RESUMEN
Chronic lung disease of prematurity/bronchopulmonary dysplasia (BPD) is the leading cause of perinatal morbidity in developed countries. Inflammation is a prominent finding. Currently available interventions have associated toxicities and limited efficacy. While BPD often resolves in childhood, survivors of preterm birth are at risk for acquired respiratory disease in early life and are more likely to develop chronic obstructive pulmonary disease (COPD) in adulthood. We previously cloned Crispld2 (Lgl1), a glucocorticoid-regulated mesenchymal secretory protein that modulates lung branching and alveogenesis through mesenchymal-epithelial interactions. Absence of Crispld2 is embryonic lethal. Heterozygous Crispld2+/- mice display features of BPD, including distal airspace enlargement, disruption of elastin, and neonatal lung inflammation. CRISPLD2 also plays a role in human fetal lung fibroblast cell expansion, migration, and mesenchymal-epithelial signaling. This study assessed the effects of endogenous and exogenous CRISPLD2 on expression of proinflammatory mediators in human fetal and adult (normal and COPD) lung fibroblasts and epithelial cells. CRISPLD2 expression was upregulated in a lipopolysaccharide (LPS)-induced human fetal lung fibroblast line (MRC5). LPS-induced upregulation of the proinflammatory cytokines IL-8 and CCL2 was exacerbated in MRC5-CRISPLD2(knockdown) cells. siRNA suppression of endogenous CRISPLD2 in adult lung fibroblasts (HLFs) led to augmented expression of IL-8, IL-6, CCL2. LPS-stimulated expression of proinflammatory mediators by human lung epithelial HAEo- cells was attenuated by purified secretory CRISPLD2. RNA sequencing results from HLF-CRISPLD2(knockdown) suggest roles for CRISPLD2 in extracellular matrix and in inflammation. Our data suggest that suppression of CRISPLD2 increases the risk of lung inflammation in early life and adulthood.
Asunto(s)
Displasia Broncopulmonar/metabolismo , Moléculas de Adhesión Celular/metabolismo , Células Epiteliales/metabolismo , Fibroblastos/metabolismo , Mediadores de Inflamación/antagonistas & inhibidores , Factores Reguladores del Interferón/metabolismo , Pulmón/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/metabolismo , Adulto , Anciano , Animales , Displasia Broncopulmonar/embriología , Displasia Broncopulmonar/patología , Moléculas de Adhesión Celular/deficiencia , Moléculas de Adhesión Celular/genética , Proliferación Celular/fisiología , Células Cultivadas , Células Epiteliales/citología , Células Epiteliales/patología , Transición Epitelial-Mesenquimal/fisiología , Fibroblastos/citología , Fibroblastos/patología , Células HEK293 , Humanos , Mediadores de Inflamación/metabolismo , Factores Reguladores del Interferón/deficiencia , Factores Reguladores del Interferón/genética , Interleucina-6/metabolismo , Lipopolisacáridos/metabolismo , Pulmón/citología , Pulmón/embriología , Pulmón/patología , Masculino , Ratones , Persona de Mediana Edad , Enfermedad Pulmonar Obstructiva Crónica/patología , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Transducción de SeñalRESUMEN
BACKGROUND: Despite the overall effectiveness of glucocorticoids (GCs) in the treatment of asthma, a large proportion of patients do not fully respond to this medication. The objective of the present study was to investigate the potential molecular mechanisms responsible for corticosteroid insensitivity in pediatric asthma. METHODS: Asthmatic children were classified as good (GSR) or poor corticosteroid responders (PSR) based on the changes in pulmonary function following GC treatment. Immortalized B-cells derived from patients at two ends of the spectrum of GC responsiveness (five each) were grown in culture and treated with hydrocortisone (10(-6)M). Baseline and temporal changes in GC receptor (GR) protein and mRNA were evaluated by western blot and quantitative reverse transcription PCR respectively. The effect of GC treatment on GR nuclear levels was assessed by western blots. RESULTS: Cells derived from PSR asthmatics displayed lower GR protein levels when compared to GSR. Moreover, in PSR cells GC-induced nuclear translocation of GR was short-lived and homologous downregulation of GR mRNA and protein was faster than in GSR. CONCLUSION: Our data demonstrate the existence of a novel mechanism of GC insensitivity resulting from limited GR nuclear bioavailability as a consequence of decreased baseline GR protein expression and more rapid hormone-induced downregulation.
