RESUMO
Green tea is a promising chemopreventive agent for lung cancer. Multiple signaling events have been reported, however, the relative importance of these mechanisms in mediating the chemopreventive function of green tea is unclear. In the present study, to examine the involvement of AP-1 in green tea polyphenols induced tumor inhibition, human NSCLC cell line H1299 and mouse SPON 10 cells were identified as AP-1 dependent, as these two lines exhibit high constitutive AP-1 activity, and when TAM67 expression was induced with doxycycline, cell growth was inhibited and correlated with suppressed AP-1 activity. RNA-seq was used to determine the global transcriptional effects of AP-1 inhibition and also uncover the possible involvement of AP-1 in tea polyphenols induced chemoprevention. TAM67 mediated changes in gene expression were identified, and within down-regulated genes, AP-1 was identified as a key transcription regulator. RNA-seq analysis revealed that Polyphenon E-treated cells shared 293 commonly down-regulated genes within TAM67 expressing H1299 cells, and by analysis of limited Chip-seq data, over 10% of the down-regulated genes contain a direct AP-1 binding site, indicating that Polyphenon E elicits chemopreventive activity by regulating AP-1 target genes. Conditional TAM67 expressing transgenic mice and NSCLC cell lines were used to further confirm that the chemopreventive activity of green tea is AP-1 dependent. Polyphenon E lost its chempreventive function both in vitro and in vivo when AP-1 was inhibited, indicating that AP-1 inhibition is a major pathway through which green tea exhibits chemopreventive effects.
Assuntos
Carcinogênese/efeitos dos fármacos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Transdução de Sinais/efeitos dos fármacos , Chá/química , Fator de Transcrição AP-1/metabolismo , Transcriptoma , Animais , Linhagem Celular Tumoral , Proliferação de Células , Análise por Conglomerados , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes , Humanos , Camundongos , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Regiões Promotoras Genéticas , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fator de Transcrição AP-1/genéticaRESUMO
We previously reported that oral administration of black raspberry powder decreased promoter methylation of tumor suppressor genes in tumors from patients with colorectal cancer. The anthocyanins (ACs) in black raspberries are responsible, at least in part, for their cancer-inhibitory effects. In the present study, we asked if ACs are responsible for the demethylation effects observed in colorectal cancers. Three days of treatment of ACs at 0.5, 5, and 25 µg/ml suppressed activity and protein expression of DNMT1 and DNMT3B in HCT116, Caco2 and SW480 cells. Promoters of CDKN2A, and SFRP2, SFRP5, and WIF1, upstream of Wnt pathway, were demethylated by ACs. mRNA expression of some of these genes was increased. mRNA expression of ß-catenin and c-Myc, downstream of Wnt pathway, and cell proliferation were decreased; apoptosis was increased. ACs were taken up into HCT116 cells and were differentially localized with DNMT1 and DNMT3B in the same cells visualized using confocal laser scanning microscopy. Although it was reported that DNMT3B is regulated by c-Myc in mouse lymphoma, DNMT3B did not bind with c-Myc in HCT116 cells. In conclusion, our results suggest that ACs are responsible, at least in part, for the demethylation effects of whole black raspberries in colorectal cancers.
Assuntos
Antocianinas/farmacologia , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Genes Supressores de Tumor/efeitos dos fármacos , Rosaceae/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Células CACO-2/efeitos dos fármacos , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Inibidores Enzimáticos/farmacologia , Proteínas do Olho/genética , Genes p16 , Humanos , Proteínas de Membrana/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Proteínas Repressoras/genética , DNA Metiltransferase 3BRESUMO
Multiple studies have shown a link between chronic inflammation and lung tumorigenesis. Inbred mouse strains vary in their susceptibility to methylcholanthrene (MCA)-initiated butylated hydroxytoluene (BHT)-promoted lung carcinogenesis. In the present study we investigated whether neutrophils play a role in strain dependent differences in susceptibility to lung tumor promotion. We observed a significant elevation in homeostatic levels of neutrophils in the lungs of tumor-susceptible BALB/cByJ (BALB) mice compared to tumor-resistant C57BL/6J (B6) mice. Additionally, BHT treatment further elevated neutrophil numbers as well as neutrophil chemoattractant keratinocyte-derived cytokine (KC)/chemokine (C-X-C motif) ligand 1 (Cxcl1) levels in BALB lung airways. Lung CD11c+ cells were a major source of KC expression and depletion of neutrophils in BALB mice resulted in a 71% decrease in tumor multiplicity. However, tumor multiplicity did not depend on the presence of T cells, despite the accumulation of T cells following BHT treatment. These data demonstrate that neutrophils are essential to promote tumor growth in the MCA/BHT two-step lung carcinogenesis model.
