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1.  Analysis of the proteome of human airway epithelial secretions 
Proteome Science  2011;9:4.
Airway surface liquid, often referred to as mucus, is a thin layer of fluid covering the luminal surface that plays an important defensive role against foreign particles and chemicals entering the lungs. Airway mucus contains various macromolecules, the most abundant being mucin glycoproteins, which contribute to its defensive function. Airway epithelial cells cultured in vitro secrete mucins and nonmucin proteins from their apical surface that mimics mucus production in vivo. The current study was undertaken to identify the polypeptide constituents of human airway epithelial cell secretions to gain a better understanding of the protein composition of respiratory mucus.
Fifty-five proteins were identified in the high molecular weight fraction of apical secretions collected from in vitro cultures of well-differentiated primary human airway epithelial cells and isolated under physiological conditions. Among these were MUC1, MUC4, MUC5B, and MUC16 mucins. By proteomic analysis, the nonmucin proteins could be classified as inflammatory, anti-inflammatory, anti-oxidative, and/or anti-microbial.
Because the majority of the nonmucin proteins possess molecular weights less than that selected for analysis, it is theoretically possible that they may associate with the high molecular weight and negatively charged mucins to form a highly ordered structural organization that is likely to be important for maintaining the proper defensive function of airway mucus.
PMCID: PMC3036598  PMID: 21251289
2.  Genes associated with MUC5AC expression in small airway epithelium of human smokers and non-smokers 
BMC Medical Genomics  2012;5:21.
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.
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.
PMCID: PMC3443416  PMID: 22676183
3.  CREB Mediates Prostaglandin F2α-Induced MUC5AC Overexpression 
Mucus secretion is an important protective mechanism for the luminal lining of open tubular organs, but mucin overproduction in the respiratory tract can exacerbate the inflammatory process and cause airway obstruction. Production of MUC5AC, a predominant gel-forming mucin secreted by airway epithelia, can be induced by various inflammatory mediators such as prostaglandins. The two major prostaglandins involved in inflammation are prostaglandin (PG) E2 and F2α. PGE2-induced mucin production has been well studied, but the effect of PGF2α on mucin production remains poorly understood. To elucidate the effect and underlying mechanism of PGF2α on MUC5AC production, we investigated the signal transduction of PGF2α associated with this effect using normal human tracheobronchial epithelial cells. Our results demonstrated that PGF2α induces MUC5AC overproduction via a signaling cascade involving protein kinase C, extracellular signal-regulated kinase, p90 ribosomal S6 protein kinase, and cAMP response element binding protein (CREB). The regulation of PGF2α-induced MUC5AC expression by CREB was further confirmed by cAMP response element-dependent MUC5AC promoter activity and by interaction between CREB and MUC5AC promoter. The abrogation of all downstream signaling activities via suppression of each signaling molecule along the pathway indicates that a single pathway from PGF2α receptor to CREB is responsible for inducing MUC5AC overproduction. As CREB also mediates mucin overproduction induced by PGE2 and other inflammatory mediators, our findings have important clinical implication for the management of airway mucus hypersecretion.
PMCID: PMC2756474  PMID: 19201889
Lung; Inflammation; Signal Transduction; Transcription Factors; Human; Gene Regulation
4.  Serine Protease(s) Secreted by the Nematode Trichuris muris Degrade the Mucus Barrier 
The polymeric mucin component of the intestinal mucus barrier changes during nematode infection to provide not only physical protection but also to directly affect pathogenic nematodes and aid expulsion. Despite this, the direct interaction of the nematodes with the mucins and the mucus barrier has not previously been addressed. We used the well-established Trichuris muris nematode model to investigate the effect on mucins of the complex mixture of immunogenic proteins secreted by the nematode called excretory/secretory products (ESPs). Different regimes of T. muris infection were used to simulate chronic (low dose) or acute (high dose) infection. Mucus/mucins isolated from mice and from the human intestinal cell line, LS174T, were treated with ESPs. We demonstrate that serine protease(s) secreted by the nematode have the ability to change the properties of the mucus barrier, making it more porous by degrading the mucin component of the mucus gel. Specifically, the serine protease(s) acted on the N-terminal polymerising domain of the major intestinal mucin Muc2, resulting in depolymerisation of Muc2 polymers. Importantly, the respiratory/gastric mucin Muc5ac, which is induced in the intestine and is critical for worm expulsion, was protected from the depolymerising effect exerted by ESPs. Furthermore, serine protease inhibitors (Serpins) which may protect the mucins, in particular Muc2, from depolymerisation, were highly expressed in mice resistant to chronic infection. Thus, we demonstrate that nematodes secrete serine protease(s) to degrade mucins within the mucus barrier, which may modify the niche of the parasite to prevent clearance from the host or facilitate efficient mating and egg laying from the posterior end of the parasite that is in intimate contact with the mucus barrier. However, during a TH2-mediated worm expulsion response, serpins, Muc5ac and increased levels of Muc2 protect the barrier from degradation by the nematode secreted protease(s).
Author Summary
Gastrointestinal parasitic worm infections cause significant morbidity, affecting up to a third of the world's populationand their domestic pets and livestock. Mucus, the gel-like material that blankets the surface of the intestine, forms a protective barrier that is an important part of our innate immune system. The whipworm Trichuris is closely associated with the intestinal mucus barrier. The major structural component of this barrier, large glycoproteins known as mucins play a significant role in the expulsion of these worms in a mouse model. Using mice that get longterm chronic infections and others able to expel the worms from the intestine, we uncover a novel role for products secreted by the worms. Enzymes secreted by whipworms can disrupt the mucin network that gives mucus its viscous properties. Moreover, we unravel that worm products are unable to degrade forms of mucins present in the mucus barrier during worm expulsion, suggesting that these enzymes may be released by the worm as part of its regime to improve its niche and survival in the host. However, the host is capable of producing mucins and other protective molecules that protect the mucus barrier from degradation and are detrimental to the viability of the worm.
PMCID: PMC3469553  PMID: 23071854
5.  Differential Muc2 and Muc5ac secretion by stimulated guinea pig tracheal epithelial cells in vitro 
Respiratory Research  2006;7(1):35.
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.
PMCID: PMC1484480  PMID: 16504136
6.  132 Characterization of 2 Epithelial Cell Air-Liquid Interface (ALI) Culture Models for Human Healthy Nasal Mucosa and Nasal Polyps 
Primary human airway epithelial cells, when submerged in culture, undergo a dedifferentiation with loss of many features of the in vivo airway epithelium. However, when cultured in an air-liquid interface (ALI), cells develop a well-differentiated, polarized, and pseudostratified epithelium. The aim of the current study was to characterize the mucociliary differentiation of human nasal mucosa and polyp epithelial cells cultured using an ALI system.
