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1.
Life Sci ; 240: 116985, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31647949

RESUMO

BACKGROUND: The infiltration and activation of macrophages play key roles in arterial restenosis, providing a promising strategy for the treatment of restenosis caused by intimal hyperplasia. Although miR-150 has been implicated in cardiovascular diseases, the individual effect of miR-150 on intimal hyperplasia remains unclear. METHODS AND RESULTS: We observed that the expression of miR-150 was robustly reduced in proinflammatory M1 macrophages and reversely induced in resolving M2 macrophages. An in vitro experiment demonstrated that miR-150 deficiency promoted extensive upregulation of the expression of M1 markers but attenuated the expression of M2 macrophage markers. MiR-150 enhanced the proliferation and migration of vascular smooth muscle cells (VSMCs) when co-cultured with conditioned medium from polarized macrophages upon LPS or IL-4 stimulation. Mechanistically, the bioinformatics analysis and luciferase assay results showed that miR-150 directly targeted STAT1 and STAT1 was required for the effect of miR-150 knockout on macrophage polarization. More importantly, we showed that knockout of miR-150 accelerated neointima formation, accompanied by the activation of M1 macrophages and the inactivation of M2 macrophages. Furthermore, miR-150 deficiency in marrow-derived cell accelerated neointima formation. CONCLUSION: Our research demonstrated that miR-150 deficiency promoted intimal hyperplasia with high ratios of M1 to M2 macrophages and subsequently increased VSMCs proliferation and migration, which were partially mediated by directly targeting to STAT1. Collectively, these results suggested that miR-150 may act as a novel therapeutic target for arterial restenosis.


Assuntos
Polaridade Celular/genética , Hiperplasia/genética , Macrófagos , MicroRNAs/genética , Neointima/genética , Animais , Movimento Celular/genética , Proliferação de Células , Biologia Computacional , Hiperplasia/patologia , Inflamação/genética , Inflamação/patologia , Interleucina-4/farmacologia , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Knockout , Músculo Liso Vascular , Neointima/patologia , Fator de Transcrição STAT1/genética
2.
Vet Microbiol ; 236: 108395, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31500730

RESUMO

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically significant pathogen that has been recognized for its genetic variation, rapid evolution, and immune suppression. Type I interferons (IFNs) play an important role in host defense against viral infection by inducing many antiviral effectors, which might be a selective pressure driving viral evolution towards IFN resistance. To investigate the IFN resistance-related variation of PRRSV genome under IFN selective pressure and explore the molecular mechanism of IFN sensitivity changes, PRRSV strain JXwn06 was serially propagated in porcine pulmonary alveolar macrophages (PAMs) with IFNα treatment for 45 passages and 3 rounds of purification. Four mutant strains named JX-αP51n (n = 1, 2, 3 and 4) with reduced IFNα sensitivity were selected; the strains showed a 100-fold higher titer than the passaging-control strain JX-P51 in IFNα-treated PAMs. IFNα-resistant strains were found to antagonize the IFNα-activated JAK-STAT signaling pathway to a greater extent than the nonresistant strain by down-regulating the expression level of IFNα-activated pJAK1 through interfering with phosphatase. Furthermore, the PRRSV genetic variations interacting with IFNα were identified by full genomic sequencing and alignment. Among these mutations, amino acid substitutions in nsp1ß (E87 G), GP3 (F143 L) and GP5 (Y136 H) were found to correlate with increased IFNα resistance by enhancing the suppression effect on pJAK1, which could be further increased if these three substitution sites were combined. These findings provide some novel evidence for understanding PRRSV genetic variation under host selective pressure and viral evolution strategies to evade the host innate immune response.


Assuntos
Farmacorresistência Viral/genética , Interferon-alfa/farmacologia , Mutação , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Regulação da Expressão Gênica , Haplorrinos , Fosforilação , Vírus da Síndrome Respiratória e Reprodutiva Suína/efeitos dos fármacos , RNA Viral , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT2/genética , Fator de Transcrição STAT2/metabolismo , Transdução de Sinais , Suínos , Proteínas Virais/genética
3.
Infect Immun ; 87(11)2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31451617

RESUMO

To date, the implications of interleukin 6 (IL-6) for immune responses in the context of Brucella infection are still unknown. In the present study, we found that Brucella abortus infection induced marked production of IL-6 in mice that was important for sufficient differentiation of CD8+ T cells, a key factor in Brucella clearance. Blocking IL-6 signaling also significantly induced serum IL-4 and IL-10, together with a decreased gamma interferon (IFN-γ) level, suggesting that IL-6 is essential for priming the T-helper (Th) 1 cell immune response during Brucella infection. The IL-6 pathway also activated the bactericidal activity of primary and cultured macrophages. Bacterial killing was markedly abrogated when IL-6 signaling was suppressed, and this phenomenon was mainly associated with decreased activity of lysosome-mediated killing. Interestingly, suppressor of cytokine signaling 3 (SOCS3) was important for regulating the IL-6-dependent anti-Brucella activity through the JAK/STAT pathway. During early infection, in the absence of SOCS3, IL-6 exhibited anti-inflammatory effects and lysosome-mediated killing inhibition; however, the increase in SOCS3 successfully shifted functional IL-6 toward proinflammatory brucellacidal activity in the late stage. Our data clearly indicate that IL-6 contributes to host resistance against B. abortus infection by controlling brucellacidal activity in macrophages and priming cellular immune responses.


