RESUMO
Inflammatory bowel disease (IBD) refers to chronic intestinal immune-mediated diseases including two main disease manifestations: ulcerative colitis (UC) and Crohn's disease (CD). Epidemiological, clinical, and preclinical evidence has highlighted the potential anti-inflammatory properties of naturally occurring alkaloids. In the present study, we investigated the potential anti-inflammatory activities of the tobacco alkaloids nicotine and anatabine in a dextran sulfate sodium (DSS)-induced UC mouse model with a fully humanized immune system. Our results show that nicotine significantly reduced all acute colitis symptoms and improved colitis-specific endpoints, including histopathologically assessed colon inflammation, tissue damage, and mononuclear cell infiltration. The tobacco alkaloid anatabine showed similar effectiveness trends, although they were generally weaker or not significant. Gene expression analysis in the context of biological network models of IBD further pinpointed a possible mechanism by which nicotine attenuated DSS-induced colitis in humanized mice. The current study enables further investigation of possible molecular mechanisms by which tobacco alkaloids attenuate UC symptoms.
Assuntos
Alcaloides , Antineoplásicos , Colite Ulcerativa , Colite , Doenças Inflamatórias Intestinais , Animais , Camundongos , Nicotiana/efeitos adversos , Nicotina/efeitos adversos , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/tratamento farmacológico , Colite Ulcerativa/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Modelos Animais de Doenças , Anti-Inflamatórios/uso terapêutico , Antineoplásicos/uso terapêutico , Alcaloides/farmacologia , Alcaloides/metabolismo , Sistema Imunitário/metabolismo , Sulfato de Dextrana/toxicidade , Camundongos Endogâmicos C57BL , Colo/metabolismoRESUMO
Mucins are gel-forming proteins that are responsible for the characteristic viscoelastic properties of mucus. Mucin overproduction is a hallmark of asthma, but the cellular requirements for airway mucin production are poorly understood. The endoplasmic reticulum (ER) protein anterior gradient homolog 2 (AGR2) is required for production of the intestinal mucin MUC2, but its role in the production of the airway mucins MUC5AC and MUC5B is not established. Microarray data were analyzed to examine the relationship between AGR2 and MUC5AC expression in asthma. Immunofluorescence was used to localize AGR2 in airway cells. Coimmunoprecipitation was used to identify AGR2-immature MUC5AC complexes. Agr2(-/-) mice were used to determine the role of AGR2 in allergic airway disease. AGR2 localized to the ER of MUC5AC- and MUC5B-producing airway cells and formed a complex with immature MUC5AC. AGR2 expression increased together with MUC5AC expression in airway epithelium from "Th2-high" asthmatics. Allergen-challenged Agr2(-/-) mice had greater than 50% reductions in MUC5AC and MUC5B proteins compared with allergen-challenged wild-type mice. Impaired mucin production in Agr2(-/-) mice was accompanied by an increase in the proportion of mucins contained within the ER and by evidence of ER stress in airway epithelium. This study shows that AGR2 increases with mucin overproduction in individuals with asthma and in mouse models of allergic airway disease. AGR2 interacts with immature mucin in the ER and loss of AGR2 impairs allergen-induced MUC5AC and MUC5B overproduction.