Asunto(s)
Antiinflamatorios/farmacología , Asma/tratamiento farmacológico , Linfocitos B/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Hidrocortisona/farmacología , Receptores de Glucocorticoides/agonistas , Transporte Activo de Núcleo Celular , Adolescente , Asma/diagnóstico , Asma/genética , Asma/metabolismo , Asma/fisiopatología , Linfocitos B/metabolismo , Línea Celular Transformada , Núcleo Celular/metabolismo , Niño , Preescolar , Regulación hacia Abajo , Resistencia a Medicamentos , Femenino , Volumen Espiratorio Forzado , Humanos , Pulmón/efectos de los fármacos , Pulmón/fisiopatología , Masculino , ARN Mensajero/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Factores de Tiempo , Resultado del TratamientoRESUMEN
As the sole E2 enzyme for SUMOylation, Ubc9 is predominantly nuclear. However, the underlying mechanisms of Ubc9 nuclear localization are still not well understood. Here we show that RNAi-depletion of Imp13, an importin known to mediate Ubc9 nuclear import, reduces both Ubc9 nuclear accumulation and global SUMOylation. Furthermore, Ubc9-R13A or Ubc9-H20D mutation previously shown to interrupt the interaction of Ubc9 with nucleus-enriched SUMOs reduces the nuclear enrichment of Ubc9, suggesting that the interaction of Ubc9 with the nuclear SUMOs may enhance Ubc9 nuclear retention. Moreover, Ubc9-R17E mutation, which is known to disrupt the interaction of Ubc9 with both SUMOs and Imp13, causes a greater decrease in Ubc9 nuclear accumulation than Ubc9-R13A or Ubc9-H20D mutation. Lastly, Ubc9-K74A/S89D mutations that perturb the interaction of Ubc9 with nucleus-enriched SUMOylation-consensus motifs has no effect on Ubc9 nuclear localization. Altogether, our results have elucidated that the amino acid residues within the N-terminal region of Ubc9 play a pivotal role in regulation of Ubc9 nuclear localization.
Asunto(s)
Núcleo Celular/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Secuencia de Aminoácidos , Citoplasma/metabolismo , Células HeLa , Humanos , Carioferinas/genética , Carioferinas/metabolismo , Datos de Secuencia Molecular , Mutación , Interferencia de ARN , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Sumoilación , Enzimas Ubiquitina-Conjugadoras/genéticaRESUMEN
Rapid growth and formation of new gas exchange units (alveogenesis) are hallmarks of the perinatal lung. Bronchopulmonary dysplasia (BPD), common in very premature infants, is characterized by premature arrest of alveogenesis. Mesenchymal cells (fibroblasts) regulate both lung branching and alveogenesis through mesenchymal-epithelial interactions. Temporal or spatial deficiency of late-gestation lung 1/cysteine-rich secretory protein LD2 (LGL1/CRISPLD2), expressed in and secreted by lung fibroblasts, can impair both lung branching and alveogenesis (LGL1 denotes late gestation lung 1 protein; LGL1 denotes the human gene; Lgl1 denotes the mouse/rat gene). Absence of Lgl1 is embryonic lethal. Lgl1 levels are dramatically reduced in oxygen toxicity rat models of BPD, and heterozygous Lgl1(+/-) mice exhibit features resembling human BPD. To explore the role of LGL1 in mesenchymal-epithelial interactions in developing lung, we developed a doxycycline (DOX)-inducible RNA-mediated LGL1 knockdown cellular model in human fetal lung fibroblasts (MRC5(LGL1KD)). We assessed the impact of LGL1 on cell proliferation, cell migration, apoptosis, and wound healing. DOX-induced MRC5(LGL1KD) suppressed cell growth and increased apoptosis of annexin V(+) staining cells and caspase 3/7 activity. LGL1-conditioned medium increased migration of fetal rat primary lung epithelial cells and human airway epithelial cells. Impaired healing by MRC5(LGL1KD) cells of a wound model was attenuated by addition of LGL1-conditioned medium. Suppression of LGL1 was associated with dysregulation of extracellular matrix genes (downregulated MMP1, ColXVα1, and ELASTIN) and proapoptosis genes (upregulated BAD, BAK, CASP2, and TNFRSF1B) and inhibition of 44/42MAPK phosphorylation. Our findings define a role for LGL1 in fibroblast expansion and migration, epithelial cell migration, and mesenchymal-epithelial signaling, key processes in fetal lung development.
Asunto(s)
Apoptosis/fisiología , Moléculas de Adhesión Celular/metabolismo , Movimiento Celular/fisiología , Proliferación Celular/fisiología , Feto/embriología , Fibroblastos/metabolismo , Factores Reguladores del Interferón/metabolismo , Pulmón/embriología , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Displasia Broncopulmonar/embriología , Displasia Broncopulmonar/genética , Displasia Broncopulmonar/metabolismo , Displasia Broncopulmonar/patología , Moléculas de Adhesión Celular/genética , Proteínas de la Matriz Extracelular/biosíntesis , Proteínas de la Matriz Extracelular/genética , Feto/citología , Fibroblastos/citología , Células HEK293 , Humanos , Factores Reguladores del Interferón/genética , Pulmón/citología , Ratones , Ratones Noqueados , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación/fisiología , Ratas , Ratas Sprague-Dawley , Mucosa Respiratoria/citología , Mucosa Respiratoria/embriología , Transducción de Señal/fisiologíaRESUMEN
The "fetal origins hypothesis" argued that physiological changes consequent to in utero exposures ultimately contribute to disease susceptibility in later life. The dramatic increase in asthma prevalence is attributed to early exposures acting on preexisting asthma-susceptible genotypes. We showed previously that distinct transcriptome signatures distinguish the developmental respiratory phenotype of atopic (Brown Norway, BN) and normoresponsive (Lewis) rats. We aimed to determine whether maternal allergen exposure would influence asthma pathogenesis by reprogramming primary patterns of developmental lung gene expression. Postnatal offspring of dams sensitized to ovalbumin before mating and challenged during pregnancy were assessed for lung function, inflammatory biomarkers, and respiratory gene expression. Although maternal ovalbumin exposure resulted in characteristic features of an allergic response (bronchoalveolar lavage neutrophils, IgE, methacholine-induced lung resistance) in offspring of both strains, substantial strain-specific differences were observed in respiratory gene expression. Of 799 probes representing the top 5% of transcriptomic variation, only 112 (14%) were affected in both strains. Strain-specific gene signatures also exhibited marked differences in enrichment for gene ontologies, with immune regulation and cell proliferation being prominent in the BN strain, cell cycle and microtubule assembly gene sets in the Lewis strain. Multiple ovalbumin-specific probes in both strains were also differentially expressed in lymphoblastoid cell lines from human asthmatic vs. nonasthmatic sibling pairs. Our data point to the existence of distinct, genetically programmed responses to maternal exposures in developing lung. These different response patterns, if recapitulated in human fetal development, can contribute to long-term pulmonary health including interindividual susceptibility to asthma.
