Airway mucus obstruction: mucin glycoproteins, MUC gene regulation and goblet cell hyperplasia.
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Airway Obstruction↗
Biomedical subjects
M C Rose
Publications and source records attributed to M C Rose.
Model systems for investigating mucin gene expression in airway diseases.
Overproduction of mucus and of mucin glycoproteins and goblet cell hyperplasia occurs in chronic obstructive airway diseases, including asthma and cystic fibrosis. Mucus overproduction results from alterations in several cellular processes, including altered regulation of airway mucin genes on exposure to environmental and infectious agents and to inflammatory mediators. Seven of the nine identified MUC genes (which encode the protein backbone of mucins) are normally expressed in human respiratory tract tissues. Several inflammatory mediators have now been shown to regulate expression of MUC2, MUC5AC, and MUC5B genes. Importantly, mucin gene expression can be regulated both transcriptionally and posttranscriptionally. Current information on airway mucin gene expression is summarized in this review along with an overview of airway epithelial model systems. In vitro model systems include airway epithelial carcinoma cell lines and primary normal human bronchial epithelial (NHBE) cells. In vivo systems include human respiratory tract tissues and rodent airways. Our laboratory has begun to investigate the role of cytokines on mucin gene expression in vitro and in vivo and on goblet cell metaplasia in vivo. Because cytokines can alter cell proliferation, we characterized the effect of interleukin (IL)-4 and IL-13 on the proliferation of NHBE cells and three human lung carcinoma cell lines--A549, NCI-H292, and Calu-3--that are frequently used for analyses of airway mucin gene expression. Both IL-4 and IL-13 had cell-specific effects. They increased proliferation moderately (1.2-3.0-fold) in NHBE and Calu-3 cells, but markedly inhibited proliferation of A549 cells in a dose-dependent manner. IL-4 increased proliferation of NCI-H292 cells moderately, although IL-13 had no significant effect. We also examined the role of IL-13 and IL-4 on MUC5AC messenger RNA (mRNA) expression in A549, Calu-3, and H292 cell lines and did not observe any significant effect. However, we recently showed an increase in Muc-5ac mRNA and protein expression in a murine model of ovalbumin-induced allergic asthma and in murine airways when IL-13 was delivered intranasally (Alimam, N.Z., et al. Am J. Respir. Cell Mol. Biol. 22:253--260). Thus, we speculate that IL-13 plays a role in the differentiation of murine airway epithelial cells into goblet cells, which then express Muc-5ac mRNA. A detailed analysis of the role of cytokines in airway cell differentiation and mucin gene expression both in vitro and in vivo is required to elucidate the roles of mucins in airway health and diseases. Identification of Muc-5ac as a major gene and gene product in goblet cell metaplasia should facilitate delineation of the molecular mechanisms underlying the induction and reversal of airway goblet cell metaplasia and goblet cell hyperplasia.
Animals↗
Muc-5/5ac mucin messenger RNA and protein expression is a marker of goblet cell metaplasia in murine airways.
