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Silencing of GATA5 gene expression as a result of promoter hypermethylation has been observed in lung, gastrointestinal and ovarian cancers. However, the regulation of GATA5 gene expression has been poorly understood. In the present study, we have demonstrated that an E (enhancer)-box in the GATA5 promoter (bp −118 to −113 in mice; bp −164 to −159 in humans) positively regulates GATA5 transcription by binding USF1 (upstream stimulatory factor 1). Using site-directed mutagenesis, EMSA (electrophoretic mobility-shift analysis) and affinity chromatography, we found that USF1 specifically binds to the E-box sequence (5′-CACGTG-3′), but not to a mutated E-box. CpG methylation of this E-box significantly diminished its binding of transcription factors. Mutation of the E-box within a GATA5 promoter fragment significantly decreased promoter activity in a luciferase reporter assay. Chromatin immunoprecipitation identified that USF1 physiologically interacts with the GATA5 promoter E-box in mouse intestinal mucosa, which has the highest GATA5 gene expression in mouse. Co-transfection with a USF1 expression plasmid significantly increased GATA5 promoter-driven luciferase transcription. Furthermore, real-time and RT (reverse transcription)–PCR analyses confirmed that overexpression of USF1 activates endogenous GATA5 gene expression in human bronchial epithelial cells. The present study provides the first evidence that USF1 activates GATA5 gene expression through the E-box motif and suggests a potential mechanism (disruption of the E-box) by which GATA5 promoter methylation reduces GATA5 expression in cancer. [ABSTRACT FROM AUTHOR]

The recent explosion of genomic data and technology points to opportunities to redefine lung diseases at the molecular level; to apply integrated genomic approaches to elucidate mechanisms of lung pathophysiology; and to improve early detection, diagnosis, and treatment of lung diseases. Research is needed to translate genomic discoveries into clinical applications, such as detecting preclinical disease, predicting patient outcomes, guiding treatment choices, and most of all identifying potential therapeutic targets for lung diseases. The Division of Lung Diseases in the National Heart, Lung, and Blood Institute convened a workshop, "Genomic Medicine and Lung Diseases," to discuss the potential for integrated genomics and systems approaches to advance 21st century pulmonary medicine and to evaluate the most promising opportunities for this next phase of genomics research to yield clinical benefit. Workshop sessions included (1) molecular phenotypes, molecular biomarkers, and therapeutics; (2) new technology and opportunity; (3) integrative genomics; (4) molecular anatomy of the lung; (5) novel data and information platforms; and (6) recommendations for exceptional research opportunities in lung genomics research. [ABSTRACT FROM AUTHOR]

Airway smooth muscle (ASM) hypertrophy is a cardinal feature of severe asthma, but the underlying molecular mechanisms remain uncertain. Forced protein kinase B/Akt 1 activation is known to induce myocyte hypertrophy in other muscle types, and, since a number of mediators present in asthmatic airways can activate Akt signaling, we hypothesized that Akt activation could contribute to ASM hypertrophy in asthma. To test this hypothesis, we evaluated whether Akt activation occurs naturally within airway myocytes in situ, whether Akt1 activation is sufficient to cause hypertrophy of normal airway myocytes, and whether such hypertrophy is accompanied by excessive accumulation of contractile apparatus proteins (contractile phenotype maturation). Immunostains of human airway sections revealed concordant activation of Akt (reflected in Ser473 phosphorylation) and of its downstream effector p70S6Kinase (reflected in Thr389 phosphorylation) within airway muscle bundles, but there was no phosphorylation of the alternative Akt downstream target glycogen synthase kinase (GSK) 3ß. Artificial overexpression of constitutively active Akt1 (by plasmid transduction or lentiviral infection) caused a progressive increase in size and protein content of cultured canine tracheal myocytes and increased p70S6Kinase phosphorylation but not GSK3ß phosphorylation; however, constitutively active Akt1 did not cause disproportionate overaccumulation of smooth muscle (sm) α-actin and SM22. Furthermore, mRNAs encoding sm-α-actin and SM22 were reduced. These results indicate that forced Akt1 signaling causes hypertrophy of cultured airway myocytes without inducing further contractile phenotypic maturation, possibly because of opposing effects on contractile protein gene transcription and translation, and suggest that natural activation of Akt1 plays a similar role in asthmatic ASM. [ABSTRACT FROM AUTHOR]

