KL-6 is a high-molecular-weight glycoprotein classified as a human MUC1 mucin. It was hypothesized that KL-6 could be detectable in the circulating blood and especially in airway secretions in lung diseases associated with mucus production such as chronic obstructive pulmonary disease (COPD). Additional aims of this study were to investigate whether the levels of KL-6 in plasma and sputum are related to ageing and smoking history.
The concentrations of KL-6 in plasma and induced sputum supernatants from young and/or middle aged/elderly non-smokers, smokers and patients with COPD were assayed by ELISA (n = 201). The subjects were classified into five groups according to age, smoking status and presence of COPD. In addition, KL-6 expression in control and diseased lung i.e. samples from patients with COPD (n = 28), were analyzed by immunohistochemistry and digital image analysis.
The plasma levels of KL-6 increased with age both in non-smokers and smokers. Among middle aged/elderly subjects, plasma KL-6 levels in all smokers regardless of COPD were significantly higher than in non-smokers, whereas sputum levels of KL-6 were significantly higher in COPD compared not only to non-smokers but also to smokers. KL-6 was more prominently expressed in the bronchiolar/alveolar epithelium in COPD than in the control lungs. Plasma and sputum KL-6 levels correlated inversely with obstruction and positively with smoking history and ageing. The linear multiple regression analysis confirmed that age and cigarette smoking had independent effects on plasma KL-6.
KL-6 increases with ageing and chronic smoking history, but prospective studies will be needed to elucidate the significance of KL-6 in chronic airway diseases.
Mucus overproduction is a characteristic of inflammatory pulmonary diseases including asthma, chronic bronchitis, and cystic fibrosis. Expression of two mucin genes, MUC2 and MUC5AC, and their protein products (mucins), is modulated in certain disease states. Understanding the signaling mechanisms that regulate the production and secretion of these major mucus components may contribute significantly to development of effective therapies to modify their expression in inflamed airways.
To study the differential expression of Muc2 and Muc5ac, a novel monoclonal antibody recognizing guinea pig Muc2 and a commercially-available antibody against human MUC5AC were optimized for recognition of specific guinea pig mucins by enzyme-linked immunosorbent assay (ELISA), Western blot, and immunohistochemistry (IHC). These antibodies were then used to analyze expression of Muc2 and another mucin subtype (likely Muc5ac) in guinea pig tracheal epithelial (GPTE) cells stimulated with a mixture of pro-inflammatory cytokines [tumor necrosis factor-α (TNF-α), interleukin 1β (IL-1β), and interferon- γ (IFN-γ)].
The anti-Muc2 (C4) and anti-MUC5AC (45M1) monoclonal antibodies specifically recognized proteins located in Muc2-dominant small intestinal and Muc5ac-dominant stomach mucosae, respectively, in both Western and ELISA experimental protocols. IHC protocols confirmed that C4 recognizes murine small intestine mucosal proteins while 45M1 does not react. C4 and 45M1 also stained specific epithelial cells in guinea pig lung sections. In the resting state, Muc2 was recognized as a highly expressed intracellular mucin in GPTE cells in vitro. Following cytokine exposure, secretion of Muc2, but not the mucin recognized by the 45M1 antibody (likely Muc5ac), was increased from the GPTE cells, with a concomitant increase in intracellular expression of both mucins.
Given the tissue specificity in IHC and the differential hybridization to high molecular weight proteins by Western blot, we conclude that the antibodies used in this study can recognize specific mucin subtypes in guinea pig airway epithelium and in proteins from GPTE cells. In addition, Muc2 is highly expressed constitutively, modulated by inflammation, and secreted differentially (as compared to Muc5ac) in GPTE cells. This finding contrasts with expression patterns in the airway epithelium of a variety of mammalian species in which only Muc5ac predominates.
Human epithelial mucin, the major glycoprotein component of mucus, plays a critical role in host innate defense response against invading microbes by facilitating the mucociliary clearance. Excess mucin production, however, overwhelms the mucociliary clearance, resulting in not only defective mucosal defense but also conductive hearing loss in the middle ear and mucus obstruction in the airway. Indeed, mucus overproduction is a hall-mark of otitis media (OM) and chronic obstructive pulmonary diseases (COPD). Thus, tight regulation of mucin production plays an important role in maintaining an appropriate balance between beneficial and detrimental outcomes. We previously reported that Streptococcus pneumoniae (S. pneumoniae) up-regulates MUC5AC mucin expression via a positive MAPK ERK1/2 and a negative JNK1/2 signaling pathway. However, the signaling components including the up-stream activators and the down-stream transcription factors involved in these two path-ways remain largely unknown. In the present study, we showed that positive regulation of MUC5AC mucin expression by ERK1/2 is dependent on Ras-Raf-1 signaling pathway, whereas the negative regulation of MUC5AC expression by JNK1/2 is dependent on MEKK3. Moreover, transcriptional factor AP-1 acts as a key regulator for both of the positive and negative regulation of MUC5AC mucin expression as evidenced by mutagenesis analysis of two AP-1 sites in the promoter region of human MUC5AC mucin gene. Ras-Raf1-ERK1/2-dependent AP-1 activation positively regulates MUC5AC mucin induction by S. pneumoniae, whereas MEKK3-JNK1/2-dependent AP-1 activation negatively regulates it. Therefore, our data unveiled a novel signaling mechanism underlying the tight regulation of MUC5AC mucin induction by S. pneumoniae and may lead to the development of new therapeutic strategy for reducing mucus overproduction in both OM and COPD.
