Characteristics of rodent intestinal mucin Muc3 and alterations in a mouse model of human cystic fibrosis

Human mucin MUC3 and rodent Muc3 are widely assumed to represent secretory mucins expressed in columnar and goblet cells of the intestine. Using a 3'-oligonucleotide probe and in situ hybridization, we observed expression of rat Muc3 mostly in columnar cells. Two antibodies specific for COOH-terminal epitopes of Muc3 localized to apical membranes and cytoplasm of columnar cells. An antibody to the tandem repeat (TR) sequence (TTTPDV)3, however, localized to both columnar and goblet cells. On CsCl gradients, Muc3 appeared in both light- and heavy-density fractions. The lighter species was immunoreactive with all three antibodies, whereas the heavier species reacted only with anti-TR antibody. Thus Muc3 is expressed in two forms, a full-length membrane-associated form found in columnar cells (light density) and a carboxyl-truncated soluble form present in goblet cells (heavy density). In a mouse model of human cystic fibrosis, both soluble Muc3 and goblet cell Muc2 were increased in amount and hypersecreted. Thus Muc2 and Muc3 contribute to the excess intestinal luminal mucus of cystic fibrosis mice.     

Altered transit and bacterial overgrowth in the cystic fibrosis mouse small intestine

Small intestinal bacterial overgrowth (SIBO) may play an important role in the gastrointestinal complications of cystic fibrosis (CF). This work explored two potential factors in development of SIBO in the CF (cftr(tm1UNC)) mouse: impaired Paneth cell innate defenses and altered gastrointestinal motility. Postnatal differentiation of Paneth cells was followed by Defcr, Lyzs, and Ang4 gene expression, and SIBO was measured by quantitative PCR of the bacterial 16S rRNA gene. Paneth cell gene expression was low in 4-day-old CF and wild-type (WT) mice and increased similarly in both groups of mice between 12 and 16 days. Peak Paneth cell gene expression was reached by 40 days of age and was less for Defcr and Lyzs in CF mice compared with WT, whereas Ang4 levels were greater in CF mice. SIBO occurred by postnatal day 8 in CF mice, which is before Paneth cell development. With the use of gavaged rhodamine-dextran to follow motility, gastric emptying in CF mice was slightly decreased compared with WT, and small intestinal transit was dramatically less. Since antibiotics improve weight gain in CF mice, their effects on gastric emptying and small intestinal transit were determined. Antibiotics did not affect gastric emptying or transit in CF mice but did significantly slow intestinal transit in WT mice, suggesting a potential role of normal microflora in regulating transit. In conclusion, small intestinal transit was significantly slower in CF mice, and this is likely a major factor in SIBO in CF.     

Disrupted tight junctions in the small intestine of cystic fibrosis mice

The tight junction (TJ) is the major determinant of paracellular permeability, which in the gut protects the body from entry of harmful substances such as microbial components. In cystic fibrosis (CF), there is increased permeability of the small intestine both in humans and in CF mice. To gain insight into the mechanisms of increased intestinal permeability in CF, I analyze the composition of the TJ in a cystic fibrosis transmembrane conductance regulator (Cftr) knockout mouse model. Significant changes in TJ gene expression in the CF intestine were found for Cldn1, Cldn7, Cldn8 and Pmp22, which were expressed at lower levels and Cldn2 that was expressed at a higher level. Protein levels of claudin-2 were increased in the CF intestine as compared to wild-type, while other TJ proteins were not significantly different. In the villus epithelium of the CF intestine, all TJ components analyzed were mislocalized to the basal cytoplasm and showed varying degrees of loss from the TJ and apico-lateral surfaces. The pore-forming claudin-2 in the CF intestine showed more intense staining but was correctly localized to the TJ, principally in the crypts that are enlarged in CF. The cytokine TNFα, known to affect TJ, was elevated to 160 % of wild-type in the CF intestine. In summary, there is a dramatic redistribution of claudin proteins from the TJ/lateral membrane to the basal cytoplasm of the villus epithelium in the CF intestine. These changes in TJ protein localization in CF are likely to be involved in the increased permeability of the CF small intestine to macromolecules and TNFα may be a causative factor.     

