Immunohistochemical staining patterns of MUC1, MUC2, MUC4, and MUC5AC mucins in hyperplastic polyps, serrated adenomas, and traditional adenomas of the colorectum.

We studied the distribution of the four human apomucins MUC1, MUC2, MUC4, and MUC5AC in hyperplastic polyps, serrated adenomas, and traditional adenomas of the colorectum using immunohistochemical techniques, with the aim of comparing and contrasting their patterns of expression. A series of 12 hyperplastic polyps, 27 serrated adenomas, and 20 traditional adenomas was studied. No significant change in apomucin expression was observed in traditional adenomas compared with normal colorectal epithelium, except for MUC5AC, which was present in 12 of the adenomas (60%) and only 20% of the normal samples. In both hyperplastic polyps and serrated adenomas, MUC2 and MUC5AC mucin expression was consistently and markedly increased. In 50% of the hyperplastic polyps, MUC4 was reduced but in the remaining cases was similar to normal. Loss of MUC4 expression was observed in all serrated adenomas. MUC1 was not increased in the hyperplastic polyps but increased expression was seen in 17 of the serrated adenomas (63%). Similar altered distribution patterns of MUC2, MUC4, and MUC5AC were seen in hyperplastic polyps and serrated adenomas, whereas traditional adenomas showed little change from normal patterns of expression. Although hyperplastic polyps are commonly defined as benign lesions without neoplastic potential, the similar phenotypes of hyperplastic and serrated adenomas and the existence of mixed polyps suggest that these lesions may represent a histogenetic continuum.     

ATP induced MUC5AC release from human airways in vitro

BACKGROUND: Chronic airway diseases are often associated with marked mucus production, however, little is known about the regulation of secretory activity by locally released endogenous mediators. AIM: This investigation was performed to determine the release of MUC5AC mucin from human bronchial preparations using the purinergic agonists adenosine 5''-triphosphate (ATP) and uridine 5''-triphosphate (UTP). METHODS: Immunohistochemical and immunoradiometric assays (IRMA) were used to detect the MUC5AC mucin. Immunohistochemical analysis were performed using individual 1-13 M1 and 21 M1 MAbs recognizing a recombinant M1 mucin partially encoded by the MUC5AC gene. IRMA measurments were performed using a mixture of eight anti-M1 mucin MAbs (PM8), which included both 1-13 M1 and 21 M1 MAbs. Lysozyme and protein were also measured in the biological fluids derived from human bronchial preparations obtained from patients who had undergone surgery for lung carcinoma. RESULTS: The anti-M1 monoclonal antibodies labelled epithelial goblet cells. After challenge of human bronchial preparations with ATP, the goblet cells exhibited less staining. In contrast, UTP did not alter the immunolabelling of goblet cells. MUC5AC mucin in the bronchial fluids derived from ATP-challenged preparations was increased while UTP had no effect on release. ATP did not alter either the quantities of lysozyme or protein detected in the biological fluids. CONCLUSION: These results suggest that ATP may regulate epithelial goblet cell secretion of MUC5AC mucin from human airways in vitro.     

Immunohistochemical study of the expression of MUC6 mucin and co-expression of other secreted mucins (MUC5AC and MUC2) in human gastric carcinomas.

To investigate the expression of MUC6 mucin in gastric carcinomas, we generated a novel monoclonal antibody (MAb CLH5) using an MUC6 synthetic peptide. MAb CLH5 reacted exclusively with the MUC6 peptide and with native and deglycosylated mucin extracts from gastric tissues. MAb CLH5 immunoreactivity was observed in normal gastric mucosa restricted to pyloric glands of the antrum and mucopeptic cells of the neck zone of the body region. In a series of 104 gastric carcinomas, 31 (29.8%) were immunoreactive for MUC6. The expression of MUC6 was not associated with histomorphological type or with clinicopathological features of the carcinomas. Analysis of the co-expression of MUC6 with other secreted mucins (MUC5AC and MUC2) in 20 gastric carcinomas revealed that different mucin core proteins are co-expressed in 55% of the cases. MUC6 was co-expressed and co-localized with MUC5AC in 45% and with MUC2 in 5% of the cases. Expression of MUC2 alone was observed in 25% of the cases. All carcinomas expressing MUC2 mucin in more than 50% of the cells were of the mucinous type according to the WHO classification. The co-expression of mucins was independent of the histomorphological type and stage of the tumors. In conclusion, we observed, using a novel well-characterized MAb, that MUC6 is a good marker of mucopeptic cell differentiation and is expressed in 30% of gastric carcinomas, independent of the clinicopathological features of the cases. Furthermore, we found that co-expression and co-localization of mucins in gastric carcinomas is independent of histomorphology and staging. Finally, we observed that intestinal mucin MUC2 is expressed as the most prominent mucin of the mucins tested in mucinous-type gastric carcinomas.     