Nasal mucosa (NM, n = 3) and nasal polyps (NP, n = 3) were obtained from patients undergoing nasal corrective surgery and endoscopic sinus surgery, respectively. Epithelial cells were obtained from the explant method, and differentiated in ALI culture during 28 days. Cultures were studied at different time points (0, 7, 14, 21, and 28 days): tissue ultrastructure by scanning electron microscopy (SEM) and transmission electron microscopy (TEM); mucous (MUC5AC, MUC5B) and serous (lactoferrin) cell secretion by ELISA; and cytokeratin 18 (epithelial marker), β-tubulin IV (cilia marker), MUC5AC (goblet cell marker), and p63 (basal cell marker) expression by immunocytochemistry.
In both NM and NP ALI cultures and at days 14 and 28, a pseudostratified epithelium with ciliated, mucus-secreting and basal cells was observed, and expression of cytokeratin 18, b-tubulin IV, MUC5AC and p63 was detected. In NP cultures, both MUC5AC (day 14: 2.2 ± 0.1-folds; day 28: 3.6-fold ± 0.7-fold) and MUC5B (day 14: 3.2-fold ± 0.6-fold; day 28: 3.1-fold ± 1-fold) increased over time compared to day 0 (P < 0.05). In NM cultures, only MUC5B (day 14: 3.9-fold ± 0.9-fold; day 28: 3.4-fold ± 0.4-fold; P < 0.05) but not MUC5AC increased over time compared to day 0 (P < 0.05). Secretion of lactoferrin was present but showed no changes over time in either NM or NP ALI cultures.
Epithelial cell ALI cultures provide a well-differentiated human nasal mucosa and polyp tissues that may be used as an in vitro model to study mucin regulation, inflammatory mechanisms of upper airways, and their regulation by antiinflammatory drugs.
PMCID: PMC3512657
7.  Hypoxia-Mediated Mechanism of MUC5AC Production in Human Nasal Epithelia and Its Implication in Rhinosinusitis 
PLoS ONE  2014;9(5):e98136.
Excessive mucus production is typical in various upper airway diseases. In sinusitis, the expression of MUC5AC, a major respiratory mucin gene, increases. However, the mechanisms leading to mucus hypersecretion in sinusitis have not been characterized. Hypoxia due to occlusion of the sinus ostium is one of the major pathologic mechanisms of sinusitis, but there have been no reports regarding the mechanism of hypoxia-induced mucus hypersecretion.
Methods and Findings
This study aims to identify whether hypoxia may induce mucus hypersecretion and elucidate its mechanism. Normal human nasal epithelial (NHNE) cells and human lung mucoepidermoid carcinoma cell line (NCI-H292) were used. Sinus mucosa from patients was also tested. Anoxic condition was in an anaerobic chamber with a 95% N2/5% CO2 atmosphere. The regulatory mechanism of MUC5AC by anoxia was investigated using RT-PCR, real-time PCR, western blot, ChIP, electrophoretic mobility shift, and luciferase assay. We show that levels of MUC5AC mRNA and the corresponding secreted protein increase in anoxic cultured NHNE cells. The major transcription factor for hypoxia-related signaling, HIF-1α, is induced during hypoxia, and transfection of a mammalian expression vector encoding HIF-1α results in increased MUC5AC mRNA levels under normoxic conditions. Moreover, hypoxia-induced expression of MUC5AC mRNA is down-regulated by transfected HIF-1α siRNA. We found increased MUC5AC promoter activity under anoxic conditions, as indicated by a luciferase reporter assay, and mutation of the putative hypoxia-response element in MUC5AC promoter attenuated this activity. Binding of over-expressed HIF-1α to the hypoxia-response element in the MUC5AC promoter was confirmed. In human sinusitis mucosa, which is supposed to be hypoxic, expression of MUC5AC and HIF-1α is higher than in control mucosa.
The results indicate that anoxia up-regulates MUC5AC by the HIF-1α signaling pathway in human nasal epithelia and suggest that hypoxia might be a pathogenic mechanism of mucus hypersecretion in sinusitis.
PMCID: PMC4026485  PMID: 24840724
8.  Modulation of MUC7 Mucin Expression by Exogenous Factors in Airway Cells In Vitro and In Vivo 
The human MUC7 gene encodes a low-molecular-mass mucin that participates in the maintenance of healthy epithelium in the oral cavity, and possibly in respiratory tracts, by promoting the clearance of various bacteria. We examined whether MUC7 gene is expressed in primary normal human tracheobronchial epithelial cells and whether the expression is modulated by exogenous factors. By assessing MUC7 transcripts, we found that the MUC7 gene was induced by culturing the normal human tracheobronchial epithelial cells at the air–liquid interface, in which the cells were well differentiated. When the cells were treated with a panel of cytokines (IL-1β, IL-4, IL-13, and TNF-α), epidermal growth factor, or a bacterial product (Pseudomonas aeruginosa lipopolysaccharide [LPS]), MUC7 transcripts and glycoprotein products were increased 1.7- to 3.2-fold. The effect of LPS on MUC7 gene expression was also studied in the airway tissues of MUC7 gene transgenic mice. In the in vitro cultured trachea and lung explants, the LPS-treated tissues showed over 2-fold increased levels of MUC7 mRNA compared with the untreated specimens. These results were confirmed by in vivo studies using the lungs and tracheas harvested from the transgenic mice irritated by LPS through the tracheal instillation. By immunohistochemistry, MUC7 glycoprotein was localized in tracheal submucosa within the serous cells. Upon LPS stimulation, the overexpressed MUC7 remains confined to the serous glands. In the lungs, MUC7 seems to be expressed within the respiratory epithelium at the level of the bronchioles. Upon stimulation with LPS, it seems to be overexpressed within the same cells and within the stromal tissue.
PMCID: PMC2658701  PMID: 16514118
intratracheal instillation; immunohistochemistry; MUC7 gene expression and regulation; MUC7 gene transgenic mice; real-time PCR; tracheobronchial epithelial cells
9.  Muc5b Is the Major Polymeric Mucin in Mucus from Thoroughbred Horses With and Without Airway Mucus Accumulation 
PLoS ONE  2011;6(5):e19678.
