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.
Microarrays were used to assess the expression of Wnt-related genes in the small airway epithelium (SAE) obtained via bronchoscopy and brushing of healthy nonsmokers, healthy smokers, and smokers with COPD. Thirty-three of 56 known Wnt-related genes were expressed in the SAE. Wnt pathway downstream mediators β-catenin and the transcription factor 7-like 1 were down-regulated in healthy smokers and smokers with COPD, as were many Wnt target genes. Among the extracellular regulators that suppress the Wnt pathway, secreted frizzled-related protein 2 (SFRP2), was up-regulated 4.3-fold in healthy smokers and 4.9-fold in COPD smokers, an observation confirmed by TaqMan Real-time PCR, Western analysis and immunohistochemistry. Finally, cigarette smoke extract mediated up-regulation of SFRP2 and down-regulation of Wnt target genes in airway epithelial cells in vitro.
Smoking down-regulates the Wnt pathway in the human airway epithelium. In the context that Wnt pathway plays an important role in differentiation of epithelial tissues, the down-regulation of Wnt pathway may contribute to the dysregulation of airway epithelium differentiation observed in smoking-related airway disorders.
Citation: Wang R, Ahmed J, Wang G, Hassan I, Strulovici-Barel Y, Hackett NR, et al. (2011) Down-Regulation of the Canonical Wnt β-Catenin Pathway in the Airway Epithelium of Healthy Smokers and Smokers with COPD. PLoS ONE6(4): e14793. https://doi.org/10.1371/journal.pone.0014793
Editor: Melanie Koenigshoff, Comprehensive Pneumology Center, Germany
Received: March 28, 2010; Accepted: February 14, 2011; Published: April 7, 2011
Copyright: © 2011 Wang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: These studies were supported, in part, by National Institutes of Health R01 HL074326, P50 HL084936; UL1-RR024996; and 1T32HL094284. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The human airway epithelium, a pseudostratified layer of cells derived from the endoderm, serves as a physical barrier against inhaled pathogens, xenobiotics, and other noxious substances in the environment , . The normal airway epithelium has the capacity of regeneration and repair –. Of the major cell types, the airway epithelial progenitor cells are in the basal cell population, cells capable of self renewal and differentiation into columnar cells and eventually, the differentiated secretory and ciliated cell populations that function to defend the airway against environmental stress , –.
There are several lines of evidence that demonstrate controlling this differentiation process, in part, is through the canonical Wnt/β-catenin signaling pathway , . The canonical Wnt pathway plays a central role in lung development and is critical for generation of the lung epithelium , –. Components of the canonical Wnt signaling pathway are expressed in embryonic and adult lung cell lines, as well as in the developing lung in a specific, spatio-temporal pattern , –. When canonical Wnt signaling is active, the pathway maintains cells in a low differentiation state –. However, when the canonical Wnt pathway is suppressed, undifferentiated progenitor cells are allowed to proceed toward differentiation –. Modulation of Wnt expression in embryonic and adult mouse lung suggests that the Wnt pathway is important for cell fate decisions and differentiation of lung cell type , . The agonists of the canonical Wnt/β-catenin pathway include multiple Wnt proteins that function through frizzled receptors to up-regulate β-catenin, resulting in activation of the T cell factor (LEF1, TCF7, TCF7L1, TCF7L2) transcription factor family , . The extracellular antagonists of Wnt signaling are the secreted fizzled-related (SFRP) and the Dickkopf (DKK) family of proteins , . When the SFRPs and/or DKKs are up-regulated, the Wnt pathway is suppressed, allowing differentiation to proceed , .
Based on this background, we hypothesized that the canonical Wnt/β-catenin signaling pathway may be deranged in the airway epithelium when it is in a state of constant stress such as in cigarette smokers. To assess this hypothesis, we used microarrays to survey the mRNA levels of the canonical Wnt/β-catenin pathway in the small airway epithelium, the initial site of smoking-induced lung disease . Pure populations of small airway epithelium were obtained by fiberoptic bronchoscopy of healthy nonsmokers, healthy smokers, and smokers with established COPD. The data demonstrate that most components of the Wnt/β-catenin pathway are expressed in the small airway epithelium. Interestingly, the downstream mediators β-catenin and the TCF transcription factor TCF7L1, as well as several downstream Wnt pathway target genes, are down-regulated in healthy smokers and smokers with COPD. Consistent with the down-regulation of Wnt/β-catenin pathway associated with smoking, the mRNA and protein levels of the Wnt inhibitor secreted frizzled-related protein 2 were greatly up-regulated in the small airway epithelium of healthy smokers and smokers with COPD, with expression primarily in ciliated cells. Finally, in vitro exposure of lung epithelium to cigarette smoke extract demonstrated similar results, with up-regulation of SFRP2 and down-regulation of the Wnt pathway. Together, these results show that smoking is associated with abnormal regulation of the Wnt pathway in the airway epithelium, an observation consistent with the disordered epithelial differentiation observed in smoking-related airway disorders.
All individuals were evaluated at the Weill Cornell NIH Clinical and Translational Science Center and Department of Genetic Medicine Clinical Research Facility using protocols approved by the Weill Cornell Medical College Institutional Review Board. All subjects provided written consent before any study procedures were undertaken. Healthy nonsmokers and healthy smokers were characterized on the basis of clinical history and physical examination, routine blood screening tests, chest X-ray, electrocardiogram, urinalysis, and pulmonary function testing. Current smoking status was confirmed by history, venous carboxyhemoglobin levels, and urinalysis for nicotine levels and its derivative cotinine. Smokers with established COPD were defined according to Global Initiative for Chronic Obstructive Lung Disease criteria , .
Collection of Small Airway Epithelium
Small airway epithelium was collected using flexible bronchoscopy as previously described , . Smokers were asked not to smoke the evening prior to the procedure. A flexible bronchoscope (Pentax, EB-1530T3) was advanced to the desired bronchus after achieving mild sedation and anesthesia of vocal cords. Small airway samples were collected from 10th to 12th order bronchi using methods previously described. The airway epithelial cells were subsequently collected separately in 5 ml of LHC8 medium (GIBO, Grand Island, NY). An aliquot of this was used for cytology and differential cell count and the remainder was processed immediately for RNA extraction. Total cell counts were obtained using a hemocytometer while differential cell counts were determined on sedimented cells prepared by centrifugation (Cytospin 11, Shandon Instruments, Pittsburg, PA) and stained with DiffQuik (Baxter Healthcare, Miami, FL).
RNA Extraction and Microarray Processing
Microarray analysis was performed using Affymetrix (Santa Clara, CA) microarray HG-U133 Plus 2.0 (54,675 probe sets) and associated protocols. Total RNA was extracted from epithelial cells using TRIzol (Invitrogen, Carlsbad, CA) followed by DNAnase (Qiagen, Valencia, CA) to remove residual DNA. An aliquot of each RNA sample was run on an Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA) to visualize and quantify the degree of RNA integrity. The concentration was determined using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). Double-stranded complementary DNA was synthesized from 3 µg of total RNA using the GeneChip One-Cycle cDNA Synthesis Kit, followed by a cleanup step using GeneChip Sample Cleanup Module. Next, an in vitro transcription (IVT) reaction was performed with GeneChip IVT Labeling Kit after which further cleanup was carried out and quantification of the resulting biotin-labeled cRNA by spectrophotometry (all reagents from Affymetrix). Hybridizations to test chips and when permissible, to the microarrays, were conducted according to Affymetrix protocols. The Affymetrix GeneChip Fluidics Station 450 was used for processing the arrays with appropriate reagents/washes, prior to scanning with an Affymetrix GeneChip Scanner 3000 7G (http://affymetrix.com/support/technical/manual/expression_manual.affx).
Captured images were analyzed using Microarray Suite version 5.0 (MAS 5.0) algorithm (Affymetrix) as previously described. Samples used for analysis were required to satisfy quality control criteria including: (1) RNA Integrity Number (RIN) ≥7.0; (2) 3′/5′ ratio for GAPDH≤3; and (3) scaling factor ≤10.0 . This data were normalized using GeneSpring version 7.3 software (Agilent Technologies, Palo Alto, CA) per array, by dividing the raw data by the 50th percentile of all measurements. The data sets were assessed for expression of 56 Wnt signaling pathway genes and 55 Wnt target genes (based on the Wnt homepage, http://www.stanford.edu/~rnusse/wntwindow.html) using criteria of present (P call) of >20% of healthy nonsmokers.