MUC5AC,but not MUC2, is a prominent mucin in respiratory secretions

Airway mucus was collected from healthy and chronic bronchitic subjects. The chronic bronchitic sputum was separated into gel and sol phase by centrifugation and mucins were isolated using isopycnic density-gradient centrfugation in CsCl. The presence of the MUC5AC and MUC2 mucins was investigated with antisera raised against synthetic peptides with sequences from the respective apoproteins. The gel and sol phase of chronic bronchitic sputum as well as healthy respiratory secretions were shown to contain MUC5AC whereas the MUC2 mucin could not be detected. Rate-zonal centrifugation showed that the MUC5AC mucin was large, polydisperse in size and that reduction yielded subunits. Ion-exchange HPLC revealed the presence of two subunit populations in all secretions, the MUC5AC subunits always being the more acidic. MUC5AC is thus the first large, subunit-based, gel-forming respiratory mucin identified and this glycoprotein is biochemically distinct from at least one other population of large, gel-forming mucins also composed of subunits but lacking a genetic identity.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate - CF cystic fibrosis - DFP diisopropylphosphofluoridate - DTT dithiothreitol - EDTA ethylenedinitrilotetraacetic acid - NEM N-ethylmaleimide - PAS periodic acid/Schiffs - PMSF phenylmethylsulphonyl fluoride - Tris Tris(hydroxymethyl)aminomethane - VNTR variable number of tandem repeats     

Mucin variable number tandem repeat polymorphisms and severity of cystic fibrosis lung disease: significant association with MUC5AC

Variability in cystic fibrosis (CF) lung disease is partially due to non-CFTR genetic modifiers. Mucin genes are very polymorphic, and mucins play a key role in the pathogenesis of CF lung disease; therefore, mucin genes are strong candidates as genetic modifiers. DNA from CF patients recruited for extremes of lung phenotype was analyzed by Southern blot or PCR to define variable number tandem repeat (VNTR) length polymorphisms for MUC1, MUC2, MUC5AC, and MUC7. VNTR length polymorphisms were tested for association with lung disease severity and for linkage disequilibrium (LD) with flanking single nucleotide polymorphisms (SNPs). No strong associations were found for MUC1, MUC2, or MUC7. A significant association was found between the overall distribution of MUC5AC VNTR length and CF lung disease severity (p = 0.025; n = 468 patients); plus, there was robust association of the specific 6.4 kb HinfI VNTR fragment with severity of lung disease (p = 6.2×10(-4) after Bonferroni correction). There was strong LD between MUC5AC VNTR length modes and flanking SNPs. The severity-associated 6.4 kb VNTR allele of MUC5AC was confirmed to be genetically distinct from the 6.3 kb allele, as it showed significantly stronger association with nearby SNPs. These data provide detailed respiratory mucin gene VNTR allele distributions in CF patients. Our data also show a novel link between the MUC5AC 6.4 kb VNTR allele and severity of CF lung disease. The LD pattern with surrounding SNPs suggests that the 6.4 kb allele contains, or is linked to, important functional genetic variation.     

