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1.
Biomed Res Int ; 2020: 8206849, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33381584

RESUMO

The role of the IκB/NF-κB signaling pathway in the uveoscleral outflow pathway was investigated with IκBα gene silencing mediated by the 3-(dimethylamino)-1-propylamine-conjugated glycogen (DMAPA-Glyp) derivative. The IκBα-siRNA-loaded DMAPA-Glyp complex was transfected into the ciliary muscles of rats by intracameral injection (labeled as the DMAPA-Glyp+siRNA group). The Lipofectamine™ 2000 (Lipo)/siRNA complex and the naked siRNA were set as the controls. The mRNA and protein expression of IκBα, NF-κBp65, and MMP-2 were analyzed by real-time PCR, western blotting, and in situ gelatin zymography. Nuclear translocation of NF-κBp65 was analyzed by immunofluorescence. Rat intraocular pressure (IOP) was monitored pre- and postinjection. Gene transfection efficiency and toxicity of the DMAPA-Glyp derivative were also evaluated. After RNA interference (RNAi), IκBα mRNA and protein expression were significantly inhibited. NF-κBp65 mRNA and protein expression showed no significant differences. Nevertheless, nuclear translocation of NF-κBp65 occurred in the DMAPA-Glyp+siRNA group. Both mRNA expression and activity of MMP-2 increased, with the largest increase in the DMAPA-Glyp+siRNA group. IOP in the DMAPA-Glyp+siRNA group fell to the lowest level on day 3 after RNAi. The levels of Cy3-siRNA in the ciliary muscle of the DMAPA-Glyp+siRNA group did not significantly decrease over time. At 7 and 14 d after RNAi, no significant pathological damage was detectable in the eyes injected with the DMAPA-Glyp derivative or the DMAPA-Glyp/siRNA complex. Taken together, our results suggest that downregulation of IκBα expression in the ciliary muscle plays a crucial role in reducing the IOP values of rats. IκBα may become a new molecular target for lowering IOP in glaucoma. The DMAPA-Glyp derivative is safe and feasible as an effective siRNA vector in rat eyes.


Assuntos
Segmento Anterior do Olho , Humor Aquoso , Inativação Gênica/fisiologia , Proteínas I-kappa B , Transdução de Sinais/genética , Animais , Segmento Anterior do Olho/metabolismo , Segmento Anterior do Olho/fisiologia , Humor Aquoso/metabolismo , Humor Aquoso/fisiologia , Diaminas/química , Sistemas de Liberação de Medicamentos , Glicogênio/química , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Masculino , NF-kappa B/genética , NF-kappa B/metabolismo , RNA Interferente Pequeno/genética , Ratos , Ratos Wistar , Transfecção
2.
Nat Commun ; 11(1): 3409, 2020 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-32641778

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is associated with high mortality and therapy resistance. Here, we show that low expression of κB-Ras GTPases is frequently detected in PDAC and correlates with higher histologic grade. In a model of KRasG12D-driven PDAC, loss of κB-Ras accelerates tumour development and shortens median survival. κB-Ras deficiency promotes acinar-to-ductal metaplasia (ADM) during tumour initiation as well as tumour progression through intrinsic effects on proliferation and invasion. κB-Ras proteins are also required for acinar regeneration after pancreatitis, demonstrating a general role in control of plasticity. Molecularly, upregulation of Ral GTPase activity and Sox9 expression underlies the observed phenotypes, identifying a previously unrecognized function of Ral signalling in ADM. Our results provide evidence for a tumour suppressive role of κB-Ras proteins and highlight low κB-Ras levels and consequent loss of Ral control as risk factors, thus emphasizing the necessity for therapeutic options that allow interference with Ral-driven signalling.


Assuntos
Células Acinares/metabolismo , Carcinoma Ductal Pancreático/genética , GTP Fosfo-Hidrolases/genética , Neoplasias Pancreáticas/genética , Pancreatite/genética , Proteínas/genética , Células Acinares/patologia , Idoso , Animais , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Feminino , GTP Fosfo-Hidrolases/metabolismo , Regulação da Expressão Gênica , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Estimativa de Kaplan-Meier , Masculino , Metaplasia/genética , Metaplasia/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Pancreatite/metabolismo , Proteínas/metabolismo , Fatores de Transcrição SOX9/genética , Fatores de Transcrição SOX9/metabolismo , Proteínas ral de Ligação ao GTP/genética , Proteínas ral de Ligação ao GTP/metabolismo , Proteínas ras/genética , Proteínas ras/metabolismo
3.
Immunology ; 160(1): 64-77, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32064589

RESUMO

Sterile inflammation contributes to many pathological states associated with mitochondrial injury. Mitochondrial injury disrupts calcium homeostasis and results in the release of CpG-rich mitochondrial DNA. The role of CpG-stimulated TLR9 innate immune signalling and sterile inflammation is well studied; however, how calcium dyshomeostasis affects this signalling is unknown. Therefore, we interrogated the relationship beτween intracellular calcium and CpG-induced TLR9 signalling in murine macrophages. We found that CpG-ODN-induced NFκB-dependent IL1α and IL1ß expression was significantly attenuated by both calcium chelation and calcineurin inhibition, a finding mediated by inhibition of degradation of the NFκB inhibitory protein IκBß. In contrast, calcium ionophore exposure increased CpG-induced IκBß degradation and IL1α and IL1ß expression. These results demonstrate that through its effect on IκBß degradation, increased intracellular Ca2+ drives a pro-inflammatory TLR9-mediated innate immune response. These results have implications for the study of innate immune signalling downstream of mitochondrial stress and injury.