TaqMan RT-PCR Confirmation of Microarray Expression Levels
To confirm the microarray findings, TaqMan real-time RT-PCR was performed on RNA samples from the small airway samples of healthy nonsmokers, healthy smokers and smokers with COPD (n = 9 each, except for β-catenin, n = 11 each) that had been used for HG-U133 Plus 2.0 microarray analysis. First, cDNA was synthesized from 2 µg RNA in a 100 µl reaction volume, using the TaqMan Reverse Transcriptase Reaction Kit (Applied Biosystems), with random hexamers as primers. Dilutions of 1∶10 and 1∶100 were made from each sample and triplicate wells were run for each dilution. TaqMan PCR reactions were carried out using primers (Applied Biosystems, Foster City, CA), and 2 µl of cDNA was used in each 25 µl reaction volume. The endogenous control was 18S ribosomal RNA and relative expression levels were determined using the ΔΔCt method (Applied Biosystems). The rRNA probe was labeled with VIC and the probe for each gene of interest (β-catenin, TCF7L1, SOX9, MMP7) was labeled with FAM. The PCR reactions were run in an Applied Biosystems Sequence Detection System 7500.
Western analysis was used to quantitatively assess SFRP2 protein expression in small airway brushing samples from healthy nonsmokers, healthy smokers and smokers with COPD. Red blood cells were lysed (red blood cell lysis buffer, eBioscience, San Diego, CA), and proteins were extracted using RIPA buffer (Sigma-Aldrich, Saint Louis, MO) following the manufacture's instructions. Protein concentrations were assessed using a bicinchoninic acid (BCA) protein concentration kit (Pierce, Rockford, IL). Equal concentration of protein mixed with NuPAGE LDS Sample Buffer and NuPAGE Reducing Agent was heated at 70°C for 10 min. Then samples were loaded on NuPAGE 4 to12% Bis-Tris Gel (Invitrogen, Carlsbad, CA). Protein electrophoresis was carried out at 200 V, 1 hr, 23°C. Sample proteins were transferred (30 V, 1 hr, 4°C) to a 0.2 µm PVDF membrane (Invitrogen, Carlsbad, CA) in NuPAGE transfer buffer. The membranes were then blocked with 5% non-fat milk in Phosphate Buffered Saline with 0.1% tween-20 (PBST) for 1 hr. The membranes were incubated with primary rabbit polyclonal anti-SFRP2 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) at 1∶200 dilution 4°C overnight. Detection was performed using secondary horseradish peroxidase-labeled goat anti-rabbit antibody (1∶2,000 dilution, Santa Cruz Biotechnology) and then enhanced chemiluminescent reagent system (GE Healthcare, Pittsburgh, PA). To assess the Western analysis quantitatively, the film was digitally imaged, maintaining exposure within the linear range of detection. The contrast was inverted, the pixel intensity of each band determined, and the background pixel intensity for a negative area of the film of identical size subtracted using MetaMorph image analysis software (Universal Imaging, Downingtown, PA). The membrane was subsequently stripped and reincubated with monoclonal anti-β-actin antibody (Sigma-Aldrich, Saint Louis, MO) as a control for equal protein concentration.
Localization of SFRP2 in the Airway Epithelium
To determine the distribution of SFRP2 expression in small airway epithelial cells, cytospin preparation of bronchial brushes obtained from the small airway epithelium of healthy nonsmoker, healthy smokers and smokers with COPD were assessed by SFRP2-specific immunofluorescence. Sections were fixed in 4% paraformaldehyde for 15 min, and washed twice in PBS. Samples were blocked in normal goat serum to reduce background staining and then incubated with the rabbit anti-human SFRP2 primary antibody (Atlas Antibodies, Stockholm, Sweden) diluted 1∶250 and rabbit IgG (Jackson Immunoresearch, West Grove, PA) as the isotype control at 4°C overnight. Goat anti-rabbit Cy3 conjugated AffiniPure IgG (Jackson ImmunoResearch) at 1∶50 dilution was used as a secondary antibody for SFRP2. Nuclei were counter stained with 4′, 6-diamidino-2-phenylindole (DAPI, 1∶2,000 dilution; Invitrogen, Carlsbad, CA). Images were captured using an Olympus IX 70 fluorescence microscope with 60-fold magnification. Images were analyzed using MetaMorph software (Universal Imaging Corporation, Downingtown, PA).
As a further assessment at the morphologic level, bronchial biopsies from large airway of healthy nonsmokers, healthy smokers and smokers with COPD were assessed by immunofluorescence for SFRP2. Sections were deparaffinized and rehydrated through a series of xylenes and alcohol. Further staining procedures were as described above.
Colocalizations of SFRP2 with a ciliated cell-specific marker β-tubulin IV, secretory cell-specific marker mucin 5AC and neuroendocrine-specific marker chromogranin A were also performed with cytospin preparations and biopsies. The following antibodies were used: for β-tubulin IV, mouse monoclonal anti-human β-tubulin IV (1∶2000; Biogenex, San Ramon, CA) and mouse IgG as the isotype control (Sigma, St Louis, MO); for mucin 5AC, mouse monoclonal (CLH2) anti-human mucin 5AC (1∶50; Vector Laboratories, Burlingame, CA) and mouse IgG as the isotype control (Sigma, St Louis, MO); for chromogranin A, mouse monoclonal (LK2H10+PHE5) anti-human chromogranin (1∶500 dilution; Thermo Scientific, Waltham, MA) and mouse IgG as the isotype control (Sigma, St Louis, MO). Following incubation with the primary antibodies, goat anti-rabbit Cy5 (Jackson ImmunoResearch) was used as a secondary antibody for SFPR2 and goat anti-mouse Cy3 (Jackson ImmunoResearch) was used as a secondary antibody for β-tubulin IV, mucin 5AC and chromogranin A. Further staining procedures were as described above.
Down-regulation of Wnt Pathway Target Genes in Human Small Airway Epithelial Cells Exposed to Cigarette Smoke Extract In Vitro
Aqueous cigarette cell extract (CSE) was generated from the combustion of 1 cigarette (Marlboro Red) bubbled through 12.5 ml of culture medium . This medium, defined as “100% CSE,” was adjusted to pH 7.4 and filtered through a 0.22 µm filter. Different concentrations of CSE diluted with the culture medium were employed, ranging from 0.1 to 20%. The human airway epithelial cell line 16HBE  was exposed to freshly prepared CSE for 72 hr. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Roche Applied Science) . Viability was expressed as percentage of the values (corresponding to 100%) of untreated cells. SFRP2, MMP7, SOX9 gene expression was assessed with TaqMan real-time PCR.
Wnt Reporter Assay
HEK293 cells were transiently transfected with the reporter construct Topflash or Fopflash (kindly provided by R. Moon, University of Washington, Seattle) using Lipofectamine LTX (Invitrogen, Carlsbad, CA) . The Topflash construct contains 8 Tcf/Lef binding sites upstream of a minimal TA viral promoter and the firefly luciferase cDNA. The Fopflash construct is identical except that it contains mutated copies of TCf/Lef binding sites and is used as a control for measuring nonspecific activation of the reporter construct. Expression of Renilla luciferase provides an internal control value to which expression of the experimental firefly luciferase reporter gene will be normalized. The experiment was divided into 6 groups: Topflash plus CMV-renilla; Fopflash plus CMV-renilla; Topflash plus CMV-renilla plus WNT1; Fopflash plus CMV-renilla plus WNT1; Topflash plus CMV-renilla plus WNT1 plus SFRP2; Fopflash plus CMV-renilla plus WNT1 plus SFRP2. After 48 hr, luciferase activities were determined using the Dual Luciferase Assay System (Promega) on a tube luminometer (Berthold detection systems).
HG-U133 Plus 2.0 microarrays were analyzed using GeneSpring software. Average expression values were calculated from normalized expression levels for healthy nonsmokers, healthy smokers, and smokers with COPD. p values were obtained using Benjamini-Hochberg correction to limit the false positive rate. Statistical comparisons between continuous variables were calculated using an unpaired, two-tailed t-test with unequal variance. Statistical comparisons for categorical data were achieved using Chi-squared test. A p value<0.05 was considered significant. p values for TaqMan data were calculated using two-tailed Student's t-test.