Airway inflammation, hyperreactivity, increased number of goblet cells, and mucus overproduction characterize asthma. Respiratory challenge with ovalbumin (OVA) of sensitized mice has been shown by several laboratories to cause pulmonary pathology similar to that observed in human allergic asthma. Recently, interleukin (IL)-13 has been shown to be a central mediator in this process. Because the airways of healthy mice have few, if any, mucus-producing cells, an increase in the number of these cells likely reflects induction of mucin-gene expression. The purpose of this study was to identify mucin genes induced as a result of airway goblet-cell metaplasia (GCM) in mice sensitized and challenged with OVA or in mice treated with IL-13 alone. BALB/c mice were sensitized by intraperitoneal injection (Days 0, 4, 7, 11, and 14) and intranasal instillation (Day 14) of 100 microg of OVA in saline, and then challenged by intranasal instillation (Days 25, 26, and 27) of the same. IL-13-treated mice received 5 microg of IL-13 by intranasal instillation on three consecutive days. Control mice were given saline alone. All mice were studied 24 h after the last challenge. Histologic analysis of the lungs revealed both a striking peribronchial and perivascular lymphocytic and eosinophilic inflammation and airway GCM in OVA-treated mice, and also airway GCM without inflammation in IL-13-treated mice. Northern blot analysis of lung RNA demonstrated (1) expression of Muc-5/5ac messenger RNA (mRNA) in OVA-treated and IL-13-treated mice, but not in control mice; (2) expression of Muc-1 mRNA at comparable levels in all mice regardless of treatment; and (3) no expression of Muc-2 or Muc-3 mRNA in control or treated mice. Western blot analysis demonstrated the expression of Muc-5/5ac protein (both apomucin and glycosylated mucin) in lung lysates of OVA-treated (but not control) mice, and also the expression of Muc-5/5ac mucins in the bronchoalveolar lavage fluid of OVA-treated and IL-13-treated mice. These findings demonstrate that airway GCM is associated with the induction of pulmonary expression of Muc-5/5ac mRNA and mucin in murine models of allergic asthma.
Alveolitis, Extrinsic Allergic↗
Neutrophil elastase increases MUC5AC mRNA and protein expression in respiratory epithelial cells.
Chronic neutrophil-predominant inflammation and hypersecretion of mucus are common pathophysiological features of cystic fibrosis, chronic bronchitis, and viral- or pollution-triggered asthma. Neutrophils release elastase, a serine protease, that causes increased mucin production and secretion. The molecular mechanisms of elastase-induced mucin production are unknown. We hypothesized that as part of this mechanism, elastase upregulates expression of a major respiratory mucin gene, MUC5AC. A549, a human lung carcinoma cell line that expresses MUC5AC mRNA and protein, and normal human bronchial epithelial cells in an air-liquid interface culture were stimulated with neutrophil elastase. Neutrophil elastase increased MUC5AC mRNA levels in a time-dependent manner in both cell culture systems. Neutrophil elastase treatment also increased MUC5AC protein levels in A549 cells. The mechanism of MUC5AC gene regulation by elastase was determined in A549 cells. The induction of MUC5AC gene expression required serine protease activity; other classes of proteases had no effect on MUC5AC gene expression. Neutrophil elastase increased MUC5AC mRNA levels by enhancing mRNA stability. This is the first report of mucin gene regulation by this mechanism.
Adenocarcinoma↗
Respiratory carcinoma cell lines. MUC genes and glycoconjugates.
Lung carcinoma cell lines are being used in many laboratories to study various airway epithelial functions, including mucin gene expression. To identify model systems for investigating regulation of MUC5/5AC gene expression and secretion of MUC5/5AC mucins in airway epithelial cells, we evaluated the expression of several mucin genes in six carcinoma cell lines of respiratory tract origin. RNA was extracted from A549, Calu-3, NCI H292, Calu-6, RPMI 2650, and A-427 cells; MUC1, MUC2, MUC4, MUC5/5AC, and MUC5B messenger RNA (mRNA) expression was determined. By Northern analyses, all cell lines expressed MUC1 mRNA, whereas MUC2 mRNA was not detectable in any of the cell lines. RPMI 2650 cell lines expressed only MUC1 mRNA. NCI-H292 cells expressed MUC4 and low levels of MUC5/5AC mRNA. Calu-3 and A549 cells expressed MUC5/5AC mRNA; A549 cells also expressed MUC5B mRNA. Glycoconjugates secreted by lung carcinoma cells were also examined. By wheat germ lectin analysis, Calu-3, H292, and A549 cells secreted high molecular weight glycoproteins having N-acetylglucosamine and/or sialic acid moieties. Western blot analyses with an anti-MUC5:TR-3A antibody demonstrated that Calu-3 and A549 cells secreted MUC5/5AC mucins. All six carcinoma cell lines secreted large, radiolabeled, sulfated macromolecules; the majority were proteoglycans that were digested by hyaluronidase. However, Calu-3 cells also secreted sulfated high molecular-weight glycoproteins that were immunoprecipitated by anti-MUC5:TR-3A antibody. These studies demonstrated that Calu-3 and A549 cell lines expressed high and moderate amounts of MUC5/5AC mRNA and MUC5/5AC mucins, whereas H292 cells expressed lesser amounts. These cell lines should prove useful for studies of MUC5/5AC gene expression and MUC5/5AC biosynthesis, trafficking, and secretions in airway epithelial cells.
