RESUMEN

Precursor B cell acute lymphoblastic leukemia (Pre-B-ALL) arises from developing B cells and frequently involves mutations in genes encoding transcription factors. In this study, we investigated the function of mutations in the transcription factor IKZF3 (Aiolos), R137* and H195Y, discovered in a mouse model of pre-B-ALL. R137* IKZF3 mutation resulted in a truncated protein, while electrophoretic mobility shift assay showed that H195Y IKZF3 mutation resulted in a protein with altered DNA binding. 38B9 pre-B cell lines were generated expressing WT and H195Y IKZF3 proteins. Anti-IKZF3 ChIP-seq showed that H195Y IKZF3 interacted with a larger number of sites that were different than WT IKZF3. Treatment with interleukin-7 induced changes in gene expression in 38B9 cells expressing WT IKZF3, but did not induce any changes in gene expression in cells expressing H195Y IKZF3. Anti-STAT5 ChIP-seq showed that expression of H195Y IKZF3 resulted in redistribution of STAT5 binding sites in the genome. H195Y IKZF3 binding sites overlapped with a subset of STAT5 binding sites, including in the promoter of the Cish gene. These findings suggest that H195Y mutation of IKZF3 results in altered DNA binding specificity and altered binding of STAT5 to target genes.

Asunto(s)

Leucemia de Células B , Leucemia-Linfoma Linfoblástico de Células Precursoras B , Animales , Ratones , Sitios de Unión , ADN , Expresión Génica , Proteínas de la Leche/genética , Mutación , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/metabolismo , Transactivadores/genética

RESUMEN

Base Excision Repair (BER) addresses base lesions and abasic sites induced by exogenous and endogenous stressors. X-ray cross complementing group 1 (XRCC1) functions as a scaffold protein in BER and single-strand break repair (SSBR), facilitating and coordinating repair through its interaction with a host of critical repair proteins. Alterations of XRCC1 protein and gene expression levels are observed in many cancers, including colorectal, ovarian, and breast cancer. While increases in the expression level of XRCC1 are reported, the transcription factors responsible for this up-regulation are not known. In this study, we identify the signal transducer and activator of transcription 3 (STAT3) as a novel regulator of XRCC1 through chromatin immunoprecipitation. Activation of STAT3 through phosphorylation at Y705 by cytokine (IL-6) signaling increases the expression of XRCC1 and the occupancy of STAT3 within the XRCC1 promoter. In triple negative breast cancer, the constitutive activation of STAT3 upregulates XRCC1 gene and protein expression levels. Increased expression of XRCC1 is associated with aggressiveness and resistance to DNA damaging chemotherapeutics. Thus, we propose that activated STAT3 regulates XRCC1 under stress and growth conditions, but constitutive activation in cancers results in dysregulation of XRCC1 and subsequently BER and SSBR.

Asunto(s)

Regiones Promotoras Genéticas/genética , Factor de Transcripción STAT3/genética , Neoplasias de la Mama Triple Negativas/genética , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X/genética , Línea Celular , Línea Celular Tumoral , Roturas del ADN de Cadena Simple , Daño del ADN/genética , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Células HEK293 , Humanos , Interleucina-6/genética , Fosforilación/genética , Regulación hacia Arriba/genética

RESUMEN

Signal transducer and activator of transcription 3 (STAT3) is an important transcription factor involved in many physiological functions including embryonic development and immune responses and is often activated under pathological conditions such as cancer. Strategies to inactivate STAT3 are being pursued as potential anticancer therapies and have led to the identification of Stattic (6-nitrobenzo[b]thiophene-1,1-dioxide) as a "specific" STAT3 inhibitor that is often used to interrogate STAT3-mediated gene expression in vitro and in vivo. Here, we show that Stattic exerts many STAT3-independent effects on cancer cells, calling for reassessment of results previously ascribed to STAT3 functions. Studies of the STAT3-deficient prostate cancer cell line PC-3 (PC3) along with STAT3-proficient breast cancer cell lines (MDA-MB-231, SUM149) revealed that Stattic attenuated histone acetylation and neutralized effects of the histone deacetylase (HDAC) inhibitor romidepsin. In PC3 cells, Stattic alone inhibited gene expression of CCL20 and CCL2, but activated expression of TNFA, CEBPD, SOX2, and MYC. In addition, we found that Stattic promoted autophagy and caused cell death. These data point to profound epigenetic effects of Stattic that are independent of its function as a STAT3 inhibitor. Our results demonstrate that Stattic directly or indirectly reduces histone acetylation and suggest reevaluation of Stattic and related compounds as polypharmacological agents through multipronged cytotoxic effects on cancer cells.