Web Deposition of Data
All MIAME-compliant microarray data have been deposited in the Gene Expression Omnibus (GEO) site (http://www.ncbi.nlm.nih.gov/geo), which is curated by the National Center for Bioinformatics. Accession number for small airways HG-U133 Plus 2.0 is GSE19407.
Small airway samples from a total of 127 individuals, including 47 healthy nonsmokers, 58 healthy smokers and 22 smokers with established COPD were analyzed with microarray HG-U133 Plus 2.0 (Table 1). There were no differences with respect to gender among the groups (p>0.3), but the COPD smokers were older than the other groups (p<0.05). There were no differences among groups with regard to ancestral background (p>0.3). All individuals were HIV negative. Smokers had urine nicotine and cotinine and venous blood carboxyhemoglobin levels confirming their current smoking status.
Sampling of Airway Epithelium
Airway epithelial cells were obtained by fiberoptic bronchoscopy and brushing of small (10th to 12th order) airways. The number of cells recovered averaged from 2.1 to 21×106, with an average of >96% epithelium (Table 1). The various categories of airway epithelial cells were, as expected, from the small airway epithelium , .
Expression of Wnt Signaling Pathway Genes in Small Epithelium of Healthy Nonsmokers, Healthy Smokers and Smokers with COPD
The genome-wide microarrays were used to analyze the gene expression profiles of canonical Wnt signaling components at the mRNA level in the small airway epithelium of healthy nonsmokers, healthy smokers, and smokers with established COPD. In the small airway epithelium of healthy nonsmokers, 33 of 56 (60%) genes in the Wnt pathway were expressed (Table S1). These expressed genes included Wnt extracellular ligands (WNT3, 4, 7B, 9A, 10A), frizzled receptors (FZD1, 3, 4, 5, 6, 7, 8), co-receptors (LRP5, LRP6), extracellular inhibitors (SFRP2, DKK1, DKK3, DKK4), intracellular mediators (β-catenin, GSK-3B, DVL1, 2, 3, AES, APC1, AXIN1, AXIN2, FRAT1, FRAT2), nuclear transcription factors (TCF7, TCF7L1, TCF7L2, LEF1). For Wnt target genes, 27 out of 55 genes are present in small airway epithelium, including MMP7, CLDN1, VEGFA, CCND1, SOX9, ID2 (Figure 1A, Table S1).
A. Schematic of the pathway, showing which (in bold) ligands, soluble antagonists, receptors, co-receptors, intracellular activators, transcription factors and downstream effectors are expressed. B. Gene expression in canonical Wnt β-catenin pathway in healthy nonsmokers in small airway epithelium. Small airway epithelium from 47 normal nonsmokers were analyzed using the HG-U133 Plus 2.0 array and expression level normalized by chip only is plotted. Error bars represent the standard error.
In healthy nonsmokers the relative expression of WNT9A was the most highly expressed Wnt ligand, with average expression approximately 3.3-fold greater than WNT3, and 4.7-fold greater than WNT10A (both p<0.0001). For receptors, the most highly expressed receptor was FZD6, which was expressed 44.4-fold higher than FZD7 (p<0.0001), the lowest expressed receptor. For transcription factors in the Wnt pathway, TCF7 was the downstream gene most highly expressed in the small airway samples, 2.3-fold more highly expressed than TCF7L1, 4.4-fold more than TCF7L2, and 2.7-fold more than LEF1 (p<0.0001, all comparisons).
Compared to healthy nonsmokers, the Wnt intracellular modulators, transcription factors and target genes were down-regulated in healthy smokers and smokers with established COPD. β-catenin, a central molecule in the Wnt signaling pathway, was down-regulated 1.5-fold in healthy smokers and 1.8-fold in smokers with COPD (both comparisons, p<0.05, Figure 2, Table 2). Consistent with this observation, TCF7L1, a transcription factor in Wnt pathway, was down-regulated 1.7-fold in smokers and 1.7-fold in smokers with established COPD (p<0.01, Figure 2, Table 2). Wnt target genes, including MMP7, VEGFA, CLDN1, CCND1, SOX9, RHOU, were also down-regulated in healthy smokers and smokers with COPD compared to healthy nonsmokers (p<0.05 for VEGFA and p<0.01 for the other genes; Figure 2, Table 3). The network of the WNT/β-catenin genes in healthy smokers and smokers with COPD compared to healthy nonsmokers were analyzed with Ingenuity Pathway Analysis (Ingenuity Systems, http://www.ingenuity.com; Figure S1).
Analysis was carried out with HG-U133 plus 2.0 microarrays normalized by chip and gene. Each bar represents mean expression with standard error; * p<0.05 compared to healthy nonsmokers, * * p<0.01 compared to healthy nonsmokers.
Modulation of the Wnt signaling pathway in the small airway epithelium of healthy smokers and smokers with COPD compared to healthy nonsmokers was verified using TaqMan PCR for selected central genes in the pathway (Figure S2). Consistent with microarray data, β-catenin was down-regulated 1.7-fold in smokers compared to nonsmokers, and 1.9-fold in smokers with COPD compared with healthy nonsmokers (p<0.05). For transcription factors, TCF7L1 was confirmed to be down-regulated in healthy smokers and smokers with COPD compared to healthy nonsmokers (p<0.05). Wnt signaling pathway target genes were also confirmed to be down-regulated. MMP7 expression decreased 7.5-fold in healthy smokers and 12.9-fold in smokers with COPD compared to healthy nonsmokers (p<0.05). SOX9, another Wnt target gene, was also confirmed to be down-regulated in the small airway epithelium of healthy smokers and smokers with COPD compared to healthy nonsmokers (3.9-fold for smokers vs nonsmokers and 3.2-fold for smokers with COPD vs nonsmokers, both p<0.01).
Expression of SFRP2 in the Small Airway Epithelium of Healthy Nonsmokers, Healthy Smokers and Smoker with COPD
The in vivo gene expression data showed that Wnt signaling pathway was down-regulated in healthy smokers and smokers with COPD compared to healthy nonsmokers in the small airway epithelial cells. As a possible mechanism to explain this down-regulation, we hypothesized that there may be smoking-induced up-regulation of the SFRP and/or DKK family of extracellular Wnt pathway inhibitors. To assess this, we analyzed Wnt inhibitors expressed in human small airway epithelium including, SFRP2, DKK1, DKK3 and DKK4. Interestingly, among these 4 inhibitors, SFRP2 showed significant gene expression change between healthy smokers and healthy nonsmokers, with SFRP2 up-regulation 4.3-fold in healthy smokers and 4.9-fold in smokers with COPD compared with healthy nonsmokers (p<0.0001, Figure 3).
Analysis was carried out with HG-U133 plus 2.0 microarrays. Each bar represents mean expression with standard error; p values are represented in brackets above the bars.
To confirm the results obtained from microarrays, anti-SFRP2 immunofluorescence and Western analysis were utilized. First, anti-SFRP2 immunofluorescence was carried out on cytospin preparations of small airway epithelial cells obtained from healthy nonsmokers, healthy smokers and smokers with COPD. In all samples, SFRP2 protein expression was detected specifically in ciliated cells; other cell types, including secretory cells, undifferentiated cells and basal cells, did not show obvious staining (Figure 4, Figure 5). Consistent with the up-regulation of SFRP2 mRNA, qualitative immunofluorescence assessment suggested that SFRP2 protein was up-regulated in the healthy smokers and COPD smokers. Second, as further confirmation, SFRP2 expression was also observed to be up-regulated in ciliated cells of large airway biopsies (Figure 6).
For Figures 4A–I, Cytospin preparations of airway epithelium from a healthy smoker were stained with antibodies against SFRP2 (green), β-tubulin IV (red), Mucin 5AC (red) and chromogranin A (red). A–C. Colocalization of SFRP2 and β-tubulin IV; D–F. Colocalization of SFRP2 and mucin 5AC; G–I. Colocalization of SFRP2 and chromogranin A. For Figure 4J–K, biopsies from a healthy smoker were stained with antibodies against SFRP2 (red) β- tubulin IV (green). J. IgG controls of SFRP2 and β-tubulin IV; and K. SFRP2 and β-tubulin IV co-localization.