Mucus accumulation is a feature of inflammatory airway disease in the horse and has been associated with reduced performance in racehorses. In this study, we have analysed the two major airways gel-forming mucins Muc5b and Muc5ac in respect of their site of synthesis, their biochemical properties, and their amounts in mucus from healthy horses and from horses with signs of airway mucus accumulation. Polyclonal antisera directed against equine Muc5b and Muc5ac were raised and characterised. Immunohistochemical staining of normal equine trachea showed that Muc5ac and Muc5b are produced by cells in the submucosal glands, as well as surface epithelial goblet cells. Western blotting after agarose gel electrophoresis of airway mucus from healthy horses, and horses with mucus accumulation, was used to determine the amounts of these two mucins in tracheal wash samples. The results showed that in healthy horses Muc5b was the predominant mucin with small amounts of Muc5ac. The amounts of Muc5b and Muc5ac were both dramatically increased in samples collected from horses with high mucus scores as determined visually at the time of endoscopy and that this increase also correlated with increase number of bacteria present in the sample. The change in amount of Muc5b and Muc5ac indicates that Muc5b remains the most abundant mucin in mucus. In summary, we have developed mucin specific polyclonal antibodies, which have allowed us to show that there is a significant increase in Muc5b and Muc5ac in mucus accumulated in equine airways and these increases correlated with the numbers of bacteria.
PMCID: PMC3094342  PMID: 21602926
10.  Effect of epithelium ATP release on cyclic pressure-induced airway mucus secretion 
Bioscience Reports  2014;34(1):e00088.
The cyclic mechanical effect of airflow during breathing creates the optimal airway hydration state. MUC (mucin) 5AC is an important component of the airway mucus. The formation of MUC5AC is related to ATP and intracellular calcium in the epithelial cells. In this study, we evaluated the effect of ATP release from intracellular calcium in epithelial cells on cyclic pressure-induced mucus secretion in the airway. 16HBE (human bronchial epithelial cells) were cultured in vitro on cyclically tilted cultured plates and divided into five groups: control, tilt, tilt and BAPTA–AM (1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid–acetoxymethyl ester), tilt and EGTA and tilt and RB-2 (reactive blue-2). The shear stress and compressive stress were induced by the surface tension of the liquid, atmospheric pressure and liquid gravity. Cell activity, MUC5AC mRNA expression level, MUC5AC protein expression level and ATP release and intracellular calcium changes were measured with the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay, RT–PCR (reverse transcription–PCR), HPLC and inverted fluorescence microscope, respectively. We detected that cyclic pressure significantly increased MUC5AC secretion and ATP release. The enhanced ATP release could be inhibited by both BAPTA–AM and RB-2, while EGTA did not have a suppressive effect. BAPTA–AM, EGTA and RB-2 did not obviously inhibit MUC5AC mRNA expression. Cyclic pressure did not induce MUC5AC secretion in the airway mucus epithelium via Ca2+-dependent ATP release, and nearly all Ca2+ was provided by stored intracellular Ca2+.
PMCID: PMC3891320  PMID: 24329423
ATP; calcium; cyclic pressure; mucins; 16HBE, human bronchial epithelial cells; AM, acetoxymethyl ester; ASL, airway surface liquid; BAPTA, 1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HRP, horseradish peroxidase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; MUC, mucin; RB-2, reactive blue-2; RT–PCR, reverse transcription–PCR
11.  Site-specific O-Glycosylation on the MUC2 Mucin Protein Inhibits Cleavage by the Porphyromonas gingivalis Secreted Cysteine Protease (RgpB)* 
The Journal of Biological Chemistry  2013;288(20):14636-14646.
Background: MUC2 polymers form the mucus layer of colon that separates luminal bacteria from the epithelium.
Results: P. gingivalis secrets a protease that cleaves the MUC2 mucin, a cleavage modulated by O-glycosylation.
Conclusion: Bacteria can disrupt the MUC2 polymer via proteolytic cleavage. However, O-glycosylation can inhibit this process.
Significance: Bacteria can dissolve the protective inner mucus layer, potentially triggering colitis.
The colonic epithelial surface is protected by an inner mucus layer that the commensal microflora cannot penetrate. We previously demonstrated that Entamoeba histolytica secretes a protease capable of dissolving this layer that is required for parasite penetration. Here, we asked whether there are bacteria that can secrete similar proteases. We screened bacterial culture supernatants for such activity using recombinant fragments of the MUC2 mucin, the major structural component, and the only gel-forming mucin in the colonic mucus. MUC2 has two central heavily O-glycosylated mucin domains that are protease-resistant and has cysteine-rich N and C termini responsible for polymerization. Culture supernatants of Porphyromonas gingivalis, a bacterium that secretes proteases responsible for periodontitis, cleaved the MUC2 C-terminal region, whereas the N-terminal region was unaffected. The active enzyme was isolated and identified as Arg-gingipain B (RgpB). Two cleavage sites were localized to IR↓TT and NR↓QA. IR↓TT cleavage will disrupt the MUC2 polymers. Because this site has two potential O-glycosylation sites, we tested whether recombinant GalNAc-transferases (GalNAc-Ts) could glycosylate a synthetic peptide covering the IRTT sequence. Only GalNAc-T3 was able to glycosylate the second Thr in IRTT, rendering the sequence resistant to cleavage by RgpB. Furthermore, when GalNAc-T3 was expressed in CHO cells expressing the MUC2 C terminus, the second threonine was glycosylated, and the protein became resistant to RgpB cleavage. These findings suggest that bacteria can produce proteases capable of dissolving the inner protective mucus layer by specific cleavages in the MUC2 mucin and that this cleavage can be modulated by site-specific O-glycosylation.
PMCID: PMC3656315  PMID: 23546879
Bacterial Pathogenesis; Glycoprotein Biosynthesis; Glycosyltransferases; Mucins; Mucus
12.  Mapping the Protein Domain Structures of the Respiratory Mucins: a mucin proteome coverage study 
Journal of proteome research  2012;11(8):4013-4023.
Mucin genes encode a family of the largest expressed proteins in the human genome. The proteins are highly substituted with O-linked oligosaccharides which greatly restrict access to the peptide backbones. The genomic organization of the N-terminal, O-glycosylated, and C-terminal regions of most of the mucins has been established and is available in the sequence databases. However, much less is known about the fate of their exposed protein regions after translation and secretion, and, to date, detailed proteomic studies complementary to the genomic studies are rather limited. Using mucins isolated from cultured human airway epithelial cell secretions, trypsin digestion and mass spectrometry, we investigated the proteome coverage of the mucins responsible for the maintenance and protection of the airway epithelia. Excluding the heavily glycosylated mucin domains, up to 85% coverage of the N-terminal region of the gel forming mucins MUC5B and MUC5AC was achieved, and up to 60% of the C-terminal regions were covered, suggesting that more N- and sparsely O-glycosylated regions as well as possible other modifications are available at the C-terminus. All possible peptides from the cysteine-rich regions that interrupt the heavily glycosylated mucin domains were identified. Interestingly, 43 cleavage sites from ten different domains of MUC5B and MUC5AC were identified, which possessed a non-tryptic cleavage site on the N-terminal end of the peptide, indicating potential exposure to proteolytic and/or “spontaneous cleavages”. Some of these non-tryptic cleavages may be important for proper maturation of the molecule, before and/or after secretion. Most of the peptides identified from MUC16 were from the SEA region. Surprisingly, three peptides were clearly identified from its heavily glycosylated regions. Up to 25% coverage of MUC4 was achieved covering seven different domains of the molecule. All peptides from the MUC1 cytoplasmic domain were detected along with the three non-tryptic cleavages in the region. Only one peptide was identified from MUC20 which led us to successful antisera raised against the molecule. Taken together, this report represents our current efforts to dissect the complexities of mucin macromolecules. Identification of regions accessible to proteolysis can help in the design of effective antibodies and points to regions that might be available for mucin-protein interactions and identification of cleavage sites will enable understanding of their pre- and post-secretory processing in normal and disease environments.