MUC5AC mucin; streptococcus pneumonia; ERK; JNK; AP-1; otitis media; COPD
Background: Airway epithelial goblet cell hyperplasia is known to occur in chronic smokers. Although the epidermal growth factor receptor has been implicated in this process, neither ErbB receptor expression nor the mucosecretory phenotype of the epithelium have been characterised in current smokers.
Methods: Bronchial biopsies obtained from non-smokers (n = 10) and current smokers, with or without chronic obstructive pulmonary disease (n = 51), were examined immunohistochemically to measure the expression of epidermal growth factor receptor, ErbB2, ErbB3, ErbB4 and mucin subtypes (MUC2, MUC5AC and MUC5B) in the bronchial epithelium. The results were correlated with neutrophil counts measured in the airway wall and induced sputum.
Results: Epidermal growth factor receptor, ErbB3 and MUC5AC expression, in addition to PAS staining, were significantly increased in all smokers compared with non-smokers, irrespective of the presence of chronic obstructive pulmonary disease. MUC5AC expression was significantly associated with both PAS staining and ErbB3 expression; no correlation was observed between either mucin or ErbB receptor expression and neutrophil counts.
Conclusions: The results suggest that long term current smoking induces enhanced epidermal growth factor receptor, ErbB3, and MUC5AC expression in vivo; these increases are not associated with the presence of neutrophilic inflammation. ErbB receptors may contribute to epithelial responses to cigarette smoke.
Rationale: Wood smoke–associated chronic obstructive pulmonary disease (COPD) is common in women in developing countries but has not been adequately described in developed countries.
Objectives: Our objective was to determine whether wood smoke exposure was a risk factor for COPD in a population of smokers in the United States and whether aberrant gene promoter methylation in sputum may modify this association.
Methods: For this cross-sectional study, 1,827 subjects were drawn from the Lovelace Smokers' Cohort, a predominantly female cohort of smokers. Wood smoke exposure was self-reported. Postbronchodilator spirometry was obtained, and COPD outcomes studied included percent predicted FEV1, airflow obstruction, and chronic bronchitis. Effect modification of wood smoke exposure with current cigarette smoke, ethnicity, sex, and promoter methylation of lung cancer-related genes in sputum on COPD outcomes were separately explored. Multivariable logistic and poisson regression models were used for binary and rate-based outcomes, respectively.
Measurements and Main Results: Self-reported wood smoke exposure was independently associated with a lower percent predicted FEV1 (point estimate [± SE] −0.03 ± 0.01) and a higher prevalence of airflow obstruction and chronic bronchitis (odds ratio, 1.96; 95% confidence interval, 1.52–2.52 and 1.64 (95% confidence interval, 1.31–2.06, respectively). These associations were stronger among current cigarette smokers, non-Hispanic whites, and men. Wood smoke exposure interacted in a multiplicative manner with aberrant promoter methylation of the p16 or GATA4 genes on lower percent predicted FEV1.
Conclusions: These studies identify a novel link between wood smoke exposure and gene promoter methylation that synergistically increases the risk for reduced lung function in cigarette smokers.
wood smoke; cigarette smokers; airflow obstruction; gene promoter methylation in sputum DNA
Background: The role of transforming growth factor-ß1 (TGF-ß1) in chronic obstructive pulmonary disease is still controversial, but it has been proposed that it may protect from mucus hypersecretion since it is able to downregulate mucin production. A study was undertaken to investigate the expression of TGF-ß1 and its type II receptor (TGF-ß RII) in the bronchial glands of smokers with COPD.
Methods: The expression of TGF-ß1 and TGF-ß RII were examined immunohistochemically in the bronchial glands of 24 smokers undergoing lung resection for solitary peripheral nodules: 12 with airflow limitation (smokers with COPD) and 12 with normal lung function.
Results: The expression of TGF-ß1 in bronchial glands was similar in the two groups of subjects while that of TGF-ß RII was lower in smokers with COPD than in smokers with normal lung function (p = 0.004). TGF-ß RII expression was inversely correlated with the values of Reid's index, a measure of gland size (p = 0.02, r = –0.50).