Small airway epithelial cell cytopreparations of healthy nonsmokers, healthy smokers and smokers with COPD were stained with anti-SFRP2 followed by a Cy3 conjugated secondary antibody (shown in red). Nuclei were stained with DAPI (shown in blue) A–D. Healthy nonsmokers. A. IgG control; B–D. Examples of anti-SFRP2. E–H. Healthy smokers. E. IgG control; F–H. Examples of anti-SFRP2. I–L. Smokers with COPD; I. IgG Control. J–L. Examples of anti-SFRP2. Bar = 10 µm.
Endobronchial biopsies from large airway of healthy nonsmoker, healthy smokers and smokers with COPD were stained with anti-SFRP2 followed by a Cy3 conjugated secondary antibody (shown in red). Nuclei were stained with DAPI (shown in blue). A–D. Healthy nonsmokers. A. IgG control; B–D. Examples of anti-SFRP2. E–H. Healthy smokers. E. IgG control; F–H. Examples of anti-SFRP2. I–L. Smokers with COPD. I. IgG Control, J–L. Examples of anti-SFRP2. Bar = 10 µm.
To quantify this observation, Western analysis of SFRP2 was carried out on small airway epithelial cell samples from healthy nonsmokers, healthy smokers and smokers with COPD. This analysis demonstrated increased SFRP2 protein expression in healthy smokers and smokers with established COPD compared to healthy nonsmokers in small airway epithelial cells (Figure 7, p<0.05).
A. Proteins were extracted from small airway epithelial cells of 3 healthy nonsmokers, 3 healthy smokers and 3 smokers with COPD. Shown is SFRP2 protein expression in healthy nonsmokers (lanes 1–3), healthy smokers (lanes 4–6) and smokers with COPD (lanes 7–9). Lower panel - same membrane probed with anti β-actin antibody, a control for protein loading. B. Quantification by densitometry of SFRP2 to β-actin. The ratio for SFRP2 to β-actin based on panel A is represented on the ordinate for the nonsmoker, smoker and smoker with COPD. Error bars represent the standard error.
Down-regulation of the Wnt Signaling Pathway in Human Airway Epithelial Cells Exposed to Cigarette Smoke Extract In Vitro
To assess whether the exposure of human airway epithelial cells to CSE would affect Wnt pathway gene expression, cultures of the human airway epithelial cell line 16HBE were exposed to freshly made CSE for 72 hr. Cell viability was analyzed with MTT assay and SFRP2, MMP7 and SOX9 gene expression was analyzed using TaqMan real-time PCR. Decreased cell viability compared to control cells was observed with concentrations of 5, 10 and 20% CSE. Cell viability was 100% with 0.1% CSE and 92% with 1% CSE medium compared with controls (Figure 8A). Based on this data, we chose 0.1% and 1% CSE for the CES in vitro exposure studies. Compared with controls, SFRP2 gene expression increased in a dose-dependent pattern, with 4.6-fold in 0.1% CSE group (p<0.01) and 10.1-fold in 1% CSE groups (p<0.001) compared to no CSE controls (Figure 7B). The Wnt target gene MMP7 decreased 2.3-fold (p<0.05) and 4.0-fold (p<0.01) respectively in 0.1% and 1% CSE groups compared to the control. SOX9, another Wnt target gene, was not significant in 0.1% CSE group, but significantly decreased in 1% CSE group (1.8-fold, p<0.05) compared to the control (Figure 8B).
After 16HBE cells were exposed to different concentrations of CSE for 3 days, cell viability (MTT) and gene expression levels of Wnt signaling pathway inhibitor SFRP2 and target genes (MMP7, SOX9) were quantified using TaqMan real-time PCR. A. MTT assessment. B. Effect of CSE on gene expression of SFRP2, MMP7, SOX9 in 16HBE cells using 0.1 and 1% CSE concentrations that do not affect viability. SFRP2 gene expression was up-regulated when treated with both 0.1 and 1% CSE (4.6-fold and 10.1-fold, p<0.01 and p<0.001). Error bars represent the standard deviation. *p<0.05 compared to no CSE controls. For TaqMan real-time PCR, the average value of 0.1% CSE treatment group for each gene is determined as the calibrator.
Wnt Reporter Assay
To demonstrate SFRP2-dependent suppression of Wnt signaling, HEK293 cells were transiently transfected with the reporter constructs. When luciferase expression was driven by a WNT1-dependent promoter, the co-transfection of SFRP2 in addition to the WNT1 plasmid decreased luciferase activity in HEK293 cells by two fold (p<0.0001, Figure S3).
In healthy individuals, as airway epithelial cells are injured or reach senescence, there is ongoing cell regeneration, with basal cell proliferation and subsequent differentiation to ciliated and secretory cells that comprise the healthy pseudostratified epithelial barrier that lines the airways –, . In cigarette smokers, the toxic components of smoke and the associated local persistent inflammatory host response to smoking place the epithelium under the constant stress of oxidants, apoptotic signals, and other mechanisms of injury –. In response, the airway epithelium regenerates at a faster rate, attempting to maintain its healthy pseudostratified character , . To assess the role of the Wnt pathway in the context of the stress of cigarette smoking, whole-genome microarray was utilized to analyze expression of the Wnt pathway genes. The data demonstrate that the majority of genes involved in the Wnt signaling pathway, representing all functional categories in the pathway, are expressed in the normal adult human small airway epithelium of healthy nonsmokers, consistent with studies showing the Wnt pathway can regulate human lung morphogenesis , , . With the stress of smoking, the Wnt pathway intracellular modulators β-catenin and transcription factor TCF7L1, as well as Wnt target genes are down-regulated, suggesting the Wnt pathway is suppressed. Assessment of the known extracellular inhibitors of the Wnt pathway revealed that smoking is associated with up-regulation of SFRP2. The up-regulated SFRP2 expression occurred in the ciliated cell population, suggesting that the differentiated cells play a role in modulating the “on” and “off” status of the Wnt pathway. Finally, in vitro exposure of human airway epithelial cells to cigarette smoke extract recapitulated the in vivo observations, with increased SFRP2 gene expression and down-regulation of Wnt target gene expression, demonstrating that cigarette smoke can directly down-regulate Wnt signaling pathway in the airway epithelium.
Wnt Signaling Pathway and Lung Development
Wnt pathway signaling is an important regulator of epithelial and mesenchymal cell biology , . To date, there are at least 19 Wnt ligands and 10 Fz receptor proteins identified, with redundancy and multiplicity of the receptor interactions and the downstream signaling , . Of the various Wnt mutant phenotypes in mice, loss of the ligands WNT7B and WNT5A results in an abnormal pulmonary phenotype , . Wnt7B lacZ−/− mice exhibit lung hypoplasia and respiratory failure , and Wnt7BlacZ−/− embryos and newborn mice exhibit severe defects in the smooth muscle component of the major pulmonary vessels, suggesting that WNT7B signaling is required for proper lung mesenchymal growth and vascular development . Mice carrying a targeted disruption of the WNT5A locus show abnormalities in distal lung morphogenesis, as manifested by truncation of the trachea and overexpansion of the distal respiratory airways . The overall architecture of these mutant lungs is characterized by overexpansion of the distal airways and inhibition of lung maturation, with a persistence of thickened intravascular interstitium . Activation of β-catenin in mice causes ectopic differentiation of alveolar type II-like cells in conducting airways, goblet cell hyperplasia, and air space enlargement, suggesting a critical role for the Wnt/β-catenin signal transduction pathway in the differentiation of the respiratory epithelium in the postnatal lung .
Wnt Signaling Pathway and Lung Disease
Loss of regulation of Wnt signaling pathways has been linked to the pathogenesis of asthma, fibrotic lung disease, nitrogen-induced pulmonary hypoplasia and lung cancer –. Konigshoff and colleagues ,  confirmed the up-regulation of WNT1, WNT7B and WNT10B, FZD2, FZD3, β-catenin, and LEF1 expression in the lungs of individuals with idiopathic pulmonary fibrosis compared to transplant donor lung. In vitro functional studies have indicated that Wnt ligands induce lung epithelial cell proliferation and (myo) fibroblast activation and collagen synthesis . Increased expression of Wnt ligands (WNT1, 2, 7A) along with decreased expression of Wnt inhibitors (DKK3 and SFRP3) was observed in non-small cell lung cancer , , –.