GATA5 is a member of the GATA zinc finger transcription factor family involved in tissue-specific transcriptional regulation during cell differentiation and embryogenesis. Previous reports indicate that null mutation of the zebrafish GATA5 gene results in embryonic lethality, whereas deletion of exon 1 from the mouse GATA5 gene causes only derangement of female urogenital development. Here, we have identified an alternate promoter within intron 1 of the mouse GATA5 gene that transcribes a 2.5-kb mRNA that lacks exon 1 entirely but includes 82 bp from intron 1 and all of exons 2-6. The alternative promoter was active during transient transfection in cultured airway myocytes and bronchial epithelial cells, and it drove reporter gene expression in gastric epithelial cells in transgenic mice. The 2.5-kb alternative transcript encodes an NH2-terminally truncated "short GATA5" comprising aa 226-404 with a single zinc finger, which retains ability to transactivate the atrial natriuretic factor promoter (albeit less efficiently than full-length GATA5). Another new GATA5 transcript contains all of exons 1-5 and the 5' portion of exon 6 but lacks the terminal 1143 bp of the 3'-untranslated region from exon 6. These findings extend current understanding of the tissue distribution of GATA5 expression and suggests that GATA5 expression and function are more complex than previously appreciated. [ABSTRACT FROM AUTHOR]

Rationale: Airway smooth muscle (SM) of patients with asthma exhibits a greater velocity of shortening (Vmax) than that of normal subjects, and this is thought to contribute to airway hyperresponsiveness. A greater Vmax can result from increased myosin activation. This has been reported in sensitized human airway SM and in models of asthma. A faster Vmax can also result from the expression of specific contractile proteins that promote faster cross-bridge cycling. This possibility has never been addressed in asthma.Objectives: We tested the hypothesis that the expression of genes coding for SM contractile proteins is altered in asthmatic airways and contributes to their increased Vmax.Methods: We quantified the expression of several genes that code for SM contractile proteins in mild allergic asthmatic and control human airway endobronchial biopsies. The function of these contractile proteins was tested using the in vitro motility assay.Measurements and Main Results: We observed an increased expression of the fast myosin heavy chain isoform, transgelin, and myosin light chain kinase in patients with asthma. Immunohistochemistry demonstrated the expression of these genes at the protein level. To address the functional significance of this overexpression, we purified tracheal myosin from the hyperresponsive Fisher rats, which also overexpress the fast myosin heavy chain isoform as compared with the normoresponsive Lewis rats, and found a faster rate of actin filament propulsion. Conversely, transgelin did not alter the rate of actin filament propulsion.Conclusions: Selective overexpression of airway smooth muscle genes in asthmatic airways leads to increased Vmax, thus contributing to the airway hyperresponsiveness observed in asthma. [ABSTRACT FROM AUTHOR]

There is abundant evidence that tidal breathing, and especially tidal breathing at elevated minute ventilation, antagonizes the development and persistence of airflow obstruction during bronchoconstrictor stimulation in normal animals and people. Here, we studied the antiobstructive effect of different tidal breathing patterns in C57B1/6J and A/J mice during bronchoconstriction induced by continuous or bolus infusion of methacholine. Anesthetized, paralyzed mice were mechanically ventilated at 1,500 ml·kg-1·min-1, using each of three breathing patterns: 5 ml/kg, 300 breath/min; 10 ml/kg, 150 breath/min; or 20 ml/kg, 75 breath/min. Changing from 10 ml/kg, 150 breath/min to 20 ml/kg, 75 breath/min, breathing functionally antagonized bronchoconstriction, reducing the level of airflow obstruction induced by methacholine infusion or boluses equivalently in both strains. In marked contrast, changing from 10 ml/kg, 150 breath/min to 5 ml/kg, 300 breath/min, breathing substantially exacerbated methacholine-induced airflow obstruction in A/J mice, whereas it had no significant effect in C57B1/6J mice. Our results therefore demonstrate that 1) even at moderate, fixed minute ventilation, the precise breathing pattern can influence the degree of airflow obstruction substantially, and 2) the influence of breathing pattern on bronchoconstriction differs considerably between genetically diverse inbred mouse strains. These findings imply that differences in antiobstructive effects of breathing can contribute to differences in apparent airway constrictor responsiveness. Much attention has been placed on dysregulation of contractile function of airway smooth muscle in human disease. We suggest that important pathophysiology might also be found in impairment of the functional antagonist effect of tidal breathing on airflow obstruction. [ABSTRACT FROM AUTHOR]

