Cigarette smoking reprograms apical junctional complex molecular architecture in the human airway epithelium in vivo

Abstract

The apical junctional complex (AJC), composed of tight and adherens junctions, maintains epithelial barrier function. Since cigarette smoking and chronic obstructive pulmonary disease (COPD), the major smoking-induced disease, are associated with increased lung epithelial permeability, we hypothesized that smoking alters the transcriptional program regulating airway epithelial AJC integrity. Transcriptome analysis revealed global down-regulation of physiological AJC gene expression in the airway epithelium of healthy smokers (n = 59) compared to nonsmokers (n = 53) in association with changes in canonical epithelial differentiation pathways such as PTEN signaling accompanied by induction of cancer-related AJC components. The overall expression of AJC-related genes was further decreased in COPD smokers (n = 23). Exposure of airway epithelial cells to cigarette smoke extract in vitro resulted in down-regulation of several AJC genes paralleled by decreased transepithelial resistance. Thus, cigarette smoking induces transcriptional reprogramming of airway epithelial AJC architecture from its physiological pattern necessary for barrier function toward a disease-associated molecular phenotype.

Fig. 1

Expression of AJC-related genes in the SAE of healthy nonsmokers. The ordinate represents P calls (percent subjects in each group expressing a given gene; yellow bars) and normalized log-transformed expression levels (means ± SD) of each gene (red genes with relatively high expression, ≥20; blue genes with relatively low expression, <20)

Fig. 2

Smoking alters SAE AJC gene expression. a Volcano plot comparing the normalized expression of all gene probe sets (left panel) or AJC-encoding genes (right panel) in the SAE of healthy smokers versus healthy nonsmokers; y-axis, negative log of P value; x-axis, log₂-transformed fold-change; red dots significant differentially expressed probe sets, grey dots not significant probe sets. b Differentially expressed physiological AJC-encoding genes (blue bars) and other genes belonging to various functional categories (black bars) in the SAE of healthy smokers versus healthy nonsmokers; y-axis, log₂-transformed fold-change. c Diagram of the AJC architecture; significantly up- and downregulated in the SAE of healthy smokers are depicted as red boxes and blue boxes, respectively; yellow boxes indicate genes with no significant change; edges indicate known interactions between individual AJC proteins based on biochemical studies [–7, 70]; the shapes of the nodes indicate different classes of AJC molecules determined based on the NCBI Entrez Gene database. d Immunofluorescence analysis of differential expression of CLDN1 and CDH1 proteins (red) in the cytospin preparations of SAE of healthy smokers versus healthy nonsmokers. Nuclei stained with DAPI (blue); scale bars 10 μm. More examples are shown in Supplementary Fig. 2

Fig. 3

Smoking-dependent changes in the SAE AJC gene expression in COPD. a Unsupervised hierarchical clustering of healthy nonsmokers (green lines), healthy smokers (orange lines) and COPD smokers (blue lines) based on the SAE expression of AJC-encoding genes. Genes expressed above the average are represented in red, below average in blue, and average in white. b PCA of healthy nonsmokers (green circles), healthy smokers (orange circles) and COPD smokers (blue circles) on all expressed gene probe sets (left panel) and on AJC-encoding gene probe sets (right panel). The percentage contributions of the first three principal components are indicated. c Distribution (percent of subjects/group; ordinate) of healthy nonsmokers (green), healthy smokers (orange) and COPD smokers (blue) based on the SAE AJC index (I_AJC; percent of abnormally expressed AJC-encoding genes; abscissa). The inserted box-plot shows the I_AJC distribution in each group: the bottom and top of each box represent the 25th and 75th percentile (the lower and upper quartiles, respectively), and the band near the middle of each box is the 50th percentile (the median); the ends of the whiskers represent the 10th percentile and the 90th percentile; points lying outside the whiskers are considered outliers; P values were determined using the Wilcoxon signed-ranks test. d Abnormal expression frequency (ordinate; percent of subjects/group having expression of a given AJC-encoding gene below the normal range)

Fig. 4

Smoking-sensitive AJC-associated molecular pathways. a IPA-based canonical pathways enriched among AJC-associated genes (smoking-sensitive genes negatively correlating with the I_AJC with r < −0.5). The ordinate indicates the degree of enrichment (percent of AJC-associated genes belonging to the pathway; orange boxes) and shows −log P values of enrichment. The axonal guidance pathway (enrichment 3.3%; P < 0.01) was excluded as being specific to neuronal signaling. b Spearman correlation (ρ) analysis of the PTEN gene normalized expression in the SAE of healthy smokers with the I_AJC. c STRING 8.2-based model of relationships between smoking-responsive physiological AJC-encoding genes and selected AJC-related genes correlating with the I_AJC with r < −0.5. Each circle corresponds to an individual gene with the size proportional to the degree of correlation (r) with the I_AJC, and the color corresponding to the clusters in which the genes were grouped based on known protein–protein interactions (Markov clustering); the form of the lines reflects the confidence of the association from thick (most confident) to dashed (least confident)

Fig. 5

Smoking-sensitive AJC-associated transcriptional network in the SAE. a Biological process-related GO categories significantly enriched in the dataset of AJC-associated genes (smoking-responsive; correlating with the I_AJC with r < −0.5); x-axis, enrichments (percent of AJC-associated genes in the particular GO category); y-axis shows –log P values of enrichment. The dashed lines indicate the arbitrarily defined area of significant enrichment (enrichment >10%; P < 0.05). b The core AJC network genes and their coexpression-based “interactions” with the intrinsic AJC network genes were identified based on their correlation with the I_AJC and each other as described in “Materials and methods”. Each circle represents an individual gene with the size differences proportional to inter-network interaction intensity, and the color corresponding to the clusters based on the unsupervised hierarchical clustering; the form of the lines reflects the strength of the correlation between the two genes (see Supplementary Table 4). c Spearman rank correlation (ρ) analysis of the PTEN gene-normalized expression and the mean AJC network gene expression in the SAE of healthy smokers. d Mean normalized expression levels of AJC network genes in the study groups; the data presented are the means±SD for each group and the P values indicate the significance of the differences between the groups

Fig. 6

Effect of the CSE on the airway epithelial AJC gene expression and barrier integrity in vitro. a Viability of differentiated 16HBE14o⁻ cells apically stimulated with 1, 2, or 4% CSE for 6 days as described in “Materials and methods” compared to unstimulated cells (n = 3 in each group); NS nonsignificant difference (P > 0.05) for all three comparisons. b Expression of AJC genes (CLDN1, CLDN8, CGN, CDH1), PTEN pathway genes (PTEN, FOXO3), and oxidative stress-related genes (CYP1A1, CYP1B1, NQO1) in 16HBE14o⁻ cells stimulated with 2% CSE for 6 days as described in “Materials and methods” compared to unstimulated cells determined by TaqMan PCR; y-axis, normalized expression levels; P values indicate the significance of differences between the groups; n = 3 in each group; c Transepithelial resistance of differentiated 16HBE14o⁻ cell monolayers (expressed as percent of initial level) was measured during 6 days of stimulation with 2% CSE, as described in “Materials and methods” compared to control (unstimulated) cells; P values indicate the significance of differences between the groups; n = 3 in each group