TNF-alpha in the regulation of MUC5AC secretion: some aspects of cytokine-induced mucin hypersecretion on the in vitro model

TNF-alpha has been implicated in the aetiology of otitis media with effusion (OME), where goblet cells proliferate in a modified respiratory epithelium, leading to the accumulation of a mucin-rich effusion in the middle-ear cleft. The MUC5AC mucin gene product has been identified as a component of these effusions. Here we have used the HT29-MTX goblet cell line, which secretes MUC5AC mucin, as a model to study the effect of TNF-alpha on goblet cells. MUC5AC mucin was identified and quantitated with a monoclonal antibody NCL-HGM-45M1. TNF-alpha stimulates MUC5AC mucin secretion in a dose-dependent manner, with 20 ng/ml producing maximal stimulation. Both pre-confluent and confluent cells showed peak stimulation after 7 h, however the pre-confluent cells showed twice the level of mucin hypersecretion. These results suggest that TNF-alpha stimulation of mucin secretion could play an important role in the early acute phase of the development of OME. This hypersecretion of mucin could then lead to the failure of the mucociliary clearance system, resulting in the accumulation of a mucin-rich effusion in the middle ear and the movement to a more chronic phase of the disease.     

Altered expression of MUC2 and MUC5AC in response to Shigella infection, an in vivo study

Infection of mucosal epithelial cells by Shigella species leads to an intense and acute inflammatory bowel disease that is characterized by watery diarrhea and purulent discharge. Mucin production is a common defense mechanism to protect the underlying mucosa against pathogens. The molecular mechanism(s) underlying mucin induction is unknown in Shigellosis. In this study, we have evaluated the relationship between Shigella infection, the expression of MUC2 and MUC5AC and the participation of signaling molecules TNF-alpha, PKC and ERK1/2. Shigella infection up-regulated MUC2 and MUC5AC expression in 6-8 h, through activation of TNF-alpha, PKC and ERK1/2. These results confirm that, in response to Shigella infection, the normal expression pattern of MUC-2 and MUC-5AC is altered. This in vivo study brings new insights into the molecular pathogenesis of Shigellosis and new potential therapeutic targets for Shigellosis.     

MCP-1/CCR2B-dependent loop upregulates MUC5AC and MUC5B in human airway epithelium

Cigarette smoke represents a major risk factor for the development of chronic obstructive pulmonary disease (COPD), a respiratory condition associated with airflow obstruction, mucus hypersecretion, chronic inflammation, and upregulation of inflammatory mediators such as the monocyte chemotactic protein-1 (MCP-1). MCP-1 through its receptor CCR2 induces chemotaxis and activates (44/42)MAPK, a kinase known to play a key role in mucin regulation in bronchial epithelium. In the present study we used differentiated primary cultures of normal human bronchial epithelial (NHBE) cells to test whether MCP-1 through its receptor CCR2 induces mucin upregulation. We have provided evidence that NHBE cells release MCP-1 to the epithelial surface and express the CCR2B isoform of the receptor mainly at the apical pole. In addition, we found that MCP-1 has a novel function in airway epithelium, increasing the two major airway mucins MUC5AC and MUC5B, an effect mediated, at least in part, by a cascade of events initiated by interaction of its receptor CCR2B with G(q) subunits in caveolae, followed by PLCβ, PKC, and (44/42)MAPK activation. We also have shown that MCP-1 is able to induce its own expression using the same receptor but through a different pathway that involves RhoA GTPase. Furthermore, we found that a single exposure to MCP-1 is enough to induce MCP-1 secretion and sustained mucin upregulation up to 7 days after initial exposure, an effect mediated by CCR2B as confirmed using short hairpin RNA. These results agree with our data in smoker's airway epithelium, where CCR2B is present in MUC5AC- and MUC5B-expressing cells and augmented MCP-1 expression is associated with increased MUC5AC and MUC5B immunolabeling, suggesting that the mechanisms described in primary cell cultures in the present study are operative in vivo. Therefore, therapeutic approaches targeting MCP-1/CCR2B may be useful in preventing not only influx of inflammatory cells to the airways but also mucus hypersecretion and goblet cell hyperplasia.     