PMCID: PMC3412937  PMID: 22663354
Mucins; respiratory; proteomics; coverage; MUC5B; MUC5AC
13.  Aberrant Mucin Assembly in Mice Causes Endoplasmic Reticulum Stress and Spontaneous Inflammation Resembling Ulcerative Colitis 
PLoS Medicine  2008;5(3):e54.
MUC2 mucin produced by intestinal goblet cells is the major component of the intestinal mucus barrier. The inflammatory bowel disease ulcerative colitis is characterized by depleted goblet cells and a reduced mucus layer, but the aetiology remains obscure. In this study we used random mutagenesis to produce two murine models of inflammatory bowel disease, characterised the basis and nature of the inflammation in these mice, and compared the pathology with human ulcerative colitis.
Methods and Findings
By murine N-ethyl-N-nitrosourea mutagenesis we identified two distinct noncomplementing missense mutations in Muc2 causing an ulcerative colitis-like phenotype. 100% of mice of both strains developed mild spontaneous distal intestinal inflammation by 6 wk (histological colitis scores versus wild-type mice, p < 0.01) and chronic diarrhoea. Monitoring over 300 mice of each strain demonstrated that 25% and 40% of each strain, respectively, developed severe clinical signs of colitis by age 1 y. Mutant mice showed aberrant Muc2 biosynthesis, less stored mucin in goblet cells, a diminished mucus barrier, and increased susceptibility to colitis induced by a luminal toxin. Enhanced local production of IL-1β, TNF-α, and IFN-γ was seen in the distal colon, and intestinal permeability increased 2-fold. The number of leukocytes within mesenteric lymph nodes increased 5-fold and leukocytes cultured in vitro produced more Th1 and Th2 cytokines (IFN-γ, TNF-α, and IL-13). This pathology was accompanied by accumulation of the Muc2 precursor and ultrastructural and biochemical evidence of endoplasmic reticulum (ER) stress in goblet cells, activation of the unfolded protein response, and altered intestinal expression of genes involved in ER stress, inflammation, apoptosis, and wound repair. Expression of mutated Muc2 oligomerisation domains in vitro demonstrated that aberrant Muc2 oligomerisation underlies the ER stress. In human ulcerative colitis we demonstrate similar accumulation of nonglycosylated MUC2 precursor in goblet cells together with ultrastructural and biochemical evidence of ER stress even in noninflamed intestinal tissue. Although our study demonstrates that mucin misfolding and ER stress initiate colitis in mice, it does not ascertain the genetic or environmental drivers of ER stress in human colitis.
Characterisation of the mouse models we created and comparison with human disease suggest that ER stress-related mucin depletion could be a fundamental component of the pathogenesis of human colitis and that clinical studies combining genetics, ER stress-related pathology and relevant environmental epidemiology are warranted.
Michael McGuckin and colleagues identify two mutations that cause aberrant mucin oligomerization in mice. The resulting phenotype, including endoplasmic reticulum stress, resembles clinical and pathologic features of human ulcerative colitis.
Editors' Summary
Inflammatory bowel diseases (IBD) are common disorders in which parts of the digestive tract become inflamed. The two main types of IBD are Crohn's disease, which mainly affects the small bowel, and ulcerative colitis (UC), which mainly affects the large bowel (colon). Both types tend to run in families and usually develop between 15 and 35 years old. Their symptoms include diarrhea, abdominal cramps, and unintentional weight loss. These symptoms can vary in severity, can be chronic (persistent) or intermittent, and may start gradually or suddenly. There is no cure for IBD (except removal of the affected part of the digestive tract), but drugs that modulate the immune system (for example, corticosteroids) or that inhibit “proinflammatory cytokines” (proteins made by the immune system that stimulate inflammation) can sometimes help.
Why Was This Study Done?
Although the clinical and pathological (disease-associated) features of Crohn's disease and UC are somewhat different, both disorders are probably caused by an immune system imbalance. Normally, the immune system protects the body from potentially harmful microbes in the gut but does not react to the many harmless bacteria that live there or to the food that passes along the digestive tract. In IBD, the immune system becomes overactive for unknown reasons, and lymphocytes (immune system cells) accumulate in the lining of the bowel and cause inflammation. In this study, the researchers use a technique called random mutagenesis (the random introduction of small changes, called mutations, into the genes of an organism using a chemical that damages DNA) to develop two mouse models that resemble human UC and that throw new light on to how this disorder develops.
What Did the Researchers Do and Find?
The researchers establish two mutant mouse strains—Winnie and Eeyore mice—that develop mild spontaneous inflammation of the colon and chronic diarrhea and that have more proinflammatory cytokines and more lymphocytes in their colons than normal mice. 25% and 40% of the Winnie and Eeyore mice, respectively, have severe clinical signs of colitis by 1 year of age. Both strains have a mutation in the Muc2 gene, which codes for MUC2 mucin, the main protein in mucus. This viscous substance (which coats the inside of the intestine) is produced by and stored in intestinal “goblet” cells. Mucus helps to maintain the intestine's immunological balance but is depleted in UC. The researchers show that the manufacture and assembly of Muc2 molecules is abnormal in Winnie and Eeyore mice, that less mucin is stored in their goblet cells than in normal mice, and that their intestinal mucus barrier is reduced. In addition, an incompletely assembled version of the molecule, called Muc2 precursor, accumulates in the endoplasmic reticulum (ER; the cellular apparatus that prepares newly manufactured proteins for release) of goblet cells, leading to overload with abnormal protein and causing a state of cellular distress known as the “ER stress response.” Finally, the researchers report that MUC2 precursor also accumulates in the goblet cells of people with UC and that even the noninflamed intestinal tissue of these patients shows signs of ER stress.
What Do These Findings Mean?