Rationale: Recent genetic studies have implicated integrins in asthma and atopy susceptibility. We therefore evaluated the integrin-beta3 gene (ITGB3), an integrin gene within an asthma linkage peak on chromosome 17, as a candidate for susceptibility to asthma- and atopy-related phenotypes.Methods and Measurements: We genotyped and performed association tests on 19 single nucleotide polymorphisms in ITGB3 in the Hutterites, a founder population, and in three outbred replication populations.Main Results: Variation in ITGB3 was strongly associated with susceptibility to bronchial hyperresponsiveness and protection from allergic sensitization to mold allergens in this population. Three independent case-control populations representing Caucasians and African Americans were used to replicate this finding, also revealing ITGB3 alleles that are associated with asthma susceptibility and protection from mold allergen sensitization.Conclusions: This study provides evidence that ITGB3 plays a role in the pathogenesis of asthma and sensitization to mold allergens. [ABSTRACT FROM AUTHOR]

1. Airway smooth muscle (ASM) has recently been termed the 'frustrated' cell of the lung given that contraction of ASM has no proven useful physiological function in adults and yet is indelibly associated with pathological conditions by virtue of its unwanted airflow-limiting actions in asthma. In contrast, pulmonary vascular smooth muscle contraction plays an essential role in the control of blood flow through the lung. 2. Little is known of the differences in phenotype between human ASM and pulmonary vascular smooth muscle (VSM) tissues, but differences in contractile protein and transcription factor expression and regulation of contractile protein promoter activity have been documented. Similarly, the embryological signals in mice required for differentiation of ASM versus pulmonary VSM are distinct. 3. Bronchoconstriction in asthma is currently treated with β2-adrenoceptor agonists, which relax contracted ASM cells. An additional approach may be to use gene therapy to render ASM unable to contract (via disruption of their contractile apparatus organization). 4. Application of ASM-specific gene therapies would rely on minimal actions on other lung smooth muscle tissues, including pulmonary and bronchial vascular smooth muscle. The combination of mRNA analysis of laser-captured micro- dissected tissue with in situ immunohistochemical staining for protein should be very useful in terms of being able to characterize definitively the differences in mRNA and protein expression between the smooth muscle species of the lung. Any discovery of an A SM-selective target could provide a novel lead for ASM-directed anti-asthma therapy. [ABSTRACT FROM AUTHOR]

Atopy is an IgE-mediated condition known to aggregate in families and is a major risk factor for asthma. As part of the Collaborative Study on the Genetics of Asthma (CSGA), a genome-wide scan for atopy, defined by skin sensitivity to one or more common environmental allergens, was conducted in 287 CSGA families (115 African American, 138 Caucasian and 34 Hispanic). Using a nonparametric genetic analysis approach, two regions were observed in the sample of all families that yielded multipoint lod scores >1.5 (chromosome 11q, lod=1.55 between D11S1986 and D11S1998; chromosome 20p between D20S473 and D20S604, lod=1.54). Modeling that included multiple genomic positions simultaneously indicated that four chromosomal regions accounted for the majority of evidence for linkage in the combined families. These four regions are on chromosomes 10p near D10S1412 (lod=0.94), 11q near D11S1986 (lod=1.76), 17q near D17S784 (lod=0.97) and 20p near D20S473 (lod=1.74). In the subset of pedigrees giving positive evidence for linkage on chromosome 11q, the evidence for linkage increased by lod scores greater than one in four other chromosomal regions: 5q (D5S1480, lod=1.65), 8p (D8S1113, lod=1.60), 12p (D12S372, lod=1.54) and 14q (D14S749, lod=1.70). These results suggest that several regions may harbor genes contributing to the risk for atopy and these may interact with one another in a complex manner. [ABSTRACT FROM AUTHOR]

Studies the effects of inflammatory mediators on airway smooth muscle contraction to airway hyperresponsiveness in asthma. Smooth muscle contractile apparatus dysfunction in asthma; Mediators involved in asthma inflammation.