Assuntos
Hidroxitolueno Butilado/toxicidade , Carcinógenos/toxicidade , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/imunologia , Metilcolantreno/toxicidade , Neutrófilos/imunologia , Animais , Transformação Celular Neoplásica , Feminino , Citometria de Fluxo , Memória Imunológica , Imunofenotipagem , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T/imunologiaRESUMO
The etiology of acute lung injury is complex and associated with numerous, chemically diverse precipitating factors. During acute lung injury in mice, one key event is epithelial cell injury that leads to reduced surfactant biosynthesis. We have previously reported that transgenic mice that express transforming growth factor alpha (TGFA) in the lung were protected during nickel-induced lung injury. Here, we find that the mechanism by which TGFA imparts protection includes maintenance of surfactant-associated protein B (SFTPB) transcript levels and epidermal growth factor receptor-dependent signaling in distal pulmonary epithelial cells. This protection is complex and not accompanied by a diminution in inflammatory mediator transcripts or additional stimulation of antioxidant transcripts. In mouse lung epithelial (MLE-15) cells, microarray analysis demonstrated that nickel increased transcripts of genes enriched in MTF1, E2F-1, and AP-2 transcription factor-binding sites and decreased transcripts of genes enriched in AP-1-binding sites. Nickel also increased Jun transcript and DNA-binding activity, but decreased SFTPB transcript. Expression of SFTPB under the control of a doxycycline-sensitive promoter increased survival during nickel-induced injury as compared with control mice. Together, these findings support the idea that maintenance of SFTPB expression is critical to survival during acute lung injury.
Assuntos
Lesão Pulmonar Aguda/induzido quimicamente , Níquel/toxicidade , Proteína B Associada a Surfactante Pulmonar/metabolismo , Administração por Inalação , Aerossóis , Animais , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Proteína B Associada a Surfactante Pulmonar/genética , Mucosa Respiratória/citologia , Taxa de Sobrevida , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Fator de Crescimento Transformador alfa/genética , Fator de Crescimento Transformador alfa/metabolismoRESUMO
Chemoprevention strategies to prevent the development of lung cancer in at-risk individuals are a key component in disease management. In addition to being highly effective, an ideal chemopreventive agent will require low toxicity as patients are likely to require treatment for several years before their risk of cancer is lowered to background levels. In principle, a combination of safe agents that work through distinct mechanisms will improve efficacy while simultaneously maintaining a favorable safety profile. Here, we describe the use of the decaffeinated green tea extract Polyphenon E (Poly E) (1% in diet) and aerosolized difluoromethylornithine (DFMO) (20 mg/kg/day, 5 days/week) in a mouse lung cancer chemoprevention study using a progression protocol. Female A/J mice were injected with benzo[a]pyrene (B[a]P) at 8 weeks of age and precancerous lesions allowed to form over a period of 21 weeks before chemoprevention treatment for an additional 25 weeks. Poly E treatment did not significantly inhibit average tumor multiplicity but reduced per animal tumor load. Analysis of tumor pathology revealed a specific inhibition of carcinomas, with the largest carcinomas significantly decreased in Poly E-treated animals. Aerosolized DFMO did not have a significant effect on lung tumor progression. Magnetic resonance imaging of B[a]P-induced lung tumors confirmed the presence of a subset of large, rapidly growing tumors in untreated mice. Our results suggest a potential role for green tea extracts in preventing the progression of large, aggressive lung adenocarcinomas.
