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1.
Exp Lung Res ; 44(1): 13-24, 2018 02.
Artículo en Inglés | MEDLINE | ID: mdl-29324052

RESUMEN

Claudins are tight junctional proteins implicated in cell polarity and epithelial barrier maintenance. Claudin misregulation adversely impacts developmental aspects of cell differentiation and proliferation. The current research evaluated transcriptional expression for Claudins 1-11 and 18 in the developing murine lung at embryonic days (E) 14.5, 16.5, and 18.5 and at post-natal day (PN) 3 and PN15. Mouse lungs were also assessed by immunohistochemical analysis to qualitatively evaluate Claudin protein expression. Pregnant dams were further exposed to secondhand smoke (SHS) from embryonic day (E)15.5 to 18.5 and Claudin mRNA was immediately screened in pup lungs. Other than Claudin-6, mRNA expression patterns for Claudin family members tended to decrease at E16.5, increase at E18.5, and decrease again at PN3 before reaching a peak of expression at PN15. Claudin-6 mRNA expression decreased through gestation and into post-natal periods. Immunohistochemical profiling implicated a subset of Claudins as plausible orchestrators of proximal vs. distal lung barrier establishment. Assessment of Claudin mRNA expression at E18.5 following SHS exposure revealed a significant reduction in transcription for all Claudins except Claudin-18 (no change). These data support the need for further studies using gene targeted mice that knock-in/out specific Claudins so that precise functions in the normal and diseased lung can be determined.


Asunto(s)
Claudinas/biosíntesis , Pulmón/crecimiento & desarrollo , Contaminación por Humo de Tabaco/efectos adversos , Transcriptoma/efectos de los fármacos , Animales , Claudinas/efectos de los fármacos , Claudinas/genética , Embrión de Mamíferos , Femenino , Pulmón/efectos de los fármacos , Pulmón/embriología , Ratones , Embarazo , ARN Mensajero/efectos de los fármacos , ARN Mensajero/metabolismo , Uniones Estrechas
2.
Exp Lung Res ; 42(8-10): 440-452, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27982694

RESUMEN

PURPOSE: Chronic obstructive pulmonary disease is a condition involving perturbed barrier integrity coincident with both emphysema and inflammation of the airways, and smoking is considered a major risk factor. Claudins (Cldns) stabilize barriers and contribute to tight junctions by preventing paracellular transport of extracellular fluid constituents. METHODS: To determine Cldn6 was differentially influenced by tobacco smoke, Cldn6 was evaluated in cells and tissues by q-PCR, immunoblotting, and immunohistochemistry following exposure. Cldn6 transcriptional regulation was also assessed using luciferase reporter constructs. RESULTS: Q-PCR and immunoblotting revealed that Cldn6 was decreased in alveolar type II-like epithelial cells (A549) and primary small airway epithelial cells when exposed to cigarette smoke extract (CSE). Cldn6 was also markedly decreased in the lungs of mice exposed to acute tobacco smoke delivered by a nose-only automated smoke machine compared to controls. Luciferase reporter assays incorporating 0.5-kb, 1.0-kb, or 2.0-kb of the Cldn6 promoter revealed decreased transcription of Cldn6 following exposure to CSE. Cldn6 transcriptional regulation was also assessed in hypoxic conditions due to low oxygen tension observed during smoking. Hypoxia and hypoxia inducible factor-1 alpha caused decreased transcription of the Cldn6 gene via interactions with putative response elements in the proximal promoter sequence. CONCLUSIONS: These data reveal that tight junctional proteins such as Cldn6 are differentially regulated by tobacco-smoke exposure and that Cldns are potentially targeted when epithelial cells respond to tobacco smoke. Further research may show that Cldns expressed in tight junctions between parenchymal cells contribute to impaired structural integrity of the lung coincident with smoking.