Assuntos
Brucella abortus/fisiologia , Citocinas/metabolismo , Interleucina-6/metabolismo , Macrófagos/microbiologia , Animais , Anticorpos , Células Apresentadoras de Antígenos , Receptor gp130 de Citocina/genética , Receptor gp130 de Citocina/metabolismo , Citocinas/genética , Interleucina-6/genética , Camundongos , Células RAW 264.7 , Interferência de RNA , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/metabolismo , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Proteína 3 Supressora da Sinalização de Citocinas/genética , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Células Th1/metabolismo
4.
J Agric Food Chem ; 67(36): 10069-10078, 2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31422663

RESUMO

Macrophage polarization has been implicated in the pathogenesis of obesity and type 2 diabetes, which are recognized as chronic proinflammatory diseases. This study investigated that high level of glucose, similar to lipopolysaccharide (LPS), activated macrophages toward M1 phenotypes and 1-20 µM asaronic acid (AA) counteracted diabetic macrophage activation. AA reduced the LPS-promoted secretion of proinflammatory interleukin (IL)-6 and monocyte chemoattractant protein-1. The LPS markedly elevated the macrophage induction of the M1 markers of Toll-like receptor 4 (TLR4), CD36, and CD68, which was attenuated by AA. Also, the LPS significantly enhanced the nuclear factor (NF)-κB transactivation, signal transducers, and activators of transcription 1 (STAT1)/STAT3 activation and suppressor of cytokine signaling 3 (SOCS3) induction in macrophages. However, AA highly suppressed the aforementioned effects of LPS. Glucose-stimulated macrophages expressed advanced glycation end products (AGEs) and receptor for AGE (RAGE). Administration of 20 µM AA to macrophages partly but significantly attenuated such effects (1.65 ± 0.12 vs 0.95 ± 0.25 times glucose control for AGE; 2.33 ± 0.31 vs 1.40 ± 0.22 times glucose control for RAGE). Furthermore, glucose enhanced the macrophage induction of TLR4 and inducible nitric oxide synthase and IL-6 production, while it demoted the production of anti-inflammatory arginase-1 and IL-10. In contrast, AA reversed the induction of these markers in glucose-loaded macrophages. AA dose-dependently and significantly encumbered NF-κB transactivation, Janus kinase 2 (JAK2) and STAT1/STAT3 activation, and SOCS3 induction upregulated in glucose-supplemented macrophages. These results demonstrated for the first time that AA may limit diabetic macrophage activation toward the M1 phenotype through the inhibition of TLR4-/IL-6-mediated NF-κB/JAK2-STAT signaling entailing AGE-RAGE interaction.


Assuntos
Glucose/imunologia , Janus Quinase 2/imunologia , Macrófagos/efeitos dos fármacos , NF-kappa B/imunologia , Extratos Vegetais/farmacologia , Fator de Transcrição STAT1/imunologia , Fator de Transcrição STAT3/imunologia , Animais , Linhagem Celular , Interleucina-6/genética , Interleucina-6/imunologia , Janus Quinase 2/genética , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , NF-kappa B/genética , Perilla/química , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT3/genética
5.
Gastroenterology ; 157(5): 1310-1322.e13, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31352002

RESUMO

BACKGROUND & AIMS: Interferon lambda (IFNL) is expressed at high levels by intestinal epithelial cells (IECs) and mucosal immune cells in response to infection and inflammation. We investigated whether IFNL might contribute to pathogenesis of Crohn's disease (CD). METHODS: We obtained serum samples and terminal ileum biopsies from 47 patients with CD and 16 healthy individuals (controls). We measured levels of IFNL by enzyme-linked immunosorbent assay and immunohistochemistry and location of expression by confocal microscopy. Activation of IFNL signaling via STAT1 was measured in areas of no, mild, moderate, and severe inflammation and correlated with Paneth cell homeostasis and inflammation. IFNL expression and function were studied in wild-type mice and mice with intestinal epithelial cell-specific (ΔIEC) disruption or full-body disruption of specific genes (Mlkl-/-, Stat1ΔIEC, Casp8ΔIEC, Casp8ΔIECRipk3-/-, Casp8ΔIECTnfr-/-, Casp8ΔIECMlkl-/-, and Nod2-/- mice). Some mice were given tail vein injections of a vector encoding a secreted form of IFNL. Intestinal tissues were collected from mice and analyzed by immunohistochemistry and immunoblots. We generated 3-dimensional small intestinal organoids from mice and studied the effects of IFNL and inhibitors of STAT-signaling pathway. RESULTS: Patients with CD had significant increases in serum and ileal levels of IFNL compared with controls. Levels of IFNL were highest in ileum tissues with severe inflammation. High levels of IFNL associated with a reduced number of Paneth cells and increased cell death at the crypt bottom in inflamed ileum samples. Intestinal tissues from the ileum of wild-type mice injected with a vector expressing IFNL had reduced numbers of Paneth cells. IFNL-induced death of Paneth cells in mice did not occur via apoptosis, but required Mixed Lineage Kinase Domain Like (MLKL) and activation of Signal transducer and activator of transcription 1 (STAT1). In organoids, inhibitors of Janus kinase (JAK) signaling via STAT1 (glucocorticoids, tofacitinib, or filgotinib) reduced expression of proteins that mediate cell death and prevented Paneth cell death. CONCLUSIONS: Levels of IFNL are increased in serum and inflamed ileal tissues from patients with CD and associated with a loss of Paneth cells. Expression of a secreted form of IFNL in mice results in loss of Paneth cells from intestinal tissues, via STAT1 and MLKL, controlled by caspase 8. Strategies to reduce IFNL or block its effects might be developed for treatment of patients with CD affecting the terminal ileum.