Assuntos
Eflornitina/farmacologia , Neoplasias Pulmonares/patologia , Extratos Vegetais/farmacologia , Chá , Aerossóis , Animais , Antineoplásicos/farmacologia , Quimioprevenção , Progressão da Doença , Eflornitina/administração & dosagem , Feminino , Humanos , Neoplasias Pulmonares/epidemiologia , Masculino , Camundongos , Estados Unidos/epidemiologiaRESUMO
PURPOSE: Available evidence suggests that fibroblast growth factor 7 (FGF7, also known as keratinocyte growth factor, KGF) serves as a paracrine growth factor modulating corneal epithelial cell proliferation. In the present study, we used a binary inducible transgenic mouse model to examine the role of FGF7 on corneal epithelium proliferation. METHODS: A keratocyte specific 3.2 kb murine keratocan promoter (Kerapr) was used to prepare Kerapr-rtTA transgenic (Kr) mice that constitutively overexpress reverse tetracycline transcription activator (rtTA) by cornea stromal keratocytes. The Kr mice were crossed with tet-O-FGF7 mice to produce Kr/tet-O-FGF7 bitransgenic mice. Expression of human FGF7 (hFGF7) was induced by the administration of doxycycline via intraperitoneal injection and/or feeding mice doxycycline in drinking water and chow. Overexpression of hFGF7 was confirmed by RT-PCR and western blot. BrdU incorporation was used to determine cell proliferation. RESULTS: The rtTA mRNA and protein were constitutively expressed by the cornea with or without doxycycline induction, whereas hFGF7 was detected only in Kr/tet-O-FGF7 bitransgenic mice upon induction by doxycycline. Examination of induction kinetics in adult Kr/tet-O-FGF7 bitransgenic mice after a single intraperitoneal injection of doxycycline revealed that hFGF7 mRNA expression was detected 12 h after doxycycline administration, peaked at 36 h, was sustained up to 48 h, and declined thereafter. The elevated level of hFGF7 expression coincided with hyperproliferation of corneal epithelial cells. In bitransgenic mice, the number of BrdU labeled cells increased after 36 and 48 h of transgene induction compared to controls of noninduced bitransgenic or doxycycline treated single transgenic mice. The BrdU labeling index was 33+/-9.2 positive cells per corneal section for Kr/tet-O-FGF7 bitransgenic mice and 25+/-9.3 for tet-O-FGF7 single transgenic mice at 36 h post-doxycycline treatment. However, the excess FGF7 driven by doxycycline induction did not produce severe perturbation of corneal epithelium homeostasis. CONCLUSIONS: Our results demonstrate that the doxycycline inducible system is effective in regulating transgene expression in corneal stroma of Kr/tet-O-FGF7 bitransgenic mice. However, the development of pathology resulting from the overexpression of transgenes may depend on whether the amount of transgene product present is sufficient to alter the homeostasis of the targeted tissues.
Assuntos
Proliferação de Células , Epitélio Corneano/citologia , Epitélio Corneano/metabolismo , Fator 7 de Crescimento de Fibroblastos/genética , Expressão Gênica , Proteoglicanas/genética , Animais , Western Blotting , Replicação do DNA , Doxiciclina/farmacologia , Feminino , Fator 7 de Crescimento de Fibroblastos/metabolismo , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Plasmídeos , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tetraciclina/biossíntese , Ativação Transcricional , CicatrizaçãoRESUMO
BACKGROUND: The development of non-small cell lung carcinoma proceeds through a series of well-defined pathological steps before the appearance of invasive lung carcinoma. The molecular changes that correspond with pathology changes are not well defined and identification of the molecular events may provide clues on the progression of intraepithelial neoplasia in the lung, as well as suggest potential targets for chemoprevention. The acquisition of anti-apoptotic signals is critical for the survival of cancer cells but the pathways involved are incompletely characterized in developing intra-epithelial neoplasia (IEN). METHODS: We used immunohistochemistry to determine the presence, relative levels, and localization of proteins that mediate anti-apoptotic pathways in developing human bronchial neoplasia. RESULTS: Bronchial epithelial protein levels of the phosphorylated (active) form of AKT kinase and the caspase inhibitor cIAP-2 were increased in more advanced grades of bronchial IEN lesions than in normal bronchial epithelium. Additionally, the percentage of biopsies with nuclear localization of p65/RELA in epithelial cells increased with advancing pathology grade, suggesting that NF-kappaB transcriptional activity was induced more frequently in advanced IEN lesions. CONCLUSION: Our results indicate that anti-apoptotic pathways are elevated in bronchial IEN lesions prior to the onset of invasive carcinoma and that targeting these pathways therapeutically may offer promise in prevention of non-small cell lung carcinoma.
