RESUMEN
Dendritic cells (DCs) are composed of multiple lineages of hematopoietic cells and orchestrate immune responses upon detecting the danger and inflammatory signals associated with pathogen and damaged tissues. Under steady-state, DCs are maintained at limited numbers and the functionally quiescent status. While it is known that a fine balance in the DC homeostasis and activation status is also important to prevent autoimmune diseases and hyperinflammation, mechanisms that control DC development and activation under stead-state remain not fully understood. Here we show that DC-specific ablation of CBL and CBL-B (CBL-/-CBL-B-/-) leads to spontaneous liver inflammation and fibrosis and early death of the mice. The mutant mice have a marked expansion of classic CD8α+/CD103+ DCs (cDC1s) in peripheral lymphoid organs and the liver. These DCs exhibit atypical activation phenotypes characterized by an increased production of inflammatory cytokines and chemokines but not the cell surface MHC-II and costimulatory ligands. While the mutant mice also have massive T cell activation, lymphocytes are not required for the disease development. The CBL-/-CBL-B-/- mutation enhances FLT3-mTOR signaling, due to defective FLT3 ubiquitination and degradation. Blockade of FLT3-mTOR signaling normalizes the homeostasis of cDC1s and attenuates liver inflammation. Our result thus reveals a critical role of CBLs in the maintenance of DC homeostasis and immune quiescence. This regulation could be relevant to liver inflammatory diseases and fibrosis in humans.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Células Dendríticas/inmunología , Proteínas Proto-Oncogénicas c-cbl/fisiología , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Presentación de Antígeno , División Celular , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/citología , Células Dendríticas/metabolismo , Hepatitis Autoinmune/genética , Hepatitis Autoinmune/inmunología , Homeostasis , Subgrupos Linfocitarios/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Mutación Puntual , Proteínas Proto-Oncogénicas c-akt/fisiología , Proteínas Proto-Oncogénicas c-cbl/deficiencia , Proteínas Proto-Oncogénicas c-cbl/genética , Sirolimus/farmacología , Tirosina Quinasa 3 Similar a fms/fisiologíaRESUMEN
Genome-wide association studies as well as lymphatic expression analyses have linked both Cbl-b and GM-CSF to human multiple sclerosis as well as other autoimmune diseases. Both Cbl-b and GM-CSF have been shown to play a prominent role in the development of murine encephalomyelitis; however, no functional connection between the two has yet been established. In this study, we show that Cblb knockout mice demonstrated significantly exacerbated severity of experimental autoimmune encephalomyelitis (EAE), augmented T cell infiltration into the central nervous system (CNS) and strongly increased production of GM-CSF in T cells in vitro and in vivo.GM-CSF neutralization demonstrated that the increased susceptibility of Cblb-/- mice to EAE was dependent on GM-CSF. Mechanistically, p50 binding to the GM-CSF promoter and the IL-3/GM-CSF enhancer element "CNSa" was strongly increased in nuclear extracts from Cbl-b-deficient T cells. This study suggests that Cbl-b limits autoimmunity by preventing the pathogenic effects of GM-CSF overproduction in T cells.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Linfocitos T CD4-Positivos/metabolismo , Encefalomielitis Autoinmune Experimental/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Proteínas Proto-Oncogénicas c-cbl/fisiología , Animales , Autoinmunidad/genética , Regulación de la Expresión Génica , Interleucina-3/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Regiones Promotoras GenéticasRESUMEN
E3 ubiquitin ligase c-Caritas B cell lymphoma (c-cbl) is associated with negative regulation of receptor tyrosine kinases, signal transduction of antigens and cytokine receptors, and immune response. However, the expression and function of c-cbl in the regulation of neuropathic pain after chronic constriction injury (CCI) are unknown. In rat CCI model, c-cbl inhibited the activation of spinal cord microglia and the release of pro-inflammatory factors including tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1ß) and interleukin 6 (IL-6), which alleviated mechanical and heat pain through down-regulating extracellular signal-regulated kinase (ERK) pathway. Additionally, exogenous TNF-α inhibited c-cbl protein level vice versa. In the primary microglia transfected with c-cbl siRNA, when treated with TNF-α or TNF-α inhibitor, the corresponding secretion of IL-1ß and IL-6 did not change. In summary, CCI down-regulated c-cbl expression and induced the activation of microglia, then activated microglia released inflammatory factors via ERK signaling to cause pain. Our data might supply a novel molecular target for the therapy of CCI-induced neuropathic pain.