These findings indicate that mucin abnormalities and ER stress can initiate colitis in mice. Results from animal studies do not always reflect what happens in people, but these findings, together with those from the small study in humans, suggest that ER stress-related mucin depletion could be a component in the development of human colitis. The results do not identify the genetic changes and/or environmental factors that might trigger ER stress in human colitis, but suggest that once initiated, ER stress might interfere with MUC2 production, which would lead to a diminished mucus barrier, expose the lining of the intestine to more toxins and foreign substances, and trigger local mucosal inflammation. The release of inflammatory cytokines would then damage the intestine's lining and exacerbate ER stress, thus setting up a cycle of intestinal damage and inflammation. Clinical studies to look for genetic changes and environmental factors capable of triggering ER stress and for ER-stress related changes in human UC should now be undertaken to test this hypothesis.
Additional Information.
Please access these Web sites via the online version of this summary at
The MedlinePlus Encyclopedia has pages on Crohn's disease and on ulcerative colitis (in English and Spanish)
The US National Institute of Diabetes and Digestive and Kidney Diseases provides information on Crohn's disease and ulcerative colitis
Information and support for patients with inflammatory bowel disease and their caregivers is provided by the Crohn's and Colitis Foundation of America and by the UK National Association for Colitis and Crohn's Disease
Wikipedia has pages on mucins and on mucus (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
PMCID: PMC2270292  PMID: 18318598
14.  118 EGCG Downregulates Mucin Gene Expression Through the Mapk Signaling Pathway in Asthma 
The World Allergy Organization Journal  2012;5(Suppl 2):S56-S57.
Mucus plays an important role in protecting human airway from external environments. Highly glycosylated mucin proteins are the major components of mucus, responsible for its viscoelastic properties. Excessive mucus is major manifestation of inflammatory respiratory diseases. Epigallocatechin-3-gallate (EGCG) is major component of green tea extract and known to provide numerous functions, such as anti-oxidant effect, anti-tumor effect, anti-diabetic effect and anti-inflammatory effect. But precise mechanisms are still unclear.
Using NCI-H292 human airway epithelial cells, we measured phorbol 12-myrisate 13-acetate (PMA)-induced MUC5B mRNA expression with the treatment of indicated doses of EGCG. We also measured PMA-induced MUC5B protein secretion with the treatment of indicated doses of EGCG using ELISA technique in NCI-H292 cells. To test the brief signaling pathways, we performed activation study of mitogen-activated protein kinase (MAPK) pathways, which is well-known to signaling the PMA-induced mucin gene over-expression, using Western blot technique in NCI-H292 cells. And then we performed in vivo study using ovalbumin-induced asthmatic mice model and control mice group. In ovalbumin-sensitized asthmatic mice model, EGCG was treated with indicated dose. And then ovalbumin was challenged and we sacrificed the mice. Tissue samples from the mice were stained with PAS (periodic acid-Schiff) for mucin distribution in bronchioles of each group. Immunocytochemical stain was performed using MUC5B specific antibody. MUC5B mRNA and protein level was measured using extracted lung tissues.
PMA-induced MUC5B mRNA and protein level was significantly decreased after treatment of EGCG at all doses in NCI-H292 cells. PMA-induced phosphorylation of p38 MAPK was significantly decreased after treatment of EGCG at all doses in NCI-H292 cells. Results from in vivo studies showed that decreased bronchiolar mucin distribution in the group of pretreated with EGCG in asthmatic mice. MUC5B mRNA and protein levels were significantly decreased in the group of pretreated with EGCG in asthmatic mice.
PMA-induced MUC5B mRNA and protein over-expression in both NCI-H292 cells and extracted tissues from asthmatic mice were significantly decreased with the treatment of EGCG. We demonstrated that EGCG downregulates mucin gene expression through the MAPK signaling pathway in asthma.
PMCID: PMC3512964
15.  Estradiol Increases Mucus Synthesis in Bronchial Epithelial Cells 
PLoS ONE  2014;9(6):e100633.
Airway epithelial mucus hypersecretion and mucus plugging are prominent pathologic features of chronic inflammatory conditions of the airway (e.g. asthma and cystic fibrosis) and in most of these conditions, women have worse prognosis compared with male patients. We thus investigated the effects of estradiol on mucus expression in primary normal human bronchial epithelial cells from female donors grown at an air liquid interface (ALI). Treatment with estradiol in physiological ranges for 2 weeks caused a concentration-dependent increase in the number of PAS-positive cells (confirmed to be goblet cells by MUC5AC immunostaining) in ALI cultures, and this action was attenuated by estrogen receptor beta (ER-β) antagonist. Protein microarray data showed that nuclear factor of activated T-cell (NFAT) in the nuclear fraction of NHBE cells was increased with estradiol treatment. Estradiol increased NFATc1 mRNA and protein in ALI cultures. In a human airway epithelial (1HAE0) cell line, NFATc1 was required for the regulation of MUC5AC mRNA and protein. Estradiol also induced post-translational modification of mucins by increasing total fucose residues and fucosyltransferase (FUT-4, -5, -6) mRNA expression. Together, these data indicate a novel mechanism by which estradiol increases mucus synthesis in the human bronchial epithelium.
PMCID: PMC4070981  PMID: 24964096
16.  Mucus hypersecretion in asthma: causes and effects 
Purpose of review
Airway mucus plugging has long been recognized as a principal cause of death in asthma. However, molecular mechanisms of mucin overproduction and secretion have not been understood until recently. These mechanisms are reviewed together with ongoing investigations relating them to lung pathophysiology.
Recent findings
Of the five secreted gel-forming mucins in mammals, only MUC5AC and MUC5B are produced in significant quantities in intrapulmonary airways. MUC5B is the principal gel-forming mucin at baseline in small airways of humans and mice, and therefore likely performs most homeostatic clearance functions. MUC5AC is the principal gel-forming mucin upregulated in airway inflammation and is under negative control by forkhead box a2 and positive control by hypoxia inducible factor-1. Mucin secretion is regulated separately from production, principally by extracellular triphosphate nucleotides that bind P2Y2 receptors on the lumenal surface of airway secretory cells, generating intracellular second messengers that activate the exocytic proteins, Munc13-2 and synaptotagmin-2.
Markedly upregulated production of MUC5AC together with stimulated secretion leads to airflow obstruction in asthma. As MUC5B appears to mediate homeostatic functions, it may be possible to selectively inhibit MUC5AC production without impairing airway function. The precise roles of mucin hypersecretion in asthma symptoms such as dyspnea and cough and in physiologic phenomena such as airway hyperresponsiveness remain to be defined.
PMCID: PMC2709596  PMID: 19077699
airway; asthma; mucin; mucous; mucus
17.  TRPM5-mediated calcium uptake regulates mucin secretion from human colon goblet cells 
eLife  2013;2:e00658.