Assuntos
Sinalização do Cálcio/imunologia , Cálcio/metabolismo , Proteínas I-kappa B/metabolismo , Imunidade Inata , Receptor Toll-Like 9/metabolismo , Animais , Inibidores de Calcineurina/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Quelantes/farmacologia , Proteínas I-kappa B/antagonistas & inibidores , Proteínas I-kappa B/genética , Interleucina-1alfa/metabolismo , Interleucina-1beta/metabolismo , Macrófagos , Masculino , Camundongos , Camundongos Knockout , Oligodesoxirribonucleotídeos/imunologia , Cultura Primária de Células , Proteólise/efeitos dos fármacos , Células RAW 264.7
4.
Sci Rep ; 10(1): 2754, 2020 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-32066788

RESUMO

The number of colon cancer cases is increasing worldwide, and type II diabetes patients have an increased risk of developing colon cancer. Diet-borne advanced glycation end-products (AGEs) may promote neoplastic transformation; however, the mechanisms involved remain elusive. The present study helped to define the relationship between dietary AGEs and cancer progression. C2BBe1 adenocarcinoma enterocytes were exposed to 200 µg/mL glycated casein (AGEs-Csn) for up to 24 h. AGEs-Csn exposure resulted in increased cell proliferation, maladaptative changes in SOD and CAT activity and moderate levels of hydrogen peroxide (H2O2) intracellular accumulation. AGEs-Csn activated pro-survival and proliferation signalling, such as the phosphorylation of mTOR (Ser2448) and Akt (Ser473). GSK-3ß phosphorylation also increased, potentially inducing extracellular matrix remodelling and thus enabling metastasis. Moreover, AGEs-Csn induced MMP-1, -3, -7, -9 and -10 expression and activated MMP-2 and MMP-9, which are regulators of the extracellular matrix and cytokine functions. AGEs-Csn induced inflammatory responses that included extracellular IL-1ß at 6 h; time-dependent increases in IL-8; RAGE and NF-κB p65 upregulation; and IκB inhibition. Co-treatment with anti-RAGE or anti-TNF-α blocking antibodies and AGEs-Csn partially counteracted these changes; however, IL-8, MMP-1 and -10 expression and MMP-9 activation were difficult to prevent. AGEs-Csn perpetuated signalling that led to cell proliferation and matrix remodelling, strengthening the link between AGEs and colorectal cancer aggressiveness.


Assuntos
Caseínas/farmacologia , Enterócitos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Produtos Finais de Glicação Avançada/farmacologia , Adenocarcinoma/etiologia , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Caseínas/química , Catalase/genética , Catalase/metabolismo , Linhagem Celular Tumoral , Neoplasias do Colo/etiologia , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Enterócitos/metabolismo , Enterócitos/patologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Glicosilação , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Biológicos , NF-kappa B/genética , NF-kappa B/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo
5.
Skelet Muscle ; 9(1): 28, 2019 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-31757224

RESUMO

BACKGROUND: Low lean body mass is the most important predictor of sarcopenia with strong genetic background. The aim of this study was to uncover genetic factors underlying lean mass development. MATERIALS AND METHODS: We performed a genome-wide association study (GWAS) of fat-adjusted leg lean mass in the Framingham Heart Study (FHS, N = 6587), and replicated in the Women's Health Initiative-African American sub-sample (WHI-AA, N = 847) and the Kansas City Osteoporosis Study (KCOS, N = 2219). We also cross-validated significant variants in the publicly available body mass index (BMI) summary results (N ~ 700,000). We then performed a series of functional investigations on the identified variants. RESULTS: Four correlated SNPs at 6p21.1 were identified at the genome-wide significance (GWS, α = 5.0 × 10-8) level in the discovery FHS sample (rs551145, rs524533, rs571770, and rs545970, p = 3.40-9.77 × 10-9), and were successfully replicated in both the WHI-AA and the KCOS samples (one-sided p = 1.61 × 10-3-0.04). They were further cross-validated by the large-scale BMI summary results (p = 7.0-9.8 × 10-3). Cis-eQTL analyses associated these SNPs with the NFKBIE gene expression. Electrophoresis mobility shift assay (EMSA) in mouse C2C12 myoblast cells implied that rs524533 and rs571770 were bound to an unknown transcription factor in an allelic specific manner, while rs551145 and rs545970 did not. Dual-luciferase reporter assay revealed that both rs524533 and rs571770 downregulated luciferase expression by repressing promoter activity. Moreover, the regulation pattern was allelic specific, strengthening the evidence towards their differential regulatory effects. CONCLUSIONS: Through a large-scale GWAS followed by a series of functional investigations, we identified 2 correlated functional variants at 6p21.1 associated with leg lean mass. Our findings not only enhanced our understanding of molecular basis of lean mass development but also provided useful candidate genes for further functional studies.