Asunto(s)
Microglía/efectos de los fármacos , Neuralgia/fisiopatología , Traumatismos de los Nervios Periféricos/fisiopatología , Proteínas Proto-Oncogénicas c-cbl/fisiología , Médula Espinal/fisiopatología , Animales , Secuencia de Bases , Constricción , Regulación hacia Abajo/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Masculino , Fosforilación/fisiología , Proteínas Proto-Oncogénicas c-cbl/genética , Ratas Sprague-Dawley , Nervio Ciático/lesiones , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
OBJECTIVE: To explore the interaction domains between BCR-ABL and E3 liagase c-CBL, so as to reveal the structure-basis for the arsenic to treat chronic myelogenous leukemia(CML). METHODS: The interactional interface of BCR-ABL and c-CBL was simulated and analyzed according to the available structure model. Based on the structural information, the WT and mutant Migr1-BCR-ABL-GFP (ΔSH2,ΔTyrKC,ΔSH2/TyrKC (S/H) and pFlag-c-CBL (ΔRF) were constructed and co-transfected into the 293T and HeLa cells. The co-immunoprecipitation (Co-IP) was performed by using M2 beads (anti-Flag), anti-GFP antibody and protein A beads, and the interaction was identified by using GFP and M2 antibody, respectively. Moreover, the colocalization of BCR-ABL and c-CBL was further evaluated by using immunofluorescent(IF) assay in transfected HeLa cells. RESULTS: Co-IP demonstrated that the TyrKC domain of BCR-ABL was primarily involved in the interaction with c-CBL, while both the SH2 domain of BCR-ABL and the RF domain of c-CBL also participated in the interaction to a certain degree, which were consistent with the structure-based simulation. IF elucidated that the colocalization of BCR-ABL and c-CBL was almost entirely vanished when the deleted TyrKC domain of BCR-ABL was co-transfected with c-CBL, which were elegantly coincident with the results from Co-IP. CONCLUSION: The TyrKC domain of BCR-ABL is sufficient and necessary to mediate the interaction between BCR-ABL and c-CBL, the SH2 domain of BCR-ABL and the RF domain of c-CBL are also involved in the association between the two proteins. It suggests that the association of BCR-ABL and c-CBL can modulate the stability and degradation of BCR-ABL, thus illustrating the molecular mechanisms of the targeting therapy for CML by arsenic.
Asunto(s)
Arsénico/uso terapéutico , Leucemia Mielógena Crónica BCR-ABL Positiva/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-cbl/fisiología , Ubiquitina-Proteína Ligasas/fisiología , Proteínas de Fusión bcr-abl , Células HeLa , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , FosforilaciónAsunto(s)
Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Resistencia a Antineoplásicos , Leucemia Mieloide Aguda/metabolismo , Proteínas Proto-Oncogénicas c-cbl/fisiología , Humanos , Transducción de Señal , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/fisiología , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidoresRESUMEN
Human epidermal growth factor receptor 2 (HER2) targeted therapy is currently considered as the standard treatment for HER2-positive advanced gastric cancer (GC). However, unsatisfactory results of recent phase III clinical trials involving lapatinib suggested biomarkers for selection of patients. The aim of this study was to identify JWA as a biomarker for lapatinib resistance in GC cells and elucidate the underlying mechanisms. Lapatinib was effective to the intrinsic cisplatin-resistant GC cells. JWA activation conferred lapatinib unresponsiveness, but reversed cisplatin resistance in GC cells. Whereas, deletion of JWA significantly restored lapatinib suppression on proliferation and lapatinib-induced apoptosis. JWA-induced down-regulation of HER2 and activation of ERK phosphorylation led to lapatinib resistance. Furthermore, c-Cbl represented a novel mechanism for HER2 degradation enhanced by JWA in GC cells. Taken together, JWA is a potential predictive marker for lapatinib resistance, targeting the patients that may benefit from lapatinib treatment in human GC.
