RESUMO
The common γ chain (γc; IL-2RG) is a subunit of the interleukin (IL) receptors for the γc cytokines IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. The lack of appropriate neutralizing antibodies recognizing IL-2RG has made it difficult to thoroughly interrogate the role of γc cytokines in inflammatory and autoimmune disease settings. Here, we generated a γc cytokine receptor antibody, REGN7257, to determine whether γc cytokines might be targeted for T cell-mediated disease prevention and treatment. Biochemical, structural, and in vitro analysis showed that REGN7257 binds with high affinity to IL-2RG and potently blocks signaling of all γc cytokines. In nonhuman primates, REGN7257 efficiently suppressed T cells without affecting granulocytes, platelets, or red blood cells. Using REGN7257, we showed that γc cytokines drive T cell-mediated disease in mouse models of graft-versus-host disease (GVHD) and multiple sclerosis by affecting multiple aspects of the pathogenic response. We found that our xenogeneic GVHD mouse model recapitulates hallmarks of acute and chronic GVHD, with T cell expansion/infiltration into tissues and liver fibrosis, as well as hallmarks of immune aplastic anemia, with bone marrow aplasia and peripheral cytopenia. Our findings indicate that γc cytokines contribute to GVHD and aplastic anemia pathology by promoting these characteristic features. By demonstrating that broad inhibition of γc cytokine signaling with REGN7257 protects from immune-mediated disorders, our data provide evidence of γc cytokines as key drivers of pathogenic T cell responses, offering a potential strategy for the management of T cell-mediated diseases.
Assuntos
Anemia Aplástica , Doença Enxerto-Hospedeiro , Subunidade gama Comum de Receptores de Interleucina , Linfócitos T , Animais , Camundongos , Anemia Aplástica/metabolismo , Anticorpos Monoclonais/metabolismo , Citocinas/metabolismo , Doença Enxerto-Hospedeiro/metabolismo , Transdução de Sinais , Linfócitos T/metabolismo , Linfócitos T/patologia , Subunidade gama Comum de Receptores de Interleucina/antagonistas & inibidores , Subunidade gama Comum de Receptores de Interleucina/metabolismo , PrimatasRESUMO
The objective was to study the safety of a Napin-Rich Canola Protein Isolate (NRCPI) fed to rats at various levels for 13-weeks. The study included four groups (20 animals/sex/group) of young Sprague Dawley rats. They were fed ad libitum with an AIN-93G based protein-free diet containing, respectively, 5%, 10% and 20% (w/w) NRCPI (test article) or 20% (w/w) vitamin-free casein (control article). Protein levels were adjusted at 18% in all groups with vitamin-free casein. Body weights, food consumption, locomotor activity and behavioral and clinical pathology parameters were recorded at various points in the study, followed by macroscopic examination, determination of organ weights and microscopic examination at termination. There were no test article-related effects on ophthalmology, functional observations, hematology, serum chemistry, urinalysis, organ weights and macroscopic or microscopic findings. Lower body weight gains were observed in the 10% NRCPI-treated males and the 20% NRCPI-treated males and females. The lower body weight gains were associated with significantly lower food consumption. Therefore, for NRCPI the No Observed Adversed Effect Level (NOAEL) was considered to be 20% (the highest fed level); equivalent to 12.46 g/kg BW/day for males and 14.95 g/kg BW/day for females. The NRCPI was considered safe under the tested conditions.