Assuntos
Cromossomos Humanos Par 6/genética , Polimorfismo de Nucleotídeo Único , Sarcopenia/genética , Magreza/genética , Idoso , Animais , Índice de Massa Corporal , Linhagem Celular , Feminino , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Proteínas I-kappa B/genética , Desequilíbrio de Ligação , Masculino , Camundongos , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas/genética , Locos de Características Quantitativas , Sarcopenia/patologia , Magreza/patologia
6.
J Agric Food Chem ; 67(39): 10871-10879, 2019 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-31517482

RESUMO

This study evaluated the effect of triterpenoids from edible mushroom Poria cocos on intestinal epithelium integrity and revealed the transcriptional regulatory pathways that underpin restorative mechanisms in the gut. Based on computational docking studies, transcriptional activation experiments and glucocorticoid receptor (GR) protein immunofluorescence localization assays in cultured cells, 16α-hydroxytrametenolic acid (HTA) was discovered as a novel GR agonist in this study. HTA ameliorates TNF-α-induced Caco-2 monolayer intestinal epithelial barrier damage and suppressed activation of phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt), which attenuated downstream IκB and nuclear factor kappa-B (NF-κB) phosphorylation through GR activation. Moreover, HTA prevented NF-κB translocation into the nucleus and binding to its cis-element and suppressed lipopolysaccharide-induced downstream NO production and pro-inflammatory cytokines at both protein and mRNA expression levels. In conclusion, HTA from P. cocos improves intestinal barrier function through a GR-mediated PI3K/Akt/NF-κB signaling pathway and may be potentially exploited as a supportive dietary therapeutic strategy for restoring gut health.


Assuntos
Mucosa Intestinal/efeitos dos fármacos , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Extratos Vegetais/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Glucocorticoides/metabolismo , Triterpenos/farmacologia , Wolfiporia/química , Células CACO-2 , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Mucosa Intestinal/metabolismo , Simulação de Acoplamento Molecular , NF-kappa B/genética , Fosfatidilinositol 3-Quinase/genética , Fosforilação , Extratos Vegetais/química , Proteínas Proto-Oncogênicas c-akt/genética , Receptores de Glucocorticoides/genética , Transdução de Sinais/efeitos dos fármacos , Triterpenos/química , Verduras/química
7.
Biochem Biophys Res Commun ; 518(4): 632-637, 2019 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-31451219

RESUMO

In the cartilage and synovial microenvironment of osteoarthritis (OA) patients, utmost changes are commonly brought upon by the inflammatory cytokines, leading to cellular dysfunction, particularly in chondrocytes. The regulation of chondrogenesis, a key part is played the microRNAs. Thus, the current study aimed to assess the function of miR-20a in osteoarthritis. The miR-20a expression was observed to increase in the tissues of OA cartilage, when compared with tissues of normal cartilage, and enhanced proliferation of chondrocyte was observed in the presence of miR-20a. Moreover, on treating the chondrocytes with LPS (lipopolysaccharide), an increase in miR-20a level was observed. On transfecting with miR-20a inhibitor, inhibition in production of LPS-induced pro-inflammatory cytokines as well as cell apoptosis were seen. The assay for luciferase activity showed that the expression of IκBß was impeded on being targeted at its 3'-UTR by miR-20a. The transfection of IκBß and inhibitor of miR-20a repressed the NF-κB pathway activation and chondrocyte cellular apoptosis. An OA model was established for in vivo studies on rats by ACLT (anterior cruciate ligament transection). In conclusion, the results demonstrate an increase in articular cavity inflammation in rats with OA in the presence of miR-20a by targeting on IκBß and activating the NF-κB signaling pathway.


Assuntos
Proteínas I-kappa B/genética , Inflamação/genética , MicroRNAs/genética , NF-kappa B/genética , Osteoartrite/genética , Regiões 3' não Traduzidas/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Cartilagem/efeitos dos fármacos , Cartilagem/metabolismo , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Citocinas/genética , Citocinas/metabolismo , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Proteínas I-kappa B/metabolismo , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , NF-kappa B/metabolismo , Osteoartrite/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
8.
Front Immunol ; 10: 1583, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31354726

RESUMO

Next to the classical developmental route, in which first CD25 and subsequently Foxp3 are induced to generate thymic regulatory T (Treg) cells, an alternative route has been described. This alternative route is characterized by reciprocal induction of Foxp3 and CD25, with CD25 induction being required to rescue developing Treg cells from Foxp3-induced apoptosis. NF-κB has been demonstrated to be crucial for the development of thymic Treg cells via the classical route. However, its impact on the alternative route is poorly characterized. Using single and double deficient mice for key regulators of the classical route, c-Rel and IκBNS, we here demonstrate that NF-κB is essential for the generation of alternative CD25-Foxp3+ precursors, as well. Thus, c-Rel and IκBNS govern both routes of thymic Treg cell development.