Asunto(s)
Alérgenos/efectos adversos , Asma/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Exposición Materna/efectos adversos , Efectos Tardíos de la Exposición Prenatal/metabolismo , Alérgenos/inmunología , Alérgenos/farmacología , Animales , Asma/etiología , Asma/inmunología , Asma/patología , Femenino , Regulación de la Expresión Génica/inmunología , Humanos , Masculino , Embarazo , Efectos Tardíos de la Exposición Prenatal/inmunología , Efectos Tardíos de la Exposición Prenatal/patología , Ratas , Ratas Endogámicas BN , Ratas Endogámicas Lew , TranscriptomaRESUMEN
BACKGROUND/PURPOSE: Fetal tracheal occlusion (TO) has been investigated as a treatment option for lung hypoplasia secondary to congenital diaphragmatic hernia. Tracheal occlusion has been shown to accelerate lung growth, but its effect on bronchial branching is unknown. In this study, we characterize the effects of in vitro TO on bronchial branch development in fetal lung explants derived from the nitrofen rat model of congenital diaphragmatic hernia. METHODS: Rat dams were gavaged nitrofen on gestational day 9.5, and fetal lungs were harvested for explant culture on gestational day 14 (term, 22 days). Four experimental groups were investigated, with TO performed ex vivo using cautery: control, control + TO, nitrofen, and nitrofen + TO. Explants were incubated for 72 hours. Representative photographs were taken at 0, 24, 48, and 72 hours from the time of culture, and the number of distal branches was counted for each explant. The Student t test was used to compare distal branch measurements. RESULTS: A minimum of 12 fetal lung explants were cultured for each group. By 24 hours, all explants undergoing TO had more branch iterations than explants that did not. Moreover, TO in nitrofen-exposed explants increased bronchial branching to control levels by 24 hours in culture. CONCLUSION: Our results suggest that TO at day 14 increases branching in normal and nitrofen-exposed lung explants. In addition, TO increases airway branching in nitrofen-exposed explants to control levels suggesting that early TO reverses the lung hypoplasia seen in this model.
Asunto(s)
Bronquios/embriología , Enfermedades Fetales/cirugía , Hernia Diafragmática/embriología , Hernia Diafragmática/cirugía , Morfogénesis , Tráquea/cirugía , Animales , Bronquios/patología , Enfermedades Fetales/patología , Hernia Diafragmática/patología , Hernias Diafragmáticas Congénitas , Técnicas In Vitro , Pulmón/embriología , Pulmón/patología , Éteres Fenílicos , RatasRESUMEN
Asthma is the leading serious pediatric chronic illness in the United States, affecting 7.1 million children. The prevalence of asthma in children under 4 years of age has increased dramatically in the last 2 decades. Existing evidence suggests that this increase in prevalence derives from early environmental exposures acting on a pre-existing asthma-susceptible genotype. We studied the origins of asthma susceptibility in developing lung in rat strains that model the distinct phenotypes of airway hyperresponsiveness (Fisher rats) and atopy (brown Norway [BN] rats). Postnatal BN rat lungs showed increased epithelial proliferation and tracheal goblet cell hyperplasia. Fisher pups showed increased lung resistance at age 2 weeks, with elevated neutrophils throughout the postnatal period. Diverse transcriptomic signatures characterized the distinct respiratory phenotypes of developing lung in both rat models. Linear regression across age and strain identified developmental variation in expression of 1,376 genes, and confirmed both strain and temporal regulation of lung gene expression. Biological processes that were heavily represented included growth and development (including the T Box 1 transcription factor [Tbx5], the epidermal growth factor receptor [Egfr], the transforming growth factor beta-1-induced transcript 1 [Tgfbr1i1]), extracellular matrix and cell adhesion (including collagen and integrin genes), and immune function (including lymphocyte antigen 6 (Ly6) subunits, IL-17b, Toll-interacting protein, and Ficolin B). Genes validated by quantitative RT-PCR and protein analysis included collagen III alpha 1 Col3a1, Ly6b, glucocorticoid receptor and Importin-13 (specific to the BN rat lung), and Serpina1 and Ficolin B (specific to the Fisher lung). Innate differences in patterns of gene expression in developing lung that contribute to individual variation in respiratory phenotype are likely to contribute to the pathogenesis of asthma.
