RESUMEN
Since its discovery over three decades ago, signal transducer and activator of transcription 1 (STAT1) has been extensively studied as a central mediator for interferons (IFNs) signaling and antiviral defense. Here, using genetic and biochemical assays, we unveil Thr748 as a conserved IFN-independent phosphorylation switch in Stat1, which restricts IFN signaling and promotes innate inflammatory responses following the recognition of the bacterial-derived toxin lipopolysaccharide (LPS). Genetically engineered mice expressing phospho-deficient threonine748-to-alanine (T748A) mutant Stat1 are resistant to LPS-induced lethality. Of note, T748A mice exhibited undisturbed IFN signaling, as well as total expression of Stat1. Further, the T748A point mutation of Stat1 recapitulates the safeguard effect of the genetic ablation of Stat1 following LPS-induced lethality, indicating that the Thr748 phosphorylation contributes inflammatory functionalities of Stat1. Mechanistically, LPS-induced Toll-like receptor 4 endocytosis activates a cell-intrinsic IκB kinase-mediated Thr748 phosphorylation of Stat1, which promotes macrophage inflammatory response while restricting the IFN and anti-inflammatory responses. Depletion of macrophages restores the sensitivity of the T748A mice to LPS-induced lethality. Together, our study indicates a phosphorylation-dependent modular functionality of Stat1 in innate immune responses: IFN phospho-tyrosine dependent and inflammatory phospho-threonine dependent. Better understanding of the Thr748 phosphorylation of Stat1 may uncover advanced pharmacologically targetable molecules and offer better treatment modalities for sepsis, a disease that claims millions of lives annually.
Asunto(s)
Lipopolisacáridos , Transducción de Señal , Animales , Ratones , Fosforilación , Lipopolisacáridos/farmacología , Interferones/metabolismo , Inflamación/metabolismo , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismoRESUMEN
Immune cells infiltrate adipose tissues and provide a framework to regulate energy homeostasis. However, the precise underlying mechanisms and signaling by which the immune system regulates energy homeostasis in metabolic tissues remain poorly understood. Here, we show that the AT-rich interactive domain 5A (Arid5a), a cytokine-induced nucleic acid binding protein, is important for the maintenance of adipose tissue homeostasis. Long-term deficiency of Arid5a in mice results in adult-onset severe obesity. In contrast, transgenic mice overexpressing Arid5a are highly resistant to high-fat diet-induced obesity. Inhibition of Arid5a facilitates the in vitro differentiation of 3T3-L1 cells and fibroblasts to adipocytes, whereas its induction substantially inhibits their differentiation. Molecular studies reveal that Arid5a represses the transcription of peroxisome proliferator activated receptor gamma 2 (Ppar-γ2) due to which, in the absence of Arid5a, Ppar-γ2 is persistently expressed in fibroblasts. This phenomenon is accompanied by enhanced fatty acid uptake in Arid5a-deficient cells, which shifts metabolic homeostasis toward prolipid metabolism. Furthermore, we show that Arid5a and Ppar-γ2 are dynamically counterregulated by each other, hence maintaining adipogenic homeostasis. Thus, we show that Arid5a is an important negative regulator of energy metabolism and can be a potential target for metabolic disorders.
Asunto(s)
Adipogénesis/genética , Tejido Adiposo/metabolismo , Proteínas de Unión al ADN/genética , Retroalimentación Fisiológica , Obesidad/genética , PPAR gamma/genética , Factores de Transcripción/genética , Células 3T3-L1 , Adipocitos/metabolismo , Adipocitos/patología , Tejido Adiposo/patología , Animales , Transporte Biológico , Diferenciación Celular , Proteínas de Unión al ADN/metabolismo , Dieta Alta en Grasa/efectos adversos , Metabolismo Energético/genética , Ácidos Grasos/metabolismo , Femenino , Regulación de la Expresión Génica , Homeostasis/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Obesidad/etiología , Obesidad/metabolismo , Obesidad/patología , PPAR gamma/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismoRESUMEN
Immunomodulatory drugs (IMiDs), including thalidomide derivatives such as lenalidomide and pomalidomide, offer therapeutic benefit in several hematopoietic malignancies and autoimmune/inflammatory diseases. However, it is difficult to study the IMiD mechanism of action in murine disease models because murine cereblon (CRBN), the substrate receptor for IMiD action, is resistant to some of IMiDs therapeutic effects. To overcome this difficulty, we generated humanized cereblon (CRBNI391V) mice thereby providing an animal model to unravel complex mechanisms of action in a murine physiological setup. In our current study, we investigated the degradative effect toward IKZF1 and CK-1α, a target substrate of IMiDs. Unlike WT mice which were resistant to lenalidomide and pomalidomide, T lymphocytes from CRBNI391V mice responded with a higher degree of IKZF1 and CK-1α protein degradation. Furthermore, IMiDs resulted in an increase in IL-2 among CRBNI391V mice but not in the WT group. We have also tested a thalidomide derivative, FPFT-2216, which showed an inhibitory effect toward IKZF1 protein level. As opposed to pomalidomide, FPFT-2216 and lenalidomide degrades CK-1α. Additionally, we assessed the potential therapeutic effects of IMiDs in dextran sodium sulfate (DSS)-induced colitis. In both WT and humanized mice, lenalidomide showed a significant therapeutic effect in the DSS model of colitis, while the effect of pomalidomide was less pronounced. Thus, while IMiDs' degradative effect on IKZF1 and CK-1α, and up-regulation of IL-2, is dependent on CRBN, the therapeutic benefit of IMiDs in a mouse model of inflammatory bowel disease occurs through a CRBN-IMiD binding region independent pathway.