Assuntos
Doença de Crohn/metabolismo , Íleo/metabolismo , Interferons/metabolismo , Interleucinas/metabolismo , Celulas de Paneth/metabolismo , Fator de Transcrição STAT1/metabolismo , Animais , Caspase 8/genética , Caspase 8/metabolismo , Morte Celular , Doença de Crohn/imunologia , Doença de Crohn/patologia , Modelos Animais de Doenças , Humanos , Íleo/imunologia , Íleo/patologia , Interferons/genética , Interleucinas/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , Celulas de Paneth/imunologia , Celulas de Paneth/patologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Fator de Transcrição STAT1/deficiência , Fator de Transcrição STAT1/genética , Transdução de Sinais , Técnicas de Cultura de Tecidos , Regulação para Cima
6.
Nat Commun ; 10(1): 3320, 2019 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-31346169

RESUMO

Activation of macrophage proinflammatory and antimicrobial phenotypes is regulated by IFN-γ and LPS via synergistic induction of canonical, inflammatory NF-κB target genes. However, whether IFN-γ negatively regulates components of the LPS response, and how this may affect macrophage activation, is still unclear. Here we use combined transcriptomic and epigenomic approaches to find that IFN-γ selectively abrogates LPS-induced feedback and alters macrophage metabolic pathways by suppressing TLR4-mediated gene activation. In contrast to superinduction of inflammatory genes via enhancers that bind IRF1 and STAT1, IFN-γ represses target enhancers that bind STAT3. TLR4-activated but IFN-γ-suppressed enhancers comprise two subsets discernable by differential regulation of histone acetylation and recruitment of STAT3, CDK8 and cohesin. Our findings thus show that IFN-γ suppresses feedback inhibitory and metabolic components of TLR responses to enhance macrophage activation; they also provide insights for IFN-γ-mediated selective inhibition of TLR4-induced transcription. Such inhibition can contribute to severe and sustained inflammatory responses.


Assuntos
Interferon gama/imunologia , Macrófagos/imunologia , Receptor 4 Toll-Like/imunologia , Regulação da Expressão Gênica , Humanos , Lipopolissacarídeos/imunologia , Ativação de Macrófagos , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/imunologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologia , Receptor 4 Toll-Like/genética
7.
Mol Immunol ; 114: 30-40, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31336247

RESUMO

Heterozygous gain-of-function (GOF) mutations in the cytokine-regulated transcription factor STAT1 (signal transducer and activator of transcription 1) lead to chronic mucocutaneous candidiasis (CMC). However, the molecular basis of these pathogenic missense mutations is largely unknown. In this study, we characterized in more detail the CMC-associated GOF substitution mutation of arginine-to-tryptophan at position 274 (R274W) and, in addition, the adjacent glutamine-to-alanine mutation at position 275 (Q275A). Both mutants displayed elevated tyrosine phosphorylation levels, prolonged nuclear accumulation, and increased transcriptional responses to interferon-γ (IFNγ) stimulation. No difference was observed between wild-type (WT) and mutant STAT1 in DNA sequence-specificity or dissociation kinetics from high-affinity DNA-binding elements known as gamma-activated sites (GAS). Furthermore, all variants exhibited similar cooperative DNA binding. Unexpectedly, in vitro dephosphorylation rates using the recombinant STAT1-inactivating Tc45 phosphatase in both the absence and presence of double-stranded GAS elements were similar in all STAT1 variants. Likewise, the rate of tyrosine phosphorylation by Janus kinase 2 (JAK2) was unaltered as compared to the WT molecule, excluding that the phenotype of these mutants is caused by either defective Tc45-catalyzed dephosphorylation or JAK2-induced hyper-activation. Interestingly, within 10 min of IFNγ exposure, the majority of R274W and Q275A molecules had entered the nucleus, whereas the wild-type protein remained predominantly cytosolic. Thus, the exchange of critical residues located at the binding interface in the antiparallel dimer conformer led to a premature accumulation of phospho-STAT1 in the nuclear compartment. In summary, our data show that the hyper-activity of the GOF mutations results, at least in part, from the premature nuclear import of the tyrosine-phosphorylated molecules and not from alterations in their phosphorylation or dephosphorylation rates.