Identification of molecular intermediates in the assembly pathway of the MUC5AC mucin

MUC5AC mucins secreted by HT-29 cells in culture are oligomeric glycoproteins with characteristics similar to the MUC5AC mucins isolated from human airway sputum (Sheehan, J. K., Brazeau, C., Kutay, S., Pigeon, H., Kirkham, S., Howard, M., and Thornton, D. J. (2000) Biochem. J. 347, 37-44). Therefore we have used this cell line as a model system to investigate the biosynthesis of this major airway mucin. Initial experiments showed that the MUC5AC mucins isolated from the cells were liable to depolymerization depending on the conditions used for their solubilization. Prevention against reduction resulted in large oligomers associated with the cells, similar to those secreted into the medium. Using a combination of density gradient centrifugation and agarose gel electrophoresis coupled with probes specific for different forms of the mucin we identified five major intracellular populations of the MUC5AC polypeptide (unglycosylated monomer and dimer, GalNAc-substituted dimer, fully glycosylated dimer, and higher order oligomers). Pulse-chase studies were performed to follow the flow of radioactivity through these various intracellular forms into the mature oligomeric mucin secreted into the medium (a process taking approximately 2-4 h). The results show that the mucin polypeptide undergoes dimerization and then becomes substituted with GalNAc residues prior to glycan elaboration to produce a mature mucin dimer, which then undergoes multimerization. These data indicate that this oligomeric mucin follows a similar assembly to the von Willebrand factor glycoprotein to yield long linear disulfide-linked chains.     

C-Mannosylation of MUC5AC and MUC5B Cys subdomains

We expressed recombinant Cys subdomains in COS-7 cells to examine the role of this highly conserved protein domain in mucin biosynthesis. The entire Cys1 and Cys5 and Cys1 and Cys3 subdomains in MUC5AC and MUC5B, respectively, each with six carboxyl terminal histidine residues, were pulse-labeled with [(35)S]cysteine/methionine, and the labeled proteins were examined in the culture medium. Under nonreducing conditions, secreted Cys subdomains were monomers, indicating the absence of interchain disulfide bonds. Cross-linking studies suggested the domains are able to interact through very weak noncovalent interactions. Though the domains had apparent M(r) consistent with the absence of N- and O-glycans, they could be purified with mannose-specific lectins. Lectin binding was prevented by mutation of the first tryptophan residue in the putative C-mannosylation acceptor motif WXXW, indicating that C-mannosylation is responsible for lectin binding. As judged by pulse-chase experiments, C-mannosylation occurred very early during the domain biosynthesis, likely in the endoplasmic reticulum (ER). Mutation of the WXXW motif or expression of the unmutated domain in CHO-Lec35.1 cells, a C-mannosylation-defective cell line, resulted in reduced secretion of the corresponding Cys subdomains. Live cell imaging of green fluorescent protein fused to the Cys subdomains clearly revealed increased presence of Cys subdomains in the ER of CHO-Lec35.1 cells when compared to the same domains expressed in CHO-K1 cells. Considered together, these studies suggest that the Cys subdomains of MUC5AC and MUC5B are C-mannosylated in their respective WXXW motifs. C-mannosylation is likely required for proper folding of the Cys subdomains and/or for some aspect of ER export during mucin biosynthesis.     

Regulation of MUC5AC mucin secretion and airway surface liquid metabolism by IL-1beta in human bronchial epithelia

Mucociliary transport in the airways significantly depends on the liquid and mucin components of the airway surface liquid (ASL). The regulation of ASL water and mucin content during pathological conditions is not well understood. We hypothesized that airway epithelial mucin production and liquid transport are regulated in response to inflammatory stimuli and tested this hypothesis by investigating the effects of the pleiotropic, early-response cytokine, IL-1beta, on cultured primary human bronchial epithelial and second-passage, normal human tracheo-bronchial epithelial (NHTBE) cell cultures. Fully differentiated NHTBE cultures secreted two major airway mucins, MUC5AC and MUC5B. IL-1beta, in a dose- and time-dependent manner, increased the secretion of MUC5AC, but not MUC5B. MUC5AC mRNA levels were only transiently increased at 1 and 4 h after the start of IL-1beta treatment and returned to control levels thereafter, even though MUC5AC mucin production remained elevated for at least 72 h. Synchronous with elevated MUC5AC secretion, ASL volume increased, its percentage of solid was reduced, and the pH/[HCO(3)(-)] of the ASL was elevated. ASL volume changes reflected altered ion transport, including an upregulation of Cl(-) secretory currents (via CFTR and Ca(2+)-activated Cl(-) conductance) and an inhibition of epithelial sodium channel (ENaC)-mediated absorptive Na(+) currents. IL-1beta increased CFTR mRNA levels without affecting those for ENaC subunits. The synchronous regulation of ASL mucin and liquid metabolism triggered by IL-1beta may be an important defense mechanism of the airway epithelium to enhance mucociliary clearance during airway inflammation.     