Carcinoma↗
Mucin gene expression (MUC1, MUC2, and MUC5/5AC) in nasal epithelial cells of cystic fibrosis, allergic rhinitis, and normal individuals.
The early pathogenic events in cystic fibrosis (CF) include colonization of Pseudomonas in the lung, airway inflammation, and mucus hypersecretion with airway obstruction. The primary mechanisms leading to chronic infection and inflammation are not well understood. One possible explanation for this cascade of events is increased or altered expression of one or more mucin (MUC) genes by CF cells in the respiratory tract. We compared expression levels of three mucin genes, MUC1, MUC2, and MUC5/5AC, known to be expressed in the respiratory tract of CF, allergic rhinitis, and normal individuals. Mucin transcript levels in nasal epithelial cells free from inflammation were quantitated by an MUC mRNA slot-blot method. This study revealed three major findings: (1) MUC5/5AC mRNA was expressed at five- to tenfold greater levels than MUC2 or MUC1 for all subjects. (2) MUC2 mRNA levels were similar among all subject groups. (3) In CF subjects, there was a trend toward reduced MUC5/5AC expression. When normalized to the levels of MUC2 expression in individual specimens, MUC5/5AC expression was reduced significantly in CF cells compared with normal cells. These data suggest that mucin gene expression is altered in noninflamed CF nasal cells.
Adolescent↗
Factitious post traumatic stress disorder: a case report.
A 24-year-old man presented with a convincing history of Post Traumatic Stress Disorder (PTSD). He claimed to be the victim of a widely publicized 'human bomb' attack by the IRA in Northern Ireland when he was serving with the armed forces. Psychometric tests for PTSD confirmed his symptoms. A subsequent check of public and military records demonstrated that he was a serviceman at that time, but showed conclusively that he could not have been present at the terrorist incident.
Adult↗
Cloning and analysis of cDNA encoding a major airway glycoprotein, human tracheobronchial mucin (MUC5).
Two unique nucleotide probes for human tracheobronchial mucin glycoprotein (TBM) were generated via polymerase chain reaction with degenerate primers deduced from the TBM:TR-3A tryptic peptide sequence and were used to isolate a 3.6 kilobase cDNA, clone NP3a, from a human nasal polyp cDNA library. Clone NP3a was localized to chromosome 11 and contained a 3168 nucleotide open reading frame which encoded three TBM peptide fragments, thus confirming that clone NP3a partially encodes TBM. TBM also contains five tandem repeats of TTVGP/S and an octapeptide GQCGTCTN, which is conserved in human intestinal mucin MUC2 and rat intestinal mucin-like protein (MLP) suggesting that this sequence has a functional significance for secreted mucins. TBM has amino acid similarity to the cysteine-rich domains at the carboxyl termini of MUC2, rat MLP, bovine and porcine submaxillary mucins, and human von Willebrand factor. Strikingly, a large percentage of the cysteine residues in the overlaps are highly conserved: 90% in MUC2 and von Willebrand factor, 80% of bovine submaxillary mucin, 70% in porcine submaxillary mucin, and 64% in rat MLP, suggesting that conserved cysteines may be important for the tertiary structure of secreted glycoproteins. These studies demonstrate that clone NP3a is a candidate for MUC5, making it the only human mucin gene reported to date whose gene product has been isolated from airway secretions.