Assuntos
Alérgenos/administração & dosagem , Asma/metabolismo , Mucina-5AC/biossíntese , Mucina-5B/biossíntese , Mucoproteínas/biossíntese , Proteínas/metabolismo , Alérgenos/imunologia , Animais , Asma/genética , Asma/imunologia , Hiper-Reatividade Brônquica/genética , Hiper-Reatividade Brônquica/imunologia , Hiper-Reatividade Brônquica/metabolismo , Linhagem Celular Tumoral , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/metabolismo , Expressão Gênica , Humanos , Hipersensibilidade Imediata/genética , Hipersensibilidade Imediata/imunologia , Hipersensibilidade Imediata/metabolismo , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Camundongos , Camundongos Transgênicos , Mucina-5AC/genética , Mucina-5AC/imunologia , Mucina-5AC/metabolismo , Mucina-5B/genética , Mucina-5B/imunologia , Mucina-5B/metabolismo , Mucoproteínas/genética , Mucoproteínas/imunologia , Mucoproteínas/metabolismo , Proteínas Oncogênicas , Proteínas/genética , Proteínas/imunologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismo , Células Th2/imunologia , Células Th2/metabolismoRESUMO
Protein disulfide isomerases (PDIs) aid protein folding and assembly by catalyzing formation and shuffling of cysteine disulfide bonds in the endoplasmic reticulum (ER). Many members of the PDI family are expressed in mammals, but the roles of specific PDIs in vivo are poorly understood. A recent homology-based search for additional PDI family members identified anterior gradient homolog 2 (AGR2), a protein originally presumed to be secreted by intestinal epithelial cells. Here, we show that AGR2 is present within the ER of intestinal secretory epithelial cells and is essential for in vivo production of the intestinal mucin MUC2, a large, cysteine-rich glycoprotein that forms the protective mucus gel lining the intestine. A cysteine residue within the AGR2 thioredoxin-like domain forms mixed disulfide bonds with MUC2, indicating a direct role for AGR2 in mucin processing. Mice lacking AGR2 were viable but were highly susceptible to colitis, indicating a critical role for AGR2 in protection from disease. We conclude that AGR2 is a unique member of the PDI family, with a specialized and nonredundant role in intestinal mucus production.
Assuntos
Dissulfetos/metabolismo , Mucosa Intestinal/metabolismo , Mucoproteínas/metabolismo , Muco/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Doença Aguda , Animais , Linhagem da Célula , Colite/induzido quimicamente , Colite/genética , Colite/metabolismo , Colite/patologia , Retículo Endoplasmático/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Predisposição Genética para Doença , Camundongos , Camundongos Knockout , Mucina-2/metabolismo , Mucoproteínas/deficiência , Mucoproteínas/genética , Proteínas Oncogênicas , Prolapso Retal/genética , Prolapso Retal/metabolismo , Prolapso Retal/patologia , Tiorredoxinas/metabolismoRESUMO
RATIONALE: Increased production of mucus is a prominent feature of asthma. IL-13-driven mucous cell metaplasia is associated with decreased expression of the transcription factor FOXA2 and increased expression of the related transcription factor FOXA3 in animal and cell culture models. OBJECTIVES: Establish how changes in FOXA2 and FOXA3 expression contribute to mucous metaplasia and determine whether FOXA2 and FOXA3 expression is altered in asthma. METHODS: Mice expressing a Foxa2 transgene in airway epithelial cells and mice deficient in Foxa3 were analyzed after allergen sensitization and challenge. Expression of FOXA2, FOXA3, MUC5AC, and the highly IL-13-inducible gene CLCA1 was analyzed in airway biopsies from subjects with asthma and control subjects. MEASUREMENTS AND MAIN RESULTS: Expression of a Foxa2 transgene reduced allergen-induced mucous metaplasia by 45% compared with control transgenic mice (P < 0.05) whereas inactivation of Foxa3 had no detectable effects on mucous metaplasia. Expression of FOXA2 was reduced in subjects with asthma and was negatively correlated with MUC5AC and CLCA1 levels in subjects with asthma. In contrast, FOXA3 expression was not significantly correlated with MUC5AC and was positively correlated with CLCA1. CONCLUSIONS: Increasing Foxa2 expression reduced mucous metaplasia in an allergic mouse model. Subjects with asthma had decreased FOXA2 expression, suggesting that therapeutic approaches that increase FOXA2 expression or function could be beneficial for reducing mucus production in asthma. Unlike FOXA2, FOXA3 did not regulate mucous metaplasia.