Altered expression of MUC2 and MUC5AC in response to Shigella infection,an in vivo study

Infection of mucosal epithelial cells by Shigella species leads to an intense and acute inflammatory bowel disease that is characterized by watery diarrhea and purulent discharge. Mucin production is a common defense mechanism to protect the underlying mucosa against pathogens. The molecular mechanism(s) underlying mucin induction is unknown in Shigellosis. In this study, we have evaluated the relationship between Shigella infection, the expression of MUC2 and MUC5AC and the participation of signaling molecules TNF-α, PKC and ERK1/2. Shigella infection up-regulated MUC2 and MUC5AC expression in 6–8 h, through activation of TNF-α, PKC and ERK1/2. These results confirm that, in response to Shigella infection, the normal expression pattern of MUC-2 and MUC-5AC is altered. This in vivo study brings new insights into the molecular pathogenesis of Shigellosis and new potential therapeutic targets for Shigellosis.     

Secreted human conjunctival mucus contains MUC5AC glycoforms.

This study addresses the extent of variation in secreted end-product mucins in human conjunctival mucus. The aim was to determine whether the variety of mucin species found was encompassed by the mucin genes which have been cloned to date. Extraction into guanidine hydrochloride and separation of mucin constituents, by a combination of cesium chloride density gradient centrifugation, size separation on Sepharose CL-2B, MonoQ ion exchange chromatography and agarose gel electrophoresis, demonstrates a complex mixture of mucins. Sample size limitations precluded compositional amino acid analysis. MUC 5AC and MUC1, 2, and 4 are all detected in the buoyant density range 1.3-1.5 g/ml by antibody binding. The mucins vary in size from >40 x 10(6)to <97 x 10(3)Da. A wide range of molecular size was confirmed using rate zonal centrifugation. The presence of smaller species contrasts with other mucous secretions similarly studied. In each size range are low, medium, and high charge mucins. Sialylation predominates in the medium charge and sulfate in the high charge. Only MUC5AC cross-reactivity is maintained throughout the analysis. It is detected in large and medium sized mucins but accounts for only the least mobile mucins within copurified species of similar density, size, and charge resolved using agarose electrophoresis. MUC5AC cross-reactivity is also detected in both medium and high charge species, indicating the presence of glycoforms.     

Cleavage in the GDPH sequence of the C-terminal cysteine-rich part of the human MUC5AC mucin

MUC5AC is the main gel-forming mucin expressed by goblet cells of the airways and stomach where it protects the underlying epithelia. We expressed the C-terminal cysteine-rich part of the human MUC5AC mucin in CHO-K1 cells (Chinese-hamster ovary K1 cells) where it formed disulfide-linked dimers in the ER (endoplasmic reticulum). After reducing the disulfide bonds of these dimers, not only the expected monomers were found, but also two smaller fragments, indicating that the protein was partially cleaved. The site of cleavage was located at an Asp-Pro bond situated in a GDPH (Gly-Asp-Pro-His) sequence found in the vWD4 (von Willebrand D4) domain. This sequence is also found in the human MUC2 mucin, previously shown to be cleaved at the same site by a slow, non-enzymatic process triggered by a pH below 6 [Lidell, Johansson and Hansson (2003) J. Biol. Chem. 278, 13944-13951]. In contrast with this, the cleavage of MUC5AC started already in the neutral ER. However, it continued and was slightly accelerated at a pH below 6.5, a pH found in the later parts of the secretory pathway. The cleavage generated a reactive group in the new C-terminus that could link the protein to a primary amine. No cleavage of MUC5AC has so far been reported. By using an antibody reacting with the C-terminal cleavage fragment, we could verify that the cleavage occurs in wild-type MUC5AC produced by HT-29 cells. The cleavage of MUC5AC and the generation of the reactive new C-terminus could contribute to the adherent and viscous mucus found at chronic lung diseases such as asthma and cystic fibrosis, characterized by mucus hypersecretion and lowered pH of the airways.     