Conclusions: In the bronchial glands of smokers with COPD there is decreased expression of TGF-ß RII which is associated with bronchial gland enlargement. These findings support the view that the absence of TGF-ß signalling may induce structural changes in the bronchial glands which, in turn, may promote mucus hypersecretion.
Background: A common pathological feature of chronic inflammatory airway diseases such as asthma and chronic obstructive pulmonary disease (COPD) is mucus hypersecretion. MUC5AC is the predominant mucin gene expressed in healthy airways and is increased in asthmatic and COPD patients. Recent clinical trials indicate that phosphodiesterase type 4 (PDE4) inhibitors may have therapeutic value for COPD and asthma. However, their direct effects on mucin expression have been scarcely investigated.
Methods: MUC5AC mRNA and protein expression were examined in cultured human airway epithelial cells (A549) and in human isolated bronchial tissue stimulated with epidermal growth factor (EGF; 25 ng/ml). MUC5AC mRNA was measured by real time RT-PCR and MUC5AC protein by ELISA (cell lysates and tissue homogenates), Western blotting (tissue homogenates) and immunohistochemistry.
Results: EGF increased MUC5AC mRNA and protein expression in A549 cells. PDE4 inhibitors produced a concentration dependent inhibition of the EGF induced MUC5AC mRNA and protein expression with potency values (–log IC50): roflumilast (∼7.5) > rolipram (∼6.5) > cilomilast (∼5.5). Roflumilast also inhibited the EGF induced expression of phosphotyrosine proteins, EGF receptor, and phospho-p38- and p44/42-MAPK measured by Western blot analysis in A549 cells. In human isolated bronchus, EGF induced MUC5AC mRNA and protein expression was inhibited by roflumilast (1 µM) as well as the MUC5AC positive staining shown by immunohistochemistry.
Conclusion: Selective PDE4 inhibition is effective in decreasing EGF induced MUC5AC expression in human airway epithelial cells. This effect may contribute to the clinical efficacy of this new drug category in mucus hypersecretory diseases.
Smoking effects on physiological and gross pathology in chronic obstructive pulmonary disease (COPD) are relatively well described. However, there is little known in COPD about the detailed interrelationships between lung function and inflammatory profiles in different airway compartments from the same individual and whether airway inflammation in these different compartments differs in ex- and current smokers with established COPD.
We compared sputum, bronchoalveolar (BAL), and airway wall inflammatory profiles in current versus ex-smokers and related this to smoking intensity and lung function in 17 current and 17 ex-smokers with mild to moderate COPD.
Current smokers had more sputum mast cells (% differential and absolute numbers), whereas ex-smokers had increased sputum neutrophils. In BAL, there was a significant increase in eosinophils in current smokers, but ex-smokers had significantly increased neutrophils, lymphocytes, and epithelial cells. There were no cell profile differences observed in airway biopsies between current and ex-smokers and there were no correlations between the individual inflammatory cell populations in any of the airway compartments. In current smokers only, smoking intensity was negatively correlated with lung function, and associated with a reduction in overall cellularity of both sputum and BAL.
Airway inflammation persists in ex-smokers with COPD, but differs from COPD current smokers. The impact of smoking appears to vary in different airway compartments and any direct relationships between cellularity and lung function tended to be negative, ie, worse lung function indicated the presence of fewer cells.
current smokers; ex-smokers; airway cellularity; sputum; BAL; endobronchial biopsies
Normally a thin layer of mucus covers the surface of the gastrointestinal tract protecting the epithelial cells from their environment. In cystic fibrosis (CF), mucus accumulation is abnormally high, resulting in severe intestinal obstruction. The major structural components of mucus are large mucin glycoproteins. We determined specific mucin RNA and protein expression in the gastrointestinal tract of inbred CF transmembrane conductance regulator (CFTR) knockout (CF) mice and correlated expression with histological analyses of tissues. Mucins were detected histochemically using general carbohydrate stains and specific mucin antibodies. Mucin RNA levels were determined by reverse transcription-PCR. Comparisons were made between CF mice and control siblings, all maintained on a liquid diet after weaning. Analyses of the mucins Muc2, Muc3, and Muc5ac showed lower levels of RNA expression in the CF mice and similar levels of protein. Significantly, there was a sixfold increase in Muc1 RNA expression in the colon of the CF mouse and a moderate increase in Muc1 protein. Further, CF mice lacking Muc1 exhibited greatly diminished intestinal mucus obstruction when compared with Muc1- expressing CF mice and had better survival on solid food. We suggest that Muc1 plays an important role in the mucus obstructions observed in the gastrointestinal tract of the CFTR knockout mouse.
Little is known about gender differences in plasma biomarker levels in patients with chronic obstructive pulmonary disease (COPD).