Asunto(s)

Antineoplásicos/farmacología , Óxidos S-Cíclicos/farmacología , Regulación Neoplásica de la Expresión Génica , Histonas/genética , Procesamiento Proteico-Postraduccional , Factor de Transcripción STAT3/genética , Acetilación/efectos de los fármacos , Autofagia/efectos de los fármacos , Autofagia/genética , Proteína delta de Unión al Potenciador CCAAT/agonistas , Proteína delta de Unión al Potenciador CCAAT/genética , Proteína delta de Unión al Potenciador CCAAT/metabolismo , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Línea Celular Tumoral , Quimiocina CCL2/antagonistas & inhibidores , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocina CCL20/antagonistas & inhibidores , Quimiocina CCL20/genética , Quimiocina CCL20/metabolismo , Femenino , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Histonas/antagonistas & inhibidores , Histonas/metabolismo , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Células PC-3 , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-myc/agonistas , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factores de Transcripción SOXB1/agonistas , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/agonistas , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Proteína Fluorescente Roja

RESUMEN

The abnormal activation and mutation of signal transducer and activator transcription (STAT) proteins has been implicated in multiple lymphomas. The research discovery and clinical application of STATs inhibitors have become an important strategy for treating lymphoma. This review introduces the abnormal activation and mutation of STATs in multiple malignant lymphomas, and focuses on reviewing the latest screening strategies targeting STATs and its clinical application in the treatment of lymphoma, providing references for the further development of STATs inhibitors.

RESUMEN

OBJECTIVE: To observe the intervention of Chushizi (Fructus Broussonetiae) (CSZ) on drug-induced liver injury (DILI) in rats, as well as indicators of liver function, serum levels of inflammatory cytokines, and expression of proteins and mRNA associated with toll-like receptor 3 (TLR3) and the signal transducer and activator of transcription 3 (STAT3) pathway in the liver [TLR3, janus protein tyrosine kinase 2 (JAK2), c-jun, c-fos, c-Jun N-terminal kinase 2 (JNK2), and STAT3]. METHODS: Forty specified pathogen free grade Sprague-Dawley rats were randomly divided into the control group, the model group, the silybin group and the CSZ group. Rats were given acetaminophen (APAP) to trigger DILI. Histopathology of the liver was observed by hematoxylin-eosin staining. The levels of alanine aminotransferase (ALT), aspartate transaminase (AST), direct bilirubin (DBIL), and total bilirubin (TBIL) in serum were detected by a semi-automatic biochemical instrument. Content of tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, IL-13, and IL-22 in serum were detected by the enzyme-linked immunosorbent assay, the expression of TLR3, phosphorylation of JAK2 (p-JAK2), while c-jun and c-fos proteins in the liver were determined by immunohistochemistry; expression of JNK2, and STAT3 in the liver were assayed by Western blot and real-time quantitative polymerase chain reaction. P-JNK2 and p-STAT3 in the liver were assayed by Western blot. RESULTS: After treatment, the activity of ALT, AST, and concentrations of TBIL, DBIL, TNF-α, IL-6, as well as IL-13 in serum, were lower than those in the model group, and expression of p-JAK2, TLR3, c-jun, c-fos, p-STAT3, and p-JNK2 could be downregulated. CONCLUSION: Our findings suggest that CSZ is a valid medicine to alleviate APAP-induced DILI, while its partial mechanism may regulate the TLR3/JNK/ c-jun/c-fos/JAK/STAT3 pathway.

Asunto(s)

Acetaminofén/efectos adversos , Hepatitis/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-jun/metabolismo , Factor de Transcripción STAT3/metabolismo , Receptor Toll-Like 3/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Hepatitis/etiología , Humanos , Hígado/efectos de los fármacos , Masculino , Fosforilación , Proteínas Proto-Oncogénicas c-fos/genética , Proteínas Proto-Oncogénicas c-jun/genética , Ratas , Ratas Sprague-Dawley , Factor de Transcripción STAT3/genética , Receptor Toll-Like 3/genética , Factor de Necrosis Tumoral alfa/genética

RESUMEN

The expression of the extracellular sulfatase SULF2 has been associated with increased hepatocellular carcinoma (HCC) growth and poor patient survival. However, the molecular mechanisms underlying SULF2-associated tumor growth remain unclear. To address this gap, here we developed a transgenic mouse overexpressing Sulf2 in hepatocytes under the control of the transthyretin promoter. In this model, Sulf2 overexpression potentiated diethylnitrosamine-induced HCC. Further analysis indicated that the transcription factor GLI family zinc finger 1 (GLI1) mediates Sulf2 expression during HCC development. A cross of the Sulf2-overexpressing with Gli1-knockout mice revealed that Gli1 inactivation impairs SULF2-induced HCC. Transcriptomic analysis revealed that Sulf2 overexpression is associated with signal transducer and activator of transcription 3 (STAT3)-specific gene signatures. Interestingly, the Gli1 knockout abrogated SULF2-mediated induction of several STAT3 target genes, including suppressor of cytokine signaling 2/3 (Socs2/3); Pim-1 proto-oncogene, Ser/Thr kinase (Pim1); and Fms-related tyrosine kinase 4 (Flt4). Human orthologs were similarly regulated by SULF2, dependent on intact GLI1 and STAT3 functions in HCC cells. SULF2 overexpression promoted a GLI1-STAT3 interaction and increased GLI1 and STAT3 enrichment at the promoters of their target genes. Interestingly, the SULF2 overexpression resulted in GLI1 enrichment at select STAT3 consensus sites, and vice versa. siRNA-mediated STAT3 or GLI1 knockdown reduced promoter binding of GLI1 and STAT3, respectively. Finally, chromatin-capture PCR confirmed long-range co-regulation of SOCS2 and FLT3 through changes in promoter conformation. These findings define a mechanism whereby SULF2 drives HCC by stimulating formation of a GLI1-STAT3 transcriptional complex.