Function of Wnt Signaling Pathway in Adult Human Airway Epithelium
In a previous study from our laboratory, the large airway epithelium of healthy individuals was denuded by bronchoscopy and brushing, and genome-wide microarrays were used to study the gene expression pattern in the same site at 0, 7 and 14 days after injury . Histologically, the injured area was completely covered by a partially redifferentiated epithelial layer after 7 days, and at this time point, a Wnt pathway inhibitor DKK1 was found to be greatly up-regulated, suggesting that the Wnt/β-catenin pathway is inhibited at this stage of airway epithelial repair .
In the present study, we found that the Wnt signaling pathway was also down-regulated in adult human airway epithelium under the stress of chronic smoking. Interestingly, using gene expression profiling with air/whole mainstream cigarette smoke to treat a three-dimensional air-liquid interface model of tracheobronchial epithelium that were grown from primary human lung epithelial cells, Maunders et al  showed that the Wnt signaling pathway was down-regulated at 1, 6, and 24 hr post-exposure. For example, at 1 hr post-treatment, 9 Wnt signaling pathway genes and 12 Wnt target genes, including AXIN2, CCND1, JAG1, RUNX2, were down-regulated, with no up-regulation of Wnt signaling pathway genes or Wnt target genes. This supports our in vivo data that smoking repressed the Wnt signaling pathway. We also showed that in vitro smokers have significant up-regulation of another Wnt pathway inhibitor, SFRP2, an extracellular mediator of the largest family of Wnt inhibitors. SFRP2 functions as a modulator of Wnt signaling through direct interaction with Wnt ligands, with consequent regulation of cell growth , –. Inactivation of SFRP2 results in subtle limb defects in mice with mesomelic shortening and consistent shortening of all autopodal elements clinically manifested as brachydactyly . SFRP2 has been shown to repress canonical Wnt signaling activated by WNT1, WNT3A, WNT4, WNT7A, and WNT9A in vitro –. Interestingly, SFRP1, another member of the SFRP family, is up-regulated in the distal epithelial cells of the mouse lung during development and in murine emphysema models . However, except for the function as the Wnt signaling pathway inhibitors, recent studies showed that SFRPs are not merely Wnt-binding proteins, but can also antagonize different SFRPs' activity, interfere with BMP signaling by acting as proteinase inhibitors, decrease susceptibility to UV-induced apoptosis in primary culture of canine mammary gland tumors by NF-κB activation or JNK suppression, and interact with other receptors or matrix molecules –. Because of the limitation of the primary human airway epithelial cells, which prohibits us from performing the functional assays of the Wnt signaling pathway in a condition similar to that in vivo, we could not be certain that the down-regulation of Wnt signaling in healthy smokers and COPD patients is the result of SFRP2 up-regulation, despite the fact that SFRP2 could decrease WNT1-induced activation of Wnt reporter luciferase activity in HEK293 cells (p<0.05, Figure S3). Another limitation of this study is, although the down-regulation of the Wnt signaling pathway might induce the human airway epithelial cells to differentiate in healthy smokers and COPD patients, in the current study, we found there were decreased ciliated cells and increased undifferentiated cells (Table 1). There are a number of reasons for this discrepancy. Although the down-regulation of the Wnt signaling pathway could promote the repair and differentiation of airway epithelial cells in healthy smokers and smokers with COPD, the effect of smoking undoubtedly has a complex effect on multiple pathways. The confirmation of this concept is limited by the lack of a proper in vitro model. Furthermore, because the airway epithelial cells in vivo are composed a mixed populations including ciliated cells, secretory cells, basal cells and other cell populations such as clara cells, the microarray data may solely reflect changes in cell populations from patient to patient. However, it is unlikely because of the up-regulation of SFRP2 (which is specifically expressed in ciliated cells) in healthy smokers and smokers with COPD as well as the fact that the healthy smokers and smokers with COPD have lower percentage of ciliated cells.
Our findings suggest there is a complex Wnt signaling system in the adult human airway epithelium, which is subjected to a tightly regulated pattern that can respond to stress. Together, the data show that smoking induces down-regulation of Wnt signaling pathway, consistent with the concept that in response to the stress of cigarette smoking, the small airway epithelium Wnt pathway is suppressed, allowing for epithelial repair and differentiation.
Expression of WNT Pathway Genes and Target Genes in Small Airway.
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Ingenuity Pathway Analysis generated WNT/β-catenin gene network. A. IPA gene network of WNT/β-catenin signaling pathway genes that displayed 1.5-fold or greater changes between healthy smokers and healthy nonsmokers. B. IPA gene network of WNT/β-catenin signaling pathway genes that displayed 1.5-fold or greater changes between smokers with COPD and healthy nonsmokers. Additional information about the genes and the indicated interactions can be found at www.ingenuity.com.
(1.79 MB TIF)
TaqMan Real-time PCR confirmation of down-regulation of selected Wnt genes and Wnt target genes in healthy nonsmokers (NS), healthy smokers (S), and smokers with COPD (COPD). Each bar represents mean expression with standard error; p values are represented in brackets above the bars. The average value of healthy nonsmokers is determined as the calibrator for each gene.
(0.43 MB TIF)
The Wnt reporter assay in HEK293 cells. HEK293 cells were transiently transfected with Wnt reporter (Topflash) or Wnt reporter control (Fopflash) constructs, and stimulated with WNT1 plasmid or WNT1 plus SFRP2 plasmids, as indicated. The relative luciferase activity is plotted. The experiment was repeated three times and representative results are presented as mean ± standard deviation.
(0.40 MB TIF)
We thank D. Dang and M. Teater for expert technical assistance; and N Mohamed for help in preparing this manuscript.
Conceived and designed the experiments: RW NH RGC. Performed the experiments: RW JA GW IH. Analyzed the data: RW JA GW YSB NH RGC. Wrote the paper: RW RGC.
- 1. Knight DA, Holgate ST (2003) The airway epithelium: structural and functional properties in health and disease. Respirology 8: 432–446.DA KnightST Holgate2003The airway epithelium: structural and functional properties in health and disease.Respirology8432446
- 2. Crystal RG, Randell SH, Engelhardt JF, Voynow J, Sunday ME (2008) Airway epithelial cells: current concepts and challenges. Proc Am Thorac Soc 5: 772–777.RG CrystalSH RandellJF EngelhardtJ. VoynowME Sunday2008Airway epithelial cells: current concepts and challenges.Proc Am Thorac Soc5772777
- 3. Bishop AE (2004) Pulmonary epithelial stem cells. Cell Prolif 37: 89–96.AE Bishop2004Pulmonary epithelial stem cells.Cell Prolif378996
- 4. Rawlins EL, Ostrowski LE, Randell SH, Hogan BL (2007) Lung development and repair: contribution of the ciliated lineage. Proc Natl Acad Sci U S A 104: 410–417.EL RawlinsLE OstrowskiSH RandellBL Hogan2007Lung development and repair: contribution of the ciliated lineage.Proc Natl Acad Sci U S A104410417