Asunto(s)
Claudinas/biosíntesis , Pulmón/metabolismo , Oxígeno/metabolismo , Humo/efectos adversos , Células A549 , Células Epiteliales Alveolares/citología , Células Epiteliales Alveolares/efectos de los fármacos , Animales , Células Cultivadas , Claudinas/efectos adversos , Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Pulmón/patología , Ratones , Tejido Parenquimatoso/citología , Tejido Parenquimatoso/efectos de los fármacos , Proteínas de Uniones Estrechas/efectos de los fármacos , Uniones Estrechas
3.
Physiol Genomics ; 47(9): 376-85, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26152686

RESUMEN

Chronic treatment with the ß-blocker carvedilol has been shown to reduce established maladaptive left ventricle (LV) hypertrophy and to improve LV function in experimental heart failure. However, the detailed mechanisms by which carvedilol improves LV failure are incompletely understood. We previously showed that carvedilol is a ß-arrestin-biased ß1-adrenergic receptor ligand, which activates cellular pathways in the heart independent of G protein-mediated second messenger signaling. More recently, we have demonstrated by microRNA (miR) microarray analysis that carvedilol upregulates a subset of mature and pre-mature miRs, but not their primary miR transcripts in mouse hearts. Here, we next sought to identify the effects of carvedilol on LV gene expression on a genome-wide basis. Adult mice were treated with carvedilol or vehicle for 1 wk. RNA was isolated from LV tissue and hybridized for microarray analysis. Gene expression profiling analysis revealed a small group of genes differentially expressed after carvedilol treatment. Further analysis categorized these genes into pathways involved in tight junction, malaria, viral myocarditis, glycosaminoglycan biosynthesis, and arrhythmogenic right ventricular cardiomyopathy. Genes encoding proteins in the tight junction, malaria, and viral myocarditis pathways were upregulated in the LV by carvedilol, while genes encoding proteins in the glycosaminoglycan biosynthesis and arrhythmogenic right ventricular cardiomyopathy pathways were downregulated by carvedilol. These gene expression changes may reflect the molecular mechanisms that underlie the functional benefits of carvedilol therapy.


Asunto(s)
Antagonistas Adrenérgicos beta/farmacología , Carbazoles/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Ventrículos Cardíacos/efectos de los fármacos , Propanolaminas/farmacología , Animales , Cardiomiopatías/genética , Carvedilol , Glicosaminoglicanos/biosíntesis , Glicosaminoglicanos/genética , Malaria/genética , Ratones Endogámicos C57BL , Miocarditis/genética , Proteínas/genética , Proteínas/metabolismo , Función Ventricular Izquierda/efectos de los fármacos
4.
Respir Res ; 15: 70, 2014 Jun 26.
Artículo en Inglés | MEDLINE | ID: mdl-24970044

RESUMEN

BACKGROUND: Claudins are transmembrane proteins expressed in tight junctions that prevent paracellular transport of extracellular fluid and a variety of other substances. However, the expression profile of Claudin-6 (Cldn6) in the developing lung has not been characterized. METHODS AND RESULTS: Cldn6 expression was determined during important periods of lung organogenesis by microarray analysis, qPCR and immunofluorescence. Expression patterns were confirmed to peak at E12.5 and diminish as lung development progressed. Immunofluorescence revealed that Cldn6 was detected in cells that also express TTF-1 and FoxA2, two critical transcriptional regulators of pulmonary branching morphogenesis. Cldn6 was also observed in cells that express Sox2 and Sox9, factors that influence cell differentiation in the proximal and distal lung, respectively. In order to assess transcriptional control of Cldn6, 0.5, 1.0, and 2.0-kb of the proximal murine Cldn6 promoter was ligated into a luciferase reporter and co-transfected with expression vectors for TTF-1 or two of its important transcriptional co-regulators, FoxA2 and Gata-6. In almost every instance, TTF-1, FoxA2, and Gata-6 activated gene transcription in cell lines characteristic of proximal airway epithelium (Beas2B) and distal alveolar epithelium (A-549). CONCLUSIONS: These data revealed for the first time that Cldn6 might be an important tight junctional component expressed by pulmonary epithelium during lung organogenesis. Furthermore, Cldn6-mediated aspects of cell differentiation may describe mechanisms of lung perturbation coincident with impaired cell junctions and abnormal membrane permeability.


