medial wall hypertrophy, intima proliferation, and various forms of obliterative and plexiform-like lesions in pulmonary arteries, similar to PAH patients. Hemodynamic measurements indicated modest increases in the right ventricular systolic pressure and right ventricle hypertrophy. Transcriptional and protein assays of lung tissue indicated p38MAPK-dependent activation of Elk-1 transcription factor and increased expression of c-Fos gene. This unique murine model of PAH-like plexiform/obliterative arteriopathy induced via a two-hit pathophysiological mechanism without hypoxia provides novel druggable targets to ameliorate and, perhaps, reverse the EC plexiform phenotype in severe human PAH.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Hipertensão Pulmonar/patologia , Pulmão/irrigação sanguínea , Pulmão/metabolismo , Artéria Pulmonar/patologia , Remodelação Vascular , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Animais , Proliferação de Células , Colágeno/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Ventrículos do Coração/patologia , Ventrículos do Coração/fisiopatologia , Hipertensão Pulmonar/fisiopatologia , Hipertrofia/patologia , Hipertrofia/fisiopatologia , Lipídeos/química , Pulmão/enzimologia , Pulmão/patologia , Camundongos , Proteínas Proto-Oncogênicas c-myc/metabolismo , Artéria Pulmonar/fisiopatologia , RNA Interferente Pequeno/metabolismo , Sístole , Transdução Genética , Proteínas Elk-1 do Domínio ets/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
PolyI:C, a synthetic double-stranded RNA analog, acts as an immune-enhancing adjuvant that regresses tumors in cytotoxic T lymphocyte (CTL)-dependent and CTL-independent manner, the latter of which remains largely unknown. Tumors contain CD11b+Ly6G+ cells, known as granulocytic myeloid-derived suppressor cells (G-MDSCs) or tumor-associated neutrophils (TANs) that play a critical role in tumor progression and development. Here, we demonstrate that CD11b+Ly6G+ cells respond to polyI:C and exhibit tumoricidal activity in an EL4 tumor implant model. PolyI:C-induced inhibition of tumor growth was attributed to caspase-8/3 cascade activation in tumor cells that occurred independently of CD8α+/CD103+ dendritic cells (DCs) and CTLs. CD11b+Ly6G+ cells was essential for the antitumor effect because depletion of CD11b+Ly6G+ cells totally abrogated tumor regression and caspase activation after polyI:C treatment. CD11b+Ly6G+ cells that had been activated with polyI:C showed cytotoxicity and inhibited tumor growth through the production of reactive oxygen species (ROS)/reactive nitrogen species (RNS). These responses were abolished in either Toll/interleukin-1 receptor domain-containing adaptor molecule-1 (TICAM-1)-/- or interferon (IFN)-αß receptor 1 (IFNAR1)-/- mice. Thus, our results suggest that polyI:C activates the TLR3/TICAM-1 and IFNAR signaling pathways in CD11b+Ly6G+ cells in tumors, thereby eliciting their antitumor activity, independent of those in CD8α+/CD103+ DCs that prime CTLs.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Antígenos Ly/metabolismo , Antígeno CD11b/metabolismo , RNA de Cadeia Dupla/metabolismo , Linfócitos T Citotóxicos/imunologia , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Antígenos Ly/imunologia , Apoptose/efeitos dos fármacos , Caspase 3/metabolismo , Caspase 8/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células Mieloides/citologia , Células Mieloides/metabolismo , Poli I-C/farmacologia , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptor de Interferon alfa e beta/deficiência , Receptor de Interferon alfa e beta/genética , Transdução de Sinais/efeitos dos fármacos , Baço/citologia , Baço/efeitos dos fármacos , Baço/metabolismo , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/efeitos dos fármacosRESUMO
Sortilin, a member of the vacuolar protein sorting 10 domain receptor family, traffics newly synthesized proteins from the trans-Golgi network to secretory pathways, endosomes, and cell surface. Sortilin-trafficked molecules, including IL-6 and acid sphingomyelinase (aSMase), mediate cholangiocyte proliferation and liver inflammation, hepatic stellate cell activation, hepatocyte apoptosis, and fibrosis. Based on these sortilin-regulated functions, we investigated its role in biliary damage leading to hepatocellular injury and fibrosis. Sortilin-/- mice displayed impaired inflammation and ductular reaction 3 days after bile duct ligation (BDL), as demonstrated by reduced cholangiocyte proliferation and activation and reduced serum IL-6. Interestingly, liver fibrosis was reduced in Sortilin-/- mice after both BDL and carbon tetrachloride treatment, in line with attenuated in vitro activation of Sortilin-/- hepatic stellate cells. Sortilin-/- hepatic aSMase activity was reduced in the BDL and carbon tetrachloride models and accompanied by reduced in vivo hepatocyte apoptosis. In addition, wild type (WT), but not Sortilin-/- hepatocytes, had increased aSMase-dependent susceptibility to bile acid-induced apoptosis in vitro. Mechanistically, short-term IL-6 neutralization in bile duct-ligated WT mice decreased hepatic inflammation and reactive cholangiocyte-derived cytokines and chemokines, without affecting fibrosis, whereas pharmacological inhibition of aSMase activity was not sufficient to attenuate hepatic fibrosis. Only combined IL-6 and aSMase inhibition significantly reduced fibrosis in bile duct-ligated WT mice. We conclude that sortilin regulates cholestatic liver damage and fibrosis via effects on both aSMase activity and serum IL-6.