Effects of laxative and N-acetylcysteine on mucus accumulation, bacterial load, transit, and inflammation in the cystic fibrosis mouse small intestine

The accumulation of mucus in affected organs is characteristic of cystic fibrosis (CF). The CF mouse small intestine has dramatic mucus accumulation and exhibits slower interdigestive intestinal transit. These factors are proposed to play cooperative roles that foster small intestinal bacterial overgrowth (SIBO) and contribute to the innate immune response of the CF intestine. It was hypothesized that decreasing the mucus accumulation would reduce SIBO and might improve other aspects of the CF intestinal phenotype. To test this, solid chow-fed CF mice were treated with an osmotic laxative to improve gut hydration or liquid-fed mice were treated orally with N-acetylcysteine (NAC) to break mucin disulfide bonds. Treatment with laxative or NAC reduced mucus accumulation by 43% and 50%, respectively, as measured histologically as dilation of the intestinal crypts. Laxative and NAC also reduced bacterial overgrowth in the CF intestine by 92% and 63%, respectively. Treatment with laxative normalized small intestinal transit in CF mice, whereas NAC did not. The expression of innate immune response-related genes was significantly reduced in laxative-treated CF mice, whereas there was no significant effect in NAC-treated CF mice. In summary, laxative and NAC treatments of CF mice reduced mucus accumulation to a similar extent, but laxative was more effective than NAC at reducing bacterial load. Eradication of bacterial overgrowth by laxative treatment was associated with normalized intestinal transit and a reduction in the innate immune response. These results suggest that both mucus accumulation and slowed interdigestive small intestinal transit contribute to SIBO in the CF intestine.     

Caloric restriction reduces IgA levels and modifies cytokine mRNA expression in mouse small intestine

The aim of this study was to determine the effect of caloric restriction (CR) in mouse small intestine on the production and secretion of immunoglobulin (Ig) A, the population of lymphocytes in the lamina propria, and the expression of cytokines that mediate and regulate innate and adaptive immunity. One group of young Balb/c mice was fed ad libitum, while the CR group was fed ad libitum and fasted on alternate days. When mice were six months old, IgA levels in the proximal small intestine were quantified by enzyme-linked immunosorbent assay, while the number of IgA containing cells, CD4⁺ T cells and CD8⁺ T cells in the duodenal mucosa was determined by immunohistochemistry. Furthermore, the expression of several intestinal cytokines, the genes for α-chain IgA, and the polymeric Ig receptor (pIgR) were analyzed by real-time polymerase chain reaction. CR decreased the levels of IgA in the intestine, apparently a consequence of a reduced number of IgA⁺ cells in the lamina propria that decrease the production and secretion of this Ig, and a reduced secretion of S-IgA into the bile, which in turn discharges into the proximal intestine. Contrarily, CR increased the expression of genes for α-chain IgA, and the pIgR, indicating that transport of IgA was not a key factor in the decrease of this Ig. Additionally, CR modified the expression of genes for tumor necrosis factor-α, interferon-γ, tumor growth factor-β, interleukin (IL)-2 and IL-10, all of which regulate the synthesis of IgA and pIgR, the inflammatory response, and the immune response in the intestine.     