Assuntos
Mutação com Ganho de Função/genética , Mutação de Sentido Incorreto/genética , Domínios Proteicos/genética , Fator de Transcrição STAT1/genética , Candidíase Mucocutânea Crônica/genética , Linhagem Celular Tumoral , Núcleo Celular/genética , Células Cultivadas , Citocinas/genética , Células HeLa , Heterozigoto , Humanos , Interferon gama/genética , Fosforilação/genética , Ligação Proteica/genética , Transdução de Sinais/genética , Transcrição Genética/genética
8.
Intervirology ; 62(2): 80-89, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31315128

RESUMO

BACKGROUND: In immunocompromised patients, human cytomegalovirus (HCMV) infection is a major cause of morbidity and mortality. Suppressor of cytokine signaling (SOCS) proteins are very potent negative regulators of the janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways. We hypothesized that HCMV exploits SOCS1 and/or SOCS3 to its advantage. METHODS: All experiments were carried out with primary human lung-derived microvascular endothelial cells (HMVEC). SOCS1 and SOCS3 were silenced by transfecting the cells with siRNA. HCMV was propagated and titered on human lung-derived fibroblasts MRC5. Real-time PCR and Western blot were used to detect mRNA and protein levels, respectively. RESULTS: The data presented show that an efficient replication of HCMV in HMVEC is dependent on SOCS3 protein. Time course analysis revealed an increase in SOCS3 protein levels in infected cells. Silencing of SOCS3 (siSOCS3) resulted in inhibition of viral immediate early, early, and late antigen production. Consistently, HCMV titers produced by siSOCS3 cultures were significantly decreased when compared to control transfected cultures (siCNTRs). STAT1 and STAT2 phosphorylation was increased in siSOCS3-infected cells when compared to siCNTR-treated cells. CONCLUSION: These findings indicate the implication of SOCS3 in the mechanism of HCMV-mediated control of cellular immune responses.


Assuntos
Citomegalovirus/fisiologia , Células Endoteliais/virologia , Imunidade Celular , Proteína 3 Supressora da Sinalização de Citocinas/imunologia , Replicação Viral , Células Cultivadas , Células Endoteliais/imunologia , Inativação Gênica , Humanos , Pulmão/citologia , Pulmão/virologia , Fosforilação , RNA Interferente Pequeno , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/imunologia , Transdução de Sinais/imunologia , Proteína 1 Supressora da Sinalização de Citocina/genética , Proteína 1 Supressora da Sinalização de Citocina/imunologia , Proteína 3 Supressora da Sinalização de Citocinas/genética
9.
Nat Commun ; 10(1): 2921, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266943

RESUMO

Cells maintain the balance between homeostasis and inflammation by adapting and integrating the activity of intracellular signaling cascades, including the JAK-STAT pathway. Our understanding of how a tailored switch from homeostasis to a strong receptor-dependent response is coordinated remains limited. Here, we use an integrated transcriptomic and proteomic approach to analyze transcription-factor binding, gene expression and in vivo proximity-dependent labelling of proteins in living cells under homeostatic and interferon (IFN)-induced conditions. We show that interferons (IFN) switch murine macrophages from resting-state to induced gene expression by alternating subunits of transcription factor ISGF3. Whereas preformed STAT2-IRF9 complexes control basal expression of IFN-induced genes (ISG), both type I IFN and IFN-γ cause promoter binding of a complete ISGF3 complex containing STAT1, STAT2 and IRF9. In contrast to the dogmatic view of ISGF3 formation in the cytoplasm, our results suggest a model wherein the assembly of the ISGF3 complex occurs on DNA.


Assuntos
Regulação da Expressão Gênica , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/metabolismo , Fator Gênico 3 Estimulado por Interferon/metabolismo , Interferons/metabolismo , Fator de Transcrição STAT2/metabolismo , Animais , Feminino , Humanos , Fator Gênico 3 Estimulado por Interferon/genética , Fator Gênico 3 Estimulado por Interferon, Subunidade gama/genética , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regiões Promotoras Genéticas , Células RAW 264.7 , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT2/genética , Transcrição Genética
10.
Nat Commun ; 10(1): 2402, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160588

RESUMO

Platelet-leukocyte interactions amplify inflammatory reactions, but the underlying mechanism is still unclear. CLEC5A and CLEC2 are spleen tyrosine kinase (Syk)-coupled C-type lectin receptors, abundantly expressed by leukocytes and platelets, respectively. Whereas CLEC5A is a pattern recognition receptor (PRR) to flaviviruses and bacteria, CLEC2 is the receptor for platelet-activating snake venom aggretin. Here we show that dengue virus (DV) activates platelets via CLEC2 to release extracellular vesicles (EVs), including exosomes (EXOs) and microvesicles (MVs). DV-induced EXOs (DV-EXOs) and MVs (DV-MVs) further activate CLEC5A and TLR2 on neutrophils and macrophages, thereby induce neutrophil extracellular trap (NET) formation and proinflammatory cytokine release. Compared to  stat1-/- mice, simultaneous blockade of CLEC5A and TLR2 effectively attenuates DV-induced inflammatory response and increases survival rate from 30 to 90%. The identification of critical roles of CLEC2 and CLEC5A/TLR2 in platelet-leukocyte interactions will support the development of novel strategies to treat acute viral infection in the future.