Asunto(s)
Proteínas de Choque Térmico/fisiología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas Proto-Oncogénicas c-cbl/fisiología , Quinazolinas/uso terapéutico , Receptor ErbB-2/análisis , Neoplasias Gástricas/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Cisplatino/uso terapéutico , Regulación hacia Abajo , Resistencia a Antineoplásicos , Humanos , Lapatinib , Proteínas de Transporte de Membrana , Receptor ErbB-2/metabolismo , Neoplasias Gástricas/química , Neoplasias Gástricas/patologíaRESUMEN
Despite the loss of Adenomatous Polyposis Coli (APC) in a majority of colorectal cancers (CRC), not all CRCs bear hallmarks of Wnt activation, such as nuclear ß-catenin. This underscores the presence of other Wnt regulators that are important to define, given the pathogenic and prognostic roles of nuclear ß-catenin in human CRC. Herein, we investigated the effect of Casitas B-lineage lymphoma (c-Cbl) on nuclear ß-catenin, which is an oncoprotein upregulated in CRC due to loss-of-function APC or gain-of-function CTNNB1 mutations. Despite mechanistic rationale and recent discoveries of c-Cbl's mutations in solid tumors, little is known about its functional importance in CRC. Our study in a cohort of human CRC patients demonstrated an inverse correlation between nuclear ß-catenin and c-Cbl. Further investigation showed that the loss of c-Cbl activity significantly enhanced nuclear ß-catenin and CRC tumor growth in cell culture and a mouse xenograft model. c-Cbl interacted with and downregulated ß-catenin in a manner that was independent of CTNNB1 or APC mutation status. This study demonstrates a previously unrecognized function of c-Cbl as a negative regulator of CRC.
Asunto(s)
Núcleo Celular/metabolismo , Neoplasias Colorrectales/etiología , Proteínas Proto-Oncogénicas c-cbl/fisiología , Proteínas Wnt/fisiología , beta Catenina/fisiología , Animales , Neoplasias Colorrectales/patología , Femenino , Células HT29 , Humanos , Masculino , Ratones , Persona de Mediana Edad , Proteínas Proto-Oncogénicas c-cbl/análisisRESUMEN
Phosphatidylinositol 3-kinaseß (PI3Kß) plays a predominant role in integrin outside-in signaling and in platelet activation by GPVI engagement. We have shown that the tyrosine kinase Pyk2 mediates PI3Kß activation downstream of integrin αIIbß3, and promotes the phosphorylation of the PI3K-associated adaptor protein c-Cbl. In this study, we compared the functional correlation between Pyk2 and PI3Kß upon recruitment of the two main platelet collagen receptors, integrin α2ß1 and GPVI. PI3Kß-mediated phosphorylation of Akt was inhibited in Pyk2-deficient platelets adherent to monomeric collagen through integrin α2ß1, but occurred normally upon GPVI ligation. Integrin α2ß1 engagement led to Pyk2-independent association of c-Cbl with PI3K. However, c-Cbl was not phosphorylated in adherent platelets, and phosphorylation of Akt occurred normally in c-Cbl-deficient platelets, indicating that the c-Cbl is dispensable for Pyk2-mediated PI3Kß activation. Stimulation of platelets with CRP, a selective GPVI ligand, induced c-Cbl phosphorylation in the absence of Pyk2, but failed to promote its association with PI3K. Pyk2 activation was completely abrogated in PI3KßKD, but not in PI3KγKD platelets, and was strongly inhibited by Src kinases and phospholipase C inhibitors, and by BAPTA-AM. The absence of PI3Kß activity also hampered GPVI-induced tyrosine-phosphorylation and activation of PLCγ2, preventing intracellular Ca2+ increase and phosphorylation of pleckstrin. Moreover, GPVI-induced intracellular Ca2+ increase and pleckstrin phosphorylation were also strongly inhibited in human platelets treated with the PI3Kß inhibitor TGX-221. These results outline important differences in the regulation of PI3Kß by GPVI and integrin α2ß1 and suggest that inhibition of Pyk2 may target PI3Kß activation in a selective context of platelet stimulation.
Asunto(s)
Quinasa 2 de Adhesión Focal/fisiología , Integrina alfa2beta1/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Glicoproteínas de Membrana Plaquetaria/fisiología , Proteínas Proto-Oncogénicas c-cbl/fisiología , Animales , Células Cultivadas , Activación Enzimática , Humanos , Ratones , Ratones Noqueados , Transducción de SeñalRESUMEN
The use of mini-implants have made a major contribution to orthodontic treatment. Demand has aroused scientific curiosity about implant placement procedures and techniques. However, the reasons for instability have not yet been made totally clear. The aim of this article is to establish a relationship between implant placement technique and mini-implant success rates by means of examining the following hypotheses: 1) Sites of poor alveolar bone and little space between roots lead to inadequate implant placement; 2) Different sites require mini-implants of different sizes! Implant size should respect alveolar bone diameter; 3) Properly determining mini-implant placement site provides ease for implant placement and contributes to stability; 4) The more precise the lancing procedures, the better the implant placement technique; 5) Self-drilling does not mean higher pressures; 6) Knowing where implant placement should end decreases the risk of complications and mini-implant loss.