Assuntos
Albuminas 2S de Plantas/toxicidade , Peso Corporal/efeitos dos fármacos , Brassica napus/química , Animais , Comportamento Animal/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Feminino , Masculino , Atividade Motora/efeitos dos fármacos , Nível de Efeito Adverso não Observado , Ratos , Ratos Sprague-Dawley , Testes de ToxicidadeRESUMO
The tumor suppressor ARF inhibits cell growth in response to oncogenic stress in a p53-dependent manner. Also, there is an increasing appreciation of ARF's ability to inhibit cell growth via multiple p53-independent mechanisms, including its ability to regulate the E2F pathway. We have investigated the interaction between the tumor suppressor ARF and DP1, the DNA binding partner of the E2F family of factors (E2Fs). We show that ARF directly binds to DP1. Interestingly, binding of ARF to DP1 results in an inhibition of the interaction between DP1 and E2F1. Moreover, ARF regulates the association of DP1 with its target gene, as evidenced by a chromatin immunoprecipitation assay with the dhfr promoter. By analyzing a series of ARF mutants, we demonstrate a strong correlation between ARF's ability to regulate DP1 and its ability to cause cell cycle arrest. S-phase inhibition by ARF is preceded by an inhibition of the E2F-activated genes. Moreover, we provide evidence that ARF inhibits the E2F-activated genes independently of p53 and Mdm2. Also, the interaction between ARF and DP1 is enhanced during oncogenic stress and "culture shock." Taken together, our results show that DP1 is a critical direct target of ARF.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fase G1/genética , Regulação da Expressão Gênica , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p14ARF/metabolismo , Animais , Ciclina A/genética , Ciclina A/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina , Regulação para Baixo , Fatores de Transcrição E2F , Fator de Transcrição E2F1 , Fase G1/fisiologia , Humanos , Camundongos , Mutação , Regiões Promotoras Genéticas/genética , Tetra-Hidrofolato Desidrogenase/genética , Fator de Transcrição DP1 , Proteína Supressora de Tumor p14ARF/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
The objective was to evaluate the safety of a cruciferin-rich canola protein isolate (Puratein) when fed as a protein source at various dietary levels to rats for 13-weeks. The study included four groups (20 animals/sex/group) of young Sprague Dawley rats. They were fed ad libitum with an AIN-93 G based protein-free diet added respectively with 5%, 10% and 20% (w/w) Puratein (test article) or 20% (w/w) vitamin-free casein (control article). Protein levels were adjusted in all groups at 18% using vitamin-free casein. Body weights, food consumption, locomotor activity and behavioral and clinical pathology parameters were recorded at various points of the study, followed by macroscopic examination, determination of organ weights and microscopic tissue examination. There were no test article-related effects on body weight, food consumption, clinical observations, functional observational battery, motor activity, clinical pathology, or ophthalmic examinations. A slightly higher thyroid/parathyroid weight (g/100g BW) noted in the 20% Puratein group was not correlated with histopathological changes. The no-observed-effect-level (NOEL) was 10%, whereas the no-observed-adverse-effect-level (NOAEL) was the highest fed level of 20%, equivalent to 11.24 g/kg BW/day for males and 14.11 g/kg BW/day for females. The cruciferin-rich canola protein isolate (Puratein) was considered safe under the conditions tested.
Assuntos
Antígenos de Plantas/toxicidade , Brassica napus/química , Proteínas de Armazenamento de Sementes/toxicidade , Ração Animal , Animais , Antígenos de Plantas/análise , Comportamento Animal/efeitos dos fármacos , Análise Química do Sangue , Peso Corporal/efeitos dos fármacos , Dieta , Ingestão de Alimentos/efeitos dos fármacos , Feminino , Testes Hematológicos , Longevidade/efeitos dos fármacos , Masculino , Atividade Motora/efeitos dos fármacos , Nível de Efeito Adverso não Observado , Tamanho do Órgão/efeitos dos fármacos , Glândulas Paratireoides/efeitos dos fármacos , Glândulas Paratireoides/patologia , Ratos , Ratos Sprague-Dawley , Proteínas de Armazenamento de Sementes/análise , Glândula Tireoide/efeitos dos fármacos , Glândula Tireoide/patologia , Testes de Toxicidade/métodos , UrináliseRESUMO
NF-kappaB is a family of transcription factors important for innate and adaptive immunity. NF-kappaB is restricted to the cytoplasm by inhibitory proteins that are degraded when specifically phosphorylated, permitting NF-kappaB to enter the nucleus and activate target genes. Phosphorylation of the inhibitory proteins is mediated by an IkappaB kinase (IKK) complex, which can be composed of two subunits with enzymatic activity, IKKalpha and IKKbeta. The preferred substrate for IKKbeta is IkappaBalpha, degradation of which liberates p65 (RelA) to enter the nucleus where it induces genes important to innate immunity. IKKalpha activates a non-canonical NF-kappaB pathway in which p100 (NF-kappaB2) is processed to p52. Once produced, p52 can enter the nucleus and induce genes important to adaptive immunity. This study shows that Akt binds to and increases the activity of IKKalpha and thereby increases p52 production in cells. Constitutively active Akt augments non-canonical NF-kappaB activity, whereas kinase dead Akt or inhibition of phosphatidylinositol 3-kinase have the opposite effect. Basal and ligand-induced p52 production is reduced in mouse embryo fibroblasts deficient in Akt1 and Akt2 compared with parental cells. These observations show that Akt plays a role in activation of basal and induced non-canonical NF-kappaB activity.