There are differences in serum biomarker levels between women and men with COPD.
Explore gender differences in plasma biomarker levels in patients with COPD and smokers without COPD.
We measured plasma levels of IL-6, IL-8, IL-16, MCP-1, MMP-9, PARC and VEGF in 80 smokers without COPD (40 males, 40 females) and 152 stable COPD patients (76 males, 76 females) with similar airflow obstruction. We determined anthropometrics, smoking history, lung function, exercise tolerance, body composition, BODE index, co-morbidities and quality of life. We then explored associations between plasma biomarkers levels and the clinical characteristics of the patients and also with the clinical and physiological variables known to predict outcome in COPD.
The plasma biomarkers level explored were similar in men and women without COPD. In contrast, in patients with COPD the median value in pg/mL of IL-6 (6.26 vs 8.0, p = 0.03), IL-16 (390 vs 321, p = 0.009) and VEGF (50 vs 87, p = 0.02) differed between women and men. Adjusted for smoking history, gender was independently associated with IL-16, PARC and VEGF levels. There were also gender differences in the associations between IL-6, IL-16 and VEGF and physiologic variables that predict outcomes.
In stable COPD patients with similar airflow obstruction, there are gender differences in plasma biomarker levels and in the association between biomarker levels and important clinical or physiological variables. Further studies should confirm our findings.
To protect the surface of the stomach, the epithelial cells secrete a mucus layer, which is mainly comprised of the MUC5AC mucin. Further protection is provided by a thick glycocalyx on the apical surface of the epithelial cell, with the cell surface mucin MUC1 as a major component. Here, we investigate the production rate and turnover of newly synthesized mucin in mice and analyze the effects of early colonization and chronic infection with H. pylori. Metabolic incorporation of an azido GalNAc analog (GalNAz) was used as a nonradioactive method to perform pulse experiments in the whole animal. First, the subcellular movement of newly synthesized mucin and mucin turnover was determined in uninfected mice. Based on the time line for mucin transport and dissemination, 2, 6, and 12 h after GalNAz injection was selected to collect the stomachs from mice infected with H. pylori strain SS1 during early colonization (7 days) and chronic infection (90 days). The results demonstrated that the speed from the start of glycosylation to the final destination is faster for the membrane-bound mucin to reach the glycocalyx (2 h) than for the secretory mucins to become secreted into the mucus layer (5 h). Furthermore, infection with H. pylori reduces the rate of mucin turnover and decreases the levels of Muc1. Since H. pylori colonizes this mucus niche, the decreased turnover rate indicates that H. pylori creates a more stable and favorable environment for itself by impairing the defense mechanism for clearing the mucosal surface of pathogens by mucus flow.
Rationale: Induced mainly by cigarette smoking, chronic obstructive pulmonary disease (COPD) is a global public health problem characterized by progressive difficulty in breathing and increased mucin production. Previously, we reported that acrolein levels found in COPD sputum could activate matrix metalloproteinase-9 (MMP9).
Objectives: To determine whether acrolein increases expression and activity of MMP14, a critical membrane-bound endopeptidase that can initial a MMP-activation cascade.
Methods: MMP14 activity and adduct formation were measured following direct acrolein treatment. MMP14 expression and activity was measured in human airway epithelial cells. MMP14 immunohistochemistry was performed with COPD tissue, and in acrolein- or tobacco-exposed mice.
Measurements and Main Results: In a cell-free system, acrolein, in concentrations equal to those found in COPD sputum, directly adducted cysteine 319 in the MMP14 hemopexin-like domain and activated MMP14. In cells, acrolein increased MMP14 activity, which was inhibited by a proprotein convertase inhibitor, hexa-d-arginine. In the airway epithelium of COPD subjects, immunoreactive MMP14 protein increased. In mouse lung, acrolein or tobacco smoke increased lung MMP14 activity and protein. In cells, acrolein-induced MMP14 transcripts were inhibited by an epidermal growth factor receptor (EGFR) neutralizing antibody, EGFR kinase inhibitor, metalloproteinase inhibitor, or mitogen-activated protein kinase (MAPK) 3/2 or MAPK8 inhibitors, but not a MAPK14 inhibitor. Decreasing the MMP14 protein and activity in vitro by small interfering (si)RNA to MMP14 diminished the acrolein-induced MUC5AC transcripts. In acrolein-exposed mice or transgenic mice with lung-specific transforming growth factor-α (an EGFR ligand) expression, lung MMP14 and MUC5AC levels increased and these effects were inhibited by a EGFR inhibitor, erlotinib.