Asunto(s)
Asma/genética , Predisposición Genética a la Enfermedad , Pulmón/crecimiento & desarrollo , Pulmón/patología , Transporte Activo de Núcleo Celular/genética , Animales , Animales Recién Nacidos , Antígenos Ly/genética , Antígenos Ly/metabolismo , Asma/patología , Asma/fisiopatología , Líquido del Lavado Bronquioalveolar/citología , Núcleo Celular/metabolismo , Proliferación Celular , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Células Caliciformes/metabolismo , Células Caliciformes/patología , Hiperplasia , Inmunidad Innata/genética , Recuento de Leucocitos , Pulmón/enzimología , Pulmón/fisiopatología , Masculino , Ratas , Pruebas de Función Respiratoria , Factores de Tiempo , Regulación hacia Arriba/genéticaRESUMEN
Alveolarization depends on circulating glucocorticoid (GC), retinoid (RA), and vitamin D (VitD). Bronchopulmonary dysplasia, a leading cause of neonatal morbidity, is associated with arrested alveolarization. In hyperoxia-exposed rats displaying features of bronchopulmonary dysplasia, reduced levels of late gestation lung 1 (Lgl1) normalize during recovery. We show that GC (100 nM) stimulates (7- to 115-fold) and VitD (100 microM) suppresses (twofold) Lgl1 expression. RA (all-trans/9-cis, 10 microM) effects are biphasic. From postnatal days 7-10, RA was stimulatory (twofold) at 24 h, after which effects were inhibitory (3- to 15-fold). Lgl1 promoter-luciferase reporter assays confirmed that these agents operated at the transcriptional level. Interestingly, the individual inhibitory effects of VitD and RA on GC induction of Lgl1 were abrogated when both agents were present, suggesting that steric hindrance may influence promoter accessibility. Analysis of the proximity (<50 base pairs) of binding sites for overlapping VitD and RA receptors to that of the GC receptor identified 81% of promoters in 66 genes (including Lgl1) important in human lung development compared with 48% in a random set of 1000 genes. Complex integration of the effects of GC, RA, and VitD on gene expression in the postnatal lung is likely to contribute to the timely advance of alveolarization without attendant inflammation.
Asunto(s)
Antineoplásicos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas/metabolismo , Alveolos Pulmonares/fisiología , Esteroides/farmacología , Tretinoina/farmacología , Vitamina D/farmacología , Animales , Sitios de Unión , Línea Celular , Femenino , Fibroblastos/citología , Fibroblastos/fisiología , Humanos , Embarazo , Regiones Promotoras Genéticas , Proteínas/genética , Alveolos Pulmonares/citología , Ratas , Ratas Sprague-Dawley , Transcripción GenéticaRESUMEN
Glucocorticoid (GC)-responsive epithelial-mesenchymal interactions regulate lung development. The GC receptor (GR) mediates GC signaling. Mice lacking GR in all tissues die at birth of respiratory failure. To determine the specific need for epithelial GR in lung development, we bred triple transgenic mice that carry SPC/rtTA, tet-O-Cre, and floxed, but not wild-type, GR genes. When exposed to doxycycline in utero, triple transgenic (GRepiâ») mice exhibit a Cre-mediated recombination event that inactivates the floxed GR gene in airway epithelial cells. Immunofluorescence confirmed the elimination of GR in Cre-positive airway epithelial cells of late gestation GRepiâ» mice. Embryonic Day 18.5 pups had a relatively immature appearance with increased lung cellularity and increased pools of glycogen in the epithelium. Postnatal Day 0.5 pups had decreased viability. We used quantitative RT-PCR to demonstrate that specific elimination of epithelial immunoreactive GR in GRepiâ» mice is associated with reduced mRNA expression for surfactant proteins (SPs) A, B, C, and D; ß- and γ-ENaC; T1α; the 10-kD Clara cell protein (CCSP); and aquaporin 5 (AQP5). Western blots confirmed reduced levels of AQP5 protein. No reduction in the levels of the GR transport protein importin (IPO)-13 was observed. Our findings demonstrate a requirement for lung epithelial cell GR in normal lung development. We speculate that impaired epithelial differentiation, leading to decreased SPs, transepithelial Na, and liquid absorption at birth, may contribute to the reduced survival of newborn mice with suppressed lung epithelial GR.