- 5. Stripp BR, Reynolds SD (2008) Maintenance and repair of the bronchiolar epithelium. Proc Am Thorac Soc 5: 328–333.BR StrippSD Reynolds2008Maintenance and repair of the bronchiolar epithelium.Proc Am Thorac Soc5328333
- 6. Zhang Y, Goss AM, Cohen ED, Kadzik R, Lepore JJ, et al. (2008) A Gata6-Wnt pathway required for epithelial stem cell development and airway regeneration. Nat Genet 40: 862–870.Y. ZhangAM GossED CohenR. KadzikJJ Lepore2008A Gata6-Wnt pathway required for epithelial stem cell development and airway regeneration.Nat Genet40862870
- 7. Rock JR, Onaitis MW, Rawlins EL, Lu Y, Clark CP, et al. (2009) Basal cells as stem cells of the mouse trachea and human airway epithelium. Proc Natl Acad Sci U S A 106: 12771–12775.JR RockMW OnaitisEL RawlinsY. LuCP Clark2009Basal cells as stem cells of the mouse trachea and human airway epithelium.Proc Natl Acad Sci U S A1061277112775
- 8. Evans MJ, Van Winkle LS, Fanucchi MV, Plopper CG (2001) Cellular and molecular characteristics of basal cells in airway epithelium. Exp Lung Res 27: 401–415.MJ EvansLS Van WinkleMV FanucchiCG Plopper2001Cellular and molecular characteristics of basal cells in airway epithelium.Exp Lung Res27401415
- 9. Snyder JC, Teisanu RM, Stripp BR (2009) Endogenous lung stem cells and contribution to disease. J Pathol 217: 254–264.JC SnyderRM TeisanuBR Stripp2009Endogenous lung stem cells and contribution to disease.J Pathol217254264
- 10. De Langhe SP, Reynolds SD (2008) Wnt signaling in lung organogenesis. Organogenesis 4: 100–108.SP De LangheSD Reynolds2008Wnt signaling in lung organogenesis.Organogenesis4100108
- 11. Pongracz JE, Stockley RA (2006) Wnt signalling in lung development and diseases. Respir Res 7: 15.JE PongraczRA Stockley2006Wnt signalling in lung development and diseases.Respir Res715
- 12. Mucenski ML, Nation JM, Thitoff AR, Besnard V, Xu Y, et al. (2005) Beta-catenin regulates differentiation of respiratory epithelial cells in vivo. Am J Physiol Lung Cell Mol Physiol 289: L971–L979.ML MucenskiJM NationAR ThitoffV. BesnardY. Xu2005Beta-catenin regulates differentiation of respiratory epithelial cells in vivo.Am J Physiol Lung Cell Mol Physiol289L971L979
- 13. Reynolds SD, Zemke AC, Giangreco A, Brockway BL, Teisanu RM, et al. (2008) Conditional stabilization of beta-catenin expands the pool of lung stem cells. Stem Cells 26: 1337–1346.SD ReynoldsAC ZemkeA. GiangrecoBL BrockwayRM Teisanu2008Conditional stabilization of beta-catenin expands the pool of lung stem cells.Stem Cells2613371346
- 14. Shu W, Guttentag S, Wang Z, Andl T, Ballard P, et al. (2005) Wnt/beta-catenin signaling acts upstream of N-myc, BMP4, and FGF signaling to regulate proximal-distal patterning in the lung. Dev Biol 283: 226–239.W. ShuS. GuttentagZ. WangT. AndlP. Ballard2005Wnt/beta-catenin signaling acts upstream of N-myc, BMP4, and FGF signaling to regulate proximal-distal patterning in the lung.Dev Biol283226239
- 15. Li C, Xiao J, Hormi K, Borok Z, Minoo P (2002) Wnt5a participates in distal lung morphogenesis. Dev Biol 248: 68–81.C. LiJ. XiaoK. HormiZ. BorokP. Minoo2002Wnt5a participates in distal lung morphogenesis.Dev Biol2486881
- 16. Wang Z, Shu W, Lu MM, Morrisey EE (2005) Wnt7b activates canonical signaling in epithelial and vascular smooth muscle cells through interactions with Fzd1, Fzd10, and LRP5. Mol Cell Biol 25: 5022–5030.Z. WangW. ShuMM LuEE Morrisey2005Wnt7b activates canonical signaling in epithelial and vascular smooth muscle cells through interactions with Fzd1, Fzd10, and LRP5.Mol Cell Biol2550225030
- 17. Weidenfeld J, Shu W, Zhang L, Millar SE, Morrisey EE (2002) The WNT7b promoter is regulated by TTF-1, GATA6, and Foxa2 in lung epithelium. J Biol Chem 277: 21061–21070.J. WeidenfeldW. ShuL. ZhangSE MillarEE Morrisey2002The WNT7b promoter is regulated by TTF-1, GATA6, and Foxa2 in lung epithelium.J Biol Chem2772106121070
- 18. Korinek V, Barker N, Moerer P, van DE, Huls G, et al. (1998) Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4. Nat Genet 19: 379–383.V. KorinekN. BarkerP. MoererDE vanG. Huls1998Depletion of epithelial stem-cell compartments in the small intestine of mice lacking Tcf-4.Nat Genet19379383
- 19. Merrill BJ, Gat U, DasGupta R, Fuchs E (2001) Tcf3 and Lef1 regulate lineage differentiation of multipotent stem cells in skin. Genes Dev 15: 1688–1705.BJ MerrillU. GatR. DasGuptaE. Fuchs2001Tcf3 and Lef1 regulate lineage differentiation of multipotent stem cells in skin.Genes Dev1516881705
- 20. Chenn A, Walsh CA (2002) Regulation of cerebral cortical size by control of cell cycle exit in neural precursors. Science 297: 365–369.A. ChennCA Walsh2002Regulation of cerebral cortical size by control of cell cycle exit in neural precursors.Science297365369
- 21. Kubo F, Takeichi M, Nakagawa S (2003) Wnt2b controls retinal cell differentiation at the ciliary marginal zone. Development 130: 587–598.F. KuboM. TakeichiS. Nakagawa2003Wnt2b controls retinal cell differentiation at the ciliary marginal zone.Development130587598
- 22. Reya T, Duncan AW, Ailles L, Domen J, Scherer DC, et al. (2003) A role for Wnt signalling in self-renewal of haematopoietic stem cells. Nature 423: 409–414.T. ReyaAW DuncanL. AillesJ. DomenDC Scherer2003A role for Wnt signalling in self-renewal of haematopoietic stem cells.Nature423409414
- 23. Sato N, Meijer L, Skaltsounis L, Greengard P, Brivanlou AH (2004) Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med 10: 55–63.N. SatoL. MeijerL. SkaltsounisP. GreengardAH Brivanlou2004Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor.Nat Med105563
- 24. Shu W, Jiang YQ, Lu MM, Morrisey EE (2002) Wnt7b regulates mesenchymal proliferation and vascular development in the lung. Development 129: 4831–4842.W. ShuYQ JiangMM LuEE Morrisey2002Wnt7b regulates mesenchymal proliferation and vascular development in the lung.Development12948314842
- 25. Li C, Hu L, Xiao J, Chen H, Li JT, et al. (2005) Wnt5a regulates Shh and Fgf10 signaling during lung development. Dev Biol 287: 86–97.C. LiL. HuJ. XiaoH. ChenJT Li2005Wnt5a regulates Shh and Fgf10 signaling during lung development.Dev Biol2878697
- 26. Van Scoyk M, Randall J, Sergew A, Williams LM, Tennis M, et al. (2008) Wnt signaling pathway and lung disease. Transl Res 151: 175–180.M. Van ScoykJ. RandallA. SergewLM WilliamsM. Tennis2008Wnt signaling pathway and lung disease.Transl Res151175180
- 27. van Amerongen R, Nusse R (2009) Towards an integrated view of Wnt signaling in development. Development 136: 3205–3214.R. van AmerongenR. Nusse2009Towards an integrated view of Wnt signaling in development.Development13632053214
- 28. Kawano Y, Kypta R (2003) Secreted antagonists of the Wnt signalling pathway. J Cell Sci 116: 2627–2634.Y. KawanoR. Kypta2003Secreted antagonists of the Wnt signalling pathway.J Cell Sci11626272634
- 29. Niehrs C (2006) Function and biological roles of the Dickkopf family of Wnt modulators. Oncogene 25: 7469–7481.C. Niehrs2006Function and biological roles of the Dickkopf family of Wnt modulators.Oncogene2574697481
- 30. Hogg JC, Chu F, Utokaparch S, Woods R, Elliott WM, et al. (2004) The nature of small-airway obstruction in chronic obstructive pulmonary disease. N Engl J Med 350: 2645–2653.JC HoggF. ChuS. UtokaparchR. WoodsWM Elliott2004The nature of small-airway obstruction in chronic obstructive pulmonary disease.N Engl J Med35026452653