Asunto(s)

Carcinoma Hepatocelular/etiología , Neoplasias Hepáticas/etiología , Factor de Transcripción STAT3/metabolismo , Sulfatasas/fisiología , Proteína con Dedos de Zinc GLI1/metabolismo , Animales , Carcinogénesis , Humanos , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas , Unión Proteica , Proto-Oncogenes Mas , Factores de Transcripción STAT , Sulfatasas/metabolismo , Transactivadores

RESUMEN

Bone morphogenetic protein 10 (BMP10) is a cardiac peptide growth factor belonging to the transforming growth factor ß superfamily that critically controls cardiovascular development, growth, and maturation. It has been shown that BMP10 elicits its intracellular signaling through a receptor complex of activin receptor-like kinase 1 with morphogenetic protein receptor type II or activin receptor type 2A. Previously, we generated and characterized a transgenic mouse line expressing BMP10 from the α-myosin heavy chain gene promoter and found that these mice have normal cardiac hypertrophic responses to both physiological and pathological stimuli. In this study, we report that these transgenic mice exhibit significantly reduced levels of cardiomyocyte apoptosis and cardiac fibrosis in response to a prolonged administration of the ß-adrenoreceptor agonist isoproterenol. We further confirmed this cardioprotective function with a newly generated conditional Bmp10 transgenic mouse line, in which Bmp10 was activated in adult hearts by tamoxifen. Moreover, the intraperitoneal administration of recombinant human BMP10 was found to effectively protect hearts from injury, suggesting potential therapeutic utility of using BMP10 to prevent heart failure. Gene profiling and biochemical analyses indicated that BMP10 activates the SMAD-mediated canonical pathway and, unexpectedly, also the signal transducer and activator of transcription 3 (STAT3)-mediated signaling pathway both in vivo and in vitro Additional findings further supported the notion that BMP10's cardioprotective function likely is due to its dual activation of SMAD- and STAT3-regulated signaling pathways, promoting cardiomyocyte survival and suppressing cardiac fibrosis.

Asunto(s)

Proteínas Morfogenéticas Óseas/metabolismo , Factor de Transcripción STAT3/metabolismo , Proteínas Smad/metabolismo , Agonistas de Receptores Adrenérgicos beta 3/farmacología , Animales , Apoptosis/efectos de los fármacos , Proteínas Morfogenéticas Óseas/genética , Matriz Extracelular/metabolismo , Corazón/efectos de los fármacos , Humanos , Isoproterenol/farmacología , Masculino , Ratones , Ratones Endogámicos C3H , Ratones Transgénicos , Miocardio/metabolismo , Miocardio/patología , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Factor de Transcripción STAT3/deficiencia , Factor de Transcripción STAT3/genética , Transducción de Señal/efectos de los fármacos

RESUMEN

Janus kinases (JAKs) and their downstream STAT proteins play key roles in cytokine signaling, tissue homeostasis, and cancer development. Using a breast cancer model that conditionally lacks Janus kinase 1, we show here that JAK1 is essential for IL-6-class inflammatory cytokine signaling and plays a critical role in metastatic cancer progression. JAK1 is indispensable for the oncogenic activation of STAT1, STAT3, and STAT6 in ERBB2-expressing cancer cells, suggesting that ERBB2 receptor tyrosine kinase complexes do not directly activate these STAT proteins in vivo. A genome-wide gene expression analysis revealed that JAK1 signaling has pleiotropic effects on several pathways associated with cancer progression. We established that FOS and MAP3K8 are targets of JAK1/STAT3 signaling, which promotes tumorsphere formation and cell migration. The results highlight the significance of JAK1 as a rational therapeutic target to block IL-6-class cytokines, which are master regulators of cancer-associated inflammation.

