RESUMO
Nicotine receptors are present in the developing lung yet their function is unknown. The transient role of nicotine receptors in lung development has not been addressed. In this study, nicotine's direct effect on smooth muscle contraction, necessary for mechanosensory-dependent fetal lung development, is examined after transient nicotine stimulation to determine the relationship between nicotine exposure, smooth muscle contraction, and fetal lung development. Rat fetuses at 16 days' gestation were exposed in utero to 5 different concentrations of nicotine or control injected directly into the amniotic fluid. Specific concentrations of in utero nicotine increased the phosphorylated Western blot analysis and immunohistochemistry of muscle contraction proteins. Respiratory function tests on nicotine-exposed rat pups showed a statistically significant decrease in airway resistance earlier in life compared to control and an upward shift of the pressure-volume curve pointing towards a structural maturation of the in utero nicotine-exposed lung. These results are consistent with transient nicotine exposure during intrauterine life stimulating stretch-induced lung organogenesis by altering phosphorylation of muscle contraction proteins. The increase in smooth muscle phosphorylation may stimulate stretch-induced lung organogenesis, which affects lung development and accelerates lung maturation in rats.
Assuntos
Desenvolvimento Fetal/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Nicotina/toxicidade , Agonistas Nicotínicos/toxicidade , Receptores Pulmonares de Alongamento/efeitos dos fármacos , Resistência das Vias Respiratórias/efeitos dos fármacos , Resistência das Vias Respiratórias/fisiologia , Animais , Animais Recém-Nascidos , Feminino , Desenvolvimento Fetal/fisiologia , Pulmão/embriologia , Pulmão/crescimento & desenvolvimento , Exposição Materna , Mecanotransdução Celular/efeitos dos fármacos , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiopatologia , Cadeias Leves de Miosina/metabolismo , Gravidez , Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Testes de Função Respiratória , Fatores de TempoRESUMO
One of the mechanisms by which adult disease can arise from a fetal origin is by in utero disruption of organogenesis. These studies were designed to examine respiratory function changes in aging rats following transient disruption of lung growth at 16 days gestation. Fetuses were treated in utero with a replication deficient adenovirus containing the cystic fibrosis conductance transmembrane regulator (CFTR) gene fragment cloned in the anti-sense direction. The in utero-treated rats demonstrated abnormal lung function beginning as early as 30 days of age and the pathology progressed as the animals aged. The pulmonary function abnormalities included decreased static compliance as well as increased conducting airway resistance, tissue damping, and elastance. Pressure volume (PV) curves demonstrated a slower early rise to volume and air trapping at end-expiration. The alterations of pulmonary function correlated with lung structural changes determined by morphometric analysis. These studies demonstrate how transient disruption of lung organogensis by single gene interference can result in progressive change in lung function and structure. They illustrate how an adult onset disease can arise from subtle changes in gene expression during fetal development.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Feto/fisiologia , Pneumopatias/fisiopatologia , Pulmão/embriologia , Adulto , Idade de Início , Animais , Diferenciação Celular , Clonagem Molecular , DNA Antissenso/genética , Feminino , Técnicas de Transferência de Genes , Humanos , Pulmão/citologia , Pneumopatias/embriologia , Fragmentos de Peptídeos/genética , Gravidez , RNA Antissenso/genética , Ratos , Reflexo de Estiramento , Testes de Função RespiratóriaRESUMO
