Genes associated with MUC5AC expression in small airway epithelium of human smokers and non-smokers

Abstract

Background: Mucus hypersecretion contributes to the morbidity and mortality of smoking-related lung diseases, especially chronic obstructive pulmonary disease (COPD), which starts in the small airways. Despite progress in animal studies, the genes and their expression pattern involved in mucus production and secretion in human airway epithelium are not well understood. We hypothesized that comparison of the transcriptomes of the small airway epithelium of individuals that express high vs low levels of MUC5AC, the major macromolecular component of airway mucus, could be used as a probe to identify the genes related to human small airway mucus production/secretion.

Methods: Flexible bronchoscopy and brushing were used to obtain small airway epithelium (10th to 12th order bronchi) from healthy nonsmokers (n=60) and healthy smokers (n=72). Affymetrix HG-U133 plus 2.0 microarrays were used to assess gene expression. Massive parallel sequencing (RNA-Seq) was used to verify gene expression of small airway epithelium from 5 nonsmokers and 6 smokers.

Results: MUC5AC expression varied 31-fold among the healthy nonsmokers. Genome-wide comparison between healthy nonsmokers (n = 60) grouped as "high MUC5AC expressors" vs "low MUC5AC expressors" identified 528 genes significantly up-regulated and 15 genes significantly down-regulated in the high vs low expressors. This strategy identified both mucus production and secretion related genes under control of a network composed of multiple transcription factors. Based on the literature, genes in the up-regulated list were used to identify a 73 "MUC5AC-associated core gene" list with 9 categories: mucus component; mucus-producing cell differentiation-related transcription factor; mucus-producing cell differentiation-related pathway or mediator; post-translational modification of mucin; vesicle transport; endoplasmic reticulum stress-related; secretory granule-associated; mucus secretion-related regulator and mucus hypersecretory-related ion channel. As a validation cohort, we assessed the MUC5AC-associated core gene list in the small airway epithelium of an independent set of healthy smokers (n = 72). There was up-regulation of MUC5AC in the small airway epithelium of smokers (2.3-fold, p < 10-8) associated with a coordinated up-regulation of MUC5AC-associated core gene expression pattern in the small airway epithelium of smokers (p < 0.01). Deep sequencing confirmed these observations.

Conclusion: The identification of the genes associated with increased airway mucin production in humans should be useful in understanding the pathogenesis of airway mucus hypersecretion and identifying therapeutic targets. AUTHOR SUMMARY: Mucus hypersecretion contributes to the morbidity and mortality of smoking-related lung diseases, especially chronic obstructive pulmonary disease (COPD), which starts in the small airways. Little is known about the gene networks associated with the synthesis and secretion of mucins in the human small airway epithelium. Taking advantage of the knowledge that MUC5AC is a major mucin secreted by the small airway epithelium, the expression of MUC5AC in small airway epithelium is highly regulated at the transcriptional level and our observation that healthy nonsmokers have variable numbers of MUC5AC+ secretory cells in the human small airway epithelium, we compared genome-wide gene expression of the small airway epithelium of high vs low MUC5AC expressors from 60 nonsmokers to identify the genes associated with MUC5AC expression. This novel strategy enabled identification of a 73 "MUC5AC-associated core gene" list with 9 categories, which control a series of processes from mucin biosynthesis to mucus secretion. The coordinated gene expression pattern of MUC5AC-associated core genes were corroborated in an independent cohort of 72 healthy smokers. Deep sequencing of small airway epithelium RNA confirmed these observations. This finding will be useful in identifying therapeutic targets to treat small airway mucus hypersecretion.

Figure 1

Variability of proportions of surface MUC5AC⁺cells in the small airway epithelium of healthy nonsmokers. Immunofluorescence of MUC5AC staining (red) was processed on cytospin slides prepared from 10^th-12^th order brushed airway epithelial cells. Shown are examples from 6 individuals. Not shown, IgG irrelevant control, negative for MUC5AC staining. Blue - DAPI. Bar = 20 μm.

Figure 2

Healthy nonsmokers were grouped based on MUC5AC gene expression in 10-12^thorder brushed airway epithelial cells. Shown is data for n = 60 nonsmokers divided into nonsmoker “high MUC5AC expressors” (MUC5AC expression, highest quartile of all healthy nonsmokers, n = 15, upper shaded region) and nonsmoker “low MUC5AC expressors” (MUC5AC expression, lowest quartile of all healthy non-smokers, n = 15, lower shaded region). Expression of 5 endogenous control genes (ACTB, GAPDH, B2M, RPLP0 and PPIA) in the same samples are used as controls to assess the expression variability in nonsmokers of housekeeping genes compared to MUC5AC. The coefficients of variation (CV) of MUC5AC and endogenous control gene expression are shown.