Cellular localization of cystic fibrosis transmembrane regulator protein in piglet and mouse intestine

Antibodies raised against the cystic fibrosis transmembrane regulator protein (CFTR) were used to localize CFTR in intestinal tissues of piglets and mice. Positive staining for CFTR was detected in goblet cells of both species. A second population of epithelial cells of unknown phenotype was also labeled by anti-CFTR antibodies. The labeling pattern was abolished by preincubation of anti-CFTR antibodies with the immunogen or when non-immune IgG was used in place of anti-CFTR antibodies. These results support other studies that suggest that alterations in goblet cell function may be involved in the intestinal abnormalities associated with cystic fibrosis. Received: 4 May 1995 / Accepted: 6 September 1995     

Glycosylation of sputum mucins is altered in cystic fibrosis patients

Cystic fibrosis (CF) is characterized by chronic lung infection and inflammation, with periods of acute exacerbation causing severe and irreversible lung tissue damage. We used protein and glycosylation analysis of high-molecular mass proteins in saline-induced sputum from CF adults with and without an acute exacerbation, CF children with stable disease and preserved lung function, and healthy non-CF adult and child controls to identify potential biomarkers of lung condition. While the main high-molecular mass proteins in the sputum from all subjects were the mucins MUC5B and MUC5AC, these appeared degraded in CF adults with an exacerbation. The glycosylation of these mucins also showed reduced sulfation, increased sialylation, and reduced fucosylation in CF adults compared with controls. Despite improvements in pulmonary function after hospitalization, these differences remained. Two CF children showed glycoprotein profiles similar to those of CF adults with exacerbations and also presented with pulmonary flares shortly after sampling, while the remaining CF children had profiles indistinguishable from those of healthy non-CF controls. Sputum mucin glycosylation and degradation are therefore not inherently different in CF, and may also be useful predictive biomarkers of lung condition.     

Muc5b and Muc5ac are the major oligomeric mucins in equine airway mucus

Horses frequently suffer from respiratory diseases, which, irrespective of etiology, are often associated with airway mucus accumulation. Studies on human airways have shown that the key structural components of the mucus layer are oligomeric mucins, which can undergo changes of expression and properties in disease. However, there is little information on these gel-forming glycoproteins in horse airways mucus. Therefore, the aims of this study were to isolate equine airways oligomeric mucins, characterize their macromolecular properties, and identify their gene products. To this end, pooled tracheal washes, collected from healthy horses and horses suffering from respiratory diseases, were solubilized with 6 M guanidinium chloride (GdmCl). The oligomeric mucins were purified by density gradient centrifugation followed by size exclusion chromatography. Biochemical and biophysical analyses showed the mucins were stiffened random coils in solution that were polydisperse in size (M(r) = 6-20 MDa, average M(r) = 14 MDa) and comprised of disulfide-linked subunits (average M(r) = 7 MDa). Agarose gel electrophoresis showed that the pooled mucus sample contained at least two populations of oligomeric mucins. Electrospray ionization tandem mass spectrometry of tryptic digests of the unfractionated mucin preparation showed that the oligomeric mucins Muc5b and Muc5ac were present. In summary, we have shown that equine airways mucus is a mixture of Muc5b and Muc5ac mucins that have a similar macromolecular organization to their human counterparts. This study will form the basis for future studies to analyze the contribution of these two mucins to equine airways pathology associated with mucus accumulation.     

Altered O-glycosylation and sulfation of airway mucins associated with cystic fibrosis

Cystic fibrosis (CF) is the most lethal genetic disorder in Caucasians and is characterized by the production of excessive amounts of viscous mucus secretions in the airways of patients, leading to airway obstruction, chronic bacterial infections, and respiratory failure. Previous studies indicate that CF-derived airway mucins are glycosylated and sulfated differently compared with mucins from nondiseased (ND) individuals. To address unresolved questions about mucin glycosylation and sulfation, we examined O-glycan structures in mucins purified from mucus secretions of two CF donors versus two ND donors. All mucins contained galactose (Gal), N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), fucose (Fuc), and sialic acid (Neu5Ac). However, CF mucins had higher sugar content and more O-glycans compared with ND mucins. Both ND and CF mucins contained GlcNAc-6-sulfate (GlcNAc-6-Sul), Gal-6-Sul, and Gal-3-Sul, but CF mucins had higher amounts of the 6-sulfated species. O-glycans were released from CF and ND mucins and derivatized with 2-aminobenzamide (2-AB), separated by ion exchange chromatography, and quantified by fluorescence. There was nearly a two-fold increase in sulfation and sialylation in CF compared with ND mucin. High performance liquid chromatography (HPLC) profiles of glycans showed differences between the two CF samples compared with the two ND samples. Glycan compositions were defined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Unexpectedly, 260 compositional types of O-glycans were identified, and CF mucins contained a higher proportion of sialylated and sulfated O-glycans compared with ND mucins. These profound structural differences in mucin glycosylation in CF patients may contribute to inflammatory responses and increased pathogenesis by Pseudomonas aeruginosa.     