Asunto(s)
Inmunomodulación/efectos de los fármacos , Inmunomodulación/fisiología , Proteínas del Tejido Nervioso/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales , Animales , Humanos , Factor de Transcripción Ikaros/efectos de los fármacos , Factor de Transcripción Ikaros/metabolismo , Factores Inmunológicos/metabolismo , Ratones , Modelos Animales , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/fisiología , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Proteolisis/efectos de los fármacos , Especificidad por Sustrato , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
AT-rich interactive domain-containing protein 5a (Arid5a) is an RNA-binding protein (RBP) required for autoimmunity via stabilization of interleukin-6 (Il6) and signal transducer and activator of transcription 3 (STAT3) mRNAs. However, the roles of Arid5a in Th17 cells and its association with autoimmunity remain unknown. Here, we show that the levels of Arid5a and OX40 are correlated in CD4+ T cells under Th17 conditions in an IL-6-dependent manner. Lack of Arid5a in T cells reduced OX40 expression levels and repressed IL-17 production in response to OX40 ligation. Arid5a stabilized OX40 mRNA by recognizing the alternative decay element (ADE)-like stem-loop (SL) in the 3' untranslated region (3'UTR). Interestingly, Arid5a impaired the RNA-destabilizing functions of Regnase-1 and Roquin-1 on OX40 ADE-like SL. In EAE, Arid5a-deficient mice exhibited resistance to EAE, with reduced OX40 expression in CD4+ T cells, and the number of CD4+ CD45+ T cells was decreased in CNS. Furthermore, ameliorated EAE was induced by adoptive transfer of Arid5a-/- encephalitogenic CD4+ T cells expressing less OX40 mRNA and producing less IL-17. In conclusion, our findings indicate that the Arid5a/OX40 axis in CD4+ T cells may have important implications in pathogenesis of autoimmune diseases such as EAE.
Asunto(s)
Autoinmunidad/inmunología , Proteínas de Unión al ADN/metabolismo , Glicoproteínas de Membrana/genética , Factor de Transcripción STAT3/inmunología , Células Th17/inmunología , Factores de Transcripción/metabolismo , Factores de Necrosis Tumoral/genética , Traslado Adoptivo , Animales , Autoinmunidad/genética , Línea Celular , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/patología , Células HEK293 , Humanos , Interleucina-17/biosíntesis , Interleucina-6/inmunología , Secuencias Invertidas Repetidas/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ligando OX40 , Interferencia de ARN , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/metabolismo , Ribonucleasas/genética , Factor de Transcripción STAT3/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
microRNA (miRNA) mediated regulation of protein expression has emerged as an important mechanism in T-cell physiology, from development and survival to activation, proliferation, and differentiation. One of the major classes of proteins involved in these processes are cytokines, which are both key input signals and major products of T-cell function. Here, we summarize the current data on the molecular cross-talk between cytokines and miRNAs: how cytokines regulate miRNA expression, and how specific miRNAs control cytokine production in T cells. We also describe the inflammatory consequences of deregulating the miRNA/cytokine axis in mice and humans. We believe this topical area will have key implications for immune modulation and treatment of autoimmune pathology.
Asunto(s)
Citocinas/genética , Citocinas/metabolismo , Regulación de la Expresión Génica , MicroARNs/genética , Transducción de Señal , Linfocitos T/inmunología , Linfocitos T/metabolismo , Animales , Humanos , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , MicroARNs/metabolismoRESUMEN
Proteins are large, complex molecules that regulate multiple functions within the cell. The protein rarely functions as a single molecule, but rather interacts with one or more other proteins forming a dynamic network. Protein-protein interactions are critical for regulating the cell's response toward various stimuli from outside and inside the cell. Identification of protein-protein interactions enhanced our understanding of various biological processes in the living cell. Immunoprecipitation (IP) has been one of the standard and most commonly used biochemical methods to identify and confirm protein-protein interactions. IP uses a target protein-specific antibody conjugated with protein A/G affinity beads to identify molecules interacting with the target protein. Here, we describe the principle, procedure and challenges of the IP assay.