Assuntos
Proteínas I-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-rel/metabolismo , Linfócitos T Reguladores/fisiologia , Timócitos/fisiologia , Animais , Diferenciação Celular , Células Cultivadas , Fatores de Transcrição Forkhead/metabolismo , Proteínas I-kappa B/genética , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-rel/genética
9.
Am J Ophthalmol ; 200: 150-160, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30653959

RESUMO

PURPOSE: To investigate the expression pattern of nucleotide-binding oligomerization domain (Nod)-like receptors that detects "danger" intracellular signaling and its correlation with clinical dry eye (DE) markers. DESIGN: Cross-sectional study. METHODS: A total of 50 participants with 50 eyes were included: 23 eyes with Sjögren syndrome (SS)-DE, 14 eyes with non-SS-DE, and 13 healthy controls with non-DE. Ocular Surface Disease Index (OSDI) was self-answered and clinical tests including the tear film breakup time (TBUT), Schirmer test, and corneal fluorescein staining (CFS) were performed. Specimens for expression pattern analysis were obtained by conjunctival impression cytology and biopsy. Nod-1, inhibitor kappa B kinase-alpha (IκKα), and nuclear factor kappa B (NF-κB) expression was determined by reverse transcription quantitative real-time polymerase chain reaction and Western blot. Correlations between Nod-1 and ocular surface parameters were determined. RESULTS: Patients with SS-DE had significantly higher OSDI and CFS scores and lower TBUT and Schirmer test scores than those with non-SS-DE patients (all P < .05). Compared with the control group, both the SS-DE and non-SS-DE groups showed significant upregulation in mRNA expression levels of Nod-1 (relative 3.48-fold and 1.72-fold upregulation, respectively, P < .01), IκKα (relative 1.83-fold and 1.24-fold upregulation, respectively, P < .01), and NF-κB (relative 1.84-fold and 1.32-fold upregulation, respectively, P < .01). Western blot analysis showed that Nod-1 protein expression increased in both the SS-DE and non-SS-DE groups (relative 2.71-fold and 1.64-fold upregulation, respectively, P < .05) compared with that in the control group. Similar findings were observed for IκKα and NF-κB. In DE participants, the expression of Nod-1 significantly correlated with the OSDI (R2 = 0.61, r = 0.78, P < .01), Schirmer test score (R2 = 0.44, r = -0.66, P < .01), and CFS (R2 = 0.46, r = 0.68, P < .01) but did not significantly correlate with TBUT (R2 < 0.01, r = 0.08, P = .66). CONCLUSIONS: Nod-1 expression was increased in the conjunctiva of DE, especially SS-DE, and was associated with disease severity. Expression of Nod-like receptors might play an important role in initiating the inflammatory response in DE.


Assuntos
Túnica Conjuntiva/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteína Adaptadora de Sinalização NOD1/genética , Síndrome de Sjogren/genética , Idoso , Western Blotting , Estudos Transversais , Feminino , Marcadores Genéticos/genética , Humanos , Proteínas I-kappa B/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Pessoa de Meia-Idade , NF-kappa B/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Síndrome de Sjogren/diagnóstico
10.
Mol Med Rep ; 19(3): 2057-2064, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30664158

RESUMO

The present study aimed to investigate the anti­arthritic effects of curculigoside isolated from the rhizome of Curculigo orchioides Gaertn in vivo and in vitro, as well as to determine the potential underlying mechanisms. A rat model of arthritis was induced with type II collagen. Arthritic rats were treated with curculigoside (50 mg/kg) and blood samples were collected to determine serum levels of tumor necrosis factor (TNF)­α, interleukin (IL)­1ß, IL­6, IL­10, IL­12 and IL­17A. Furthermore, indices of the thymus and spleen were determined. The anti­proliferative effects of curculigoside were detected with Cell Counting kit­8 assays in rheumatoid arthritis­derived fibroblast­like synoviocyte MH7A cells. In addition, expression levels of Janus kinase (JAK)1, JAK3, signal transducer and activator of transcription (STAT)3, nuclear factor (NF)­κB p65 and its inhibitor (IκB) were determined by western blotting. The results revealed that curculigoside inhibited paw swelling and arthritis scores in type II collagen­induced arthritic (CIA) rats. Additionally, curculigoside decreased serum levels of TNF­α, IL­1ß, IL­6, IL­10, IL­12 and IL­17A in CIA rats. Curculigoside also significantly inhibited MH7A cell proliferation in a time and concentration­dependent manner. Furthermore, treatment downregulated the expression of JAK1, JAK3 and STAT3, and upregulated cytosolic nuclear factor (NF)­κB p65 and IκB. In conclusion, the results of the present study indicated that curculigoside exhibited significant anti­arthritic effects in vivo and in vitro, and the molecular mechanism may be associated with the JAK/STAT/NF­κB signaling pathway.