Proliferation stage-dependent expression of DNA methyltransferase (Dnmt1) in mouse small intestine

In cultured cells, the maintenance-type DNA methyltransferase (Dnmt1) is highly expressed during the proliferation stage. In the present study, we detected significant expression of Dnmt1 protein in the nuclear fraction of mouse small intestine. From its mobility in SDS polyacrylamide gel electrophoresis and the specific antibodies against the somatic cell-type Dnmt1, Dnmt1 was determined as a somatic cell type. Immunofluorescence study revealed that the Dnmt1 was highly expressed in the proliferating stem cells in crypts, and was localized in the nuclei. The present results indicate that the expression of Dnmt1 in vivo is also under the control of cell proliferation as in cultured cells.     

Acidic duodenal pH alters gene expression in the cystic fibrosis mouse pancreas

The duodenum is abnormally acidic in cystic fibrosis (CF) due to decreased bicarbonate ion secretion that is dependent on the CF gene product CFTR. In the CFTR null mouse, the acidic duodenum results in increased signaling from the intestine to the exocrine pancreas in an attempt to stimulate pancreatic bicarbonate ion secretion. Excess stimulation is proposed to add to the stress/inflammation of the pancreas in CF. DNA microarray analysis of the CF mouse revealed altered pancreatic gene expression characteristic of stress/inflammation. When the duodenal pH was corrected genetically (crossing CFTR null with gastrin null mice) or pharmacologically (use of the proton pump inhibitor omeprazole), expression levels of genes measured by quantitative RT-PCR were significantly normalized. It is concluded that the acidic duodenal pH in CF contributes to the stress on the exocrine pancreas and that normalizing duodenal pH reduces this stress.     

Detection of circulating mammary mucin (Muc1) and MUC1 immune complexes (Muc1-CIC) in healthy women

There is convincing epidemiological evidence that multiparity provides protection against the development of breast cancer. In the present study we evaluated the levels of MUC1 and MUC1 circulating immune complexes (MUC1-CIC) in 135 serum samples obtained from healthy women. The study population included 13 women who had never been pregnant, 31 primiparous pregnant women, 36 multiparous pregnant women who had lactated, 5 multiparous pregnant women who had never lactated, 24 multiparous non-pregnant women who were lactating at the time of the study, 24 multiparous non-pregnant women who had lactated, and 2 multiparous non-pregnant women who had never lactated. The purpose of this work was to detect MUC1 variations during pregnancy and lactation as well as to study the possible induction of a humoral immune response against MUC1 in these conditions. We employed ELISA techniques to measure MUC1 (CASA test) and MUC1-CIC (IgM and IgG) using two anti-MUC1 monoclonal antibodies (MAbs): C595 and SM3. Statistical analysis was performed using the ANOVA test. The pooled results pertaining to pregnant versus non-pregnant women were compared and significant differences were observed in MUC1 and MUC1-CIC-lgM levels detected with both MAbs; the MUC1-CIC-lgG levels detected with C595 were increased in the pregnant group while the MUC1-CIC-lgG levels detected with SM3 did not show any significant differences. When the results were compared between lactating and non-lactating women, no significant differences were found. In conclusion, MUC1 and MUC1-CIC-lgM, detected with both MAbs, and MUC1-CIC-4gG levels detected with the MAb C595 are apparently induced by pregnancy.     