Assuntos
Apoptose , Carcinoma in Situ/genética , Proteínas Inibidoras de Apoptose/análise , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/fisiopatologia , Proteína Oncogênica v-akt/análise , Fator de Transcrição RelA/análise , Biópsia , Carcinoma in Situ/fisiopatologia , Carcinoma Pulmonar de Células não Pequenas , Sobrevivência Celular , Humanos , Imuno-Histoquímica , Proteínas Inibidoras de Apoptose/biossíntese , Invasividade Neoplásica , Proteína Oncogênica v-akt/biossíntese , Transdução de Sinais , Fator de Transcrição RelA/biossínteseRESUMO
Primary pulmonary malignancies remain the major source of cancer-related deaths in the Western World. While surgical resection is an efficacious therapy for those with early stage disease, the majority of patients present with advanced malignancies and systemic treatments, such as cytotoxic chemotherapy, have only limited efficacy in lung cancer. Furthermore, chemoprevention for current or former smokers has demonstrated only limited success using available agents. The mouse model of primary lung carcinogenesis represents a very valuable tool for the study of tumor initiation, promotion, and therapy. Here we discuss several models of chemically-induced murine lung cancer with a specific emphasis on translational and clinically-relevant lines of investigation. We emphasize the pros and cons of currently available models in order to facilitate further investigations into the development and treatment of primary pulmonary malignancies.
Assuntos
Modelos Animais de Doenças , Neoplasias Pulmonares/induzido quimicamente , Animais , Carcinogênese , Carcinógenos/toxicidade , Humanos , CamundongosRESUMO
Alveolar type II epithelial or other pulmonary cells secrete GM-CSF that regulates surfactant catabolism and mucosal host defense through its capacity to modulate the maturation and activation of alveolar macrophages. GM-CSF enhances expression of scavenger receptors MARCO and SR-A. The alveolar macrophage SP-R210 receptor binds the surfactant collectin SP-A mediating clearance of respiratory pathogens. The current study determined the effects of epithelial-derived GM-CSF in host resistance to influenza A pneumonia. The results demonstrate that GM-CSF enhanced resistance to infection with 1.9×10(4) ffc of the mouse-adapted influenza A/Puerto Rico/8/34 (PR8) H1N1 strain, as indicated by significant differences in mortality and mean survival of GM-CSF-deficient (GM(-/-)) mice compared to GM(-/-) mice in which GM-CSF is expressed at increased levels. Protective effects of GM-CSF were observed both in mice with constitutive and inducible GM-CSF expression under the control of the pulmonary-specific SFTPC or SCGB1A1 promoters, respectively. Mice that continuously secrete high levels of GM-CSF developed desquamative interstitial pneumonia that impaired long-term recovery from influenza. Conditional expression of optimal GM-CSF levels at the time of infection, however, resulted in alveolar macrophage proliferation and focal lymphocytic inflammation of distal airways. GM-CSF enhanced alveolar macrophage activity as indicated by increased expression of SP-R210 and CD11c. Infection of mice lacking the GM-CSF-regulated SR-A and MARCO receptors revealed that MARCO decreases resistance to influenza in association with increased levels of SP-R210 in MARCO(-/-) alveolar macrophages. In conclusion, GM-CSF enhances early host resistance to influenza. Targeting of MARCO may reinforce GM-CSF-mediated host defense against pathogenic influenza.
Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H1N1/patogenicidade , Infecções por Orthomyxoviridae/prevenção & controle , Animais , Feminino , Pulmão/imunologia , Pulmão/patologia , Linfócitos/imunologia , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Orthomyxoviridae/mortalidade , Infecções por Orthomyxoviridae/patologia , Análise de SobrevidaRESUMO
Lung cancer is the leading cause of cancer mortality in the United States and worldwide. The identification of key regulatory and molecular mechanisms involved in lung tumorigenesis is therefore critical to increase our understanding of this disease and could ultimately lead to targeted therapies to improve prevention and treatment. Induction of members of the activator protein-1 (AP-1) transcription factor family has been described in human non-small cell lung carcinoma. Activation of AP-1 can either stimulate or repress transcription of multiple gene targets, ultimately leading to increased cell proliferation and inhibition of apoptosis. In the present study, we show induction of AP-1 in carcinogen-induced mouse lung tumors compared with surrounding normal lung tissue. We then used a transgenic mouse model directing conditional expression of the dominant-negative c-jun mutant TAM67 in lung epithelial cells to determine the effect of AP-1 inhibition on mouse lung tumorigenesis. Consistent with low AP-1 activity in normal lung tissue, TAM67 expression had no observed effects in adult mouse lung. TAM67 decreased tumor number and overall lung tumor burden in chemically induced mouse lung tumor models. The most significant inhibitory effect was observed on carcinoma burden compared with lower-grade lesions. Our results support the concept that AP-1 is a key regulator of mouse lung tumorigenesis, and identify AP-1-dependent transcription as a potential target to prevent lung tumor progression.