Quantitative MUC5AC and MUC6 mucin estimations in gastric mucus by a least-squares minimization method

We have determined the molar proportions of the MUC5AC and MUC6 mucus glycoproteins (mucins) in mucus from the normal and pathological human gastric antrum using a least-squares minimization analysis applied to amino acid compositions. We noted that the content of MUC5AC mucin in mucus from individuals without gastroduodenal disease was very high, suggesting that the integrity and barrier properties of the adherent gastric mucus layer are normally maintained by building-block structures formed from this mucin alone. We observed that the molar content of MUC6 mucin doubled (without significance) in mucus from patients with duodenal ulcer, and increased five times (with high significance) in mucus from patients with gastric ulcer, when compared with that in mucus from individuals without gastroduodenal disease.     

Fenretinide favorably affects mucins (MUC5AC/MUC5B) and fatty acid imbalance in a manner mimicking CFTR-induced correction

Cystic fibrosis (CF) is the most common genetic disease in Caucasians. CF is manifested by abnormal accumulation of mucus in the lungs, which serves as fertile ground for the growth of microorganisms leading to recurrent infections and ultimately, lung failure. Mucus in CF patients consists of DNA from dead neutrophils as well as mucins produced by goblet cells. MUC5AC mucin leads to pathological plugging of the airways whereas MUC5B has a protective role against bacterial infection. Therefore, decreasing the level of MUC5AC while maintaining MUC5B intact would in principle be a desirable mucoregulatory treatment outcome.Fenretinide prevented the lipopolysaccharide-induced increase of MUC5AC gene expression, without affecting the level of MUC5B, in a lung goblet cell line. Additionally, fenretinide treatment reversed the pro-inflammatory imbalance of fatty acids by increasing docosahexaenoic acid and decreasing the levels of arachidonic acid in a lung epithelial cell line and primary leukocytes derived from CF patients. Furthermore, for the first time we also demonstrate the effect of fenretinide on multiple unsaturated fatty acids, as well as differential effects on the levels of long- compared to very-long-chain saturated fatty acids which are important substrates of complex phospholipids. Finally, we demonstrate that pre-treating mice with fenretinide in a chronic model of P. aeruginosa lung infection efficiently decreases the accumulation of mucus. These findings suggest that fenretinide may offer a new approach to therapeutic modulation of pathological mucus production in CF.     

Neutrophil elastase induces MUC5AC secretion via protease-activated receptor 2

Mucus hypersecretion is a major manifestation in patients with chronic inflammatory airway diseases, and mucin5AC (MUC5AC) protein is a major component of airway mucus. Previous studies have demonstrated that neutrophil elastase (NE) stimulates the secretion of MUC5AC from airway epithelial cells, however, the mechanism is poorly understood. NE is a known ligand for protein active receptors (PARs), which have been confirmed to participate in releasing MUC5AC in the airways. However, the role of PARs in NE-induced MUC5AC secretion remains unclear. We demonstrated that airway goblet-like Calu-3 cells express PAR1, PAR2, and PAR3 with a predominant level of PAR2. NE can increase PAR2 expression and MUC5AC release. In our study, we showed that NE binding to PAR2 can increase the cytosolic calcium concentration and subsequently activate PKC, leading to MUC5AC secretion. In order to investigate the mechanism of increased cytosolic calcium in Calu-3 cells, thapsigargin was used to exhaust the endoplasmic reticulum (ER) calcium pools, and 2-aminoethoxydiphenyl borate was used to inhibit the function of the store-operated calcium entry (SOCE) channels in the plasma membrane. We found that the NE-induced increase in intracellular calcium concentration is derived from release of the ER calcium pool and its subsequent calcium internal flux from the extracellular space via SOCE channels, which is dependent on sufficient levels of extracellular calcium.