Asunto(s)

Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Progresión de la Enfermedad , Janus Quinasa 1/metabolismo , Animales , Carcinogénesis/metabolismo , Carcinogénesis/patología , Línea Celular Tumoral , Movimiento Celular/genética , Epitelio/metabolismo , Epitelio/patología , Femenino , Humanos , Janus Quinasa 1/deficiencia , Ligandos , Ratones Noqueados , Metástasis de la Neoplasia , Proteínas de Neoplasias/metabolismo , Fosforilación , Receptor ErbB-2/metabolismo , Factores de Transcripción STAT/metabolismo , Esferoides Celulares/metabolismo , Esferoides Celulares/patología

RESUMEN

Mice depleted of hepatic stellate cells (HSCs) are protected from concanavalin A (ConA)-induced liver injury that is mediated by the activation of interferon regulatory factor 1 (IRF1). The aim of this study was to determine the mechanisms of ConA-mediated signaling and synthesis/release of mediators by HSCs that damage hepatocytes. Primary cultures of wildtype (WT) and IRF1-knockout (KO) HSCs and hepatocytes were used, and ConA-induced liver damage in interferon (IFN)αß receptor-deficient (IFNαßR-KO) mice was determined. Specific binding of ConA to HSCs induced rapid activation of JAK2 and STAT1. ConA-induced expression of IRF1, IFNß, tumor necrosis factor α, and CXCL1 was abrogated by selective inhibition of JAK2 and STAT1. Despite activating JAK2/STAT1, ConA failed to stimulate expression of inflammatory cytokines in HSCs from IRF1-KO mice. ConA-conditioned WT-HSC medium caused activation of JNK and caspase 3, and apoptosis of hepatocytes from WT but not from IRF1-KO or IFNαßR-KO mice. Conversely, ConA-conditioned medium of IRF1-KO HSCs failed to cause apoptosis of WT or IRF1-KO hepatocytes. IFNαßR-KO mice were protected from ConA-induced liver damage, and ConA-induced hepatic expression of IRF1 and pro-inflammatory cytokines and chemokines, and infiltration of neutrophils were significantly lower in IFNαßR-KO than in WT mice. These results demonstrate distinct roles of IRF1 in hepatic inflammation (HSCs) and injury (hepatocytes) and can be an important target for intervention in acute liver injury.

Asunto(s)

Enfermedad Hepática Inducida por Sustancias y Drogas/fisiopatología , Concanavalina A/farmacología , Citocinas/biosíntesis , Células Estrelladas Hepáticas/efectos de los fármacos , Factor 1 Regulador del Interferón/fisiología , Animales , Células Cultivadas , Medios de Cultivo Condicionados , Citocinas/metabolismo , Células Estrelladas Hepáticas/metabolismo , Factor 1 Regulador del Interferón/genética , Interferón gamma/metabolismo , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Receptor de Interferón alfa y beta/genética , Transducción de Señal , Superóxido Dismutasa-1/metabolismo

RESUMEN

SEC2, a major histocompatibility complex class II (MHC II)-dependent T-cell mitogen, binds MHC II and T-cell receptor (TCR) Vßs to induce effective co-stimulating signals for clonal T-cell expansion. We previously characterized a SEC2 mutant with increased recognition of TCR Vßs, ST-4, which could intensify NF-κB signaling transduction, leading to IL-2 production and T-cell activation. In this study, we found that in contrast to SEC2, ST-4 could induce murine CD4+ T-cell proliferation in a Vß8.2- and Vß8.3-specific manner in the absence of MHC II+ antigen-presenting cells (APCs). Furthermore, although IL-2 secretion in response to either SEC2 or ST-4 stimulation was accompanied by up-regulation of protein kinase Cθ (PKCθ), inhibitor of κB (IκB), α and ß IκB kinase (IKKα/ß), IκBα, and NF-κB in mouse splenocytes, only ST-4 could activate CD4+ T cells in the absence of MHC II+ APCs through the PKCθ/NF-κB signaling pathway. The PKCθ inhibitor AEB071 significantly suppressed SEC2/ST-4-induced T-cell proliferation, CD69 and CD25 expression, and IL-2 secretion with or without MHC II+ APCs. Further, SEC2/ST-4-induced changes in PKCθ/NF-κB signaling were significantly relieved by AEB071 in a dose-dependent manner. Using Lck siRNA, we found that Lck controlled SEC2/ST-4-induced phosphorylation of PKCθ. We also demonstrated that the IL-2R/STAT5 pathway is essential for SEC2/ST-4-induced T-cell activation. Collectively, our data demonstrate that an enhanced ST-4-TCR interaction can compensate for lack of MHC II and stimulate MHC II-free CD4+ T-cell proliferation via PKCθ/NF-κB and IL-2R/STAT5 signaling pathways. Compared with SEC2, intensified PKCθ/NF-κB and IL-2R/STAT5 signals induced by ST-4 lead to enhanced T-cell activation. The results of this study will facilitate better understanding of TCR-based immunotherapies for cancer.