Mucin 5AC (MUC5AC) is secreted by goblet cells of the respiratory tract and, surprisingly, also expressed de novo in mucus secreting cancer lines. siRNA-mediated knockdown of 7343 human gene products in a human colonic cancer goblet cell line (HT29-18N2) revealed new proteins, including a Ca2+-activated channel TRPM5, for MUC5AC secretion. TRPM5 was required for PMA and ATP-induced secretion of MUC5AC from the post-Golgi secretory granules. Stable knockdown of TRPM5 reduced a TRPM5-like current and ATP-mediated Ca2+ signal. ATP-induced MUC5AC secretion depended strongly on Ca2+ influx, which was markedly reduced in TRPM5 knockdown cells. The difference in ATP-induced Ca2+ entry between control and TRPM5 knockdown cells was abrogated in the absence of extracellular Ca2+ and by inhibition of the Na+/Ca2+ exchanger (NCX). Accordingly, MUC5AC secretion was reduced by inhibition of NCX. Thus TRPM5 activation by ATP couples TRPM5-mediated Na+ entry to promote Ca2+ uptake via an NCX to trigger MUC5AC secretion.
eLife digest
Goblet cells are specialized cells that produce proteins called mucins, which combine with water, salt and other proteins to form mucus, the slippery fluid that protects the respiratory and digestive tracts from bacteria, viruses and other pathogens. However, a defect in the production of one particular type of mucin—Mucin 5AC—can result in diseases such as cystic fibrosis, chronic obstructive pulmonary disease and Crohn’s disease, so there is a clear need to understand the production of mucus in detail.
Before they are secreted, the mucins are packaged inside granules in the goblet cells. When a certain extracellular signal arrives at a goblet cell, these granules move through the cell, fuse with the cell membrane and release the mucins, which then expand their volume by a factor of up to a 1000. Calcium ions (Ca2+) have a critical role in the signal that leads to the secretion of mucins, but many details about the signalling and secretion processes are poorly understood.
Now, Mitrovic et al. have used genetic methods to study 7343 gene products in goblet cells derived from a human colon. They identified 16 new proteins that are involved in the secretion of Mucin 5AC, including a channel protein called TRPM5. This protein is activated when the concentration of Ca2+ inside the cell increases, and its activation allows sodium (Na+) ions to enter the cells. These intracellular Na+ ions are then exchanged for Ca2+ ions from outside the cell, and these Ca2+ ions then couple to the molecular machinery that is responsible for the secretion of the mucins.
By using electrophysiological and Ca2+ imaging approaches, Mitrovic et al. were able to visualize and measure TRPM5-mediated Na+ currents and the subsequent Ca2+ uptake by the cells, and confirmed that extracellular Ca2+ ions were responsible for stimulating the secretion of mucins. The next step is to determine how the other 15 genes are involved in mucin secretion and, in the longer term, explore how these insights might be translated into treatments for cystic fibrosis and other conditions associated with defective mucus secretion.
PMCID: PMC3667631  PMID: 23741618
Mucin5AC; TRPM5; Secretion; Human
18.  Genome wide analysis of the bovine mucin genes and their gastrointestinal transcription profile 
BMC Genomics  2011;12:140.
Mucins are large glycoproteins implicated in protection of all mucosal surfaces. In humans and rodents, the mucin gene family has been well described and previous studies have investigated the distribution and function of mucins in the gastrointestinal (GI) tract. In contrast, little data is available on the mucin gene family in polygastric species, such as cattle. The aim of the current study was to identify all members of the bovine mucin family by genome mining and subsequently investigate the transcription pattern of these mucins in the GI tract.
Nine bovine membrane-associated mucins (MUC1, MUC3A, MUC4, MUC12, MUC13, MUC15, MUC16, MUC20 and MUC21) and six secreted mucins (MUC2, MUC5AC, MUC5B, MUC6, MUC7 and MUC19) were identified in the bovine genome. No homologues could be identified for MUC3B, MUC8 and MUC17. In general, domain architecture of the membrane-associated mucins was found to be similar between humans and cattle, while the protein architecture of the gel-forming mucins appeared to be less conserved. Further analysis of the genomic organization indicated that the previously reported bovine submaxillary mucin (BSM) may be part of a larger gene encoding for MUC19. Analysis of the transcription profile showed that the secreted mucins were transcribed from the abomasum onwards, whereas the membrane associated mucins MUC1 and MUC20 were transcribed throughout the whole GI tract. In contrast to humans, MUC5B transcript was found in both the small and large intestine, but was absent in oesophageal tissue.
This study provides the first characterization of the mucin gene family in cattle and their transcriptional regulation in the GI tract. The data presented in this paper will allow further studies of these proteins in the physiology of the GI tract in ruminants and their interactions with pathogens.
PMCID: PMC3056801  PMID: 21385362
19.  Human fetal ductal plate revisited: II. MUC1, MUC5AC, and MUC6 are expressed in human fetal ductal plate and MUC1 is expressed also in remodeling ductal plate, remodeled ductal plate and mature bile ducts of human fetal livers 
Mucins are high-molecular-weight glycoproteins, which are heavily decorated with a large number of O-linked oligosaccharides and a few N-glycan chains, linked to a protein backbone. The protein backbone is called mucin core protein or MUC apomucins. MUC expression is down-regulated or up-regulated in malignant neoplasms. These alterations of MUC apomucins, which are regulated by MUC genes, are associated with carcinogenesis and malignant potentials of cancers. MUC expression during human fetal intrahepatic bile duct (IBD) development has been studied only once, and there has been only one histochemical study of mucins in human fetal IBD development. The author herein immunohistochemically investigated the expression of MUC1, MUC2, MUC5AC, and MUC6, and histochemically investigated carbohydrate component of mucins in human fetal cholangiocytes with the use of 32 human fetal livers of various gestational ages. MUC1 is a transmembranous apomucin, while MUC2, MUC5AC and MUC6 are secretory apomucins. Under normal conditions, MUC1 (polymorphic epithelial mucin) is present mainly in the pancreatic epithelium. MUC2 (goblet cell mucin) is mainly located in goblet cells. MUC5AC (gastric foveolar mucin) and MUC6 (pyloric gland-type mucin) are located in the stomach. In the present study, the processes of the human IBD development could be categorized into four stages; ductal plate (DP), remodeling DP, remodeled DP, and mature IBDs. The author identified that MUC1 was present in ductal plate (DP), remodeling DP, remodeled DP, and mature IBD in human fetal livers. MUC5AC and MUC6 were present only in the DP. MUC5AC and MUC6 were absent in remodeling DP, remodeled DP, and mature IBD in human fetal livers. No expression of MUC2 was seen throughout the fetal IBD development. Histochemically, no carbohydrate component of mucins were seen in the remodeling DP and remodeled DP, while neutral and acidic mucins (carboxylated and sulfated mucins) were seen in mature IBD in human fetal livers. The DP showed frequently neutral mucins and less frequently acidic mucins (carboxylated and sulfated mucins residues). These findings suggest that the DP cells have MUC1, MUC5AC and MUC6, and that remodeling DP, remodeled DP, and mature IBDs have MUC1, but not MUC5AC and MUC6. The presence of neutral and acidic carbohydrates in DP suggests that these carbohydrates of mucin are attached to the MUC5AC and MUC6 mucin core proteins. Although the implications are unclear, the expression of these MUC apomucins and their carbohydrate residues are associated with normal development of IBDs in human fetal livers.