Assuntos
Artrite Reumatoide/tratamento farmacológico , Benzoatos/administração & dosagem , Glucosídeos/administração & dosagem , Janus Quinase 1/genética , Janus Quinase 3/genética , Fator de Transcrição STAT3/genética , Animais , Artrite Reumatoide/induzido quimicamente , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo II/toxicidade , Modelos Animais de Doenças , Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas I-kappa B/genética , Interleucinas/genética , Ratos , Transdução de Sinais , Sinoviócitos/efeitos dos fármacos , Fator de Transcrição RelA/genética , Fator de Necrose Tumoral alfa/genética
11.
Nat Commun ; 9(1): 4001, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30275490

RESUMO

Diffuse large B-cell lymphoma (DLBCL) is an aggressive cancer originating from mature B-cells. Prognosis is strongly associated with molecular subgroup, although the driver mutations that distinguish the two main subgroups remain poorly defined. Through an integrative analysis of whole genomes, exomes, and transcriptomes, we have uncovered genes and non-coding loci that are commonly mutated in DLBCL. Our analysis has identified novel cis-regulatory sites, and implicates recurrent mutations in the 3' UTR of NFKBIZ as a novel mechanism of oncogene deregulation and NF-κB pathway activation in the activated B-cell (ABC) subgroup. Small amplifications associated with over-expression of FCGR2B (the Fcγ receptor protein IIB), primarily in the germinal centre B-cell (GCB) subgroup, correlate with poor patient outcomes suggestive of a novel oncogene. These results expand the list of subgroup driver mutations that may facilitate implementation of improved diagnostic assays and could offer new avenues for the development of targeted therapeutics.


Assuntos
Regulação Neoplásica da Expressão Gênica , Genes Reguladores/genética , Variação Genética , Genoma Humano/genética , Linfoma Difuso de Grandes Células B/genética , Regiões 3' não Traduzidas/genética , Proteínas Adaptadoras de Transdução de Sinal , Linfócitos B/metabolismo , Linfócitos B/patologia , Linhagem Celular Tumoral , Exoma/genética , Estudo de Associação Genômica Ampla , Centro Germinativo/metabolismo , Centro Germinativo/patologia , Humanos , Proteínas I-kappa B/genética , Linfoma Difuso de Grandes Células B/metabolismo , Linfoma Difuso de Grandes Células B/patologia , Mutação , Proteínas Nucleares/genética , Receptores de IgG/genética , Análise de Sequência de DNA , Transcriptoma
12.
J Immunol ; 201(7): 1907-1917, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30127089

RESUMO

In both NOD mice and humans, the development of type 1 diabetes (T1D) is dependent in part on autoreactive CD8+ T cells recognizing pancreatic ß cell peptides presented by often quite common MHC class I variants. Studies in NOD mice previously revealed that the common H2-Kd and/or H2-Db class I molecules expressed by this strain aberrantly lose the ability to mediate the thymic deletion of pathogenic CD8+ T cell responses through interactions with T1D susceptibility genes outside the MHC. A gene(s) mapping to proximal chromosome 7 was previously shown to be an important contributor to the failure of the common class I molecules expressed by NOD mice to mediate the normal thymic negative selection of diabetogenic CD8+ T cells. Using an inducible model of thymic negative selection and mRNA transcript analyses, we initially identified an elevated Nfkbid expression variant as a likely NOD-proximal chromosome 7 region gene contributing to impaired thymic deletion of diabetogenic CD8+ T cells. CRISPR/Cas9-mediated genetic attenuation of Nfkbid expression in NOD mice resulted in improved negative selection of autoreactive diabetogenic AI4 and NY8.3 CD8+ T cells. These results indicated that allelic variants of Nfkbid contribute to the efficiency of intrathymic deletion of diabetogenic CD8+ T cells. However, although enhancing thymic deletion of pathogenic CD8+ T cells, ablating Nfkbid expression surprisingly accelerated T1D onset that was associated with numeric decreases in both regulatory T and B lymphocytes in NOD mice.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Cromossomos Humanos Par 7/genética , Diabetes Mellitus Tipo 1/imunologia , Proteínas I-kappa B/genética , Timo/imunologia , Alelos , Animais , Autoantígenos/imunologia , Diferenciação Celular , Células Cultivadas , Deleção Clonal , Modelos Animais de Doenças , Suscetibilidade a Doenças , Humanos , Proteínas I-kappa B/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Polimorfismo Genético
14.
Exp Dermatol ; 27(9): 1048-1052, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29938836