O uso de mini-implantes trouxe grandes contribuições ao tratamento ortodôntico. Essa demanda gerou curiosidade científica sobre os procedimentos e técnicas de implantação. Entretanto, instabilidades desses dispositivos ocorrem por motivos ainda não totalmente esclarecidos. Objetiva-se, com esse trabalho, relacionar a técnica de implantação com a taxa de sucesso dos mini-implantes por meio das seguintes hipóteses: 1) áreas com osso alveolar pobre e com pouco espaço inter-radicular levam à inadequada implantação; 2) diferentes áreas requerem distintos tamanhos de mini-implantes! O tamanho do implante deve acompanhar o diâmetro do osso alveolar; 3) a correta determinação do local em que será colocado o mini-implante facilita a instalação e contribui para a estabilidade; 4) quanto mais precisa for a lancetagem, melhor será a técnica de implantação; 5) autoperfuração não significa alta pressão; 6) saber onde finalizar a implantação diminui a incidência de complicações e de perda dos mini-implantes.
Asunto(s)
Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Discapacidades del Desarrollo/genética , Mutación de Línea Germinal , Leucemia Mielomonocítica Juvenil/genética , Proteínas Proto-Oncogénicas c-cbl/genética , Criptorquidismo/complicaciones , Criptorquidismo/genética , Análisis Mutacional de ADN , Discapacidades del Desarrollo/complicaciones , Predisposición Genética a la Enfermedad , Mutación de Línea Germinal/fisiología , Leucemia Mielomonocítica Juvenil/complicaciones , Linaje , Proteínas Proto-Oncogénicas c-cbl/fisiologíaRESUMEN
SIRT2 is a member of the sirtuin family of NAD(+)-dependent protein deacetylases. It is involved in metabolic homeostasis and has been linked to the progression of age-related diseases. Casitas B-lineage lymphoma (Cbl) proteins regulate signal transduction through many pathways and, consequently, regulate cell function and development. Cbl proteins are ubiquitin ligases that ubiquitinate and target many signaling molecules for degradation. The function of SIRT2 is modulated by post-translational modifications. However, the precise molecular signaling mechanism of SIRT2 through interactions with Cbl proteins has not yet been established. In this study, we investigated the potential regulation of SIRT2 function by the Cbl mammalian family members Cbl-b and c-Cbl. We found that Cbl-b and c-Cbl increased the protein level and stability of SIRT2 and that Cbl-b and c-Cbl interact with SIRT2. They were also found to regulate the deacetylase activity of SIRT2. Further investigation revealed that Cbl-mediated SIRT2 regulation occurred via ubiquitination of SIRT2.
Asunto(s)
Proteínas Proto-Oncogénicas c-cbl/fisiología , Sirtuina 2/fisiología , Línea Celular , Humanos , Procesamiento Proteico-Postraduccional , Proteína p53 Supresora de Tumor/metabolismo , UbiquitinaciónRESUMEN
BACKGROUND: The ubiquitin ligase Cbl-b potently modulates T lymphocyte immune responses and is critical in modulating tumor-induced immunosuppression. The influence of Cbl-b in modulating T lymphocyte activity against prostate cancer remains ill defined. We have determined the effects of silencing Cbl-b expression in T lymphocytes and their subsequent cytotoxic activity against prostate cancer cells. METHODS: T lymphocytes were isolated from the spleens of C57BL/6 mice. Lipofectamine-directed transfection of T lymphocytes with specific small interfering RNA (siRNA) silenced Cbl-b expression, which was confirmed by Western immunoblotting. The siRNA species were chosen that promoted the greatest transfection efficiency and dampened Cbl-b expression in T lymphocytes. The expression of CD69, CD25, and CD71 by the transfected T lymphocytes was determined by flow cytometry. T lymphocyte proliferation was assessed by CCK-8 assay. Enzyme-linked immunosorbent assay (ELISA) was used to measure the secretion of interleukin (IL)-2, interferon (IFN)-γ, and tumor necrosis factor (TNF)-ß. The objective was to compare the cytotoxic activity of transfected T lymphocytes and nontransfected (i.e., negative control) T lymphocytes against the murine prostate cancer cell line target RM-1 in vitro. RESULTS: We selected a specific siRNA that decreased T lymphocyte Cbl-b expression to 15%. The siRNA-transfected T lymphocytes showed higher proliferation; higher CD69, CD25, and CD71 expression (p < 0.001); and higher IL-2, IFN-γ, and TNF-ß secretion (p < 0.05), compared to the nontransfected cells. Transfected T lymphocytes were also more potent at killing RM-1 prostate cancer cells, compared to the negative control in vitro. CONCLUSION: Silencing Cbl-b significantly enhanced T lymphocyte function and T lymphocyte cytotoxicity activity against a model prostate cancer cell line in vitro. This study suggests a potentially novel immunotherapeutic strategy against prostate cancer.