Asunto(s)
Claudinas/fisiología , Proteínas de Unión al ADN/fisiología , Factor de Transcripción GATA6/fisiología , Factor Nuclear 3-beta del Hepatocito/fisiología , Pulmón/crecimiento & desarrollo , Transcripción Genética/fisiología , Animales , Animales Recién Nacidos , Regulación del Desarrollo de la Expresión Génica , Pulmón/embriología , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Mucosa Respiratoria/crecimiento & desarrollo , Mucosa Respiratoria/metabolismo , Factores de Transcripción
5.
Respir Res ; 15: 133, 2014 Oct 29.
Artículo en Inglés | MEDLINE | ID: mdl-25359169

RESUMEN

BACKGROUND: Receptors for advanced glycation end-products (RAGE) are multiligand cell-surface receptors expressed abundantly by distal pulmonary epithelium. Our lab has discovered RAGE-mediated effects in the orchestration of lung inflammation induced by tobacco smoke and environmental pollutants; however, the specific contribution of RAGE to the progression of proximal airway inflammation is still inadequately characterized. METHODS AND RESULTS: We generated a Tet-inducible transgenic mouse that conditionally overexpressed RAGE using the club cell (Clara) secretory protein (CCSP) promoter expressed by club (Clara) cells localized to the proximal airway. RAGE was induced for 40 days from weaning (20 days of age) until sacrifice date at 60 days. Immunohistochemistry, immunoblotting, and qPCR revealed significant RAGE up-regulation when compared to non-transgenic controls; however, H&E staining revealed no detectible morphological abnormalities and apoptosis was not enhanced during the 40 days of augmentation. Freshly procured bronchoalveolar lavage fluid (BALF) from CCSP-RAGE TG mice had significantly more total leukocytes and PMNs compared to age-matched control littermates. Furthermore, CCSP-RAGE TG mice expressed significantly more tumor necrosis factor alpha (TNF-α), interleukin 7 (IL-7), and interleukin 14 (IL-14) in whole lung homogenates compared to controls. CONCLUSIONS: These data support the concept that RAGE up-regulation specifically in lung airways may function in the progression of proximal airway inflammation.


Asunto(s)
Células Epiteliales Alveolares/metabolismo , Neumonía/metabolismo , Receptor para Productos Finales de Glicación Avanzada/metabolismo , Células Epiteliales Alveolares/inmunología , Animales , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/inmunología , Genotipo , Mediadores de Inflamación/metabolismo , Interleucina-7/metabolismo , Interleucinas/metabolismo , Ratones Transgénicos , Infiltración Neutrófila , Fenotipo , Neumonía/genética , Neumonía/inmunología , Regiones Promotoras Genéticas , Receptor para Productos Finales de Glicación Avanzada/genética , Factor de Necrosis Tumoral alfa/metabolismo , Regulación hacia Arriba , Uteroglobina/genética , Proteínas de Transporte Vesicular
6.
Respir Res ; 15: 129, 2014 Oct 23.
Artículo en Inglés | MEDLINE | ID: mdl-25338658

RESUMEN

BACKGROUND: Receptors for advanced glycation end-products (RAGE) are immunoglobulin-like pattern recognition receptors abundantly localized to lung epithelium. Our research demonstrated that primary tobacco smoke exposure increases RAGE expression and that RAGE partly mediates pro-inflammatory signaling during exposure. However, the degree to which RAGE influences developing lungs when gestating mice are exposed to secondhand smoke (SHS) has not been determined to date. METHODS: Timed pregnant RAGE null and wild type control mice were exposed to 4 consecutive days of SHS from embryonic day (E) 14.5 through E18.5 using a state of the art nose-only smoke exposure system (Scireq, Montreal, Canada). RAGE expression was assessed using immunofluorescence, immunoblotting, and quantitative RT-PCR. TUNEL immunostaining and blotting for caspase-3 were performed to evaluate effects on cell turnover. Matrix abnormalities were discerned by quantifying collagen IV and MMP-9, a matrix metalloprotease capable of degrading basement membranes. Lastly, TNF-α and IL-1ß levels were assessed in order to determine inflammatory status in the developing lung. RESULTS: Pulmonary RAGE expression was elevated in both dams exposed to SHS and in fetuses gestating within mothers exposed to SHS. Fetal weight, a measure of organismal health, was decreased in SHS-exposed pups, but unchanged in SHS-exposed RAGE null mice. TUNEL assessments suggested a shift toward pulmonary cell apoptosis and matrix in SHS-exposed pups was diminished as revealed by decreased collagen IV and increased MMP-9 expression. Furthermore, SHS-exposed RAGE null mice expressed less TNF-α and IL-1ß when compared to SHS-exposed controls. CONCLUSIONS: RAGE augmentation in developing pups exposed to maternal SHS weakens matrix deposition and influences lung inflammation.


