Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Smoking-induced Wnt pathway downregulation

ABSTRACT: The Wnt pathway plays a central role in controlling differentiation of epithelial tissues; when Wnt is on, differentiation is suppressed, but when Wnt is off, differentiation is allowed to proceed. Based on this concept, we hypothesized that expression of key genes in the Wnt pathway are suppressed in the human airway epithelium under the stress of cigarette smoking, a stress associated with dysregulation of the differentiated state of the airway epithelium. For this purpose, HG-U133 Plus 2.0 microarrays were used to assess the expression of Wnt-related genes in the small airway (10th-12th generation) epithelium (SAE) obtained via bronchoscopy and brushing of healthy nonsmokers (n=47), healthy smokers (n=58), and smokers with established COPD (n=22). With expression defined as present in >20% of samples, microarray analysis demonstrated that 35 of 57 known Wnt-related genes are expressed in the adult SAE. Wnt pathway downstream targets β-catenin (p<0.05) and the transcription factor 7-like 1 were down-regulated in healthy smokers, and smokers with COPD, as were a number of Wnt target genes, including VEGFA, CCND1, MMP7, CLDN1, SOX9, RHOU (all p<0.05 compared to healthy nonsmokers). As a mechanism to explain this broad, smoking-induced suppression of the Wnt pathway, we assessed expression of the DKK and SFRP families, extracellular regulators that suppress the Wnt pathway. Among these, secreted frizzled-related protein 2 (SFRP2), was up-regulated 4.3-fold (p<0.0001) in healthy smokers and 4.9-fold (p<0.0001) in COPD smokers, an observation confirmed by TaqMan Real-time PCR. AT the protein levels, Western analysis demonstrated SFRP2 up-regulation, and immunohistochemistry demonstrated that the smoking-induced SFRP2 upregulation occurred in differentiated ciliated cells. Finally, cigarette smoke extract mediated up-regulation of SFRP2 and downregulation of Wnt target genes in airway epithelial cells in vitro. These observations are consistent with the hypothesis that the Wnt pathway plays a role in airway epithelial cell differentiation in the adult human airway epithelium, with smoking associated with down-regulation of Wnt pathway, contributing to the dysregulation of airway epithelial differentiation observed in the smoking-related airway disorders. Affymetrix arrays were used to assess gene expression data of genes in the Wnt pathway in small airway epithelium obtained by fiberoptic bronchoscopy of 47 healthy non-smokers and 58 healthy smokers and 22 smokers with COPD.

ORGANISM(S): Homo sapiens

SUBMITTER: Yael Strulovici-Barel

PROVIDER: E-GEOD-19407 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Down-regulation of the canonical Wnt β-catenin pathway in the airway epithelium of healthy smokers and smokers with COPD.

Wang Rui R Ahmed Joumana J Wang Guoqing G Hassan Ibrahim I Strulovici-Barel Yael Y Hackett Neil R NR Crystal Ronald G RG

PloS one 20110407 4

<h4>Background</h4>The Wnt pathway mediates differentiation of epithelial tissues; depending on the tissue types, Wnt can either drive or inhibit the differentiation process. We hypothesized that key genes in the Wnt pathway are suppressed in the human airway epithelium under the stress of cigarette smoking, a stress associated with dysregulation of the epithelial differentiated state.<h4>Methodology/principal findings</h4>Microarrays were used to assess the expression of Wnt-related genes in th ...[more]