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/deficiência , Apoptose , Ductos Biliares/patologia , Colestase/complicações , Hepatócitos/patologia , Cirrose Hepática/patologia , Fígado/lesões , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Proliferação de Células , Quimiocinas/metabolismo , Colestase/patologia , Células Estreladas do Fígado/metabolismo , Células Estreladas do Fígado/patologia , Hepatócitos/metabolismo , Inflamação/patologia , Interleucina-6/metabolismo , Ligadura , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/complicações , Cirrose Hepática/metabolismo , Camundongos Endogâmicos C57BL , Testes de Neutralização , Fenótipo , Esfingomielina Fosfodiesterase/metabolismoRESUMO
Receptor-interacting protein kinase 1 (RIPK1) promotes cell survival-mice lacking RIPK1 die perinatally, exhibiting aberrant caspase-8-dependent apoptosis and mixed lineage kinase-like (MLKL)-dependent necroptosis. However, mice expressing catalytically inactive RIPK1 are viable, and an ill-defined pro-survival function for the RIPK1 scaffold has therefore been proposed. Here we show that the RIP homotypic interaction motif (RHIM) in RIPK1 prevents the RHIM-containing adaptor protein ZBP1 (Z-DNA binding protein 1; also known as DAI or DLM1) from activating RIPK3 upstream of MLKL. Ripk1RHIM/RHIM mice that expressed mutant RIPK1 with critical RHIM residues IQIG mutated to AAAA died around birth and exhibited RIPK3 autophosphorylation on Thr231 and Ser232, which is a hallmark of necroptosis, in the skin and thymus. Blocking necroptosis with catalytically inactive RIPK3(D161N), RHIM mutant RIPK3, RIPK3 deficiency, or MLKL deficiency prevented lethality in Ripk1RHIM/RHIM mice. Loss of ZBP1, which engages RIPK3 in response to certain viruses but previously had no defined role in development, also prevented perinatal lethality in Ripk1RHIM/RHIM mice. Consistent with the RHIM of RIPK1 functioning as a brake that prevents ZBP1 from engaging the RIPK3 RHIM, ZBP1 interacted with RIPK3 in Ripk1RHIM/RHIMMlkl-/- macrophages, but not in wild-type, Mlkl-/- or Ripk1RHIM/RHIMRipk3RHIM/RHIM macrophages. Collectively, these findings indicate that the RHIM of RIPK1 is critical for preventing ZBP1/RIPK3/MLKL-dependent necroptosis during development.
Assuntos
Apoptose , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Glicoproteínas/antagonistas & inibidores , Glicoproteínas/metabolismo , Necrose , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Motivos de Aminoácidos , Animais , Animais Recém-Nascidos , Caspase 8/genética , Caspase 8/metabolismo , Embrião de Mamíferos/citologia , Feminino , Glicoproteínas/química , Glicoproteínas/deficiência , Macrófagos/metabolismo , Masculino , Camundongos , Mutação , Fosforilação , Ligação Proteica , Proteínas Quinases/deficiência , Proteínas Quinases/metabolismo , Proteínas de Ligação a RNA , Proteína Serina-Treonina Quinases de Interação com Receptores/química , Proteína Serina-Treonina Quinases de Interação com Receptores/deficiência , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Disabled-2 (Dab2) is a widely expressed clathrin binding endocytic adaptor protein and known for the endocytosis of the low-density lipoprotein (LDL) family receptors. Dab2 also modulates endosomal Ras/MAPK (Erk1/2) activity by regulating the disassembly of Grb2/Sos1 complexes associated with clathrin-coated vesicles. We found that the most prominent phenotype of Dab2 knockout mice was their striking lean body composition under a high fat and high caloric diet, although the weight of the mutant mice was indistinguishable from wild-type littermates on a regular chow. The remarkable difference in resistance to high caloric diet-induced weight gain of the dab2-deleted mice was presented only in juvenile but not in mature mice. Investigation using Dab2-deficient embryonic fibroblasts and mesenchymal stromal cells indicated that Dab2 promoted adipogenic differentiation by modulation of MAPK (Erk1/2) activity, which otherwise suppresses adipogenesis through the phosphorylation of PPARγ. The results suggest that Dab2 is required for the excessive calorie-induced differentiation of an adipocyte progenitor cell population that is present in juvenile but depleted in mature animals. The finding provides evidence for a limited pre-adipocyte population in juvenile mammals and the requirement of Dab2 in the regulation of Ras/MAPK signal in the commitment of the precursor cells to adipose tissues.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Adipócitos/fisiologia , Diferenciação Celular , Fibroblastos/fisiologia , Células-Tronco Mesenquimais/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Animais , Proteínas Reguladoras de Apoptose , Peso Corporal , Células Cultivadas , Dieta Hiperlipídica , Regulação da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Camundongos KnockoutRESUMO