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Activación de Linfocitos , Neoplasias de la Próstata/inmunología , Proteínas Proto-Oncogénicas c-cbl/fisiología , Linfocitos T/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Antígenos CD/análisis , Antígenos de Diferenciación de Linfocitos T/análisis , Línea Celular Tumoral , Citocinas/metabolismo , Lectinas Tipo C/análisis , Masculino , Ratones , Ratones Endogámicos C57BL , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-cbl/genética , ARN Interferente Pequeño/genética , Receptores de Transferrina/análisisRESUMEN
Obesity causes type 2 diabetes, atherosclerosis and cardiovascular diseases by inducing systemic insulin resistance. It is now recognized that obesity is related to chronic low-grade inflammation in adipose tissue. Specifically, activated immune cells infiltrate adipose tissue and cause inflammation. There is increasing evidence that activated macrophages accumulate in the hypertrophied adipose tissue of rodents and humans and induce systemic insulin resistance by secreting inflammatory cytokines. Accordingly, a better understanding of the molecular mechanisms underlying macrophage activation in adipose tissue will facilitate the development of new therapeutic strategies. Currently, little is known about the regulation of macrophage activation, although E3 ubiquitin ligase Casitas B-lineage lymphoma (Cbl)-b was identified recently as a novel negative regulator of macrophage activation in adipose tissue. Cbl-b, which is a suppressor of T- and B-cell activation, inhibits intracellular signal transduction by targeting some tyrosine kinases. Notably, preventing Cbl-b-mediated macrophage activation improves obesity-induced insulin resistance in mice. c-Cbl is another member of the Cbl family that is associated with insulin resistance in obesity. These reports suggest that Cbl-b and c-Cbl are potential therapeutic targets for treating obesity-induced insulin resistance. In this review, we focus on the importance of Cbl-b in macrophage activation in aging-induced and high-fat diet-induced obesity.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Resistencia a la Insulina/genética , Obesidad/metabolismo , Proteínas Proto-Oncogénicas c-cbl/fisiología , Envejecimiento/fisiología , Animales , Dieta Alta en Grasa , Humanos , Sistema Inmunológico/enzimología , Sistema Inmunológico/metabolismo , Activación de Macrófagos/genética , Ratones , Obesidad/complicaciones , Obesidad/genéticaRESUMEN
BACKGROUND: The proto-oncogene Casitas b-lineage lymphoma (c-Cbl) is an adaptor protein with an intrinsic E3 ubiquitin ligase activity that targets receptor and nonreceptor tyrosine kinases, resulting in their ubiquitination and downregulation. However, the function of c-Cbl in the control of cardiac function is currently unknown. In this study, we examined the role of c-Cbl in myocyte death and cardiac function after myocardial ischemia. METHODS AND RESULTS: We show increased c-Cbl expression in human ischemic and dilated cardiomyopathy hearts and in response to pathological stress stimuli in mice. c-Cbl-deficient mice demonstrated a more robust functional recovery after myocardial ischemia/reperfusion injury and significantly reduced myocyte apoptosis and improved cardiac function. Ubiquitination and downregulation of key survival c-Cbl targets, epidermal growth factor receptors and focal adhesion kinase, were significantly reduced in c-Cbl knockout mice. Inhibition of c-Cbl expression or its ubiquitin ligase activity in cardiac myocytes offered protection against H2O2 stress. Interestingly, c-Cbl deletion reduced the risk of death and increased cardiac functional recovery after chronic myocardial ischemia. This beneficial effect of c-Cbl deletion was associated with enhanced neoangiogenesis and increased expression of vascular endothelial growth factor-a and vascular endothelial growth factor receptor type 2 in the infarcted region. CONCLUSIONS: c-Cbl activation promotes myocyte apoptosis, inhibits angiogenesis, and causes adverse cardiac remodeling after myocardial infarction. These findings point to c-Cbl as a potential therapeutic target for the maintenance of cardiac function and remodeling after myocardial ischemia.