RESUMO

Antagonists of IL-17A and its receptor have proven to be highly effective in the treatment of psoriasis. However, many of the underlying molecular mechanisms involved in the pathogenesis of psoriasis are still to be determined. IκBζ (encoded by the NFKBIZ gene) plays a key role in the development of psoriasis by mediating IL-17A- and IL-17F-driven effects. Both IL-17A and IL-17F expression are increased in lesional psoriatic skin. IL-17A/A and IL-17F/F homodimers as well as the IL-17A/F heterodimer signal through the same receptors. The aim of this study was to characterize the role of the IL-17A/F heterodimer in the regulation of NFKBIZ expression and in the regulation of selected psoriasis-associated genes. We demonstrated that IL-17A/F stimulation of human keratinocytes significantly induced NFKBIZ expression. Moreover, silencing IκBζ by siRNA revealed that IκBζ is a key regulator of IL-17A/F-inducible psoriasis-associated genes, including CCL20, DEFB4, IL-8, CHI3L1 and S100A7. In addition, IL-17A/F-induced NFKBIZ expression was mediated by a mechanism involving the p38 MAPK and NF-κB signalling pathways. In conclusion, we present IκBζ as a novel key regulator of IL-17A/F-driven effects in psoriasis. Thus, antagonists to IL-17A/F or IκBζ may present a targeted approach for treating psoriasis.


Assuntos
Expressão Gênica/efeitos dos fármacos , Proteínas I-kappa B/genética , Interleucina-17/farmacologia , Proteínas Nucleares/genética , Psoríase/genética , Proteínas Adaptadoras de Transdução de Sinal , Células Cultivadas , Quimiocina CCL20/genética , Proteína 1 Semelhante à Quitinase-3/genética , Inativação Gênica , Humanos , Interleucina-8/genética , Queratinócitos , Sistema de Sinalização das MAP Quinases , NF-kappa B/metabolismo , Proteína A7 Ligante de Cálcio S100/genética , Fator de Necrose Tumoral alfa/farmacologia , beta-Defensinas/genética
15.
Int J Mol Sci ; 19(6)2018 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-29899297

RESUMO

Primary cutaneous diffuse large B-cell lymphoma, leg type (PCDLBCL-LT) is one of the well-recognized extranodal lymphomas commonly addicted to the B-cell receptor-MYD88 superpathway. We aimed to describe the genomic changes in a patient who progressed through treatment with ibrutinib, a Bruton's tyrosine kinase (BTK) inhibitor. An 80-year-old woman presented with multiply relapsed PCDLBCL-LT after multiple lines of chemoimmunotherapy and radiotherapy. Pre-treatment testing of the localized cutaneous tumor lesion on a lymphoid amplicon panel demonstrated an MYD88 p.L265P mutation. Ibrutinib therapy was subsequently commenced, resulting in complete resolution of the skin disease. Despite an ongoing skin response, the patient developed progressive nodal disease at two months. Genomic analysis of the cutaneous tumor sample at baseline was compared to that of the inguinal lymph node upon progression, and revealed the acquisition of multiple genomic changes. These included several aberrations expected to bypass BTK inhibition, including two CARD11-activating mutations, and a deleterious mutation in the nuclear factor kappa B (NF-κB) negative regulator, NFKBIE. In addition, an IgH-IRF8 translocation was detected (which brings the IRF8 transcription factor under control of the immunoglobulin heavy chain locus), representing a third plausible mechanism contributing to ibrutinib resistance. Several copy-number changes occurred in both samples, including an amplification of 18q, which encodes the anti-apoptotic protein BCL2. We describe the first case of novel genomic changes of PCDLBCL-LT that occurred while on ibrutinib, providing important mechanistic insights into both pathogenesis and drug resistance.


Assuntos
Antineoplásicos/uso terapêutico , Linfoma Difuso de Grandes Células B/genética , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Neoplasias Cutâneas/genética , Idoso de 80 Anos ou mais , Antineoplásicos/administração & dosagem , Proteínas Adaptadoras de Sinalização CARD/genética , Progressão da Doença , Feminino , Instabilidade Genômica , Guanilato Ciclase/genética , Humanos , Proteínas I-kappa B/genética , Fatores Reguladores de Interferon/genética , Metástase Linfática , Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/patologia , Mutação , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Pirazóis/administração & dosagem , Pirimidinas/administração & dosagem , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/patologia
16.
Biochem Biophys Res Commun ; 500(4): 944-951, 2018 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-29705698

RESUMO

The pro-inflammatory vasoconstrictor Angiotensin II can cause endothelial dysfunction and is considered to be one of the mediators of atherosclerosis. Our former results demonstrated that polysaccharides derived from the red alga Porphyridium sp. attenuate inflammatory processes by interfering with tumor necrosis factor-alpha-induced inflammation, in human coronary artery endothelial cells. However, the anti-inflammatory effect of these polysaccharides on inflammation processes occurring under Angiotensin II stimulation is yet unknown. Herein, we studied the polysaccharide's anti-inflammatory effect by quantification of inflammatory markers in Angiotensin II- stimulated Human Coronary Artery Endothelial Cells following pre-treatment with polysaccharides. Inflammatory atherosclerotic pathways up-regulated by Angiotensin II, including adhesion molecule expression and nuclear factor kappa-light-chain-enhancer of activated B cells translocation, were significantly attenuated or diminished in cells pre-treated with the polysaccharides. In addition, the polysaccharides increased the antioxidant response elements activity through the nuclear factor-E2-related factor 2- antioxidant protection system. These polysaccharide's promising abilities may be considered as a basis for future use as a therapeutic agent aimed at improving vascular health by attenuation of the inflammatory atherosclerotic process.