Objective -- To evaluate in humans the mechanisms underlying the increase in nasal mucosal temperature following immersion of the feet in warm water (42°C). Material and Methods -- The nasal mucosal temperature of subjects was measured whilst their feet were immersed in warm water. Results -- The nasal mucosal temperature rose quickly on immersion and dissipated equally fast on removal of the feet from warm water. Intranasal lidocaine raised the mucosal temperature slightly after application, but also blocked the feet warming-induced increase in nasal mucosal temperature, suggesting a neural reflex. Whereas the cutaneous-nasal reflex stimulates a transient parasympathetic response, acetylcholine does not seem to contribute to the more prolonged increase in nasal mucosal temperature following immersion of the feet in warm water. Warming of the feet probably leads to a loss of α-sympathetic activity of nasal blood vessels and an increase in nasal mucosal temperature because application of phenoxybenzamine, an α-sympathetic blocking agent, to the nasal mucosa increased the nasal mucosal temperature. Conclusions -- Our data suggest that the increase in nasal mucosal temperature after warming of the feet is mediated by a neural reflex, which is caused by loss of sympathetic activity of the nasal vasculature and a possible additional contribution of a long-acting parasympathetic mediator. [ABSTRACT FROM AUTHOR]

Background: Increasing functional residual capacity (FRC) or tidal volume (VT) reduces airway resistance and attenuates the response to bronchoconstrictor stimuli in animals and humans. What is unknown is which one of the above mechanisms is more effective in modulating airway caliber and whether their combination yields additive or synergistic effects. To address this question, we investigated the effects of increased FRC and increased VT in attenuating the bronchoconstriction induced by inhaled methacholine (MCh) in healthy humans. Methods: Nineteen healthy volunteers were challenged with a single-dose of MCh and forced oscillation was used to measure inspiratory resistance at 5 and 19 Hz (R5 and R19), their difference (R5-19), and reactance at 5 Hz (X5) during spontaneous breathing and during imposed breathing patterns with increased FRC, or VT, or both. Importantly, in our experimental design we held the product of VT and breathing frequency (BF), i.e, minute ventilation (VE) fixed so as to better isolate the effects of changes in VT alone. Results: Tripling VT from baseline FRC significantly attenuated the effects of MCh on R5, R19, R5-19 and X5. Doubling VT while halving BF had insignificant effects. Increasing FRC by either one or two VT significantly attenuated the effects of MCh on R5, R19, R5-19 and X5. Increasing both VT and FRC had additive effects on R5, R19, R5-19 and X5, but the effect of increasing FRC was more consistent than increasing VT thus suggesting larger bronchodilation. When compared at iso-volume, there were no differences among breathing patterns with the exception of when VT was three times larger than during spontaneous breathing. Conclusions: These data show that increasing FRC and VT can attenuate induced bronchoconstriction in healthy humans by additive effects that are mainly related to an increase of mean operational lung volume. We suggest that static stretching as with increasing FRC is more effective than tidal stretching at constant VE, possibly through a combination of effects on airway geometry and airway smooth muscle dynamics. [ABSTRACT FROM AUTHOR]

Background: Genome-wide association studies have yet to identify the majority of genetic variants involved in asthma. We hypothesized that expression quantitative trait locus (eQTL) mapping can identify novel asthma genes by enabling prioritization of putative functional variants for association testing. Objective: We evaluated 6,706 cis-acting expression-associated variants (eSNP) identified through a genome-wide eQTL survey of CD4+ lymphocytes for association with asthma. Methods: eSNP were tested for association with asthma in 359 asthma cases and 846 controls from the Childhood Asthma Management Program, with verification using family-based testing. Significant associations were tested for replication in 579 parent-child trios with asthma from Costa Rica. Further functional validation was performed by Formaldehyde Assisted Isolation of Regulatory Elements (FAIRE)-qPCR and Chromatin-Immunoprecipitation (ChIP)-PCR in lung derived epithelial cell lines (Beas-2B and A549) and Jurkat cells, a leukemia cell line derived from T lymphocytes. Results: Cis-acting eSNP demonstrated associations with asthma in both cohorts. We confirmed the previously-reported association of ORMDL3/GSDMB variants with asthma (combined p=2.9 × 108). Reproducible associations were also observed for eSNP in three additional genes: FADS2 (p=0.002), NAGA (p=0.0002), and F13A1 (p=0.0001). We subsequently demonstrated that FADS2 mRNA is increased in CD4+ lymphocytes in asthmatics, and that the associated eSNPs reside within DNA segments with histone modifications that denote open chromatin status and confer enhancer activity. Conclusions: Our results demonstrate the utility of eQTL mapping in the identification of novel asthma genes, and provide evidence for the importance of FADS2, NAGA, and F13A1 in the pathogenesis of asthma.

Investigates epithelial modulation of tracheal smooth muscle (TSM) responsiveness in vitro from guinea pigs receiving active immune sensitization in vivo. Effect of ovalbumin on muscle contraction; TSM's response to electric field stimulation; Evaluation of sympathomimetic and muscarinic responsiveness.