Asunto(s)

Enterotoxinas/genética , Activación de Linfocitos , Mutación , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T/inmunología , Animales , Proliferación Celular , Enterotoxinas/metabolismo , Femenino , Regulación de la Expresión Génica/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Interleucina-2/metabolismo , Ratones , Ratones Endogámicos BALB C , FN-kappa B/metabolismo , Fosforilación , Proteína Quinasa C-theta/metabolismo , Receptores de Interleucina-2/metabolismo , Factor de Transcripción STAT5/metabolismo , Bazo/inmunología , Linfocitos T/citología

RESUMEN

Interleukin (IL)-13 is a signature cytokine of type 2 inflammation important for the pathogenesis of various diseases, including allergic diseases. Signal transducer and activator of transcription (STAT) 6 is a critical transcriptional factor for the IL-13 signals; however, it remains unknown how expression of the IL-13-induced genes is differentiated by the transcriptional machineries. In this study, we identified IL-13-induced transcriptional factors in lung fibroblasts using DNA microarrays in which SOX11 was included. Knockdown of SOX11 down-regulated expression of periostin and CCL26, both of which are known to be downstream molecules of IL-13, whereas enforced expression of SOX11 together with IL-13 stimulation enhanced expression of periostin. Moreover, we found that in DNA microarrays combining IL-13 induction and SOX11 knockdown there exist both SOX11-dependent and -independent molecules in IL-13-inducible molecules. In the former, many inflammation-related and fibrosis-related molecules, including periostin and CCL26, are involved. These results suggest that SOX11 acts as a trans-acting transcriptional factor downstream of STAT6 and that in lung fibroblasts the IL-13 signals are hierarchically controlled by STAT6 and SOX11.

Asunto(s)

Interleucina-13/metabolismo , Pulmón/metabolismo , Factores de Transcripción SOXC/fisiología , Factor de Transcripción STAT6/fisiología , Transducción de Señal/fisiología , Moléculas de Adhesión Celular/metabolismo , Línea Celular , Quimiocina CCL26/metabolismo , Regulación hacia Abajo , Fibroblastos/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Pulmón/citología , Análisis de Secuencia por Matrices de Oligonucleótidos , Factores de Transcripción SOXC/genética , Transactivadores/metabolismo , Transcripción Genética , Regulación hacia Arriba

RESUMEN

Macrophages are essential for innate immunity and inflammatory responses and differentiate into various functional phenotypes. Tribbles homolog 1 (Trib1), a member of the mammalian Tribbles homolog pseudokinase family, has been implicated in regulation of cell differentiation, proliferation, and metabolism, but its role in macrophage biology has not been fully elucidated. Here, we investigated the consequences of Trib1 deficiency on macrophage functions and M1/M2 polarization. Bone marrow-derived macrophages (BMDMs) from Trib1-deficient (Trib1-/-) mice exhibited elevated phagocytic capacity, correlating with up-regulation of several scavenger receptors. Concomitantly, uptake of modified low-density lipoprotein was increased in Trib1-/- BMDMs. Trib1-/- macrophages also exhibited diminished migration in the presence of the chemokine MCP-1, associated with reduced expression of the MCP-1 receptor Ccr2 Furthermore, Trib1 deficiency attenuated the response of BMDMs to both M1 and M2 stimuli; induction of the M1-marker genes Il6, Il1b, and Nos2 upon LPS/IFNγ stimulation and of the M2-marker genes Cd206, Fizz1, and Arg1 upon IL-4 stimulation was reduced. Functionally, Trib1 deficiency decreased secretion of proinflammatory cytokines (IL-6, TNFα, IL-1ß, and CXCL1) and reduced nitric oxide and reactive oxygen species production in M1-polarized macrophages. Supporting the attenuated M2 phenotype, IL-4-stimulated Trib1-/- macrophages secreted less IL-10 and TGFß. Mechanistically, Trib1-/- BMDMs displayed lower levels of Janus kinase 1 (JAK1), resulting in reduced activation of LPS/IFNγ-mediated STAT1 signaling. Likewise, decreased levels of JAK1 along with lower activation of STAT6 and STAT3 were observed in M2-polarized Trib1-/- BMDMs. Our findings suggest that Trib1 extensively controls macrophage M1/M2 polarization via the JAK/STAT signaling pathway.

Asunto(s)

Movimiento Celular , Polaridad Celular , Eliminación de Gen , Péptidos y Proteínas de Señalización Intracelular/genética , Macrófagos/citología , Fagocitosis , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Animales , Células de la Médula Ósea/citología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Janus Quinasa 1/metabolismo , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción STAT/metabolismo , Transducción de Señal

RESUMEN

Interleukin 4 (IL4) is generally viewed as a Th2 cytokine capable of polarizing macrophages into an anti-inflammatory phenotype, whereas granulocyte macrophage-colony-stimulating factor (GM-CSF) is often viewed as a proinflammatory cytokine with part of this function due to its action on monocytes/macrophages. Paradoxically, these two cytokines act additively to enhance the in vitro differentiation of dendritic cells from precursors such as monocytes. One up-regulated marker of an IL4-polarized M2 macrophage is the chemokine (C-C motif) ligand 17 (CCL17), which we have recently reported to be induced by GM-CSF in monocytes/macrophages in an interferon regulatory factor 4 (IRF4)-dependent manner. In this study, we report that IL4 also induces CCL17 production by acting through IRF4 in human monocytes and murine macrophages. Furthermore, evidence is presented that IL4 up-regulates IRF4 expression at the epigenetic level by enhancing the expression and activity of jumonji domain-containing protein 3 (JMJD3) demethylase. Intriguingly, silencing the signal transducer and activator of transcription 6 (STAT6) gene led to a decrease in not only CCL17 formation, but also in that of its upstream regulators, JMJD3 and IRF4. Moreover, IL4 treatment of human monocytes resulted in an increased association of STAT6 to the promoter regions of the CCL17, IRF4, and JMJD3 genes. Thus, despite their vastly different functions, IL4 and GM-CSF appear to share elements of a common signaling pathway in regulating CCL17 production in human monocytes and murine macrophages.