Amino Acid Sequence↗
Quantitation of mucin mRNA in respiratory and intestinal epithelial cells.
Mucin glycoproteins (mucins) are the major macromolecular constituents of mucus gels in mammalian respiratory, gastrointestinal, and reproductive tracts. Disorders of mucin glycosylation, which may result from either abnormal post-translational processing or differences in mucin protein gene expression, have been indicated in several diseases. Quantitation of mucin gene expression has been hindered by two features of human mucin genes: variable numbers of tandemly repeating nucleotides per mRNA molecule and polydisperse mRNA transcripts. We report here a method to quantitate mucin mRNA levels in epithelial cells and have evaluated three mucin genes, MUC1, MUC2, and MUC5, which are expressed in respiratory epithelium. The method uses the 3' non-tandem repeat mucin cDNA sequences, as they were shown to have a single-size transcript when amplified by the polymerase chain reaction, consistent with a one-to-one relationship with the mRNA molecule. The 3' non-tandem repeat cDNA sequences were cloned and transcribed in vitro to prepare complementary RNA (cRNA) standards. By comparison to a cRNA standard curve, mucin gene expression was evaluated in colon adenocarcinoma, pancreatic adenocarcinoma, and transformed respiratory epithelial cells and in nasal polyp tissue by slot blot analysis. CFPAC-1, a pancreatic adenocarcinoma cell line, expressed the highest MUC1 transcript levels. Colon adenocarcinoma cell lines varied in MUC2 expression levels, and one colon adenocarcinoma cell line, HT-29, had higher levels of MUC5 than MUC2. Nasal polyp tissue expressed more MUC5 mRNA than MUC1 or MUC2 mRNA. This mucin mRNA slot blot method provides a quantitative method for investigating the regulation of mucin gene expression in health and disease.
Adenocarcinoma↗
Mucins: structure, function, and role in pulmonary diseases.
Mucins, major components of the extracellular mucus blanket that protect and lubricate mammalian epithelia, are high-molecular-mass glycoconjugates (154 to > or = 7,000 kDa) with hundreds of oligosaccharide chains in O-glycosidic linkages to a protein backbone. The apparent expression of more than one type of oligosaccharide core structure in mucins isolated from pathological material may reflect either inherent limitations in analysis, disease-related alterations in parameters affecting glycosylation and post-translational modifications (e.g., nucleotide-sugar concentrations, expression of specific glycosyltransferases, rates of transport through the endoplasmic reticulum and Golgi) or the activation of mucin protein genes that are more highly expressed in disease states with different glycosylation patterns. Recent studies have revealed the existence of a family of at least four human mucin proteins; MUC1, MUC2, MUC3, MUC4, each of which contains a variable number of tandem repeats that differ in sequence and size. Full-length sequences of cDNA clones encoding human mucin proteins are currently available only for MUC1 which, in contrast to most airway and intestinal mucins, is membrane associated and not secreted. Current information on mucin oligosaccharides and proteins is reviewed herein. More detailed knowledge of the protein and oligosaccharide structures of mucins will be important in identifying specific role(s) in health and disease, i.e., in the physiological functions of mucus.
Amino Acid Sequence↗
Isolation and characterization of mucin from the serum of cystic fibrosis patients.