Asunto(s)
Inmunoprecipitación , Mapeo de Interacción de Proteínas , Inmunoprecipitación/métodos , Humanos , Animales , Mapeo de Interacción de Proteínas/métodos , Ratones , Unión Proteica , Xenoinjertos , Proteínas/metabolismoRESUMEN
Interleukin (IL-6) is a pleotropic cytokine with both tumor-promoting and -inhibitory effects on breast cancer growth. However, the mechanisms governing the outcome of IL-6 on cancer progression remain to be clarified. Our study unraveled a novel long noncoding RNA (lncRNA) AU021063 downstream of IL-6 signaling. We found that IL-6 induced the expression of AU021063 predominantly in breast cancer compared to other cancer types. Mechanistically, IL-6 induced AT-rich interactive domain 5a (Arid5a) expression, which promotes the transcription of AU021063. In turn, AU021063 promotes breast cancer metastasis through stabilizing tribbles homolog 3 (Trib3) and activating Mek/Erk signaling pathway. Genetic ablation of either Arid5a, AU021063 or Trib3 abolished breast cancer metastasis in vitro and in vivo. Overall, our study highlights the importance of IL-6-Arid5a-AU021063 axis in regulating breast cancer invasiveness and metastasis, which may provide potential novel therapeutics for breast cancer.
Asunto(s)
Neoplasias de la Mama/genética , Proteínas de Ciclo Celular/genética , Proteínas de Unión al ADN/genética , Interleucina-6/genética , ARN Largo no Codificante/genética , Factores de Transcripción/genética , Animales , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Modelos Animales de Enfermedad , Femenino , Humanos , Sistema de Señalización de MAP Quinasas/genética , Ratones , Invasividad Neoplásica/genética , Invasividad Neoplásica/patología , Metástasis de la Neoplasia , Transducción de SeñalRESUMEN
The acquisition of mesenchymal traits leads to immune evasion in various cancers, but the underlying molecular mechanisms remain unclear. In this study, we found that the expression levels of AT-rich interaction domain-containing protein 5a (Arid5a), an RNA-binding protein, were substantially increased in mesenchymal tumor subtypes. The deletion of Arid5a in tumor cell lines enhanced antitumor immunity in immunocompetent mice, but not in immunodeficient mice, suggesting a role for Arid5a in immune evasion. Furthermore, an Arid5a-deficient tumor microenvironment was shown to have robust antitumor immunity, as manifested by suppressed infiltration of granulocytic myeloid-derived suppressor cells and regulatory T cells. In addition, infiltrated T cells were more cytotoxic and less exhausted. Mechanistically, Arid5a stabilized Ido1 and Ccl2 mRNAs and augmented their expression, resulting in enhanced tryptophan catabolism and an immunosuppressive tumor microenvironment. Thus, our findings demonstrate the role of Arid5a beyond inflammatory diseases and suggest Arid5a as a promising target for the treatment of immunotolerant malignant tumors.See related Spotlight by Van den Eynde, p. 854.
Asunto(s)
Quimiocinas/metabolismo , Proteínas de Unión al ADN/metabolismo , Evasión Inmune/inmunología , Inmunoterapia/métodos , Factores de Transcripción/metabolismo , Triptófano/metabolismo , Animales , Femenino , Humanos , Ratones , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Interleukin-6 (IL-6) has been identified as a 26-kD secreted protein that stimulates B cells to produce antibodies. Later, IL-6 was revealed to have various functions that overlap with other IL-6 family cytokines and use the common IL-6 signal transducer gp130. IL-6 stimulates cells through multiple pathways, using both membrane and soluble IL-6 receptors. As indicated by the expanding market for IL-6 inhibitors, it has become a primary therapeutic target among IL-6 family cytokines. Here, we revisit the discovery of IL-6; discuss insights regarding the roles of this family of cytokines; and highlight recent advances in our understanding of regulation of IL-6 expression.
Asunto(s)
Interleucina-6/historia , Animales , Pleiotropía Genética , Historia del Siglo XX , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Modelos Biológicos , Mutación/genética , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/metabolismoRESUMEN
The lipopolysaccharide (LPS)-induced endocytosis of Toll-like receptor 4 (TLR4) is an essential step in the production of interferon-ß (IFN-ß), which activates the transcription of antiviral response genes by STAT1 phosphorylated at Tyr701 Here, we showed that STAT1 regulated proinflammatory cytokine production downstream of TLR4 endocytosis independently of IFN-ß signaling and the key proinflammatory regulator NF-κB. In human macrophages, TLR4 endocytosis activated a noncanonical phosphorylation of STAT1 at Thr749, which subsequently promoted the production of interleukin-6 (IL-6) and IL-12p40 through distinct mechanisms. STAT1 phosphorylated at Thr749 activated the expression of the gene encoding ARID5A, which stabilizes IL6 mRNA. Moreover, STAT1 phosphorylated at Thr749 directly enhanced transcription of the gene encoding IL-12p40 (IL12B). Instead of affecting STAT1 nuclear translocation, phosphorylation of Thr749 facilitated the binding of STAT1 to a noncanonical DNA motif (5'-TTTGANNC-3') in the promoter regions of ARID5A and IL12B The endocytosis of TLR4 induced the formation of a complex between the kinases TBK1 and IKKß, which mediated the phosphorylation of STAT1 at Thr749 Our data suggest that noncanonical phosphorylation in response to LPS confers STAT1 with distinct DNA binding and gene-regulatory properties that promote both IL12B expression and IL6 mRNA stabilization. Thus, our study provides a potential mechanism for how TLR4 endocytosis might regulate proinflammatory cytokine production.