Conclusions: Taken together, these findings implicate acrolein-induced MMP14 expression and activity in mucin production in COPD.
cigarette smoke; acrolein; erlotinib; mucous cell metaplasia; chronic obstructive pulmonary disease
Persistent respiratory syncytial virus (RSV) infections have been associated with the exacerbation of chronic inflammatory diseases, including chronic obstructive pulmonary disease (COPD). This virus infects the respiratory epithelium, leading to chronic inflammation, and induces the release of mucins and the loss of cilia activity, two factors that determine mucus clearance and the increase in sputum volume. These alterations involve reactive oxygen species-dependent mechanisms. The antioxidant N-acetylcysteine (NAC) has proven useful in the management of COPD, reducing symptoms, exacerbations, and accelerated lung function decline. NAC inhibits RSV infection and mucin release in human A549 cells. The main objective of this study was to analyze the effects of NAC in modulating ciliary activity, ciliagenesis, and metaplasia in primary normal human bronchial epithelial cell (NHBEC) cultures infected with RSV. Our results indicated that RSV induced ultrastructural abnormalities in axonemal basal bodies and decreased the expression of β-tubulin as well as two genes involved in ciliagenesis, FOXJ1 and DNAI2. These alterations led to a decrease in ciliary activity. Furthermore, RSV induced metaplastic changes to the epithelium and increased the number of goblet cells and the expression of MUC5AC and GOB5. NAC restored the normal functions of the epithelium, inhibiting ICAM1 expression, subsequent RSV infection through mechanisms involving nuclear receptor factor 2, and the expression of heme oxygenase 1, which correlated with the restoration of the antioxidant capacity, the intracellular H2O2 levels and glutathione content of NHBECs. The results presented in this study support the therapeutic use of NAC for the management of chronic respiratory diseases, including COPD.
Chronic obstructive pulmonary disease (COPD) is associated with increased oxidative and nitrosative stress. The aim of our study was to assess the importance of these factors in the airways of healthy smokers and symptomatic smokers without airway obstruction, i.e. individuals with GOLD stage 0 COPD.
Exhaled NO (FENO) and induced sputum samples were collected from 22 current smokers (13 healthy smokers without any respiratory symptoms and 9 with symptoms i.e. stage 0 COPD) and 22 healthy age-matched non-smokers (11 never smokers and 11 ex-smokers). Sputum cell differential counts, and expressions of inducible nitric oxide synthase (iNOS), myeloperoxidase (MPO), nitrotyrosine and 4-hydroxy-2-nonenal (4-HNE) were analysed from cytospins by immunocytochemistry. Eosinophil cationic protein (ECP) and lactoferrin were measured from sputum supernatants by ELISA.
FENO was significantly decreased in smokers, mean (SD) 11.0 (6.7) ppb, compared to non-smokers, 22.9 (10.0), p < 0.0001. Induced sputum showed increased levels of neutrophils (p = 0.01) and elevated numbers of iNOS (p = 0.004), MPO (p = 0.003), nitrotyrosine (p = 0.003), and 4-HNE (p = 0.03) positive cells in smokers when compared to non-smokers. Sputum lactoferrin levels were also higher in smokers than in non-smokers (p = 0.02). Furthermore, we noted four negative correlations between FENO and 1) total neutrophils (r = -0.367, p = 0.02), 2) positive cells for iNOS (r = -0.503, p = 0.005), 3) MPO (r = -0.547, p = 0.008), and 4) nitrotyrosine (r = -0.424, p = 0.03). However, no major differences were found between never smokers and ex-smokers or between healthy smokers and stage 0 COPD patients.
Our results clearly indicate that several markers of oxidative/nitrosative stress are increased in current cigarette smokers compared to non-smokers and no major differences can be observed in these biomarkers between non-symptomatic smokers and subjects with GOLD stage 0 COPD.
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.
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.
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.
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.
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.
Introduction: The presence of MUC5AC (M1 antigen) and MUC6 have previously been found in ovarian mucinous cyst. We characterized the mucins in the crude mucus and tissue of a mature ovarian teratoma in an 8 year old girl. Materials and Methods: Mucins were purified from crude mucus by density gradient ultra-centrifugation in CsCl and analysed by gel-filtration and SDS-PAGE analysis. Mucin identification and expression was by western blotting and immunohistochemistry. Results: Histology showed a tumour with solid and cystic areas, with the cysts lined by colonic and respiratory mucosae. Equal volumes of 'sol' and 'gel' phases of approximately 10.0ml of crude mucus were obtained. Gel filtration and SDS-PAGE analyses suggested that the mucin was mainly of the large polymeric type which dissociated upon reduction of disulphide bonds with DTT. The colonic and respiratory epithelia predominantly expressed acidic mucin of the sialated and sulphated types respectively. MUC1 and MUC1c were expressed exclusively in respiratory epithelium, MUC2 and some MUC6 (focal) in the colonic tissue and MUC5AC in both tissues. Western blotting confirmed the presence of MUC2, MUC5AC and MUC5B in the secreted gel. Serine, threonine and proline made up the bulk of the amino acids in the sample. Discussion: Ovarian teratoma produced a highly viscous mucus secretion in which the mucin was largely polymeric and of the MUC2, MUC5AC and MUC5B type. The respiratory component of the teratoma expressed MUC1 and MUC1c and the colonic components of the teratoma expressed MUC2 and some MUC6. MUC5AC was expressed in both components.