Assuntos
Plaquetas/metabolismo , Vírus da Dengue/imunologia , Dengue/imunologia , Vesículas Extracelulares/imunologia , Lectinas Tipo C/imunologia , Macrófagos/imunologia , Neutrófilos/imunologia , Receptores de Superfície Celular/imunologia , Receptor 2 Toll-Like/imunologia , Animais , Micropartículas Derivadas de Células/imunologia , Micropartículas Derivadas de Células/metabolismo , Citocinas/imunologia , Dengue/virologia , Exossomos/imunologia , Exossomos/metabolismo , Armadilhas Extracelulares/imunologia , Vesículas Extracelulares/metabolismo , Humanos , Inflamação , Lectinas Tipo C/genética , Camundongos , Camundongos Knockout , Ativação Plaquetária , Receptores de Superfície Celular/genética , Fator de Transcrição STAT1/genética , Taxa de Sobrevida
11.
J Exp Clin Cancer Res ; 38(1): 279, 2019 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-31242951

RESUMO

BACKGROUND: A better understanding of locally advanced cervical cancer (LACC) is mandatory for further improving the rates of disease control, since a significant proportion of patients still fail to respond or undergo relapse after concurrent chemoradiation treatment (CRT), and survival for these patients has generally remained poor. METHODS: To identify specific markers of CRT response, we compared pretreatment biopsies from LACC patients with pathological complete response (sensitive) with those from patients showing macroscopic residual tumor (resistant) after neoadjuvant CRT, using a proteomic approach integrated with gene expression profiling. The study of the underpinning mechanisms of chemoradiation response was carried out through in vitro models of cervical cancer. RESULTS: We identified annexin A2 (ANXA2), N-myc downstream regulated gene 1 (NDRG1) and signal transducer and activator of transcription 1 (STAT1) as biomarkers of LACC patients' responsiveness to CRT. The dataset collected through qPCR on these genes was used as training dataset to implement a Random Forest algorithm able to predict the response of new patients to this treatment. Mechanistic investigations demonstrated the key role of the identified genes in the balance between death and survival of tumor cells. CONCLUSIONS: Our results define a predictive gene signature that can help in cervical cancer patient stratification, thus providing a useful tool towards more personalized treatment modalities.


Assuntos
Anexina A2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fator de Transcrição STAT1/metabolismo , Neoplasias do Colo do Útero/terapia , Adulto , Idoso , Anexina A2/genética , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais , Proteínas de Ciclo Celular/genética , Quimiorradioterapia , Cisplatino/uso terapêutico , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Pessoa de Meia-Idade , Terapia Neoadjuvante , Poli(ADP-Ribose) Polimerase-1/metabolismo , Tolerância a Radiação , Fator de Transcrição STAT1/genética , Transcriptoma , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Adulto Jovem
12.
J Exp Clin Cancer Res ; 38(1): 206, 2019 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-31113461

RESUMO

BACKGROUND: The aberrant expression of myotubularin-related protein 2 (MTMR2) has been found in some cancers, but little is known about the roles and clinical relevance. The present study aimed to investigate the roles and clinical relevance of MTMR2 as well as the underlying mechanisms in gastric cancer (GC). METHODS: MTMR2 expression was examined in 295 GC samples by using immunohistochemistry (IHC). The correlation between MTMR2 expression and clinicopathological features and outcomes of the patients was analyzed. The roles of MTMR2 in regulating the invasive and metastatic capabilities of GC cells were observed using gain-and loss-of-function assays both in vitro and in vivo. The pathways involved in MTMR2-regulating invasion and metastasis were selected and identified by using mRNA expression profiling. Functions and underlying mechanisms of MTMR2-mediated invasion and metastasis were further investigated in a series of in vitro studies. RESULTS: MTMR2 was highly expressed in human GC tissues compared to adjacent normal tissues and its expression levels were significantly correlated with depth of invasion, lymph node metastasis, and TNM stage. Patients with MTMR2high had significantly shorter lifespan than those with MTMR2low. Cox regression analysis showed that MTMR2 was an independent prognostic indicator for GC patients. Knockdown of MTMR2 significantly reduced migratory and invasive capabilities in vitro and metastases in vivo in GC cells, while overexpressing MTMR2 achieved the opposite results. MTMR2 knockdown and overexpression markedly inhibited and promoted the epithelial-mesenchymal transition (EMT), respectively. MTMR2 mediated EMT through the IFNγ/STAT1/IRF1 pathway to promote GC invasion and metastasis. Phosphorylation of STAT1 and IRF1 was increased by MTMR2 knockdown and decreased by MTMR2 overexpression accompanying with ZEB1 down-regulation and up-regulation, respectively. Silencing IRF1 upregulated ZEB1, which induced EMT and consequently enhanced invasion and metastasis in GC cells. CONCLUSIONS: Our findings suggest that MTMR2 is an important promoter in GC invasion and metastasis by inactivating IFNγ/STAT1 signaling and may act as a new prognostic indicator and a potential therapeutic target for GC.