BACKGROUND: Mechanicosensory mechanisms regulate cell differentiation during lung organogenesis. We have previously demonstrated that cystic fibrosis transmembrane conductance regulator (CFTR) was integral to stretch-induced growth and development and that transient expression of antisense-CFTR (ASCFTR) had negative effects on lung structure and function. In this study, we examined adult alveolar type II (ATII) cell phenotype after transient knock down of CFTR by adenovirus-directed in utero expression of ASCFTR in the fetal lung. RESULTS: In comparison to (reporter gene-treated) Controls, ASCFTR-treated adult rat lungs showed elevated phosphatidylcholine (PC) levels in the large but not in the small aggregates of alveolar surfactant. The lung mRNA levels for SP-A and SP-B were lower in the ASCFTR rats. The basal PC secretion in ATII cells was similar in the two groups. However, compared to Control ATII cells, the cells in ASCFTR group showed higher PC secretion with ATP or phorbol myristate acetate. The cell PC pool was also larger in the ASCFTR group. Thus, the increased surfactant secretion in ATII cells could cause higher PC levels in large aggregates of surfactant. In freshly isolated ATII cells, the expression of surfactant proteins was unchanged, suggesting that the lungs of ASCFTR rats contained fewer ATII cells. Gene array analysis of RNA of freshly isolated ATII cells from these lungs showed altered expression of several genes including elevated expression of two calcium-related genes, Ca2+-ATPase and calcium-calmodulin kinase kinase1 (CaMkk1), which was confirmed by real-time PCR. Western blot analysis showed increased expression of calmodulin kinase I, which is activated following phosphorylation by CaMkk1. Although increased expression of calcium regulating genes would argue in favor of Ca2+-dependent mechanisms increasing surfactant secretion, we cannot exclude contribution of alternate mechanisms because of other phenotypic changes in ATII cells of the ASCFTR group. CONCLUSION: Developmental changes due to transient disruption of CFTR in fetal lung reflect in altered ATII cell phenotype in the adult life.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Células Epiteliais/fisiologia , Alvéolos Pulmonares/fisiologia , Animais , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Células Cultivadas , Células Epiteliais/citologia , Técnicas de Silenciamento de Genes , Pulmão/metabolismo , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Fosfatidilcolinas/metabolismo , Proteína A Associada a Surfactante Pulmonar/metabolismo , Proteína B Associada a Surfactante Pulmonar/metabolismo , RNA Antissenso/metabolismo , Ratos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
To understand the role of reactive oxygen species in mechanosensory control of lung development a new approach to interfere with protein-protein interactions by means of a short interacting peptide was developed. This technology was used in the developing rodent lung to examine the role of NADPH oxidase (NOX), casein kinase 2 (CK2), and the cystic fibrosis transmembrane conductance regulator (CFTR) in stretch-induced differentiation. Interactions between these molecules was targeted in an in utero system with recombinant adeno-associated virus (rAAV) containing inserted DNA sequences that express a control peptide or small interfering peptides (siPs) specific for subunit interaction or phosphorylation predicted to be necessary for multimeric enzyme formation. In all cases only siPs with sequences necessary for a predicted normal function were found to interfere with assembly of the multimeric enzyme. A noninterfering control siP to nonessential regions or reporter genes alone had no effect. Physiologically, it was shown that siPs that interfered with the NOX-CFTR-CK2 complex that we call an "interactonome" affected markers of stretch-induced lung organogenesis including Wnt/beta-catenin signaling.