Mucus; mucins; ovary; teratoma
Mucus hypersecretion contributes to morbidity and mortality in many obstructive lung diseases. Gel-forming mucins are the chief glycoprotein components of airway mucus, and elevated expression of these during mucous metaplasia precedes the hypersecretory phenotype. Five orthologous genes (MUC2, MUC5AC, MUC5B, MUC6, and MUC19) encode the mammalian gel-forming mucin family, and several have been implicated in asthma, cystic fibrosis, and chronic obstructive pulmonary disease pathologies. However, in the absence of a comprehensive analysis, their relative contributions remain unclear. Here, we assess the expression of the entire gel-forming mucin gene family in allergic mouse airways and show that Muc5ac is the predominant gel-forming mucin induced. We previously showed that the induction of mucous metaplasia in ovalbumin-sensitized and -challenged mouse lungs occurs within bronchial Clara cells. The temporal induction and localization of Muc5ac transcripts correlate with the induced expression and localization of mucin glycoproteins in bronchial airways. To better understand the tight regulation of Muc5ac expression, we analyzed all available 5′-flanking sequences of mammalian MUC5AC orthologs and identified evolutionarily conserved regions within domains proximal to the mRNA coding region. Analysis of luciferase reporter gene activity in a mouse transformed Clara cell line demonstrates that this region possesses strong promoter activity and harbors multiple conserved transcription factor–binding motifs. In particular, SMAD4 and HIF-1α bind to the promoter, and mutation of their recognition motifs abolishes promoter function. In conclusion, Muc5ac expression is the central event in antigen-induced mucous metaplasia, and phylogenetically conserved 5′ noncoding domains control its regulation.
mucin; metaplasia; airway; lung; epithelium
Mucins are gel-forming proteins that are responsible for the characteristic viscoelastic properties of mucus. Mucin overproduction is a hallmark of asthma, but the cellular requirements for airway mucin production are poorly understood. The endoplasmic reticulum (ER) protein anterior gradient homolog 2 (AGR2) is required for production of the intestinal mucin MUC2, but its role in the production of the airway mucins MUC5AC and MUC5B is not established. Microarray data were analyzed to examine the relationship between AGR2 and MUC5AC expression in asthma. Immunofluorescence was used to localize AGR2 in airway cells. Coimmunoprecipitation was used to identify AGR2-immature MUC5AC complexes. Agr2−/− mice were used to determine the role of AGR2 in allergic airway disease. AGR2 localized to the ER of MUC5AC- and MUC5B-producing airway cells and formed a complex with immature MUC5AC. AGR2 expression increased together with MUC5AC expression in airway epithelium from “Th2-high” asthmatics. Allergen-challenged Agr2−/− mice had greater than 50% reductions in MUC5AC and MUC5B proteins compared with allergen-challenged wild-type mice. Impaired mucin production in Agr2−/− mice was accompanied by an increase in the proportion of mucins contained within the ER and by evidence of ER stress in airway epithelium. This study shows that AGR2 increases with mucin overproduction in individuals with asthma and in mouse models of allergic airway disease. AGR2 interacts with immature mucin in the ER and loss of AGR2 impairs allergen-induced MUC5AC and MUC5B overproduction.
asthma; airway epithelium; mucus; endoplasmic reticulum stress; protein disulfide isomerase
The pathogenesis of exercise-induced bronchoconstriction (EIB) involves the release of mediators from several airway cells in response to exercise challenge, but the mechanism leading to airflow obstruction during EIB is incompletely understood.
To evaluate the role of secreted mucin in the pathogenesis of EIB.
Induced sputum was collected at baseline and 30 minutes after exercise challenge in patients with asthma with EIB. The expression of gel-forming mucins and epidermal growth factor receptor ligands were assessed by quantitative polymerase chain reaction. Secreted mucin 5AC (MUC5AC), the eicosanoids cysteinyl leukotrienes (cysLTs) and 15S-hydroxyeicosatetraenoic acid (15S-HETE), and tachykinins neurokinin A (NKA) and substance P (SP) were measured in induced sputum supernatant.
Among the gel-forming mucins, MUC5AC was expressed at the highest level. The gene expression of MUC5AC increased after exercise challenge compared with baseline and was associated with EIB severity by regression analysis. The relative levels of MUC5AC in induced sputum increased from a geometric mean of 9.5 at baseline to 18.4 postexercise challenge. Associations between the levels of MUC5AC and cysLTs and between the levels of cysLTs and NKA postexercise challenge were identified by regression analysis.