Differential binding of Pseudomonas aeruginosa to normal and cystic fibrosis tracheobronchial mucins

Pseudomonas aeruginosa infection is a leading cause of deteriorationof pulmonary function in patients with cystic fibrosis (CF).The interaction of the bacterium with CF and non-CF tracheobronchialmucins was examined to understand the biochemical basis forthe high susceptibility of the lungs of CF patients to infectionby P.aeruginosa. The binding of radiolabelled bacteria to puremucins in solid-phase assays was not significantly above non-specificbinding to various blocking agents, such as bovine serum albumin,Tween 20, milk powder and polyvinyl pyrrolidine. Further, therewas a tendency for the bacteria to be excluded from plasticwells and membranes coated with mucin. Therefore, an indirectapproach involving the binding of radiolabelled P.aeruginosato asialo GM₁ ganglioside, the putative receptor for the bacteriaon tracheal cells, was used to compare the interaction of CFand non-CF mucins with the bacteria. Highly purified preparationsof CF mucin were consistently better inhibitors of the bindingof the bacteria to asialo GM₁ ganglioside than non-CF mucinpreparations. In the case of the binding of a stable mucoidstrain, the difference was statistically significant (P <0.001) at all concentrations of mucin tested. For the non-mucoidstrain, the difference was significant only at the higher concentrations.Of the saccharides tested similarly, sialyl lactose and theoligosaccharide portion of asialo GM₁ were found to be goodinhibitors. The increased binding of the bacteria to CF mucinwas further confirmed by a solution binding assay in which thebinding of ¹²⁵I-labelled mucin to unlabelled bacteria was determined.The binding of the bacteria to labelled CF and non-CF mucincould be inhibited by an excess of unlabelled human tracheobronchialmucin, but not by unrelated mucins, hyaluronic acid, alginicacid, bovine serum albumin and tetramethyl urea. The higherbinding of CF mucin, particularly to the mucoid strain of P.aeruginosa,is interesting and provides a model system to further investigatethe biochemical parameters of the interaction. asialo GM₁ ganglioside cystic fibrosis Pseudomonas aeruginosa respiratory mucins saccharide inhibitors.     

The structure of tracheobronchial mucins from cystic fibrosis and control patients.

Tracheobronchial mucin samples from control and cystic fibrosis patients were purified by gel filtration chromatography on Sephacryl S-1000 and by density gradient centrifugation. Normal secretions contained high molecular weight (approximately 10(7] mucins, whereas the cystic fibrosis secretions contained relatively small amounts of high molecular weight mucin together with larger quantities of lower molecular weight mucin fragments. These probably represent products of protease digestion. Reducing the disulfide bonds in either the control or cystic fibrosis high molecular weight mucin fractions released subunits of approximately 2000 kDa. Treating these subunits with trypsin released glycopeptides of 300 kDa. Trypsin treatment of unreduced mucin also released fragments of 2000 kDa that could be converted into 300-kDa glycopeptides upon disulfide bond reduction. Thus, protease-susceptible linkages within these mucins must be cross-linked by disulfide bonds so that the full effects of proteolytic degradation of mucins remain cryptic until disulfide bonds are reduced. Since various combinations of protease treatment and disulfide bond reduction release either 2000- or 300-kDa fragments, these fragments must represent important elements of mucin structure. The high molecular weight fractions of cystic fibrosis mucins appear to be indistinguishable from control mucins. Their amino acid compositions are the same, and various combinations of disulfide bond reduction and protease treatment release products of identical size and amino acid composition. Sulfate and carbohydrate compositions did vary considerably from sample to sample, but the limited number of samples tested did not demonstrate a cystic fibrosis-specific pattern. Thus, tracheobronchial mucins from cystic fibrosis and control patients are very similar, and both share the same generalized structure previously determined for salivary, cervical, and intestinal mucins.     