Assuntos
Fator 3-beta Nuclear de Hepatócito/metabolismo , Fator 3-gama Nuclear de Hepatócito/metabolismo , Hipersensibilidade Respiratória/genética , Animais , Asma/genética , Asma/imunologia , Asma/patologia , Modelos Animais de Doenças , Células Epiteliais/imunologia , Células Epiteliais/patologia , Expressão Gênica , Fator 3-beta Nuclear de Hepatócito/imunologia , Fator 3-gama Nuclear de Hepatócito/imunologia , Pulmão/citologia , Pulmão/imunologia , Camundongos , Camundongos Transgênicos , Muco/imunologia , Hipersensibilidade Respiratória/imunologia , Hipersensibilidade Respiratória/patologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The 16-kDa N-terminal fragment of human prolactin (16K hPRL) is a potent angiostatic factor that inhibits tumor growth in mouse models. Using microarray experiments, we have dissected how the endothelial-cell genome responds to 16K hPRL treatment. We found 216 genes that show regulation by 16K hPRL, of which a large proportion turned out to be associated with the process of immunity. 16K hPRL induces expression of various chemokines and endothelial adhesion molecules. These expressions, under the control of nuclear factor-kappaB, result in an enhanced leukocyte-endothelial cell interaction. Furthermore, analysis of B16-F10 tumor tissues reveals a higher expression of adhesion molecules (intercellular adhesion molecule 1, vascular cell adhesion molecule 1, or E-selectin) in endothelial cells and a significantly higher number of infiltrated leukocytes within the tumor treated with 16K hPRL compared with the untreated ones. In conclusion, this study describes a new antitumor mechanism of 16K hPRL. Because cellular immunity against tumor cells is a crucial step in therapy, the discovery that treatment with 16K hPRL overcomes tumor-induced anergy may become important for therapeutic perspectives.
Assuntos
Proteínas Angiostáticas/farmacologia , Anergia Clonal/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , NF-kappa B/fisiologia , Fragmentos de Peptídeos/farmacologia , Prolactina/farmacologia , Animais , Adesão Celular , Anergia Clonal/genética , Endotélio Vascular/imunologia , Humanos , Leucócitos/efeitos dos fármacos , Leucócitos/imunologia , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , NF-kappa B/genética , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/patologiaRESUMO
In cystic fibrosis (CF) patients, pulmonary inflammation is a major cause of morbidity and mortality and may precede bacterial colonization. The aim of the present study was to investigate the molecular mechanisms underlying intrinsic inflammation in cystic fibrosis airways. Using different cystic fibrosis cell models, we first demonstrated that, beside a high constitutive nuclear factor of kappaB (NF-kappaB) activity, CF cells showed a higher activator protein-1 (AP-1) activity as compared to their respective control cells. Gene expression profiles, confirmed by RT-PCR and ELISA, showed over-expression of numerous NF-kappaB and AP-1-dependent pro-inflammatory genes in CF cells in comparison with control cells. Activation of NF-kappaB was correlated with higher inhibitor of kappaB kinase (IKK) activity. In addition, Bio-plex phosphoprotein assays revealed higher extracellular signal-regulated kinase (ERK) phosphorylation in CFT-2 cells. Inhibition of this kinase strongly decreased expression of pro-inflammatory genes coding for growth-regulated proteins (Gro-alpha, Gro-beta and Gro-gamma) and interleukins (IL-1beta, IL-6 and IL-8). Moreover, inhibition of secreted interleukin-1beta (IL-1beta) and basic fibroblast growth factor (bFGF) with neutralizing antibodies reduced pro-inflammatory gene expression. Our data thus demonstrated for the first time that the absence of functional cystic fibrosis transmembrane conductance regulator (CFTR) at the plasma membrane leads to an intrinsic AP-1, in addition to NF-kappaB, activity and consequently to a pro-inflammatory state sustained through autocrine factors such as IL-1beta and bFGF.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Quinase I-kappa B/metabolismo , Inflamação , Linhagem Celular Transformada , Quimiocina CXCL1 , Quimiocina CXCL2 , Quimiocinas CXC/metabolismo , Fibrose Cística/patologia , Ensaio de Imunoadsorção Enzimática , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Fator 2 de Crescimento de Fibroblastos/metabolismo , Expressão Gênica , Genes Reporter , Células HeLa , Homozigoto , Humanos , Quinase I-kappa B/antagonistas & inibidores , Inflamação/patologia , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Luciferases/metabolismo , Modelos Biológicos , Mutação , NF-kappa B/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Traqueia/citologia , Traqueia/embriologia , Fator de Transcrição AP-1/metabolismoRESUMO
In cystic fibrosis patients, inflammation is often considered to be secondary to chronic infections. In the present study, we show increased levels of pro-inflammatory proteins in the lungs of a cystic fibrosis foetus compared to the lungs of two normal foetuses. Our findings suggest therefore the existence of an early intrinsic pro-inflammatory state in cystic fibrosis airways.