Assuntos
Pulmão/metabolismo , Peptídeos/metabolismo , Sequência de Aminoácidos , Animais , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Dependovirus/genética , Pulmão/embriologia , Camundongos , Dados de Sequência Molecular , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Peptídeos/genética , Fosforilação , Ligação Proteica , Multimerização Proteica , Ratos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Proteínas Wnt/fisiologia , beta Catenina/fisiologiaRESUMO
BACKGROUND: Cystic fibrosis transmembrane conductance regulator (CFTR) was shown previously to modify stretch induced differentiation in the lung. The mechanism for CFTR modulation of lung development was examined by in utero gene transfer of either a sense or antisense construct to alter CFTR expression levels. The BAT-gal transgenic reporter mouse line, expressing beta-galactosidase under a canonical Wnt/beta-catenin-responsive promoter, was used to assess the relative roles of CFTR, Wnt, and parathyroid hormone-related peptide (PTHrP) in lung organogenesis. Adenoviruses containing full-length CFTR, a short anti-sense CFTR gene fragment, or a reporter gene as control were used in an intra-amniotic gene therapy procedure to transiently modify CFTR expression in the fetal lung. RESULTS: A direct correlation between CFTR expression levels and PTHrP levels was found. An inverse correlation between CFTR and Wnt signaling activities was demonstrated. CONCLUSION: These data are consistent with CFTR participating in the mechanicosensory process essential to regulate Wnt/beta-Catenin signaling required for lung organogenesis.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Pulmão/crescimento & desenvolvimento , Proteína Relacionada ao Hormônio Paratireóideo/genética , Transdução de Sinais , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Animais , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Embrião de Mamíferos , Regulação da Expressão Gênica/fisiologia , Camundongos , Organogênese , TransfecçãoRESUMO
The proinflammatory effects of particulate pollutants, including diesel exhaust particles (DEP), are related to their content of redox cycling chemicals and their ability to generate oxidative stress in the respiratory tract. An antioxidant defense pathway, which involves phase II enzyme expression, protects against the pro-oxidative and proinflammatory effects of DEP. The expression of enzymes, including heme oxygenase-1 (HO-1) and GST, is dependent on the activity of a genetic antioxidant response element in their promoters. In this study we investigated the mechanism by which redox cycling organic chemicals, prepared from DEP, induce phase II enzyme expression as a protective response. We demonstrate that aromatic and polar DEP fractions, which are enriched in polycyclic aromatic hydrocarbons and quinones, respectively, induce the expression of HO-1, GST, and other phase II enzymes in macrophages and epithelial cells. We show that HO-1 expression is mediated through accumulation of the bZIP transcription factor, Nrf2, in the nucleus, and that Nrf2 gene targeting significantly weakens this response. Nrf2 accumulation and subsequent activation of the antioxidant response element is regulated by the proteasomal degradation of Nrf2. This pathway is sensitive to pro-oxidative and electrophilic DEP chemicals and is also activated by ambient ultrafine particles. We propose that Nrf2-mediated phase II enzyme expression protects against the proinflammatory effects of particulate pollutants in the setting of allergic inflammation and asthma.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Epitélio/metabolismo , Inflamação/induzido quimicamente , Macrófagos/metabolismo , Estresse Oxidativo/fisiologia , Transativadores/metabolismo , Emissões de Veículos/efeitos adversos , Regulação da Expressão Gênica , Heme Oxigenase (Desciclizante)/biossíntese , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase-1 , Inflamação/metabolismo , Microscopia Confocal , Fator 2 Relacionado a NF-E2 , RNA Mensageiro/metabolismo , Elementos de RespostaRESUMO
The transcription factor Nrf2, which normally exists in an inactive state as a consequence of binding to a cytoskeleton-associated protein Keap1, can be activated by redox-dependent stimuli. Alteration of the Nrf2-Keap1 interaction enables Nrf2 to translocate to the nucleus, bind to the antioxidant-responsive element (ARE) and initiate the transcription of genes coding for detoxifying enzymes and cytoprotective proteins. This response is also triggered by a class of electrophilic compounds including polyphenols and plant-derived constituents. Recently, the natural antioxidants curcumin and caffeic acid phenethyl ester (CAPE) have been identified as potent inducers of haem oxygenase-1 (HO-1), a redox-sensitive inducible protein that provides protection against various forms of stress. Here, we show that in renal epithelial cells both curcumin and CAPE stimulate the expression of Nrf2 in a concentration- and time-dependent manner. This effect was associated with a significant increase in HO-1 protein expression and haem oxygenase activity. From several lines of investigation we also report that curcumin (and, by inference, CAPE) stimulates ho-1 gene activity by promoting inactivation of the Nrf2-Keap1 complex, leading to increased Nrf2 binding to the resident ho-1 AREs. Moreover, using antibodies and specific inhibitors of the mitogen-activated protein kinase (MAPK) pathways, we provide data implicating p38 MAPK in curcumin-mediated ho-1 induction. Taken together, these results demonstrate that induction of HO-1 by curcumin and CAPE requires the activation of the Nrf2/ARE pathway.