Asunto(s)
Cardiomiopatía Dilatada/fisiopatología , Insuficiencia Cardíaca/fisiopatología , Isquemia Miocárdica/fisiopatología , Proteínas Proto-Oncogénicas c-cbl/fisiología , Adulto , Anciano , Animales , Apoptosis/fisiología , Cateterismo Cardíaco , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/patología , Ecocardiografía , Electrocardiografía , Femenino , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/patología , Humanos , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Isquemia Miocárdica/genética , Isquemia Miocárdica/patología , Miocitos Cardíacos/citología , Miocitos Cardíacos/fisiología , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-cbl/genética , Ratas , Ratas Sprague-Dawley , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Discoidin domain receptor 2 (DDR2), a collagen receptor tyrosine kinase, initiates signal transduction upon collagen binding, but little is known as to how DDR2 signaling is negatively regulated. Herein we demonstrate that Cbl family member Cbl-b predominantly promotes the ubiquitination of DDR2 upon collagen II stimulation. Cbl-b-mediated ubiquitination accelerates the degradation of activated DDR2. Finally, the production of MMP-13, a downstream target of DDR2, is enhanced in Cbl-b-knocked down MC3T3-E1 cells and Cbl-b-deficient mouse primary synovial fibroblasts. Thus, Cbl-b, by promoting the ubiquitination and degradation of DDR2, functions as a negative regulator in the DDR2 signaling pathway.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Proteínas Proto-Oncogénicas c-cbl/fisiología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Mitogénicos/metabolismo , Ubiquitinación , Células 3T3 , Animales , Receptores con Dominio Discoidina , Estabilidad de Enzimas , Fibroblastos/enzimología , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Metaloproteinasa 13 de la Matriz/metabolismo , Ratones , Osteoblastos/enzimología , Transducción de Señal , Líquido Sinovial/citologíaRESUMEN
BACKGROUND: T-cell targeted peptide epitope tolerogens from grass pollen allergens may be useful in treating seasonal allergic rhinitis, but there is urgent need for optimisation of approaches from improved understanding of mechanism. OBJECTIVE: We sought to identify human leukocyte antigen (HLA)-DR1-restricted epitopes from the Timothy grass pollen allergen, Phleum pratense, and characterise T-cell immune regulation following intranasal administration of a single, immunodominant epitope. METHODS: T-cell epitopes within P pratense were identified using HLA-DR1 transgenic mice and tetramer-guided epitope mapping (TGEM) in HLA-DR1-positive individuals with grass allergy. An immunodominant epitope was tested in HLA-DR1 transgenics for impact on responses to whole Phl p5 b or peptide. Microarrays and quantitative PCR were used to characterise T-cell immunity. RESULTS: Peptide 26 (p26) was identified in HLA-DR1 transgenic mice and by TGEM analysis of HLA-DR1-positive individuals with grass allergy. p26 shows promiscuous binding to a wide range of HLA class II alleles, making it of relevance across immunogenetically diverse patients. The epitope is conserved in rye and velvet grass, making it applicable across a spectrum of grass pollen allergy. Intranasal pretreatment of mice with p26 results in significantly reduced T-cell responses. Transcriptomic array analysis in mice showed T-cell regulation in the intranasal treatment group associated with increased expression of members of the Cbl-b and Itch E3 ubiquitin ligase pathway. CONCLUSIONS: We defined an immunodominant P pratense epitope, p26, with broad binding across multiple HLA class II alleles. Intranasal treatment of mice with p26 results in T-cell regulation to whole allergen, involving the Cbl-b and Itch regulatory pathway.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Alérgenos/inmunología , Linfocitos T CD4-Positivos/inmunología , Antígeno HLA-DR1/inmunología , Epítopos Inmunodominantes/inmunología , Proteínas de Plantas/inmunología , Polen/inmunología , Proteínas Proto-Oncogénicas c-cbl/fisiología , Rinitis Alérgica Estacional/inmunología , Ubiquitina-Proteína Ligasas/fisiología , Adulto , Animales , Femenino , Humanos , Inmunidad Celular , Masculino , Ratones , Ratones Transgénicos , Análisis por Micromatrices , Persona de Mediana Edad , Phleum/inmunología , Reacción en Cadena en Tiempo Real de la Polimerasa , Reino Unido , Adulto JovenRESUMEN