Assuntos
Bronquite/embriologia , Fibrose Cística/embriologia , Citocinas/metabolismo , Doenças Fetais/metabolismo , NF-kappa B/metabolismo , Biomarcadores/metabolismo , Bronquite/metabolismo , Bronquite/patologia , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Feminino , Doenças Fetais/patologia , Imunofluorescência , Idade Gestacional , Humanos , Imuno-Histoquímica , Pulmão/metabolismo , Pulmão/patologia , Microscopia Confocal , Gravidez , Resultado da GravidezRESUMO
Peroxiredoxin 5 is a mammalian thioredoxin peroxidase ubiquitously expressed in tissues. Peroxiredoxin 5 can be intracellularly localized to mitochondria, peroxisomes, the cytosol, and, to a lesser extent, the nucleus. This remarkably wide subcellular distribution compared with the five other mammalian peroxiredoxins prompted us to further investigate the antioxidant protective function of peroxiredoxin 5 in mammalian cells according to its subcellular localization. Chinese hamster ovary cells overexpressing human peroxiredoxin 5 in the cytosol, in mitochondria, or in the nucleus were established by stable transfection. Cells overexpressing peroxiredoxin 5 were exposed for 1 h to low or acute oxidative stress with exogenously added hydrogen peroxide or tert-butylhydroperoxide. Cell protection conferred by peroxiredoxin 5 was evaluated by clonogenicity and lactate dehydrogenase assays. Overexpressing peroxiredoxin 5 in either the cytosolic, mitochondrial, or nuclear compartment significantly reduced cell death, with more effective protection with overexpression of peroxiredoxin 5 in mitochondria, confirming that this organelle is a major target of peroxides. Moreover, we evaluated, with the comet assay, nuclear DNA damage induced by hydrogen peroxide or tert-butylhydroperoxide. Overexpression of peroxiredoxin 5 in the nucleus significantly decreased DNA damage induced by both peroxides. In conclusion, the present study suggests that multiple subcellular targeting of peroxiredoxin 5 in mammalian cells can be implicated in antioxidant protective mechanisms under nonpathological conditions but also during acute oxidative stress caused by peroxides occurring in pathophysiological situations.
Assuntos
Dano ao DNA/efeitos dos fármacos , Peroxidases/genética , Peroxidases/metabolismo , Peróxidos/farmacologia , Animais , Células CHO , Catalase/metabolismo , Núcleo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Citosol/metabolismo , Expressão Gênica , Vetores Genéticos/genética , Glutationa Peroxidase/metabolismo , Humanos , Peróxido de Hidrogênio/farmacologia , Imuno-Histoquímica , Mitocôndrias/metabolismo , Peroxirredoxinas , terc-Butil Hidroperóxido/farmacologiaRESUMO
In tumor cells, the transcription factor NF-kappaB has been described to be antiapoptotic and proproliferative and involved in the production of angiogenic factors such as vascular endothelial growth factor. From these data, a protumorigenic role of NF-kappaB has emerged. Here, we examined in endothelial cells whether NF-kappaB signaling pathway is involved in mediating the angiostatic properties of angiogenesis inhibitors. The current report describes that biochemically unrelated agents with direct angiostatic effect induced NF-kappaB activation in endothelial cells. Our data showed that endostatin, anginex, angiostatin, and the 16-kDa N-terminal fragment of human prolactin induced NF-kappaB activation in endothelial cells in both cultured human endothelial cells and in vivo in a mouse tumor model. It was also found that NF-kappaB activity was required for the angiostatic activity, because inhibition of NF-kappaB in endothelial cells impaired the ability of angiostatic agents to block sprouting of endothelial cells and to overcome endothelial cell anergy. Therefore, activation of NF-kappaB in endothelial cells can result in an unexpected antitumor outcome. Based on these data, the current approach of systemic treatment with NF-kappaB inhibitors may therefore be revisited because NF-kappaB activation specifically targeted to endothelial cells might represent an efficient strategy for the treatment of cancer.