Assuntos
Subunidade p52 de NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Linhagem Celular , Citoplasma/metabolismo , Embrião de Mamíferos/metabolismo , Humanos , Imunidade , Camundongos , Subunidade p52 de NF-kappa B/química , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Recombinantes/química , Especificidade por SubstratoRESUMO
The capacity of DNA damaging agents to induce apoptosis is regulated by target gene induction by p53. We found that p53 targeted MDM2 in cells in which DNA repair was occurring, but persistent DNA damage induced by chemotherapy led p53 to selectively target PTEN. High dose chemotherapy induced the phosphorylation of p53 on serine 46, whereas low dose chemotherapy did not. A nonphosphorylatable serine 46 to alanine p53 mutant (S46A) targeted the MDM2 promoter in preference to that for PTEN. A serine 46 to aspartate mutant (S46D, a phosphorylation mimic) targeted PTEN in preference to MDM2. These observations show that phosphorylation of serine 46 in p53 is sufficient for it to induce the PTEN (phosphatase and tensin homolog deleted on chromosome ten) tumor suppressor protein in preference to MDM2. S46A induced significantly less cell death than the S46D in cells. The phosphorylation-induced change of p53 promoter targeting suppresses the induction of MDM2 and the formation of the autoregulatory feedback loop. Induction of PTEN by p53 followed by expression of PTEN inhibits AKT-induced translocation of MDM2 into the nucleus and sustains p53 function. The protection of p53 from MDM2 by PTEN and the damage-induced activation of PTEN by phosphorylated p53 leads to the formation of an apoptotic amplification cycle in which p53 and PTEN coordinately increase cellular apoptosis.
Assuntos
Apoptose , Regulação Neoplásica da Expressão Gênica , Proteínas Nucleares/fisiologia , Monoéster Fosfórico Hidrolases/fisiologia , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/fisiologia , Serina/química , Proteína Supressora de Tumor p53/química , Proteínas Supressoras de Tumor/fisiologia , Antineoplásicos/farmacologia , Morte Celular , Linhagem Celular Tumoral , Ensaio Cometa , Dano ao DNA , Etoposídeo/farmacologia , Retroalimentação Fisiológica , Genes Reporter , Humanos , Immunoblotting , Mutação , Proteínas Nucleares/química , PTEN Fosfo-Hidrolase , Fosforilação , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas c-mdm2 , RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Transcrição Gênica , Ativação Transcricional , Transfecção , Proteína Supressora de Tumor p53/metabolismoRESUMO
Tumor necrosis factor (TNF) promotes immunity and modulates cell viability, in part, by promoting alterations of cellular gene expression. The mechanisms through which TNF communicates with the nucleus and alters gene expression are incompletely understood. Incubation of human umbilical vein endothelial cells (HUVEC) with TNF induces phosphorylation of the CRE-binding protein (CREB) transcription factor on serine 133 and increases CREB DNA binding and transactivation. Dominant negative CREB, an antagonist antibody directed against the type 1 TNF receptor, or pharmacological inhibition of p38 MAPK signaling blocked TNF-induced CREB activation as determined by phosphorylation and gene reporter assays. From among the kinases that can activate CREB, we found that downstream of p38 MAPK, MSK1 is activated by TNF to promote CREB activation. These observations show that CREB is activated by TNF/TNFR1 signaling through a p38MAPK/MSK1 signaling pathway.
Assuntos
Antineoplásicos/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Endotélio Vascular/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Antígenos CD/metabolismo , Antineoplásicos/farmacologia , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Endotélio Vascular/citologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Fosforilação , Receptores do Fator de Necrose Tumoral/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral , Serina/metabolismo , Ativação Transcricional/efeitos dos fármacos , Ativação Transcricional/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Veias Umbilicais/citologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Phosphatidylinositol (PI) 3-kinase/Akt signaling activates NF-kappa B through pleiotropic, cell type-specific mechanisms. This study investigated the significance of PI 3-kinase/Akt signaling to tumor necrosis factor (TNF)-induced NF-kappa B activation in transformed, immortalized, and primary cells. Pharmacological inhibition of PI 3-kinase blocked TNF-induced NF-kappa B DNA binding in the 293 line of embryonic kidney cells, partially affected binding in MCF-7 breast cancer cells, HeLa and ME-180 cervical carcinoma cells, and NIH 3T3 cells but was without significant effect in H1299 and human umbilical vein endothelial cells, cell types in which TNF activated Akt. NF-kappa B is retained in the cytoplasm by inhibitory proteins, I kappa Bs, which are phosphorylated and targeted for degradation by I kappa B kinases (IKK alpha and IKK beta). Expression and the ratios of IKK alpha and IKK beta, which homo- and heterodimerize, varied among cell types. Cells with a high proportion of IKK alpha (the IKK kinase activated by Akt) to IKK beta were most sensitive to PI 3-kinase inhibitors. Consequently, transient expression of IKK beta diminished the capacity of the inhibitors to block NF-kappa B DNA binding in 293 cells. Also, inhibitors of PI 3-kinase blocked NF-kappa B DNA binding in Ikk beta-/- but not Ikk alpha-/- or wild-type cells in which the ratio of IKK alpha to IKK beta is low. Thus, noncoordinate expression of I kappa B kinases plays a role in determining the cell type-specific role of Akt in NF-kappa B activation.