Asunto(s)
Feto/metabolismo , Pulmón/metabolismo , Neumonía/metabolismo , Efectos Tardíos de la Exposición Prenatal/metabolismo , Receptores Inmunológicos/biosíntesis , Contaminación por Humo de Tabaco/efectos adversos , Animales , Femenino , Feto/patología , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Neumonía/patología , Embarazo , Efectos Tardíos de la Exposición Prenatal/patología , Receptor para Productos Finales de Glicación Avanzada
7.
Int J Dev Biol ; 59(10-12): 479-85, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26198145

RESUMEN

Claudin 6 (Cldn6) is a tetraspanin protein expressed by barrier epithelial cells. In order to assess the effects of persistent tight junctions involving Cldn6 during lung development, a doxycycline (dox)-inducible conditional transgenic mouse was generated that up-regulates Cldn6 in the distal lung. Pups had unlimited access to dox from conception until sacrifice date at embryonic day (E) 18.5. Quantitative PCR, immunoblotting, and immunohistochemistry revealed significantly elevated Cldn6 expression in transgenic mice compared to non-transgenic controls. There were no differences in terms of lung size, lung weight, or whole body weight at the time of necropsy. Histological evaluations led to the discovery that E18.5 Cldn6 transgenic pups appeared to be in the early canalicular stage of development coincident with fewer, thickened respiratory airspaces. In contrast, controls appeared to have entered the saccular stage characterized by thin airspace walls and spherical architecture. Immunostaining for transcriptional regulators including TTF-1 and FoxA2 was conducted to assess cell differentiation and specific cell types were identified via staining for pro-surfactant protein C (alveolar type II epithelial cells) or Clara Cell Secretory Protein (cub or Clara cells). Lastly, cell turnover was qualitatively measured via staining for cell proliferation or apoptosis. These data suggest that Cldn6 is an important junctional protein potentially involved in the programming of epithelial cells during lung development. Furthermore, genetic down-regulation of Cldn6 as development proceeds may influence differentiation observed in the transition from the canalicular to the saccular lung.


Asunto(s)
Apoptosis , Proliferación Celular , Claudinas/fisiología , Embrión de Mamíferos/patología , Pulmón/embriología , Pulmón/patología , Animales , Western Blotting , Células Cultivadas , Embrión de Mamíferos/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Técnicas para Inmunoenzimas , Inmunoprecipitación , Integrasas/metabolismo , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
8.
Appl Plant Sci ; 2(10)2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25309841

RESUMEN

PREMISE OF THE STUDY: The American Cross Timbers forest ecosystem runs from southeastern Kansas to Central Texas and is primarily composed of post oak (Quercus stellata). This old-growth forest currently occupies only about 2% of its ancestral range. To facilitate genetic research on this species, we developed microsatellite primers specific to post oak from reduced genomic libraries. • METHODS AND RESULTS: Two Q. stellata individuals, sampled from the northern and southern range of the post oak forest, were subject to genomic reduction and 454 pyrosequencing. Bioinformatic analysis identified putative microsatellites from which 12 polymorphic primer sets were screened on three populations. The number of alleles observed ranged from five to 20 across all populations, while observed and expected heterozygosity values ranged from 0.05 to 0.833 and 0.236 to 0.893, respectively, within individual populations. • CONCLUSIONS: We report the development of microsatellite markers, specific to post oak, to aid the study of genetic diversity and population structure of extant forest remnants.

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