BACKGROUND: The mechanisms involved in lung cancer (LC) progression are poorly understood making discovery of successful therapies difficult. Adaptor proteins play a crucial role in cancer as they link cell surface receptors to specific intracellular pathways. Intersectin-1s (ITSN-1s) is an important multidomain adaptor protein implicated in the pathophysiology of numerous pulmonary diseases. To date, the role of ITSN-1s in LC has not been studied. METHODS: Human LC cells, human LC tissue and A549 LC cells stable transfected with myc-ITSN-1s construct (A549 + ITSN-1s) were used in correlation with biochemical, molecular biology and morphological studies. In addition scratch assay with time lapse microscopy and in vivo xenograft tumor and mouse metastasis assays were performed. RESULTS: ITSN-1s, a prevalent protein of lung tissue, is significantly downregulated in human LC cells and LC tissue. Restoring ITSN-1s protein level decreases LC cell proliferation and clonogenic potential. In vivo studies indicate that immunodeficient mice injected with A549 + ITSN-1s cells develop less and smaller metastatic tumors compared to mice injected with A549 cells. Our studies also show that restoring ITSN-1s protein level increases the interaction between Cbl E3 ubiquitin ligase and Eps8 resulting in enhanced ubiquitination of the Eps8 oncoprotein. Subsequently, downstream unproductive assembly of the Eps8-mSos1 complex leads to impaired activation of the small GTPase Rac1. Impaired Rac1 activation mediated by ITSN-1s reorganizes the cytoskeleton (increased thick actin bundles and focal adhesion (FA) complexes as well as collapse of the vimentin filament network) in favor of decreased LC cell migration and metastasis. CONCLUSION: ITSN-1s induced Eps8 ubiquitination and impaired Eps8-mSos1 complex formation, leading to impaired activation of Rac1, is a novel signaling mechanism crucial for abolishing the progression and metastatic potential of LC cells.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Neoplasias Pulmonares/patologia , Proteínas Proto-Oncogênicas c-cbl/metabolismo , Proteína SOS1/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Células A549 , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Camundongos , Metástase Neoplásica , Transplante de Neoplasias , Proteínas Proto-Oncogênicas c-cbl/genética , Proteína SOS1/genética , Imagem com Lapso de Tempo , Ubiquitinação , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
Skeletal muscle regeneration requires coordination between dynamic cellular populations and tissue microenvironments. Macrophages, recruited via CCR2, are essential for regeneration; however, the contribution of macrophages and the role of CCR2 on nonhematopoietic cells has not been defined. In addition, aging and sex interactions in regeneration and sarcopenia are unclear. Muscle regeneration was measured in young (3-6 mo), middle (11-15 mo), old (24-32 mo) male and female CCR2-/- mice. Whereas age-related muscle atrophy/sarcopenia was present, regenerated myofiber cross-sectional area (CSA) in CCR2-/- mice was comparably impaired across all ages and sexes, with increased adipocyte area compared with wild-type (WT) mice. CCR2-/- mice myofibers achieved approximately one third of baseline CSA even 84 d after injury. Regenerated CSA and clearance of necrotic tissue were dependent on bone marrow-derived cellular expression of CCR2. Myogenic progenitor cells isolated from WT and CCR2-/- mice exhibited comparable proliferation and differentiation capacity. The most striking cellular anomaly in injured muscle of CCR2-/- mice was markedly decreased macrophages, with a predominance of Ly6C- anti-inflammatory monocytes/macrophages. Ablation of proinflammatory TLR signaling did not affect muscle regeneration or resolution of necrosis. Of interest, many proinflammatory, proangiogenic, and chemotactic cytokines were markedly elevated in injured muscle of CCR2-/- relative to WT mice despite impairments in macrophage recruitment. Collectively, these results suggest that CCR2 on bone marrow-derived cells, likely macrophages, were essential to muscle regeneration independent of TLR signaling, aging, and sex. Decreased proinflammatory monocytes/macrophages actually promoted a proinflammatory microenvironment, which suggests that inflammaging was present in young CCR2-/- mice.