Asunto(s)
Epitelio/metabolismo , Epitelio/patología , Pulmón/metabolismo , Pulmón/patología , Receptores de Glucocorticoides/deficiencia , Animales , Animales Recién Nacidos , Biomarcadores/metabolismo , Doxiciclina/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/patología , Canales Epiteliales de Sodio/genética , Canales Epiteliales de Sodio/metabolismo , Epitelio/efectos de los fármacos , Epitelio/embriología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Pulmón/embriología , Ratones , Ratones Noqueados , Especificidad de Órganos/efectos de los fármacos , Organogénesis/efectos de los fármacos , Alveolos Pulmonares/efectos de los fármacos , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/patología , Proteínas Asociadas a Surfactante Pulmonar/genética , Proteínas Asociadas a Surfactante Pulmonar/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Análisis de Supervivencia , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Neonatal lung injury, a leading cause of morbidity in prematurely born infants, has been associated with arrested alveolar development and is often accompanied by goblet cell hyperplasia. Genes that regulate alveolarization and inflammation are likely to contribute to susceptibility to neonatal lung injury. We previously cloned Lgl1, a developmentally regulated secreted glycoprotein in the lung. In rat, O2 toxicity caused reduced levels of Lgl1, which normalized during recovery. We report here on the generation of an Lgl1 knockout mouse in order to determine whether deficiency of Lgl1 is associated with arrested alveolarization and contributes to neonatal lung injury. METHODS: An Lgl1 knockout mouse was generated by introduction of a neomycin cassette in exon 2 of the Lgl1 gene. To evaluate the pulmonary phenotype of Lgl1+/- mice, we assessed lung morphology, Lgl1 RNA and protein, elastin fibers and lung function. We also analyzed tracheal goblet cells, and expression of mucin, interleukin (IL)-4 and IL-13 as markers of inflammation. RESULTS: Absence of Lgl1 was lethal prior to lung formation. Postnatal Lgl1+/- lungs displayed delayed histological maturation, goblet cell hyperplasia, fragmented elastin fibers, and elevated expression of TH2 cytokines (IL-4 and IL-13). At one month of age, reduced expression of Lgl1 was associated with elevated tropoelastin expression and altered pulmonary mechanics. CONCLUSION: Our findings confirm that Lgl1 is essential for viability and is required for developmental processes that precede lung formation. Lgl1+/- mice display a complex phenotype characterized by delayed histological maturation, features of inflammation in the post-natal period and altered lung mechanics at maturity. Lgl1 haploinsufficiency may contribute to lung disease in prematurity and to increased risk for late-onset respiratory disease.
Asunto(s)
Glicoproteínas/metabolismo , Células Caliciformes/metabolismo , Factores Inmunológicos/metabolismo , Pulmón/metabolismo , Ratones Noqueados/metabolismo , Mecánica Respiratoria , Animales , Células Cultivadas , Citocinas , Glicoproteínas/genética , Lesión Pulmonar , RatonesRESUMEN
BACKGROUND: A precise balance exists between the actions of endogenous glucocorticoids (GC) and retinoids to promote normal lung development, in particular during alveolarization. The mechanisms controlling this balance are largely unknown, but recent evidence suggests that midkine (MK), a retinoic acid-regulated, pro-angiogenic growth factor, may function as a critical regulator. The purpose of this study was to examine regulation of MK by GC and RA during postnatal alveolar formation in rats. METHODS: Newborn rats were treated with dexamethasone (DEX) and/or all-trans-retinoic acid (RA) during the first two weeks of life. Lung morphology was assessed by light microscopy and radial alveolar counts. MK mRNA and protein expression in response to different treatment were determined by Northern and Western blots. In addition, MK protein expression in cultured human alveolar type 2-like cells treated with DEX and RA was also determined. RESULTS: Lung histology confirmed that DEX treatment inhibited and RA treatment stimulated alveolar formation, whereas concurrent administration of RA with DEX prevented the DEX effects. During normal development, MK expression was maximal during the period of alveolarization from postnatal day 5 (PN5) to PN15. DEX treatment of rat pups decreased, and RA treatment increased lung MK expression, whereas concurrent DEX+RA treatment prevented the DEX-induced decrease in MK expression. Using human alveolar type 2 (AT2)-like cells differentiated in culture, we confirmed that DEX and cAMP decreased, and RA increased MK expression. CONCLUSION: We conclude that MK is expressed by AT2 cells, and is differentially regulated by corticosteroid and retinoid treatment in a manner consistent with hormonal effects on alveolarization during postnatal lung development.
Asunto(s)
Proteínas Angiogénicas/metabolismo , Citocinas/metabolismo , Dexametasona/farmacología , Células Epiteliales/efectos de los fármacos , Alveolos Pulmonares/efectos de los fármacos , Tretinoina/farmacología , Factores de Edad , Proteínas Angiogénicas/genética , Animales , Animales Recién Nacidos , Northern Blotting , Western Blotting , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , AMP Cíclico/metabolismo , Citocinas/genética , Células Epiteliales/metabolismo , Humanos , Midkina , Alveolos Pulmonares/crecimiento & desarrollo , Alveolos Pulmonares/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
BACKGROUND: Glucocorticoid function is dependent on efficient translocation of the glucocorticoid receptor (GR) from the cytoplasm to the nucleus of cells. Importin-13 (IPO13) is a nuclear transport receptor that mediates nuclear entry of GR. In airway epithelial cells, inhibition of IPO13 expression prevents nuclear entry of GR and abrogates anti-inflammatory effects of glucocorticoids. Impaired nuclear entry of GR has been documented in steroid-non-responsive asthmatics. We hypothesize that common IPO13 genetic variation influences the anti-inflammatory effects of inhaled corticosteroids for the treatment of asthma, as measured by change in methacholine airway hyperresponsiveness (AHR-PC20). METHODS: 10 polymorphisms were evaluated in 654 children with mild-to-moderate asthma participating in the Childhood Asthma Management Program (CAMP), a clinical trial of inhaled anti-inflammatory medications (budesonide and nedocromil). Population-based association tests with repeated measures of PC20 were performed using mixed models and confirmed using family-based tests of association. RESULTS: Among participants randomized to placebo or nedocromil, IPO13 polymorphisms were associated with improved PC20 (i.e. less AHR), with subjects harboring minor alleles demonstrating an average 1.51-2.17 fold increase in mean PC20 at 8-months post-randomization that persisted over four years of observation (p = 0.01-0.005). This improvement was similar to that among children treated with long-term inhaled corticosteroids. There was no additional improvement in PC20 by IPO13 variants among children treated with inhaled corticosteroids. CONCLUSION: IPO13 variation is associated with improved AHR in asthmatic children. The degree of this improvement is similar to that observed with long-term inhaled corticosteroid treatment, suggesting that IPO13 variation may improve nuclear bioavailability of endogenous glucocorticoids.