Assuntos
Angiotensina II/farmacologia , Anti-Inflamatórios/farmacologia , Células Endoteliais/efeitos dos fármacos , NF-kappa B/genética , Polissacarídeos/farmacologia , Rodófitas/química , Anti-Inflamatórios/isolamento & purificação , Elementos de Resposta Antioxidante/efeitos dos fármacos , Linhagem Celular , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Inflamação , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Luciferases/genética , Luciferases/metabolismo , Modelos Biológicos , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Polissacarídeos/isolamento & purificação , Transdução de Sinais , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo
17.
Hum Immunol ; 79(6): 494-498, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29601852

RESUMO

The NF-kappaB pathway might play a role in the pathogenesis of renal disease and type 2 diabetes (T2DM). Our aim was to determine whether common polymorphisms in NF-kappaB genes were associated with impaired renal function and T2DM in a cohort of healthy elderly individuals. We studied 487 individuals, all Caucasian and aged 65-85 years. A total of 104 (21%) had impaired renal function (estimated glomerular filtration rate, eGFR < 60) and 146 (30%) were classified as diabetics. The genotypes of 4 common variants were determined through PCR-RFLP or fluorescent capillary electrophoresis. The NFKB1 variants were significantly associated with T2DM: rs7667496 p = 0.01, OR = 1.68; and rs28362491 p = 0.02, OR = 1.67. They remained significantly associated in a multiple logistic regression with age, gender, hypertension, body mass index, and cholesterol. There was a trend toward the association of these variants with eGFR < 60. The two NFKB1 variants were in linkage disequilibrium (D' = -0.86), and homozygous for the two non-risk alleles (rs7667496 CC + rs28362491 II), were significantly more common in the non-diabetics (p = 0.02). In our cohort the NFKB1 variation was an independent risk factor for developing T2DM. Additional studies to confirm this association are of special interest, as well as studies to give a functional explanation to the genetic association.


Assuntos
Diabetes Mellitus Tipo 2/genética , Genótipo , Proteínas I-kappa B/genética , Rim/metabolismo , Inibidor de NF-kappaB alfa/genética , Subunidade p50 de NF-kappa B/genética , Proteínas Nucleares/genética , Proteínas Adaptadoras de Transdução de Sinal , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Grupo com Ancestrais do Continente Europeu , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Rim/patologia , Masculino , Polimorfismo de Nucleotídeo Único , Fatores Sexuais
18.
Food Funct ; 9(3): 1713-1725, 2018 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-29485160

RESUMO

We conducted the present study to determine the gastric injury preventive effects of polyphenols in Kuding tea (KTPs) in Kunming (KM) mice through the inhibition of gastric-acid secretion and the protection of the gastric mucosa. Mice treated with a high concentration of Kuding tea polyphenols (HKTP) had lower serum levels of interleukin-6 (IL-6), IL-12, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), motilin (MOT), substance P (SP), and endothelin-1 (ET-1), and higher serum levels of somatostatin (SS) and vasoactive intestinal peptide (VIP) than did the mice in the control group. Serum and gastric tissue levels of nitrous oxide (NO), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and GSH were higher in the HKTP-treated mice than in the control mice, but malondialdehyde (MDA) levels were lower in the HKTP-treated mice than in the control mice. The expression of occludin, epidermal growth factor (EGF), EGF receptor (EGFR), vascular endothelial growth factor (VEGF), nuclear factor of κ-light polypeptide gene enhancer in B-cells inhibitor-α (IκBα), Cu/Zn-SOD (cuprozinc-superoxide dismutase), Mn-SOD (manganese-superoxide dismutase), GSH-Px (glutathione peroxidase), neuronal nitric oxide synthase (nNOS), endothelial NOS (eNOS), messenger RNA (mRNA) and protein in gastric tissue was stronger in the HKTP-treated mice than in the control mice, while the expression of p38 mitogen-activated protein kinase (P38MAPK, or p38), nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB), inducible NOS (iNOS), and cyclooxygenase-2 (COX-2) was weaker in the HKTP group than in the control group. And HKTP also could reduce the TNF-α, IL-1ß (interleukin-1 beta), and IL-6 mRNA expression in gastric injury mice. A high performance liquid chromatography (HPLC) assay showed that Kuding tea polyphenols (KTPs) contained chlorogenic acid, cryptochlorogenic acid, and isochlorogenic acids A, B, and C. These constituents contributed to the preventive effects of KTPs on gastric injury. According to these results, KTPs are a kind of active component that have a strong preventive effect on gastric injury.