These data indicate that (1) the predominant gel-forming mucin expressed in induced sputum of patients with asthma with EIB is MUC5AC; (2) an increase in MUC5AC gene expression and release of MUC5AC protein occurs after exercise challenge; and (3) MUC5AC release may occur through the cysLT-associated activation of sensory airway nerves.
Asthma; exercise-induced bronchoconstriction; epithelial cell; goblet cell; mucin
Mucociliary clearance is a critical innate defense system responsible for clearing up invading pathogens including bacteria and virus. Although the right amount of mucus is good, excessive mucus causes airway obstruction and tends to precipitate disease symptoms. Rhinovirus (RV) is a common cold virus that causes asthma and chronic obstructive pulmonary disease exacerbation. Mucus overproduction has been linked to the pathogenesis of RV-induced diseases and disease exacerbations. However, the molecular mechanism is not clear. In this study, using one of the major airway mucin-MUC5AC as marker, we found that both major and minor groups of RV induced mucin production in primary human epithelial cells and cell line. RV1A (a minor group of RV) could induce mucous cell metaplasia in vivo. Viral replication was needed for RV-induced mucin expression, and this induction was also dependent on TLR3, suggesting the involvement of double-stranded (ds) RNA signaling. Indeed, dsRNA alone could also induce mucin expression. TLR3-mediated mucin induction was negatively regulated by MyD88, and only partially dependent on TRIF, which suggests a departure from well-documented TLR3 signaling paradigm that mediates inflammatory and other innate defense gene inductions. In addition, TLR3 signaling activated epidermal growth factor receptor (EGFR) through inductions of the expression of EGFR ligands (transforming growth factor-α and amphiregulin), which in turn activated EGFR-ERK signaling and mucin expression through an autocrine/paracrine loop. This novel coupling of antiviral defense machinery (i.e., TLR3) and major epithelial proliferation/repair pathway (i.e., EGFR) might play an important role in viral-induced airway remodeling and airway disease exacerbation.
mucin; airway epithelium; TLR3; rhinovirus
Chronic obstructive pulmonary disease (COPD) is characterized by inflammation and remodeling of the lungs. This results in alterations in extracellular matrix (ECM) and structural changes leading to airflow obstruction. We studied the expression of tenascin-C (Tn-C) and alpha smooth muscle actin (α-SMA), which act as a marker of myofibroblasts, in large airways from COPD patients. Our aim was to elucidate whether this expression correlated with smoking or with disease development.
Bronchoscopy was performed on 20 COPD patients (mean age 56 years; range 39-61; FEV1/FVC < 70% and FEV1 median 53% (range 33-69) of predicted). Age and smoking matched smokers (S) without COPD (n = 13) and age matched non-smokers (NS) (n = 14) served as controls. Bronchial mucosal biopsies were analyzed by immunohistochemistry. The distribution of Tn-C expression was assessed and graded in three levels, and the number of spindle shaped cells staining positive for α-SMA were counted.
Biopsies from COPD patients had more (P < 0.001) Tn-C expression than the two control groups. A significantly (P < 0.05) increased number of spindle shaped cells expressing α-SMA was observed in COPD patients compared with the controls. Smokers and nonsmokers did not differ in this respect. The expression of Tn-C correlated positively (P < 0.001) to the number of α-SMA positive cells.
We demonstrate increased expression of Tn-C and α-SMA positive cells in the large airways in COPD. This was not associated to smoking per se, but to the presence of airway obstruction. Our findings add new information regarding remodeling characteristics and highlight the large airways as a potential site for airways obstruction in COPD.
Mucin genes MUC2, MUC5AC and MUC5B have been identified as major gel-forming mucins in the middle ear (ME). This study compared polymorphisms in MUC2, MUC5AC and MUC5B genes in otitis media (OM) patients and controls.
Cross-sectional case-control study. Patients age 6 months to 14 years undergoing routine tympanostomy tube (TT) insertion for recurrent otitis media (RecOM) or chronic otitis media with effusion (OME) were compared to control patients with no history of OM undergoing an unrelated procedure.
Genomic DNA extracted from peripheral blood samples was analyzed by Southern blots using gene-specific probes to determine sizes of MUC2 and MUC5AC genes. Polymerase chain reaction (PCR) was used to determine the size of MUC5B genes.
Twenty RecOM patients, twenty OME patients, and forty control patients were analyzed. There were no statistically significant differences in polymorphism expression identified between groups for MUC2 and MUC5B genes. OME patients were more likely than Controls or RecOM patients to carry the longer MUC5AC-b allele.