Asunto(s)

Quimiocina CCL17/genética , Epigénesis Genética , Interleucina-4/genética , Macrófagos/metabolismo , Monocitos/metabolismo , Activación Transcripcional , Animales , Células Cultivadas , Humanos , Factores Reguladores del Interferón/genética , Histona Demetilasas con Dominio de Jumonji/genética , Ratones , Ratones Endogámicos C57BL , Regulación hacia Arriba

RESUMEN

Transcriptional activators bind DNA and recruit cofactors to modify chromatin. The extent to which these two events are separable is unclear. Here, using a custom ChIP tiling array to map chromatin modifications, we show that interferon-γ-induced DNA binding of signal transducer and activator of transcription 1 (STAT1), typically associated with the transcription factor interferon regulatory factor 1 (IRF1), causes histone acetylation (H3ac, H4ac). In contrast, among IRF1 sites lacking concomitant STAT1 recruitment, only 25% underwent inducible histone acetylation, 31% exhibited constitutive histone acetylation, and 44% had no histone acetylation. These latter "orphan sites" also lacked other activating modifications (e.g. H3K4me1, H3K4me2) and were typically remote from transcription start sites. In these cases the closest gene was typically an IFNγ-inducible locus that did not respond to IFNγ in this setting. Orphan sites were detected in different cell types, suggesting broad relevance. Despite an atypical downstream response (i.e. no histone modifications), IRF1 binding depended on SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 4 (SMARCA4 or BRG1), as is typical of active IRF1 enhancers. Although SMARCA4 permitted IRF1 access to the orphan sites, there was no corecruitment of the histone acetyltransferases CREB-binding protein (CBP) and p300. Orphan sites were constitutively unacetylated, and several were marked with repressive chromatin modifications (e.g. H3K27me3). In conclusion, although IRF1 can trigger enhanceosome formation independently of STAT1, its ability to do so depends on local chromatin cues.

Asunto(s)

Histonas/metabolismo , Factor 1 Regulador del Interferón/química , Factor 1 Regulador del Interferón/metabolismo , Acetilación , Sitios de Unión , Cromatina/metabolismo , Células HeLa , Humanos , Factor de Transcripción STAT1/metabolismo

RESUMEN

Asthma is a chronic inflammatory disease of the lungs and airways and one of the most burdensome of all chronic maladies. Previous studies have established that expression of experimental and human asthma requires the IL-4/IL-13/IL-4 receptor α (IL-4Rα) signaling pathway, which activates the transcription factor STAT6. However, no small molecules targeting this important pathway are currently in clinical development. To this end, using a preclinical asthma model, we sought to develop and test a small-molecule inhibitor of the Src homology 2 domains in mouse and human STAT6. We previously developed multiple peptidomimetic compounds on the basis of blocking the docking site of STAT6 to IL-4Rα and phosphorylation of Tyr641 in STAT6. Here, we expanded the scope of our initial in vitro structure-activity relationship studies to include central and C-terminal analogs of these peptides to develop a lead compound, PM-43I. Conducting initial dose range, toxicity, and pharmacokinetic experiments with PM-43I, we found that it potently inhibits both STAT5- and STAT6-dependent allergic airway disease in mice. Moreover, PM-43I reversed preexisting allergic airway disease in mice with a minimum ED50 of 0.25 µg/kg. Of note, PM-43I was efficiently cleared through the kidneys with no long-term toxicity. We conclude that PM-43I represents the first of a class of small molecules that may be suitable for further clinical development against asthma.

Asunto(s)

Asma/tratamiento farmacológico , Terapia Molecular Dirigida , Factor de Transcripción STAT5/química , Factor de Transcripción STAT5/metabolismo , Factor de Transcripción STAT6/química , Factor de Transcripción STAT6/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Asma/inmunología , Asma/metabolismo , Línea Celular , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Pulmón/efectos de los fármacos , Pulmón/inmunología , Pulmón/metabolismo , Ratones , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Relación Estructura-Actividad , Dominios Homologos src