Assuntos
Carcinoma/genética , Transformação Celular Neoplásica/genética , Genes jun , Neoplasias Pulmonares/genética , Proteínas Mutantes/fisiologia , Animais , Benzo(a)pireno , Carcinógenos , Carcinoma/induzido quimicamente , Carcinoma/patologia , Citoproteção/efeitos dos fármacos , Citoproteção/genética , Regulação para Baixo/fisiologia , Regulação Neoplásica da Expressão Gênica , Genes Dominantes/fisiologia , Genes Supressores de Tumor/fisiologia , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Transgênicos , Proteínas Mutantes/genética , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/fisiologia , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/fisiologia , Fator de Transcrição AP-1/genéticaAssuntos
Anticarcinógenos/uso terapêutico , Catequina/análogos & derivados , Neoplasias Pulmonares/metabolismo , NF-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Budesonida/uso terapêutico , Catequina/uso terapêutico , Linhagem Celular Tumoral , Eflornitina/uso terapêutico , Humanos , Neoplasias Pulmonares/prevenção & controle , Proteínas Proto-Oncogênicas c-aktRESUMO
Chronic obstructive pulmonary disease (COPD) is a lethal progressive lung disease culminating in permanent airway obstruction and alveolar enlargement. Previous studies suggest CTL involvement in COPD progression; however, their precise role remains unknown. Here, we investigated whether the CTL activation receptor NK cell group 2D (NKG2D) contributes to the development of COPD. Using primary murine lung epithelium isolated from mice chronically exposed to cigarette smoke and cultured epithelial cells exposed to cigarette smoke extract in vitro, we demonstrated induced expression of the NKG2D ligand retinoic acid early transcript 1 (RAET1) as well as NKG2D-mediated cytotoxicity. Furthermore, a genetic model of inducible RAET1 expression on mouse pulmonary epithelial cells yielded a severe emphysematous phenotype characterized by epithelial apoptosis and increased CTL activation, which was reversed by blocking NKG2D activation. We also assessed whether NKG2D ligand expression corresponded with pulmonary disease in human patients by staining airway and peripheral lung tissues from never smokers, smokers with normal lung function, and current and former smokers with COPD. NKG2D ligand expression was independent of NKG2D receptor expression in COPD patients, demonstrating that ligand expression is the limiting factor in CTL activation. These results demonstrate that aberrant, persistent NKG2D ligand expression in the pulmonary epithelium contributes to the development of COPD pathologies.
Assuntos
Pulmão/fisiopatologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Mucosa Respiratória/fisiopatologia , Fumaça/efeitos adversos , Fumar/efeitos adversos , Animais , Linfócitos T CD8-Positivos/imunologia , Modelos Animais de Doenças , Enfisema/etiologia , Enfisema/imunologia , Regulação da Expressão Gênica , Células Matadoras Naturais/imunologia , Ativação Linfocitária , Proteínas de Membrana/genética , Camundongos , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Doença Pulmonar Obstrutiva Crônica/etiologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologiaRESUMO
Acrolein exposure represents a significant human health hazard. Repeated acrolein exposure causes the accumulation of monocytes/macrophages and lymphocytes, mucous cell metaplasia, and epithelial injury. Currently, the mechanisms that control these events are unclear, and the relative contribution of T-cell subsets to pulmonary pathologies following repeated exposures to irritants is unknown. To examine whether lymphocyte subpopulations regulate inflammation and epithelial cell pathology, we utilized a mouse model of pulmonary pathology induced by repeated acrolein exposures. The role of lymphocyte subsets was examined by utilizing transgenic mice genetically deficient in either alphabeta T cells or gammadelta T cells, and changes in cellular, molecular, and pathologic outcomes associated with repeated inhalation exposure to 2.0 and 0.5 ppm acrolein were measured. To examine the potential functions of lymphocyte subsets, we purified these cells from the lungs of mice repeatedly exposed to 2.0 ppm acrolein, isolated and amplified messenger RNA, and performed microarray analysis. Our data demonstrate that alphabeta T cells are required for macrophage accumulation, whereas gammadelta T cells are critical regulators of epithelial cell homeostasis, as identified by epithelial cell injury and apoptosis, following repeated acrolein exposure. This is supported by microarray analyses that indicated the T-cell subsets are unique in their gene expression profiles following acrolein exposures. Microarray analyses identified several genes that may contribute to phenotypes mediated by T-cell subpopulations including those involved in cytokine receptor signaling, chemotaxis, growth factor production, lymphocyte activation, and apoptosis. These data provide strong evidence that T-cell subpopulations in the lung are major determinants of pulmonary pathology and highlight the advantages of dissecting their effector functions in response to toxicant exposures.