Differences in mucin polymorphisms have been demonstrated in other diseases. In otitis media, OME patients are more likely than Controls or RecOM to carry the longer MUC5AC-b allele. MUC5AC is a primary innate defense mechanism for ME epithelium and alterations in protein structure have the potential to affect that defense and predispose patients to disease. This study demonstrates that the properties of post-transcriptionally modified MUC5AC deserve further study and specific pathogen-host interactions studies should explore the impact of this polymorphism.
mucin gene; otitis media; polymorphism
Mucins are large, hydrophilic glycoproteins that protect wet-surfaced epithelia from pathogen invasion as well as provide lubrication. At least 17 mucin genes have been cloned to date. This study sought to determine the mucin gene expression profile of the human male urogenital tract epithelia, to determine if mucins are present in seminal fluid, and to assess the effect of androgens on mucin expression.
METHODS AND RESULTS
Testis, epididymis, vas deferens, seminal vesicle, prostate, bladder, urethra and foreskin were assessed for mucin expression by RT-PCR and immunohistochemistry. Epithelia of the vas deferens, prostate and urethra expressed the greatest number of mucins, each expressing 5–8 mucins. Messenger RNA of MUC1 and MUC20, both membrane-associated mucins, were detected in most tissues analyzed. Conversely, MUC6 was predominantly detected in seminal vesicle. MUC1, MUC5B and MUC6 were detected in seminal fluid samples by immunoblot analysis. Androgens had no effect on mucin expression by cultured human prostatic epithelial cells.
Each region of urogenital tract epithelium expressed a unique mucin gene repertoire. Secretory mucins are present in seminal fluid, and androgens do not appear to regulate mucin gene expression.
Mucin; MUC; Male urogenital tract; male reproductive tract epithelia
The predominant emphysema phenotype is associated with more severe airflow limitation in patients with chronic obstructive pulmonary disease (COPD). A study was undertaken to investigate whether COPD patients, with or without emphysema quantitatively confirmed by high resolution computed tomography (HRCT), have different COPD severity as assessed by the BODE index (body mass index, airflow obstruction, dyspnoea, exercise performance) and inspiratory capacity to total lung capacity ratio (IC/TLC), and by different biological markers of lung parenchymal destruction.
Twenty six outpatients with COPD and eight healthy non‐smokers were examined. Each subject underwent HRCT scanning, pulmonary function tests, cell counts, and measurements of neutrophil elastase, matrix metalloproteinase (MMP)‐9 and tissue inhibitor of metalloproteinase (TIMP)‐1 in induced sputum, as well as measurement of desmosine, a marker of elastin degradation in urine, plasma and sputum.
Patients with HRCT confirmed emphysema had a higher BODE index and lower IC/TLC ratio than subjects without HRCT confirmed emphysema and controls. Forced expiratory volume in 1 second (FEV1), FEV1/forced vital capacity ratio, and carbon monoxide transfer coefficient were lower, whereas the number of eosinophils, MMP‐9, and the MMP‐9/TIMP‐1 ratio in sputum were higher in patients with emphysema. In COPD patients the number of sputum eosinophils was the biological variable that correlated positively with the HRCT score of emphysema (p = 0.04).
These results suggest that COPD associated with HRCT confirmed emphysema is characterised by more severe lung function impairment, more intense airway inflammation and, possibly, more serious systemic dysfunction than COPD not associated with HRCT confirmed emphysema.
chronic obstructive pulmonary disease; emphysema; biological markers; outcomes
Several matrix metalloproteinases (MMPs) are involved in the pathogenesis of chronic obstructive pulmonary disease (COPD). In mice, MMP‐12 plays a crucial role in the development of cigarette smoke induced emphysema. A study was undertaken to investigate the role of MMP‐12 in the development of COPD in human smokers.
Induced sputum samples were collected from patients with stable COPD (n = 28), healthy smokers (n = 14), never smokers (n = 20), and former smokers (n = 14). MMP‐12 protein levels in induced sputum were determined by ELISA and compared between the four study groups. MMP‐12 enzymatic activity in induced sputum was evaluated by casein zymography and by cleaving of a fluorescence quenched substrate.
Median (IQR) MMP‐12 levels were significantly higher in COPD patients than in healthy smokers, never smokers, and former smokers (17.5 (7.1–42.1) v 6.7 (3.9–10.4) v 4.2 (2.4–11.3) v 6.1 (4.5–7.6) ng/ml, p = 0.0002). MMP‐12 enzymatic activity was significantly higher in patients with COPD than in controls (4.11 (1.4–8.0) v 0.14 (0.1–0.2) μg/μl, p = 0.0002).
MMP‐12 is markedly increased in induced sputum from patients with stable COPD compared with controls, suggesting a role for MMP‐12 in the development of COPD in smokers.
chronic obstructive pulmonary disease; matrix metalloproteinases; induced sputum; MMP‐12; smoking