RESUMEN

Interferon consensus sequence-binding protein (Icsbp) is required for terminating emergency granulopoiesis, an episodic event responsible for granulocyte production in response to infections and a key component of the innate immune response. Icsbp inhibits the expression of Stat3 and C/ebpß, transcription factors essential for initiating and sustaining granulopoiesis, and activates transcription of Fanconi C (FANCC), a DNA repair protein. In prior studies, we noted accelerated bone marrow failure in Fancc-/- mice undergoing multiple episodes of emergency granulopoiesis, associated with apoptosis of bone marrow cells with unrepaired DNA damage. Additionally, we found increased expression of Fanconi C and F proteins during emergency granulopoiesis. These findings suggest that Icsbp protects the bone marrow from DNA damage by increasing activity of the Fanconi DNA repair pathway, but the mechanisms for FANCC activation during initiation of emergency granulopoiesis are unclear. In this study, we observed that Stat3 and C/ebpß activate FANCC transcription and contribute to DNA repair. Our findings indicate that FancC expression is increased during Stat3- and C/ebpß-induced initiation of emergency granulopoiesis by these transcription factors and is maintained through termination by Icsbp. Our work reveals that Stat3- and C/ebpß-mediated FancC expression is a critical component for initiating and sustaining key innate immune responses.

Asunto(s)

Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Proteína del Grupo de Complementación C de la Anemia de Fanconi/genética , Regulación de la Expresión Génica , Granulocitos/patología , Factor de Transcripción STAT3/metabolismo , Transcripción Genética , Animales , Apoptosis , Secuencia de Bases , Proteína beta Potenciadora de Unión a CCAAT/genética , Reparación del ADN , Proteína del Grupo de Complementación C de la Anemia de Fanconi/metabolismo , Granulocitos/metabolismo , Hematopoyesis , Humanos , Ratones , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas , Factor de Transcripción STAT3/genética , Homología de Secuencia , Células U937

RESUMEN

E2F transcription factors are regulated by binding to the retinoblastoma (Rb) tumor suppressor family of proteins. Previously, we reported an E2FLQ mutation that disrupts the binding with Rb proteins without affecting the transcriptional activity of E2F. We also showed that mouse embryonic fibroblasts with an E2F3LQ mutation exhibit increased E2F activity and more rapid cell proliferation. In this report, we analyzed E2F3LQ mice to further characterize the in vivo consequences of Rb family-independent E2F3 activity. We found that homozygous E2F3LQ mice were viable and had no obvious developmental defects or tumor growth. Our results also indicated that E2F3LQ cells largely retain normal control of cell proliferation in vivo However, female E2F3LQ mice had partial nursing defects. Examination of the E2F3LQ mammary glands revealed increased caveolin-1 (CAV1) expression, reduced prolactin receptor/Stat5 signaling, and impaired pregnancy-induced cell proliferation and differentiation. Of note, ChIP experiments disclosed that E2F3 binds the CAV1 promoter. Furthermore, E2F3 overexpression induced CAV1 expression, and CRISPR/CAS9-mediated E2F3 knockout reduced CAV1 levels and also increased prolactin receptor-induced Stat5 signaling in mammary epithelial cells. Our results suggest that the Rb family-independent E2F3 LQ variant inhibits pregnancy-induced mammary gland cell proliferation and differentiation by up-regulating CAV1 expression and inhibiting Stat5 signaling.

Asunto(s)

Factor de Transcripción E2F3/genética , Factor de Transcripción E2F3/metabolismo , Glándulas Mamarias Animales/citología , Mutación , Factor de Transcripción STAT5/metabolismo , Transducción de Señal , Animales , Secuencia de Bases , Caveolina 1/genética , Diferenciación Celular/genética , Proliferación Celular/genética , Femenino , Regulación de la Expresión Génica/genética , Ratones , Embarazo

RESUMEN

The peptide hormone prolactin (PRL) and certain members of the epidermal growth factor (EGF) family play central roles in mammary gland development and physiology, and their dysregulation has been implicated in mammary tumorigenesis. Our recent studies have revealed that the CUB and zona pellucida-like domain-containing protein 1 (CUZD1) is a critical factor for PRL-mediated activation of the transcription factor STAT5 in mouse mammary epithelium. Of note, CUZD1 controls production of a specific subset of the EGF family growth factors and consequent activation of their receptors. Here, we found that consistent with this finding, CUZD1 overexpression in non-transformed mammary epithelial HC11 cells increases their proliferation and induces tumorigenic characteristics in these cells. When introduced orthotopically in mouse mammary glands, these cells formed adenocarcinomas, exhibiting elevated levels of STAT5 phosphorylation and activation of the EGF signaling pathway. Selective blockade of STAT5 phosphorylation by pimozide, a small-molecule inhibitor, markedly reduced the production of the EGF family growth factors and inhibited PRL-induced tumor cell proliferation in vitro Pimozide administration to mice also suppressed CUZD1-driven mammary tumorigenesis in vivo Analysis of human MCF7 breast cancer cells indicated that CUZD1 controls the production of the same subset of EGF family members in these cells as in the mouse. Moreover, pimozide treatment reduced the proliferation of these cancer cells. Collectively, these findings indicate that overexpression of CUZD1, a regulator of growth factor pathways controlled by PRL and STAT5, promotes mammary tumorigenesis. Blockade of the STAT5 signaling pathway downstream of CUZD1 may offer a therapeutic strategy for managing these breast tumors.