Assuntos
Interferon gama/genética , Proteínas Tirosina Fosfatases não Receptoras/genética , Fator de Transcrição STAT1/genética , Neoplasias Gástricas/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica , Humanos , Fator Regulador 1 de Interferon/genética , Metástase Linfática , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Fosforilação , Prognóstico , Transdução de Sinais , Neoplasias Gástricas/patologia , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética
13.
Fish Shellfish Immunol ; 89: 411-419, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30978449

RESUMO

The dark sleeper, Odontobutis obscura (O. obscura), is a commercially important species of freshwater sleeper native to East Asia. However, its molecular biology system is unexplored, including the interferon (IFN) signaling pathway, which is crucial to the antiviral response. In this study, we characterised the IFN regulation pattern of dark sleeper interferon regulatory factor 3 (OdIRF3), supplementing evidence of the conservation of this classical pathway in fish. First, the open reading frame (ORF) of OdIRF3 was cloned from the liver tissue by Rapid amplification of cDNA ends (RACE). Amino acid sequence analysis suggested that OdIRF3 is homologous with other fish IRF3 and that the N-terminal DNA-binding domain (DBD) and the C-terminal IRF-association domain (IAD) are conserved. Then, the cellular distribution demonstrated that OdIRF3 is located in the cytoplasm region and transfers into the nuclear region under stimulation. For the function identification, OdIRF3 activated several types of IFN promoters and induced downstream interferon stimulated genes (ISGs) expression. Finally, the overexpression of OdIRF3 significantly decreased viral proliferation. Taken together, these data systematically characterised the sequence, cellular location, and function in IFN expression of OdIRF3, shedding light on the molecular biology mechanism of the dark sleeper.


Assuntos
Doenças dos Peixes/imunologia , Peixes/genética , Peixes/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Fator Regulador 3 de Interferon/química , Interferons/genética , Filogenia , Fator de Transcrição STAT1/genética , Alinhamento de Sequência/veterinária
14.
J Neurooncol ; 143(1): 35-47, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30993511

RESUMO

PURPOSE: Glioma is a highly aggressive and lethal brain tumor. Signal transducers and activators of transcription (STAT) pathway are widely implicated in glioma carcinogenesis. Our previous study found that the Fynrelated kinase (FRK) gene, plays as a tumor suppressor in the development and progression of glioma. This study aimed to investigate the role of FRK in the activation pathway of STATs and its effect on the growth of glioma. METHODS: The U251 and U87 cells with stable FRK overexpression were generated by lentivirus technique. The effects of FRK on the related proteins of STAT signaling pathway were detected by western blotting. Coimmunoprecipitation was used to detect the association of FRK and STAT1. The effects of STAT1 on the proliferation of glioma cells were detected by CCK8 or Edu cell proliferation assays. The expressions and correlation of FRK and p-STAT1 in glioma tissues were detectd by western blotting or immunohistochemistry. The effect of FRK on the growth of glioma was investigated in vivo mouse model. RESULTS: The level of p-JAK2 and p-STAT1 increased after FRK overexpression, while they decreased after FRK downregulation both in U251 and U87 cells. However, FRK had no effect on STAT3 phosphorylation. FRK-induced STAT1 activation was not dependent on JAK2. FRK associated with STAT1, induced STAT1 nuclear translocation and regulated the expressions of STAT1-related target genes. STAT1 overexpression suppressed the proliferation of glioma cells. In contrast, STAT1 knockdown by siRNA promoted glioma cell growth. Importantly, down-regulation of STAT1 partially attenuated FRK-induced growth suppression. The clinical sample-based study indicated that the expression of FRK was significantly correlated with the expression of p-STAT1. FRK significantly inhibited glioma tumor growth in vivo. CONCLUSIONS: Our findings highlighted a critical role of FRK in tumor suppression ability through promoting STAT1 activation, and provided a potential therapeutic target for glioma.


Assuntos
Neoplasias Encefálicas/metabolismo , Proliferação de Células/fisiologia , Glioma/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Quinases/metabolismo , Fator de Transcrição STAT1/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Progressão da Doença , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glioma/tratamento farmacológico , Glioma/patologia , Células HEK293 , Humanos , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/metabolismo , Masculino , Camundongos Nus , Transplante de Neoplasias , Fosforilação , Fator de Transcrição STAT1/genética , Transdução de Sinais , Sincalida/metabolismo , Carga Tumoral/fisiologia
15.
J Immunol ; 202(10): 3076-3086, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30936295

RESUMO

Why some tumors remain indolent and others progress to clinical relevance remains a major unanswered question in cancer biology. IFN signaling in nascent tumors, mediated by STAT1, is a critical step through which the surveilling immune system can recognize and destroy developing tumors. In this study, we have identified an interaction between the progesterone receptor (PR) and STAT1 in breast cancer cells. This interaction inhibited efficient IFN-induced STAT1 phosphorylation, as we observed a decrease in phospho-STAT1 in response to IFN treatment in PR-positive breast cancer cell lines. This phenotype was further potentiated in the presence of PR ligand. In human breast cancer samples, PR-positive tumors exhibited lower levels of phospho-STAT1 as compared with their PR-negative counterparts, indicating that this phenotype translates to human tumors. Breast cancer cells lacking PR exhibited higher levels of IFN-stimulated gene (ISG) RNA, the transcriptional end point of IFN activation, indicating that unliganded PR alone could decrease transcription of ISGs. Moreover, the absence of PR led to increased recruitment of STAT1, STAT2, and IRF9 (key transcription factors necessary for ISG transcription) to ISG promoters. These data indicate that PR, both in the presence and absence of ligand, attenuates IFN-induced STAT1 signaling, culminating in significantly abrogated activation of genes transcribed in response to IFNs. PR-positive tumors may use downregulation of STAT1-mediated IFN signaling to escape immune surveillance, leading to the development of clinically relevant tumors. Selective immune evasion of PR-positive tumors may be one explanation as to why over 65% of breast cancers are PR positive at the time of diagnosis.