Assuntos
Acroleína/toxicidade , Pulmão/efeitos dos fármacos , Receptores de Antígenos de Linfócitos T alfa-beta/fisiologia , Receptores de Antígenos de Linfócitos T gama-delta/fisiologia , Linfócitos T/fisiologia , Animais , Apoptose/efeitos dos fármacos , Separação Celular , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Perfilação da Expressão Gênica , Interleucina-18/fisiologia , Pulmão/imunologia , Pulmão/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Mucina-5AC , Mucinas/análise , Análise de Sequência com Séries de OligonucleotídeosRESUMO
Fibroblast growth factor-7 (FGF7) is a lung epithelial cell mitogen that is cytoprotective during injury. Transgenic mice that conditionally expressed FGF7 were used to dissect the mechanisms of FGF7 protection during lung injury. FGF7 improved survival when induced 3 days prior to acute lung injury. In contrast, FGF7 caused pulmonary inflammation and lung injury after 7 days or longer. Gene expression analysis of mouse lung mRNA identified mRNAs that contribute to the protective effects of FGF7. FGF7 improved survival during acute lung injury in adult mouse lung after short-term expression, but paradoxically induced inflammation and injury after persistent expression.
Assuntos
Citoproteção , Fator 7 de Crescimento de Fibroblastos/fisiologia , Inflamação/etiologia , Pulmão/citologia , Animais , Células Epiteliais/fisiologia , Fator 7 de Crescimento de Fibroblastos/análise , Perfilação da Expressão Gênica , Camundongos , Camundongos Transgênicos , Níquel/toxicidade , Proteínas/genética , RNA Mensageiro/análise , Síndrome do Desconforto Respiratório/prevenção & controle , Fatores de TempoRESUMO
BACKGROUND: Persistent macrophage accumulation and alveolar enlargement are hallmark features of chronic obstructive pulmonary disease (COPD). A role for CD8(+) lymphocytes in the development of COPD is suggested based on observations that this T cell subset is increased in the airways and parenchyma of smokers that develop COPD with airflow limitation. In this study, we utilize a mouse model of COPD to examine the contributions of CD8(+) T cells in the persistent macrophage accumulation and airspace enlargement resulting from chronic irritant exposure. METHODS: We analyzed pulmonary inflammation and alveolar destruction in wild-type and Cd8-deficient mice chronically exposed to acrolein, a potent respiratory tract irritant. We further examined cytokine mRNA expression levels by RNase protection assay, matrix metalloproteinase (MMP) activity by gelatin zymography, and epithelial cell apoptosis by active caspase3 immunohistochemistry in wild-type and Cd8-deficient mice exposed chronically to acrolein. RESULTS: These studies demonstrate that CD8(+) T cells are important mediators of macrophage accumulation in the lung and the progressive airspace enlargement in response to chronic acrolein exposures. The expression of several inflammatory cytokines (IP-10, IFN-gamma, IL-12, RANTES, and MCP-1), MMP2 and MMP9 gelatinase activity, and caspase3 immunoreactivity in pulmonary epithelial cells were attenuated in the Cd8-deficient mice compared to wild-type. CONCLUSIONS: These results indicate that CD8(+) T cells actively contribute to macrophage accumulation and the development of irritant-induced airspace enlargement.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Irritantes , Pulmão , Macrófagos/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Acroleína/imunologia , Acroleína/toxicidade , Animais , Líquido da Lavagem Broncoalveolar/citologia , Antígenos CD8/genética , Antígenos CD8/imunologia , Citocinas/genética , Citocinas/imunologia , Humanos , Irritantes/imunologia , Irritantes/toxicidade , Pulmão/anatomia & histologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fumar , Subpopulações de Linfócitos T/imunologiaAssuntos
Variação Genética , Estudo de Associação Genômica Ampla , Inositol/genética , Catecóis , Expressão Gênica , Predisposição Genética para Doença , Modelos Biológicos , Polimorfismo de Nucleotídeo Único , Locos de Características Quantitativas , Sequências Reguladoras de Ácido Nucleico , RiscoRESUMO