Lack of the intestinal Muc1 mucin impairs cholesterol uptake and absorption but not fatty acid uptake in Muc1-/- mice

Before cholesterol and fatty acid molecules in the small intestinal lumen can interact with their possible transporters for uptake and absorption, they must pass through a diffusion barrier, which may modify the kinetics of nutrient assimilation. This barrier includes an unstirred water layer and a surface mucous coat, which is located at the intestinal lumen-membrane interface. In the present study, we investigated whether disruption of the mucin gene (Muc)1 may influence intestinal uptake and absorption of cholesterol and fatty acid in male Muc1(-/-) mice. The wild-type mice displayed relatively high levels of Muc1, Muc2, Muc3, and Muc4 mRNAs and relatively low levels of Muc5ac and Muc5b mRNAs in the small intestine. The absence of Muc1 mRNA and protein in the small intestines of Muc1(-/-) mice confirmed complete knockout of the Muc1 gene, but the mRNA expression for other mucin genes remained unchanged. Intestinal uptake and absorption of cholesterol but not palmitic acid were significantly reduced in Muc1(-/-) mice compared with the wild-type mice. However, knockout of the Muc1 gene did not impair either expression levels of the genes that encode intestinal sterol efflux transporters Abcg5 and Abcg8 and fatty acid transporter Fatp4 or small intestinal transit rates. We conclude that physiological levels of the epithelial mucin produced by the Muc1 gene are necessary for normal intestinal uptake and absorption of cholesterol in mice. Our study implies that because cholesterol absorption efficiency is reduced by approximately 50% in Muc1-deficient mice, there may be one or more additional pathways for cholesterol absorption.     

Increased FGF1-FGFRc expression in idiopathic pulmonary fibrosis

Background

Recent clinical studies show that tyrosine kinase inhibitors slow the rate of lung function decline and decrease the number of acute exacerbations in patients with Idiopathic Pulmonary Fibrosis (IPF). However, in the murine bleomycin model of fibrosis, not all tyrosine kinase signaling is detrimental. Exogenous ligands Fibroblast Growth Factor (FGF) 7 and 10 improve murine lung repair and increase survival after injury via tyrosine kinase FGF receptor 2b-signaling. Therefore, the level and location of FGF/FGFR expression as well as the exogenous effect of the most highly expressed FGFR2b ligand, FGF1, was analyzed on human lung fibroblasts.

Methods

FGF ligand and receptor expression was evaluated in donor and IPF whole lung homogenates using western blotting and qPCR. Immunohistochemistry for FGF1 and FGFR1/2/3/4 were performed on human lung tissue. Lastly, the effects of FGF1, a potent, multi-FGFR ligand, were studied on primary cultures of IPF and non-IPF donor fibroblasts. Western blots for pro-fibrotic markers, proliferation, FACS for apoptosis, transwell assays and MetaMorph analyses on cell cultures were performed.

Results

Whole lung homogenate analyses revealed decreased FGFR b-isoform expression, and an increase in FGFR c-isoform expression. Of the FGFR2b-ligands, FGF1 was the most significantly increased in IPF patients; downstream targets of FGF-signaling, p-ERK1/2 and p-AKT were also increased. Immunohistochemistry revealed FGF1 co-localization within basal cell sheets, myofibroblast foci, and Surfactant protein-C positive alveolar epithelial type-II cells as well as co-localization with FGFR1, FGFR2, FGFR3, FGFR4 and myofibroblasts expressing the migratory marker Fascin. Both alone and in the presence of heparin, FGF1 led to increased MAPK-signaling in primary lung fibroblasts. While smooth muscle actin was unchanged, heparin + FGF1 decreased collagen production in IPF fibroblasts. In addition, FGF1 + heparin increased apoptosis and cell migration. The FGFR inhibitor (PD173074) attenuated these effects.

Conclusions

Strong expression of FGF1/FGFRs in pathogenic regions of IPF suggest that aberrant FGF1-FGFR signaling is increased in IPF patients and may contribute to the pathogenesis of lung fibrosis by supporting fibroblast migration and increased MAPK-signaling.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0242-2) contains supplementary material, which is available to authorized users.