Assuntos
Neoplasias da Mama/imunologia , Interferon gama/imunologia , Proteínas de Neoplasias/imunologia , Receptores de Progesterona/imunologia , Fator de Transcrição STAT1/imunologia , Evasão Tumoral , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Feminino , Humanos , Interferon gama/genética , Proteínas de Neoplasias/genética , Fosforilação/genética , Fosforilação/imunologia , Receptores de Progesterona/genética , Fator de Transcrição STAT1/genética
16.
Vet Microbiol ; 230: 78-85, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30827409

RESUMO

Retinoic acid-inducible gene I (RIG-I) is a nucleic acid sensor that plays a key role in host antiviral defenses. Duck viral enteritis (DEV) is a DNA virus that causes significant economic losses to the poultry industry worldwide. Although RIG-I is known to be involved in a common antiviral signaling pathway triggered by RNA viruses, its role in DEV infection remains unclear. In this study, we demonstrated that DEV infection increased the expression levels of interferon ß (IFN-ß) and RIG-I in ducks both in vivo and in vitro. Furthermore, overexpression of duck RIG-I significantly upregulated the expression of interferon-stimulated genes, including myxovirus resistance protein (Mx), Interferon-induced oligodenylate synthetase-like (OASL) and IFN-ß. We therefore used overexpression and knockdown methods to determine if RIG-I affected DEV infection in ducks. Viral infection was inhibited by RIG-I, and enhanced by knockdown of RIG-I expression using small interfering RNA. RIG-I overexpression also activated signal transducer and activator of transcription 1 (STAT1), as a member of the JAK-STAT family. The combined results following STAT1 knockdown and RIG-I overexpression suggested that the antiviral activity of RIG-I was STAT1-dependent. Overall, these findings indicate that RIG-I effectively restricts DEV replication and may play a vital role in the host immune response to DEV infection in ducks.


Assuntos
Proteína DEAD-box 58/genética , Interferon beta/imunologia , Mardivirus/genética , Replicação Viral , 2',5'-Oligoadenilato Sintetase/genética , Animais , Linhagem Celular , Patos/virologia , Imunidade Inata , Mardivirus/fisiologia , Proteínas de Resistência a Myxovirus/genética , RNA Interferente Pequeno , Fator de Transcrição STAT1/genética , Transdução de Sinais
17.
J Immunol ; 202(8): 2332-2347, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30804041

RESUMO

Epithelial barrier cells are proposed to be critical for host defense, and airway epithelial cell capacity for IFN signal transduction is presumed to protect against respiratory viral infection. However, it has been difficult to fully test these concepts given the absence of tools to analyze IFN signaling specific to airway epithelial cells in vivo. To address these issues, we generated a new line of transgenic mice with Cre-driver genes (Foxj1 and Scgb1a1) for a floxed-Stat1 allele (designated Foxj1-Scgb1a1-Cre-Stat1f/f mice) to target the master IFN signal regulator STAT1 in airway epithelial cells and tested these mice for control of infection because of mouse parainfluenza (Sendai) virus and human enterovirus D68 (EV-D68). Indeed, both types of infections showed increases in viral titers and severity of acute illness in Foxj1-Scgb1a1-Cre-Stat1f/f mice and conventional Stat1-/- mice compared with wild-type mice. In concert, the chronic lung disease that develops after Sendai virus infection was also increased in Foxj1-Scgb1a1-Cre-Stat1f/f and Stat1-/ - mice, marked by airway and adjacent parenchymal immune cell infiltration and mucus production for at least 7 wk postinfection. Unexpectedly, relatively mild EV-D68 infection also progressed to chronic lung disease in Foxj1-Scgb1a1-Cre-Stat1f/f and Stat1 -/- mice but was limited (like viral replication) to airways. The results thereby provide proof-of-concept for a critical role of barrier epithelial cells in protection from acute illness and chronic disease after viral infection and suggest a specific role for airway epithelial cells given the limitation of EV-D68 replication and acute and chronic manifestations of disease primarily to airway tissue.


Assuntos
Células Epiteliais/imunologia , Pneumopatias/imunologia , Infecções por Respirovirus/imunologia , Fator de Transcrição STAT1/imunologia , Vírus Sendai/imunologia , Animais , Doença Crônica , Células Epiteliais/virologia , Pneumopatias/genética , Pneumopatias/virologia , Camundongos , Camundongos Knockout , Infecções por Respirovirus/genética , Fator de Transcrição STAT1/genética
18.
J Toxicol Sci ; 44(2): 83-92, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30726814