The mTOR positively regulates cell proliferation and survival through forming 2 complexes with raptor (mTOR complex 1; mTORC1) or rictor (mTOR complex 2; mTORC2). Compared with the mTORC1, relatively little is known about the biologic functions of mTORC2. This study focuses on addressing whether mTORC2 regulates apoptosis, particularly induced by TRAIL (TNFSF10). Using the mTOR kinase inhibitor, PP242, as a research tool, we found that it synergized with TRAIL to augment apoptosis of cancer cells. PP242 reduced the abundance of the short form of c-FLIP (FLIP(S), CFLAR(S)) and survivin (BIRC5). Enforced expression of ectopic FLIP(S), but not survivin, attenuated augmented apoptosis induced by PP242 plus TRAIL. Thus, it is FLIP(S) downregulation that contributes to synergistic induction of apoptosis by PP242 plus TRAIL. PP242 decreased FLIP(S) stability, increased FLIP(S) ubiquitination, and facilitated FLIP(S) degradation. Moreover, knockdown of the E3 ligase Cbl (CBL) abolished PP242-induced FLIP(S) reduction. Thus, PP242 induces Cbl-dependent degradation of FLIP(S), leading to FLIP(S) downregulation. Consistently, knockdown of rictor or mTOR, but not raptor, mimicked PP242 in decreasing FLIP(S) levels and sensitizing cells to TRAIL. Rictor knockdown decreased FLIP(S) stability, whereas enforced expression of rictor stabilized FLIP(S). Moreover, silencing of Cbl abrogated FLIP(S) reduction induced by rictor knockdown. Collectively we conclude that it is mTORC2 inhibition that results in FLIP(S) downregulation and subsequent sensitization of TRAIL-induced apoptosis. Our findings provide the first evidence showing that mTORC2 stabilizes FLIP(S), hence connecting mTORC2 signaling to the regulation of death receptor-mediated apoptosis.
Asunto(s)
Apoptosis/fisiología , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Complejos Multiproteicos/fisiología , Proteínas Proto-Oncogénicas c-cbl/fisiología , Ligando Inductor de Apoptosis Relacionado con TNF/fisiología , Serina-Treonina Quinasas TOR/fisiología , Secuencia de Bases , Western Blotting , Línea Celular Tumoral , Humanos , Indoles/farmacología , Diana Mecanicista del Complejo 2 de la Rapamicina , Complejos Multiproteicos/antagonistas & inhibidores , Proteolisis , Proteínas Proto-Oncogénicas c-akt/metabolismo , Purinas/farmacología , ARN Interferente Pequeño , Serina-Treonina Quinasas TOR/antagonistas & inhibidoresRESUMEN
One of the causes of breast cancer is overexpression of the human epidermal growth factor receptor 2 (HER2). Enhanced receptor autophosphorylation and resistance to activation-induced downregulation have been suggested as mechanisms for HER2-induced sustained signaling and cell transformation. However, the molecular mechanisms underlying these possibilities remain incompletely understood. In the current report, we present evidence that show that HER2 overexpression does not lead to receptor hyper-autophosphorylation, but alters patterns in a manner that favors receptor stability and sustained signaling. Specifically, HER2 overexpression blocks epidermal growth factor receptor (EGFR) tyrosine phosphorylation on Y1045 and Y1068, the known docking sites of c-Cbl and Grb2, respectively, whereas promoting phosphorylation on Y1173, the known docking site of the Gab adaptor proteins and phospholipase C gamma. Under these conditions, HER2 itself is phosphorylated on Y1221/1222, with no known role, and on Y1248 that corresponds to Y1173 of EGFR. Interestingly, suppressed EGFR autophosphorylation on the Grb2 and c-Cbl-binding sites correlated with receptor stability and sustained signaling, suggesting that HER2 accomplishes these tasks by altering autophosphorylation patterns. In conformity with these findings, mutation of the Grb2-binding site on EGFR (Y1068F-EGFR) conferred resistance to ligand-induced degradation, which in turn induced sustained signaling, and increased cell proliferation and transformation. These findings suggest that the Grb2-binding site on EGFR is redundant for signaling, but critical for receptor regulation. On the other hand, mutation of the putative Grb2-binding site in HER2 (Y1139) did not affect stability, signaling or transformation, suggesting that Y1139 in HER2 may not serve as a Grb2-binding site. In agreement with the role of EGFR in HER2 signaling, inhibition of EGFR expression reduced HER2-induced anchorage-independent growth and tumorigenesis. These results imply that complementing HER2-targeted therapies with anti-EGFR drugs may be beneficial in HER2-positive breast cancer.