Asunto(s)
Asma/tratamiento farmacológico , Asma/genética , Carioferinas/genética , Antiasmáticos/uso terapéutico , Asma/fisiopatología , Hiperreactividad Bronquial/genética , Hiperreactividad Bronquial/fisiopatología , Budesonida/uso terapéutico , Niño , Método Doble Ciego , Variación Genética , Genotipo , Haplotipos , Humanos , Desequilibrio de Ligamiento/genética , Nedocromil/uso terapéutico , Polimorfismo Genético , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
Importin 13 is a member of the importin beta superfamily of nuclear transport proteins and is expressed in multiple tissues at high levels both in humans and rodents, including fetal lung, brain, and heart. In order to elucidate potential functions of imp13 in the heart, we have used rat imp13 as bait to screen a human heart cDNA library and identified an interaction with the C-terminal peptide of myopodin (a.a. 360-698), an actin-bundling protein, associated with tumor-suppressor activity that localizes to both the cytoplasm and the nucleus. We have used GST-pull down assays and co-immunoprecipitation experiments to demonstrate an interaction between imp13 and full-length myopodin and observed that RanGTP dissociates the myopodin-imp13 complex. In studies of cultured cells, we show that both imp13 siRNA and a C-terminal fragment of imp13 protein prevent nuclear localization of myopodin. We, therefore, conclude that imp13 functions in myopodin import and we suggest that the regulation of these events is critical for normal and abnormal cellular differentiation.
Asunto(s)
Carioferinas/metabolismo , Proteínas de Microfilamentos/metabolismo , Transducción de Señal/fisiología , Transporte Activo de Núcleo Celular , Diferenciación Celular/fisiología , Células Cultivadas , Células HeLa , Humanos , Unión ProteicaRESUMEN
In order to better understand the regulation of lung maturation by glucocorticoid-glucocorticoid receptor signaling, we studied glucocorticoid receptor (GR) hypomorphic mice with a mixed C57Bl6/129 sv background, in which disruption of exon 2 of the GR gene produces an N-terminal truncated GR protein. Four groups of mice were compared: homozygous mice that die at birth (non-survivors), homozygous mice that survive the neonatal period (survivors), heterozygotes and wild-type mice. Newborn non-survivors had 50% thicker airspace walls and a 46% decrease in the formation of secondary crests (the beginning of alveolar secondary septation) compared to either survivor or wild-type littermates (n = 9 mice in each group). The lung tissue to airspace ratio in homozygous mice not expressing wild-type GR (non-survivor and survivor) was increased compared to heterozygotes and wild-type mice that do express wild-type GR (0.91 +/- 0.08 vs. 0.49 +/- 0.02, n = 4 in each of the four subgroups), suggesting that complete morphological maturation of the lung is dependent on effective glucocorticoid signaling through a fully functional GR. Moreover, the relatively mature lung morphology of survivor versus non-survivor newborns suggests that a partial reduction in mesenchymal thickness is compatible with capillary remodeling, alveolar septation, and viable respiratory function after birth. Our findings suggest that in mice homozygous for disrupted GR, the severity of newborn respiratory insufficiency correlates with the degree of lung structural immaturity.
Asunto(s)
Glucocorticoides/metabolismo , Pulmón/patología , Receptores de Glucocorticoides/genética , Insuficiencia Respiratoria/genética , Insuficiencia Respiratoria/patología , Animales , Animales Recién Nacidos , Capilares/patología , Células Endoteliales/patología , Glucocorticoides/genética , Heterocigoto , Homocigoto , Inmunohistoquímica , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL/genética , Músculo Liso/patología , Insuficiencia Respiratoria/metabolismo , Índice de Severidad de la Enfermedad , Transducción de SeñalRESUMEN
Late-gestation lung protein 1 (LGL1) is a glycoprotein secreted by fetal lung mesenchyme that stimulates branching morphogenesis of the developing lung bud. We show that Lgl1 mRNA and protein are also expressed in mesenchymally derived lineages of fetal kidney. Although Lgl1 expression is stimulated by glucocorticoids in kidney cells, cortisol (10(-7) M) actually suppresses ureteric bud branching of fetal kidneys from HoxB7/GFP mice in explant culture. However, early branching morphogenesis in the lung and kidney is stimulated by retinoic acid, and we identified putative retinoic acid response elements in the Lgl1 promoter. All-trans-retinoic acid (10(-6) M) stimulated Lgl1 promoter activity and endogenous Lgl1 mRNA expression in vitro. Branching of cultured fetal kidney explants was increased in the presence of all-trans retinoic acid (10(-6) M). Heterozygous Lgl1 knockout mice were crossed to HoxB7/GFP mice to visualize the extent of ureteric bud branching at fetal stages. At embryonic (E) days E12.5-E13.0, mutant Lgl1(+/-) embryos showed a 20% reduction in ureteric bud branching compared with wild-type littermates. We propose a model in which retinoic acid stimulates branching morphogenesis by activating Lgl1 early in development. The prominent effects of glucocorticoids on Lgl1 expression in late lung development suggest a second role for LGL1 in alveolar maturation.