Using monoclonal antibody 19-9, elevated levels of the sialyl Lea antigen (NeuAc alpha 2-3Gal beta 1-3[Fuc alpha 1-4]GlcNac beta 1-3R) are detected in serum from most cystic fibrosis patients. We now report further characterization of the serum antigen and evidence that it is on a mucin glycoprotein and not on glycolipids. The antigen has an apparent molecular weight greater than 2 X 10(6) by gel filtration on Sephacryl S-400. On density gradient centrifugation, the antigen has a density of 1.54 g/ml in cesium chloride and 1.42 g/ml in cesium chloride/4 M guanidine HCl. Immunostaining with monoclonal antibody 19-9 of lipid extracts from cystic fibrosis patient serum and erythrocytes does not detect any antigen on glycolipids. The antigen was purified by gel filtration and density gradient centrifugation. After tritium labeling of the sialic acid residues, sodium dodecyl sulfate gel electrophoresis separates two subunits with apparent molecular weights of 200,000 and 400,000. All of the labeled sialic acid is released as low molecular weight oligosaccharides after mild alkaline borohydride degradation. The purified antigen contains fucose, galactosamine, glucosamine, and galactose but no mannose and is enriched in the amino acids threonine, serine, glycine, proline, and alanine. The purified antigen binds several antibodies recognizing epitopes common to many mucins. Thus, the physical, biochemical, and immunochemical properties of the purified antigen indicate that the sialyl Lea antigen is present on mucins in the serum of cystic fibrosis patients.
Adolescent↗
Proteolytic fragmentation and peptide mapping of human carboxyamidomethylated tracheobronchial mucin.
Human tracheobronchial mucin was isolated from lung mucosal gel by chromatography on Sepharose 4B in the presence of dissociating and reducing agents, and its thiol residues were carboxyamidomethylated with iodo[1(-14)C]acetamide. The 14C-carboxyamido-methylated mucin was purified by chromatography on Sepharose 2B. No low molecular weight components were detected by molecular sieve chromatography or polyacrylamide gel electrophoresis in the presence of dissociating and reducing agents or by analytical density centrifugation in CsCl/guanidinium chloride. After digestion of the purified 14C-mucin with trypsin-L-1-tosylamido-2-phenylethyl chloromethyl ketone, three fractions (TR-1, TR-2, and TR-3) were observed by chromatography on Sepharose 4B. TR-1, a 260-kDa mucin glycopeptide fragment, contained all of the neutral hexose and blood group activity and 20% of the radioactivity in the undigested mucin. TR-1 was refractory to a second incubation with trypsin but could be digested by papain or Pronase to a smaller mucin glycopeptide fraction, as judged by the slight decrease in apparent molecular weight on Sepharose CL-4B. These mucin glycopeptides contained approximately 50% of the radioactivity in the TR-1 fraction, indicating that the glycosylated domains of carboxyamidomethylated tracheobronchial mucin contained thiol residues. The remainder of the radioactivity from papain or Pronase digests of TR-1 eluted, like the TR-3 fractions, in the salt fraction on Sepharose CL-4B. Peptide mapping of the nonglycosylated TR-3 fraction by TLC and high voltage electrophoresis yielded six principal and several less intensely stained ninhydrin reactive components, with the radiolabel concentrated in one of the latter peptides. Peptide purification of the TR-3 fraction by high pressure liquid chromatography on a C18 reverse phase column demonstrated the presence of four major peptides, with TR-3A being the dominant component. The TR-3D peptide contained S-carboxy-aminomethylcysteine and had 69% sequence similarity to the sgs-7 salivary glue protein of Drosophila.
Amino Acids↗
Characterization of human tracheobronchial mucin glycoproteins.
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Bronchi↗
Epithelial mucous glycoproteins and cystic fibrosis.
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Cystic Fibrosis↗
Biochemical properties of tracheobronchial mucins from cystic fibrosis and non-cystic fibrosis individuals.