Asunto(s)

Neoplasias de la Mama/metabolismo , Carcinogénesis/metabolismo , Regulación Neoplásica de la Expresión Génica , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Transducción de Señal , Animales , Anticarcinógenos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Carcinogénesis/efectos de los fármacos , Carcinogénesis/patología , Línea Celular , Proliferación Celular/efectos de los fármacos , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Células MCF-7 , Glándulas Mamarias Animales/efectos de los fármacos , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/patología , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , Interferencia de ARN , Receptores de Prolactina/antagonistas & inhibidores , Receptores de Prolactina/genética , Receptores de Prolactina/metabolismo , Factor de Transcripción STAT5/antagonistas & inhibidores , Factor de Transcripción STAT5/metabolismo , Transducción de Señal/efectos de los fármacos

RESUMEN

Type I (α and ß) and type III (λ) IFNs are induced upon viral infection through host sensory pathways that activate IFN regulatory factors (IRFs) and nuclear factor κB. Secreted IFNs induce autocrine and paracrine signaling through the JAK-STAT pathway, leading to the transcriptional induction of hundreds of IFN-stimulated genes, among them sensory pathway components such as cGAS, STING, RIG-I, MDA5, and the transcription factor IRF7, which enhance the induction of IFN-αs and IFN-λs. This positive feedback loop enables a very rapid and strong host response that, at some point, has to be controlled by negative regulators to maintain tissue homeostasis. Type I IFN signaling is controlled by the inducible negative regulators suppressor of cytokine signaling 1 (SOCS1), SOCS3, and ubiquitin-specific peptidase 18 (USP18). The physiological role of these proteins in IFN-γ signaling has not been clarified. Here we used knockout cell lines and mice to show that IFN-λ signaling is regulated by SOCS1 but not by SOCS3 or USP18. These differences were the basis for the distinct kinetic properties of type I and III IFNs. We found that IFN-α signaling is transient and becomes refractory after hours, whereas IFN-λ provides a long-lasting IFN-stimulated gene induction.

Asunto(s)

Regulación de la Expresión Génica/fisiología , Interferones/metabolismo , Transducción de Señal/fisiología , Proteína 1 Supresora de la Señalización de Citocinas/metabolismo , Animales , Línea Celular Tumoral , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/metabolismo , Endopeptidasas/genética , Endopeptidasas/metabolismo , Humanos , Helicasa Inducida por Interferón IFIH1/genética , Helicasa Inducida por Interferón IFIH1/metabolismo , Interferones/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Receptores Inmunológicos , Proteína 1 Supresora de la Señalización de Citocinas/genética , Proteína 3 Supresora de la Señalización de Citocinas/genética , Proteína 3 Supresora de la Señalización de Citocinas/metabolismo , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismo

RESUMEN

To examine the biochemical influences that may contribute to the success of gene therapy for ocular disorders, the role of versican, a vitreous component, in adenoviral-mediated transgene expression was examined. Versican is a large chondroitin sulfate-containing, hyaluronic acid-binding proteoglycan present in the extracellular matrix and in ocular vitreous body. Y79 retinoblastoma cells and CD44-negative SK-N-DZ neuroblastoma cells transduced with adenoviral vectors in the presence of versican respond with an activation of transgene expression. Proteolysis of versican generates a hyaluronan-binding G1 domain. The addition of recombinant versican G1 to SK-N-DZ cells results in a similar activation of transgene expression, and treatment with dasatinib, an inhibitor of Src family kinases, also mimics the effects of versican. Enhancement is accompanied by an increase in signal transducer and activator of transcription 5 (STAT5) phosphorylation and is abrogated by treatment with C188-9, a STAT3/5 inhibitor, or with ruxolitinib, a Janus kinase 1/2 (JAK1/2) inhibitor. These data implicate versican G1 in enhancing adenoviral vector transgene expression in a hyaluronic acid-CD44 independent manner that is down-regulated by inhibitors of the JAK/STAT pathway and enhanced by inhibitors of the Src kinase pathway.

Asunto(s)

Antineoplásicos/farmacología , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/terapia , Inhibidores de Proteínas Quinasas/farmacología , Versicanos/metabolismo , Adenoviridae/crecimiento & desarrollo , Adenoviridae/fisiología , Animales , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , ADN Recombinante/metabolismo , ADN Viral/metabolismo , Genes Reporteros/efectos de los fármacos , Vectores Genéticos , Humanos , Quinasas Janus/antagonistas & inhibidores , Quinasas Janus/metabolismo , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/virología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción STAT/antagonistas & inhibidores , Factores de Transcripción STAT/metabolismo , Transducción de Señal/efectos de los fármacos , Versicanos/química , Versicanos/genética , Replicación Viral/efectos de los fármacos , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/metabolismo