PMCID: PMC3606847  PMID: 23573304
Ductal plate; human fetal liver; intrahepatic bile duct development; mucins; MUC apomucins; histochemistry; immunohistochemistry
20.  Humanized Mouse Model Used to Monitor MUC Gene Expression in Nasal Polyps and to Preclinically Evaluate the Efficacy of Montelukast in Reducing Mucus Production 
To determine whether MUC gene expression could be down-regulated in nasal polyps by the leukotriene receptor antagonist montelukast, we developed a system in which nondisrupted human nasal polyps could be successfully implanted into severely immunocompromised mice, and in which the histopathology of the original nasal polyp tissue could be preserved for long periods. In addition, the histopathologic changes in the human nasal polyps were carefully examined to determine the origin of the submucosal glands (SMGs) that develop in true nasal polyps found in the anterior third of the nose.
Small, nondisrupted pieces of human nasal polyp tissues were subcutaneously implanted into NOD-scid IL-2rγnull mice. Xenograft-bearing mice were treated with either montelukast or saline solution. Xenografts at 8 to 12 weeks after implantation were examined histologically, and expression of MUC genes 4, 5AC, and 7 was studied in the polyps before implantation and in the 8-week xenograft. Alzet pumps were inserted into the mice, and montelukast (Singulair) was continuously delivered to determine its effect on goblet cell hyperplasia, mucus production, and the enlargement of nasal polyps over an 8-week period.
The xenografts were maintained in a viable and functional state for up to 3 months and retained a histopathology similar to that of the original tissue, but with a noticeable increase in goblet cell hyperplasia and marked mucus accumulation in the SMGs. MUC4 and MUC5AC were significantly increased in the xenograft 8 weeks after implantation, but MUC7 was significantly decreased compared to the preimplantation polyps. Inasmuch as MUC7 is found exclusively in serous glands, the findings suggest that serous glands are not found in polyps in the anterior third of the nose. The histopathologic findings confirm the original findings of Tos et al suggesting that the SMGs are derived from pinching-off of the epithelium of the enlarging polyp following inflammatory changes. These SMGs have the same epithelium as surface epithelium and consist of multiple goblet cells that secrete periodic acid Schiff stain–positive mucin into the interior of the SMGs. A progressive increase in the volume of the xenografts was observed, with little or no evidence of mouse cell infiltration into the human leukocyte antigen–positive human tissue. An average twofold increase in polyp volume was found 2 months after engraftment. Montelukast did not decrease the growth of the xenograft in the 8-week NOD-scid mice, nor did it affect MUC gene expression.
The use of innate and adaptive immunodeficient NOD-scid mice homozygous for targeted mutations in the IL-2 gamma-chain locus NOD-scid IL-2rγnull for establishing engraftment of nondisrupted pieces of human nasal polyp tissues represents a significant advancement in studying chronic inflammation over a long period of time. In the present study, we utilized this humanized mouse model to confirm our prediction that MUC genes 4 and 5AC are highly expressed and significantly increased over those of preimplanted polyps. The overexpression of these 2 MUC genes correlates with both the goblet cell hyperplasia and the excessive mucus production that are found in nasal polyp xenografts. MUC7, which is primarily associated with the submucosa, as opposed to MUC4 and MUC5AC, which are primarily expressed in the epithelium, was significantly decreased in the nasal polyp xenografts. Montelukast had no significant effect on MUC gene expression in the xenografts. In addition to the MUC gene expression patterns, the histology of the xenografts supports the concept that mucinous glands that are characteristic of true nasal polyps are significantly different from those in the mucosa found in the lateral wall of the nose in patients with chronic sinusitis without nasal polyps. The mucinous glands seen in nasal polyps (which appear to be derived from an invagination of hyperplastic epithelial mucosa containing large numbers of goblet cells) are histologically distinct from the seromucinous glands found in the submucosa of hyperplastic middle turbinates. The data presented here establish a humanized mouse model as a viable approach to study nasal polyp growth, to assess the therapeutic efficacy of various drugs in this chronic inflammatory disease, and to contribute to our understanding of the pathogenesis of this disease.
PMCID: PMC3621975  PMID: 22724276
montelukast; mucous gland; nasal polyp; SCID mouse
21.  Extracellular MUC3 mucin secretion follows adherence of Lactobacillus strains to intestinal epithelial cells in vitro 
Gut  2003;52(6):827-833.
Background: Mucins are large complex glycoproteins that protect intestinal mucosal surfaces by limiting access of environmental matter to their epithelial cells. Several mucin genes have been described, including MUC3 that is a membrane associated mucin of the small intestine. Increased MUC3 mRNA transcription is induced by incubation of intestinal epithelial cells with a Lactobacillus strain known to be adherent to them.
Aims: To determine whether increased epithelial cell MUC3 mucin expression in response to Lactobacillus strains results in increased extracellular secretion of MUC3 mucins and the importance of epithelial cell adherence in modulation of MUC3 mucin expression.
Methods: HT29 cells grown to enhance expression of MUC3 mucins were incubated with selected Lactobacillus strains. Spent cell culture medium was collected for detection of secreted MUC3 mucins using dot blot immunoassay with a generated MUC3 antibody. Post-incubation HT29 cell RNA was collected for analysis of MUC3 expression by northern blot analysis using a MUC3 cDNA probe. In vitro binding studies using Lactobacillus strains incubated alone or coincubated with enteropathogenic Escherichia coli strain E2348/69 were used for adherence and inhibition of adherence studies, respectively.
Results: Lactobacillus strains with minimal ability to adhere to HT29 cells failed to induce upregulation of mucin gene expression. There was a direct correlation between upregulation of MUC3 mucin mRNA expression and extracellular secretion of MUC3 mucin. The same Lactobacillus strains that increased extracellular secretion of MUC3 mucin led to reduced adherence of enteropathogen E coli E2348/69 during coincubation experiments.