PMID: 21490961

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Project description:The apical junctional complex (AJC), composed of tight junctions and adherens junctions, is essential for maintaining epithelial barrier function. Since cigarette smoking and chronic obstructive pulmonary disease (COPD), the major smoking-induced disease, are both associated with increased lung epithelial permeability, we hypothesized that smoking alters the transcriptional program regulating AJC integrity in the small airway epithelium (SAE), the primary site of pathological changes in COPD. Transcriptome analysis revealed a global down-regulation of physiological AJC gene expression in the SAE of healthy smokers (n=53) compared to healthy nonsmokers (n=59), an observation associated with changes in molecular pathways regulating epithelial differentiation such as PTEN signaling and accompanied by induction of cancer-related AJC genes. Genome-wide co-expression analysis identified a smoking-sensitive AJC transcriptional network. The overall expression of AJC-associated genes was further decreased in COPD smokers (n=23). Exposure of human airway epithelial cells to cigarette smoke extract in vitro resulted in down-regulation of several AJC-related genes, accompanied by decreased transepithelial resistance. Thus, cigarette smoking alters the AJC gene expression architecture in the human airway epithelium, providing a molecular basis for the dysregulation of airway epithelial barrier function during the development of smoking-induced lung disease. The apical junctional complex (AJC), composed of tight junctions and adherens junctions, is essential for maintaining epithelial barrier function. Since cigarette smoking and chronic obstructive pulmonary disease (COPD), the major smoking-induced disease, are both associated with increased lung epithelial permeability, we hypothesized that smoking alters the transcriptional program regulating AJC integrity in the small airway epithelium (SAE), the primary site of pathological changes in COPD. In this study, microarray analysis of the SAE obtained from 53 healthy nonsmokers, 59 healthy smokers, and 23 smokers with COPD was performed to determine physiological AJC gene expression architecture in the SAE and its modification by cigarette smoking and during the development of COPD.

Project description:Nuclear factor erythroid 2-related factor 2 (NFE2L2, Nrf2) is an oxidant responsive transcription factor known to induce phase 2 detoxifying and antioxidant genes to protect cells from oxidative stress. Cigarette smoke, with its large oxidant content, is a major stressor to the small airway epithelium, the cells of which are vulnerable to oxidant damage and consequent malignant transformation. In this study, we assessed the role of cigarette smoke in activation of Nrf2 in the human small airway epithelium in vivo. Fiberoptic bronchoscopy was used to sample a pure population of small airway epithelium in 38 healthy nonsmokers and 45 healthy smokers, and gene expression was assessed using Affymetrix HG-U133 Plus 2.0 microarrays. Compared to that of healthy nonsmokers, Nrf2 protein was significantly activated in the small airway epithelium of healthy normal smokers and localized in the nucleus (p<0.05). Of the human homologs of 201 known murine Nrf2-mediated genes, 13 highly smoking-responsive genes were identified (p<10-4, all comparisons smokers to nonsmokers). Using a “Nrf2-index” to quantify the extent of expression in the small airway epithelium of these 13 known Nrf2 genes, variability in the level of expression was observed among the 45 healthy smokers, but the variability was coordinately modulated among the 13 genes, an observation confirmed by TaqMan quantitative PCR. This variability in the coordinate level of expression of the 13 Nrf2-mediated genes was independent of the smoking history. Based on these observations, the “Nrf2 index” was used to evaluate whether other genes modulated by smoking in the small airway epithelium were also coordinately up- or down- modulated among the 45 healthy smokers. Two genes, pirin (PIR) and UDP glucuronosyltransferase 1 family polypeptide A4 (UGT1A4), not previously known to be modulated by Nrf2 were identified as being coordinately modulated among the 45 smokers. Both genes contain several functional antioxidant response elements in the promoter region. Using an electrophoretic mobility shift assay, these antioxidant response elements in the promoters of PIR and UGT1A4 responded in vitro to activated Nrf2. These observations are consistent with the concept that Nrf2 plays an important role in regulating cellular defenses against smoking in the highly vulnerable small airway epithelium cell population, and that there is variability among the population in the relative Nrf2 responsiveness to a similar oxidant burden. Affymetrix arrays were used to assess gene expression data in small airway epithelium obtained by fiberoptic bronchoscopy of 38 healthy non-smokers and 45 healthy smokers