Figure 3

Identification of MUC5AC-associated core genes. Shown is a volcano plot comparing the normalized expression of gene probe sets in the 10-12th order brushed airway epithelial cells of nonsmoker-high MUC5AC expressors vs low MUC5AC expressors; y-axis - negative log of p value; x-axis - log2-transformed fold-change; red dots - probe sets with significant p value; blue dots - probe sets with non-significant p value. Differentially expressed genes were determined by unequal variance Student’s t-test followed by Benjamini-Hochberg correction (p < 0.05). There were 528 genes identified genome-wide that were significantly correlated with MUC5AC high expressors. Of these, 73 genes were identified as MUC5AC-associated core genes based on literature mining (Table1, Additional file 1: Table S1, S2).

Figure 4

cMUC5AC expression and MUC5AC-core genes in the small airway epithelium of healthy smokers. A. MUC5AC gene expression distribution in healthy smokers (blue, n = 60) compared to healthy nonsmokers (red, n = 72). Y-axis, number of subjects; X-axis, normalized MUC5AC expression. B. Volcano plot of MUC5AC-core genes comparing healthy smokers and healthy nonsmokers. Differentially expressed genes between smokers (n = 72) and nonsmokers (n = 60) were determined by unequal variance Student’s t-test followed by Benjamini-Hochberg correction (p < 0.05). Only MUC5AC-core genes were plotted. y-axis - negative log of p value; x-axis - log2-transformed fold-change; red dots – smoking up-regulated MUC5AC-core genes; blue dots – smoking down-regulated MUC5AC-core genes; grey dots – smoking unchanged MUC5AC-core genes. Numbers of each group are shown. C. Index of expression of the MUC5AC-core genes (% of abnormally expressed MUC5AC-core genes beyond that of healthy nonsmoker MUC5AC low-expressors in healthy nonsmokers (blue) compared to healthy smokers (red). Fold-change represents the average index difference between the groups, p value indicates significant differences between the groups. D. Unsupervised hierarchical clustering analysis of expression of MUC5AC-core genes in nonsmoker-high MUC5AC expressors (black, highest quartile of all healthy non-smokers, n = 15), nonsmoker-low MUC5AC expressors (green, lowest quartile of all healthy non-smokers, n = 15), smoker-high MUC5AC expressors (red, highest quartile of all healthy smokers, n = 18) and smoker-low MUC5AC expressors (blue, lowest quartile of all healthy smokers, n = 18). Genes expressed above the average are represented in red, below average in blue, and average in white.

Figure 5

MUC5AC-core genes assessed by RNA-Seq analysis in healthy smokers and healthy nonsmokers. Shown is a hierarchical cluster analysis of RNA-Seq data of expression of the MUC5AC-associated core genes from 5 healthy nonsmokers and 6 healthy smokers. Smoking status was represented by different colors (nonsmoker, blue; smoker, brown). In the heat map, gene expression above the average is represented in red, below average in blue, and average in white. The color schemes represent the relative expression level across each row (each gene). Dendrogram color denotes there are two different groups independent of smoking status. The expression level (RPKM) of MUC5AC is shown under the label of each individual (NS = nonsmoker; S = smoker).

Figure 6

Proposed gene network of the MUC5AC-core genes. From the MUC5AC-core gene list, genes with at least one literature supported connection to other MUC5AC-core genes were selected (see Methods for detail). The connection denotes that the expression level of one gene is affected by the other gene; the data is derived from literature with in vitro gene overexpression/activation/knockdown experiments or transgenic mice data (see Additional file 1: Table S5 for references). Protein to protein interactions are not included. Genes in the brown solid circle include, transcription factors (SPDEF, SOX2, KLF4) and the Wnt, Notch, MAPK pathways, which can affect MUC5AC-producing cell differentiation. A red dashed circle was used to emphasize their important roles. Genes in green solid circle represent transcription factors in the MUC5AC-core gene list with literature supported potential role in MUC5AC-producing cells. Genes in green rectangles represent non-transcription factors in the MUC5AC-core gene list. Arrows indicate upstream gene can regulate downstream gene expression. Dashed arrow, based on mouse data [31], KLF4 can affect mucus-producing cell differentiation, but there is no direct evidence showing KLF4 can affect MUC5AC expression. The solid line linked to pathway indicates that MAPK13, KRAS and RPS6KA3 belong to “MAPK pathway”; HES1 and TSTA3 belong to “Notch pathway”; LRRFIP2 belong to “Wnt pathway”. The “T” like symbol indicates a potential blocking effect.