Conclusion: Probiotic microbes induce MUC3 mucin transcription and translation with extracellular secretion of the MUC3 mucins. Epithelial cell adherence enhances the effects of probiotics on eukaryotic mucin expression.
PMCID: PMC1773687  PMID: 12740338
Lactobacillus; Escherichia coli; mucin; adherence
22.  Assay of Mucins in Human Tear Fluid 
Experimental eye research  2007;84(5):939-950.
Mucin genes, both secreted (MUC2, MUC5AC, MUC5B, MUC7) and membrane associated (MUC1, MUC4, MUC16), have been reported to be expressed by ocular surface epithelia. The purpose of this study was to comprehensively assay the mucin content of human tear fluid using multiple antibodies for each mucin and to develop a sensitive, semi-quantitative method for the assay of mucins in tears. Tear washes were obtained by instillation of saline onto the ocular surface, followed by collection from the inferior fornix. Tear proteins were separated in 1% agarose gels, transferred to nitrocellulose membrane by vacuum blotting and probed with multiple antibodies recognizing MUC1, MUC2, MUC4, MUC5AC, MUC5B, MUC7 and MUC16. Binding was detected using chemiluminescence, and quantity was determined by densitometry. Serial dilutions of pooled tears from normal individuals were assayed to determine the linear range of detectability. MUC1, MUC4, MUC16, MUC5AC and low levels of MUC2 were consistently detected in human tear fluid, while MUC5B and MUC7 were not. Use of several antibodies recognizing different epitopes on the same mucin confirmed these findings. The antibodies to mucins bound to serial dilutions of tears in a linear fashion (r2 >0.9), indicating the feasibility of semi-quantitation. MUC5AC in tear fluid had an increased electrophoretic mobility compared to MUC5AC isolated from conjunctival tissue. This study provides clear evidence that the mucin component of tears is a mixture of secreted and shed membrane-associated mucins, and for the first time demonstrates MUC16 in tear fluid. Immunoblots of tears using agarose gel electrophoresis and chemiluminescence detection provide a semi-quantitative assay for mucin protein that will be useful for comparisons with tears from diseased eyes or after pharmacological intervention.
PMCID: PMC1950265  PMID: 17399701
Tears; ocular surface; mucins; membrane mucins; MUC16; MUC1; MUC5AC; MUC4
23.  Chronic Intermittent Mechanical Stress Increases MUC5AC Protein Expression 
Increased abundance of mucin secretory cells is a characteristic feature of the epithelium in asthma and other chronic airway diseases. We showed previously that the mechanical stresses of airway constriction, both in the intact mouse lung and a cell culture model, activate the epidermal growth factor receptor (EGFR), a known modulator of mucin expression in airway epithelial cells. Here we tested whether chronic, intermittent, short-duration compressive stress (30 cm H2O) is sufficient to increase the abundance of MUC5AC-positive cells and intracellular mucin levels in human bronchial epithelial cells cultured at an air–liquid interface. Compressive stress applied for 1 hour per day for 14 days significantly increased the percentage of cells staining positively for MUC5AC protein (22.0 ± 3.8%, mean ± SD) relative to unstimulated controls (8.6 ± 2.6%), and similarly changed intracellular MUC5AC protein levels measured by Western and slot blotting. The effect of compressive stress was gradual, with significant changes in MUC5AC-positive cell numbers evident by Day 7, but required as little as 10 minutes of compressive stress daily. Daily treatment of cells with an EGFR kinase inhibitor (AG1478, 1 μM) significantly but incompletely attenuated the response to compressive stress. Complete attenuation could be accomplished by simultaneous treatment with the combination of AG1478 and a transforming growth factor (TGF)-β2 (1 μg/ml)–neutralizing antibody, or with anti–TGF-β2 alone. Our findings demonstrate that short duration episodes of mechanical stress, representative of those occurring during bronchoconstriction, are sufficient to increase goblet cell number and MUC5AC protein expression in bronchial epithelial cells in vitro. We propose that the mechanical environment present in asthma may fundamentally bias the composition of airway epithelial lining in favor of mucin secretory cells.
PMCID: PMC2746990  PMID: 19168703
asthma; mechanotransduction; EGFR; TGF-β; bronchial epithelium
24.  AGR2 Is Induced in Asthma and Promotes Allergen-Induced Mucin Overproduction 
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.
PMCID: PMC3423459  PMID: 22403803
asthma; airway epithelium; mucus; endoplasmic reticulum stress; protein disulfide isomerase
25.  Reconstituted Human Upper Airway Epithelium as 3-D In Vitro Model for Nasal Polyposis 
PLoS ONE  2014;9(6):e100537.
Primary human airway epithelial cells cultured in an air-liquid interface (ALI) develop a well-differentiated epithelium. However, neither characterization of mucociliar differentiation overtime nor the inflammatory function of reconstituted nasal polyp (NP) epithelia have been described.
1st) To develop and characterize the mucociliar differentiation overtime of human epithelial cells of chronic rhinosinusitis with nasal polyps (CRSwNP) in ALI culture system; 2nd) To corroborate that 3D in vitro model of NP reconstituted epithelium maintains, compared to control nasal mucosa (NM), an inflammatory function.
Epithelial cells were obtained from 9 NP and 7 control NM, and differentiated in ALI culture for 28 days. Mucociliary differentiation was characterized at different times (0, 7, 14, 21, and 28 days) using ultrastructure analysis by electron microscopy; ΔNp63 (basal stem/progenitor cell), β-tubulin IV (cilia), and MUC5AC (goblet cell) expression by immunocytochemistry; and mucous (MUC5AC, MUC5B) and serous (Lactoferrin) secretion by ELISA. Inflammatory function of ALI cultures (at days 0, 14, and 28) through cytokine (IL-8, IL-1β, IL-6, IL-10, TNF-α, and IL-12p70) and chemokine (RANTES, MIG, MCP-1, IP-10, eotaxin-1, and GM-CSF) production was analysed by CBA (Cytometric Bead Array).
In both NP and control NM ALI cultures, pseudostratified epithelium with ciliated, mucus-secreting, and basal cells were observed by electron microscopy at days 14 and 28. Displaying epithelial cell re-differentation, β-tubulin IV and MUC5AC positive cells increased, while ΔNp63 positive cells decreased overtime. No significant differences were found overtime in MUC5AC, MUC5B, and lactoferrin secretions between both ALI cultures. IL-8 and GM-CSF were significantly increased in NP compared to control NM regenerated epithelia.
Reconstituted epithelia from human NP epithelial cells cultured in ALI system provides a 3D in vitro model that could be useful both for studying the role of epithelium in CRSwNP while developing new therapeutic strategies, including cell therapy, for CRSwNP.
PMCID: PMC4063947  PMID: 24945146

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