The production of the inflammatory cytokine interleukin (IL)-1 is increased in lungs of patients with chronic obstructive pulmonary disease (COPD) or asthma. To characterize the in vivo actions of IL-1 in the lung, transgenic mice were generated in which human IL-1beta was expressed in the lung epithelium with a doxycycline-inducible system controlled by the rat Clara cell secretory protein (CCSP) promoter. Induction of IL-1beta expression in the lungs of adult mice caused pulmonary inflammation characterized by neutrophil and macrophage infiltrates. IL-1beta caused distal airspace enlargement, consistent with emphysema. IL-1beta caused disruption of elastin fibers in alveolar septa and fibrosis in airway walls and in the pleura. IL-1beta increased the thickness of conducting airways, enhanced mucin production, and caused lymphocytic aggregates in the airways. Decreased immunostaining for the winged helix transcription factor FOXA2 was associated with goblet cell hyperplasia in IL-1beta-expressing mice. The production of the neutrophil attractant CXC chemokines KC (CXCL1) and MIP-2 (CXCL2), and of matrix metalloproteases MMP-9 and MMP-12, was increased by IL-1beta. Chronic production of IL-1beta in respiratory epithelial cells of adult mice causes lung inflammation, enlargement of distal airspaces, mucus metaplasia, and airway fibrosis in the adult mouse.
Assuntos
Interleucina-1/fisiologia , Doença Pulmonar Obstrutiva Crônica/etiologia , Enfisema Pulmonar/etiologia , Animais , Asma/etiologia , Asma/imunologia , Asma/patologia , Sequência de Bases , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Quimiocinas CXC/biossíntese , Colágeno/metabolismo , DNA Complementar/genética , Modelos Animais de Doenças , Elastina/metabolismo , Expressão Gênica , Humanos , Inflamação/etiologia , Inflamação/imunologia , Inflamação/patologia , Interleucina-1/genética , Linfócitos/patologia , Metaloproteinases da Matriz/biossíntese , Camundongos , Camundongos Transgênicos , Tamanho do Órgão , Especificidade de Órgãos , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/patologia , Enfisema Pulmonar/imunologia , Enfisema Pulmonar/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sistema Respiratório/imunologia , Sistema Respiratório/patologia , Fator de Necrose Tumoral alfa/fisiologiaRESUMO
To determine the effects of expression of mutant Ki-ras on lung tumorigenesis, we developed a bitransgenic mouse model that expresses the human Ki-ras(G12C) allele in alveolar type II and/or Clara cells in a tetracycline-inducible, lung-specific manner. Expression of Ki-ras(G12C) caused multiple, small lung tumors over a 12-month time period. Although tumor multiplicity increased upon continued Ki-ras expression, most lung lesions were hyperplasias or well-differentiated adenomas. This is in contrast to the more severe phenotypes observed in other transgenic mouse models in which different mutant Ki-ras alleles were expressed in the lung. Expression of Ki-ras(G12C) was associated with a 2-fold increase in the activation of the Ras and Ral signaling pathways and increased phosphorylation of Ras downstream effectors, including Erk, p90 ribosomal S6 kinase, ribosomal S6 protein, p38 and MAPKAPK-2. In contrast, expression of the transgene had no effect on the activation of the JNK and Akt signaling pathways. Withdrawal of doxycycline for 1 month resulted in almost a complete absence of proliferative pulmonary lesions, suggesting tumor regression in the absence of Ki-ras expression. Mutant Ki-ras(G12C) expression was sufficient for initial lung tumor transformation, required for maintenance of tumor phenotype, and induced transformation of lung epithelial cells by the activation of multiple effector pathways. These results describe a novel mouse lung tumor model demonstrating benign tumor development in the absence of tumor progression, which will provide a new tool for understanding the early stages of lung tumor pathogenesis.
