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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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE19407 | GEO | 2011/04/20

SECONDARY ACCESSION(S): PRJNA121779

REPOSITORIES: GEO

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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. 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.

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:Cystatin A (gene: CSTA), is up-regulated in non-small-cell lung cancer (NSCLC) and dysplastic vs normal human bronchial epithelium. In the context that chronic obstructive pulmonary disease (COPD), a small airway epithelium (SAE) disorder, is independently associated with NSCLC (especially squamous cell carcinoma, SCC), but only occurs in a subset of smokers, we hypothesized that genetic variation, smoking and COPD modulate CSTA gene expression levels in SAE, with further up-regulation in SCC. Gene expression was assessed by microarray in SAE of 178 individuals [healthy nonsmokers (n=60), healthy smokers (n=82), and COPD smokers (n=36)], with corresponding large airway epithelium (LAE) data in a subset (n=52). Blood DNA was genotyped by SNP microarray. Twelve SNPs upstream of the CSTA gene were all significantly associated with CSTA SAE gene expression (p<0.04 to 5 x 10-4). CSTA gene expression levels in SAE were higher in COPD smokers (28.4 Â± 2.0) than healthy smokers (19.9 Â± 1.4, p<10-3), who in turn had higher levels than nonsmokers (16.1 Â± 1.1, p<0.04). CSTA LAE gene expression was also smoking-responsive (p<10-3). Using comparable publicly available NSCLC expression data, CSTA was up-regulated in SCC vs LAE (p<10-2) and down-regulated in adenocarcinoma vs SAE (p<10-7). All phenotypes were associated with significantly different proportional gene expression of CSTA to cathepsins. The data demonstrate that regulation of CSTA expression in human airway epithelium is influenced by genetic variability, smoking, and COPD, and is further up-regulated in SCC, all of which should be taken into account when considering the role of CSTA in NSCLC pathogenesis. CSTA gene expression was assessed in the small airway epithelium obtained by bronchoscopy from 178 individuals, including healthy nonsmokers (n= 60) and healthy smokers (n= 118) and the large airway epithelium from healthy nonsmokers (n=21) and healthy smokers(n=31). *** Processed data not provided for all gene expression records. *** Blood DNA from the majority of these individuals was genotyped and an association analysis of gene expression with genotypes of all 48 SNPs within 100 kb of CSTA was performed in PLINK, and tested for significance following 103 permutations within ancestral clusters. Supplementary file (linked below) reports genotypes of all 48 SNPs within 100 kb of the CSTA gene for the 112 genotyping Samples.

Project description:Study Smoking and COPD are associated with decreased mucociliary clearance and healthy smokers have shorter cilia in the large airway than nonsmokers. Intraflagellar transport (IFT) is the process by which cilia are produced and maintained. We assessed expression of IFT-related genes in smokers and nonsmokers and evaluated cilia length in the large and small airway of nonsmokers, healthy smokers, and smokers with COPD. Methods Airway epithelium was obtained via bronchoscopic brushing. Affymetrix microarrays were used to evaluate IFT gene expression in 2 independent data sets from large and small airway. Cilia length was assessed by measuring 100 cilia (10 cilia on each of 10 cells) per subject. Results All 40 IFT genes were expressed in the human large and small airway epithelium. In the large airway, 10 IFT genes were down-regulated and 1 up-regulated in smokers. In the small airway, 11 genes were down-regulated and 3 up-regulated in smokers. A set of 8 IFT genes was down-regulated in both data sets. In the large and small airway epithelium, cilia were significantly shorter in healthy smokers than nonsmokers, and significantly shorter in COPD smokers than in both healthy smokers and nonsmokers. Answer to the Question These results support the concept that loss of cilia length contributes to defective mucociliary clearance in COPD, and that smoking-induced changes in expression of IFT genes may be one mechanism of abnormally short cilia in smokers. Strategies to normalize cilia length may be an important avenue for novel COPD therapies. Gene expression was assessed for 40 intraflagellar transport related genes in the LAE of nonsmokers (n=21) and healthy smokers (n=31) and the SAE of an independent group of nonsmokers (n=28) and healthy smokers (n=69). Cilia length was assessed in a total of 228 airway epithelium samples, including 120 LAE samples and 108 SAE samples; a subset of the 228 samples is represented among the 149 samples in this Series.

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Smoking-induced Wnt pathway downregulation

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