Asunto(s)
Neoplasias de la Mama/patología , Proliferación Celular , Receptores ErbB/fisiología , Receptor ErbB-2/fisiología , Transducción de Señal , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Transformación Celular Neoplásica , Femenino , Proteína Adaptadora GRB2/metabolismo , Humanos , Fosforilación , Multimerización de Proteína , Proteínas Proto-Oncogénicas c-cbl/fisiología , Receptor ErbB-2/química , UbiquitinaciónRESUMEN
Platelets play a fundamental role in hemostasis. Their functional responses have to be tightly controlled as any disturbance may lead to bleeding disorders or thrombosis. It is thus important to clearly identify and understand the signaling mechanisms involved in platelet function. An important role of c-Cbl and Cbl-b ubiquitin ligases in platelet functional responses and in hematological malignancies has been recently described. Cbl proteins perform negative and positive regulation of several signaling pathways in platelets. In this review, we explore the role of Cbl proteins in platelet functional responses.
Asunto(s)
Activación Plaquetaria/fisiología , Proteínas Proto-Oncogénicas c-cbl/fisiología , Plaquetas/fisiología , HumanosRESUMEN
BCR-ABL induces chronic myeloid leukemia (CML) through the aberrant regulation of multiple signaling substrates. Previous research has shown that BCR-ABL mediates down-modulation of CBL-B protein levels. A murine bone marrow transplantation (BMT) study was performed to assess the contribution of Cbl-b to BCR-ABL-induced disease. The predominant phenotype in the Cbl-b(-/-) recipients was a CML-like myeloproliferative disease (MPD) similar to that observed in the wild-type animals, but with a longer latency, diminished circulating leukocyte numbers and reduced spleen weights. Despite the decreased leukemic burden in comparison to their wild-type counterparts, the Cbl-b(-/-) animals displayed enhanced numbers of Gr-1(+)/Mac-1(+) spleen cells and neutrophilia. On the basis of prior evidence of CBL-B-dependent motility toward SDF-1α, we hypothesized that Cbl-b deficiency might impair bone marrow localization during transplantation. Homing experiments showed reduced migration of Cbl-b(-/-) cells to the bone marrow. Intrafemoral transplantation of BCR-ABL-transduced Cbl-b(-/-) cells revealed equivalent latency of disease development to the wild-type transplants, supporting the conclusion that Cbl-b deficiency diminishes homing of leukemic cells to the bone marrow, and perturbs the proliferation of BCR-ABL-expressing malignant clones during CML development.
Asunto(s)
Células de la Médula Ósea/fisiología , Proteínas de Fusión bcr-abl/fisiología , Leucemia Mielógena Crónica BCR-ABL Positiva/etiología , Proteínas Proto-Oncogénicas c-cbl/fisiología , Animales , Trasplante de Médula Ósea , Movimiento Celular , Células Cultivadas , Ratones , Ratones Endogámicos BALB C , Receptores de Quimiocina/análisisRESUMEN
Protein tyrosine kinases (PTKs) coordinate a broad spectrum of cellular responses to extracellular stimuli and cell-cell interactions during development, tissue homeostasis, and responses to environmental challenges. Thus, an understanding of the regulatory mechanisms that ensure physiological PTK function and potential aberrations of these regulatory processes during diseases such as cancer are of broad interest in biology and medicine. Aside from the expected role of phospho-tyrosine phosphatases, recent studies have revealed a critical role of covalent modification of activated PTKs with ubiquitin as a critical mechanism of their negative regulation. Members of the Cbl protein family (Cbl, Cbl-b and Cbl-c in mammals) have emerged as dominant "activated PTK-selective" ubiquitin ligases. Structural, biochemical and cell biological studies have established that Cbl protein-dependent ubiquitination targets activated PTKs for degradation either by facilitating their endocytic sorting into lysosomes or by promoting their proteasomal degradation. This mechanism also targets PTK signaling intermediates that become associated with Cbl proteins in a PTK activation-dependent manner. Cellular and animal studies have established that the relatively broadly expressed mammalian Cbl family members Cbl and Cbl-b play key physiological roles, including their critical functions to prevent the transition of normal immune responses into autoimmune disease and as tumor suppressors; the latter function has received validation from human studies linking mutations in Cbl to human leukemia. These newer insights together with embryonic lethality seen in mice with a combined deletion of Cbl and Cbl-b genes suggest an unappreciated role of the Cbl family proteins, and by implication the ubiquitin-dependent control of activated PTKs, in stem/progenitor cell maintenance. Future studies of existing and emerging animal models and their various cell lineages should help test the broader implications of the evolutionarily-conserved Cbl family protein-mediated, ubiquitin-dependent, negative regulation of activated PTKs in physiology and disease.