Assuntos
Transformação Celular Neoplásica , Genes ras/fisiologia , Neoplasias Pulmonares/etiologia , Mutação/genética , Transdução de Sinais , Adenoma/etiologia , Adenoma/metabolismo , Adenoma/patologia , Alelos , Animais , Brônquios , Doxiciclina/farmacologia , Genes ras/genética , Humanos , Hiperplasia/etiologia , Hiperplasia/metabolismo , Hiperplasia/patologia , Peptídeos e Proteínas de Sinalização Intracelular , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Transgênicos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Alvéolos Pulmonares/citologia , Alvéolos Pulmonares/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Células Tumorais Cultivadas , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Proteínas ral de Ligação ao GTPRESUMO
RATIONALE: Acute lung injury can occur from multiple causes, resulting in high mortality. The pathophysiology of nickel-induced acute lung injury in mice is remarkably complex, and the molecular mechanisms are uncertain. OBJECTIVES: To integrate molecular pathways and investigate the role of transforming growth factor beta (TGF-beta) in acute lung injury in mice. METHODS: cDNA microarray analyses were used to identify lung gene expression changes after nickel exposure. MAPPFinder analysis of the microarray data was used to determine significantly altered molecular pathways. TGF-beta1 protein in bronchoalveolar lavage fluid, as well as the effect of inhibition of TGF-beta, was assessed in nickel-exposed mice. The effect of TGF-beta on surfactant-associated protein B (Sftpb) promoter activity was measured in mouse lung epithelial cells. MEASUREMENTS AND MAIN RESULTS: Genes that decreased the most after nickel exposure play important roles in lung fluid absorption or surfactant and phospholipid synthesis, and genes that increased the most were involved in TGF-beta signaling. MAPPFinder analysis further established TGF-beta signaling to be significantly altered. TGF-beta-inducible genes involved in the regulation of extracellular matrix function and fibrinolysis were significantly increased after nickel exposure, and TGF-beta1 protein was also increased in the lavage fluid. Pharmacologic inhibition of TGF-beta attenuated nickel-induced protein in bronchoalveolar lavage. In addition, treatment with TGF-beta1 dose-dependently repressed Sftpb promoter activity in vitro, and a novel TGF-beta-responsive region in the Sftpb promoter was identified. CONCLUSIONS: These data suggest that TGF-beta acts as a central mediator of acute lung injury through the alteration of several different molecular pathways.
Assuntos
Expressão Gênica , Proteína B Associada a Surfactante Pulmonar/genética , RNA/genética , Síndrome do Desconforto Respiratório/genética , Fator de Crescimento Transformador beta/metabolismo , Animais , Líquido da Lavagem Broncoalveolar/química , Células Cultivadas , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Feminino , Fibrinólise/efeitos dos fármacos , Fibrinólise/genética , Camundongos , Níquel/toxicidade , Análise de Sequência com Séries de Oligonucleotídeos , Proteína B Associada a Surfactante Pulmonar/metabolismo , Síndrome do Desconforto Respiratório/tratamento farmacológico , Síndrome do Desconforto Respiratório/metabolismo , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Mucosa Respiratória/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/uso terapêutico , Fator de Crescimento Transformador beta1RESUMO
Transgenic mouse models mediating conditional temporal and spatial regulation of gene expression to the respiratory epithelium were developed utilizing the reverse tetracycline transactivator (rtTA) expressed under the control of SP-C and CCSP promoters. Luciferase activity was detected in the lungs of fetal and adult double transgenic mice but was not detected in other tissues or in single transgenic mice. In adult mice, maximal luciferase activity was detected 16 h after the administration of doxycycline in the drinking water, or 2 h after the injection of doxycycline. Activation of the transgene was observed after the administration of doxycycline in food pellets. After prolonged exposure to doxycycline, luciferase activity decreased slowly following removal of doxycycline, suggesting the importance of tissue pools which maintained expression of the transgene. In SP-C-rtTA mice, exposure of the pregnant dam to doxycycline induced luciferase activity in fetal lung tissue as early as E10.5. Luciferase activity was maintained in the lung tissue of pups during the period of lactation when the mother received doxycycline in the drinking water. In the CCSP-rtTA mice, luciferase was not detected in the absence of doxycycline. In the SP-C-rtTA mice, luciferase activity was detected in the absence of doxycycline but was enhanced approximately 10-fold by administration of drugs. The SP-C-rtTA and CCSP-rtTA activator mice control the expression of transgenes in the developing and mature respiratory epithelium, and will be useful for the study of gene function in the lung.