Asunto(s)
Glicoproteínas/fisiología , Riñón/embriología , Riñón/crecimiento & desarrollo , Morfogénesis/fisiología , Proteínas/fisiología , Tretinoina/farmacología , Animales , Línea Celular , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/fisiología , Glucocorticoides/farmacología , Glicoproteínas/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Humanos , Hidrocortisona/farmacología , Ratones , Ratones Endogámicos C3H , Ratones Noqueados , Morfogénesis/efectos de los fármacos , Proteínas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , TransfecciónRESUMEN
Antiinflammatory effects of glucocorticoids are critical to treatment of airway inflammation in such common disorders as asthma. There is considerable variation in responsiveness to glucocorticoid, and prolonged exposure can result in glucocorticoid resistance. We cloned LGL2, a glucocorticoid-inducible gene in fetal rat lung. We described the characterization of lgl2 as a nuclear transport protein, classified as importin 13 (IPO13), and demonstrated developmental regulation of IPO13 nucleocytoplasmic shuttling. We now report on the identification of the glucocorticoid receptor (GR) as a cargo substrate for IPO13. Binding of GR and IPO13 was demonstrated by GR-GST pulldown and coimmunoprecipitation. To investigate the role of IPO13 in modulating GR signaling in the lung, we studied IPO13-regulated GR transport in airway epithelial cells. Small interfering RNAs that inhibited IPO13 synthesis prevented nuclear translocation of GR. Silencing of IPO13 also abrogated the ability of cortisol to inhibit synthesis of the inflammatory cytokine IL-8 after stimulation with TNF-alpha. Our findings support a role for IPO13 in promoting nuclear occupancy of GR in a way that strongly potentiates the antiinflammatory effects of glucocorticoids. We speculate that variation in cellular levels of IPO13 and intracellular IPO13 shuttling rates may contribute to glucocorticoid resistance.
Asunto(s)
Núcleo Celular/metabolismo , Receptores de Glucocorticoides/metabolismo , Mucosa Respiratoria/metabolismo , beta Carioferinas/metabolismo , Transporte Activo de Núcleo Celular/efectos de los fármacos , Animales , Antiinflamatorios/farmacología , Núcleo Celular/efectos de los fármacos , Resistencia a Medicamentos , Células HeLa , Humanos , Hidrocortisona/farmacología , Inmunoprecipitación , Interleucina-8/biosíntesis , Unión Proteica , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Wistar , Receptores de Glucocorticoides/efectos de los fármacos , Receptores de Glucocorticoides/genética , Proteínas Recombinantes de Fusión/metabolismo , Mucosa Respiratoria/citología , Mucosa Respiratoria/efectos de los fármacos , Transfección , Factor de Necrosis Tumoral alfa/farmacología , Regulación hacia Arriba/efectos de los fármacos , beta Carioferinas/genéticaRESUMEN
BACKGROUND: Congenital truncation of the glucocorticoid receptor (GR) is known to lead to lethal lung immaturity in newborn mice associated with increased lung cellularity (ratio of tissue to airspace) and, as we previously showed, prolonged expression of the retinoid-responsive growth factor midkine. OBJECTIVES: We sought to determine if these changes would be reversed by transgenic expression of GR exclusively in the distal airway epithelium. METHODS: Mice were generated with expression of transgenic rat (r) GR driven by the human (h) SP-C promoter, on a background of congenital GR truncation. RESULTS: Transgenic epithelial GR expression reduced lung cellularity and midkine expression to levels comparable to wild-type littermates. Nevertheless, the newborn transgenic mice still displayed respiratory failure. Moreover, epithelial expression of the GR transgene did not alter expression of a number of important markers of lung maturation. CONCLUSIONS: Our data demonstrating normalization of the lung tissue to airspace ratio in neonatal mice expressing transgenic GR in the distal airway epithelium is consistent with the concept that normal mesenchymal cell loss is due to GR-responsive stimulation from epithelial cells. However, we could find no evidence of altered apoptotic activity between the groups of mice. We speculate that correction of the severe neonatal lung phenotype of GR-deficient mice will require expression of normal GR in non-epithelial as well as epithelial tissues.