Assuntos
Camellia sinensis/química , Extratos Vegetais/administração & dosagem , Polifenóis/administração & dosagem , Gastropatias/tratamento farmacológico , Estômago/lesões , Animais , Etanol/efeitos adversos , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/metabolismo , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Humanos , Ácido Clorídrico/efeitos adversos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Malondialdeído/metabolismo , Camundongos , Estômago/efeitos dos fármacos , Gastropatias/induzido quimicamente , Gastropatias/genética , Gastropatias/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Chá/química , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
19.
Nucleic Acids Res ; 46(8): 4256-4270, 2018 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-29471506

RESUMO

The expression of proteins during inflammatory and immune reactions is coordinated by post-transcriptional mechanisms. A particularly strong suppression of protein expression is exerted by a conserved translational silencing element (TSE) identified in the 3' UTR of NFKBIZ mRNA, which is among the targets of the RNA-binding proteins Roquin-1/2 and MCPIP1/Regnase-1. We present evidence that in the context of the TSE MCPIP1, so far known for its endonuclease activity toward mRNAs specified by distinct stem-loop (SL) structures, also suppresses translation. Overexpression of MCPIP1 silenced translation in a TSE-dependent manner and reduced ribosome occupancy of the mRNA. Correspondingly, MCPIP1 depletion alleviated silencing and increased polysomal association of the mRNA. Translationally silenced NFKBIZ or reporter mRNAs were mostly capped, polyadenylated and ribosome associated. Furthermore, MCPIP1 silenced also cap-independent, CrPV-IRES-dependent translation. This suggests that MCPIP1 suppresses a post-initiation step. The TSE is predicted to form five SL structures. SL4 and 5 resemble target structures reported for MCPIP1 and together were sufficient for MCPIP1 binding and mRNA destabilization. Translational silencing, however, required SL1-3 in addition. Thus the NFKBIZ TSE functions as an RNA element in which sequences adjacent to the site of interaction with MCPIP1 and dispensable for accelerated mRNA degradation extend the functional repertoire of MCPIP1 to translational silencing.


Assuntos
Inativação Gênica , Proteínas I-kappa B/genética , Proteínas Nucleares/genética , Biossíntese de Proteínas , Sequências Reguladoras de Ácido Ribonucleico , Ribonucleases/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sítios de Ligação , Células HeLa , Humanos , Elongação Traducional da Cadeia Peptídica , Domínios Proteicos , RNA Mensageiro/metabolismo , Receptor EphB3 , Ribonucleases/química , Ribossomos/metabolismo , Fatores de Transcrição/química
20.
Mol Med Rep ; 17(3): 4376-4382, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29344652

RESUMO

Chrysophanol is an anthraquinone compound, which exhibits anticancer effects on certain types of cancer cells. However, the effects of chrysophanol on human breast cancer remain to be elucidated. The aim of the present study was to clarify the role of chrysophanol on breast cancer cell lines MCF­7 and MDA­MB­231, and to identify the signal transduction pathways regulated by chrysophanol. MTT assay and flow cytometric analysis demonstrated that chrysophanol inhibited cell proliferation, and cell cycle progression in a dose­dependent manner. The expression of cell cycle­associated cyclin D1 and cyclin E were downregulated while p27 expression was upregulated following chrysophanol treatment at the mRNA, and protein levels. The Annexin V/propidium iodide staining assay results revealed that apoptosis levels increased following chrysophanol treatment. Chrysophanol upregulated caspase 3 and poly (ADP­ribose) polymerase cleavage in both cell lines. Furthermore, chrysophanol enhanced the effect of paclitaxel on breast cancer cell apoptosis. In addition, chrysophanol downregulated apoptosis regulator Bcl­2 protein, and transcription factor p65 and IκB phosphorylation. Inhbition of nuclear factor (NF)­κB by ammonium pyrrolidine dithiocarbamate diminished the effect of chrysophanol on apoptosis and associated proteins. In conclusion, the results of the current study demonstrated that chrysophanol effectively suppresses breast cancer cell proliferation and facilitates chemosentivity through modulation of the NF-κB signaling pathway.


Assuntos
Antraquinonas/farmacologia , Antineoplásicos/farmacologia , Ciclina D1/genética , Regulação Neoplásica da Expressão Gênica , Proteínas I-kappa B/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Protocolos de Quimioterapia Combinada Antineoplásica , Apoptose/efeitos dos fármacos , Caspase 3/genética , Caspase 3/metabolismo , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ciclina D1/metabolismo , Ciclina E/genética , Ciclina E/metabolismo , Inibidor de Quinase Dependente de Ciclina p27/genética , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Humanos , Proteínas I-kappa B/metabolismo , Células MCF-7 , Paclitaxel/farmacologia , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo
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