Assuntos
Antioxidantes/metabolismo , Curcumina/farmacologia , Proteínas de Ligação a DNA/fisiologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Heme Oxigenase (Desciclizante)/genética , Álcool Feniletílico/análogos & derivados , Transativadores/fisiologia , Animais , Western Blotting , Ácidos Cafeicos/farmacologia , Proteínas de Transporte/fisiologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Ensaio de Desvio de Mobilidade Eletroforética , Regulação Enzimológica da Expressão Gênica/fisiologia , Células LLC-PK1 , Fator 2 Relacionado a NF-E2 , Álcool Feniletílico/farmacologia , Regiões Promotoras Genéticas , Transativadores/antagonistas & inibidoresRESUMO
Nrf2 mediates inducer-dependent activation of the heme oxygenase-1 (HO-1) gene (Alam, J., Stewart, D., Touchard, C., Boinapally, S., Choi, A. M., and Cook, J. L. (1999) J. Biol. Chem. 274, 26071-26078), but the mechanism by which HO-1 inducers regulate Nrf2 function is not known. Treatment of mouse hepatoma (Hepa) cells with 50 microm CdCl(2) increased the amount of Nrf2 protein in a time-dependent manner; induction was observed within 30 min, prior to the accumulation of HO-1 mRNA. Cadmium did not significantly affect the steady-state level of Nrf2 mRNA or the initial rate of Nrf2 protein synthesis but increased the half-life of Nrf2 from approximately 13 to 100 min. Proteasome inhibitors, but not other protease inhibitors, enhanced the expression of Nrf2, and ubiquitinylated Nrf2 was detected after proteasome inhibition. Cycloheximide inhibited cadmium-stimulated Nrf2 expression and DNA binding activity and attenuated HO-1 mRNA accumulation. Conversely, proteasome inhibitors enhanced HO-1 mRNA and protein accumulation by a Nrf2-dependent mechanism. Together, these results indicate that Nrf2 is targeted for rapid degradation by the ubiquitin-proteasome pathway and that cadmium delays the rate of Nrf2 degradation leading to ho-1 gene activation.
Assuntos
Cádmio/farmacologia , Cisteína Endopeptidases/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Complexos Multienzimáticos/metabolismo , Transativadores/química , Transativadores/metabolismo , Ubiquitina/metabolismo , Animais , Western Blotting , Cádmio/metabolismo , Cicloeximida/farmacologia , Relação Dose-Resposta a Droga , Camundongos , Fator 2 Relacionado a NF-E2 , Complexo de Endopeptidases do Proteassoma , Ligação Proteica , Inibidores da Síntese de Proteínas/farmacologia , RNA/metabolismo , RNA Mensageiro/metabolismo , Fatores de Tempo , Células Tumorais CultivadasRESUMO
The mechanism of heme oxygenase-1 gene (ho-1) activation by heme in immortalized rat proximal tubular epithelial cells was examined. Analysis of the ho-1 promoter identified the heme-responsive sequences as the stress-response element (StRE), multiple copies of which are present in two enhancer regions, E1 and E2. Electrophoretic mobility shift assays identified Nrf2, MafG, ATF3, and Jun and Fos family members as StRE-binding proteins; binding of Nrf2, MafG, and ATF3 was increased in response to heme. Dominant-negative mutants of Nrf2 and Maf, but not of c-Fos and c-Jun, inhibited basal and heme-induced expression of an E1-controlled luciferase gene. Heme did not affect the transcription activity of Nrf2, dimerization between Nrf2 and MafG, or the level of MafG, but did stimulate expression of Nrf2. Heme did not influence the level of Nrf2 mRNA but increased the half-life of Nrf2 protein from approximately 10 min to nearly 110 min. These results indicate that heme promotes stabilization of Nrf2, leading to accumulation of Nrf2. MafG dimers that bind to StREs to activate the ho-1 gene.