Assuntos
Inibidores da Angiogênese/farmacologia , Endotélio Vascular/metabolismo , NF-kappa B/metabolismo , Animais , Sequência de Bases , Primers do DNA , Ensaio de Desvio de Mobilidade Eletroforética , Endotélio Vascular/citologia , Citometria de Fluxo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/irrigação sanguínea , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
BACKGROUND: The defect in chloride and sodium transport in cystic fibrosis (CF) patients is a consequence of CF transmembrane conductance regulator (CFTR) loss of function and an abnormal interaction between CFTR and the epithelial sodium channel (ENaC). A few patients were described with CF-like symptoms, a single CFTR mutation, and an ENaC mutation. METHODS: To study African patients with CF-like symptoms and to relate the disease to gene mutations of both CFTR and ENaC genes, we collected clinical data and DNA samples from 60 African patients with a CF phenotype. The CFTR gene was first analyzed in all patients by denaturing high-performance liquid chromatography followed by direct sequencing; whereas, the sodium channel non-voltage-gated 1 alpha (SCNN1A), sodium channel non-voltage-gated 1 beta (SCNN1B), and sodium channel non-voltage-gated 1 gamma (SCNN1G) subunits of the ENaC gene were analyzed by sequencing in the five patients who carried only one CF mutation. The frequency of all identified ENaC variants was established in a control group of 200 healthy individuals and in the 55 CF-like patients without any CFTR mutation. RESULTS: Three CFTR mutants, including one previously undescribed missense mutation (p.A204T), and a 5T/7T variant were identified in five patients. ENaC gene sequencing in these five patients detected the following eight ENaC variants: c.72T>C and p.V573I in SCNN1A; p.V348M, p.G442V, c.1473 + 28C>T, and p.T577T in SCNN1B; and p.S212S and c.1176 + 30G>C in SCNN1G. In the 55 CF-like patients without any CFTR mutation, we identified five of these eight ENaC variants, including the frequent p.G442V polymorphism, but we did not detect the presence of the p.V348M, p.T577T, and c.1176 + 30G>C ENaC variants. Moreover, these last three ENaC variants, p.V348M, p.T577T, and c.1176 + 30G>C, were not found in the control group. CONCLUSION: Our data suggest that CF-like syndrome in Africa could be associated with CFTR and ENaC mutations.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Canais Epiteliais de Sódio/genética , Adolescente , População Negra/genética , Criança , Pré-Escolar , Cromatografia Líquida de Alta Pressão , Feminino , Variação Genética , Humanos , Masculino , Mutação , Fenótipo , Polimorfismo Genético , RuandaRESUMO
Cystic fibrosis is a common genetic disorder characterized by a severe lung inflammation and fibrosis leading to the patient's death. Enhanced angiogenesis in cystic fibrosis (CF) tissue has been suggested, probably caused by the process of inflammation, as similarly described in asthma and chronic bronchitis. The present study demonstrates an intrinsic pro-angiogenic status of cystic fibrosis airway epithelial cells. Microarray experiments showed that CF airway epithelial cells expressed several angiogenic factors such as VEGF-A, VEGF-C, bFGF, and PLGF at higher levels than control cells. These data were confirmed by real-time quantitative PCR and, at the protein level, by ELISA. Conditioned media of these cystic fibrosis cells were able to induce proliferation, migration and sprouting of cultured primary endothelial cells. This report describes for the first time that cystic fibrosis epithelial cells have an intrinsic angiogenic activity. Since excess of angiogenesis is correlated with more severe pulmonary disease, our results could lead to the development of new therapeutic applications.