- 31. Pauwels RA, Buist AS, Calverley PM, Jenkins CR, Hurd SS (2001) Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease. NHLBI/WHO Global Initiative for Chronic Obstructive Lung Disease (GOLD) Workshop summary. Am J Respir Crit Care Med 163: 1256–1276.RA PauwelsAS BuistPM CalverleyCR JenkinsSS Hurd2001Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease. NHLBI/WHO Global Initiative for Chronic Obstructive Lung Disease (GOLD) Workshop summary.Am J Respir Crit Care Med16312561276
- 32. Rabe KF, Hurd S, Anzueto A, Barnes PJ, Buist SA, et al. (2007) Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: GOLD executive summary. Am J Respir Crit Care Med 176: 532–555.KF RabeS. HurdA. AnzuetoPJ BarnesSA Buist2007Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: GOLD executive summary.Am J Respir Crit Care Med176532555
- 33. Harvey BG, Heguy A, Leopold PL, Carolan BJ, Ferris B, et al. (2007) Modification of gene expression of the small airway epithelium in response to cigarette smoking. J Mol Med 85: 39–53.BG HarveyA. HeguyPL LeopoldBJ CarolanB. Ferris2007Modification of gene expression of the small airway epithelium in response to cigarette smoking.J Mol Med853953
- 34. Tilley AE, Harvey BG, Heguy A, Hackett NR, Wang R, et al. (2009) Down-regulation of the notch pathway in human airway epithelium in association with smoking and chronic obstructive pulmonary disease. Am J Respir Crit Care Med 179: 457–466.AE TilleyBG HarveyA. HeguyNR HackettR. Wang2009Down-regulation of the notch pathway in human airway epithelium in association with smoking and chronic obstructive pulmonary disease.Am J Respir Crit Care Med179457466
- 35. Raman T, O'Connor TP, Hackett NR, Wang W, Harvey BG, et al. (2009) Quality control in microarray assessment of gene expression in human airway epithelium. BMC Genomics 10: 493.T. RamanTP O'ConnorNR HackettW. WangBG Harvey2009Quality control in microarray assessment of gene expression in human airway epithelium.BMC Genomics10493
- 36. Ning W, Li CJ, Kaminski N, Feghali-Bostwick CA, Alber SM, et al. (2004) Comprehensive gene expression profiles reveal pathways related to the pathogenesis of chronic obstructive pulmonary disease. Proc Natl Acad Sci U S A 101: 14895–14900.W. NingCJ LiN. KaminskiCA Feghali-BostwickSM Alber2004Comprehensive gene expression profiles reveal pathways related to the pathogenesis of chronic obstructive pulmonary disease.Proc Natl Acad Sci U S A1011489514900
- 37. Cozens AL, Yezzi MJ, Kunzelmann K, Ohrui T, Chin L, et al. (1994) CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells. Am J Respir Cell Mol Biol 10: 38–47.AL CozensMJ YezziK. KunzelmannT. OhruiL. Chin1994CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells.Am J Respir Cell Mol Biol103847
- 38. Berridge MV, Tan AS (1993) Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction. Arch Biochem Biophys 303: 474–482.MV BerridgeAS Tan1993Characterization of the cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): subcellular localization, substrate dependence, and involvement of mitochondrial electron transport in MTT reduction.Arch Biochem Biophys303474482
- 39. Jaluria P, Betenbaugh M, Konstantopoulos K, Shiloach J (2007) Enhancement of cell proliferation in various mammalian cell lines by gene insertion of a cyclin-dependent kinase homolog. BMC Biotechnol 7: 71.P. JaluriaM. BetenbaughK. KonstantopoulosJ. Shiloach2007Enhancement of cell proliferation in various mammalian cell lines by gene insertion of a cyclin-dependent kinase homolog.BMC Biotechnol771
- 40. Cosio MG, Hale KA, Niewoehner DE (1980) Morphologic and morphometric effects of prolonged cigarette smoking on the small airways. Am Rev Respir Dis 122: 265–21.MG CosioKA HaleDE Niewoehner1980Morphologic and morphometric effects of prolonged cigarette smoking on the small airways.Am Rev Respir Dis12226521
- 41. van der Vaart H, Postma DS, Timens W, Ten Hacken NH (2004) Acute effects of cigarette smoke on inflammation and oxidative stress: a review. Thorax 59: 713–721.H. van der VaartDS PostmaW. TimensNH Ten Hacken2004Acute effects of cigarette smoke on inflammation and oxidative stress: a review.Thorax59713721
- 42. Kinnula VL (2005) Focus on antioxidant enzymes and antioxidant strategies in smoking related airway diseases. Thorax 60: 693–700.VL Kinnula2005Focus on antioxidant enzymes and antioxidant strategies in smoking related airway diseases.Thorax60693700
- 43. Rennard SI (2007) Inflammation in COPD: a link to systemic comorbidities. Eur Respir Rev 16: 91–97.SI Rennard2007Inflammation in COPD: a link to systemic comorbidities.Eur Respir Rev169197
- 44. Demoly P, Simony-Lafontaine J, Chanez P, Pujol JL, Lequeux N, et al. (1994) Cell proliferation in the bronchial mucosa of asthmatics and chronic bronchitics. Am J Respir Crit Care Med 150: 214–217.P. DemolyJ. Simony-LafontaineP. ChanezJL PujolN. Lequeux1994Cell proliferation in the bronchial mucosa of asthmatics and chronic bronchitics.Am J Respir Crit Care Med150214217
- 45. Morrisey EE (2003) Wnt signaling and pulmonary fibrosis. Am J Pathol 162: 1393–1397.EE Morrisey2003Wnt signaling and pulmonary fibrosis.Am J Pathol16213931397
- 46. Takayasu H, Nakazawa N, Montedonico S, Puri P (2007) Down-regulation of Wnt signal pathway in nitrofen-induced hypoplastic lung. J Pediatr Surg 42: 426–430.H. TakayasuN. NakazawaS. MontedonicoP. Puri2007Down-regulation of Wnt signal pathway in nitrofen-induced hypoplastic lung.J Pediatr Surg42426430
- 47. Huang CL, Liu D, Ishikawa S, Nakashima T, Nakashima N, et al. (2008) Wnt1 overexpression promotes tumour progression in non-small cell lung cancer. Eur J Cancer 44: 2680–2688.CL HuangD. LiuS. IshikawaT. NakashimaN. Nakashima2008Wnt1 overexpression promotes tumour progression in non-small cell lung cancer.Eur J Cancer4426802688
- 48. Konigshoff M, Balsara N, Pfaff EM, Kramer M, Chrobak I, et al. (2008) Functional Wnt signaling is increased in idiopathic pulmonary fibrosis. PLoS One 3: e2142.M. KonigshoffN. BalsaraEM PfaffM. KramerI. Chrobak2008Functional Wnt signaling is increased in idiopathic pulmonary fibrosis.PLoS One3e2142
- 49. Akiri G, Cherian MM, Vijayakumar S, Liu G, Bafico A, et al. (2009) Wnt pathway aberrations including autocrine Wnt activation occur at high frequency in human non-small-cell lung carcinoma. Oncogene 28: 2163–2172.G. AkiriMM CherianS. VijayakumarG. LiuA. Bafico2009Wnt pathway aberrations including autocrine Wnt activation occur at high frequency in human non-small-cell lung carcinoma.Oncogene2821632172
- 50. Konigshoff M, Eickelberg O (2009) WNT signaling in lung disease: a failure or a regeneration signal? Am J Respir Cell Mol Biol. M. KonigshoffO. Eickelberg2009WNT signaling in lung disease: a failure or a regeneration signal?Am J Respir Cell Mol Biol
- 51. You L, He B, Xu Z, Uematsu K, Mazieres J, et al. (2004) Inhibition of Wnt-2-mediated signaling induces programmed cell death in non-small-cell lung cancer cells. Oncogene 23: 6170–6174.L. YouB. HeZ. XuK. UematsuJ. Mazieres2004Inhibition of Wnt-2-mediated signaling induces programmed cell death in non-small-cell lung cancer cells.Oncogene2361706174
- 52. He B, Barg RN, You L, Xu Z, Reguart N, et al. (2005) Wnt signaling in stem cells and non-small-cell lung cancer. Clin Lung Cancer 7: 54–60.B. HeRN BargL. YouZ. XuN. Reguart2005Wnt signaling in stem cells and non-small-cell lung cancer.Clin Lung Cancer75460