Asunto(s)
Citocinas/genética , Pulmón/fisiología , Péptidos/genética , Regiones Promotoras Genéticas , Receptores de Glucocorticoides/genética , Animales , Animales Modificados Genéticamente , Animales Recién Nacidos , Secuencia de Bases , Cartilla de ADN , Regulación de la Expresión Génica , Genotipo , Humanos , Péptidos y Proteínas de Señalización Intercelular , Pulmón/citología , Ratones , Ratones Noqueados , Ratones Transgénicos , Midkina , Proteína C Asociada a Surfactante Pulmonar , Ratas , Receptores de Glucocorticoides/deficienciaRESUMEN
Bronchopulmonary dysplasia (BPD), a major cause of morbidity in premature infants, is characterized by arrest of lung growth and inhibited alveologenesis. We had earlier cloned late-gestation lung 1 (LGL1), a glucocorticoid (GC)-induced, developmentally regulated gene in lung mesenchyme, and showed that reduced levels of late-gestation lung 1 protein (lgl1) inhibit lung branching. Maximal fetal expression of LGL1 is concordant with the onset of alveolar septation, suggesting an additional role for lgl1 in alveologenesis. At postnatal d 7, during the period of maximal septation in postnatal rat lung, lgl1 concentrates at the tips of budding secondary alveolar septa. We studied two models of impaired postnatal alveologenesis generated by exposure of newborn rats to 60% O2 for 2 wk or 95% O2 for 1 wk. A profound decrease of lgl1 expression with oxygen exposure was observed in both animal models. Animals exposed to 95% O2 for 1 wk recovered in air over a 3-wk period, associated with normalization of lgl1 levels. Changes in lung levels of alpha-actin (a marker of myofibroblast differentiation associated with alveologenesis) and the mesenchymal marker vimentin were significant but less marked. Our findings support a role for lgl1 in postnatal lung development. We speculate that deficiency of lgl1 contributes to the arrested alveolar partitioning observed in BPD and that recovery is associated with normalization of lgl1 levels.
Asunto(s)
Aire , Displasia Broncopulmonar/fisiopatología , Pulmón , Oxígeno/toxicidad , Proteínas/metabolismo , Actinas/genética , Actinas/metabolismo , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , Femenino , Humanos , Recién Nacido , Pulmón/anatomía & histología , Pulmón/crecimiento & desarrollo , Pulmón/metabolismo , Pulmón/patología , Embarazo , Proteínas/genética , Ratas , Ratas Sprague-Dawley , Vimentina/genética , Vimentina/metabolismoRESUMEN
To investigate molecular mechanisms of lung organogenesis, we searched for glucocorticoid-inducible genes in developing lung. We cloned LGL2, a developmentally and hormonally regulated gene in fetal lung (Zhang, C., N. B. Sweezey, S. Gagnon, B. Muskat, D. Koehler, M. Post, and F. Kaplan. 2000. A novel karyopherin-beta homolog is developmentally and hormonally regulated in fetal lung. Am. J. Respir. Cell Mol. Biol. 22:451-459). A comparison of lgl2 protein to sequences in the genome database suggested that lgl2 is a nuclear transport receptor. We report on the functional characterization of lgl2 as an importin beta protein and on the developmental regulation of its nucleocytoplasmic shuttling in fetal lung. We investigated the subcellular localization and Ran-binding properties of lgl2 and its N- and C-terminal regions. We used fluorescence recovery after photobleaching and fluorescence loss in photobleaching to study nucleocytoplasmic shuttling of lgl2. We showed that N-terminal lgl2 supports shuttling at a reduced rate. We showed that the nucleocytoplasmic distribution of lgl2 favors the nucleus in fetal lung and that lgl2 enters the nucleus much more rapidly at fetal Day 18 than at Day 21. Total nuclear recovery of lgl2 was dramatically different at the two time points. Early in development, nuclear import of transcription factors in response to hormones and growth agonists regulates prominent signal transduction pathways that govern lung organogenesis. We speculate that lgl2 may be one important modulator of this process.
Asunto(s)
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Pulmón/embriología , beta Carioferinas/metabolismo , Transporte Activo de Núcleo Celular , Animales , Transporte Biológico , Desarrollo Embrionario y Fetal , Células HeLa , Humanos , Pulmón/fisiología , Señales de Localización Nuclear , Ratas , Ratas Wistar , Transducción de Señal , Fracciones Subcelulares , beta Carioferinas/genética , Proteína de Unión al GTP ran/metabolismoRESUMEN
Although Africa has played a central role in human evolutionary history, certain studies have suggested that not all contemporary human genetic diversity is of recent African origin. We investigated 35 simple polymorphic sites and one T(n) microsatellite in an 8-kb segment of the dystrophin gene. We found 86 haplotypes in 1,343 chromosomes from around the world. Although a classical out-of-Africa topology was observed in trees based on the variant frequencies, the tree of haplotype sequences reveals three lineages accounting for present-day diversity. The proportion of new recombinants and the diversity of the T(n) microsatellite were used to estimate the age of haplotype lineages and the time of colonization events. The lineage that underwent the great expansion originated in Africa prior to the Upper Paleolithic (27,000-56,000 years ago). A second group, of structurally distinct haplotypes that occupy a central position on the tree, has never left Africa. The third lineage is represented by the haplotype that lies closest to the root, is virtually absent in Africa, and appears older than the recent out-of-Africa expansion. We propose that this lineage could have left Africa before the expansion (as early as 160,000 years ago) and admixed, outside of Africa, with the expanding lineage. Contemporary human diversity, although dominated by the recently expanded African lineage, thus represents a mosaic of different contributions.