RESUMO

Immunological functions are disturbed in humans who have been chronically exposed to arsenic via contaminated groundwater. Little is known about the specific mechanisms underlying the impairment of immunological defense system caused by arsenic. The activation of macrophage cells upon infection with bacteria and viruses plays important roles in the defense against these pathogens. Here we show that exposure to arsenite (As(III)) suppresses nitric oxide (NO) production in murine RAW264.7 macrophage cells stimulated with lipopolysaccharide (LPS) and poly(I:C), the compounds mimicking bacterial and viral infection, respectively. As(III) suppressed the LPS- or poly(I:C)-evoked induction of inducible NO synthase (iNOS) without affecting the transactivation of NF-κB. As the interferon (IFN)-ß/STAT1 pathway is also involved in the induction of iNOS in addition to NF-κB, we examined the effects of As(III) on the expression and secretion of IFN-ß, the expression of the components of IFN-α/ß receptor, the phosphorylation of STAT1, and the levels of cytokines involved in STAT1 activation. The results showed that the expression and secretion of IFN-ß were specifically suppressed by As(III) treatment in RAW264.7 cells stimulated with LPS or poly(I:C). These results suggest that As(III) suppresses the expression and secretion of IFN-ß, leading to the reduced STAT1 activation and consequently the reduced iNOS induction in macrophage cells. Our data suggest an important role of the arsenic-induced suppression of IFN-ß on the disturbances in immunological defense against both bacteria and viruses.


Assuntos
Arsenitos/toxicidade , Interferon beta/metabolismo , Óxido Nítrico/metabolismo , Animais , Interferon beta/genética , Lipopolissacarídeos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Óxido Nítrico Sintase Tipo II/metabolismo , Poli I-C , Células RAW 264.7 , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo
19.
Fish Shellfish Immunol ; 87: 386-394, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30703549

RESUMO

Signal transducer and activator of transcription 1 (STAT1) plays an important role in the Janus kinase (JAK)-STAT signaling of human and mammals; however, the mechanism of STAT1 in innate immune activation of teleost fishes remains largely unknown. In this study, two STAT1 homologues (bcSTAT1a and bcSTAT1b) of black carp (Mylopharyngodon piceus) have been cloned and characterized. Both bcSTAT1a and bcSTAT1b transcription in host cells was obviously increased in response to the stimulation of poly (I:C), lipopolysaccharide (LPS), grass carp reovirus (GCRV) and interferon (IFN); however, the increase rate of bcSTAT1b transcription post stimulation was obviously higher than that of bcSTAT1a. bcSTAT1a and bcSTAT1b were distributed in both cytoplasm and nucleus in the immunofluorescence staining assay. Self-association of bcSTAT1a and bcSTAT1b, and the interaction between bcSTAT1a and bcSTAT1b have been detected through co-immunoprecipitation (co-IP) assay; and the data of native polyacrylamide gel electrophoresis (PAGE) implied that bcSTAT1a and bcSTAT1b might form homodimer and heterodimer in vivo like their mammalian counterparts. Both bcSTAT1a and bcSTAT1b presented IFN-inducing ability in report assay, and both bcSTAT1a and bcSTAT1b showed antiviral activities against GCRV in EPC cells. Our data support the conclusion that both bcSTAT1a and bcSTAT1b play important roles in host antiviral innate immune activation initiated by GCRV.


Assuntos
Carpas/genética , Carpas/imunologia , Doenças dos Peixes/imunologia , Expressão Gênica/imunologia , Imunidade Inata/genética , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Perfilação da Expressão Gênica/veterinária , Filogenia , Fator de Transcrição STAT1/química , Alinhamento de Sequência/veterinária
20.
Food Chem Toxicol ; 126: 67-71, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30769049

RESUMO

Aloin is the major anthraquinone glycoside obtained from the Aloe species and exhibits anti-inflammatory and anti-oxidative activities. Here, we aimed to determine the effects of aloin on heme oxygenase-1 (HO-1) induction and on the expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX) 2 in lipopolysaccharide (LPS)-activated human umbilical vein endothelial cells (HUVECs). To the end, aloin was tested whether aloin reduces iNOS protein expression and inflammatory markers (interleukin (IL)-1ß and tumor necrosis factor (TNF)-α) in LPS-treated mice lung tissue. The results indicated that aloin affected HO-1 induction and reduced LPS-activated NF-κB-luciferase activity showed to preferential inhibition of iNOS/NO and COX-2/PGE2 that was partly related to inhibition of STAT-1 phosphorylation. In particular, aloin induced translocation of Nrf2 from cytosol into the nucleus by an increased Nrf2-ARE binding activity, and reduced IL-1ß production in LPS-activated HUVECs. The reduced expression of iNOS/NO by aloin was reversed by siHO-1RNA-transfection. In LPS-treated mice, aloin significantly reduced iNOS protein in lung tissues, and TNF-α levels in the BALF. We concluded that aloin may be beneficial for treatment of lung injury.


Assuntos
Anti-Inflamatórios/administração & dosagem , Emodina/análogos & derivados , Pneumopatias/tratamento farmacológico , NF-kappa B/imunologia , Óxido Nítrico Sintase Tipo II/genética , Extratos Vegetais/administração & dosagem , Fator de Transcrição STAT1/imunologia , Animais , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/imunologia , Emodina/administração & dosagem , Heme Oxigenase-1/genética , Heme Oxigenase-1/imunologia , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/imunologia , Humanos , Pneumopatias/genética , Pneumopatias/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/genética , Óxido Nítrico Sintase Tipo II/imunologia , Fator de Transcrição STAT1/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia
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