- 53. He B, Jablons DM (2006) Wnt signaling in stem cells and lung cancer. Ernst Schering Found Symp Proc 27–58.B. HeDM Jablons2006Wnt signaling in stem cells and lung cancer.Ernst Schering Found Symp Proc2758
- 54. Heguy A, Harvey BG, Leopold PL, Dolgalev I, Raman T, et al. (2007) Responses of the human airway epithelium transcriptome to in vivo injury. Physiol Genomics 29: 139–148.A. HeguyBG HarveyPL LeopoldI. DolgalevT. Raman2007Responses of the human airway epithelium transcriptome to in vivo injury.Physiol Genomics29139148
- 55. Maunders H, Patwardhan S, Phillips J, Clack A, Richter A (2007) Human bronchial epithelial cell transcriptome: gene expression changes following acute exposure to whole cigarette smoke in vitro. Am J Physiol Lung Cell Mol Physiol 292: L1248–L1256.H. MaundersS. PatwardhanJ. PhillipsA. ClackA. Richter2007Human bronchial epithelial cell transcriptome: gene expression changes following acute exposure to whole cigarette smoke in vitro.Am J Physiol Lung Cell Mol Physiol292L1248L1256
- 56. Yoshino K, Rubin JS, Higinbotham KG, Uren A, Anest V, et al. (2001) Secreted Frizzled-related proteins can regulate metanephric development. Mech Dev 102: 45–55.K. YoshinoJS RubinKG HiginbothamA. UrenV. Anest2001Secreted Frizzled-related proteins can regulate metanephric development.Mech Dev1024555
- 57. Chen Y, Stump RJ, Lovicu FJ, McAvoy JW (2004) Expression of Frizzleds and secreted frizzled-related proteins (Sfrps) during mammalian lens development. Int J Dev Biol 48: 867–877.Y. ChenRJ StumpFJ LovicuJW McAvoy2004Expression of Frizzleds and secreted frizzled-related proteins (Sfrps) during mammalian lens development.Int J Dev Biol48867877
- 58. Hayashi K, Spencer TE (2006) WNT pathways in the neonatal ovine uterus: potential specification of endometrial gland morphogenesis by SFRP2. Biol Reprod 74: 721–733.K. HayashiTE Spencer2006WNT pathways in the neonatal ovine uterus: potential specification of endometrial gland morphogenesis by SFRP2.Biol Reprod74721733
- 59. Wagatsuma A (2007) Adipogenic potential can be activated during muscle regeneration. Mol Cell Biochem 304: 25–33.A. Wagatsuma2007Adipogenic potential can be activated during muscle regeneration.Mol Cell Biochem3042533
- 60. Alfaro MP, Pagni M, Vincent A, Atkinson J, Hill MF, et al. (2008) The Wnt modulator sFRP2 enhances mesenchymal stem cell engraftment, granulation tissue formation and myocardial repair. Proc Natl Acad Sci U S A 105: 18366–18371.MP AlfaroM. PagniA. VincentJ. AtkinsonMF Hill2008The Wnt modulator sFRP2 enhances mesenchymal stem cell engraftment, granulation tissue formation and myocardial repair.Proc Natl Acad Sci U S A1051836618371
- 61. Cho SW, Her SJ, Sun HJ, Choi OK, Yang JY, et al. (2008) Differential effects of secreted frizzled-related proteins (sFRPs) on osteoblastic differentiation of mouse mesenchymal cells and apoptosis of osteoblasts. Biochem Biophys Res Commun 367: 399–405.SW ChoSJ HerHJ SunOK ChoiJY Yang2008Differential effects of secreted frizzled-related proteins (sFRPs) on osteoblastic differentiation of mouse mesenchymal cells and apoptosis of osteoblasts.Biochem Biophys Res Commun367399405
- 62. Das AV, Bhattacharya S, Zhao X, Hegde G, Mallya K, et al. (2008) The canonical Wnt pathway regulates retinal stem cells/progenitors in concert with Notch signaling. Dev Neurosci 30: 389–409.AV DasS. BhattacharyaX. ZhaoG. HegdeK. Mallya2008The canonical Wnt pathway regulates retinal stem cells/progenitors in concert with Notch signaling.Dev Neurosci30389409
- 63. Descamps S, Arzouk H, Bacou F, Bernardi H, Fedon Y, et al. (2008) Inhibition of myoblast differentiation by Sfrp1 and Sfrp2. Cell Tissue Res 332: 299–306.S. DescampsH. ArzoukF. BacouH. BernardiY. Fedon2008Inhibition of myoblast differentiation by Sfrp1 and Sfrp2.Cell Tissue Res332299306
- 64. Matsuyama M, Aizawa S, Shimono A (2009) Sfrp controls apicobasal polarity and oriented cell division in developing gut epithelium. PLoS Genet 5: e1000427.M. MatsuyamaS. AizawaA. Shimono2009Sfrp controls apicobasal polarity and oriented cell division in developing gut epithelium.PLoS Genet5e1000427
- 65. Morello R, Bertin TK, Schlaubitz S, Shaw CA, Kakuru S, et al. (2008) Brachy-syndactyly caused by loss of Sfrp2 function. J Cell Physiol 217: 127–137.R. MorelloTK BertinS. SchlaubitzCA ShawS. Kakuru2008Brachy-syndactyly caused by loss of Sfrp2 function.J Cell Physiol217127137
- 66. Deb A, Davis BH, Guo J, Ni A, Huang J, et al. (2008) SFRP2 regulates cardiomyogenic differentiation by inhibiting a positive transcriptional autofeedback loop of Wnt3a. Stem Cells 26: 35–44.A. DebBH DavisJ. GuoA. NiJ. Huang2008SFRP2 regulates cardiomyogenic differentiation by inhibiting a positive transcriptional autofeedback loop of Wnt3a.Stem Cells263544
- 67. Hayashi K, Burghardt RC, Bazer FW, Spencer TE (2007) WNTs in the ovine uterus: potential regulation of periimplantation ovine conceptus development. Endocrinology 148: 3496–3506.K. HayashiRC BurghardtFW BazerTE Spencer2007WNTs in the ovine uterus: potential regulation of periimplantation ovine conceptus development.Endocrinology14834963506
- 68. Imai K, D'Armiento J (2002) Differential gene expression of sFRP-1 and apoptosis in pulmonary emphysema. Chest 121: 7S.K. ImaiJ. D'Armiento2002Differential gene expression of sFRP-1 and apoptosis in pulmonary emphysema.Chest1217S
- 69. Bovolenta P, Rodriguez J, Esteve P (2006) Frizzled/RYK mediated signalling in axon guidance. Development 133: 4399–4408.P. BovolentaJ. RodriguezP. Esteve2006Frizzled/RYK mediated signalling in axon guidance.Development13343994408
- 70. Bovolenta P, Esteve P, Ruiz JM, Cisneros E, Lopez-Rios J (2008) Beyond Wnt inhibition: new functions of secreted Frizzled-related proteins in development and disease. J Cell Sci 121: 737–746.P. BovolentaP. EsteveJM RuizE. CisnerosJ. Lopez-Rios2008Beyond Wnt inhibition: new functions of secreted Frizzled-related proteins in development and disease.J Cell Sci121737746
- 71. Esteve P, Trousse F, Rodriguez J, Bovolenta P (2003) SFRP1 modulates retina cell differentiation through a beta-catenin-independent mechanism. J Cell Sci 116: 2471–2481.P. EsteveF. TrousseJ. RodriguezP. Bovolenta2003SFRP1 modulates retina cell differentiation through a beta-catenin-independent mechanism.J Cell Sci11624712481
- 72. Kim AS, Anderson SA, Rubenstein JL, Lowenstein DH, Pleasure SJ (2001) Pax-6 regulates expression of SFRP-2 and Wnt-7b in the developing CNS. J Neurosci 21: RC132.AS KimSA AndersonJL RubensteinDH LowensteinSJ Pleasure2001Pax-6 regulates expression of SFRP-2 and Wnt-7b in the developing CNS.J Neurosci21RC132
- 73. Lee HX, Ambrosio AL, Reversade B, De Robertis EM (2006) Embryonic dorsal-ventral signaling: secreted frizzled-related proteins as inhibitors of tolloid proteinases. Cell 124: 147–159.HX LeeAL AmbrosioB. ReversadeEM De Robertis2006Embryonic dorsal-ventral signaling: secreted frizzled-related proteins as inhibitors of tolloid proteinases.Cell124147159
- 74. Lee JL, Chang CJ, Chueh LL, Lin CT (2006) Secreted frizzled related protein 2 (sFRP2) decreases susceptibility to UV-induced apoptosis in primary culture of canine mammary gland tumors by NF-kappaB activation or JNK suppression. Breast Cancer Res Treat 100: 49–58.JL LeeCJ ChangLL ChuehCT Lin2006Secreted frizzled related protein 2 (sFRP2) decreases susceptibility to UV-induced apoptosis in primary culture of canine mammary gland tumors by NF-kappaB activation or JNK suppression.Breast Cancer Res Treat1004958