Project description:The Wnt pathway plays a central role in controlling differentiation of epithelial tissues; when Wnt is on, differentiation is suppressed, but when Wnt is off, differentiation is allowed to proceed. Based on this concept, we hypothesized that expression of key genes in the Wnt pathway are suppressed in the human airway epithelium under the stress of cigarette smoking, a stress associated with dysregulation of the differentiated state of the airway epithelium. For this purpose, HG-U133 Plus 2.0 microarrays were used to assess the expression of Wnt-related genes in the small airway (10th-12th generation) epithelium (SAE) obtained via bronchoscopy and brushing of healthy nonsmokers (n=47), healthy smokers (n=58), and smokers with established COPD (n=22). With expression defined as present in >20% of samples, microarray analysis demonstrated that 35 of 57 known Wnt-related genes are expressed in the adult SAE. Wnt pathway downstream targets β-catenin (p<0.05) and the transcription factor 7-like 1 were down-regulated in healthy smokers, and smokers with COPD, as were a number of Wnt target genes, including VEGFA, CCND1, MMP7, CLDN1, SOX9, RHOU (all p<0.05 compared to healthy nonsmokers). As a mechanism to explain this broad, smoking-induced suppression of the Wnt pathway, we assessed expression of the DKK and SFRP families, extracellular regulators that suppress the Wnt pathway. Among these, secreted frizzled-related protein 2 (SFRP2), was up-regulated 4.3-fold (p<0.0001) in healthy smokers and 4.9-fold (p<0.0001) in COPD smokers, an observation confirmed by TaqMan Real-time PCR. AT the protein levels, Western analysis demonstrated SFRP2 up-regulation, and immunohistochemistry demonstrated that the smoking-induced SFRP2 upregulation occurred in differentiated ciliated cells. Finally, cigarette smoke extract mediated up-regulation of SFRP2 and downregulation of Wnt target genes in airway epithelial cells in vitro. These observations are consistent with the hypothesis that the Wnt pathway plays a role in airway epithelial cell differentiation in the adult human airway epithelium, with smoking associated with down-regulation of Wnt pathway, contributing to the dysregulation of airway epithelial differentiation observed in the smoking-related airway disorders.

Project description:Disparate Oxidant-related Gene Expression of Human Small Airway Epithelium Compared to Autologous Alveolar Macrophages in Response to the In Vivo Oxidant Stress of Cigarette Smoking The oxidant burden of cigarette smoking induces lung cell dysfunction, and play a significant role in the pathogenesis of lung disease. Two cell populations directly exposed to the oxidants in cigarette smoke are the small airway epithelium and alveolar macrophages. Of these, the epithelium appears to be more vulnerable to smoking, becoming disordered in differentiation, repair and function, while alveolar macrophages become activated, without becoming diseased. In this context, we asked: for the same individuals, what is the baseline trancriptome of oxidant-related genes in small airway epithelium compared to alveolar macrophages and do the responses of the transcriptome of these 2 cell populations differ substantially to inhaled cigarette smoke? To address these questions we used microarray gene expression and TaqMan analysis to assess the gene expression profile of known oxidant-related genes in paired samples recovered by bronchoscopy from small airway epithelium and alveolar macrophages from the same healthy nonsmokers and normal smokers. Of the 155 oxidant-related genes surveyed, 122 (77%) were expressed in both cell populations in nonsmokers. However, of the genes expressed by both cell populations, oxidant related gene expression levels were higher in alveolar macrophages (67 genes, 43%) than small airway epithelium (37 genes, 24%). There were more oxidant-related genes uniquely expressed in the small airway epithelium (17%), than in alveolar macrophages (5%). In healthy smokers, the majority of oxidant-related genes were expressed in both cell populations, but there were marked differences in the numbers of oxidant-related genes that smoking up- or down-regulated. While smoking up-regulated 15 genes (10%) and down-regulated 7 genes (5%) in the small airway epithelium, smoking had far less effect on alveolar macrophages [only 4 (3%) genes up-regulated, and only 1 (0.6%) down-regulated]. Only a small number of smoking responsive oxidant-related genes overlapped between the two cell types (2 up-regulated, and no down-regulated genes). Consistent with this observation, pathway analysis of smoking-responsive genes in the small airway epithelium showed oxidant-related pathways dominated, but in alveolar macrophages immune-response pathways dominated. Thus, the responses of the oxidant-related transcriptome of cells with an identical genome and exposed to the same oxidant stress of cigarette smoking are very different, with responses of oxidant-related genes of alveolar macrophages far more subdued than that of small airway epithelium, consistent with the clinical observation that, while the small airway epithelium is vulnerable, alveolar macrophages are not "diseased" in response to the oxidant stress of cigarette smoking. Gene expression profiles of known oxidant-related genes in paired samples recovered by bronchoscopy from small airway epithelium and alveolar macrophages from the same healthy nonsmokers and normal smokers.