Tracheobronchial mucins from healthy individuals and from patients with bronchial asthma or cystic fibrosis (CF) were isolated from lung mucus, purified, and their chemical and physical properties compared. Normal and asthmatic mucins required both a dissociating and a reducing agent for solubilization and exhibited identical chromatographic behavior on Sepharose 4B, Sepharose 2B, and hydroxylapatite and similar amino acid and carbohydrate compositions. In contrast, 1) CF lung mucins were solubilized in the absence of dissociating and/or reducing agents and 2) the majority of the CF mucins analyzed was eluted in the included volume of Sepharose 4B with Kd values of 0.3 +/- 0.1 rather than in the void volume and thus appeared smaller than normal and asthmatic mucins. The lower molecular weight mucins in CF sputum apparently are produced by bacterial or inflammatory cell proteinases since radiolabelled asthmatic mucin was digested to smaller fragments when incubated with crude CF lung mucosal samples. Furthermore, mucins secreted by tracheal explants from CF and from non-CF individuals eluted in the void volume on Sepharose 4B, suggesting that CF tracheobronchial mucins were not inherently smaller than non-CF mucins.
Adolescent↗
Intravenous mushroom poisoning.
Mushrooms of the genus Psilocybe frequently are ingested by recreational drug users for their hallucinogenic effects. We present the case of a 30-year-old man who allegedly received an intravenous injection of an extract of Psilocybe mushrooms. His clinical course was characterized in part by vomiting, severe myalgias, hyperpyrexia, hypoxemia, and mild methemoglobinemia, and it was similar to two previously reported cases. The patient improved rapidly with supportive care.
Adult↗
Structural features of human tracheobronchial mucus glycoprotein.
Electron microscopy of platinum-shadowed preparations of human tracheobronchial mucins showed very flexible filamentous structures that frequently occurred in an intricate random-coiled pattern of filament(s) surrounding a dense core-like domain. The filament(s) associated with cores accounted for 70-80% of the mass of the mucin preparation, the remainder being accounted for by free filaments. On aggregation, the molecules formed a large interwoven network quite different from the massive rope-like structures characteristic of sheep submaxillary mucin aggregates [Rose, Voter, Sage, Brown & Kaufman (1984) J. Biol. Chem. 259, 3167-3172]. Mild sonication resulted in extensive fragmentation of the tracheobronchial mucin molecules and yielded short filaments of various lengths, free cores and some cores associated with short filaments. Mucin glycopeptide fragments obtained by proteolytic digestion were flexible, core-free, filaments. The glycopeptides obtained by Pronase digestion were shorter than those obtained by tryptic digestion. The intricate structures of human tracheobronchial mucin differ markedly from the extended filaments reported for sheep submaxillary and human ovarian-cyst mucins but agree with the roughly spherical expanded model proposed for mucins by Creeth & Knight [(1967) Biochem. J. 105, 1135-1145] on the basis of hydrodynamic measurements.
Bronchi↗
Effects of deglycosylation on the architecture of ovine submaxillary mucin glycoprotein.
The structural features of native and deglycosylated ovine submaxillary mucin (OSM) were determined by electron microscopy of platinum unidirectionally shadowed preparations and by ultracentrifugation. Thin filamentous molecules, of which 90% were 100-230 nm in length with estimated diameters of 1.0-1.4 nm, were observed with dilute samples of OSM in high ionic strength solvents (5-30 micrograms/ml in 0.8 M NaCl or NH4Ac). Ultracentrifugation studies indicated that these filamentous structures were monomers and/or dimers. At higher mucin concentrations or in lower ionic strength solvents, OSM molecules were oligomers that appeared as long rope-like strands. Removal of sialic acid residues by incubation with Clostridium perfringens neuraminidase yielded filamentous structures similar to those observed with OSM and some smaller less extended structures. Subsequent removal of the GalNAc residues of asialo-OSM with C. perfringens alpha-N-acetylgalactosaminidase resulted in a dramatic change in appearance, from an extended filament to a globular form. The frictional ratios of OSM and deglycosylated OSM were consistent with the marked structural differences of these molecules. Native OSM had a frictional ratio of 3.09, comparable to that of highly asymmetric tropomyosin (3.22); deglycosylated OSM had a frictional ratio of 1.11, comparable to that of globular ovalbumin (1.08).
Amino Acids↗