Project description:Down-regulation of the Notch Differentiation Pathway in the Human Airway Epithelium in Normal Smokers and Smokers with Chronic Obstructive Lung Disease; In cigarette smokers, the toxic components of smoke place the epithelium under the constant stress of a variety of mechanisms of injury, with consequent modulation of airway epithelial regeneration and disordered differentiation. Based on the underlying hypothesis that these airway epithelial changes must involve quantitative changes in genes involved with the regulation of differentiation, we assessed the expression of the Notch pathway, a signaling pathway known to play a fundamental role in the embryonic lung as a gatekeeper for differentiation, in the small airway epithelium of non-smokers, normal smokers, and smokers with COPD. Microarray analysis demonstrated that 45 of the 55 Notch pathway-related genes are expressed in the human adult small airway epithelium and TaqMan quantitative PCR confirmed the expression of key genes in the pathway. TaqMan quantitative PCR analysis of the normal small airway epithelium demonstrated that Delta-like ligand 1 is the most highly expressed Notch ligand, Notch2 and 3 the most highly expressed receptor genes, and Hes1 the predominant downstream effector gene. TaqMan PCR was used to compare gene expression in nonsmokers vs healthy smokers vs smokers with COPD. The data show that some key genes in the ligands, receptors and downstream effectors in the Notch pathway are differentially expressed in smokers, with significant downregulation of a greater number of Notch-related genes in smokers with COPD compared to healthy smokers. These observations are consistent with the hypothesis that the Notch pathway, known to play an important role in lung morphogenesis, also likely plays a role in the adult human airway epithelium, with at least some of the Notch pathway gene expression dysregulated in association with smoking and its related disorder, COPD. Experiment Overall Design: Gene expression in airway epithelial cells of normal non-smokers.

Project description:Microarray technology provides a powerful tool for defining gene expression profiles of airway epithelium that lend insight into the pathogenesis of human airway disorders. The focus of this study was to establish rigorous quality control parameters to ensure that microarray assessment of the airway epithelium is not confounded by experimental artifact. Samples (total n=223) of trachea, large and small airway epithelium were collected by fiberoptic bronchoscopy of 144 individuals (42 healthy non-smokers, 49 healthy smokers, 11 symptomatic smokers, 22 smokers with lone emphysema with normal spirometry, and 20 smokers with COPD) were processed and hybridized to Affymetrix HG-U133 2.0 Plus microarrays. The pre- and post-chip quality control (QC) criteria established, included: (1) RNA quality, assessed by RNA Integrity Number (RIN) ≥7.0 using Agilent 2100 Bioanalyzer software; (2) cRNA transcript integrity, assessed by signal intensity ratio of GAPDH 3' to 5' probe sets ≤3.0; and (3) the multi-chip normalization scaling factor ≤10.0 Of the 223 samples, 213 (95.5%) passed the QC criteria. In a data set of 34 arrays (10 samples failing QC criteria, 24 randomly chosen samples passing QC criteria), correlation coefficients for pairwise comparisons of expression levels for 100 housekeeping genes in which at least one array failed the QC criteria were significantly lower (average Pearson r = 0.90 ± 0.04) and more broadly dispersed than correlation coefficients for pairwise comparisons between any two arrays that passed the QC criteria (average Pearson r = 0.97 ± 0.01). By using the QC cutoff criteria, the inter-array variability, as assessed by the coefficient of variation in the expression levels for 100 housekeeping genes, was reduced from 35.7% to 21.7%. Based on the aberrant housekeeping gene data generated from samples failing the established QC criteria, we propose that the QC criteria outlined in this study can accurately distinguish high quality from low quality data and can be used to delete poor quality microarray samples before proceeding to higher-order biological analyses and interpretation. Affymetrix arrays were used to assess the quality of gene expression data in trachea, large airway and small airway epithelium obtained by fiberoptic bronchoscopy of 42 healthy non-smokers, 49 healthy smokers, 11 symptomatic smokers, 22 smokers with lone emphysema with normal spirometry, and 20 smokers with COPD.

Dataset Information

Smoking-induced Wnt pathway downregulation

Publications

Down-regulation of the canonical Wnt β-catenin pathway in the airway epithelium of healthy smokers and smokers with COPD.

Similar Datasets

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